Yoshinobu Shiose

Antitumor Activity and Induction of TP53-Dependent Apoptosis toward Ovarian Clear Cell Adenocarcinoma by the Dual PI3K/mTOR Inhibitor DS-7423

DS-7423, a novel, small-molecule dual inhibitor of phosphatidylinositol-3-kinase (PI3K) and mammalian target of rapamycin (mTOR), is currently in phase I clinical trials for solid tumors. Although DS-7423 potently inhibits PI3Kα (IC50 = 15.6 nM) and mTOR (IC50 = 34.9 nM), it also inhibits other isoforms of class I PI3K (IC50 values: PI3Kβ = 1,143 nM; PI3Kγ = 249 nM; PI3Kδ = 262 nM). The PI3K/mTOR pathway is frequently activated in ovarian clear cell adenocarcinomas (OCCA) through various mutations that activate PI3K-AKT signaling. Here, we describe the anti-tumor effect of DS-7423 on a panel of nine OCCA cell lines. IC50 values for DS-7423 were <75 nM in all the lines, regardless of the mutational status of PIK3CA. In mouse xenograft models, DS-7423 suppressed the tumor growth of OCCA in a dose-dependent manner. Flow cytometry analysis revealed a decrease in S-phase cell populations in all the cell lines and an increase in sub-G1 cell populations following treatment with DS-7423 in six of the nine OCCA cell lines tested. DS-7423-mediated apoptosis was induced more effectively in the six cell lines without TP53 mutations than in the three cell lines with TP53 mutations. Concomitantly with the decreased phosphorylation level of MDM2 (mouse double minute 2 homolog), the level of phosphorylation of TP53 at Ser46 was increased by DS-7423 in the six cell lines with wild-type TP53, with induction of genes that mediate TP53-dependent apoptosis, including p53AIP1 and PUMA at 39 nM or higher doses. Our data suggest that the dual PI3K/mTOR inhibitor DS-7423 may constitute a promising molecular targeted therapy for OCCA, and that its antitumor effect might be partly obtained by induction of TP53-dependent apoptosis in TP53 wild-type OCCAs.

Small molecule agonists of PPAR-γ exert therapeutic effects in esophageal cancer

The transcription factor PPAR-γ plays various roles in lipid metabolism, inflammation, cellular differentiation, and apoptosis. PPAR-γ agonists used to treat diabetes may have utility in cancer treatment. Efatutazone is a novel later generation PPAR-γ agonist that selectively activates PPAR-γ target genes and has antiproliferative effects in a range of malignancies. In this study, we investigated PPAR-γ status in esophageal squamous cell carcinoma (ESCC) and investigated the antiproliferative effects of efatutazone. PPAR-γ was expressed heterogeneously in ESCC, in which it exhibited an inverse relationship with Ki-67 expression. PPAR-γ expression was associated independently with good prognosis in ESCC. Efatutazone, but not the conventional PPAR-γ agonist troglitazone, inhibited ESCC cell proliferation in vitro and in vivo. Mechanistic investigations suggested that efatutazone acted by upregulating p21Cip1 protein in the nucleus through inactivation of the Akt pathway and dephosphorylation of p21Cip1 at Thr145 without affecting the transcriptional activity of p21Cip1. We also found that treatment with efatutazone led to phosphorylation of the EGF receptor and activation of the mitogen-activated protein kinase (MAPK) pathway. Accordingly, the combination of efatutazone with the antiepithelial growth factor receptor antibody cetuximab synergized to negatively regulate the phosphoinositide 3-kinase-Akt and MAPK pathways. Together, our results suggest that efatutazone, alone or in combination with cetuximab, may offer therapeutic effects in ESCC.

A possible mechanism for the long-lasting antitumor effect of the macromolecular conjugate DE-310: mediation by cellular uptake and drug release of its active camptothecin analog DX-8951

DE-310, a new macromolecular prodrug, was designed to enhance the pharmacological profiles of a novel camptothecin analog (DX-8951f), and a single treatment with DE-310 exhibits a similar or greater therapeutic effect than do optimally scheduled multiple administrations of DX-8951f in several types of tumors. In this study, the drug-release mechanism by which DE-310 excites antitumor activity was investigated in Meth A cells, a malignant ascites model of murine fibrosarcoma. A single i.v. injection of DE-310 at the maximum tolerated dose (MTD) prolonged survival of Meth A-bearing mice by 300%. DX-8951 and glycyl-8951 (G-DX-8951), enzymatic cleavage products of DE-310, were detected in serum and ascites fluid, and also in the culture medium of Meth A ascites cells incubated in vitro with DE-310. The total amounts of DX-8951, G-DX-8951, and conjugated DX-8951 in Meth A tumor cells were three times higher than that in macrophages. Furthermore, DX-8951-related fluorescence was observed in Meth A ascites cells obtained from Meth A-bearing mice that had received DE-310 or CM-Dex-PA-DX-8951 that does not release free DX-8951. DX-8951-related fluorescence was also observed at the site of lysosomes in cells incubated in vitro with DE-310 at 37°C, but not in those incubated at 4°C. Drugs were released from DE-310 by cysteine proteinase prepared from Meth A tumor tissue. These results suggest that the mechanism by which DX-8951 is released from DE-310 in vivo is involved in the process of uptake of DE-310 into tumor or macrophages, digestion by intracellular lysosomal cysteine proteinase, and subsequent secretion of the drugs.

Systematic research of peptide spacers controlling drug release from macromolecular prodrug system, carboxymethyldextran polyalcohol-peptide-drug conjugates.

The primary purpose of this study was to comprehensively delineate specificity of the peptide spacer sequence to tumor-expressed proteases for the design of macromolecular carrier-peptide spacer-drug conjugate system. 225 conjugates of carboxymethyldextran polyalcohol (CM-Dex-PA) as water-soluble carrier and a dansyl derivative (N-(4-aminobutyl)-5-(dimethylamino)-1-naphthalenesulfonamide, DNS) as the model drug linked with different tetrapeptide spacers (Gly-Gly-P(2)-P(1), P(2), P(1): Ala, Asn, Gly, Cit, Gln, Ile, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val) were combinatorially synthesized. First, the drug release assay of all of the fluorogenic model conjugates was performed in murine Meth A solid tumor homogenates. The drug release rate was higher with conjugates having hydrophobic amino acids at P(2). It was also found that conjugates with Asn release the drug rapidly and, in contrast, those with Pro does not. Second, we selected three peptide spacers (Gly-Gly-Phe-Gly, Gly-Gly-Ile-Gly, Gly-Gly-Pro-Leu), which release only DNS at different rates, and applied them to doxorubicin (DXR) conjugates. These three DXR conjugates were used for investigating relationships with drug release, pharmacokinetics, and antitumor activity against Meth A bearing mice of these conjugates. The release of DXR from the conjugates corresponded well with that of DNS conjugates in tumor homogenates. CM-Dex-PA-Gly-Gly-Phe-Gly-DXR and CM-Dex-PA-Gly-Gly-Ile-Gly-DXR indicated strong antitumor activity, with the comparable pharmacokinetic profile of released DXR in tumor. Taken with the fact that the drug release rate in tumor homogenates was approximately 10-fold different between these two DXR conjugates, it is likely that cellular uptake of the conjugate would be rate-limiting, rather than the drug release process under the in vivo situation. However, much weaker antitumor activity was observed with CM-Dex-PA-Gly-Gly-Pro-Leu-DXR, of which the drug release was extremely slow.

Preclinical therapeutic efficacy of a novel blood-brain barrier-penetrant dual PI3K/mTOR inhibitor with preferential response in PI3K/PTEN mutant glioma

Glioblastoma (GBM) is an ideal candidate disease for signal transduction targeted therapy because the majority of these tumors harbor genetic alterations that result in aberrant activation of growth factor signaling pathways. Loss of heterozygosity of chromosome 10, mutations in the tumor suppressor gene PTEN, and PI3K mutations are molecular hallmarks of GBM and indicate poor prognostic outcomes in many cancers. Consequently, inhibiting the PI3K pathway may provide therapeutic benefit in these cancers. PI3K inhibitors generally block proliferation rather than induce apoptosis. To restore the sensitivity of GBM to apoptosis induction, targeted agents have been combined with conventional therapy. However, the molecular heterogeneity and infiltrative nature of GBM make it resistant to traditional single agent therapy. Our objectives were to test a dual PI3K/mTOR inhibitor that may cross the blood–brain barrier (BBB) and provide the rationale for using this inhibitor in combination regimens to chemotherapy-induced synergism in GBM. Here we report the preclinical potential of a novel, orally bioavailable PI3K/mTOR dual inhibitor, DS7423 (hereafter DS), in in-vitro and in-vivo studies. DS was tested in mice, and DS plasma and brain concentrations were determined. DS crossed the BBB and led to potent suppression of PI3K pathway biomarkers in the brain. The physiologically relevant concentration of DS was tested in 9 glioma cell lines and 22 glioma-initiating cell (GIC) lines. DS inhibited the growth of glioma tumor cell lines and GICs at mean 50% inhibitory concentration values of less than 250 nmol/L. We found that PI3K mutations and PTEN alterations were associated with cellular response to DS treatment; with preferential inhibition of cell growth in PI3KCA-mutant and PTEN altered cell lines. DS showed efficacy and survival benefit in the U87 and GSC11 orthotopic models of GBM. Furthermore, administration of DS enhanced the antitumor efficacy of temozolomide against GBM in U87 glioma models, which shows that PI3K/mTOR inhibitors may enhance alkylating agent-mediated cytotoxicity, providing a novel regimen for the treatment of GBM. Our present findings establish that DS can specifically be used in patients who have PI3K pathway activation and/or loss of PTEN function. Further studies are warranted to determine the potential of DS for glioma treatment.

Abstract 525: Induction of apoptosis in ovarian clear cell carcinomas with wild-type TP53 by inhibiting PI3K/mTOR signaling pathway.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Background:Epithelial ovarian cancer is a leading cause of death from gynecological malignancies.Ovarian clear cell adenocarcinoma (OCCA) is the second most common cause of death from ovarian cancer with a higher incidence in Asia, especially Japan (>25%). Due to low response rates to conventional platinum-based chemotherapy, the clinical outcome is generally poor. PIK3CA mutations are frequent (>40%) in OCCA compared with other histology types and overexpression of receptor tyrosine kinases (RTKs), including HER2 and cMET, is also frequently observed in OCCA. Thus, the RTK-PI3K (phosphatidylinositol 3-kinase)/mTOR (mammalian target of rapamycin) signaling axis is broadly activated in OCCA. This study evaluated the anti-tumor efficacy of DS-7423, a novel PI3K/mTOR dual inhibitor, in a panel of OCCA cell lines. Material and methods: We evaluated the phosphorylation status of RTK-PI3K/mTOR signaling pathway in 9 OCCA cell lines. Four of those cell lines possessed PIK3CA mutations; 3 of the 5 remaining cell lines overexpressed HER2, HER3 and/or cMET. We determined the effect of DS-7423 by MTT assay, FACS analysis, and Western blotting. Apoptosis induction was detected by annexin-V and propidium iodide (PI) staining. Induction of p53 target genes, including p53AIP1, was evaluated by RT-PCR. Results: IC50 values for DS-7423 were <100 nM for all 9 OCCA cell lines. Efficacy did not significantly differ between PIK3CA mutant and wild type OCCA cells. AKT and its downstream targets (GSK3β, FOXO1/3a, MDM2, 4EBP1, and S6) were phosphorylated in OCCA cell lines regardless of PIK3CA mutational status. DS-7423 suppressed the phosphorylation levels of AKT and its target proteins in all these OCCA cell lines. FACS analysis revealed an increase in sub-G1 cell populations by treatment at ≥156 nM DS-7423 in 6 out of 9 OCCA cell lines. In these 6 cell lines, the number of apoptotic cells, as determined by annexin-V and PI staining, increased in a dose-dependent manner and DS-7423 induced apoptosis more effectively in TP53 wild type OCCA cell lines than in TP53 mutant OCCA cell lines. In accordance with decreased phospho-MDM2 levels, phosphorylation levels of p53 (Ser46) were increased and expression of p53AIP1 and PUMA, genes regulating p53-dependent apoptosis, were induced by DS-7423 in these p53 wild type cells. We also evaluated the expression status of bcl-2 family proteins in the OCCA cell line OVISE, and observed that DS-7423 treatment induced up-regulation of proapoptotic Bim and down-regulation of anti-apoptotic Mcl-1. Conclusions: Dual PI3K/mTOR inhibitors such as DS-7423 may constitute a promising molecular targeted therapy for OCCA, regardless of PIK3CA status. DS-7423 may induce apoptosis via activation of p53 and bcl-2 family members. As p53 mutations ar generally rare in OCCA, DS-7423 might induce p53-dependent apoptosis in this cancer type. Citation Format: Tomoko Kashiyama, Katsutoshi Oda, Yuji Ikeda, Yoshinobu Shiose, Yasuhide Hirora, Aki Miyasaka, Takahiro Koso, Kanako Inaba, Tomohiko Fukuda, Keiko Shoji, Michihiro Tanikawa, Kazunori Nagasaka, Osamu Hiraike-Wada, Kei Kawana, Hiroyuki Aburatani, Shunsuke Nakagawa, Tomoyuki Fujii, Tetsu Yano, Shiro Kozuma. Induction of apoptosis in ovarian clear cell carcinomas with wild-type TP53 by inhibiting PI3K/mTOR signaling pathway. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 525. doi:10.1158/1538-7445.AM2013-525

Abstract 5511: The proliferation-inhibiting effects of Efatutazone, a novel third-generation peroxisome proliferator-activated receptor gamma agonist, on esophageal squamous cell carcinoma in vitro and in vivo.

Background: Peroxisome proliferator-activated receptor gamma (PPARγ) inhibits the proliferation of several cancers. Efatutazone, a novel third-generation thiazolidinedione PPARγ agonist, demonstrated acceptable tolerability with evidence of disease control in patients with advanced alignancies in the phase 1 study. However, the status and function of PPARγ in esophageal squamous cell carcinoma (ESCC) remains unclear and the effects of the PPARγ agonist are unknown. Methods: Immunohistochemical staining was performed using paraffin sections of specimens obtained from 145 ESCC patients who underwent curative resection. The anti-tumor effects of Efatutazone were investigated in ESCC cell lines both in vitro and in vivo. Results: The expression of PPARγ was detected in the nucleus of the normal esophageal squamous epithelium in all patients, while positive staining in ESCC was observed in 24.8% of the patients. The expression of PPARγ decreases according to the depth of invasion and exhibits an inverse relationship with the expression of ki-67. Efatutazone inhibited the proliferation of the ESCC cell lines in a time- and dose-dependent manner in vitro. The anti-proliferation effect of Efatutazone was more evident in cell lines with high PPARγ expression than in those with low PPARγ expression. Efatutazone inhibited the sphere formation of TE-4, which is one of the high PPARγ expression cell lines. Efatutazone upregulated the cell cycle kinase inhibitor p21 WAF1 / CIP1 and induced G1 arrest. We confirmed that the antitumor effect of this drug occurred in a PPARγ dependent manner using si-RNA for PPARγ. In a mouse xenograft model established with TE-4, we compared three arms: the control group (n=8), the Troglitazone group (10 mg/kg, n=8) and the Efatutazone group (10 mg/kg, n=8). Although tumor volume did not differ between the control group and the Troglitazone group, 50.4±13.6% (average ± SD) reduction in tumor volume was observed in the Efatutazone group compared with the control group. The mRNA expression of PLIN-2, a transcription product of PPARγ, was two-fold higher in the Efatutazone group than in the control group. Immunohistochemical staining demonstrated enhanced p21 WAF1 / CIP1 expression and decreased the ki-67 expression in the Efatutazone group in comparison to either the control or Troglitazone group. Conclusions: The expression of PPARγ decreased in ESCC compared with normal esophageal epithelium. The anti-proliferation effect of the third-generation PPARγ agonist, Efatutazone, for ESCC was observed both in vitro and in vivo. Efatutazone is a candidate for novel anti-cancer agent against ESCC. Citation Format: Hiroshi Sawayama, Takatsugu Ishimoto, Masayuki Watanabe, Naoya Yoshida, Yoshifumi Baba, Masaaki Iwatsuki, Hidetaka Sugihara, Hiroshi Onuma, Yoshinobu Shiose, Hideo Baba. The proliferation-inhibiting effects of Efatutazone, a novel third-generation peroxisome proliferator-activated receptor gamma agonist, on esophageal squamous cell carcinoma in vitro and in vivo . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5511. doi:10.1158/1538-7445.AM2013-5511