Yurie Kura

Efficacy of targeted AKT inhibition in genetically engineered mouse models of PTEN -deficient prostate cancer

The PI3K/AKT pathway is frequently altered in advanced human prostate cancer mainly through the loss of functional PTEN, and presents as potential target for personalized therapy. Our aim was to determine the therapeutic potential of the pan-AKT inhibitor, AZD5363, in PTEN-deficient prostate cancer. Here we used a genetically engineered mouse (GEM) model of PTEN-deficient prostate cancer to evaluate the in vivo pharmacodynamic and antitumor activity of AZD5363 in castration-naïve and castration-resistant prostate cancer. An additional GEM model, based on the concomitant inactivation of PTEN and Trp53 (P53), was established as an aggressive model of advanced prostate cancer and was used to further evaluate clinically relevant endpoints after treatment with AZD5363. In vivo pharmacodynamic studies demonstrated that AZD5363 effectively inhibited downstream targets of AKT. AZD5363 monotherapy significantly reduced growth of tumors in castration-naïve and castration-resistant models of PTEN-deficient prostate cancer. More importantly, AZD5363 significantly delayed tumor growth and improved overall survival and progression-free survival in PTEN/P53 double knockout mice. Our findings demonstrate that AZD5363 is effective against GEM models of PTEN-deficient prostate cancer and provide lines of evidence to support further investigation into the development of treatment strategies targeting AKT for the treatment of PTEN-deficient prostate cancer.

Abstract 4702: PD-L1 blockade in preclinical models ofPTEN-deficient prostate cancer

Immune checkpoint blockade with antibodies aimed at enhancing antitumor immunity have become an attractive therapeutic option. Establishing immunocompetent and clinically relevant models that predict treatment response is essential for the development of novel immunotherapies. To gain better insights into the preclinical evaluation of novel immunotherapeutic strategies, we used genetically engineered mouse models of PTEN-deficient prostate cancer. We first analyzed the transcriptome in castration-naive prostate tumors and the progression to castration-resistant disease. Comparative analyses were performed between age-matched normal prostate and PTEN-/- prostate tumor samples from castration-naive mice and, at 4 weeks (castration-sensitive) and 10 weeks (castration-resistant) post-surgical castration. Pathway and gene-set enrichment analysis indicated that abnormal tumor immunity was strongly associated with the progression to castration resistance. Chemokine signaling, B cell receptor and T cell receptor signaling pathways were among the top dysregulated pathways, and gene signatures of suppressed tumor immunity were enriched in castration-resistant tumors. Higher expression patterns of the programmed cell death protein 1(PD-1) and its ligand (PD-L1) were observed in castration-resistant tumors compared to castration-naive tumors. We also characterized immune expression profiles in more aggressive tumors from PTEN/P53 double knockout (DKO) mice. PD-L1 was strongly expressed cancer cells, but a higher presence of PD-1+, CD45+ and F4/80+ stromal infiltrating immune cells in was observed in castration-resistant tumors. PD-1/PD-L1 blockade with antibodies against mouse PD-L1 increased CD45+ tumor infiltrating lymphocytes (TILs) in an early model of PTEN/P53-deficient castration-resistant prostate cancer. PTEN/P53-DKO mice with advanced castration-naive prostate cancer tended to experienced longer overall survival after treatment with PD-1/PD-L1 blockade compared to vehicle and isotype controls, median survival 23, 14 and 11 days, respectively, P=0.458. A higher number of CD45+ TILs was observed in tumors of mice receiving the anti-PD-L1 treatment. Interim analysis of PD-1/PD-L1 blockade on a model of advanced PTEN/P53-deficient castration resistant prostate cancer (CRPC) has thus far shown that mice receiving treatment experience significantly longer survival (P=0.017, median survival not yet reached). Our findings show that castration-resistance promotes tumor immune suppression in mouse PTEN-deficient prostate cancer and suggest that this phenomenon can be reversed pharmacologically with anti-PD-L1 blockade. Thus, we provide preclinical evidence for immune checkpoint blockade as a potentially promising prostate cancer therapy. Citation Format: Marco A. De Velasco, Yurie Kura, Naomi Ando, Noriko Sato, Kazuko Sakai , Barry R. Davies, Koichi Sugimoto, Masahiro Nozawa, Kazuhiro Yoshimura, Kazuhiro Yoshikawa, Kazuto Nishio , Hirotsugu Uemura. PD-L1 blockade in preclinical models of PTEN-deficient prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4702. doi:10.1158/1538-7445.AM2017-4702

Conditional PTEN-deficient Mice as a Prostate Cancer Chemoprevention Model

BACKGROUND We generated a mouse model of prostate cancer based on the adult-prostate-specific inactivation of phosphatase and tensin homolog (PTEN) using the Cre-loxP system. The potential of our mice as a useful animal model was examined by evaluating the chemopreventive efficacy of the anti-androgen, chlormadinone acetate (CMA). MATERIALS AND METHODS Six-week-old mice were treated subcutaneously with 50 μg/g of CMA three times a week for 9 or 14 weeks and sacrificed at weeks 15 and 20. Macroscopic change of the entire genitourinary tract (GUT) and histologically evident prostate gland tumor development were evaluated. Proliferation and apoptosis status in the prostate were examined by immunohistochemistry. RESULTS CMA triggered significant shrinkage of not only the GUT but also prostate glands at 15 weeks compared to the control (p=0.017 and p=0.010, respectively), and the trend became more marked after a further five-weeks of treatment. The onset of prostate adenocarcinoma was not prevented but the proliferation of cancer cells was inhibited by CMA, which suggested the androgen axis is critical for cancer growth in these mice. CONCLUSIONS Conditional PTEN-deficient mice are useful as a preclinical model for chemoprevention studies and serve as a valuable tool for the future screening of potential chemopreventive agents.

Abstract 1226: Interleukin-6 (IL-6) as a therapeutic target in prostate cancer.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Interleukin-6 or IL-6 is a pleiotropic cytokine which is implicated in the regulation of immune response, but is also involved in regulating the growth of various malignant tumors. Mounting evidence suggests that IL-6 contributes to prostate cancer progression. Activated IL-6 has been shown to be elevated in the sera from patients with metastatic prostate cancer and it appears that the prostate tissue itself is a source of IL-6 and its receptor. Experimental data suggests that the IL-6/IL-6R signaling axis is an autocrine and paracrine growth factor in androgen independent prostate cancer cell lines and can activate a variety of signal transduction cascades, some of which may activate androgen receptor activity. In the present study we examined the role of IL6-induced Stat3 activation in the development and progression of prostate cancer and its possible role as a target for the treatment of prostate cancer using the prostate-specific conditional targeting PTEN knockout mouse model. In PTEN-mutant mice, elevated levels of IL6/IL-6R correlated with increased cellular proliferation in non-castrate and castrate prostate cancer (CRPC). Similar to human prostate cancer, mice with CPRC showed elevated levels of IL-6 and IL6-R in both serum and prostate tissue. To establish a therapeutic role for IL6/IL6R inhibition, we conducted a series of pharmacodynamic studies using an anti-mouse IL-6R antibody (MR-16) in tumor bearing homozygous PTEN-mutant mice. Our data shows that STAT3 activation was inhibited in a dose dependent manner during acute IL6-R blockade. Inhibition of STAT3 activity after chronic IL-6R blockade was transient. However, at day 45 of treatment, tumor cell proliferation was inhibited and apoptosis was induced. These findings show that this mouse model is relevant for the study of Stat3 signaling and tumor progression and inhibition of Stat3 transcriptional activation by upstream signal blockade may be a promising approach for molecularly targeted therapeutic strategies in human prostate cancer. Thus this study will serve as the foundation for future drug intervention studies designed to assess the efficacy of targeting the JAK/STAT signal pathway using targeted monotherapy and combination drug therapy models. Citation Format: Yurie Kura, Marco A. De Velasco, Yasuyuki Kobayashi, Naomi Ando, Emiko Fukushima, Yutaka Yamamoto, Yuji Hatanaka, Nobutaka Shimizu, Kazuhiro Yoshimura, Masahiro Nozawa, Kazuhiro Yoshikawa, Kazuto Nishio, Hirotsugu Uemura. Interleukin-6 (IL-6) as a therapeutic target in prostate cancer. [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 1226. doi:10.1158/1538-7445.AM2013-1226

Abstract 4439: Genetic alterations in specific RNA processing genes is associated with poor patient outcome in prostate cancer

Altered expression of splicing factors has been implicated in human cancers but their role in disease progression is complex due to the magnitude of potential protein products. We previously showed that androgen withdrawal led to increased alternatively spliced products in mouse Pten-deficient prostate tumors. Here, we perform gene expression analysis this mouse prostate cancer model to identify candidate mRNA processing genes implicated in the progression to castration-resistant disease. Genome-wide analysis revealed a set of 43 unique RNA processing and splicing factor genes associated with the progression to castration-resistant disease. Expression analysis of this gene set was examined in a pan-cancer analysis of 30 TCGA cancer studies covering 26 human cancer types. Alterations of this gene set (z-score threshold variances >2) were found in 70.5% (6978/9896) of cases. The median percentage of affected cases across the studies ranged from 28.2-98.2% with a median of 78.3%. In prostate cancer patients (TCGA-Prostate Adenocarcinoma (PRAD)), 65.7% (327/497) of the cases had mRNA alterations of this gene set. Twenty-seven of the 30 studies had survival data, of these only three studies indicated poor disease-free survival (DSF) outcomes with mRNA alterations: TCGA-PRAD, P=0.00616; TCGA-Brain Lower Grade Glioma (BLGG), P=0.0088; TCGA-Thymoma, P=0.0265. In the TCGA-PRAD study, the median time to DSF was 81.2 months in altered cases vs. median not reached in non-altered cases. In three studies, favorable outcomes were seen in cases with alterations in the gene set: TCGA-Uveal melanoma, TCGA- Glioblastoma Multiforme and TCGA-Lung Squamous Cell Carcinoma. TCGA-BLGG was the only study to show a statistically significant association between gene set alterations and overall survival (OS), P=0.0206. In TCGA-PRAD, median OS was 115 months in altered cases vs. median not reached in non-altered cases P=0.262. We further analyzed the scope of molecular alterations of this gene set in prostate cancer patients, overall, 78.9% (393/498) of the cases had gene alterations consisting of mutations, amplifications, deletions, and mRNA downregulation in six (1.2%), two (0.4%), 49 (9.8%), 193 (38.8%), and 143 (28.7%) cases, respectively. Notably a strong association was revealed for poor OS for cases with mutations/copy number alterations (P=0.00621). These data suggest that molecular alterations in specific RNA processing genes can cooperate to promote specific cancer types and further studies will be required to fully understand the prostate cancer-driving events in this gene set. Citation Format: Koichi Sugimoto, Marco A. De Velasco, Yurie Kura, Kazuko Sakai, Masahiro Nozawa, Kazuhiro Yoshimura, Kazuhiro Yoshikawa, Kazuto Nishio, Hirotsugu Uemura. Genetic alterations in specific RNA processing genes is associated with poor patient outcome in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4439.

Abstract 751: Characterization of STAT3 activation in human prostate cancer

Signal transducers and activators of transcription (STATs) were initially associated with cytokine signal transduction pathways but are now also recognized as key modulators of key survival processes in in various cancers. Activation of STAT3 occurs by the binding of various cytokines to its receptors leading to the activation of the JAK/ STAT3 signaling pathway. Interleukin-6 (IL-6) has been implicated in regulating growth of various malignant tumors. Activated IL-6 has also been shown to be elevated in the sera from patients with metastatic prostate cancer, and persistent activation of STAT3 is a common feature. To better characterize the potential role of JAK/STAT3 as a therapeutic target for advanced prostate cancer, we examined expression patterns of activated STAT3 in 111 cases of localized prostate cancer from patients who underwent radical prostatectomy. Association studies were conducted with clinicopathological characteristics and biochemical recurrence. We also analyzed the expression of IL-6/KAK/STAT3 genes from RNA-sequencing data form the Cancer Genome Atlas Prostate Adenocarcinoma (TCGA-PRAD) data set and its association to long-term outcomes. Immunohistochemical analysis revealed that 11.7%, 42.3% and 45.9% of patients had high, moderate or low/negative expression of phosphorylated-STAT3 (Tyr705). Cancerous glands tended to have higher STAT3 expression compared to paired normal adjacent tissue, P=0.088. No correlations were observed between STAT3 expression and clinicopathological parameters. Patients with moderate-high p-STAT3 expression tended to experience shorter times to biochemical recurrence (median time, 15 months) compared to patients with low/negative STAT3 activity, (median time to recurrence not reached, P=0.064). Cluster analysis suggested a trend for decreased disease-free survival for patients with high IL-6/KAK/STAT3 gene expression signature. Our data suggests an association between STAT3 signaling and prostate cancer recurrence and provides clinical evidence to support JAK/STAT3 as a potential therapeutic target for patients with advanced prostate cancer. Citation Format: Marco A. De Velasco, Yuji Hatanaka, Yurie Kura, Naomi Ando, Kazuko Sakai, Koichi Sugimoto, Masahiro Nozawa, Kazuhiro Yoshimura, Kazuhiro Yoshikawa, Kazuto Nishio, Hirotsugu Uemura. Characterization of STAT3 activation in human prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 751. doi:10.1158/1538-7445.AM2017-751

Abstract 1582: Therapeutic potential of combination therapy using a next generation antisense oligonucleotide targeting the androgen receptor and AKT inhibition with AZD5363 in genetically engineered mouse models of prostate cancer

Prostate cancer is highly dependent on androgen receptor (AR) and PI3K/AKT signaling pathways for survival and disease progression. Preclinical evidence suggests that combinatorial approaches targeting both AR and PI3K/AKT activity improves treatment efficacy. However, sustained responses from traditional and next-generation anti-androgen therapies targeting AR remain elusive in clinical practice due to inherent/acquired resistance resulting in lethal castration-resistant prostate cancer (CRPC). Mechanisms for continued AR transcriptional activity may be ligand dependent or independent but still require AR gene expression. Persistent AR gene expression is a key feature of CRPC. Thus, blocking AR gene expression by antisense oligonucleotides (ASO) is a logical approach to CRPC. We previously showed that monotherapy with ISIS581088, a generation 2.5 ASO targeting mouse AR, demonstrated strong antitumor activity in a transgenic mouse model of PTEN-deficient prostate cancer. In this study we show the antitumor effects of combined therapy of ISIS581088 and AZD5363, a potent AKT inhibitor and demonstrate the therapeutic benefit of combination therapy in a clinically relevant mouse model of CRPC. Sixteen-week-old mice with PTEN-/- castration-naive prostate tumors were treated with ISIS581088 and AZD5363 alone or in combination for four weeks. Tumor growth inhibition rates were 41.2%, 20.2% and 54.4% for ISIS581088, AZD5363 and ISIS581088/AZD5363 treatment groups, respectively, P Citation Format: Marco A. De Velasco, Yurie Kura, Naomi Ando, Koichi Sugimoto, Kazuko Sakai, Barry R. Davies, Youngsoo Kim, A. Robert MacLeod, Masahiro Nozawa, Kazuhiro Yoshimura, Kazuhiro Yoshikawa, Kazuto Nishio , Hirotsugu Uemura. Therapeutic potential of combination therapy using a next generation antisense oligonucleotide targeting the androgen receptor and AKT inhibition with AZD5363 in genetically engineered mouse models of prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1582. doi:10.1158/1538-7445.AM2017-1582

Abstract 1096: Co-targeting of AKT and Pim kinases in mousePTEN-deficient prostate cancer

AKT and Pim kinases modulate programmed cell death by the phosphorylation of common substrates that regulate apoptosis and other survival processes. Evidence suggests that the antitumor effects of targeted Akt inhibition can be inhibited or diminished by the JAK/STAT-dependent induction of Pim kinases. In this study we examined the therapeutic potential of co-targeting AKT and Pim in a genetically engineered mouse model of prostate cancer driven by the conditional inactivation of PTEN. The antitumor effects of AZD5363, a pan AKT inhibitor, and AZD1208, a highly potent Pim kinase inhibitor, were investigated as monotherapy or in combination on mice harboring castration-naive prostate tumors and mice that developed castration-resistant disease. Mice were randomized treated for four weeks. Safety and tolerability was assessed by bodyweight changes. Antitumor activity was determined by differences in tumor burden, proliferation and apoptosis and histology. Molecular activity was assessed by examining the phosphorylation of common substrates by western blot analysis. Treatments were well-tolerated and no significant differences in bodyweight changes were observed. In castration-naive prostate tumors, treatments with AZD5363, AZD1208 and AZD5363/AZD1208 resulted in 11.9%, 13.5% and 36.9% reductions of tumor burden compared to vehicle treated controls, respectively, P Citation Format: Marco A. De Velasco, Koichi Sugimoto, Yurie Kura, Naomi Ando, Noriko Sato, Kazuko Sakai, Barry R. Davies, Dennis Huszar, Masahiro Nozawa, Kazuhiro Yoshimura, Kazuhiro Yoshikawa, Kazuto Nishio, Hirotsugu Uemura. Co-targeting of AKT and Pim kinases in mouse PTEN-deficient prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1096. doi:10.1158/1538-7445.AM2017-1096

Abstract 4315: Effects of increased dietary fat consumption on prostate cancer progression in genetically engineered mice

Prostate cancer is a highly heterogeneous disease and evidence indicates that lifestyle and environmental factors can contribute to cancer-related deaths. Accumulating data also suggests that increased dietary fat consumption may promote and increase the risk of prostate cancer-related morbidity and mortality. However, data between studies is inconsistent and a direct link between increased consumption of dietary fat and the promotion of prostate cancer remains unclear. Here, we present our findings from a preclinical study of increased dietary fat consumption and its effects on prostate tumor growth and progression using genetically engineered mouse models of prostate cancer. We determined the effects of a high fat diet on tumor cell proliferation and apoptosis in vivo using a well-established mouse model of prostate cancer. Twenty-week-old mice, with conditional biallelic deletion of PTEN, harboring castration-naive or castration-resistant prostate tumors were randomized and assigned to a standard laboratory diet (4% fat) or a high-fat diet (HFD, 13.6% fat) for 8 weeks. In both tumor models, mice fed a HFD had increased expression levels of proteins associated with proliferation and decreased levels of apoptosis markers. To determine the long term effects of a HFD, we used a mouse model of advanced prostate cancer. Mice with concomitant inactivation of PTEN and p53 develop tumors that progress quickly and are characterized with increased metastatic development and decreased survival compared to PTEN knockout mice. PTEN/p53 double knockout mice were randomized and assigned to control or HFD experimental groups after weaning. Our findings revealed that mice fed a HFD resulted in obesity that led to decreased progression-free survival and overall survival rates compared to mice fed a standard diet. Our results provide evidence to support the effects of high dietary fat consumption and increased prostate cancer risk, and provide a platform to investigate chemoprevention strategies. Citation Format: Marco A. De Velasco, Yurie Kura, Yuji Hatanaka, Takashi Oki, Yutaka Yamamoto, Koichi Sugimoto, Yasunori Mori, Kazuhiro Yoshimura, Masahiro Nozawa, Kazuhiro Yoshikawa, Kazuto Nishio, Hirotsugu Uemura. Effects of increased dietary fat consumption on prostate cancer progression in genetically engineered mice. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4315.

Abstract 954: Analysis of noncoding RNA expression in a mouse model of PTEN-deficient prostate cancer

Prostate cancer is a heterogeneous disease that is driven by combined genetic and epigenetic alterations. A significant portion of the mammalian genome consists of non-coding regions of RNA. Increasing evidence has shown that these noncoding RNAs (ncRNAs) have significant roles in the epigenetic regulation of several cellular processes, and their dysregulation can contribute to tumorigenesis and promote disease progression in many cancer types. In order to gain better insights into the potential roles of ncRNAs in prostate cancer, we used a genetically engineered mouse model of prostate cancer to perform a comparative analysis of the cancer transcriptome and the landscape of ncRNAs in castration-naive prostate tumors and the progression to castration-resistant disease. Whole transcription arrays were used to explore both messenger (mRNA) and long intergenic non-coding RNA (LincRNA) transcript expression in normal prostate tissue from wildtype mice and prostate tumors from conditional PTEN-knockout mice. Comparative analyses were performed between prostate tumor samples from castration-naive mice and, at 4 weeks and 10 weeks post-surgical castration. Altered expression of ncRNAs was most commonly observed and accounted for 56.2% (2370/4216), 47.6% (4460/9366) and 41.57% (1545/3717) of the total genes differentially expressed between normal mouse prostate and prostate tumors from castration-naive, 4 weeks post-castration and 10 weeks post-castration, respectively. We also profile the expression of relevant cancer-related lincRNAs present in these models. Overall, our analyses provide data to support a role of LincRNA dysregulation in the pathogenesis of PTEN-deficient prostate cancer, and suggest that these mouse models might provide a potential preclinical tool to test candidate prospective therapies targeting ncRNAs. Citation Format: Marco A. De Velasco, Yurie Kura, Kazuko Sakai, Yuji Hatanaka, Yoshihiko Fujita, Yosuke Togashi, Masato Terashima, Kazuhiro Yoshikawa, Kazuto Nishio, Hirotsugu Uemura. Analysis of noncoding RNA expression in a mouse model of PTEN-deficient prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 954.