Biochemistry, Genetics and Molecular Biology › Molecular Biology

PI3K/AKT/mTOR signaling in cancer

Works Count: 34547

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Description

This cluster of papers focuses on the mTOR signaling pathway, particularly its role in growth control, metabolism, and disease, with a specific emphasis on cancer. The PI3K/AKT pathway, Raptor, TSC2, and rapamycin are central to the research discussed in these papers.

Keywords

mTOR; signaling; PI3K/AKT pathway; cancer; growth control; metabolism; Raptor; TSC2; rapamycin; cell growth

Oncogenic kinase signalling

2001-05-01

Peter Blume‐Jensen , Tony Hunter

A coding-independent function of gene and pseudogene mRNAs regulates tumour biology

2010-06-01

Laura Poliseno , Leonardo Salmena , Jiangwen Zhang +3 more

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Targeting PI3K signalling in cancer: opportunities, challenges and limitations

2009-07-24

Jeffrey A. Engelman

mTOR: a pharmacologic target for autophagy regulation

2015-01-01

Young Chul Kim , Kun‐Liang Guan

mTOR, a serine/threonine kinase, is a master regulator of cellular metabolism. mTOR regulates cell growth and proliferation in response to a wide range of cues, and its signaling pathway is … mTOR, a serine/threonine kinase, is a master regulator of cellular metabolism. mTOR regulates cell growth and proliferation in response to a wide range of cues, and its signaling pathway is deregulated in many human diseases. mTOR also plays a crucial role in regulating autophagy. This Review provides an overview of the mTOR signaling pathway, the mechanisms of mTOR in autophagy regulation, and the clinical implications of mTOR inhibitors in disease treatment.

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Targeting the phosphoinositide 3-kinase pathway in cancer

2009-07-31

Pixu Liu , Hailing Cheng , Thomas M. Roberts +1 more

Prolonged Rapamycin Treatment Inhibits mTORC2 Assembly and Akt/PKB

2006-04-01

Dos D. Sarbassov , Siraj M. Ali , Shomit Sengupta +5 more

New insights into tumor suppression: PTEN suppresses tumor formation by restraining the phosphoinositide 3-kinase/AKT pathway

1999-04-13

Lewis C. Cantley , Benjamin G. Neel

The most recently discovered PTEN tumor suppressor gene has been found to be defective in a large number of human cancers. In addition, germ-line mutations in PTEN result in the … The most recently discovered PTEN tumor suppressor gene has been found to be defective in a large number of human cancers. In addition, germ-line mutations in PTEN result in the dominantly inherited disease Cowden syndrome, which is characterized by multiple hamartomas and a high proclivity for developing cancer. A series of publications over the past year now suggest a mechanism by which PTEN loss of function results in tumors. PTEN appears to negatively control the phosphoinositide 3-kinase signaling pathway for regulation of cell growth and survival by dephosphorylating the 3 position of phosphoinositides.

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AKT/PKB Signaling: Navigating Downstream

2007-06-01

Brendan D. Manning , Lewis C. Cantley

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TOR, a Central Controller of Cell Growth

2000-10-01

Tobias Schmelzle , Michael N. Hall

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Human insulin receptor and its relationship to the tyrosine kinase family of oncogenes

1985-02-01

A Ullrich , Jeffrey R. Bell , E. Y. Chen +7 more

Negative Regulation of PKB/Akt-Dependent Cell Survival by the Tumor Suppressor PTEN

1998-10-01

Vuk Stambolic , Akira Suzuki , José Luís de la Pompa +7 more

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Molecular mechanisms of mTOR-mediated translational control

2009-04-02

Xiaoju Max , John Blenis

The protein kinase encoded by the Akt proto-oncogene is a target of the PDGF-activated phosphatidylinositol 3-kinase

1995-06-01

Thomas Franke , Sung‐Il Yang , Tung O. Chan +5 more

mTOR Interacts with Raptor to Form a Nutrient-Sensitive Complex that Signals to the Cell Growth Machinery

2002-07-01

Do‐Hyung Kim , Dos D. Sarbassov , Siraj M. Ali +5 more

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The evolution of phosphatidylinositol 3-kinases as regulators of growth and metabolism

2006-07-18

Jeffrey A. Engelman , Ji Luo , Lewis C. Cantley

The functions and regulation of the PTEN tumour suppressor

2012-04-04

Min Sup Song , Leonardo Salmena , Pier Paolo Pandolfi

The Rag GTPases Bind Raptor and Mediate Amino Acid Signaling to mTORC1

2008-05-23

Yasemin Sancak , Timothy R. Peterson , Yoav D. Shaul +4 more

The multiprotein mTORC1 protein kinase complex is the central component of a pathway that promotes growth in response to insulin, energy levels, and amino acids and is deregulated in common … The multiprotein mTORC1 protein kinase complex is the central component of a pathway that promotes growth in response to insulin, energy levels, and amino acids and is deregulated in common cancers. We find that the Rag proteins—a family of four related small guanosine triphosphatases (GTPases)—interact with mTORC1 in an amino acid–sensitive manner and are necessary for the activation of the mTORC1 pathway by amino acids. A Rag mutant that is constitutively bound to guanosine triphosphate interacted strongly with mTORC1, and its expression within cells made the mTORC1 pathway resistant to amino acid deprivation. Conversely, expression of a guanosine diphosphate–bound Rag mutant prevented stimulation of mTORC1 by amino acids. The Rag proteins do not directly stimulate the kinase activity of mTORC1, but, like amino acids, promote the intracellular localization of mTOR to a compartment that also contains its activator Rheb.

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Protein kinase B (c-Akt) in phosphatidylinositol-3-OH kinase signal transduction

1995-08-01

Boudewijn Burgering , Paul J. Coffer

Regulation of Neuronal Survival by the Serine-Threonine Protein Kinase Akt

1997-01-31

Henryk Dudek , Sandeep Robert Datta , Thomas Franke +6 more

A signaling pathway was delineated by which insulin-like growth factor 1 (IGF-1) promotes the survival of cerebellar neurons. IGF-1 activation of phosphoinositide 3-kinase (PI3-K) triggered the activation of two protein … A signaling pathway was delineated by which insulin-like growth factor 1 (IGF-1) promotes the survival of cerebellar neurons. IGF-1 activation of phosphoinositide 3-kinase (PI3-K) triggered the activation of two protein kinases, the serine-threonine kinase Akt and the p70 ribosomal protein S6 kinase (p70 S6K ). Experiments with pharmacological inhibitors, as well as expression of wild-type and dominant-inhibitory forms of Akt, demonstrated that Akt but not p70 S6K mediates PI3-K-dependent survival. These findings suggest that in the developing nervous system, Akt is a critical mediator of growth factor-induced neuronal survival.

The activation of Akt/PKB signaling pathway and cell survival

2005-01-01

Gang Song , Gaoliang Ouyang , Shideng Bao

Akt/PKB is a serine/threonine protein kinase that functions as a critical regulator of cell survival and proliferation. Akt/PKB family comprises three highly homologous members known as PKBα/Akt1, PKBβ/Akt2 and PKBγ/Akt3 … Akt/PKB is a serine/threonine protein kinase that functions as a critical regulator of cell survival and proliferation. Akt/PKB family comprises three highly homologous members known as PKBα/Akt1, PKBβ/Akt2 and PKBγ/Akt3 in mammalian cells. Similar to many other protein kinases, Akt/PKB contains a conserved domain structure including a specific PH domain, a central kinase domain and a carboxyl-terminal regulatory domain that mediates the interaction between signaling molecules. Akt/PKB plays important roles in the signaling pathways in response to growth factors and other extracellular stimuli to regulate several cellular functions including nutrient metabolism, cell growth, apoptosis and survival. This review surveys recent developments in understanding the molecular mechanisms of Akt/PKB activation and its roles in cell survival in normal and cancer cells.

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PTEN activation contributes to tumor inhibition by trastuzumab, and loss of PTEN predicts trastuzumab resistance in patients

2004-08-01

Yoichi Nagata , Keng‐Hsueh Lan , Xiaoyan Zhou +7 more

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Rictor, a Novel Binding Partner of mTOR, Defines a Rapamycin-Insensitive and Raptor-Independent Pathway that Regulates the Cytoskeleton

2004-07-01

Dos D. Sarbassov , Siraj M. Ali , Do‐Hyung Kim +5 more

The Tumor Suppressor, PTEN/MMAC1, Dephosphorylates the Lipid Second Messenger, Phosphatidylinositol 3,4,5-Trisphosphate

1998-05-01

Tomohiko Maehama , Jack E. Dixon

Phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) is a key molecule involved in cell growth signaling. We demonstrated that overexpression of PTEN, a putative tumor suppressor, reduced insulin-induced PtdIns(3,4,5)P3 production in human 293 cells … Phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) is a key molecule involved in cell growth signaling. We demonstrated that overexpression of PTEN, a putative tumor suppressor, reduced insulin-induced PtdIns(3,4,5)P3 production in human 293 cells without effecting insulin-induced phosphoinositide 3-kinase activation. Further, transfection of the catalytically inactive mutant of PTEN (C124S) caused PtdIns(3,4,5)P3 accumulation in the absence of insulin stimulation. Purified recombinant PTEN catalyzed dephosphorylation of PtdIns(3,4,5)P3, specifically at position 3 on the inositol ring. PTEN also exhibited 3-phosphatase activity toward inositol 1,3,4,5-tetrakisphosphate. Our results raise the possibility that PTEN acts in vivo as a phosphoinositide 3-phosphatase by regulating PtdIns(3,4,5)P3 levels. As expected, the C124S mutant of PTEN was incapable of catalyzing dephosphorylation of PtdIns(3,4,5)P3 consistent with the mechanism observed in protein-tyrosine phosphatase-catalyzed reactions. Phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) is a key molecule involved in cell growth signaling. We demonstrated that overexpression of PTEN, a putative tumor suppressor, reduced insulin-induced PtdIns(3,4,5)P3 production in human 293 cells without effecting insulin-induced phosphoinositide 3-kinase activation. Further, transfection of the catalytically inactive mutant of PTEN (C124S) caused PtdIns(3,4,5)P3 accumulation in the absence of insulin stimulation. Purified recombinant PTEN catalyzed dephosphorylation of PtdIns(3,4,5)P3, specifically at position 3 on the inositol ring. PTEN also exhibited 3-phosphatase activity toward inositol 1,3,4,5-tetrakisphosphate. Our results raise the possibility that PTEN acts in vivo as a phosphoinositide 3-phosphatase by regulating PtdIns(3,4,5)P3 levels. As expected, the C124S mutant of PTEN was incapable of catalyzing dephosphorylation of PtdIns(3,4,5)P3 consistent with the mechanism observed in protein-tyrosine phosphatase-catalyzed reactions. A recently identified candidate tumor suppressor gene, PTEN/MMAC1, shares sequence identity with the family of protein-tyrosine phosphatases (PTPases) 1The abbreviations used are: PTPase, protein-tyrosine phosphatase; PI, phosphoinositide; PtdIns(3,4,5)P3, phosphatidylinositol 3,4,5-trisphosphate; PtdIns(4,5)P2, phosphatidylinositol 4,5-bisphosphate; Ins(1,3,4,5)P4, inositol 1,3,4,5-tetrakisphosphate; Ins(1,4,5)P3, inositol 1,4,5-trisphosphate; DTT, dithiothreitol; VHR, VH1-related. (1Fauman E.B. Saper M.A. Trends Biochem. Sci. 1996; 21: 413-417Abstract Full Text PDF PubMed Scopus (319) Google Scholar). Deletions and mutations within the PTEN gene have been observed in several cancer cell types and tumor cell lines (2Li J. Yen C. Liaw D. Podsypanina K. Bose S. Wang S.I. Puc J. Miliaresis C. Rodgers L. McCombie R. Bigner S.H. Giovanella B.C. Ittmann M. Tycko B. Hibshoosh H. Wigler M.H. Parsons R. Science. 1997; 275: 1943-1947Crossref PubMed Scopus (4315) Google Scholar, 3Steck P.A. Pershouse M.A. Jasser S.A. Yung W.K. Lin H. Ligon A.H. Langford L.A. Baumgard M.L. Hattier T. Davis T. Frye C. Hu R. Swedlund B. Teng D.H. Tavtigian S.V. Nat. Genet. 1997; 15: 356-362Crossref PubMed Scopus (2526) Google Scholar). Additional evidence that PTEN functions as a tumor suppressor was obtained by Furnari et al. (4Furnari F.B. Lin H. Huang H.S. Cavenee W.K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12479-12484Crossref PubMed Scopus (382) Google Scholar), who showed that PTEN had a growth suppressor activity in glioma cells. PTEN encodes the active site consensus motif HCXXGXXR(S/T) found in all PTPases. In contrast, the recombinant protein is a poor catalyst toward both phosphoproteins and peptide substrates with the highest activity of PTEN observed toward the highly negatively charged, multiply phosphorylated polymer of (Glu-Tyr)n (5Li D.M. Sun H. Cancer Res. 1997; 57: 2124-2129PubMed Google Scholar, 6Myers M.P. Stolarov J.P. Eng C. Li J. Wang S.I. Wigler M.H. Parsons R. Tonks N.K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9052-9057Crossref PubMed Scopus (738) Google Scholar). Based on these observations we thought it possible that PTEN could catalyze the dephosphorylation of acidic nonproteinaceous substrate. Identification of possible in vivo substrates would not only suggest a possible physiological function of PTEN, but they might also provide insight into how PTEN functions as a tumor suppressor. PtdIns(3,4,5)P3 is an important second messenger involved in cell growth signaling (7Stephens L.R. Jackson T.R. Hawkins P.T. Biochim. Biophys. Acta. 1993; 1179: 27-75Crossref PubMed Scopus (426) Google Scholar). PtdIns(3,4,5)P3 is specifically produced from PtdIns(4,5)P2 by PI 3-kinase upon stimulation by a variety of ligands (7Stephens L.R. Jackson T.R. Hawkins P.T. Biochim. Biophys. Acta. 1993; 1179: 27-75Crossref PubMed Scopus (426) Google Scholar). Recent studies have identified that PtdIns(3,4,5)P3 can directly activate Akt, which in turn activates p70 S6 kinase and inhibits glycogen synthase kinase-3 (8Marte B.M. Downward J. Trends Biochem. Sci. 1997; 22: 355-358Abstract Full Text PDF PubMed Scopus (649) Google Scholar, 9Downward J. Science. 1998; 279: 673-674Crossref PubMed Scopus (181) Google Scholar). Although there are several phosphoinositide 5-phosphatases, the mechanism of regulation and particularly the degradation pathway of PtdIns(3,4,5)P3 in vivois still unclear (10Woscholski R. Parker P.J. Trends Biochem. Sci. 1997; 22: 427-431Abstract Full Text PDF PubMed Scopus (71) Google Scholar, 11Guilherme A. Klarlund J.K. Krystal G. Czech M.P. J. Biol. Chem. 1996; 271: 29533-29536Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). In the present study we demonstrate that recombinant PTEN has PtdIns(3,4,5)P3 3-phosphatase activity. In addition, we provide evidence that PTEN may act in vivo as a regulator of PtdIns(3,4,5)P3, which produces a substrate that can be recycled by PI 3-kinase. The coding sequence of human PTEN and the C124S mutant of PTEN (gift from Yi Zhao) were amplified by polymerase chain reaction using 5′ primer (5′-CCGGTACCGCCACCATGGACTACAAGGACGACGATGACAAGACAGCCATCATCAAAGAG-3′) and 3′ primer (5′-CCGTCGACTCAGACTTTTGTAATTTGTG-3′). The product was cleaved with KpnI and SalI and ligated into the KpnI/SalI sites of pCMV5 (gift from David W. Russell) to produce FLAG-tagged PTEN/pCMV5 and PTEN(CS)/pCMV5. Human 293 cells were cultured on a 6-well plate, and transfection of the cells were performed as described (12Li L. Ernsting B.R. Wishart M.J. Lohse D.L. Dixon J.E. J. Biol. Chem. 1997; 272: 29403-29406Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar) using 1 μg of the constructs. The efficiency of transfection was about 80% in this condition. 48 h after the transfection, the cells were serum-starved and labeled with [32P]Pi (100 μCi/ml) for 4 h. The cells were stimulated by incubation with insulin (0.1 μg/ml) for 2.5 min at 37 °C, and the stimulation was quenched by the addition of 0.93 ml of CH3OH/CHCl3/1% HClO4 (50/25/18, v/v/v). The lipids were extracted and separated on a TLC plate as described (13Okada T. Hazeki O. Ui M. Katada T. Biochem. J. 1996; 317: 475-480Crossref PubMed Scopus (49) Google Scholar) to determine the amount of [32P]PtdIns(3,4,5)P3. To analyze the expression of FLAG-tagged PTEN protein, the transfected cells were lysed in Laemmli sample buffer and subjected to SDS-polyacrylamide gel electrophoresis. The samples were transferred to Immobilon filter (Millipore) and immunoblotted with anti-FLAG M2 antibody (Kodak), and the signal was visualized by Enhanced Chemiluminescence (Amersham Pharmacia Biotech) using the manufacturer's recommended protocols. Transfection, starvation and stimulation of human 293 cells were carried out as described above in the absence of radiolabel. After the stimulation, the cells were lysed, followed by immunoprecipitation as described (14Kurosu H. Maehama T. Okada T. Yamamoto T. Hoshino S. Fukui Y. Ui M. Hazeki O. Katada T. J. Biol. Chem. 1997; 272: 24252-24256Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar) using 4G10 anti-phosphotyrosine antibody (Upstate Biotechnology, Inc.). To analyze the PI 3-kinase activity of the immunoprecipitants, the sample was incubated for 10 min at 37 °C in 33 mm Tris-HCl (pH 7.4), 2.5 mm EGTA, 5 mm MgCl2, 30 mm NaCl, 0.1 mm [γ-32P]ATP (20 μCi), 0.1 mg/ml PtdIns(4,5)P2, and 0.15 mg/ml phosphatidylserine. The reaction was terminated by the addition of 0.47 ml of CH3OH/CHCl3/6% HClO4(30/15/2, v/v/v), and the phospholipids were extracted and separated on a TLC plate as described (13Okada T. Hazeki O. Ui M. Katada T. Biochem. J. 1996; 317: 475-480Crossref PubMed Scopus (49) Google Scholar). The expression vector for PTEN was constructed by ligating a bluntedNdeI/SalI fragment from PTEN/pT7–7 (12Li L. Ernsting B.R. Wishart M.J. Lohse D.L. Dixon J.E. J. Biol. Chem. 1997; 272: 29403-29406Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar) into theSmaI site of pGEX-KG (15Guan K.L. Dixon J.E. Anal. Biochem. 1991; 192: 262-267Crossref PubMed Scopus (1641) Google Scholar). This vector was used to transformEscherichia coli strain JM109. Protein expression in 4-liter culture was carried out as described (16Lohse D.L. Denu J.M. Santoro N. Dixon J.E. Biochemistry. 1997; 36: 4568-4575Crossref PubMed Scopus (160) Google Scholar). All of the following procedures were performed at 4 °C. Cells were harvested, resuspended in 80 ml of lysis buffer (20 mm Tris-HCl (pH 8), 2 mm EDTA, 2 mm DTT, 300 mm NaCl, and 1 mm phenylmethylsulfonyl fluoride) and lysed by sonication. The crude lysate was diluted by the addition of 720 ml of the lysis buffer containing 1% (w/v) Triton X-100 and stirred for 30 min. Cell debris was removed by centrifugation at 27,000 ×g for 20 min. A 5-ml slurry of glutathione-Sepharose 4B (Amersham Pharmacia Biotech) was then added to the supernatant. After the incubation for 2 h, the resin was packed in a column and washed with 100 ml of the lysis buffer, and the glutathioneS-transferase-fusioned PTEN was eluted with 10 ml of the lysis buffer containing 10 mm glutathione. After overnight incubation with thrombin, the eluate was dialyzed against the lysis buffer and passed through glutathione-Sepharose 4B column, followed by a p-aminobezamidine-agarose column for adsorption of glutathione S-transferase and thrombin, respectively. Then the eluate was diluted with equal volume of TED buffer (20 mm Tris-HCl (pH 8), 2 mm EDTA, 2 mmDTT) and applied to MonoQ HR5/5 (Amersham Pharmacia Biotech) column equilibrated with TED buffer containing 150 mm NaCl. PTEN was eluted with a linear gradient of NaCl (150–500 mm, 20 ml), followed by concentration using Centricon-30 (Amicon) and stored at −80 °C until use. PtdIns(3,4,5)P3 phosphatase assay was performed at 37 °C in a buffer (20 μl) consisting of 100 mm Tris-HCl (pH 8), 10 mm DTT, [32P]PtdIns(3,4,5)P3 and 1 μg of purified PTEN. The reaction was terminated by the addition of 0.47 ml of CH3OH/CHCl3/6% HClO4 (30/15/2, v/v/v). Then the phospholipids were extracted and separated on a TLC plate as described (13Okada T. Hazeki O. Ui M. Katada T. Biochem. J. 1996; 317: 475-480Crossref PubMed Scopus (49) Google Scholar). To prepare [32P]PtdIns(3,4,5)P3, the phosphorylation of PtdIns(4,5)P2 by PI 3-kinase using [γ-32P]ATP was carried out as described above. Then the phospholipids were extracted as described (13Okada T. Hazeki O. Ui M. Katada T. Biochem. J. 1996; 317: 475-480Crossref PubMed Scopus (49) Google Scholar) and stored at −20 °C until use. The PI 3-kinase used was prepared by immunoprecipitation from the 293 cell lysate with anti-p85 antibody (Upstate Biotechnology, Inc.) as described (14Kurosu H. Maehama T. Okada T. Yamamoto T. Hoshino S. Fukui Y. Ui M. Hazeki O. Katada T. J. Biol. Chem. 1997; 272: 24252-24256Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar). For identification of the dephosphorylation site (see Fig. 2 B), dephosphorylation of PtdIns(3,4,5)P3 by PTEN was carried out in a buffer (20 μl) consisting of 100 mm Tris-HCl (pH 8), 10 mm DTT, 0.1 mg/ml of PtdIns(3,4,5)P3 (BIOMOL), 0.15 mg/ml of phosphatidylserine, and 1 μg of purified PTEN. The reaction was terminated by the addition of 0.47 ml of CH3OH/CHCl3/6% HClO4 (30/15/2, v/v). The phospholipids were extracted as described (13Okada T. Hazeki O. Ui M. Katada T. Biochem. J. 1996; 317: 475-480Crossref PubMed Scopus (49) Google Scholar), dried, and then used for PI 3-kinase-catalyzed reaction. Inositol phosphatase assays were performed using commercially available [3H]inositol phosphate (NEN) as substrates. Assay was carried out at 37 °C in a buffer (20 μl) consisting of 100 mm Tris-HCl (pH 8), 10 mm DTT, 60 μm [3H]inositol phosphate (0.01 μCi) and 1 μg of enzyme. After an incubation of 30 min, the reaction was terminated by the addition of 1 ml of stop solution. Then, to separate the dephosphorylated product from the substrate, the sample was applied to AG1-X8 column (0.5 ml) equilibrated with the stop solution. Dephosphorylated [3H]inositol phosphate was eluted with 5 ml of the stop solution, whereas the substrate remained in the column, and the radioactivity of the eluate was measured. The stop solutions used were 0.2 m HCOONH4/0.1 mHCOOH, 0.4 m HCOONH4/0.1 m HCOOH, and 0.7 m HCOONH4/0.1 m HCOOH for [3H]inositol 1, 4-bisphosphate, [3H]Ins(1,4,5)P3, and [3H]Ins(1,3,4,5)P4, respectively. Recombinant human VHR, Cdc25B, and PTP1D were kindly gifts from Harris Vikis, Elizabeth Gottlin, and Jin Zhou, respectively. For kinetic analysis (see Fig. 3 B), inositol phosphatase activity was assayed in the same buffer as described above using 10–150 μmIns(1,3,4,5)P4 instead of [3H]inositol phosphate. After an incubation of 1 min at 37 °C, the reaction was terminated by the addition of 1 ml of ice-cold water, and the amount of Ins(1,4,5)P3 produced was estimated using BIOTRAK Ins(1,4,5)P3 detection kit (Amersham Pharmacia Biotech) following the manufacturer's recommended protocol. Kinetic constants were determined using KaleidaGraph software (Abelbech).Figure 3Inositol phosphatase activity of PTEN. A, inositol phosphatase activity of various enzymes was assayed using [3H]inositol phosphate as indicated. After the incubation for 30 min, dephosphorylated [3H]inositol phosphate was separated as described under "Experimental Procedures," and then the radioactivity of the dephosphorylated products was counted. Similar results were obtained in a repeated experiment. WT, wild type; CS, C124S mutant.B, initial rate of PTEN-catalyzed dephosphorylation of Ins(1,3,4,5)P4 was determined as described under "Experimental Procedures" using various concentration of Ins(1,3,4,5)P4. The Lineweaver-Burk plot is shown ininset. The data are presented as the means ± S.E. of triplicate determinations.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Although PTEN has the consensus sequence of a PTPase, it dephosphorylates p-nitrophenylphosphate and other artificial protein substrates poorly, having the highest catalytic activity with the highly negatively charged, multiply phosphorylated polymer of (Glu-Tyr)n (5Li D.M. Sun H. Cancer Res. 1997; 57: 2124-2129PubMed Google Scholar, 6Myers M.P. Stolarov J.P. Eng C. Li J. Wang S.I. Wigler M.H. Parsons R. Tonks N.K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9052-9057Crossref PubMed Scopus (738) Google Scholar). This observation raised the distinct possibility that PTEN might utilize acidic substrates other than Tyr or Ser/Thr phosphoproteins. In order to explore this possibility, we transfected PTEN into 293 cells and analyzed the changes in cellular phospholipids. PtdIns(3,4,5)P3is an important second messenger in the regulation of cell growth (7Stephens L.R. Jackson T.R. Hawkins P.T. Biochim. Biophys. Acta. 1993; 1179: 27-75Crossref PubMed Scopus (426) Google Scholar). In human 293 cells, insulin stimulates PI 3-kinase activity (Fig.1 C, lanes 1 and2), resulting in an increase in PtdIns(3,4,5)P3(Fig. 1 A, lanes 1 and 2). When PTEN was overexpressed in the 293 cells, the insulin-induced PtdIns(3,4,5)P3 levels were significantly reduced in a dose-dependent manner (Fig. 1, A andB), whereas no effect on the activation of PI 3-kinase was observed (Fig. 1 C, lanes 2 and 4). Because PtdIns(3,4,5)P3 is specifically produced by PI 3-kinase, this result suggests that PTEN directly effects the turnover of PtdIns(3,4,5)P3. Additionally, overexpression of the catalytically inactive mutant (C124S) of PTEN (see Fig. 3 A) caused an accumulation of PtdIns(3,4,5)P3 in the absence of insulin stimulation (Fig. 1 D, lanes 1 and3), whereas overexpression of the mutant did not affect PI 3-kinase activity (data not shown). These results suggest that PTEN potentially regulates PtdIns(3,4,5)P3 levels in cells without alteration of the insulin-stimulated PI 3-kinase activity. To investigate the possibility that PTEN has PtdIns(3,4,5)P3phosphatase activity, the recombinant enzyme was expressed inE. coli and purified to homogeneity (data not shown). Radiolabel was incorporated in position 3 of the substrate, [32P]PtdIns(3,4,5)P3, using PtdIns(4,5)P2, PI 3-kinase and [γ-32P]ATP. When [32P]PtdIns(3,4,5)P3 was incubated with the purified PTEN, the radiolabel of [32P]PtdIns(3,4,5)P3 rapidly disappeared from the lipid phase (Fig. 2 A,inset) while coincidentally appearing in the aqueous phase (Fig. 2 A), suggesting the release of inorganic phosphate. In order to conclusively prove that the only phosphate that had been cleaved was at position 3 of the phosphoinositide, as opposed to other possible cleavages that could also generate a water-soluble radiolabel, we used the product of the reaction of PTEN as a substrate for PI 3-kinase. When the product was treated with PI 3-kinase, [32P]PtdIns(3,4,5)P3 was reformed, thereby providing further evidence that the two products generated by PTEN were inorganic phosphate and PtdIns(4,5)P2 (Fig. 2 B). Under similar conditions, PTEN also exhibited 3-phosphatase activity on phosphatidylinositol 3-monophosphate and phosphatidylinositol 3,4-bisphosphate; however, dephosphorylation of these phosphoinositides occurred at ~20% the rate observed with PtdIns(3,4,5)P3(data not shown). PTPases including PTP1D and dual-specific phosphatases (VHR, Cdc25B) exhibited no phosphoinositide phosphatase activity (data not shown). To more carefully dissect the specific nature of the catalytic activity of PTEN toward PtdIns(3,4,5)P3, we asked if PTEN displayed activity toward inositol phosphates. PTEN can dephosphorylate Ins(1,3,4,5)P4, whereas tyrosine-specific (PTP1D) and the dual-specific phosphatases (Cdc25B, VHR) exhibited no activity toward this inositol phosphate (Fig.3 A). Again, the PTEN-catalyzed reaction was specific for the position 3 of Ins(1,3,4,5)P4. Other inositol phosphates that do not have a phosphate at the position 3 on the inositol ring were not dephosphorylated by PTEN (Fig.3 A). The dephosphorylated product was identified as Ins(1,4,5)P3 using the Ins(1,4,5)P3-binding protein (Fig. 3 B). These results demonstrate that PTEN also has 3-phosphatase activity toward inositol phosphate. Both Ins(1,4,5)P3 and Ins(1,3,4,5)P4 have been proposed to be a functional second messenger responsible for the intracellular calcium signaling (17Putney J.W. Bird G.S.J. Endocr. Rev. 1993; 14: 610-631Crossref PubMed Scopus (486) Google Scholar). Interestingly, Ins(1,3,4,5)P4 can associate and activate a GTPase-activating protein (18Cullen P.J. Hsuan J.J. Truong O. Letcher A.J. Jackson T.R. Dawson A.P. Irvine R.F. Nature. 1995; 376: 527-530Crossref PubMed Scopus (286) Google Scholar). In contrast to PtdIns(3,4,5)P3, Ins(1,3,4,5)P4 is water-soluble and therefore was used to assess the significance of ourin vitro observations by determining the kinetic parameters for the PTEN-catalyzed dephosphorylation occurred at position 3 on the inositol ring. The K m andV max values for Ins(1,3,4,5)P4 were 98.9 μm and 8.49 nmol/min/mg (k cat, 0.49 min−1), respectively (Fig. 3 B). The K m value of 98.9 μm is 250-fold lower than the K m ofp-nitrophenylphosphate, which is 25.6 mm. Similar comparisons with the phosphorylated polymer (Glu-Tyr)nwere difficult to assess because a detailed kinetic analysis was not performed with this substrate (6Myers M.P. Stolarov J.P. Eng C. Li J. Wang S.I. Wigler M.H. Parsons R. Tonks N.K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9052-9057Crossref PubMed Scopus (738) Google Scholar), and it is likely to be phosphorylated at more than one site. The lowV max for the PTEN-catalyzed dephosphorylation of Ins(1,3,4,5)P4 implies that this may not be the preferred substrate in vitro for this enzyme. These in vitro assays may not be reflective of the in vivoactivity of PTEN because the phosphatase could be regulated by phosphorylation, subcellular localization, and/or interaction with other cellular proteins. It is noteworthy that the C terminus of the phosphatase has a consensus PDZ binding site, and we have recently shown that PTEN interacts with several PDZ-containing proteins. 2Y. Zhao and J. E. Dixon, unpublished data. The activity of PTPases and dual-specific phosphatases toward all protein substrates is dependent upon an essential cysteine residue that forms a phosphoenzyme intermediate during catalysis (1Fauman E.B. Saper M.A. Trends Biochem. Sci. 1996; 21: 413-417Abstract Full Text PDF PubMed Scopus (319) Google Scholar). Because PTEN has the HCXXGXXR(S/T) motif conserved in all tyrosine or dual-specific phosphatases, we were interested in determining whether the cysteine was essential for the Ins(1,3,4,5)P4 phosphatase activity. Mutation at Cys124 of PTEN (C124S) resulted in a complete loss of enzyme activity toward Ins(1,3,4,5)P4 (Fig. 3 A). This mutation also resulted in a loss of phosphoinositide phosphatase activity (data not shown). Additionally, PTEN is extremely labile in the absence of thiols in the assay buffer. Optimum concentration of DTT for the PTEN-catalyzed reactions was 10 mm (data not shown). Therefore, we propose that PTEN-catalyzed dephosphorylation of inositol phosphate and phosphoinositide proceeds via a mechanism that is consistent with that described for other PTPases (1Fauman E.B. Saper M.A. Trends Biochem. Sci. 1996; 21: 413-417Abstract Full Text PDF PubMed Scopus (319) Google Scholar). We have established that the recombinant PTEN has phosphoinositide 3-phosphatase and inositol phosphate 3-phosphatase activities. The data shown in Fig. 1 suggest that suppression of insulin-induced PtdIns(3,4,5)P3 production by overexpression of PTEN is due to its phosphoinositide phosphatase activity. In addition, as shown in Fig. 1 D, overexpression of the CS mutant caused PtdIns(3,4,5)P3 accumulation without insulin stimulation. These results strongly suggest that PTEN can act as a regulator of PtdIns(3,4,5)P3 in vivo. Insulin activates PI 3-kinase via tyrosine phosphorylation of insulin receptor substrate-1 catalyzed by the insulin receptor (Fig.4). PtdIns(3,4,5)P3 produced by the PI 3-kinase can then activate Akt-mediated signals (Fig. 4). PtdIns(3,4,5)P3 levels reached a plateau within 3–5 min after the stimulation, and then PtdIns(3,4,5)P3 is degraded by unknown mechanisms (data not shown). Although our results suggest that PTEN can alter PtdIns(3,4,5)P3 levels in 293 cells, it is clear that there are other cellular mechanisms that can also alter phosphoinositide concentrations. For example, Guilherme et al. (11Guilherme A. Klarlund J.K. Krystal G. Czech M.P. J. Biol. Chem. 1996; 271: 29533-29536Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar) reported that a phosphoinositide 5-phosphatase was activated by insulin stimulation, and SHIP (SH2 domain-containing inositol 5-phosphatase) is also well known as a regulator of PtdIns(3,4,5)P3 (10Woscholski R. Parker P.J. Trends Biochem. Sci. 1997; 22: 427-431Abstract Full Text PDF PubMed Scopus (71) Google Scholar, 19Scharenberg A.M. Kinet J.P. Cell. 1996; 87: 961-964Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). If PTEN functions in vivo as a PtdIns(3,4,5)P3phosphatase, it follows that a homozygous deletion/mutation of this tumor suppressor gene could lead to a tumorigenic state through activation of the proto-oncogene, Akt. Specifically, loss of PTEN function would increase cellular levels of PtdIns(3,4,5)P3, thereby resulting in enhanced activation of Akt. In conclusion, although the physiological function of PTEN needs further clarification, we propose that: (i) members of the PTPase family of enzymes having an active site motif HCXXGXXR(S/T) such as PTEN are candidates to regulate intracellular levels of nonproteinaceous substrates as has also been reported for the RNA 5′-triphosphatase activity of CEL-1 (20Takagi T. Moore C.R. Diehn F. Buratowski S. Cell. 1997; 89: 867-873Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar); (ii) the dephosphorylation of phosphoinositide and inositol phosphate by PTEN is specific for position 3 on the inositol ring; and (iii) there are likely to be additional activating and/or localization mechanisms for PTEN within the cell regulating the catalytic activity of this enzyme. We thank Dr. Kim Orth for critical reading of the manuscript.

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The Phosphoinositide 3-Kinase Pathway

2002-05-31

Lewis C. Cantley

Phosphorylated lipids are produced at cellular membranes during signaling events and contribute to the recruitment and activation of various signaling components. The role of phosphoinositide 3-kinase (PI3K), which catalyzes the … Phosphorylated lipids are produced at cellular membranes during signaling events and contribute to the recruitment and activation of various signaling components. The role of phosphoinositide 3-kinase (PI3K), which catalyzes the production of phosphatidylinositol-3,4,5-trisphosphate, in cell survival pathways; the regulation of gene expression and cell metabolism; and cytoskeletal rearrangements are highlighted. The PI3K pathway is implicated in human diseases including diabetes and cancer, and understanding the intricacies of this pathway may provide new avenues for therapuetic intervention.

Prevalence of ras gene mutations in human colorectal cancers

1987-05-01

Johannes L. Bos , Eric R. Fearon , Stanley R. Hamilton +4 more

Defining the Role of mTOR in Cancer

2007-07-01

David A. Guertin , David M. Sabatini

Targeting RAS signalling pathways in cancer therapy

2003-01-01

Julian Downward

Exploiting the PI3K/AKT Pathway for Cancer Drug Discovery

2005-12-01

Bryan T. Hennessy , Debra L. Smith , Prahlad T. Ram +2 more

mTOR Inhibition Induces Upstream Receptor Tyrosine Kinase Signaling and Activates Akt

2006-02-01

Kathryn O’Reilly , F. Rojo , Qing‐Bai She +7 more

Abstract Stimulation of the insulin and insulin-like growth factor I (IGF-I) receptor activates the phosphoinositide-3-kinase/Akt/mTOR pathway causing pleiotropic cellular effects including an mTOR-dependent loss in insulin receptor substrate-1 expression leading … Abstract Stimulation of the insulin and insulin-like growth factor I (IGF-I) receptor activates the phosphoinositide-3-kinase/Akt/mTOR pathway causing pleiotropic cellular effects including an mTOR-dependent loss in insulin receptor substrate-1 expression leading to feedback down-regulation of signaling through the pathway. In model systems, tumors exhibiting mutational activation of phosphoinositide-3-kinase/Akt kinase, a common event in cancers, are hypersensitive to mTOR inhibitors, including rapamycin. Despite the activity in model systems, in patients, mTOR inhibitors exhibit more modest antitumor activity. We now show that mTOR inhibition induces insulin receptor substrate-1 expression and abrogates feedback inhibition of the pathway, resulting in Akt activation both in cancer cell lines and in patient tumors treated with the rapamycin derivative, RAD001. IGF-I receptor inhibition prevents rapamycin-induced Akt activation and sensitizes tumor cells to inhibition of mTOR. In contrast, IGF-I reverses the antiproliferative effects of rapamycin in serum-free medium. The data suggest that feedback down-regulation of receptor tyrosine kinase signaling is a frequent event in tumor cells with constitutive mTOR activation. Reversal of this feedback loop by rapamycin may attenuate its therapeutic effects, whereas combination therapy that ablates mTOR function and prevents Akt activation may have improved antitumor activity. (Cancer Res 2006; 66(3): 1500-8)

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mTOR Signaling in Growth Control and Disease

2012-04-01

Mathieu Laplante , David M. Sabatini

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High Frequency of Mutations of the <i>PIK3CA</i> Gene in Human Cancers

2004-03-15

Yardena Samuels , Zhenghe Wang , Alberto Bardelli +7 more

3,1232,680MetricsTotal Downloads3,123Last 6 Months470Last 12 Months1,013Total Citations2,680Last 6 Months51Last 12 Months112View all metrics 3,1232,680MetricsTotal Downloads3,123Last 6 Months470Last 12 Months1,013Total Citations2,680Last 6 Months51Last 12 Months112View all metrics

Upstream and downstream of mTOR

2004-08-15

Nissim Hay , Nahum Sonenberg

The evolutionarily conserved checkpoint protein kinase, TOR (target of rapamycin), has emerged as a major effector of cell growth and proliferation via the regulation of protein synthesis. Work in the … The evolutionarily conserved checkpoint protein kinase, TOR (target of rapamycin), has emerged as a major effector of cell growth and proliferation via the regulation of protein synthesis. Work in the last decade clearly demonstrates that TOR controls protein synthesis through a stunning number of downstream targets. Some of the targets are phosphorylated directly by TOR, but many are phosphorylated indirectly. In this review, we summarize some recent developments in this fast-evolving field. We describe both the upstream components of the signaling pathway(s) that activates mammalian TOR (mTOR) and the downstream targets that affect protein synthesis. We also summarize the roles of mTOR in the control of cell growth and proliferation, as well as its relevance to cancer and synaptic plasticity.

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TOR Signaling in Growth and Metabolism

2006-02-01

Stephan Wullschleger , Robbie Loewith , Michael N. Hall

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Protein kinases — the major drug targets of the twenty-first century?

2002-04-01

Philip Cohen

mTOR signaling at a glance

2009-10-07

Mathieu Laplante , David M. Sabatini

The mammalian target of rapamycin (mTOR) signaling pathway integrates both intracellular and extracellular signals and serves as a central regulator of cell metabolism, growth, proliferation and survival. Discoveries that have … The mammalian target of rapamycin (mTOR) signaling pathway integrates both intracellular and extracellular signals and serves as a central regulator of cell metabolism, growth, proliferation and survival. Discoveries that have been made over the last decade show that the mTOR pathway is activated during various cellular processes (e.g. tumor formation and angiogenesis, insulin resistance, adipogenesis and T-lymphocyte activation) and is deregulated in human diseases such as cancer and type 2 diabetes. These observations have attracted broad scientific and clinical interest in mTOR. This is highlighted by the growing use of mTOR inhibitors [rapamycin and its analogues (rapalogues)] in pathological settings, including the treatment of solid tumors, organ transplantation, coronary restenosis and rheumatoid arthritis. Here, we highlight and summarize the current understanding of how mTOR nucleates distinct multi-protein complexes, how intra- and extracellular signals are processed by the mTOR complexes, and how such signals affect cell metabolism, growth, proliferation and survival.The mTOR protein is a 289-kDa serine-threonine kinase that belongs to the phospho-inositide 3-kinase (PI3K)-related kinase family and is conserved throughout evolution. The poster depicts an overview of mTOR structural domains. mTOR nucleates at least two distinct multi-protein complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) (reviewed by Guertin and Sabatini, 2007).mTORC1 has five components: mTOR, which is the catalytic subunit of the complex; regulatory-associated protein of mTOR (Raptor); mammalian lethal with Sec13 protein 8 (mLST8, also known as GβL); proline-rich AKT substrate 40 kDa (PRAS40); and DEP-domain-containing mTOR-interacting protein (Deptor) (Peterson et al., 2009). The exact function of most of the mTOR-interacting proteins in mTORC1 still remains elusive. It has been proposed that Raptor might affect mTORC1 activity by regulating assembly of the complex and by recruiting substrates for mTOR (Hara et al., 2002; Kim et al., 2002). The role of mLST8 in mTORC1 function is also unclear, as deletion of this protein does not affect mTORC1 activity in vivo (Guertin et al., 2006). PRAS40 and Deptor have been characterized as distinct negative regulators of mTORC1 (Peterson et al., 2009; Sancak et al., 2007; Vander Haar et al., 2007). When the activity of mTORC1 is reduced, PRAS40 and Deptor are recruited to the complex, where they promote the inhibition of mTORC1. It was proposed that PRAS40 regulates mTORC1 kinase activity by functioning as a direct inhibitor of substrate binding (Wang et al., 2007). Upon activation, mTORC1 directly phosphorylates PRAS40 and Deptor, which reduces their physical interaction with mTORC1 and further activates mTORC1 signaling (Peterson et al., 2009; Wang et al., 2007).mTORC2 comprises six different proteins, several of which are common to mTORC1 and mTORC2: mTOR; rapamycin-insensitive companion of mTOR (Rictor); mammalian stress-activated protein kinase interacting protein (mSIN1); protein observed with Rictor-1 (Protor-1); mLST8; and Deptor. There is some evidence that Rictor and mSIN1 stabilize each other, establishing the structural foundation of mTORC2 (Frias et al., 2006; Jacinto et al., 2006). Rictor also interacts with Protor-1, but the physiological function of this interaction is not clear (Thedieck et al., 2007; Woo et al., 2007). Similar to its role in mTORC1, Deptor negatively regulates mTORC2 activity (Peterson et al., 2009); so far, Deptor is the only characterized endogenous inhibitor of mTORC2. Finally, mLST8 is essential for mTORC2 function, as knockout of this protein severely reduces the stability and the activity of this complex (Guertin et al., 2006).Now that many mTOR-interacting proteins have been identified, additional biochemical studies will be needed to clarify the functions of these proteins in mTOR signaling and their potential implications in health and disease. Below, we discuss current understanding of the functions of mTORC1 and mTORC2.mTORC1 positively regulates cell growth and proliferation by promoting many anabolic processes, including biosynthesis of proteins, lipids and organelles, and by limiting catabolic processes such as autophagy. Much of the knowledge about mTORC1 function comes from the use of the bacterial macrolide rapamycin. Upon entering the cell, rapamycin binds to FK506-binding protein of 12 kDa (FKBP12) and interacts with the FKBP12-rapamycin binding domain (FRB) of mTOR, thus inhibiting mTORC1 functions (reviewed by Guertin and Sabatini, 2007). In contrast to its effect on mTORC1, FKBP12-rapamycin cannot physically interact with or acutely inhibit mTORC2 (Jacinto et al., 2004; Sarbassov et al., 2004). On the basis of these observations, mTORC1 and mTORC2 have been respectively characterized as the rapamycin-sensitive and rapamycin-insensitive complexes. However, this paradigm might not be entirely accurate, as chronic rapamycin treatment can, in some cases, inhibit mTORC2 activity by blocking its assembly (Sarbassov et al., 2006). In addition, recent reports suggest that important mTORC1 functions are resistant to inhibition by rapamycin (Choo et al., 2008; Feldman et al., 2009; Garcia-Martinez et al., 2009; Thoreen et al., 2009).mTORC1 positively controls protein synthesis, which is required for cell growth, through various downstream effectors. mTORC1 promotes protein synthesis by phosphorylating the eukaryotic initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1) and the p70 ribosomal S6 kinase 1 (S6K1). The phosphorylation of 4E-BP1 prevents its binding to eIF4E, enabling eIF4E to promote cap-dependent translation (reviewed by Richter and Sonenberg, 2005). The stimulation of S6K1 activity by mTORC1 leads to increases in mRNA biogenesis, cap-dependent translation and elongation, and the translation of ribosomal proteins through regulation of the activity of many proteins, such as S6K1 aly/REF-like target (SKAR), programmed cell death 4 (PDCD4), eukaryotic elongation factor 2 kinase (eEF2K) and ribosomal protein S6 (reviewed by Ma and Blenis, 2009). The activation of mTORC1 has also been shown to promote ribosome biogenesis by stimulating the transcription of ribosomal RNA through a process involving the protein phosphatase 2A (PP2A) and the transcription initiation factor IA (TIF-IA) (Mayer et al., 2004).Autophagy – that is, the sequestration of intra - cellular components within autophagosomes and their degradation by lysosomes – is a catabolic process that is important in organelle degradation and protein turnover. When nutrient availability is limited, the degradation of organelles and protein complexes through autophagy provides biological material to sustain anabolic processes such as protein synthesis and energy production. Studies have shown that mTORC1 inhibition increases autophagy, whereas stimulation of mTORC1 reduces this process (reviewed by Codogno and Meier, 2005). We have observed that mTORC1 controls autophagy through an unknown mechanism that is essentially insensitive to inhibition by rapamycin (Thoreen et al., 2009). It was recently shown by three independent groups that mTORC1 controls autophagy through the regulation of a protein complex composed of unc-51-like kinase 1 (ULK1), autophagy-related gene 13 (ATG13) and focal adhesion kinase family-interacting protein of 200 kDa (FIP200) (Ganley et al., 2009; Hosokawa et al., 2009; Jung et al., 2009). These studies have revealed that mTORC1 represses autophagy by phosphorylating and thereby repressing ULK1 and ATG13.The role of mTORC1 in regulating lipid synthesis, which is required for cell growth and proliferation, is beginning to be appreciated. It has been demonstrated that mTORC1 positively regulates the activity of sterol regulatory element binding protein 1 (SREBP1) (Porstmann et al., 2008) and of peroxisome proliferator-activated receptor-γ (PPARγ) (Kim and Chen, 2004), two transcription factors that control the expression of genes encoding proteins involved in lipid and cholesterol homeostasis. Blocking mTOR with rapamycin reduces the expression and the transactivation activity of PPARγ (Kim and Chen, 2004). The molecular mechanism of SREBP1 activation by mTORC1 is unknown. Additionally, rapamycin reduces the phosphorylation of lipin-1 (Huffman et al., 2002), a phosphatidic acid (PA) phosphatase that is involved in glycerolipid synthesis and in the coactivation of many transcription factors linked to lipid metabolism, including PPARγ, PPARα and PGC1-α. The precise impact of lipin-1 phosphorylation on lipid synthesis remains to be established.Mitochondrial metabolism and biogenesis are both regulated by mTORC1. Inhibition of mTORC1 by rapamycin lowers mitochondrial membrane potential, oxygen consumption and cellular ATP levels, and profoundly alters the mitochondrial phosphoproteome (Schieke et al., 2006). Recently, it has been observed that mitochondrial DNA copy number, as well as the expression of many genes encoding proteins involved in oxidative metabolism, are reduced by rapamycin and increased by mutations that activate mTORC1 signaling (Chen et al., 2008; Cunningham et al., 2007). Additionally, conditional deletion of Raptor in mouse skeletal muscle reduces the expression of genes involved in mitochondrial biogenesis (Bentzinger et al., 2008). Cunningham and colleagues have discovered that mTORC1 controls the transcriptional activity of PPARγ coactivator 1 (PGC1-α), a nuclear cofactor that plays a key role in mitochondrial biogenesis and oxidative metabolism, by directly altering its physical interaction with another transcription factor, namely yin-yang 1 (YY1) (Cunningham et al., 2007).mTORC1 integrates four major signals – growth factors, energy status, oxygen and amino acids – to regulate many processes that are involved in the promotion of cell growth. One of the most important sensors involved in the regulation of mTORC1 activity is the tuberous sclerosis complex (TSC), which is a heterodimer that comprises TSC1 (also known as hamartin) and TSC2 (also known as tuberin). TSC1/2 functions as a GTPase-activating protein (GAP) for the small Ras-related GTPase Rheb (Ras homolog enriched in brain). The active, GTP-bound form of Rheb directly interacts with mTORC1 to stimulate its activity (Long et al., 2005; Sancak et al., 2007). The exact mechanism by which Rheb activates mTORC1 remains to be determined. As a Rheb-specific GAP, TSC1/2 negatively regulates mTORC1 signaling by converting Rheb into its inactive GDP-bound state (Inoki et al., 2003; Tee et al., 2003). Consistent with a role of TSC1/2 in the negative regulation of mTORC1, inactivating mutations or loss of heterozygosity of TSC1/2 give rise to tuberous sclerosis, a disease associated with the presence of numerous benign tumors that are composed of enlarged and disorganized cells (reviewed by Crino et al., 2006).Growth factors stimulate mTORC1 through the activation of the canonical insulin and Ras signaling pathways. The stimulation of these pathways increases the phosphorylation of TSC2 by protein kinase B (PKB, also known as AKT) (Inoki et al., 2002; Potter et al., 2002), by extracellular-signal-regulated kinase 1/2 (ERK1/2) (Ma et al., 2005), and by p90 ribosomal S6 kinase 1 (RSK1) (Roux et al., 2004), and leads to the inactivation of TSC1/2 and thus to the activation of mTORC1. Additionally, AKT activation by growth factors can activate mTORC1 in a TSC1/2-independent manner by promoting the phosphorylation and dissociation of PRAS40 from mTORC1 (Sancak et al., 2007; Vander Haar et al., 2007; Wang et al., 2007).The binding of insulin to its cell-surface receptor promotes the tyrosine kinase activity of the insulin receptor, the recruitment of insulin receptor substrate 1 (IRS1), the production of phosphatidylinositol (3,4,5)-triphosphate [PtdIns(3,4,5)P3] through the activation of PI3K, and the recruitment and activation of AKT at the plasma membrane. In many cell types, activation of mTORC1 strongly represses the PI3K-AKT axis upstream of PI3K. Activation of S6K1 by mTORC1 promotes the phosphorylation of IRS1 and reduces its stability (reviewed by Harrington et al., 2005). This auto-regulatory pathway, characterized as the S6K1-dependent negative feedback loop, has been shown to have profound implications for both metabolic diseases and tumorigenesis (reviewed by Manning, 2004). Other pathways that are independent of IRS1 are also likely to contribute to the retro-inhibition of mTORC1. For example, loss of TSC1/2 suppresses platelet-derived growth factor receptor (PDGFR) expression in a rapamycin-sensitive manner (Zhang et al., 2007). How mTOR signaling controls PDGFR expression remains to be determined.The energy status of the cell is signaled to mTORC1 through AMP-activated protein kinase (AMPK), a master sensor of intracellular energy status (reviewed by Hardie, 2007). In response to energy depletion (low ATP:ADP ratio), AMPK is activated and phosphorylates TSC2, which increases the GAP activity of TSC2 towards Rheb and reduces mTORC1 activation (Inoki et al., 2003). Additionally, AMPK can reduce mTORC1 activity in response to energy depletion by directly phosphorylating Raptor (Gwinn et al., 2008).Oxygen levels affect mTORC1 activity through multiple pathways (reviewed by Wouters and Koritzinsky, 2008). Under conditions of mild hypoxia, the reduction in ATP levels activates AMPK, which promotes TSC1/2 activation and inhibits mTORC1 signaling as described in the previous section (Arsham et al., 2003; Liu et al., 2006). Hypoxia can also activate TSC1/2 through transcriptional regulation of DNA damage response 1 (REDD1) (Brugarolas et al., 2004; Reiling and Hafen, 2004). REDD1 blocks mTORC1 signaling by releasing TSC2 from its growth-factor-induced association with 14-3-3 proteins (DeYoung et al., 2008). This ability of REDD1 to reduce mTORC1 signaling by disrupting the interaction of TSC2 and 14-3-3 has probably evolved to limit energy-consuming processes when oxygen, but not growth factors, is scarce. Additionally, promyelocytic leukemia (PML) tumor suppressor and BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3) reduce mTORC1 signaling during hypoxia by disrupting the interaction between mTOR and its positive regulator Rheb (Bernardi et al., 2006; Li et al., 2007).Amino acids represent a strong signal that positively regulates mTORC1 (reviewed by Guertin and Sabatini, 2007). It was recently shown that leucine, an essential amino acid required for mTORC1 activation, is transported into cells in a glutamine-dependent fashion (Nicklin et al., 2009). Glutamine, which is imported into cells through SLC1A5 [solute carrier family 1 (neutral amino acid transporter) member 5], is exchanged to import leucine via a heterodimeric system composed of SLC7A5 [antiport solute carrier family 7 (cationic amino acid transporter, y+ system, member 5] and SLC3A2 [solute carrier family 3 (activators of dibasic and neutral amino acid transport) member 2]. The mechanism by which intracellular amino acids then signal to mTORC1 remained obscure for many years. The activation of mTORC1 by amino acids is known to be independent of TSC1/2, because the mTORC1 pathway remains sensitive to amino acid deprivation in cells that lack TSC1 or TSC2 (Nobukuni et al., 2005). Some studies have implicated human vacuolar protein-sorting-associated protein 34 (VPS34) in nutrient sensing (Nobukuni et al., 2005); however, the precise role of human VPS34 in this process still remains to be established (Juhasz et al., 2008).Recently, two independent teams, including ours, have shown that the Rag proteins, a family of four related small GTPases, interact with mTORC1 in an amino acid-sensitive manner and are necessary for the activation of the mTORC1 pathway by amino acids (Kim et al., 2008; Sancak et al., 2008). In the presence of amino acids, Rag proteins bind to Raptor and promote the relocalization of mTORC1 from discrete locations throughout the cytoplasm to a perinuclear region that contains its activator Rheb (Sancak et al., 2008). The physical dissociation of mTORC1 and Rheb with amino acid deprivation might explain why activators of Rheb, such as growth factors, cannot stimulate mTORC1 signaling in the absence of amino acids.In addition to the key signals described above, other cellular conditions and signals, such as genotoxic stress, inflammation, Wnt ligand and PA, have all been shown to regulate mTORC1 signaling. Genotoxic stress reduces mTORC1 activity through many mechanisms. For instance, the activation of p53 in response to DNA damage rapidly activates AMPK through an unknown process, which in turn phosphorylates and thereby activates TSC2 (Feng et al., 2005). Additionally, p53 negatively controls mTORC1 signaling by increasing the transcription of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and TSC2, two negative regulators of the pathway (Feng et al., 2005; Stambolic et al., 2001). Inflammatory mediators also signal to mTORC1 via the TSC1/2 complex. Pro-inflammatory cytokines, such as TNFα, activate IκB kinase-β (IKKβ), which physically interacts with and inactivates TSC1, leading to mTORC1 activation (Lee et al., 2007). This positive relationship between inflammation and mTORC1 activation is thought to be important in tumor angiogenesis (Lee et al., 2007) and in the development of insulin resistance (Lee et al., 2008). Wnt signaling also increases mTORC1 activity through the inactivation of TSC1/2. Stimulation of the Wnt pathway inhibits glycogen synthase kinase 3 (GSK3), a kinase that promotes TSC1/2 activity by directly phosphorylating TSC2 (Inoki et al., 2006). Finally, PA has been identified as another activator of mTORC1. Many groups have shown that exogenous PA or overexpression of PA-producing enzymes such as phospholipase D1 (PLD1) and PLD2 significantly increases mTORC1 signaling (reviewed by Foster, 2007). A recent study suggests that PA affects mTOR signaling by facilitating the assembly of mTOR complexes, or stabilizing the complexes (Toschi et al., 2009).In contrast to mTORC1, for which many upstream signals and cellular functions have been defined (see above), relatively little is known about mTORC2 biology. The early lethality caused by the deletion of mTORC2 components in mice, as well as the absence of mTORC2 inhibitors, have complicated the study of this protein complex. Nonetheless, many important discoveries have been made over the last few years. Using various genetic approaches, it has been demonstrated that mTORC2 plays key roles in various biological processes, including cell survival, metabolism, proliferation and cytoskeleton organization. The role of mTORC2 in these processes is discussed in more detail below.Cell survival, metabolism and proliferation are all highly dependent on the activation status of AKT, which positively regulates these processes through the phosphorylation of various effectors (reviewed by Manning and Cantley, 2007). Full activation of AKT requires its phosphorylation at two sites: Ser308, by phosphoinositide-dependent kinase 1 (PDK1), and Ser473, by a kinase that remained unidentified for many years, but was demonstrated to be mTORC2 by our group in 2005 (Sarbassov et al., 2005). Other studies have subsequently observed that ablation of various mTORC2 components specifically blocks AKT phosphorylation at Ser473 and the downstream phosphorylation of some, but not all, AKT substrates (Guertin et al., 2006; Jacinto et al., 2006). Inhibition of AKT following mTORC2 depletion reduces the phosphorylation of, and therefore activates, the forkhead box protein O1 (FoxO1) and FoxO3a transcription factors, which control the expression of genes involved in stress resistance, metabolism, cell-cycle arrest and apoptosis (reviewed by Calnan and Brunet, 2008). By contrast, the phosphorylation state of TSC2 and GSK3 is not affected by mTORC2 inactivation. Recently, serum- and glucocorticoid-induced protein kinase 1 (SGK1), which shares homology with AKT, was also shown to be regulated by mTORC2 (Garcia-Martinez and Alessi, 2008). In contrast to AKT, which retains a basal activity when mTORC2 is inhibited, SGK1 activity is totally abrogated under these conditions. Because SGK1 and AKT phosphorylate FoxO1 and FoxO3a on common sites, it is possible that the lack of SGK1 activity in mTORC2-deficient cells is responsible for the inhibition of phosphorylation of FoxO1 and FoxO3a.mTORC2 regulates cytoskeletal organization. Many independent groups have observed that knocking down mTORC2 components affects actin polymerization and perturbs cell morphology (Jacinto et al., 2004; Sarbassov et al., 2004). These studies have suggested that mTORC2 controls the actin cytoskeleton by promoting protein kinase Cα (PKCα) phosphorylation, phosphorylation of paxillin and its relocalization to focal adhesions, and the GTP loading of RhoA and Rac1. The molecular mechanism by which mTORC2 regulates these processes has not been determined.The signaling pathways that lead to mTORC2 activation are not well characterized. Because growth factors increase mTORC2 kinase activity and AKT phosphorylation at Ser473, they are considered to be a plausible signal for regulating this pathway (reviewed by Guertin and Sabatini, 2007). With growth-factor stimulation, AKT is phosphorylated at the cell membrane through the binding of PtdIns(3,4,5)P3 to its pleckstrin homology (PH) domain. Under these conditions, PDK1 is also recruited to the membrane through its PH domain and phosphorylates AKT at Ser308 (reviewed by Lawlor and Alessi, 2001). Interestingly, the mTORC2 component mSIN1 possesses a PH domain at its C-terminus, suggesting that mSIN1 can promote the translocation of mTORC2 to the membrane and the phosphorylation of AKT at Ser473. Additional work is needed to support this model and to identify other cellular signals that play a role in the regulation of mTORC2.Over the last decade, knowledge of the mTOR signaling pathway has greatly progressed, enabling researchers to better understand the mechanism of diseases such as cancer and type 2 diabetes. Despite these advances, our understanding of this signaling network is far from complete and many important questions remain to be answered. For example, how is mTORC2 regulated and which biological processes does it control? How are the mTORC1 and mTORC2 signaling pathways integrated with each other? What are the functions of these complexes in adult tissues and organs and what are the implications of their dysfunction or dysregulation in health and disease? Are there additional mTOR complexes that regulate other biological processes? Finding answers to these important questions will advance our understanding of cellular biology, and will also help the development of therapeutic avenues to treat many human diseases.

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Ras, PI(3)K and mTOR signalling controls tumour cell growth

2006-05-01

Reuben J. Shaw , Lewis C. Cantley

PI3K pathway alterations in cancer: variations on a theme

2008-09-15

T L Yuan , Lewis C. Cantley

Cellular survival: a play in three Akts

1999-11-15

Soma Datta , Anne Brunet , Michael E. Greenberg

The programmed cell death that occurs as part of normal mammalian development was first observed nearly a century ago (Collin 1906). It has since been established that approximately half of … The programmed cell death that occurs as part of normal mammalian development was first observed nearly a century ago (Collin 1906). It has since been established that approximately half of all neurons in the neuroaxis and >99.9% of the total number of cells generated during the course of a human lifetime go on to die through a process of apoptosis (for review, see Datta and Greenberg 1998; Vaux and Korsmeyer 1999). The induction of developmental cell death is a highly regulated process and can be suppressed by a variety of extracellular stimuli. The purification in the 1950s of the nerve growth factor (NGF), which promotes the survival of sympathetic neurons, set the stage for the discovery that peptide trophic factors promote the survival of a wide variety of cell types in vitro and in vivo (Levi-Montalcini 1987). The profound biological consequences of growth factor (GF) suppression of apoptosis are exemplified by the critical role of target-derived neurotrophins in the survival of neurons and the maintenance of functional neuronal circuits. (Pettmann and Henderson 1998). Recently, the ability of trophic factors to promote survival have been attributed, at least in part, to the phosphatidylinositide 38-OH kinase (PI3K)/c-Akt kinase cascade. Several targets of the PI3K/c-Akt signaling pathway have been recently identified that may underlie the ability of this regulatory cascade to promote survival. These substrates include two components of the intrinsic cell death machinery, BAD and caspase 9, transcription factors of the forkhead family, and a kinase, IKK, that regulates the NF-kB transcription factor. This article reviews the mechanisms by which survival factors regulate the PI3K/c-Akt cascade, the evidence that activation of the PI3K/c-Akt pathway promotes cell survival, and the current spectrum of c-Akt targets and their roles in mediating c-Akt-dependent cell survival.

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PI3K/Akt signalling pathway and cancer

2003-11-24

Juan Ángel Fresno Vara , E. Casado , Javier de Castro +3 more

Germline mutations of the PTEN gene in Cowden disease, an inherited breast and thyroid cancer syndrome

1997-05-01

Danny Liaw , Deborah J. Marsh , Jing Li +7 more

Mammalian TOR complex 2 controls the actin cytoskeleton and is rapamycin insensitive

2004-10-03

Estela Jacinto , Robbie Loewith , Anja Schmidt +4 more

mTOR: from growth signal integration to cancer, diabetes and ageing

2010-12-15

Roberto Zoncu , Alejo Efeyan , David M. Sabatini

Everolimus in Postmenopausal Hormone-Receptor–Positive Advanced Breast Cancer

2011-12-07

José Baselga , Mario Campone , Martine Piccart +7 more

Resistance to endocrine therapy in breast cancer is associated with activation of the mammalian target of rapamycin (mTOR) intracellular signaling pathway. In early studies, the mTOR inhibitor everolimus added to … Resistance to endocrine therapy in breast cancer is associated with activation of the mammalian target of rapamycin (mTOR) intracellular signaling pathway. In early studies, the mTOR inhibitor everolimus added to endocrine therapy showed antitumor activity.In this phase 3, randomized trial, we compared everolimus and exemestane versus exemestane and placebo (randomly assigned in a 2:1 ratio) in 724 patients with hormone-receptor-positive advanced breast cancer who had recurrence or progression while receiving previous therapy with a nonsteroidal aromatase inhibitor in the adjuvant setting or to treat advanced disease (or both). The primary end point was progression-free survival. Secondary end points included survival, response rate, and safety. A preplanned interim analysis was performed by an independent data and safety monitoring committee after 359 progression-free survival events were observed.Baseline characteristics were well balanced between the two study groups. The median age was 62 years, 56% had visceral involvement, and 84% had hormone-sensitive disease. Previous therapy included letrozole or anastrozole (100%), tamoxifen (48%), fulvestrant (16%), and chemotherapy (68%). The most common grade 3 or 4 adverse events were stomatitis (8% in the everolimus-plus-exemestane group vs. 1% in the placebo-plus-exemestane group), anemia (6% vs. <1%), dyspnea (4% vs. 1%), hyperglycemia (4% vs. <1%), fatigue (4% vs. 1%), and pneumonitis (3% vs. 0%). At the interim analysis, median progression-free survival was 6.9 months with everolimus plus exemestane and 2.8 months with placebo plus exemestane, according to assessments by local investigators (hazard ratio for progression or death, 0.43; 95% confidence interval [CI], 0.35 to 0.54; P<0.001). Median progression-free survival was 10.6 months and 4.1 months, respectively, according to central assessment (hazard ratio, 0.36; 95% CI, 0.27 to 0.47; P<0.001).Everolimus combined with an aromatase inhibitor improved progression-free survival in patients with hormone-receptor-positive advanced breast cancer previously treated with nonsteroidal aromatase inhibitors. (Funded by Novartis; BOLERO-2 ClinicalTrials.gov number, NCT00863655.).

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The phosphatidylinositol 3-Kinase–AKT pathway in human cancer

2002-07-01

Igor Vivanco , Charles L. Sawyers

mTOR Signaling in Growth, Metabolism, and Disease

2017-03-01

Robert A. Saxton , David M. Sabatini

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AKT/PKB Signaling: Navigating the Network

2017-04-01

Brendan D. Manning , Alex Toker

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The PI3K Pathway in Human Disease

2017-08-01

David A. Fruman , Honyin Chiu , Benjamin D. Hopkins +3 more

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mTOR Signaling in Growth, Metabolism, and Disease

2017-04-01

Robert A. Saxton , David M. Sabatini

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mTOR at the nexus of nutrition, growth, ageing and disease

2020-01-14

Grace Y. Liu , David M. Sabatini

Phosphorylation and Regulation of Akt/PKB by the Rictor-mTOR Complex

2005-02-17

Dos D. Sarbassov , David A. Guertin , Siraj M. Ali +1 more

Deregulation of Akt/protein kinase B (PKB) is implicated in the pathogenesis of cancer and diabetes. Akt/PKB activation requires the phosphorylation of Thr308 in the activation loop by the phosphoinositide-dependent kinase … Deregulation of Akt/protein kinase B (PKB) is implicated in the pathogenesis of cancer and diabetes. Akt/PKB activation requires the phosphorylation of Thr308 in the activation loop by the phosphoinositide-dependent kinase 1 (PDK1) and Ser473 within the carboxyl-terminal hydrophobic motif by an unknown kinase. We show that in Drosophila and human cells the target of rapamycin (TOR) kinase and its associated protein rictor are necessary for Ser473 phosphorylation and that a reduction in rictor or mammalian TOR (mTOR) expression inhibited an Akt/PKB effector. The rictor-mTOR complex directly phosphorylated Akt/PKB on Ser473 in vitro and facilitated Thr308 phosphorylation by PDK1. Rictor-mTOR may serve as a drug target in tumors that have lost the expression of PTEN, a tumor suppressor that opposes Akt/PKB activation.

A mouse model of PTEN Hamartoma Tumour Syndrome reveals that loss of the nuclear function of PTEN drives macrocephaly, lymphoid overgrowth, and late-onset cancer

2025-06-25

Priyanka Tibarewal , Victoria Rathbone , Sarah E. Conduit +7 more | bioRxiv (Cold Spring Harbor Laboratory)

PTEN Hamartoma Tumour Syndrome (PHTS) is a rare disorder characterized by germline heterozygous mutations in the PTEN tumour suppressor gene, leading to multi-organ/tissue overgrowth, autism spectrum disorder and increased cancer … PTEN Hamartoma Tumour Syndrome (PHTS) is a rare disorder characterized by germline heterozygous mutations in the PTEN tumour suppressor gene, leading to multi-organ/tissue overgrowth, autism spectrum disorder and increased cancer risk. PHTS individuals display heterogeneity in phenotypes, which has been linked in part to the diverse genetic alterations in the PTEN gene and the multifaceted functions of this protein. Indeed, while PTEN primarily functions as a PIP3 lipid phosphatase in the cytosol, regulating PI3K/AKT signalling, a pathway commonly deregulated in cancer, it also plays crucial roles in maintaining chromosomal stability through nuclear activities such as double strand (ds) DNA damage repair. Recent studies have identified a subset of missense PHTS variants that cause nuclear exclusion of PTEN, impairing its nuclear functions. Here, we present our findings from one such pathogenic variant, PTEN-R173C, frequently found in PHTS and somatic cancers. Using cell biological and mouse modelling approaches, we show that PTEN-R173C has higher PIP3 phosphatase activity than wild-type PTEN, resulting in effective regulation of canonical PI3K/AKT signalling. However, PTEN-R173C is unstable and excluded from the nucleus. Aligning with their near normal PI3K/AKT signalling, Pten+/R173C mice display a low incidence of solid tumours compared to Pten+/- mice. Pten+/R173C mice also exhibit lymphoid hyperplasia and macrocephaly which correlates with compromised nuclear functions of PTEN-R173C. That nuclear functions are compromised is demonstrated by reduced dsDNA damage repair in Pten+/R173C mice. Integrating PHTS patient data with findings from our mouse model, our study indicates that nuclear dysfunction of pathogenic PTEN variants is a key factor in predicting the onset of the different PHTS-associated phenotypes. We speculate that late-onset cancer in individuals with nuclear-excluded PTEN results from genetic alterations unrelated to PTEN itself, facilitated by impaired PTEN-mediated dsDNA damage repair.

CRISPR RiPCA for Investigating eIF4E-m7GpppX Capped mRNA Interactions

2025-06-23

Gabriela Vega-Hernández , Juan Carlos Duque , Barbara Klein +5 more | bioRxiv (Cold Spring Harbor Laboratory)

Post-transcriptional modifications expand the information encoded by an mRNA. These dynamic and reversible modifications are specifically recognized by reader RNA-binding proteins (RBPs), which mediate the regulation of gene expression, RNA … Post-transcriptional modifications expand the information encoded by an mRNA. These dynamic and reversible modifications are specifically recognized by reader RNA-binding proteins (RBPs), which mediate the regulation of gene expression, RNA processing, localization, stability, and translation. Given their crucial functions, any disruptions in the normal activity of these readers can have significant implications for cellular health. Consequently, the dysregulation of these RBPs has been associated with neurodegenerative disorders, cancers, and viral infections. Therefore, there has been growing interest in targeting reader RBPs as a potential therapeutic strategy since developing molecules that restore proper RNA processing and function may offer a promising avenue for treating diseases. In this work, we coupled our previously established live-cell RNA-protein interaction (RPI) assay, RNA interaction with Protein-mediated Complementation Assay (RiPCA), with CRISPR technology to build a new platform, CRISPR RiPCA. As a model for development, we utilized the interaction of eukaryotic translation initiation factor 4E (eIF4E), a reader RBP that binds to the m7GpppX cap present at the 5 terminus of coding mRNAs, with an m7G capped RNA substrate. Using eIF4E CRISPR RiPCA, we demonstrate our technologys potential for measuring on-target activity of inhibitors of the eIF4E RPI of relevance to cancer drug discovery.

The mTOR pathway in Gliomas: From molecular insights to targeted therapies

2025-06-21

Safura Pournajaf , Mohammad H. Pourgholami | Biomedicine & Pharmacotherapy

The Src family kinase inhibitor drug Dasatinib and glucocorticoids display synergistic activity against tongue squamous cell carcinoma and reduce MET kinase activity

2025-06-19

Ali Hmedat , Jessica Doondeea , Daniel Ebner +2 more | Cell Communication and Signaling

Tongue squamous cell carcinoma (TSCC) is an aggressive cancer associated with a poor prognosis and limited treatment options, necessitating new drug targets to improve therapeutic outcomes. Our current work studies … Tongue squamous cell carcinoma (TSCC) is an aggressive cancer associated with a poor prognosis and limited treatment options, necessitating new drug targets to improve therapeutic outcomes. Our current work studies protein tyrosine kinases as well-known targets for successful cancer therapies. It focuses on Src family kinases (SFK), which are known to play a critical role in some head and neck tumors. Western blot analyses of phospho-tyrosine protein patterns in 34 TSCC lines facilitated the investigation of SFK as contributors to these phosphorylations. The SFK inhibitors PP2 and Dasatinib were utilized to determine SFK contributions to cell motility and survival. A high-throughput screen with 1600 FDA-approved drugs was performed with three TSCC lines to discover drugs that act synergistically with Dasatinib against TSCC cell viability. Glucocorticoids emerged as potential candidates and were further investigated in 2D culture and by 3D soft agar colony formation. Dexamethasone was chosen as the major tool for our analyses of synergistic effects of Dasatinib and glucocorticoids on TSCC lines. Effects on the cell cycle were investigated by flow cytometry and expression levels of cell cycle regulators. Senescence was analyzed by senescence-associated β galactosidase detection and p27Kip1 protein expression. Autophagy was measured by Acridine Orange staining. A panel of 34 TSCC lines showed a surprisingly homogenous pTyr-protein pattern and a prominent 130 kDa pTyr-protein. Inhibition of SFK activity greatly reduced overall pTyr-protein levels and p130Cas tyrosine phosphorylation. It also impaired TSCC viability in 2D cell culture and 3D soft agar colony formation. A high-throughput drug combination screen with Dasatinib identified glucocorticoids as promising candidates for synergistic activity. Dasatinib and Dexamethasone combination treatment showed strong synergistic effects on Src and p130Cas phosphorylation and led to reduced p130Cas expression. Dexamethasone also suppressed phosphorylation of the MET kinase and its key substrate Gab1. On the cellular level, Dasatinib combination with glucocorticoids led to G1 cell cycle arrest, appeared to increase senescence and enhanced autophagy. This was also reflected by effects on cell cycle regulatory proteins, including CDKs and cyclins. This work is the first to show a strong synergistic activity of Dasatinib in combination with clinically used glucocorticoids in solid tumors. Furthermore, the tyrosine kinase MET and its effector protein Gab1 are newly identified glucocorticoid targets. Given the extensive research on MET as a drug target in various cancers, our findings have the potential to advance future cancer treatments.

Clinicogenomic landscape and function of PIK3CA, AKT1, and PTEN mutations in breast cancer

2025-06-18

Jacqueline J. Tao , Saumya D. Sisoudiya , Hanna Tukachinsky +4 more | medRxiv (Cold Spring Harbor Laboratory)

Abstract Purpose To comprehensively characterize the clinical and genomic landscapes of PIK3CA, AKT1, and PTEN alterations and examine their functional implications in AKT-driven breast cancer. Experimental Design Comprehensive genomic profiling … Abstract Purpose To comprehensively characterize the clinical and genomic landscapes of PIK3CA, AKT1, and PTEN alterations and examine their functional implications in AKT-driven breast cancer. Experimental Design Comprehensive genomic profiling of 51,767 breast tumors was performed with FoundationOne ® CDx or FoundationOne ® . We examined the genomic landscape of PIK3CA , PTEN , and AKT1 alterations and their distribution across clinical variables of interest. Prior deep mutational scanning (DMS) data was used to functionally characterize clinical PTEN variants. Results There were 29,157 total variants across PIK3CA, AKT1, and PTEN , including pathogenic variants and VUS. The most frequently altered gene was PIK3CA (37.4% of cases), followed by PTEN (13.5%), then AKT1 (5.4%). The most common alterations in each gene were PIK3CA H1047R (35.6% of PIK3CA -altered cases), E545K (19.7%), and E542K (11.7%); AKT1 E17K (69.7%); and PTEN homozygous copy number deletion (37.3%). PIK3CA alterations were less prevalent in patients of African genetic ancestry (27.1% vs 38.6% in European genetic ancestry), while AKT1 and PTEN alterations were balanced across ancestries. PIK3CA , AKT1 , and PTEN pathogenic alterations were all mutually exclusive to each other. Using available DMS data on missense PTEN mutations, we found that 32.5% showed discordant effects on protein stability and phosphatase activity, underscoring the need for functional validation beyond predicted loss-of-function. Conclusions Here we present the landscape of PIK3CA , AKT1 , and PTEN alterations in the largest clinical cohort examined to date. The functional implications of lesser-known variants in each gene warrant further investigation by tools such as deep mutational scanning.

PTEN neddylation aggravates CDK4/6 inhibitor resistance in breast cancer

2025-06-18

Fan Liu , Weixiao Liu , Yawen Tan +6 more | Oncogene

Recombinant chromosome 6 open reading frame 120 protein promotes angiogenesis and endothelial‑to‑mesenchymal transition in human umbilical vein endothelial cells via the PI3K/Akt signaling pathway

2025-06-17

Yingying Lin , Xin Wang , Yanyan Li +3 more | Molecular Medicine Reports

The vascular endothelium plays a pivotal role in modulating various physiological processes and its dysfunction is fundamental to the development of numerous vascular and non‑vascular diseases. Chromosome 6 open reading … The vascular endothelium plays a pivotal role in modulating various physiological processes and its dysfunction is fundamental to the development of numerous vascular and non‑vascular diseases. Chromosome 6 open reading frame 120 (C6ORF120) has been implicated in cellular processes such as apoptosis, inflammation, immunomodulation and fibrosis. However, the specific effects of C6ORF120 on endothelial cell function remain unclear. The present study aimed to explore the potential role of C6ORF120 in endothelial dysfunction and its underlying molecular mechanisms. It synthesized recombinant C6ORF120 protein (rC6ORF120) and assessed its effects on human umbilical vein endothelial cells (HUVECs) through various functional assays, including the CCK‑8 assay for proliferation, scratch assay for migration and tube formation assay for angiogenesis. Additionally, immunofluorescence (IF) and western blotting (WB) were employed to evaluate endothelial‑mesenchymal transition (EndMT). The present study also quantified the expression of key proteins within the PI3K/Akt signaling pathway to elucidate its role in mediating the effects of rC6ORF120 on HUVECs. Treatment with rC6ORF120 significantly enhanced HUVEC proliferation (200 ng/ml vs. control at 72 h, 1.14±0.01 vs. 1.05±0.02; t=8.15; P<0.001) and induced phenotypic changes. In migration and angiogenesis assays, rC6ORF120‑treated HUVECs exhibited increased wound closure (37.69±2.74% vs. 66.16±6.13%; t=7.35; P=0.002) and angiogenesis assays showed significant improvements in tube formation parameters such as total tubule length (77,199.67±4,684.88 µm vs. 96,203.00±3,354.89 µm; t=5.71; P=0.002). WB and IF analyses both indicated that rC6ORF120 promotes EndMT in HUVECs. Furthermore, rC6ORF120 treatment increased PI3K/Akt phosphorylation significantly compared with controls (p‑PI3K; 1.57±0.18 vs. 1.00±0.00; t=5.64; P=0.005). LY294002 significantly reversed these effects on EndMT and angiogenesis (P<0.05), while the effect on cell migration was less pronounced (P=0.565). Our study highlights the critical role of C6ORF120 in HUVECs, promoting proliferation, migration, angiogenesis and EndMT, which are mediated, at least in part, by the PI3K/Akt pathway.

Editor’s Note: Lactate Dehydrogenase B Is Critical for Hyperactive mTOR-Mediated Tumorigenesis

2025-06-16

Xiaojun Zha , Fang Wang , Ying Wang +4 more | Cancer Research

Antitumor Effect of mTOR1/2 Dual Inhibitor AZD8055 in Canine Pulmonary Carcinoma

2025-06-14

Tomokazu Nagashima , Kazuhiko Ochiai , Yoshinori Takizawa +7 more | Cancers

Background/Objectives: Primary pulmonary carcinoma (PC) is a malignant neoplasm that occurs in humans, dogs, and other species. In canine PC, palliative care remains the most practical approach for dogs with … Background/Objectives: Primary pulmonary carcinoma (PC) is a malignant neoplasm that occurs in humans, dogs, and other species. In canine PC, palliative care remains the most practical approach for dogs with inoperable PC. Methods: We investigated the effectiveness of mammalian target of rapamycin (mTOR) inhibitors in canine lung cancer upon PI3K/AKT/mTOR activation. Three canine PC cell lines (AZACL1, AZACL2, and cPAC-1) were treated with three mTOR inhibitors (AZD8055, temsirolimus, and everolimus). In vitro, sensitivity assays were conducted to evaluate proliferation and Western blotting was used to examine pathway activation and phosphorylation of mTOR-related protein. Results: AZD8055 had a stronger inhibitory effect on cell proliferation than temsirolimus and everolimus in all three PC cell lines. The IC50 for AZD8055 in the AZACL1, AZACL2, and cPAC-1 cell lines were 23.8 μM, 95.8 nM, and 237 nM, for temsirolimus they were 34.6 μM, 11.5 μM, and 11.2 μM, and for everolims they were 36.6 μM, 33.4 μM, and 33.0 μM, respectively. Western blotting revealed PI3K/AKT/mTOR pathway activation and differential phosphorylation of mTOR signal-related proteins across the three PC cell lines. In xenograft mice injected with the AZACL1 and AZACL2 cell lines we showed that the AZD8055-treated group exhibited a significant reduction in tumor volume via the inhibition of tumor growth compared to the control group. Conclusions: These findings reveal that the PI3K/AKT/mTOR pathway plays a key role in canine PC and that AZD8055 may be a novel therapeutic agent for PC-bearing dogs.

Abstract P4-12-19: Preclinical Characterization of a Novel PI3Kα H1047R Mutant-Selective Inhibitor

2025-06-13

Aaron Smith , Ben Arwood-Levine , Abiezer Blandon +7 more | Clinical Cancer Research

Abstract PIK3CA encodes the p110α catalytic subunit of PI3-kinase alpha (PI3Kα) and is the most frequently mutated kinase in human cancer with common mutations occurring in the kinase domain (H1047R) … Abstract PIK3CA encodes the p110α catalytic subunit of PI3-kinase alpha (PI3Kα) and is the most frequently mutated kinase in human cancer with common mutations occurring in the kinase domain (H1047R) and helical domain (E542K/E545K). The approved PI3Kα inhibitor, alpelisib, shows promise for this targeted class of agents with improvements in progression-free survival in ER+/Her2- breast cancer patients in combination with fulvestrant. However, toxicities attributed to the inhibition of wild-type PI3Kα, such as hyperglycemia, gastrointestinal issues, and skin reactions, lead to sub-optimal target engagement due to requisite dosing modifications. A PI3K mutant inhibitor that spares WT PI3K is predicted to be better tolerated, require fewer dosing modifications, and therefore, have the potential to provide improved clinical benefit. Herein, we present the preclinical in vitro and in vivo activity of a novel, wild type sparing PI3Kα inhibitor series which is potent against the oncogenic H1047R mutation. Citation Format: Aaron C. Smith, Ben Arwood-Levine, Abiezer Blandon, Alexandra Born, Richard Brizendine, Payal Chatterjee, Mark J. Chicarelli, Michael L. Conner, Brad Fell, Jennifer Fulton, Anna Guarnieri, Hannah Hubert, Ravi Jalluri, Hailey J. Knox, Keith Koch, Daniel Krischlunas, Vijay Kumar, Sara Kuzbiel, Colin McHugh, Brent Mclean, Kelsey W. Nassar, Brad Newhouse, Scott Niman, Rob Rieger, John Robinson, Marelí Rodriguez, Leah Salituro, Vincent Scarato, Lee Stunkard, Francis Sullivan, Patrick Sutter, Roy Turton, Robb Van Gulick, Brooklynn Venteicher, Logan E. Vine, Shannon Winski, Yeyun Zhou. Preclinical Characterization of a Novel PI3Kα H1047R Mutant-Selective Inhibitor [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P4-12-19.

Abstract P3-08-19: Preliminary results from PIKture-01, a First-in-Human Study of OKI-219, a mutant selective inhibitor of PI3Kα-H1047R, in mutant selected solid tumors including breast cancer

2025-06-13

Samuel Agresta , Alexander I. Spira , Andreas Varkaris +7 more | Clinical Cancer Research

Abstract Background: Aberrant activation of the PI3Ka pathway contributes to tumorigenesis and is associated with resistance to anticancer therapies, making this pathway an attractive target for new therapies[1]. PI3Kα is … Abstract Background: Aberrant activation of the PI3Ka pathway contributes to tumorigenesis and is associated with resistance to anticancer therapies, making this pathway an attractive target for new therapies[1]. PI3Kα is one of the most commonly mutated oncogenes, found in approximately 13% of human cancers and 29% of breast cancer [2]. There are three predominate mutations in PI3Kα important in cancer. The H1047R mutation in the kinase domain is the most common of the three, found in 40% of HR+/HER2– breast cancers and 10-15% of HER2+ breast cancers [3]. The currently approved PI3Kα inhibitors such as alpelisib target both wild-type and mutant forms, leading to significant on-target toxicities, including hyperglycemia, rash, and diarrhea [4,5]. OKI-219, a mutant-selective PI3Kα inhibitor, has shown preclinical efficacy in PI3Kα-H1047R-mutated models, without the metabolic dysfunction associated with wild-type inhibition, supporting a potential improved therapeutic profile. We hypothesize OKI-219 may achieve greater mutant target coverage with a wider therapeutic window compared to other non-selective PI3Kα inhibitors. Methods: PIKture-01 is a global, multi-center, first-in-human phase 1a/1b study evaluating OKI-219 as monotherapy and in combination with fulvestrant or trastuzumab in subjects with advanced solid tumors including breast cancer harboring a PI3Kα-H1047R mutation. In Phase 1a, subjects receive escalating oral doses of OKI-219 starting at 300 mg BID continuously. Phase 1b will assess OKI-219 in combination with fulvestrant in patients with HR+ breast cancer, or with trastuzumab in patients with HER2+ breast cancer. The study also includes a dose optimization phase to evaluate the optimal combination doses of OKI-219 with fulvestrant or trastuzumab. Results: As of 30 September 2024, OKI-219 has been dosed at three dose levels as a single agent: 300 mg BID, 600 mg BID, and 900 mg BID, continuously. Across all dose levels to date, a total of ten subjects have been dosed: six subjects with HR+/HER2- breast cancer, two with HER2+ positive breast cancer, and two with colorectal cancer in the single agent dose escalation. Eight of the ten subjects remain on study. OKI-219 has been very well tolerated, with no dose-limiting toxicities, dose interruptions, or dose reductions required. The most common treatment-emergent adverse events (TEAE) that occurred in &gt;15% of subjects were urinary tract infection, upper extremity cellulitis and pruritus. The most common treatment-related adverse events (TRAE) were grade 1 pruritus. Single-dose pharmacokinetic (PK) results of OKI-219 are consistent with predicted human exposures. At steady state, the exposures of OKI-219 exceed exposures associated with robust antitumor activity in preclinical models. Conclusion: OKI-219 has been very well tolerated with a favorable safety profile, and only Grade 1 TRAEs observed at exposures that are consistent with preclinical activity, even at the lowest dose level. As a single agent, OKI-219 has shown favorable PK that support pharmacologically relevant exposures, even at the lowest assessed dose levels, with a safety profile that suggests little or no inhibition of WT PI3Kα. We anticipate near completion of enrollment of the single agent portion of the study as well as the initiation of combination expansion of OKI-219 with fulvestrant by the end of the year. Data will be updated accordingly. References: 1. Martini, M., et al., PI3K/AKT signaling pathway and cancer: an updated review. Ann Med, 2014. 46(6): p. 372-83. 2. Millis, S.Z., et al., Landscape of phosphatidylinositol-3-kinase pathway alterations across 19 784 diverse solid tumors. JAMA Oncol, 2016. 2(12): p. 1565-1573. 3. COSMIC database https://cancer.sanger.ac.uk/cosmic/gene/analysis?all_data=&coords=AA%3AAA&dr=&end=1069&gd=&id=276592&ln=PIK3CA&seqlen=1069&sn=breast&start=1#ts 4. Narayan, P., et al., FDA approval summary: alpelisib plus fulvestrant for patients with HR-positive, HER2-negative, PIK3CA-mutated, advanced or metastatic breast cancer. Clin Cancer Res, 2021. 27(7): p. 1842-1849. 5. Shields, M., et al., A systematic review and meta-analysis of selected toxicity endpoints of alpelisib. Oncotarget, 2020. 11(42): p. 3793-3799. Citation Format: Samuel Agresta, Alexander Spira, Andreas Varkaris, Seock-Ah Im, Peter Kabos, Ramon Yarza, Yeon Hee Park, Kevin Litwiler, Guy Gammon, Amy Heim, Brian Tunquist, Robbie Alton, Duncan Walker. Preliminary results from PIKture-01, a First-in-Human Study of OKI-219, a mutant selective inhibitor of PI3Kα-H1047R, in mutant selected solid tumors including breast cancer [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P3-08-19.

Abstract P4-12-24: Preclinical characterization of LY4045004, a next-generation, mutant-selective PI3Kα inhibitor

2025-06-13

Raymond Gilmour , Andrew L. Faber , Weihua Shen +7 more | Clinical Cancer Research

Abstract Introduction: PI3Kα mutations are oncogenic drivers found in approximately 40% of HR+ breast cancers as well as multiple other solid tumors. Approved treatments for PI3Kα mutant-driven HR+ breast cancers … Abstract Introduction: PI3Kα mutations are oncogenic drivers found in approximately 40% of HR+ breast cancers as well as multiple other solid tumors. Approved treatments for PI3Kα mutant-driven HR+ breast cancers include direct inhibitors of either PIK3CA or downstream AKT. However, these inhibitors block PI3K pathway signaling in unmutated host tissues resulting in hyperglycemia, rash, and GI toxicity. Herein we describe the preclinical profile of LY4045004, a next-generation, mutant-selective inhibitor of PI3Kα. The compound exhibits potent inhibition across the most common PI3Kα mutations in breast cancer (H1047R, E545K, and E542K) while sparing wild type (WT) PI3Kα. Strong efficacy in multiple PI3Kα-mutant breast cancer models is demonstrated while avoiding the hyperglycemia and insulin increases characteristic of non-selective inhibitors. Methods: PI3Kα biochemical potency was measured using ADP Glo kinase assay. PI3Kα on-rate and off-rate were measured using Transcreener FI assay. PI3Kα cell potency was measured using cell viability and signal transduction assays. Tumor growth inhibition, pharmacokinetic (PK), and pharmacodynamic effects were assessed in in vivo studies using PI3Kα mutant cell-derived xenograft (CDX) and patient-derived xenograft (PDX)-models. Plasma insulin and C-peptide levels were measured using ELISA. Results: LY4045004 is a potent, allosteric inhibitor of both kinase and helical PI3Kα mutations in enzyme and cell-based assays, inhibiting growth and signaling responses in multiple mutant-driven breast cancer cell lines. The compound is selective for mutant PI3Kα over WT PI3Kα in both enzyme and cell-based assays and exhibits a very slow off-rate from PI3Kα. LY4045004 demonstrates favorable in vitro ADME properties and excellent PK with high oral bioavailability across preclinical species. In vivo, orally administered LY4045004 demonstrated dose-dependent tumor regressions in PI3Kα H1047R-driven and E545K-driven breast cancer models both as monotherapy and in combination with fulvestrant without inducing hyperglycemia (no significant increase in insulin or C-peptide) or body weight change. Tumor pharmacodynamic analyses confirmed strong pathway inhibition at doses that caused regressions. Additionally, LY4045004 demonstrated tumor regressions in PI3Kα H1047R and E545K PDX models. LY4045004 was well-tolerated at efficacious doses in mice both as a single agent and in combination, and well-tolerated in rat, dog, and cynomolgus monkey toxicology studies. Conclusions: The favorable potency, selectivity, and PK properties of LY4045004 are predicted to result in efficacy with improved tolerability in patients with prevalent PI3Kα helical- and kinase-domain mutant HR+ breast cancers. Global regulatory submissions are planned in the first half of 2025. Citation Format: Raymond Gilmour, Andrew L. Faber, Weihua Shen, Harold B. Brooks, Lisa J. Kindler, Sarah M. Bogner, Loredana Puca, Viviana Volta, Michele Dowless, Jennifer R. Stephens, Anke Klippel, Rui Wang, Divya Ramchandani, Parisa Zolfaghari, Kannan Karukurichi, Alex Gousie, Robert Bondi, Gereint Sis, Ross Wallace, Ronee Baracani, Nathan Wright, Gabrielle Kolakowski, Laurie LeBrun, Steven W. Andrews. Preclinical characterization of LY4045004, a next-generation, mutant-selective PI3Kα inhibitor [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P4-12-24.

Abstract P2-03-16: Prevalence of PIK3CA/AKT1/PTEN and other genomic alterations in primary and recurrent tumor tissue: exploratory analysis from the Phase 3 CAPItello-291 clinical trial

2025-06-13

Javier Cortés , Hope S. Rugo , Mafalda Oliveira +7 more | Clinical Cancer Research

Abstract Background: Based on the positive results from the CAPItello-291 trial, capivasertib plus fulvestrant is a treatment option for adult patients with HR+/HER2− locally advanced or metastatic breast cancer who … Abstract Background: Based on the positive results from the CAPItello-291 trial, capivasertib plus fulvestrant is a treatment option for adult patients with HR+/HER2− locally advanced or metastatic breast cancer who have progressed on an endocrine-based regimen and whose tumors harbor one or more alterations in PIK3CA, AKT1, or PTEN. In CAPItello-291, participants were requested to provide a formalin-fixed, paraffin-embedded tumor sample from the most recently collected tumor tissue for next-generation sequencing (NGS) testing at the stage of either primary or recurrent disease. Overall, 48% of samples with a valid NGS test result had a PIK3CA/AKT1/PTEN alteration. In this exploratory analysis, we assessed the prevalence of PIK3CA/AKT1/PTEN alterations in tissue samples from primary versus recurrent disease and explored the broader molecular profiles between cohorts. Methods: A retrospective review of molecular profiles of non-matched primary and recurrent breast cancer samples from CAPItello-291 was performed to assess the prevalence of PIK3CA/AKT1/PTEN alterations in tissue from the primary tumor versus recurrent disease. Based on the tissue requisition forms, samples were classified as primary if the type of tissue provided was reported as ‘primary’ and had breast/lymph node as the anatomic site, while samples were classified as ‘recurrent’ if the tissue provided was reported as ‘recurrence’ and the anatomic site was not breast. Analysis for pathogenic/likely pathogenic genes with alteration prevalence of ≥2% captured by FoundationOne®CDx was carried out by primary/recurrent and PIK3CA/AKT1/PTEN-altered/non-altered status. Statistical analyses were performed using chi-square or Fisher exact test. All analyses were exploratory. Results: A total of 381/594 (64.1%) of the analyzed samples were defined as primary, 156/594 (26.3%) were defined as recurrent, and 57/594 (9.6%) were defined as unknown/not valid and excluded from further analysis. Prevalence of PIK3CA/AKT1/PTEN alterations was similar in primary and recurrent cohorts, overall (50.7% primary vs 46.2% recurrent, p=0.39) and by gene (PIK3CA: 36.2% primary vs 30.8% recurrent, p=0.27; AKT1: 5.5% primary vs 6.4% recurrent, p=0.84; PTEN: 5.7% primary vs 6.4% recurrent, p=0.94). Compared with primary samples, recurrent samples were enriched for alterations in ESR1 (5% vs 17%, p≤0.001), BRCA2 (5% vs 11%, p=0.02), GATA3 (17% vs 24%, p=0.04), CDKN2A (2% vs 6%, p=0.04), and SMAD4 (1% vs 4%, p=0.04). Notably, ESR1 mutations were enriched at recurrence in both the PIK3CA/AKT1/PTEN-altered and non-altered cohorts, whereas GATA3 alterations were enriched in the PIK3CA/AKT1/PTEN non-altered cohort and CDKN2A alterations in the PIK3CA/AKT1/PTEN-altered cohort at recurrence. When comparing the overall PIK3CA/AKT1/PTEN-altered versus the non-altered cohorts, the altered cohort had more alterations in CDH1, MAP3K1, TBX3, CBFB, and BCL6, whereas the non-altered cohort had more alterations in genes associated with cell cycle, such as GATA3, CCND1, and MYC. Conclusions: Overall, the prevalence of PIK3CA/AKT1/PTEN alterations was comparable between non-matched tumor tissue samples collected at primary or recurrent disease used for NGS testing in CAPItello-291, in line with their role as oncogenic drivers in HR+/HER2− breast cancer. Consistent with the literature, recurrent tissues were enriched for ESR1, BRCA2, GATA3, CDKN2A, and SMAD4 alterations, indicating a potential role for these gene alterations in driving disease recurrence. The PIK3CA/AKT1/PTEN-altered cohort was enriched for CDH1 mutations while the non-altered cohort was enriched for alterations in cell cycle-related genes previously associated with endocrine and cyclin-dependent kinase 4/6 inhibitor resistance. Citation Format: Javier Cortés, Hope S. Rugo, Mafalda Oliveira, Sacha J. Howell, Florence Dalenc, Henry L. Gomez, Xichun Hu, Komal Jhaveri, Petr Krivorotko, Sibylle Loibl, Meena Okera, Yeon Hee Park, Joo-Hyuk Sohn, Masakazu Toi, Eriko Tokunaga, Lyudmila Zhukova, Agostina Nardone, Elza C. de Bruin, Robert McEwen, Marta Fulford, Nicholas C. Turner. Prevalence of PIK3CA/AKT1/PTEN and other genomic alterations in primary and recurrent tumor tissue: exploratory analysis from the Phase 3 CAPItello-291 clinical trial [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P2-03-16.

Abstract P5-02-20: Clinicogenomic landscape and function of PIK3CA, AKT1, and PTEN mutations in breast cancer

2025-06-13

Jacqueline J. Tao , Saumya D. Sisoudiya , Smruthy Sivakumar +2 more | Clinical Cancer Research

Abstract Background: The AKT inhibitor capivasertib was recently approved in the 2L+ setting for patients (pts) with hormone receptor-positive metastatic breast cancer with alterations in PIK3CA, AKT1, and/or PTEN. There … Abstract Background: The AKT inhibitor capivasertib was recently approved in the 2L+ setting for patients (pts) with hormone receptor-positive metastatic breast cancer with alterations in PIK3CA, AKT1, and/or PTEN. There is a need to further characterize the landscape of PIK3CA, AKT1, and PTEN mutations in breast cancer and their functions, to improve our understanding and therapeutic targeting of PI3K-driven breast cancer. Methods: 51,767 pts with breast cancer who underwent tumor sequencing using FoundationOne®CDx or FoundationOne® were included in this analysis. We assessed the frequency, type, and pathogenicity of genomic alterations found in PIK3CA, PTEN, and AKT1 overall and stratified by receptor subtype, patient ancestry, and sample type. Co-occurrence with other pathogenic variants was explored using a Fisher’s exact test with a FDR correction. We correlated PTEN mutations with functional data from deep mutational scanning. Results: The most frequently altered PI3K-pathway gene was PIK3CA (37.4%, 19,384/51,767), followed by PTEN (13.5%) then AKT1 (5.4%), including both pathogenic and VUS alterations in all genes. Among 1,024 distinct PIK3CA alterations identified, 690 (67.4%) were missense mutations. The most common missense mutations were H1047R (35.6% of PIK3CA-altered cases), E545K (19.7%), and E542K (11.7%). Among 288 AKT1 alterations identified, 205 (71.2%) were missense mutations. The most common missense mutation was E17K (69.7% of AKT1-altered cases) followed by gene amplification (14.0%). The most common alteration among patients bearing PTEN alterations was gene deletion (37.3% of PTEN-altered cases). Among 1,723 distinct PTEN alterations identified, 721 (41.9%) were frameshift mutations, 494 (28.7%) were missense mutations, and 246 (14.3%) were splice site mutations. To determine function, PTEN mutations were correlated with existing PTEN deep mutational scanning datasets; these data will be presented. PIK3CA pathogenic alterations were more prevalent in estrogen receptor (ER)+/HER2- and HER2+ cases (40.3% and 37.6% of cases) versus triple negative breast cancer (TNBC; 20.9%), consistent with previously described data. Among all pathogenic PIK3CA alterations, those outside the 542, 545, and 1047 codons were more common in TNBC (42%) compared to ER+/HER2- or HER2+ (33%, p=0.002 and 33%, p=0.002). AKT1 mutations were more prevalent in ER+/HER2- disease (6.0%) than HER2+ or TNBC (1.7%, p=4.7x10^-20 and 3.0%, p=3.5x10^-5). In contrast, PTEN alterations were more commonly found in TNBC (17.9%) than in ER+/HER2- and HER2+ cases (11.3%, p=1.8x10^-8 and 3.9%, p=2x10^-57). PIK3CA alterations were enriched in metastases compared to local tumors (39.6% vs 33.2%, p=6x10^-41) and were less prevalent in African American pts than other groups (27.1% v 38.6% in Europeans, p=9.6x10^-81), while PTEN and AKT alterations were similar across ancestries. PIK3CA, AKT1, and PTEN pathogenic alterations are all mutually exclusive to each other. Statistically significant co-occurring mutations included PIK3CA with CDH1, MAP3K1, SOX2, TBX3, and PRKCI; PTEN with FAS, TP53, and RB1; and AKT1 with NF1. Conclusions: Here we describe the clinicogenomic landscape of PIK3CA, AKT1, and PTEN alterations in a large breast cancer cohort. Our results corroborate previously reported findings that PIK3CA alterations are present in around 40% of ER+/HER2- breast cancer and concentrated in 3 hotspots. However, one-third of patients have variants beyond E542, E545, and H1047 with less certainty about targetability. The preponderance of rare PIK3CA mutations and co-occurrences between PIK3CA, AKT1, and PTEN with genes outside the PI3K pathway merits functional investigation. Citation Format: Jacqueline Tao, Saumya Sisoudiya, Smruthy Sivakumar, Ethan Sokol, Neil Vasan. Clinicogenomic landscape and function of PIK3CA, AKT1, and PTEN mutations in breast cancer [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P5-02-20.

Abstract P2-03-19: Capivasertib-fulvestrant for patients w/ HR-pos/HER2-negative advanced breast cancer who had relapsed or progressed during or after aromatase inhibitor treatment: exploratory analysis of PTEN deficiency by IHC from phase III CAPItello-291 trial

2025-06-13

Komal Jhaveri , Hope S. Rugo , Javier Cortés +7 more | Clinical Cancer Research

Abstract Background: In the phase III CAPItello-291 trial in patients with HR-positive/HER2-negative advanced breast cancer (ABC) who had relapsed or progressed during or after aromatase inhibitor (AI) treatment, the addition … Abstract Background: In the phase III CAPItello-291 trial in patients with HR-positive/HER2-negative advanced breast cancer (ABC) who had relapsed or progressed during or after aromatase inhibitor (AI) treatment, the addition of capivasertib (a potent, selective pan-AKT inhibitor) to fulvestrant significantly improved progression-free survival (PFS) compared with placebo-fulvestrant in the overall population (hazard ratio: 0.60; 95% confidence interval [CI]: 0.51–0.71; p&lt;0.001) and in patients with PIK3CA/AKT1/PTEN-altered tumors detected by next-generation sequencing (NGS; hazard ratio: 0.50; 95% CI: 0.38–0.65; p&lt;0.001). Previous exploratory analysis also showed consistent PFS benefit across each alteration detected, including in patients with PTEN-altered tumors (n=50; hazard ratio: 0.45; 95% CI: 0.24–0.84). Here, we report a prespecified exploratory analysis of alteration prevalence and PFS in patients with deficient PTEN expression as detected by immunohistochemistry (IHC). Methods: In CAPItello-291, eligible pre-/peri- or postmenopausal women or men with HR-positive/HER2-negative ABC that had recurred or progressed on or after AI treatment with or without a cyclin-dependent kinase 4/6 inhibitor (CDK4/6i) were randomized 1:1 to receive fulvestrant (500 mg intramuscularly on days 1 and 15 of cycle 1, and day 1 of each subsequent 28-day cycle) with either placebo or capivasertib (400 mg twice daily; 4 days on, 3 days off). PIK3CA/AKT1/PTEN alteration status was determined post-randomization using NGS in tumor tissue collected prior to study enrollment. Available samples were processed centrally to determine deficient PTEN protein expression by IHC using the VENTANA® PTEN SP218 antibody and a prespecified cutoff for this study of &lt;10% staining of tumor cells. Hazard ratios for PFS were calculated using Cox proportional hazards models stratified by prior use of CDK4/6i. Data cutoff: August 15, 2022. Results: In total, PTEN results by IHC were obtained from 373/708 (53%) patient tumor samples. Baseline characteristics were broadly balanced between those with and without PTEN testing results. 71/373 (19%) patient tumor samples were identified as PTEN deficient by IHC; of these, 55% (n=39) had PIK3CA/AKT1/PTEN-altered tumors by NGS (26% PIK3CA [PIK3CA only n=15; PIK3CA and PTEN n=4]; 1% AKT1 [AKT only n=1]; 32% PTEN [PTEN only n=19; PIK3CA and PTEN n=4]), 30% (n=21) had PIK3CA/AKT1/PTEN-non-altered tumors by NGS, and 15% (n=11) had unknown NGS results. For tumors PTEN proficient by IHC 302/373 (81%), 164 (55%) were non-altered and 128 (42%) were altered by NGS (107/302, 35% PIK3CA, 6% AKT1, 3% PTEN) and 10 (3%) had unknown NGS result. Within samples with both NGS and IHC data, all samples with homozygous deletions or large rearrangements of PTEN by NGS were PTEN deficient by IHC. In patients with PTEN-deficient tumors by IHC, 34/71 (48%) received capivasertib-fulvestrant and 37/71 (52%) received placebo-fulvestrant. In this group, PFS benefit was observed with capivasertib-fulvestrant versus placebo-fulvestrant: median PFS: 9.3 months versus 3.7 months; hazard ratio: 0.52 (95% CI: 0.28–0.93). Conclusions: In this CAPItello-291 exploratory analysis, 19% of patient tumor samples that were available for central IHC testing were PTEN deficient by IHC. Within this subgroup of PTEN-deficient tumors, over half also had PIK3CA/AKT1/PTEN alterations detected by NGS. In the PTEN-deficient by IHC cohort, PFS benefit was noted with capivasertib-fulvestrant versus placebo-fulvestrant, although results are exploratory. Citation Format: Komal Jhaveri, Hope S. Rugo, Javier Cortes, Mafalda Oliveira, Sacha J. Howell, Florence Dalenc, Henry L. Gomez, Xichun Hu, Petr Krivorotko, Sibylle Loibl, Meena Okera, Yeon Hee Park, Joo-Hyuk Sohn, Masakazu Toi, Eriko Tokunaga, Lyudmila Zhukova, Agostina Nardone, Elza C. de Bruin, Ian Wadsworth, Celina D'Cruz, Nicholas C. Turner. Capivasertib-fulvestrant for patients w/ HR-pos/HER2-negative advanced breast cancer who had relapsed or progressed during or after aromatase inhibitor treatment: exploratory analysis of PTEN deficiency by IHC from phase III CAPItello-291 trial [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P2-03-19.

Abstract P1-05-27: Analytical comparison of tissue-based next-generation sequencing assays for the detection of PIK3CA, AKT1, and PTEN tumor alterations in breast cancer

2025-06-13

Xiaodun Li , Alexander Yarunin , Ben T. Chaffey +6 more | Clinical Cancer Research

Abstract Background: Next-generation sequencing (NGS) testing in patients with advanced breast cancer (ABC) enables genomic biomarker interrogation, potentially guiding clinical management. Results from the CAPItello-291 Phase 3 randomized trial led … Abstract Background: Next-generation sequencing (NGS) testing in patients with advanced breast cancer (ABC) enables genomic biomarker interrogation, potentially guiding clinical management. Results from the CAPItello-291 Phase 3 randomized trial led to the approval of the first-in-class pan-AKT inhibitor capivasertib in combination with fulvestrant as a treatment option in patients with hormone receptor (HR)-positive/human epidermal growth factor receptor 2 (HER2)-negative ABC, with one or more PIK3CA/AKT1/PTEN tumor alterations following disease progression on/after prior aromatase inhibitor therapy. PIK3CA/AKT1/PTEN alterations are present in approximately 50% of all HR-positive/HER2-negative breast cancers and also in around 30% of all triple-negative breast cancers (TNBC). This study aimed to analytically compare the ability of commercially available tissue-based NGS assays to detect PIK3CA/AKT1/PTEN tumor genomic alterations in breast cancer samples. Methods: Formalin-fixed, paraffin-embedded tumor samples collected from patients with TNBC were analyzed at three different sites using the following commercial NGS assays in accordance with the manufacturers’ instructions: AVENIO Tumor Tissue CGP (Roche), TruSight Oncology 500 (Illumina), oncoReveal Core LBx (Pillar Biosciences), Oncomine Comprehensive Assay v3 (ThermoFisher Scientific), AmoyDx HANDLE Classic (Amoy Diagnostics), and SOPHiA ExtHRS (SOPHiA GENETICS). Detection of single nucleotide variants, insertions/deletions, and copy number variants in PIK3CA, AKT1, and PTEN approved by the FDA as genomic alterations which determine eligibility for treatment with capivasertib in combination with fulvestrant in patients with HR-positive/HER2-negative ABC was recorded and compared. Positive percent agreement (PPA), negative percent agreement (NPA), and overall percent agreement (OPA) were calculated for all assays. AVENIO Tumor Tissue CGP was used as the reference assay, as the content is broadly comparable to the FDA-approved FoundationOne CDx assay. Results: Overall, 45 samples were processed, and all samples were included in the final analysis. PPA, NPA, and OPA for any alteration(s) (PIK3CA, AKT1 or PTEN) versus AVENIO (reference) were: TruSight: 86.0%, 100.0%, and 86.7%; oncoReveal: 81.4%, 100.0%, and 82.2%; Oncomine: 81.4%, 100.0%, and 82.2%; AmoyDx: 86.0%, 100.0%, and 86.7%; SOPHiA: 88.4%, 100.0%, and 88.9%. There was 100.0% PPA, NPA, and OPA for both PIK3CA and AKT1 between TruSight, Oncomine, AmoyDx, SOPHiA and AVENIO. For oncoReveal, PPA, NPA, and OPA were 100.0%, 94.1%, and 97.8% for PIK3CA and 100.0%, 100.0%, and 100.0% for AKT1 alterations, respectively. For PTEN alterations, PPA, NPA, and OPA per assay versus reference were: TruSight: 58.8%, 96.4%, and 82.2%; oncoReveal: 17.6%, 100.0%, and 68.9%; Oncomine: 35.3%, 100.0%, and 75.6%; AmoyDx: 47.1%, 100.0%, and 80.0%; SOPHiA: 58.8%, 100.0%, and 84.4%. Lower agreement for PTEN alterations was due to differences in gene coverage and ability of some assays to detect complex PTEN genomic alterations, such as large rearrangements and copy number variations. Conclusions: All assays evaluated demonstrated good concordance with the AVENIO Tumor Tissue CGP test, especially for detecting capivasertib treatment eligible alterations in AKT1 and PIK3CA. Further improvement on detection of PTEN structural and copy number alterations is needed for some assays in order to maximise patient identification for capivasertib in combination with fulvestrant. These data can help clinicians make informed decisions regarding suitable diagnostic tests to determine patient eligibility for breast cancer therapies. Citation Format: Xiaodun Li, Alexander Yarunin, Benjamin Chaffey, Manisha Maurya, Peter Stewart, Fionn Corr, Efstratios Efstratiou, Kirsty Trewellard, David Gonzalez. Analytical comparison of tissue-based next-generation sequencing assays for the detection of PIK3CA, AKT1, and PTEN tumor alterations in breast cancer [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P1-05-27.

Abstract P4-01-17: Uncovering of NRAGE/JNK and PI3K/AKT pathways through serial WGS of breast cancer patients

2025-06-13

Byeongju Kang , In Hee Lee , Soo Jung Lee +7 more | Clinical Cancer Research

Abstract Purpose: Cancer genomic sequencing has fundamentally advanced our understanding of the basic biology of this disease, and more recently, has offered method to guide and evaluate treatment in clinic. … Abstract Purpose: Cancer genomic sequencing has fundamentally advanced our understanding of the basic biology of this disease, and more recently, has offered method to guide and evaluate treatment in clinic. We performed whole-genome sequencing (WGS) on three consecutive tissue and blood samples from breast cancer patients: diagnosis, post-neoadjuvant chemotherapy, recurrence after curative resection. Methods: We performed WGS to compared the genetic profiles of the primary, surgical and recurrent samples (tissue and blood) to determine: (1) how closely related the genetics are among the primary , post- neoadjuvant chemotherapy, and the recurrent tumors, (2) whether there are variations in mutational processes among the primary, post- neoadjuvant chemotherapy, and recurrent tumor. WGS was performed on three patients for whom both tissue and blood were available at diagnosis, at surgery after neoadjuvant chemotherapy and at a recurrence. Results: As a result of somatic protein coding variant analysis, PIK3CA mutation was confirmed at the time of diagnosis tissue in all cases. Mutations were found at p.E545K in patient 14, p.R916C in patient 18, and p.R916C in patient 3. Among these, patients 14 and 18 had PIK3CA mutations even at the time of recurrence. In patient 14, the same PIK3CA mutation was found in 3 consecutive samples, but in patient 18, it changed from p.R916C at diagnosis to p.G480E at relapse. Furthermore, functional enrichment analysis identified a common NRAGE signaling system in the cfDNA. The NRAG pathway was found in the blood at diagnosis, surgery, and recurrence in all three patients. Conclusion: This study was a proof-of-concept study, in which we hypothesized that NRAGE/JNK and PI3K/AKT signaling pathways are involved in breast cancer recurrence and treatment response through WGS of consecutive samples. Further research is ongoing with larger numbers of patients. Citation Format: Byeongju Kang, In Hee Lee, Soo Jung Lee, Jeeyeon Lee, Ho Yong Park, Ji-Young Park, Nora Jee-Young Park, Eun Ae Kim, Seolhwa Jeong, Jieun Kang, Yee Soo Chae. Uncovering of NRAGE/JNK and PI3K/AKT pathways through serial WGS of breast cancer patients [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P4-01-17.

Abstract P5-06-16: Preclinical Characterization of LAE118, a Novel Allosteric Pan-mutant Selective PI3Kα Inhibitor

2025-06-13

Ming Li , Ruipeng Zhang , Junyan Chen +3 more | Clinical Cancer Research

Abstract PIK3CA is one of the most frequently mutated oncogenes in cancer, found in approximately 13% human cancers. PI3Kα mutations are prevalent in patients with breast, colorectal, lung, endometrial, and … Abstract PIK3CA is one of the most frequently mutated oncogenes in cancer, found in approximately 13% human cancers. PI3Kα mutations are prevalent in patients with breast, colorectal, lung, endometrial, and numerous other cancers. Three hotspot mutations (H1047R, E545K, E542K) on the p110α subunit of PI3Kα are recognized activating mutations accounting for approximately 1/3 of all PI3Kα mutations. Targeting PI3Kα in cancer has been validated as a therapeutic strategy, as evidenced by the approved drug Alpelisib and the positive clincal trial result of Inavolisib, demonstrating clinical efficacy either alone or in combincation with other therapies. However, treament with non-mutant selective inhibitors raises tolerability concerns such as hyperglycimea, due to the inhibition of wild type PI3Kα. Moreover, the clinical use of PI3Kα orthosteric inhibitors, such as Alpelisib and Invaolvisib, has led to the emergency of secondary mutations (e.g. Q859K & W780R) in the ATP binding pocket resulting in resistance to these drugs. Therefore, there is a pressing need to develop novel PI3Kα-targeted therapies that can minimize on-target toxicities and overcome drug ressistance. LAE118 is a novel allosteric pan-mutant selective PI3Kα inhibitor. LAE118 demonstrates excellent in vitro anti-proliferation activities in PI3Kα mutant cell lines and remains active against cells resistant to orthosteric PI3Kα inhibitors. LAE118 shows strong anti-tumor growth effect in Xenograft models at a dose that is much lower than other allosteric inhibitors and has less effect on glucose homeostasis compared to orthosteric inhibitors. In pre-clinical toxicology studies, LAE118 also demonstrated good tolerability. These data indicate that LAE118 offers improved efficacy and larger safety window. LAE118 is currently in IND enabling stage. Citation Format: Ming Li, Ruipeng Zhang, Junyan Chen, Meijuan Hu, Xiaofen Lin, Justin Gu. Preclinical Characterization of LAE118, a Novel Allosteric Pan-mutant Selective PI3Kα Inhibitor [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P5-06-16.

Abstract P4-12-18: Discovery and characterization of ETX-636, a potential best-in-class, oral, small molecule, pan-mutant-selective PI3Kα inhibitor

2025-06-13

Robert F. Koncar , Mingzong Li , Jingyan Gao +7 more | Clinical Cancer Research

Abstract PIK3CA, which encodes p110α, the catalytic subunit of phosphatidylinositol 3-kinase alpha (PI3Kα), is one of the most frequently mutated oncogenes, with activating mutations seen in ∼16% of all solid … Abstract PIK3CA, which encodes p110α, the catalytic subunit of phosphatidylinositol 3-kinase alpha (PI3Kα), is one of the most frequently mutated oncogenes, with activating mutations seen in ∼16% of all solid tumors and up to 40% of breast tumors, including hormone receptor-positive/HER2-negative, advanced breast cancer. The most frequent gain-of-function PI3Kα hotspot mutations, H1047R, E542K, and E545K, are well-recognized oncogenic drivers. Cancer cells with PIK3CA activating mutations are dependent on PI3Kα signaling and HR-positive, HER2-negative breast cancer patients with PIK3CA mutations respond to alpelisib, an approved PI3Kα inhibitor. While PI3Kα is important for cancer cell proliferation and tumor growth, it is also a critical component of the insulin signaling pathway. Therefore, the use of orthosteric inhibitors like alpelisib, which inhibit both the wildtype and mutant proteins, often results in significant hyperglycemia limiting their clinical utility. We report the discovery and pre-clinical characterization of an allosteric, pan-mutant-selective PI3Kα inhibitor, ETX-636, which was designed leveraging our Kinetic Ensemble® platform for optimal binding properties. Compared to other allosteric, pan-mutant-selective PI3Kα inhibitors (ie RLY-2608 and STX-478), ETX-636 has stronger target binding affinity, better on-target potency in biochemical and cellular pharmacodynamic and viability assays, and demonstrates superior anti-tumor activity in vivo. More specifically, ETX-636 inhibits both kinase (H1047X) and helical domain (E542K and E545K) mutant PI3Kα biochemical activity with single digit nM potency and 8-10-fold selectivity relative to wildtype PI3Kα protein. ETX-636 shows greater than 1000-fold selectivity over the β, δ, and γ class I PI3K isoforms in biochemical assays and the selectivity extends more broadly across a panel of ∼350 kinases. In cellular assays, ETX-636 potently inhibits proliferation and PI3Kα signaling specifically in PIK3CA-mutant cell lines. Mechanistically, ETX-636 induces proteasome-dependent degradation of mutant p110α protein in vitro and in vivo. Consistent with the compound-mediated decrease of mutant p110α protein and the high binding affinity (slow off-rate) of the compound, ETX-636 achieves sustained PI3Kα pathway inhibition in cellular washout assays and in cell-derived xenograft (CDX) tumor models. Once daily, oral dosing of ETX-636 shows single agent efficacy at well-tolerated doses in both PI3Kα kinase and helical domain-mutant breast cancer CDX models, significantly inhibiting tumor growth or inducing regression, while suppressing PI3K pathway activity in a dose-dependent manner. In an ER-positive, HER2-negative, PI3Kα-mutant breast cancer xenograft model, ETX-636 is efficacious as a single agent and shows enhanced activity in combination with fulvestrant, inducing consistent tumor regression while being well-tolerated. ETX-636 demonstrates superior magnitude and duration of PI3Kα pathway inhibition in vivo, compared to known pan-mutant-selective allosteric PI3Kα inhibitors, as well as orthosteric inhibitors. At efficacious doses, ETX-636 has significantly less of an effect on blood glucose in mice compared to orthosteric inhibitors, demonstrating that ETX-636 can achieve potent anti-tumor activity by targeting mutant PI3Kα protein without significantly affecting the activity of the wildtype protein. In addition, based on pharmacokinetic/pharmacodynamic/efficacy and toxicology studies, ETX-636 is unlikely to pose a significant risk of hyperglycemia at predicted human efficacious doses. These data support clinical exploration of ETX-636 in mutant PI3Kα solid tumors and, potentially, mutant PI3Kα-driven rare diseases. Citation Format: Robert Koncar, Mingzong Li, Jingyan Gao, Fei Pang, Ying Lin, Raj Nagaraja, Yong Tang, Hannah Szeto, Zipeng Fan, Karan Kapoor, Robbie Chen, Eric Simone, Minghong Hao, Shengfang Jin, Tao Liu, Tai Wong, Meghana Kulkarni, Jeffery Kutok. Discovery and characterization of ETX-636, a potential best-in-class, oral, small molecule, pan-mutant-selective PI3Kα inhibitor [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P4-12-18.

Abstract P3-10-13: A Comprehensive Analysis of Dysregulation in the PTEN/PI3K/AKT Pathway in Breast Cancer Among the Chinese Population

2025-06-13

Ziang Li , Meizhen Hu , Fei Pang +3 more | Clinical Cancer Research

Abstract Background: Breast cancer (BC) has the highest incidence rate among cancers in females in China. The PTEN/PI3K/AKT pathway regulates key biological processes, including apoptosis, metabolism, cell proliferation and growth … Abstract Background: Breast cancer (BC) has the highest incidence rate among cancers in females in China. The PTEN/PI3K/AKT pathway regulates key biological processes, including apoptosis, metabolism, cell proliferation and growth in BC. This is the first comprehensive study to explore the association between these pathway gene alterations and clinical outcomes in a large cohort of Chinese BC patients. Methods: We retrospectively reviewed 1018 Chinese BC patients with clinical information from OrigiMed Chinese Real-World Database between 2016 and 2023. Tumor tissue samples and paired peripheral blood leukocytes were analyzed using an OrigiMed 450 gene next-generation sequencing panel. Chi-square test and Fisher’s exact test assessed differences in these genetic alterations across different subgroups and to examine the co-occurrence of alterations between these genes and other genes. Results: A total of 38.5%, 5.3% and 8.4% of Chinese BC patients exhibited alterations in PIK3CA, AKT1 and PTEN genes, respectively, which is similar to the Caucasian-dominated MSK-IMPACT cohort (PIK3CA: 38.5% vs. 35.2%, P =0.102; AKT1: 5.3% vs. 5.3%, P =0.995; PTEN : 8.4% vs. 7.0%, P =0.232). Subgroup analysis revealed higher PTEN alteration in stage Ⅳ patients compared to stage I-III (12.7% vs. 6.4%, P =0.001). Furthermore, HR+/HER2- BC patients exhibited a significantly higher incidence of PIK3CA (45.2% vs. 30.8%, P &lt;0.001) and AKT1 (7.7% vs. 2.5%, P &lt;0.001) gene alterations, compared to other subtypes. The overall incidence of alterations in the PTEN/PI3K/AKT pathway was 50.9%, which was higher among HR+/HER2- BC patients compared to other subtypes (57.0% vs. 43.9%, P &lt;0.001). PIK3CA alterations were associated with lower AKT1 alterations (3.1% vs. 6.7%, P =0.011). ERBB2 alterations were associated with lower AKT1 (0.4% vs. 6.9%, P&lt;0.001) and PTEN (4.4% vs. 9.7%, P =0.009) alterations. TP53 alterations were associated with higher PTEN/PI3K/AKT pathway alterations (55.4% vs. 45.9%, P =0.002). No significant differences in PIK3CA (25.6% vs. 21.4%), AKT1 (7.0% vs. 9.5%), and PTEN (14.0% vs. 23.8%) gene alterations were observed between primary and metastatic lesions in the same patients (N=41, P &gt;0.05). No significant differences were found in the alterations of PIK3CA (38.7% vs. 41.1%), AKT1 (5.8% vs. 5.4%), and PTEN (10.4% vs. 7.6%) between samples taken before and after treatments such as chemo-immunotherapy and targeted therapy (N=840, P &gt;0.05). Survival analysis (median follow-up: 32.7 months) showed PTEN alterations correlated with higher mortality risk (HR 2.779, 95% CI 1.083-7.132, P =0.034), while PIK3CA alterations were associated with reduced mortality risk (HR 0.338, 95% CI 0.141-0.820, P =0.016), with overall survival (OS) as the outcome. Conclusion: This study highlights the significant role of PIK3CA, AKT1 and PTEN gene alterations in Chinese BC patients. In Chinese BC patients, alterations in the PTEN/PI3K/AKT signaling pathway are common, and the frequencies of PIK3CA, AKT1 and PTEN gene are similar to that observed in Western countries. The gene alterations in this pathway are associated with clinicopathological features as well as prognosis, such as disease stage and BC subtypes, but not with primary/metastatic status or treatment. PIK3CA alterations were a benign predictor of survival, whereas PTEN alterations were a negative predictor. These findings suggest that genetic profiling of the PTEN/PI3K/AKT pathway could guide treatment strategies and prognosis prediction in Chinese BC patients. Future studies should focus on validating these findings in prospective cohorts and exploring targeted therapies based on these genetic alterations. Citation Format: Ziang Li, Meizhen Hu, Fei Pang, Bo Peng, Aodi Wang, Huanwen Wu. A Comprehensive Analysis of Dysregulation in the PTEN/PI3K/AKT Pathway in Breast Cancer Among the Chinese Population [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P3-10-13.

Cancer stem cells and the SHH pathway in malignant melanoma: Therapeutic implications

2025-06-10

Berrin Özdil , Hüseyin Aktuğ | Ege Tıp Dergisi

Malignant melanoma is an aggressive skin cancer arising from melanocytes. Melanoma is a complex disease both sourced from genetics and environmental factors. Within melanoma, cancer stem cells (CSCs) play a … Malignant melanoma is an aggressive skin cancer arising from melanocytes. Melanoma is a complex disease both sourced from genetics and environmental factors. Within melanoma, cancer stem cells (CSCs) play a crucial role in tumor progression, therapeutic resistance, and recurrence due to their capabilities for self-renewal and differentiation. The Sonic Hedgehog (SHH) signaling pathway is an important regulator of CSCs and is essential for cell differentiation and proliferation. Due to it is known role in embryonic development and involvement in cancers, SHH pathway significantly affects CSC behavior in malignant melanoma, promoting tumorigenicity, metastasis, and resistance to therapies. This pathway coordinates canonical mechanisms involving Gli transcription factors and non-canonical mechanisms affecting cell migration and cytoskeletal organization. Targeting the SHH pathway has emerged as a promising therapeutic strategy, with inhibitors focusing on components like Smoothened (Smo) and Gli proteins. However, resistance to these inhibitors necessitates further exploration of novel therapeutic combinations. Current research focuses on combining SHH inhibitors with immunotherapies for more effective, long-lasting responses. Targeted medicines, which disrupt SHH processes, attempt to eliminate the fundamental causes of carcinogenesis and increase melanoma patient survival rates.

EIF4A3 enhances the viability, invasion and osteogenic differentiation of BMSCs via the USP53/SMAD5 pathway

2025-06-06

Gang Cheng , Xiaoling Yang , Yulan Tang +3 more | Scientific Reports

SMAD5 has been demonstrated to promote osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) through the circ_0001825/miR-1270/SMAD5 axis or KCNQ1OT1/miR-320a/SMAD5 axis. Therefore, SMAD5 may be a key regulator of … SMAD5 has been demonstrated to promote osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) through the circ_0001825/miR-1270/SMAD5 axis or KCNQ1OT1/miR-320a/SMAD5 axis. Therefore, SMAD5 may be a key regulator of BMSCs osteogenic differentiation, and its more related molecular mechanisms are worth further revealing. Western blot analysis was used to detect the protein levels of SMAD5, ubiquitin-specific peptidase 53 (USP53), eukaryotic translation initiation factor 4A3 (EIF4A3), and osteogenic differentiation-related markers. Cell counting kit 8 and transwell assay were performed to measure cell viability and invasion. Alkaline phosphatase (ALP) activity detection and Alizarin red S staining were employed to assess osteogenic differentiation. The interactions between USP53 and SMAD5/EIF4A3 were confirmed by Co-immunoprecipitation assay. The mRNA levels of SMAD5 and USP53 were examined using quantitative real-time PCR. SMAD5 silencing suppressed viability, invasion and osteogenic differentiation of BMSCs, while its overexpression had opposite effects. USP53 deubiquitinated SMAD5 to stabilize its protein expression. Moreover, USP53 knockdown inhibited viability, invasion and osteogenic differentiation of BMSCs, while these effects were reverted by SMAD5 overexpression. EIF4A3 stabilized USP53 mRNA expression, and the inhibitory effect of EIF4A3 silencing on viability, invasion and osteogenic differentiation of BMSCs was abolished by USP53 overexpression. Furthermore, EIF4A3 enhanced SMAD5 expression by interacting with USP53. EIF4A3-stabilized USP53 promotes SMAD5 deubiquitination to enhance viability, invasion and osteogenic differentiation of BMSCs.

RHCG inhibition promotes the sensitivity of PIK3CA-mutant non-small lung cancer to PI3K inhibitor.

2025-06-06

Haitao Liu , Muqun Wang , Yingying Pang | PubMed

PIK3CA, the gene encoding the catalytic subunit of the PI3K complex, is significantly amplified and mutated in several cancer forms, including non-small cell lung cancer (NSCLC). Although follicular lymphoma and … PIK3CA, the gene encoding the catalytic subunit of the PI3K complex, is significantly amplified and mutated in several cancer forms, including non-small cell lung cancer (NSCLC). Although follicular lymphoma and chronic lymphocytic leukemia have been treated clinically with PI3K inhibitors, there are currently no FDA-approved medications targeting PI3K mutations in lung cancer. In this study, by TCGA database analysis, we found that RHCG was highly expressed in PIK3CA-mutated NSCLC and was associated with poor prognosis. Knockdown of RHCG in PIK3CA-mutated NSCLC inhibited cell proliferation and promoted apoptosis, thereby sensitizing cells to PI3K inhibitors. Mechanistically, the knockdown of RHCG acts synergistically with PI3K inhibitors to produce more effective inhibition of AKT and S6RP phosphorylation downstream of PI3K. This finding suggests a potential NSCLC target with PIK3CA mutations.

Retraction: Activated α2-macroglobulin binding to cell surface GRP78 induces T-loop phosphorylation of Akt1 by PDK1 in association with raptor

2025-06-05

| PLoS ONE

Cancer prognosis and treatment results in patients with PTEN Hamartoma Tumour Syndrome (PHTS)—a European cohort study

2025-06-04

Linda A.J. Hendricks , Katja C J Verbeek , Janneke Schuurs-Hoeijmakers +7 more | BJC Reports

Abstract Background PTEN hamartoma tumour syndrome (PHTS) patients have a high hereditary risk of cancer, especially breast (BC), endometrial (EC), and thyroid cancer (TC). However, the prognosis of PHTS-related cancers … Abstract Background PTEN hamartoma tumour syndrome (PHTS) patients have a high hereditary risk of cancer, especially breast (BC), endometrial (EC), and thyroid cancer (TC). However, the prognosis of PHTS-related cancers is unknown. Methods This European cohort study included adult PHTS patients with data from medical files, registries, and/or questionnaires. Overall survival (OS) was assessed using Kaplan-Meier analyses and were compared with sporadic cancer and the general population using standardized mortality (SMR) and relative survival rates (RSR). Survival bias was addressed using left-truncation. Results Overall, 147 BC patients were included. The 10y-OS was 77% (95%CI = 66–90), decreasing with increasing stage from 90% (95%CI = 73–100) for stage 0 to 0% (95%CI = 0–0) for stage IV. BC relative survival was comparable to sporadic BC in the first two years (2y-RSR = 1.1; 95%CI = 1.1–1.1) and increasing thereafter (5y-RSR = 1.7; 95%CI = 1.6–1.7). For TC ( N = 56) and EC ( N = 35), 10y-OS was 87% (95%CI = 74–100) and 64% (95%CI = 38–100), respectively. Overall and cancer-specific mortality in female PHTS patients exceeded general population rates (SMR = 3.7; 95%CI = 2.6–5.0 and SMR = 2.7; 95%CI = 1.6–4.4). Conclusions The prognosis of PHTS-related cancers was comparable to the general population. The higher overall mortality in PHTS patients is presumably related to their higher cancer incidence. These findings, and the high survival observed in early-stage cancer, emphasise the importance of recognising PHTS early to facilitate cancer surveillance.

A Dual-Functional Mitochondrial Probe for Melanoma Visualization and PI3K/AKT/mTOR-EMT Signaling Axis-Mediated Antitumor Therapy

2025-06-03

Tong Xia , Hongye Liao , Ziyuan Zeng +7 more | Colloids and Surfaces B Biointerfaces

Increased AKT1 Immunostaining in Colorectal Carcinoma is Associated with Poor Prognosis

2025-06-02

Omar J. Mohammed , Wafaey Gomaa , rabab ahmed safwat +1 more | El-Minia Medical Bulletin

Identifying C1orf122 as a potential HCC exacerbated biomarker dependently of SRPK1 regulates PI3K/AKT/GSK3β signaling pathway

2025-06-01

Jing Cai , Rong Li , Runzhi Wang +7 more | Genes & Diseases

FHL2 Facilitates LUSC Growth and Therapy Resistance through PI3K/AKT/mTOR Activation

2025-06-01

Lingxian Zhang , Dingguo Wang , Lei Zeng +7 more | Journal of Biological Chemistry

ABS0048 DIFFERENTIAL ACTIVATION OF LYSOPHOSPHATIDIC ACID REGULATED GENES IN DIFFUSE AND LIMITED CUTANEOUS SYSTEMIC SCLEROSIS

2025-06-01

Madhuri Kanitkar , Philip Yee , Stefano Rodolfi +2 more | Annals of the Rheumatic Diseases

Effects of PIK3CA Gene Modifications on Radiosensitivity in Colorectal Cancer Cells

2025-06-01

Ghazi Alsbeih , Khaled Al-Hadyan | Asian Pacific Journal of Cancer Prevention

The phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) plays a critical role in cell growth and survival. PIK3CA somatic mutations are linked to the PIK3CA-related overgrowth spectrum (PROS) syndrome. Cells from … The phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) plays a critical role in cell growth and survival. PIK3CA somatic mutations are linked to the PIK3CA-related overgrowth spectrum (PROS) syndrome. Cells from those patients appeared to be associated with a moderate but significant radiosensitivity. Mutations or amplifications in this gene are common in breast, colorectal, and lung cancers. Alterations in the PIK3CA gene, including amplification and mutation, are common in cancer, but their influence on radiotherapy is not yet fully understood. This report reveals a potential association between PIK3CA gene modifications and radiosensitivity (p < 0.05) deducted from 8 established colorectal cancer cell lines (HT-29, DLD-1, HCT-116, SW480, HCT-15, Colo-320, and LoVo). Meanwhile gene amplification (> 2) seems to be linked to increased radiation sensitivity, mutations appear to be associated with increased radioresistance in colorectal cancer cells. Leveraging this relationship, PIK3CA amplification and mutations could act as biomarker to pinpoint patients who might benefit from more personalized radiotherapy regimens. However, this association is preliminary and hypothesis-generating in view of the limited number of cases. Further studies are needed to confirm this conclusion. By uncovering the distinct mechanistic effects of these PIK3CA alterations on radiosensitivity phenotype in both normal and cancerous cells, researchers can lay the groundwork for tailored radiotherapy strategies in colorectal cancer. This insight could enhance treatment effectiveness while reducing side effects, ultimately leading to improved patient outcomes.

First Screening For Germline Variations In Exon 5 PTEN Gene and Their Contribution to Triple Negative Breast Cancer in Eastern Algeria

2025-06-01

Souad Haddad , Karim Chekroud , Housna Zidoune +5 more | Asian Pacific Journal of Cancer Prevention

Triple negative breast cancer (TNBC) is the most aggressive breast cancer subtype associated with younger age, bigger tumor size, high grade tumor, high risk of tumor recurrence and death. PTEN … Triple negative breast cancer (TNBC) is the most aggressive breast cancer subtype associated with younger age, bigger tumor size, high grade tumor, high risk of tumor recurrence and death. PTEN is one of the tumor suppressor genes that have been analyzed to provide its role in predisposition to TNBC. The aim of this study was to screen germline variants in exon 5 of the PTEN gene in Algerian TNBC patients and to assess their association with the clinical characteristics of TNBC. 69 TNBC patients coming from different regions of eastern Algeria were analyzed for germline variants in exon 5 of the PTEN gene, among them 6 patients (8.69%) had a family history of breast cancer. Peripheral blood samples were obtained and genomic DNA was extracted from leukocytes using the salt-saturation method. Exon 5 was amplified by PCR and then sequenced. The resulted sequences were aligned against the reference sequence available in GenBank. All detected variants were annotated using the Ensembl database and their pathogenicity was predicted according to their REVEL scores. 30 different variants in 27 (39.13%) of the 69 patients were identified. 6 missense variants were predicted to be likely benign and 24 variants were predicted to be pathogenic. Among them, 19 were missense variants, 2 were nonsense variants and 3 were frameshift variants, including 1 deletion and 2 novel insertions. The pathogenic variants occurred in 17 patients, who harbored between 1 and 4 pathogenic variants. No pathogenic variants were found in patients with a family history of breast cancer. The correlation between pathogenic variants and the clinical characteristics of TNBC patients was statistically insignificant. Conclusion: The frequency of pathogenic variants identified in the Algerian population is higher than that in other populations; however, they are not associated with susceptibility to TNBC.

Development of Benzoxazole Derivatives Targeting mTOR: A Promising Approach for Breast Cancer Therapy

2025-05-31

Mehmet Abdullah Alagöz , Meryem Temiz Resitoglu , Burak Kuzu +7 more | ChemistrySelect

Abstract Clinical use of mTOR inhibitors in cancer treatment is well established due to the critical role of mTOR signaling in tumor progression. In this study, we report the structure‐based … Abstract Clinical use of mTOR inhibitors in cancer treatment is well established due to the critical role of mTOR signaling in tumor progression. In this study, we report the structure‐based design and biological evaluation of a series of benzoxazole derivatives as potential mTOR inhibitors. Cytotoxicity studies using MTT assays showed that compounds B4, B11, B12, and B20 exhibited significant antiproliferative effects against breast cancer cell lines with IC₅₀ values between 4.96 and 9.82 µM. Colorimetric enzymatic assays further revealed that among these, only B12 and B20 effectively inhibited mTOR phosphorylation at Ser2448 in MCF‐7 cells. Additionally, both compounds modulated the expression of key apoptotic proteins, including Bax, caspase‐3, p53, and Bcl2. Molecular docking studies against the 4JT5 protein demonstrated binding affinities with docking scores ranging from −7.084 to −7.426 kcal/mol, comparable to the reference compound P2X (−7.309 kcal/mol). Molecular dynamics simulations over 150 ns confirmed the stability of B12 and B20 in the active site, with an average RMSD of 2.8 Å and 3.0 Å, respectively. The absorption, distribution, metabolism, excretion, and toxicity (ADMET) properties of the synthesized compounds were evaluated in silico. Among them, B4, B11, B12, and B20 exhibited drug‐like characteristics and showed no undesirable toxic effects. These findings highlight the potential of B12 and B20 as lead compounds for the development of novel mTOR inhibitors in breast cancer therapy.