Mutant Of Phosphatidylinositol 3-kinase Research Articles

Discovery of GDC-0077 (Inavolisib), a Highly Selective Inhibitor and Degrader of Mutant PI3Kα.

Small molecule inhibitors that target the phosphatidylinositol 3-kinase (PI3K) signaling pathway have received significant interest for the treatment of cancers. The class I isoform PI3Kα is most commonly associated with solid tumors via gene amplification or activating mutations. However, inhibitors demonstrating both PI3K isoform and mutant specificity have remained elusive. Herein, we describe the optimization and characterization of a series of benzoxazepin-oxazolidinone ATP-competitive inhibitors of PI3Kα which also induce the selective degradation of the mutant p110α protein, the catalytic subunit of PI3Kα. Structure-based design informed isoform-specific interactions within the binding site, leading to potent inhibitors with greater than 300-fold selectivity over the other Class I PI3K isoforms. Further optimization of pharmacokinetic properties led to excellent in vivo exposure and efficacy and the identification of clinical candidate GDC-0077 (inavolisib, 32), which is now under evaluation in a Phase III clinical trial as a treatment for patients with PIK3CA-mutant breast cancer.

Abstract P3-11-23: GDC-0077 is a selective PI3K alpha inhibitor with robust efficacy in PIK3CA mutant hormone-positive breast cancer models

Abstract The phosphatidylinositol 3kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathway is a major regulator of tumor cell growth, proliferation and survival. Hotspot mutations of PIK3CA are common in all subtypes of breast cancer with a prevalence of approximately 30%. GDC-0077 is a potent ATP-competitive inhibitor of PI3K alpha (IC50 = 0.038 ± 0.003 nM), thereby inhibiting the phosphorylation of membranebound PIP2 to PIP3. Biochemically, GDC-0077 is more than 300fold selective over the other Class I PI3K isoforms, beta, delta, and gamma, and more than 2000-fold more selective over PI3K class II and III family members. Importantly, GDC-0077 is more selective for mutant versus wild-type PI3K alpha in cell-based assays. Mechanism of action studies indicate that GDC-0077 treatment leads to degradation of the mutant PI3K alpha protein in a proteasome dependent fashion, without changing WT PI3K alpha levels. Drug treatment results in sustained reduction of PI3K pathway biomarkers pAkt and pPRAS40, inhibition of cell proliferation, and increased apoptosis in human PIK3CA mutant breast cancer cell lines, to a greater extent than other non-degrading inhibitors such as alpelisib. Combination studies of GDC-0077 with the CDK4/6 inhibitor palbociclib demonstrate a stronger effect in estrogen-depleted HR+ PIK3CA mutant breast cell lines, compared to cells grown in the presence of estrogen. Daily dosing of GDC-0077 in PIK3CA mutant breast cancer patient derived xenograft (PDX) models (n=4) resulted in tumor regressions, induction of apoptosis and a reduction of pAkt, pPRAS40, and pS6RP in a dose-dependent fashion. In vivo combination efficacy studies of GDC-0077 with fulvestrant and palbociclib also indicate the triple combination results in the greatest efficacy. Collectively, our preclinical data support evaluation of GDC-0077 in a triple combination with endocrine therapy and CDK4/6 inhibition in the clinic, in patients with locally advanced or metastatic hormone receptor positive breast cancer whose tumors harbor mutant PIK3CA. Citation Format: Kyle Edgar, Rebecca Hong, Kyung Song, Steven Schmidt, Marc Hafner, Alfonso Arrazate, Erin Williams, Cecile De La Cruz, Jason Oeh, Deepak Sampath, Steve Staben, Lori Friedman. GDC-0077 is a selective PI3K alpha inhibitor with robust efficacy in PIK3CA mutant hormone-positive breast cancer models [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-11-23.

Abstract 156: Preclinical characterization of GDC-0077, a specific PI3K alpha inhibitor in early clinical development

Abstract The phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/ mammalian target of rapamycin (mTOR) signaling pathway is a major regulator of tumor cell growth, proliferation and survival. Dysregulation of the PI3K/Akt/mTOR signaling pathway through multiple mechanisms has been described in solid tumor malignancies, including activating and transforming “hotspot” mutations as well as amplification of PIK3CA that encodes the p110 alpha subunit of PI3K. Hotspot mutations of PIK3CA mutation are frequently observed in breast cancer with a prevalence of approximately 30%. GDC-0077 is a potent inhibitor of PI3K alpha (IC50 = 0.038 + 0.003 nM) and exerts its activity by binding to the ATP binding site of PI3K, thereby inhibiting the phosphorylation of PIP2 to PIP3. Biochemically, GDC-0077 is more than 300-fold selective over other Class I PI3K isoforms such as beta, delta, and gamma and more than 2000 fold more selective over PI3K class II and III family members. Importantly, GDC-0077 is more selective for mutant versus wild-type PI3K alpha in cell based assays. Compared to the PI3K inhibitor, taselisib, the improved biochemical selectivity of GDC-0077 against PI3K delta is demonstrated in human CD69+ B-cells, which are primarily dependent on PI3K delta for proliferation and survival and were more sensitive to taselisib than GDC-0077. Mechanism of action studies indicate that GDC-0077 induces depletion of mutant PI3K alpha protein resulting in reduction of PI3K pathway biomarkers such as pAkt and pPRAS40, inhibition of cell proliferation and increased apoptosis in human PIK3CA mutant breast cancer cell lines to a greater extent when compared to PIK3CA wild-type cells. In vivo, daily oral treatment with GDC-0077 in cell-culture-derived and patient derived PIK3CA mutant breast cancer xenograft models, resulted in tumor regressions, induction of apoptosis and a reduction of pAkt, pPRAS40, and pS6RP in a dose-dependent fashion. In vivo efficacy in a PIK3CA-mutant human breast cancer xenograft model was also improved when GDC-0077 was combined with standard-of-care therapies for hormone-receptor positive (HR+) breast cancer such as anti-estrogens (fulvestrant) or CDK4/6 inhibitor (palbociclib). Collectively, the preclinical data provide rationale for evaluating GDC-0077, a PI3K alpha mutant selective inhibitor, as a single agent and in combination with standard-of-care endocrine and targeted therapies that may provide additional benefit to patients that harbor PIK3CA mutations. Citation Format: Kyle Edgar, Emily Hanan, Steven Staben, Stephen Schmidt, Rebecca Hong, Kyung Song, Amy Young, Patricia Hamilton, Alfonso Arrazate, Cecile de la Cruz, Marcia Belvin, Michelle Nannini, Lori S. Friedman, Deepak Sampath. Preclinical characterization of GDC-0077, a specific PI3K alpha inhibitor in early clinical development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 156. doi:10.1158/1538-7445.AM2017-156

Abstract A118: PIKHER2: A phase Ib study evaluating oral BKM120 in combination with lapatinib in trastuzumab-resistant HER2-positive advanced breast cancer

Abstract Background: Phosphatidylinositol-3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR)-pathway is frequently activated in HER2-positive breast cancer and may play a major role in resistance to trastuzumab. Lapatinib is a dual anti-HER2/EGFR tyrosine kinase inhibitor with clinical activity after trastuzumab failure in HER2-positive advanced breast cancer (ABC). BKM120 is a pan-class I PI3K inhibitor with potent and selective activity against wild-type and mutant PI3K p110α. PIKHER2 phase Ib study aimed primarily to determine maximum tolerated dose (MTD) and propose a recommended phase II dose (RP2D) for BKM120 in combination with lapatinib in HER2-positive, trastuzumab-resistant, ABC. Secondary objectives included safety, antitumor activity, pharmacokinetics and biomarker assessments. Methods: PIKHER2 was a multi-center study, enrolling HER2 positive (IHC 3+ or FISH positive) ABC, with disease progressing either while on trastuzumab for metastatic disease or within 12 months of the last infusion for patients who received trastuzumab as adjuvant/neoadjuvant treatment. Oral BKM120 (B; 40, 60 or 80 mg) and lapatinib (L; 750, 1000 or 1250 mg) were administered daily. A modified CRM using an adaptive Bayesian model guided the dose escalation of both agents. PIK3CA mutational status and PTEN/hormone receptor expression IHC was evaluated on available tumor tissue. Results: A total of 24 HER2-positive ABC pts, with a median number of previous lines of cytotoxics = 2 (1-5) and previous lines of anti-HER2 = 2 (1-6) for advanced stage, were treated across 5 dose-levels (B,40 + L,750; B, 60 + L,750; B,80 + L,750; B,80 + L,1000; B,80 + L,1250). Following cycle 1, 5 pts experienced DLTs: G3 ALT elevation, G3 vomiting, G3 stomatitis, G3 hyperglycemia and G3 diarrhea. MTD was reached at B,80 + L,1250 but toxicities and early treatment discontinuation beyond cycle 1 led us to select B,80 + L,1000 as the RP2D. Main drug-related adverse events were: diarrhea (83% of pts, G3 in 21%), nausea/vomiting (83% of pts, G3 in 4%), skin toxicity (75% of pts, G3 in 21%), asthenia (70% of patients, no G3), depression (58% of pts, G3 in 4%), anxiety (42% of pts, no G3), transaminases increase (29% of pts, G3 in 17%). B and L PK parameters values were consistent with those already published for both drugs. A large inter-individual variability was observed for both drugs. There was no significant evidence for drug-drug PK interaction. Disease control rate (DCR) was 79% [57-92%], one patient obtained a complete remission and 6 additional patients experienced stable disease for ≥ 24 weeks (clinical benefit rate, CBR of 29% [12-51%]). PIK3CA mutations and PTEN loss were observed in 4 of 14 and 1 of 21 patients, respectively. DCR and CBR were higher in hormone receptor-negative tumors. Conclusion: Combining BKM120 and lapatinib in HER2-positive trastuzumab-resistant was feasible. Preliminary evidences of antitumor activity were observed in this heavily pre-treated population. Citation Format: Anthony Gonçalves, Mathilde Guerin, Nicolas Isambert, Mario Campone, Keyvan Resai, Aurélie Autret, Jihane Pakradouni, Alexie Robert, Magali Provansal, Emmanuelle Charafe-Jauffret, Renaud Sabatier, Alice Hervieu, Jean-Marc Extra, Patrice Viens, François Lokiec, Jean-Marie Boher. PIKHER2: A phase Ib study evaluating oral BKM120 in combination with lapatinib in trastuzumab-resistant HER2-positive advanced breast cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A118.

Abstract A007: Mutant PIK3CA accelerates HER2-driven transgenic mammary tumor progression, enhances cancer stem cell features, and induces resistance to combinations of anti-HER2 therapies

Abstract The HER2 (ERBB2) oncogene is amplified in 20-25% of breast cancers and is associated with poor patient outcome. Mutational activation of PIK3CA, the gene encoding the p110α catalytic subunit of phosphatidylinositol 3-kinase (PI3K), occurs in ~30% of breast cancers. HER2 amplification and PIK3CA mutations often co-occur in breast cancer. Aberrant activation of the PI3K pathway has been shown to correlate with a diminished response to HER2-directed therapies. We sought to directly determine whether mutant PI3K enhances HER2-induced tumor progression and promotes resistance to HER2 inhibition in vivo. For this purpose, we generated a conditional transgenic mouse model of HER2-overexpressing (HER2+), PIK3CAH1047R-mutant breast cancer. Mice expressing both human HER2 and mutant PIK3CA in the mammary epithelium developed tumors with shorter latencies compared to mice expressing PIK3CA alone (p=0.0012) or HER2 alone (p<0.0001). HER2 and mutant PIK3CA also strongly cooperated to promote lung metastases following tail vein injection of cells derived from transgenic tumors (p<0.01). PIK3CA and HER2+/PIK3CA tumors were histologically diverse (i.e., adenocarcinoma, adenomyoepithelioma, adenocarcinoma with squamous metaplasia, and metaplastic carcinoma), expressed luminal and basal cytokeratins by IHC, and were associated with the claudin-low subtype of human breast cancers by microarray analysis. Accordingly, HER2+/PIK3CA tumors expressed elevated transcripts encoding EMT and stem cell markers, such as Snail, Slug, Twist, Bmi1, ITGB1 (CD29), and Procr. Further, single cells from HER2+/PIK3CA tumors formed more and larger mammospheres compared to cells from HER2-driven tumors. HER2+/PIK3CA mammospheres and tumors were completely resistant to the HER2 monocolonal antibody trastuzumab, alone and in combination with the HER2 tyrosine kinase inhibitor lapatinib or with pertuzumab, an antibody that inhibits ligand-induced HER2 dimerization. Both drug resistance and enhanced mammosphere formation were reversed by treatment with the pan-PI3K inhibitor BKM120. BKM120 treatment strongly inhibited T308 and S473 pAKT, whereas pAKT levels were unaffected by trastuzumab in combination with lapatinib or with pertuzumab. We further generated a model of resistance to the combination of trastuzumab, pertuzumab, and BKM120 by long-term treatment of HER2+/PIK3CA tumor transplants. Tumors initially responded, but a subset (3/11) resumed growth in the presence of drugs within 2 weeks of treatment initiation. Resistance was maintained following passaging in mice. We are currently performing whole-exome sequencing on resistant vs. untreated tumors in order to determine the mechanism(s) of resistance. In sum, PIK3CAH1047R accelerates HER2-mediated breast epithelial transformation and metastatic progression, alters the intrinsic phenotype of HER2-overexpressing cancers and generates resistance to FDA-approved combinations of anti-HER2 therapies. We propose the mouse model presented herein will be a valuable tool to investigate the underlying biology of HER2+/PI3K-mutant tumors and for the preclinical testing of therapeutic strategies against this subtype of breast cancer. Citation Format: Ariella B. Hanker, Adam Pfefferle, Justin M. Balko, Maria Gabriela Kuba, Christian D. Young, Violeta Sanchez, Cammie R. Sutton, Hailing Cheng, Charles M. Perou, Jean J. Zhao, Rebecca S. Cook, Carlos L. Arteaga. Mutant PIK3CA accelerates HER2-driven transgenic mammary tumor progression, enhances cancer stem cell features, and induces resistance to combinations of anti-HER2 therapies. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A007.

PIKHER2: A phase Ib/II study evaluating safety and efficacy of oral BKM120 in combination with lapatinib in HER2-positive, PI3K-activated, trastuzumab-resistant advanced breast cancer.

TPS663 Background: Phosphatidylinositol-3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR)-pathway is frequently activated in HER2-positive breast cancer and may play a major role in resistance to trastuzumab. BKM120 is an oral pan-class I PI3K inhibitor with potent and selective activity against wild-type and mutant PI3K p110α. PIKHER2 study will evaluate safety and efficacy of oral, daily lapatinib-BKM120 combination in HER2-positive, trastuzumab-resistant advanced breast cancer (ABC) with activated PI3K/AKT/mTOR pathway. Methods: This open label, single arm, multi-center study includes a phase Ib dose-escalation part and a phase II expansion part at the recommended dose (RP2D). Primary objectives include determination of the maximum-tolerated dose (MTD) of BKM120 in combination with lapatinib in trastumuzab-resistant HER2-positive ABC (phase Ib part) and evaluation of the activity of BKM120-lapatinib combination at RP2D as measured by objective response rate (ORR) in patients with activated PI3K/AKT/mTOR pathway, as defined by PTEN-negative by IHC and/or somatic mutations (exons 9 and 20) of PIK3CA and/or overexpression of phospho-AKT by IHC (phase II part). Secondary objectives include safety and tolerability of the combination, clinical benefit and progression-free survival, pharmacokinetic profiles, biological and pharmacodynamic correlates. Main eligibility criteria are PS ≤ 1, HER2-positive ABC resistant to trastuzumab, documented activated PI3K/AKT/mTOR pathway (phase II part only). A modified CRM using an adaptive Bayesian model guides the dose escalation of both agents with a maximum of 24 patients planned to be enrolled in phase Ib part. Efficacy will be tested according to a standard 2-stage Simon design under the following unacceptable and desirable hypotheses: H0, ORR <= 10% (unacceptable rate) vs. H1, ORR >=30% (desirable rate). A maximum of 35 patients with activated PI3K/AKT/mTOR pathway is planned to be enrolled in phase II part with an interim look after 18 evaluable patients will have been recruited. Clinical trial information: NCT01589861.

Abstract LB-103: Conditional loss of ErbB3 delays mammary gland hyperplasias induced by mutant PIK3CA in transgenic mice, but does not affect mammary tumor latency, gene expression or signaling

Abstract The phosphatidylinositol-3-kinase (PI3K) pathway is the most frequently mutated pathway in breast cancer. Signaling through the ErbB family of receptor tyrosine kinases (RTKs) strongly activates PI3K. Six YXXM PI3K binding motifs in ErbB3 make this RTK a strong upstream activator of PI3K. Mutations in PIK3CA, the gene encoding the p110α catalytic subunit of PI3K, are associated with increased PI3K activity, enhanced transformation, and drug resistance. We hypothesized that upstream receptors, such as ErbB3, are still required to localize PI3K to the plasma membrane and maximally drive tumor formation induced by mutant PI3K. To test this hypothesis, we generated a doxycycline (Dox)-inducible system in triple transgenic mice (MMTV-rtTA, Tet-Op-HA-PIK3CAH1047R-IRES-Luc and Tet-Op-Cre) which are homozygous or heterozygous for floxed ErbB3 alleles. In this model, referred to as iPI3K.iCre.ErbB3FL/FL, Dox induces the expression of PIK3CAH1047R and Cre recombinase. This results in Cre-mediated deletion of floxed ErbB3 alleles in mammary cells expressing PIK3CAH1047R. Loss of ErbB3 delayed mammary hyperplasias induced by PIK3CAH1047R in 12-week old animals. The mean mammary tumor latency of iPI3K.iCre.ErbB3FL/+ animals was 398 days compared to 419 days in iPI3K.iCre.ErbB3FL/FL animals (p=0.93). Both ErbB3FL/Fl and ErbB3+ tumors showed mixed pathologies of solid, papillary or cribiform adenocarcinomas (with and without squamous metaplasia). The heterogeneity of PI3K mutant tumors was also shown by the mixed expression of cytokeratin 8-positive (luminal-like) and cytokeratin 5-positive (basal-like) epithelial cells, which was not altered by loss of ErbB3. Microarray analysis of tumors revealed similar gene expression patterns in tumors with or without ErbB3. Immunoblot analysis of tumor lysates confirmed the absence of ErbB3 protein in iPI3K.iCre.ErbB3FL/FL tumors. However, downstream effectors of PI3K such as Akt, PDK1, SGK1, GSK3, 4EBP1 and S6 were similarly phosphorylated in tumors with and without ErbB3 expression. RTKs other than ErbB3, such as ErbB2, PDGFR, EGFR and MSPR, were phosphorylated in both tumor types. In ErbB3+ tumors, P-ErbB3 associated with p85, the regulatory subunit of PI3K, and coprecipitated with p85 antibodies. In ErbB3-deficient tumors and in breast cancer cell lines where ErbB3 was knocked down with RNAi, several P-Tyr proteins remained bound to p85, including the adaptor molecules IRS-1 and Gab2. We speculate these molecules circumvent a potential requirement for ErbB3 in cancers induced and driven by mutant PIK3CA. However, ErbB3 is required for the early mouse mammary hyperplasias induced by mutant PI3K. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-103. doi:1538-7445.AM2012-LB-103

Abstract 4825: Inhibition of AKT abrogates resistance to endocrine therapy in estrogen receptor positive breast cancer

Abstract Estrogen receptor α-positive (ER+) breast cancers eventually adapt to hormone deprivation and acquire resistance to aromatase inhibitors. Upon acquisition of hormone independence, ER+ breast cancer cells increase their dependence on the phosphatidylinositol-3 kinase (PI3K)/AKT signaling pathway. Herein we investigated the effects of AKT inhibition on the hormone-independent growth of four ER+ breast cancer cell lines (MCF-7, ZR75-1, MDA-MB-361 and HCC-1428) with acquired resistance to estrogen deprivation (termed long-term estrogen-deprived cells, LTED). Treatment with the ATP-competitive AKT inhibitor AZD5363 inhibited phosphorylation of the AKT/mTOR substrates PRAS40, GSK3β/α, and S6K while inducing hyperphosphorylation of AKT at S473 and T308. AZD5363 suppressed the hormone-independent growth of 3/4 LTED lines. AZD5363 prevented the emergence of hormone-independent MCF-7, ZR75-1, and MDA-361 cells. Further, treatment of ovariectomized athymic mice with AZD5363 suppressed the hormone-independent growth of ER+/PI3K mutant MCF-7 xenografts. Combined treatment with AZD5363 and the ER downregulator fulvestrant more effectively suppressed xenograft growth than either drug alone. AZD5363 also caused dose-dependent inhibition of an ER+ breast cancer explant model unresponsive to fulvestrant or tamoxifen. We next examined whether AKT inhibition results in feedback upregulation of mitogenic signaling pathways. Treatment with AZD5363 resulted in upregulation and phosphorylation of the HER3 receptor tyrosine kinase (RTK), as well as upregulation of insulin receptor (InsR) protein levels and phosphorylation of Src at Y416. A Src family kinase inhibitor suppressed AZD5363-induced upregulation of P-HER3 in MCF-7/LTED cells. Src inhibition or siRNA-mediated knockdown of InsR or IGF-IR significantly enhanced the growth inhibitory effects of AZD5363. Following treatment with AZD5363, phospho-RTK array analysis revealed increased phosphorylation of multiple RTKs in MCF-7/LTED and/or ZR75-1/LTED cells, including InsR, IGF-IR, EGFR, HER2, HER3, HER4, Dtk, and FGFR3. Treatment with AZD5363 also upregulated RTK mRNA levels. Further, treatment of MCF-7/LTED cells with AZD5363 resulted in marked translocation of AKT PH-GFP to the membrane, reflective of increased PIP3 production and, thus, AKT phosphorylation at T308. Pre-treatment with the PI3K inhibitor BKM120 or the IGF-IR/InsR TKI AEW541 prevented AZD5363-induced membrane localization of PH-GFP. These results suggest that 1) AKT signaling is critical for hormone-independent growth of ER+ breast cancer cells in vitro and in vivo; and 2) AKT inhibition in these cells induces feedback upregulation of RTK expression and activity. Thus, inhibitors of AKT merit evaluation as a potential treatment for endocrine-resistant breast cancer. The combination of these agents with inhibitors of upstream RTKs are under evaluation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4825. doi:1538-7445.AM2012-4825

HMGB-1 induces cell motility and α5β1 integrin expression in human chondrosarcoma cells

Chondrosarcoma is a type of highly malignant tumor with a potent capacity to invade locally and cause distant metastasis. High mobility group box chromosomal protein 1 (HMGB)-1 is a widely studied, ubiquitous nuclear protein that is present in eukaryotic cells, and plays a crucial role in inflammatory response. However, the effects of HMGB-1 on human chondrosarcoma cells are largely unknown. In this study, we found that HMGB-1 increased the migration and the expression of α5β1 integrin in human chondrosarcoma cells. Transfection of cells with receptor for advanced glycation end products (RAGE) receptor siRNA reduced HMGB-1-induced cell migration and integrin expression. Activations of phosphatidylinositol 3-kinase (PI3K), Akt, and AP-1 pathways after HMGB-1 treatment were demonstrated, and HMGB-1-induced expression of integrin and migration activity was inhibited by the specific inhibitor and mutant of PI3K, Akt, and AP-1 cascades. Taken together, our results indicated that HMGB-1 enhances the migration of chondrosarcoma cells by increasing α5β1 integrin expression through the RAGE receptor/PI3K/Akt/c-Jun/AP-1 signal transduction pathway.

Caffeine induces endothelial tissue factor expression via phosphatidylinositol 3-kinase inhibition

Tissue factor (TF) is the key activator of coagulation and is involved in acute coronary syndromes. Caffeine is often reported to increase cardiovascular risk; however, its effect on cardiovascular morbidity and mortality is controversial. Hence, this study was designed to investigate the impact of caffeine on endothelial TF expression in vitro. Caffeine concentration-dependently enhanced TF protein expression and surface activity in human endothelial cells stimulated by tumour necrosis factor (TNF)-α or thrombin. Caffeine inhibited phosphatidylinositol 3-kinase (PI3K) activity and this effect was comparable to that of the known PI3K inhibitor LY294002. Consistently, treatment of endothelial cells with LY294002 enhanced TNF-α induced TF expression to a similar extent as caffeine, and adenoviral expression of the active PI3K mutant (p110) reversed the effect of both caffeine and LY294002 on TF expression. Caffeine and LY294002 increased DNA binding capacity of the transcription factor nuclear factor κB, whereas the activation pattern of mitogen-activated protein kinases (MAPK) remained unaltered. Luciferase reporter assay revealed a caffeine dependent activation of the TF promoter, and RT-PCR revealed a dose dependent increase in TF mRNA levels when stimulated with caffeine in the presence of TNF-α. In conclusion, caffeine enhances TNF-α-induced endothelial TF protein expression as well as surface activity by inhibition of PI3K signalling. Since the caffeine concentrations applied in the present study are within the plasma range measured in humans, our findings indicate that caffeine enhances the prothrombotic potential of endothelial cells and underscore the importance of PI3K in mediating these effects.

IGF-I enhances α5β1 integrin expression and cell motility in human chondrosarcoma cells

Chondrosarcoma is a type of highly malignant tumor with a potent capacity to invade locally and cause distant metastasis. Chondrosarcoma shows a predilection for metastasis to the lungs. Integrins are the major adhesive molecules in mammalian cells and have been associated with metastasis of cancer cells. Insulin-like growth factor-I (IGF)-I plays an important role in regulating cell growth, proliferation, survival, and metabolism. However, the effects of IGF-I in migration and integrin expression in chondrosarcoma cells are largely unknown. In this study, we found that IGF-I increased the migration and the expression of α5β1 integrin in human chondrosarcoma cells. Pretreatment of cells with IGF-I receptor antibody reduced IGF-I-induced cell migration and integrin expression. Activations of phosphatidylinositol 3-kinase (PI3K), Akt, and nuclear factor-κB (NF-κB) pathways after IGF-I treatment were demonstrated, and IGF-I-induced expression of integrin and migration activity was inhibited by the specific inhibitor and mutant of PI3K, Akt, and NF-κB cascades. Taken together, our results indicated that IGF-I enhances the migration of chondrosarcoma cells by increasing α5β1 integrin expression through the IGF-I receptor/PI3K/Akt/NF-κB signal transduction pathway.

AKT induces senescence in human cells via mTORC1 and p53 in the absence of DNA damage: implications for targeting mTOR during malignancy

The phosphatidylinositol 3-kinase (PI3K)/AKT and RAS oncogenic signalling modules are frequently mutated in sporadic human cancer. Although each of these pathways has been shown to play critical roles in driving tumour growth and proliferation, their activation in normal human cells can also promote cell senescence. Although the mechanisms mediating RAS-induced senescence have been well characterised, those controlling PI3K/AKT-induced senescence are poorly understood. Here we show that PI3K/AKT pathway activation in response to phosphatase and tensin homolog (PTEN) knockdown, mutant PI3K, catalytic, α polypeptide (PIK3CA) or activated AKT expression, promotes accumulation of p53 and p21, increases cell size and induces senescence-associated β-galactosidase activity. We demonstrate that AKT-induced senescence is p53-dependent and is characterised by mTORC1-dependent regulation of p53 translation and stabilisation of p53 protein following nucleolar localisation and inactivation of MDM2. The underlying mechanisms of RAS and AKT-induced senescence appear to be distinct, demonstrating that different mediators of senescence may be deregulated during transformation by specific oncogenes. Unlike RAS, AKT promotes rapid proliferative arrest in the absence of a hyperproliferative phase or DNA damage, indicating that inactivation of the senescence response is critical at the early stages of PI3K/AKT-driven tumourigenesis. Furthermore, our data imply that chronic activation of AKT signalling provides selective pressure for the loss of p53 function, consistent with observations that PTEN or PIK3CA mutations are significantly associated with p53 mutation in a number of human tumour types. Importantly, the demonstration that mTORC1 is an essential mediator of AKT-induced senescence raises the possibility that targeting mTORC1 in tumours with activated PI3K/AKT signalling may exert unexpected detrimental effects due to inactivation of a senescence brake on potential cancer-initiating cells.

IL‐8 increases integrin expression and cell motility in human chondrosarcoma cells

Chondrosarcoma is a type of highly malignant tumor with a potent capacity to invade locally and cause distant metastasis. Chondrosarcoma shows a predilection for metastasis to the lungs. Interleukin-8 (IL-8), a chemokine with a defining CXC amino acid motif, is known to possess tumorigenic and proangiogenic properties. Over-expression of IL-8 has been detected in many human tumors. However, the effects of IL-8 in migration and integrin expression in chondrosarcoma cells are largely unknown. In this study, we found that IL-8 increased the migration and the expression of αvβ3 integrin in human chondrosarcoma cells. Activations of phosphatidylinositol 3-kinase (PI3K), Akt, and AP-1 pathways after IL-8 treatment were demonstrated, and IL-8-induced expression of integrin and migration activity was inhibited by the specific inhibitor and mutant of PI3K, Akt, and AP-1 cascades. Taken together, our results indicated that IL-8 enhances the migration of chondrosarcoma cells by increasing αvβ3 integrin expression through the PI3K/Akt/AP-1 signal transduction pathway.

The Selective Class I PI3K Inhibitor CH5132799 Targets Human Cancers Harboring Oncogenic PIK3CA Mutations

The phosphatidylinositol 3-kinase (PI3K) pathway plays a central role in cell proliferation and survival in human cancer. PIK3CA mutations, which are found in many cancer patients, activate the PI3K pathway, resulting in cancer development and progression. We previously identified CH5132799 as a novel PI3K inhibitor. Thus, this study aimed to clarify the biochemical and antitumor activity of CH5132799 and elucidate the correlation between CH5132799 response and genetic alterations in the PI3K pathway. Kinase inhibitory activity was profiled in cell-free assays. A large panel of human breast, ovarian, prostate, and endometrial cancer cell lines, as well as xenograft models, were used to evaluate the antitumor activity of CH5132799, followed by analysis for genetic alterations. Effects on Akt phosphorylation induced by mTORC1 inhibition were tested with CH5132799 and compared with mTORC1 and PI3K/mTOR inhibitors. CH5132799 selectively inhibited class I PI3Ks and PI3Kα mutants in in vitro kinase assays. Tumors harboring PIK3CA mutations were significantly sensitive to CH5132799 in vitro and were remarkably regressed by CH5132799 in in vivo mouse xenograft models. In combination with trastuzumab, tumors disappeared in the trastuzumab-insensitive breast cancer model with the PIK3CA mutation. Moreover, CH5132799 did not reverse a negative feedback loop of PI3K/Akt/mTOR signaling and induced regression against tumors regrown after long-term mTORC1 inhibitor treatment. CH5132799 is a selective class I PI3K inhibitor with potent antitumor activity against tumors harboring the PIK3CA mutations. Prediction of CH5132799 response on the basis of PIK3CA mutations could enable patient stratification in clinical settings.

Abstract 5486: Combined targeting of the PI3K and MAPK pathways is required for efficacy in treating colorectal cancer of the wild and mutant types

Abstract The KRAS oncogene is commonly mutated in human colon cancer. The downstream pathways of mutant KRAS, including the RAF/MEK/ERK, mitogen-activated protein kinase (MAPK), and phosphatidylinositol 3-kinase (PI3K)-AKT signaling are indispensible in order to understand how to pharmacologically target them when treating colon cancer with the KRAS mutant. However, the heterogeneous responses of these effector pathways remain unclear. Recent studies have indicated that mutational activation of PI3K (PIK3CA) in KRAS mutant tumor cells induces resistance to the inhibition of MEK, one of the MAPK signal cascades. Wild-type p53 also suppresses the oncogenic potential of the MAPK pathway. Our study shows that bortezomib inhibits the PI3K-AKT pathway and that nutlin-3A used as a p53-inducing agent blocks the MAPK pathway. The high doses of bortezomib induced apoptosis through complete suppression of AKT phosphorylation in KRAS and PI3K mutant colon cancer cells. Treating patients using high doses of nutlin-3A resulted in apoptosis by inducing p53 phosphorylation in colon cancer cells with wild-type p53. We also showed that the combination of bortezomib and nutlin-3A exhibited a synergistic effect on KRAS mutant colon cancer cells with PI3K mutant and wild-type p53, whereas there are no combined effect in KRAS mutant colon cancer cells with the p53 mutant. In addition, bortezomib and nutlin-3A exhibited combined effect in KRAS wild-type colon cancer cells with PI3K mutant and wild-type p53, although not in p53 mutant cells. We consistently noted that treatment with bortezomib and nutlin-3A after p53 silencing using small, interfering RNA (siRNA) in p53 wild-type colon cancer cells, resulted in a decrease of the populations of dead cells. Therefore, these results suggest that a combination of bortezomib and nutlin-3A can overcome the heterogeneous responses of the effector pathway in colon cancer with KRAS mutant; they also provide a new basis for the clinical testing of PI3K- and MAPK-targeted therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5486. doi:10.1158/1538-7445.AM2011-5486

Stromal cell‐derived factor‐1/CXCR4 promotes IL‐6 production in human synovial fibroblasts

The production of chemokine stromal cell-derived factor (SDF)-1 is significantly higher in synovial fluid of patients with osteoarthritis (OA). IL-6 is a multifunctional cytokine that plays a central role in both OA and rheumatoid arthritis. However, the effects of SDF-1α on human synovial fibroblasts are largely unknown. In this study, we investigated the intracellular signaling pathway involved in SDF-1α-induced IL-6 production in human synovial fibroblast cells. SDF-1α caused concentration- and time-dependent increases in IL-6 production. SDF-1α also increased the mRNA and surface expression of CXCR4 receptor in human synovial fibroblasts. CXCR4-neutralizing antibody, CXCR4-specific inhibitor (AMD3100), or small interfering RNA against CXCR4 inhibited the SDF-1α-induced increase of IL-6 expression. The transcriptional regulation of IL-6 by SDF-1α was mediated by phosphorylation of phosphatidylinositol 3-kinase (PI3K)/Akt and activation of the activator protein (AP)-1 component of c-Jun. The binding of c-Jun to the AP-1 element on the IL-6 promoter and the increase in AP-1 luciferase activity was enhanced by SDF-1α. Co-transfection with CXCR4, PI3K, Akt, and c-Jun mutants or siRNA inhibited the potentiating action of SDF-1α on AP-1 promoter activity. Taken together, our results suggest that SDF-1α-increased IL-6 production in human synovial fibroblasts via the CXCR4 receptor, PI3K, Akt, c-Jun, and AP-1 signaling pathways.

Phosphatidylinositol 3-kinase interacts with the glucocorticoid receptor upon TLR2 activation

Airway inflammation is a common condition where glucocorticoids (GC) are a well-established therapy. It has been demonstrated that GC stimulate components of innate immunity. Specifically, GC up-regulate TLR2 expression and activation upon inflammatory stimuli; however, little is known about the signalling involved in this process. To determine the mechanism by which dexamethasone modulates TLR2-induced cytokine production this signalling pathway was monitored in a lung epithelial cell line exposed to the TLR2 synthetic agonist, Pam3-Cys-Ser-Lys4. These experiments demonstrate that phosphatidylinositol 3-kinase (PI3K) is critical for the TLR2 downstream effects of GC. Cells expressing a PI3K mutant (p85-dominant negative, DN; p85 Δ478–511) and exposed to Pam3-Cys-Ser-Lys4 in the presence or absence of dexamethasone, showed enhanced tumour necrosis factor (TNF)α expression while AP-1 and NF-κB transcriptional activity were repressed. We provide experimental evidence that PI3K physically interacts with the glucocorticoid receptor (GR) through two putative PI3K recruitment consensus YxxM binding motifs in the GR, suggesting that some functions regulated by this receptor might occur through kinase interaction. Mutations of two tyrosine residues in the GR, 598 and 663, to phenylalanine significantly reduced interaction with PI3K and the GC effects on TLR2-induced TNF-α expression. However, these mutations did not alter GR transcriptional activity nor affect cellular localization of the expressed mutant GR in COS-1 cells. Therefore, the PI3K-GR interaction may contribute to the effects of GC on the TLR2 pro-inflammatory signalling cascade, thus defining a novel signalling mechanism with a profound impact on innate immune responses.

H1047R phosphatidylinositol 3-kinase mutant enhances HER2-mediated transformation by heregulin production and activation of HER3

Hyperactivation of phosphatidylinositol-3 kinase (PI3K) can occur as a result of somatic mutations in PIK3CA, the gene encoding the p110α subunit of PI3K. The HER2 oncogene is amplified in 25% of all breast cancers and some of these tumors also harbor PIK3CA mutations. We examined mechanisms by which mutant PI3K can enhance transformation and confer resistance to HER2-directed therapies. We introduced the PI3K mutations E545K and H1047R in MCF10A human mammary epithelial cells that also overexpress HER2. Both mutants conferred a gain of function to MCF10A/HER2 cells. Expression of H1047R PI3K but not E545K PI3K markedly upregulated the HER3/HER4 ligand heregulin (HRG). HRG siRNA inhibited growth of H1047R but not E545K-expressing cells and synergized with the HER2 inhibitors trastuzumab and lapatinib. The PI3K inhibitor BEZ235 markedly inhibited HRG and pAKT levels and, in combination with lapatinib, completely inhibited growth of cells expressing H1047R PI3K. These observations suggest that PI3K mutants enhance HER2-mediated transformation by amplifying the ligand-induced signaling output of the ErbB network. This also counteracts the full effect of therapeutic inhibitors of HER2. These data also suggest that mammary tumors that contain both HER2 gene amplification and PIK3CA mutations should be treated with a combination of HER2 and PI3K inhibitors.

Roles of phosphatidylinositol 3 kinase in silica-induced DNA double strand breaks damage repair in human embryo lung fibroblasts

To study the role of Phosphatidylinositol 3 kinase (PI3K) in silica-induced DNA double strand break repair in human embryo lung fibroblasts (HELF). Control HELF cells and DN-Deltap85 (HELF transfected with Dominant negative mutant of PI3K) were treated with 200 microg/ml silica for different times. The expression levels of phosphor-H2AX (H2AX), Ku70, Ku80 and DNA-PKcs were determined by Western blot. Furthermore, DNA double strand breaks were measured by neutral comet assay after cells were treated with 200 microg/ml silica for 0, 12 and 24 h. After treatment with 200 microg/ml silica for different times, the levels of H2AX were increased in a time-dependent manner and the expression levels of H2AX were obviously suppressed in DN-Deltap85 compared with control cells. The levels of Ku70 and Ku80 were also significantly suppressed in DN-Deltap85 (0.37 +/- 0.14, 0.55 +/- 0.17) compared with control cells (0.58 +/- 0.09, 0.95 +/- 0.21) after treatment with 200 microg/ml silica for 12 h (P < 0.05). Both the percentage of tail DNA in HELF and DN-Deltap85 increased significantly at 12 h (9.78 +/- 1.15, 11.79 +/- 4.90) compared with groups without treatment with silica (2.40 +/- 0.69, 3.31 +/- 1.35) and then decreased at 24 h (4.19 +/- 0.47, 7.58 +/- 4.32), but only the decrease of HELF at 24 h was significant compared with HELF at 12 h (P < 0.05). DNA repair competence of HELF was 75.74% and that of DN-Deltap85 declined to 49.64%. Silica dust can induce DNA double strand breaks in human embryo lung fibroblasts. PI3K might play a role in silica-induced DNA double strand break repair by regulating the expression levels of Ku70 and Ku80.

Knockdown of Ron Kinase Inhibits Mutant Phosphatidylinositol 3-Kinase and Reduces Metastasis in Human Colon Carcinoma

Abnormal accumulation and activation of receptor tyrosine kinase Ron (recepteur d'origine nantais) has been demonstrated in a variety of primary human cancers. We show that RNA interference-mediated knockdown of Ron kinase in a highly tumorigenic colon cancer cell line led to reduced proliferation as compared with the control cells. Decreased Ron expression sensitized HCT116 cells to growth factor deprivation stress-induced apoptosis as reflected by increased DNA fragmentation and caspase 3 activation. In addition, cell motility was decreased in Ron knockdown cells as measured by wound healing assays and transwell assays. HCT116 cells are heterozygous for gain of function mutant PIK3CA H1047R. Analysis of signaling proteins that are affected by Ron knockdown revealed that phosphatidylinositol 3-kinase (PI3K) activity of the mutant PI3K as well as AKT phosphorylation was substantially reduced in the Ron knockdown cells compared with the control cells. Moreover, we demonstrated in vivo that knockdown of Ron expression significantly reduced lung metastasis as compared with the control cells in the orthotopic models. In summary, our results demonstrate that Ron plays an essential role in maintaining malignant phenotypes of colon cancer cells through regulating mutant PI3K activity. Therefore, targeting Ron kinase could be a potential strategy for colon cancer treatment, especially in patients bearing gain of function mutant PI3K activity.