Antitumor Efficacy And Predictive Biomarker Of PI3Kα Isoform Inhibitor In Breast Cancer Treatment

The PI3 K signaling pathway regulates multiple physiological functions and cellular processes,including survival,growth,proliferation and metabolism.Aberrant activation of the PI3 K pathway is commonly observed in many cancer types.Activating mutation of PIK3 CA,encoding the p110α catalytic unit,occurs almost in all types of breast cancer with an overall frequency of over 30%.PIK3 CA activation has been found to induce initiation and development of mammary tumors,which makes PI3Kα being the desirable target for breast cancer therapy.A number of pan-PI3 K inhibitors and four PI3Kα isoform inhibitors are currently in clinical trials as single agent or combination regimen for the therapy of breast cancer.The number of PI3Kα-selective inhibitors is very limited and there is no such inhibitor in clinical trial in China.It is of urgent need to discover and develop PI3Kα-selective inhibitors with self-owned intellectual property in China.Variable response of breast cancer patients to PI3Kα inhibitors clinically has been observed.Thus,identifying and validating predictive biomarker of PI3Kα inhibitors is critical for the development of PI3Kα inhibitor as well as improving anti-tumor efficacy.In this study,we systematically investigated the anti-tumor efficacy of a novel PI3Kα inhibitor CYH33 against breast cancer.In addition,we identified and validated the predictive biomarker for the efficacy of PI3Kα inhibitors in the therapy of breast cancer.CYH33 is a novel PI3Kα-selective inhibitor synthesized and discovered by collaboration between Dr.Chunhao Yang’s group and our lab,which displays potent antitumor activity against a variety of tumor cells originated from different tissue types.Specifically,CYH33 possesses significant anti-proliferative activity in a panel of human breast tumor cells,which is comparable to a pan-PI3 K inhibitor GDC0941,but more potent than a PI3Kα-selective inhibitor BYL719.CYH33 inhibited phosphorylation of AKT in T47 D,MCF-7 and MDA-MB-231 cells in a dose-dependent manner,while suppressed phosphorylation of p70S6 K and S6 just in T47 D and MCF-7 cells.CYH33 arrested sensitive cells in G1 arrest,which is accompanied with downregulation of Rb,Cyclin D1 and accumulation of p27,while this observation failed to be obtained in CYH33 resistant breast cancer cells.In addition,we discovered that CYH33 failed to induce apoptosis both in sensitive and resistant cells.Thus,CYH33 exerts its anti-tumor efficacy via G1 arrest.CYH33 significantly suppressed the tumor growth in mice bearing T47 D xenograft and in PIK3CAH1047 R genetically engineered mice.In addition,CYH33 had little effect on the body weight and blood glucose regulation during administration.We preliminarily analyzed the response of breast cancer cells to CYH33 based on molecular subtypes of breast cancer,and found that luminal and HER2-enriched breast cancer cell lines were relatively sensitive to CYH33 and basal-like breast cancer cell lines were resistant to CYH33.In addition,statistical analysis of the association between aberrant alteration of PI3 K pathway and response of breast cancer cells to CYH33 revealed that breast cancer cells bearing PIK3 CA mutation and/or HER2 amplification were sensitive to CYH33,while breast cancer cells harboring PTEN loss displayed resistance to CYH33.Though molecular subtypes of breast cancer and aberrant alteration of PI3 K pathway reflected the sensitivity of breast cancer cells to CYH33 to some extent,we found a few basal-like breast cancer cells and those with normal PI3 K pathway were sensitive to CYH33.Thus,exploring the biomarker predicting the efficacy of PI3Kα inhibitors based on their mechanism of action might provide a useful strategy to achieve precise prediction.We then sought to discover predictive biomarker for the efficacy of PI3Kα inhibitors by dissecting the proliferative signaling pathway mediated by PI3 K in breast cancer.PI3Kα inhibitors concurrently inhibited the phosphorylation of AKT and ERK in PIK3CA-mutated human breast cancer cells.PI3K-regulated ERK phosphorylation was independent of canonical PDK1/AKT/mTOR pathway,while it was associated with RAF/MEK.Hyper-activation of EGFR or RAS abrogated inhibition of ERK phosphorylation by PI3Kα inhibitors.Furthermore,hyper-activation of receptor tyrosine kinases(RTKs)including EGFR,c-MET,FGFR and HER3 but not IGF-1R restored ERK phosphorylation and cell viability suppressed by BYL719,suggesting the discriminative functions of RTKs in cell signaling and proliferation.By profiling 22 breast cancer cell lines,we found that PI3Kα inhibitors were more potent in cell lines where phosphorylation of both AKT and ERK was attenuated than those where only AKT phosphorylation was inhibited.The potency of PI3Kα inhibitors was further found to be significantly correlated with the expression profile of RTKs in breast cancer cells.Specifically,overexpression of EGFR,c-MET and/or FGFR1 forecasted resistance,while overexpression of IGF-1R and/or HER2 predicted sensitivity to PI3Kα inhibitors in breast cancer cells.Similar correlation between PI3Kα inhibitors efficacy and expression profile of RTKs was found in patient-derived xenograft models of breast cancer.In summary,CYH33 displays superior activity than BYL719 against breast cancer in vitro and in vivo,which promised its advancement to clinical trials.We discovered and validated that inhibition of ERK phosphorylation by PI3Kα inhibitors contributed to its antitumor efficacy and was determined by the converged signaling from RTKs.The expression profile of RTKs in breast cancer tissue could be potentially developed as a predictive biomarker for the efficacy of PI3Kα inhibitors.