PIK3CA Mutant Breast Cancer Cell Research Articles

Abstract 2634: Investigating the chemotherapeutic effects of aspirin in mutant PIK3CA breast cancer

Abstract Although aspirin is primarily administered as an antipyretic and analgesic, numerous observational and randomized clinical trials have suggested a potential chemotherapeutic use. Recently, two independent studies demonstrated that aspirin use is associated with superior survival in colorectal cancer patients with mutant-PIK3CA but not among those with the wild-type gene. Despite these seminal observations, the molecular basis for this phenomenon remains poorly understood. Furthermore, whether this observation occurs in breast cancer is unknown. Given that (1) mutation of PIK3CA, the gene encoding the catalytic subunit of the phosphoinositide 3-kinase (PI 3-K) is highly prevalent in breast cancer and that (2) aspirin use is associated with a decrease in breast cancer mortality and distant recurrence, the relationship between PI 3-K pathway activation and the protective benefit of aspirin in breast cancer was investigated. Here, we utilize both PIK3CA mutant breast cancer cell lines and a well-characterized system of immortalized mammary epithelial cells stably expressing either wild-type and mutant PIK3CA E545K and H1047R respectively. Mutant PIK3CA breast cancer cells show a dose-dependent decrease in cell viability with increasing concentration of aspirin/salicylate. In these cells, co-treatment of aspirin with PI 3-K pathway inhibitors BYL719 or BKM120 caused a more significant decrease on cell growth compared to each single agent alone. In 3D culture, MCF10A cells expressing mutant PIK3CA-H1047R showed greater sensitivity to aspirin compared to cells expressing wild-type PIK3CA. Gene expression profiling and quantitative qRT-PCR was employed to identify and validate mutant PIK3CA-regulated genes respectively. Many of these genes were associated with inflammation; among them was the PTGS2 gene which encodes for cyclooxygenase-2 (COX-2). We observed that mutant PIK3CA regulates cyclooxygenase-2 (COX-2) mRNA and protein expression as well as production of prostaglandin E2α (PGE2α). Expression of mutant PIK3CA also increases phosphorylation of IKKβ and IκB; increases mTORC1 signaling and reduces AMPK activity. This is abrogated upon pharmacologic inhibition of mutant PIK3CA with BYL719 and BKM120. Notably, both aspirin and its metabolite salicylate can inhibit these PIK3CA-induced pathways and activate AMPK signaling. An understanding of these key survival and metabolic signaling pathways, which are modulated by aspirin, may help to explain aspirin's beneficial effect in PIK3CA-mutant breast cancers. Given the growing interest in personalized medicine, this pre-clinical study may provide insight for the stratification of patients who are most likely to benefit from adjuvant aspirin therapy. Citation Format: Whitney Henry, Alex Toker. Investigating the chemotherapeutic effects of aspirin in mutant PIK3CA breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2634. doi:10.1158/1538-7445.AM2015-2634

Abstract 3683: Targeting PIK3CA mutant breast cancer with the combination of PIK3CA-specific inhibitor, BYL719, and IGF1-R antibody, ganitumab

Abstract Background: PI3K/AKT signaling is a critical growth and survival pathway in many human cancers. PI3Ks are lipid kinases that are important in controlling signaling pathways involved in cell proliferation, motility, cell death and cell invasion. The α isoform of PI3K (PIK3CA) is linked upstream mainly to receptor tyrosine kinases. Various solid tumor types such as colorectal cancers, gastric cancers, brain cancers, glioblastomas, breast and lung cancers have been found to contain gain-of-function mutations of PIK3CA. The existing scientific evidence strongly supports the concept of targeting PIK3CA in human cancers, especially those harboring PIK3CA mutations. Sustained PIK3CA inhibition may lead to aberrant RTK signaling to attenuate PIK3CA inhibition. IGF1R is a RTK commonly expressed in human neoplasia. Its signaling, primarily through AKT, has been implicated in tumor cell survival. Additionally, IGF1R/IRS signaling has been shown to be a feedback pathway to down-modulate mTOR inhibition by RAD001 in cancer cells. We speculate that the efficacy of PIK3CA inhibition might be enhanced if PIK3CA inhibitor is combined with a targeted agent against a key RTK in pAKT signaling. Methods: With pATK signaling closely linked to mTOR signaling, we set out to examine whether PIK3CA inhibition would also trigger IGF1-R/IRS signaling. In addition, we explored the combination of PIK3CA-specific inhibitor, BYL719, and a fully human antibody against IGF1-R, AMG 479 (ganitumab), preclinically against a PIK3CA mutant breast cancer model, MCF7. Results: Our data indicate that IGF1-R/IRS signaling is activated upon PIK3CA inhibition. BYL719 exhibited concentration-dependent tumor growth inhibition in vitro. Ganitumab alone had modest inhibitory activity. The combination of BYL719 and ganitumab inhibited MCF7 growth synergistically in vitro. This combination was further tested in an MCF7 xenograft in mice. BYL719 monotherapy resulted in tumor stasis. Ganitumab alone had marginal growth inhibition. The combination of BYL719 and ganitumab led to tumor regression. Further more, the combination of BYL719 and Ganitumumab lead to increased suppression of pS6RP in vivo. Conclusions: Our data suggest that the combination of BYL719 and ganitumab would act synergistically to inhibit PIK3CA mutant breast cancer cells by blocking two inter-connected pathways. The combination of BYL719 and ganitumab has now entered into clinical trial (NCT01708161). It represents a promising approach against PI3KCA mutant breast cancers. Citation Format: Z. Alexander Cao, Maria Pinzon-Ortiz, Yan Chen, Xiaoyan Li, Pedro J. Beltran, Jennifer Gansert, Malte Peters, Robert Schlegel, Karl M. Schumacher, Alan Huang. Targeting PIK3CA mutant breast cancer with the combination of PIK3CA-specific inhibitor, BYL719, and IGF1-R antibody, ganitumab. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3683. doi:10.1158/1538-7445.AM2014-3683

Abstract P2-17-01: The PI3K inhibitor GDC-0032 is selectively potent against PIK3CA mutant breast cancer cell lines and tumors

Abstract Mutations in the phosphoinositide-3 kinase alpha isoform (PIK3CA) are frequent in breast cancer and activate the PI3K signaling pathway. We discovered GDC-0032, a selective, potent, orally bioavailable inhibitor of PI3Ka with a Ki = 0.2nM, and with reduced inhibitory activity against PI3Kβ. This selectivity profile, and excellent pharmacokinetic and pharmaceutical properties, allowed for greater efficacy in vivo at the maximum tolerated dose relative to a pan Class I PI3K inhibitor in PIK3CA mutant xenografts. Notably, GDC-0032 preferentially inhibited PIK3CA mutant cells relative to cells with wild-type PI3K. GDC-0032 potently inhibits signal transduction downstream of PI3K and induces apoptosis at low concentrations in breast cancer cell lines and xenograft models that harbor PIK3CA mutations. The mutant-bias of GDC-0032 is linked to unique properties of GDC-0032, including cellular potency against the mutant isoform and reduction of receptor tyrosine kinase (RTK) signaling. Endocrine therapies such as letrozole are commonly used treatment options for metastatic Hormone Receptor positive (HR+) breast cancer but many patients ultimately relapse. Due to the importance of PI3K in breast cancer, PI3K inhibitors such as GDC-0032 are attractive for combination with endocrine therapies. GDC-0032 was evaluated in breast cancer lines and models in combination with letrozole, and assayed for cellular viability, modulation of PI3K pathway, modulation of ER pathway markers, and apoptosis induction. The combination of GDC-0032 and letrozole decreased cellular viability and increased apoptosis relative to either single agent. We observed cross-talk between the PI3K and ER pathways that suggest a mechanism of action for the combination. In a secreted factor screen we found that multiple soluble factors render breast cancer cells non-responsive to letrozole. It was discovered that many of these factors signal through the PI3K pathway and GDC-0032 in combination with letrozole was able to overcome the growth inhibition caused by the soluble factor. We also established letrozole resistant cell lines that grow independently of any estrogen source. These letrozole resistant lines have elevated PI3K pathway signaling and are still sensitive to GDC-0032. Taken together, these data provide rationale for evaluating GDC-0032 in combination with endocrine therapies for ER+ breast cancer treatment in the clinic. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-17-01.

Acquired PIK3CA amplification causes resistance to selective phosphoinositide 3-kinase inhibitors in breast cancer

Agents targeting the PI3K/mTOR signaling axis have shown promise in early-phase clinical trials and are currently being studied in later stages of clinical development in multiple indications. Experience with other targeted agents suggests that clinical responses may be short-lived because of acquired resistance to therapy. Here, we report preclinical modeling of acquired resistance in a HER2-positive, PIK3CA mutant breast cancer cell line, KPL-4. We identified a heretofore-unreported mechanism of resistance, specifically high-level amplification of the mutant allele of PIK3CA, which resulted in a marked upregulation of PI3K signaling, enabling resistant cells to regain proliferative capacity at clinically relevant concentrations of the PI3K inhibitor, GDC-0941. We show that knockdown of the amplified PIK3CA mutant allele in these cells by small interfering RNA restored pathway signaling and sensitivity to PI3K inhibition at levels comparable to parental cells. These novel preclinical findings suggest that, in addition to assessment of other previously reported mechanisms of resistance, evaluation of PI3K copy number variation should be integrated into the exploratory analysis of biopsies obtained at disease progression.

Abstract B106: mTORC1 inhibition is required for sensitivity to PI3K p110α inhibitors in PIK3CA-mutant breast cancer.

Abstract Activating mutations of the PIK3CA gene occur frequently in breast cancer and inhibitors that are specific for PI3K p110α, such as BYL719, are being investigated in clinical trials. In a search for correlates of sensitivity to p110α inhibition among PIK3CA-mutant breast cancer cell lines, we observed that sensitivity to BYL719 (as assessed by cell proliferation) was associated with full inhibition of signaling through the TORC1 pathway. Conversely, cancer cells that were resistant to BYL719 had persistently active mTORC1 signaling, although Akt phosphorylation was inhibited. Similarly in patients, pS6 (residues 240/4) expression (a marker of mTORC1 signaling) was associated with tumor response to BYL719, and mTORC1 was found to be reactivated in tumors from patients whose disease progressed after treatment. In PIK3CA-mutant cancer cell lines with persistent mTORC1 signaling despite PI3K p110α blockade (that is, resistance), the addition of the allosteric mTORC1 inhibitor RAD001 to the cells along with BYL719 resulted in reversal of resistance in vitro and in vivo. Finally, we found that growth factors such as IGF1 and neuregulin1 can activate mTOR and mediate resistance to BYL719. Our findings suggest that simultaneous administration of mTORC1 inhibitors may enhance the clinical activity of p110α targeted drugs and delay the appearance of resistance. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B106. Citation Format: Moshe Elkabets, Sadhna Vora, Dejan Juric, Natasha Morse, Mari Mino-Kenudson, Taru Muranen, Jessica Tao, Ana Bosch Campos, Jordi Rodon, Yasir H. Ibrahim, Violeta Serra, Vanessa Rodrik-Outmezguine, Saswati Hazra, Sharat Singh, Phillip Kim, Cornelia Quadt, Liu Manway, Alan Huang, Neal Rosen, Jeffrey A. Engelman, Maurizio Scaltriti, José Baselga. mTORC1 inhibition is required for sensitivity to PI3K p110α inhibitors in PIK3CA-mutant breast cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B106.

Dual PI3K/mTOR Inhibitor NVP-BEZ235 Sensitizes Docetaxel in Castration Resistant Prostate Cancer

Effective therapeutic strategies that can achieve long-term improvement in patients with castration resistant prostate cancer are urgently needed. We recently reported that the activated PI3K/Akt/mTOR signaling pathway induced by docetaxel explains resistance to docetaxel in castration resistant prostate cancer. In this study we explored the efficacy of NVP-BEZ235, a dual PI3K and mTORC1/2 inhibitor, for docetaxel resistant castration resistant prostate cancer. We used the 2 human castration resistant prostate cancer cell lines C4-2 and C4-2AT6. At our laboratory C4-2AT6 cells were established from C4-2 under androgen ablated treatment for 6 months. We investigated the efficacy of NVP-BEZ235 monotherapy and NVP-BEZ235 combined with docetaxel in vitro and in vivo. Increased phosphorylated Akt in C4-2AT6 cells was significantly inhibited by NVP-BEZ235 in a dose and time dependent manner. WST cell proliferation assay results in C4-2AT6 cells revealed that combined administration of NVP-BEZ235 and docetaxel had significant, synergistically greater cytotoxicity than NVP-BEZ235 or docetaxel monotherapy. Combined NVP-BEZ235 (40 mg/kg) and docetaxel (4 mg/kg) in vivo in a castrated mouse xenograft model inhibited C4-2AT6 tumor growth to a greater degree than in the monotherapy groups. Also, NVP-BEZ235 showed significant efficacy with docetaxel at a low concentration in vivo, suggesting that NVP-BEZ235 effectively decreased resistance to docetaxel. Results suggest that inhibition of the PI3K/Akt/mTOR signaling pathway by NVP-BEZ235 can overcome docetaxel resistance in human castration resistant prostate cancer. Our findings provide a molecular basis for the clinical use of combined administration of NVP-BEZ235 and docetaxel in patients with castration resistant prostate cancer.

MTORC1 Inhibition Is Required for Sensitivity to PI3K p110α Inhibitors in PIK3CA -Mutant Breast Cancer

Activating mutations of the PIK3CA gene occur frequently in breast cancer, and inhibitors that are specific for phosphatidylinositol 3-kinase (PI3K) p110α, such as BYL719, are being investigated in clinical trials. In a search for correlates of sensitivity to p110α inhibition among PIK3CA-mutant breast cancer cell lines, we observed that sensitivity to BYL719 (as assessed by cell proliferation) was associated with full inhibition of signaling through the TORC1 pathway. Conversely, cancer cells that were resistant to BYL719 had persistently active mTORC1 signaling, although Akt phosphorylation was inhibited. Similarly, in patients, pS6 (residues 240/4) expression (a marker of mTORC1 signaling) was associated with tumor response to BYL719, and mTORC1 was found to be reactivated in tumors from patients whose disease progressed after treatment. In PIK3CA-mutant cancer cell lines with persistent mTORC1 signaling despite PI3K p110α blockade (that is, resistance), the addition of the allosteric mTORC1 inhibitor RAD001 to the cells along with BYL719 resulted in reversal of resistance in vitro and in vivo. Finally, we found that growth factors such as insulin-like growth factor 1 and neuregulin 1 can activate mammalian target of rapamycin (mTOR) and mediate resistance to BYL719. Our findings suggest that simultaneous administration of mTORC1 inhibitors may enhance the clinical activity of p110α-targeted drugs and delay the appearance of resistance.

Targeting PIK3CA mutant breast cancer with the combination of PIK3CA-specific inhibitor, BYL719, and IGF1-R antibody, ganitumab.

e13525 Background: PI3K/AKT signaling is a critical growth and survival pathway in many human cancers. PI3Ks are lipid kinases that are important in controlling signaling pathways involved in cell proliferation, motility, cell death and cell invasion. The α isoform of PI3K (PIK3CA) is linked upstream mainly to receptor tyrosine kinases and is commonly mutated in various solid tumor types. The existing scientific evidence strongly supports the concept of targeting PIK3CA in human cancers, especially those harboring PIK3CA mutations. Sustained PIK3CA inhibition may lead to aberrant RTK signaling to attenuate PIK3CA inhibition. IGF1R is a RTK commonly expressed in human neoplasia. Its signaling has been implicated in tumor cell survival. Additionally, IGF1R/IRS signaling has been shown to be a feedback pathway to down-modulate mTOR inhibition by RAD001 in cancer cells. We reason that the efficacy of PIK3CA inhibition would be enhanced if PIK3CA inhibitor is combined with a targeted agent against a key RTK in pAKT signaling. Methods: We set out to examine whether PIK3CA inhibition would also trigger IGF1-R/IRS signaling. In addition, we explored the combination of PIK3CA-specific inhibitor, BYL719, and a fully human antibody against IGF1-R, ganitumab, preclinically against a PIK3CA mutant breast cancer model, MCF7. Results: Our data indicate that IGF1-R/IRS signaling is activated upon PIK3CA inhibition. BYL719 exhibited concentration-dependent tumor growth inhibition in vitro. Ganitumab alone had modest inhibitory activity. The combination of BYL719 and ganitumab inhibited MCF7 growth synergistically in vitro. This combination was further tested in an MCF7 xenograft in mice. BYL719 monotherapy resulted in tumor stasis. Ganitumab alone had marginal growth inhibition. The combination of BYL719 and ganitumab led to tumor regression. Conclusions: Our data suggest that the combination of BYL719 and ganitumab would act synergistically to inhibit PIK3CA mutant breast cancer cells by blocking two inter-connected pathways. The combination of BYL719 and ganitumab has now entered into clinical trial (NCT01708161). It represents a promising approach against PI3KCA mutant breast cancers.

Specific apoptosis induction by the dual PI3K/mTor inhibitor NVP-BEZ235 in HER2 amplified and PIK3CA mutant breast cancer cells

NVP-BEZ235 is a dual PI3K/mTOR inhibitor currently in phase I clinical trials. We profiled this compound against a panel of breast tumor cell lines to identify the patient populations that would benefit from such treatment. In this setting, NVP-BEZ235 selectively induced cell death in cell lines presenting either HER2 amplification and/or PIK3CA mutation, but not in cell lines with PTEN loss of function or KRAS mutations, for which resistance could be attributed, in part to ERK pathway activity. An in depth analysis of death markers revealed that the cell death observed upon NVP-BEZ235 treatment could be recapitulated with other PI3K inhibitors and that this event is linked to active PARP cleavage indicative of an apoptotic process. Moreover, the effect seemed to be partly independent of the caspase-9 executioner and mitochondrial activated caspases, suggesting an alternate route for apoptosis induction by PI3K inhibitors. Overall, this study will provide guidance for patient stratification for forthcoming breast cancer phase II trials for NVP-BEZ235.