Mutsumi Okamura

Correlating Phosphatidylinositol 3-Kinase Inhibitor Efficacy with Signaling Pathway Status: In silico and Biological Evaluations

The phosphatidylinositol 3-kinase (PI3K) pathway is frequently activated in human cancers, and several agents targeting this pathway including PI3K/Akt/mammalian target of rapamycin inhibitors have recently entered clinical trials. One question is whether the efficacy of a PI3K pathway inhibitor can be predicted based on the activation status of pathway members. In this study, we examined the mutation, expression, and phosphorylation status of PI3K and Ras pathway members in a panel of 39 pharmacologically well-characterized human cancer cell lines (JFCR39). Additionally, we evaluated the in vitro efficacy of 25 PI3K pathway inhibitors in addition to conventional anticancer drugs, combining these data to construct an integrated database of pathway activation status and drug efficacies (JFCR39-DB). In silico analysis of JFCR39-DB enabled us to evaluate correlations between the status of pathway members and the efficacy of PI3K inhibitors. For example, phospho-Akt and KRAS/BRAF mutations prominently correlated with the efficacy and the inefficacy of PI3K inhibitors, respectively, whereas PIK3CA mutation and PTEN loss did not. These correlations were confirmed in human tumor xenografts in vivo, consistent with their ability to serve as predictive biomarkers. Our findings show that JFCR39-DB is a useful tool to identify predictive biomarkers and to study the molecular pharmacology of the PI3K pathway in cancer.

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.

AMF-26, a Novel Inhibitor of the Golgi System, Targeting ADP-ribosylation Factor 1 (Arf1) with Potential for Cancer Therapy

Background: Golgi is a potential target for cancer treatment, but no inhibitor became an anticancer drug. Results: Using a unique bioinformatics approach, we identified a novel Golgi inhibitor, AMF-26, targeting Arf1 activation and possessing potent antitumor activity. Conclusion: AMF-26 is a promising new anticancer drug lead. Significance: Our data indicate that Arf1 activation is a promising target for cancer treatment. ADP-ribosylation factor 1 (Arf1) plays a major role in mediating vesicular transport. Brefeldin A (BFA), a known inhibitor of the Arf1-guanine nucleotide exchange factor (GEF) interaction, is highly cytotoxic. Therefore, interaction of Arf1 with ArfGEF is an attractive target for cancer treatment. However, BFA and its derivatives have not progressed beyond the pre-clinical stage of drug development because of their poor bioavailability. Here, we aimed to identify novel inhibitors of the Arf1-ArfGEF interaction that display potent antitumor activity in vivo but with a chemical structure distinct from that of BFA. We exploited a panel of 39 cell lines (termed JFCR39) coupled with a drug sensitivity data base and COMPARE algorithm, resulting in the identification of a possible novel Arf1-ArfGEF inhibitor AMF-26, which differed structurally from BFA. By using a pulldown assay with GGA3-conjugated beads, we demonstrated that AMF-26 inhibited Arf1 activation. Subsequently, AMF-26 induced Golgi disruption, apoptosis, and cell growth inhibition. Computer modeling/molecular dynamics (MD) simulation suggested that AMF-26 bound to the contact surface of the Arf1-Sec7 domain where BFA bound. AMF-26 affected membrane traffic, including the cis-Golgi and trans-Golgi networks, and the endosomal systems. Furthermore, using AMF-26 and its derivatives, we demonstrated that there was a significant correlation between cell growth inhibition and Golgi disruption. In addition, orally administrated AMF-26 (83 mg/kg of body weight; 5 days) induced complete regression of human breast cancer BSY-1 xenografts in vivo, suggesting that AMF-26 is a novel anticancer drug candidate that inhibits the Golgi system, targeting Arf1 activation.

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.

Identification of Cyproheptadine as an Inhibitor of SET Domain Containing Lysine Methyltransferase 7/9 (Set7/9) That Regulates Estrogen-Dependent Transcription

SET domain containing lysine methyltransferase 7/9 (Set7/9), a histone lysine methyltransferase (HMT), also methylates non-histone proteins including estrogen receptor (ER) α. ERα methylation by Set7/9 stabilizes ERα and activates its transcriptional activities, which are involved in the carcinogenesis of breast cancer. We identified cyproheptadine, a clinically approved antiallergy drug, as a Set7/9 inhibitor in a high-throughput screen using a fluorogenic substrate-based HMT assay. Kinetic and X-ray crystallographic analyses revealed that cyproheptadine binds in the substrate-binding pocket of Set7/9 and inhibits its enzymatic activity by competing with the methyl group acceptor. Treatment of human breast cancer cells (MCF7 cells) with cyproheptadine decreased the expression and transcriptional activity of ERα, thereby inhibiting estrogen-dependent cell growth. Our findings suggest that cyproheptadine can be repurposed for breast cancer treatment or used as a starting point for the discovery of an anti-hormone breast cancer drug through lead optimization.

Thiophene-3-carboxamide analogue of annonaceous acetogenins as antitumor drug lead

Five novel acetogenin analogues with a furan, thiophene, or thiazole ring were synthesized, and their inhibitory activities toward human cancer cell lines were evaluated. The analogues showed more potent activities than natural acetogenin. One of them, the thiophene-3-carboxamide analogue, strongly inhibited the growth of human lung cancer cell line NCI-H23 in the xenograft mouse assay without critical toxicity.

M-COPA, a Golgi disruptor, inhibits cell surface expression of MET protein and exhibits antitumor activity against MET-addicted gastric cancers

The Golgi apparatus is responsible for transporting, processing, and sorting numerous proteins in the cell, including cell surface-expressed receptor tyrosine kinases (RTK). The small-molecule compound M-COPA [2-methylcoprophilinamide (AMF-26)] disrupts the Golgi apparatus by inhibiting the activation of Arf1, resulting in suppression of tumor growth. Here, we report an evaluation of M-COPA activity against RTK-addicted cancers, focusing specifically on human gastric cancer (GC) cells with or without MET amplification. As expected, the MET-addicted cell line MKN45 exhibited a better response to M-COPA than cell lines without MET amplification. Upon M-COPA treatment, cell surface expression of MET was downregulated with a concurrent accumulation of its precursor form. M-COPA also reduced levels of the phosphorylated form of MET along with the downstream signaling molecules Akt and S6. Similar results were obtained in additional GC cell lines with amplification of MET or the FGF receptor FGFR2 MKN45 murine xenograft experiments demonstrated the antitumor activity of M-COPA in vivo Taken together, our results offer an initial preclinical proof of concept for the use of M-COPA as a candidate treatment option for MET-addicted GC, with broader implications for targeting the Golgi apparatus as a novel cancer therapeutic approach. Cancer Res; 76(13); 3895-903. ©2016 AACR.

A novel thiophene‐3‐carboxamide analog of annonaceous acetogenin exhibits antitumor activity via inhibition of mitochondrial complex I

Previously we synthesized JCI‐20679, a novel thiophene‐3‐carboxamide analog of annonaceous acetogenins which have shown potent antitumor activity, with no serious side effects, in mouse xenograft models. In this study, we investigated the antitumor mechanism of JCI‐20679. The growth inhibition profile (termed “fingerprint”) of this agent across a panel of 39 human cancer cell lines (termed “JFCR39”) was measured; this fingerprint was analyzed by the COMPARE algorithm utilizing the entire drug sensitivity database for the JFCR39 panel. The JCI‐20679‐specific fingerprint exhibited a high similarity to those of two antidiabetic biguanides and a natural rotenoid deguelin which were already known to be mitochondrial complex I inhibitors. In addition, the fingerprint exhibited by JCI‐20679 was not similar to that displayed by any typical anticancer drugs within the database, suggesting that it has a unique mode of action. In vitro experiments using bovine heart‐derived mitochondria showed direct inhibition of mitochondrial complex I by JCI‐20679 and associated derivatives. This inhibition of enzymatic activity positively correlated with tumor cell growth inhibition. Furthermore, a fluorescently labeled derivative of JCI‐20679 localized to the mitochondria of live cancer cells in vitro. These results suggest that JCI‐20679 can inhibit cancer cell growth by inhibiting mitochondrial complex I. Our results show that JCI‐20679 is a novel anticancer drug lead with a unique mode of action.

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)