Hisashi Yoshimi

Antiangiogenic effect of ZSTK474, a novel phosphatidylinositol 3-kinase inhibitor

Angiogenesis is known to be required for tumour growth and metastasis. Recent reports indicated that phosphatidylinositol 3-kinase (PI3K) promoted angiogenesis by inducing expressions of HIF-1alpha and vascular endothelial growth factor (VEGF). The present study aims to investigate the antiangiogenic effect of ZSTK474, a novel pan-PI3K inhibitor. ZSTK474 significantly inhibited tumour growth in the RXF-631L xenograft model. Immunohistochemical staining of the tumour tissue with anti-von Willebrand Factor antibody showed a significantly reduced number of microvessels in the ZSTK474-treated mice, suggesting the highly promising antiangiogenic activity in vivo. In human umbilical vein endothelial cells (HUVECs), submicromolar concentrations of ZSTK474 inhibited cell growth, blocked VEGF-induced cell migration and the tube formation, and thus revealed potent in vitro antiangiogenic activity. Furthermore, ZSTK474 inhibited phosphorylation of Akt at submicromolar concentrations. In RXF-631L cancer cells, on the other hand, ZSTK474 treatment inhibited the expression of HIF-1alpha and secretion of VEGF. Together, these results suggest that ZSTK474 has potent antiangiogenic activity, which could be attributed to dual-target inhibitory properties: inhibition of VEGF secretion by cancer cells and inhibition of PI3K in endothelial cells.

Inhibition of PI3K by ZSTK474 suppressed tumor growth not via apoptosis but G0/G1 arrest.

Phosphoinositide 3-kinase (PI3K) is a potential target in cancer therapy. Inhibition of PI3K is believed to induce apoptosis. We recently developed a novel PI3K inhibitor ZSTK474 with antitumor efficacy. In this study, we have examined the underlying mode of action by which ZSTK474 exerts its antitumor efficacy. In vivo, ZSTK474 effectively inhibited the growth of human cancer xenografts. In parallel, ZSTK474 treatment suppressed the expression of phospho-Akt, suggesting effective PI3K inhibition, and also suppressed the expression of nuclear cyclin D1 and Ki67, both of which are hallmarks of proliferation. However, ZSTK474 treatment did not increase TUNEL-positive apoptotic cells. In vitro, ZSTK474 induced marked G(0)/G(1) arrest, but did not increase the subdiploid cells or activate caspase, both of which are hallmarks of apoptosis. These results clearly indicated that inhibition of PI3K by ZSTK474 did not induce apoptosis but rather induced strong G(0)/G(1) arrest, which might cause its efficacy in tumor cells.

ZSTK474, a specific phosphatidylinositol 3-kinase inhibitor, induces G1 arrest of the cell cycle in vivo

Phosphatidylinositol 3-kinase (PI3K) is regarded as a promising therapeutic target because it is often activated in cancer. We previously reported that ZSTK474, a specific PI3K inhibitor, inhibits tumour cell proliferation via G1 arrest of the cell cycle without inducing apoptosis in vitro. However, it remained unclear whether ZSTK474 induces G1 arrest to exert antitumour efficacy in vivo. We recently developed a live imaging system, named Fluorescent Ubiquitination-based Cell Cycle Indicator (Fucci), to visualise cell cycle distribution. Here, by using this system, we tested whether ZSTK474 induces G1 arrest in tumour cells in vivo, as well as in vitro. Fucci-introduced human breast cancer MCF-7 cells and cervical cancer HeLa cells were subcutaneously xenografted in nude mice. ZSTK474 was administered to the tumour-bearing mice for 5 days, and the cell cycle distribution in the xenografted tumours were analysed by monitoring fluorescence in live mice. We demonstrate that ZSTK474 induces G1arrest along with tumour suppression in vivo. Moreover, we show that ZSTK474 suppresses the tumour growth without inducing apoptosis. Interestingly, such increase in G1 cells and tumour suppression was maintained during long-term (3-month) administration of ZSTK474. These results suggest that ZSTK474 exerts its in vivo antitumour efficacy via G1 arrest but not via apoptosis as long as it is administered, and could be used for months as maintenance therapy for patients with advanced cancers.

Effectiveness of combined treatment using X-rays and a phosphoinositide 3-kinase inhibitor, ZSTK474, on proliferation of HeLa cells in vitro and in vivo.

ZSTK474 is a novel orally applicable phosphoinositide 3‐kinase‐specific inhibitor that strongly inhibits cancer cell proliferation. To further explore the antitumor effect of ZSTK474 for future clinical usage, we studied its combined effects with radiation. The proliferation of HeLa cells was inhibited by treatment with X‐rays alone or ZSTK474 alone. Combination treatment using X‐rays then ZSTK474 given orally for 8 days, starting 24 h post‐irradiation, significantly enhanced cell growth inhibition. The combined effect was also observed for clonogenic survival with continuous ZSTK474 treatment. Western blot analysis showed enhanced phosphorylation of Akt and GSK‐3β by X‐irradiation, whereas phosphorylation was inhibited by ZSTK474 treatment alone. Treatment with ZSTK474 after X‐irradiation also inhibited phosphorylation, and remarkably inhibited xenograft tumor growth. Combined treatment with X‐rays and ZSTK474 has greater therapeutic potential than radiation or drug therapy alone, both in vitro and in vivo. (Cancer Sci 2011; 102: 1176–1180)

Basal expression of insulin-like growth factor 1 receptor determines intrinsic resistance of cancer cells to a phosphatidylinositol 3-kinase inhibitor ZSTK474

Drug resistance often critically limits the efficacy of molecular targeted drugs. Although pharmacological inhibition of phosphatidylinositol 3‐kinase (PI3K) is an attractive therapeutic strategy for cancer therapy, molecular determinants for efficacy of PI3K inhibitors (PI3Kis) remain unclear. We previously identified that overexpression of insulin‐like growth factor 1 receptor (IGF1R) contributed to the development of drug resistance after long‐term exposure to PI3Kis. In this study, we examined the involvement of basal IGF1R expression in intrinsic resistance of drug‐naïve cancer cells to PI3Kis and whether inhibition of IGF1R overcomes the resistance. We found that cancer cells highly expressing IGF1R showed resistance to dephosphorylation of Akt and subsequent antitumor effect by ZSTK474 treatment. Knockdown of IGF1R by siRNAs facilitated the dephosphorylation and enhanced the drug efficacy. These cells expressed tyrosine‐phosphorylated insulin receptor substrate 1 at high levels, which was dependent on basal IGF1R expression. In these cells, the efficacy of ZSTK474 in vitro and in vivo was improved by its combination with the IGF1R inhibitor OSI‐906. Finally, we found a significant correlation between the basal expression level of IGF1R and the inefficacy of ZSTK474 in an in vivo human cancer panel, as well as in vitro. These results suggest that basal IGF1R expression affects intrinsic resistance of cancer cells to ZSTK474, and IGF1R is a promising target to improve the therapeutic efficacy. The current results provide evidence of combination therapy of PI3Kis with IGF1R inhibitors for treating IGF1R‐positive human cancers.

Abstract A43: Basal expression of insulin-like growth factor 1 receptor determines intrinsic resistance of cancer cells to a PI3K inhibitor ZSTK474

Drug resistance often critically limits the efficacy of molecular targeted drugs. Although pharmacological inhibition of phosphatidylinositol 3-kinase (PI3K) is an attractive therapeutic strategy for cancer therapy, molecular determinants for efficacy of PI3K inhibitors remain unclear. We previously identified that overexpression of insulin-like growth factor 1 receptor (IGF1R) contributed to the development of drug resistance after long-term exposure to PI3K inhibitors. In this study, we examined the involvement of basal IGF1R expression in intrinsic resistance of drug-naive cancer cells to PI3K inhibitors and whether inhibition of IGF1R overcomes the resistance. We found that cancer cells highly expressing IGF1R exhibited resistance to dephosphorylation of Akt and subsequent antitumor effect by ZSTK474 treatment. Knockdown of IGF1R by siRNAs facilitated the dephosphorylation and enhanced the drug efficacy. These cells expressed tyrosine-phosphorylated IRS-1 in high levels, which was dependent on basal IGF1R expression. In these cells, combination with the IGF1R-TKI, OSI-906, improved the efficacy of ZSTK474 in vitro and in vivo. Finally, we found a significant correlation between the basal expression level of IGF1R and the inefficacy of ZSTK474 in in vivo human cancer panel, as well as in vitro. These results suggest that basal IGF1R expression affects intrinsic resistance of cancer cells to ZSTK474, and IGF1R is a promising target to improve the therapeutic efficacy. The current results provide evidence of combination therapy of PI3K inhibitors with IGF1R inhibitors for treating IGF1R-positive human cancers. Citation Format: Sho Isoyama, Gensei Kajiwara, Naomi Tamaki, Okamura Mutsumi, Hisashi Yoshimi, Naoki Nakamura, Yumiko Nishimura, Takao Yamori, Shingo Dan. Basal expression of insulin-like growth factor 1 receptor determines intrinsic resistance of cancer cells to a PI3K inhibitor ZSTK474. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr A43.

Abstract C96: Development of combination therapy with an IGF1R inhibitor and a PI3K inhibitor ZSTK474 in cancer cells exhibiting the intrinsic resistance to PI3K inhibitors.

Background: Tumor cells can vary markedly in their response to anti-cancer drugs. Therefore predicting sensitivity or resistance of tumor cells to the drug and developing therapeutic strategies for overcoming the resistance is required to realize personalized medicine for cancer treatment. Although multiple drugs targeting phosphatidylinositol 3-kinase (PI3K) including ZSTK474 are undergoing clinical trials, little is known about the resistance. We previously reported that overexpression of insulin-like growth factor 1 receptor (IGF1R) contributed to acquired resistance to PI3K inhibitors (PI3Kis). In this study, we examined whether IGF1R contributes to intrinsic resistance to PI3Kis and whether combination of IGF1R tyrosine kinase inhibitor (IGF1R-TKI) and PI3Ki is an effective strategy for cancer cells exhibiting the intrinsic resistance to PI3Kis. Materials and Methods: Correlation analyses between the expression level of IGF1R with the efficacy of PI3Ki across an in vitro panel of 39 human cancer cell lines (termed JFCR39) and a panel of 24 human tumor xenografts implanted in nude mice (termed JFCR24 xenografts) were performed. Specific siRNAs against IGF1R were used to examine the functional involvement of IGF1R in intrinsic resistance. Combination effect of ZSTK474 and an IGF1R-TKI OSI-906 was examined in vitro using isobologram analysis and in vivo using human tumor xenografts implanted in nude mice. Results: Correlation analysis revealed that PI3Ki-naive cancer cells expressing high levels of IGF1R exhibited resistance to ZSTK474 in vitro and in vivo. Targeted knockdown of IGF1R expression using specific siRNAs enhanced the efficacy of ZSTK474 in intrinsic resistant cell lines overexpressing IGF1R. In these cell lines, higher concentration of ZSTK474 was needed to dephosphorylate Akt compared to IGF1R-negative cell lines, and specific knockdown of IGF1R made it easier to dephosphorylate Akt after ZSTK474 treatment. Finally, we found a synergistic therapeutic effect of ZSTK474 in combination with OSI-906 in vivo, as well as in vitro. Conclusion: PI3Ki-naive cancer cells overexpressing IGF1R exhibited resistance to ZSTK474, but combination of ZSTK474 with an IGF1R-TKI exerted a remarkable therapeutic response in such cancer cells. We hope that our findings would contribute to personalized medicine using PI3Kis in future clinical application. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C96. Citation Format: Sho Isoyama, Gensei Kajiwara, Naomi Tamaki, Hisashi Yoshimi, Naoki Nakamura, Mutsumi Okamura, Yumiko Nishimura, Takao Yamori, Shingo Dan. Development of combination therapy with an IGF1R inhibitor and a PI3K inhibitor ZSTK474 in cancer cells exhibiting the intrinsic resistance to PI3K inhibitors. [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 C96.

Abstract 2493: Inhibition of phosphoinositide 3-kinase suppressed growth of cancer cells with PIK3CA mutations or loss of PTEN expression via G0/G1 arrest of cell cycle but not apoptosis

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Background: The phosphoinositide 3-kinase (PI3K) pathway is frequently activated in cancer by gain-of-function mutation of PIK3CA and loss of PTEN expression, and mediates growth- and anti-apoptotic signals. Therefore, PI3K is considered as a promising therapeutic target to inhibit growth and to induce apoptosis in such cancers. We previously developed a novel PI3K-specific inhibitor ZSTK474 with antitumor efficacy (Yaguchi et al, J. Natl. Cancer Inst. 98: 545-56, 2006; Kong and Yamori, Curr. Med. Chem. 16: 2839-54, 2009). In this study, we have examined the cellular mechanism by which ZSTK474 exerts its antitumor efficacy in human cancer cells with or without PIK3CA/PTEN aberrations. Methods: We examined the antitumor effect of ZSTK474 on various human cancer cell lines with or without PIK3CA/PTEN aberration in vitro and in vivo. Protein expression and apoptosis in ZSTK474-treated and untreated tumor sections were analyzed by immunohistochemistry and TUNEL assay, respectively. Analysis of cell cycle in vitro was determined by flow cytometry and by Fucci (Fluorescent Ubiquitination-based Cell Cycle Indicator) system introduced to cancer cells. Induction of apoptosis in vitro was estimated by caspase activation using a substrate cleavage assay and by detection of subdiploid cells using flow cytometry. Results: In vivo, ZSTK474 effectively inhibited the growth of human tumor xenograft derived from a PTEN-null prostate cancer cell line PC3. In parallel, ZSTK474 treatment suppressed the expression of phospho-Akt, suggesting effective PI3K inhibition, and also suppressed the expression of nuclear cyclin D1 and Ki67, both of which are hallmarks of proliferation. However, ZSTK474 treatment did not increase TUNEL-positive apoptotic cells. Similar result was obtained in another PTEN-negative cell line KM-12 and three PIK3CA-mutated cell lines HCT-15 (E545K), MKN1 (E545K) and SK-OV3 (H1047R). In vitro, ZSTK474 induced marked G/G1 arrest accompanied by a decrease of nuclear cyclin D1, induction of p27kip1 and p15ink4b, and hypophosphorylation of pRB in PC-3 cells. However, ZSTK474 did not increase the subdiploid cells or activate caspase, both of which are hallmarks of apoptosis. Similar result was obtained other cell lines with or without PIK3CA /PTEN aberrations. Conclusion: These results clearly indicated that ZSTK474 did not induce apoptosis but rather induced strong G/G1 arrest in cancer cells with or without PIK3CA/PTEN aberrations, which might cause its therapeutic efficacy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2493.

Abstract C65: Enhanced antitumor effect by the combination of a PI3K inhibitor ZSTK474 with an mTOR inhibitor rapamycin

PI3K is a promising molecular target for the therapy of cancer. Various PI3K inhibitors are now under development. We developed a novel PI3K inhibitor ZSTK474 and demonstrated its potent antitumor efficacy. ZSTK474 is highly specific to PI3K and does not inhibit other kinases including mTOR that is a down stream component of PI3K pathway. The purpose of this study was to examine whether dual inhibition of PI3K and mTOR further improved antitumor efficacy. To assess the effect of the combination of ZSTK474 and rapamycin on prostate cancer PC‐3 cells and lung cancer A549 cells in vitro , we used the isobologram method. The combination of ZSTK474 and rapamycin showed a supra‐additive effect (synergism) on both PC‐3 and A549. We then evaluated the efficacy of the combination of ZSTK474 with an mTOR inhibitor rapamycin against the xenografts of PC‐3 and A549. Suboptimal doses of ZSTK474 and rapamycin were determined as 100 mg/kg/day (p.o., from day 0 to day 14, except days 3 and 10) and 0.1 mg/kg/day (i.p., from day 0 to day 5 except day 3), respectively. Then, the combination of the two drugs was compared to the single drug treatments. The combination of ZSTK474 and rapamycin showed almost complete inhibition of the tumor growth whereas each single drug treatment showed partial inhibition. The synergy in the toxicity was not observed. We examined the effects of the combination on the PI3K/Akt downstream molecules in the PC‐3 tumor tissues by immunohistochemical technique. The single treatment of ZSTK474 reduced the phosphorylation of Akt (Ser473), GSK3‐beta and 4E‐BP1, and partially reduced the phosphorylation of S6 and the expression of Cyclin D1. The single treatment of rapamycin reduced the phosphorylation of 4E‐BP1, and partially reduced the phosphorylation of S6 and the expression of Cyclin D1, but did not reduce the phosphorylation of Akt (Ser473) or that of GSK3‐beta. The combination of ZSTK474 and rapamycin reduced the phosphorylation of Akt, GSK3‐beta, 4E‐BP1 and S6, and the expression of Cyclin D1. These results confirmed the therapeutic efficacy of the combination of ZSTK474 and rapamycin against PC‐3 xenografts. Therefore, the combination of PI3K inhibitor and mTOR inhibitor may be effective for cancer therapy. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C65.