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Background: Prostate epithelium is exquisitely sensitive to the overexpression of the proto-oncogene MYC which causes neoplastic transformation. Indeed, MYC protein is almost universally overexpressed in metastatic castration-resistant prostate cancer (CRPC) making targeting MYC an attractive option for treating advanced stage disease. Unfortunately, the development of therapeutic agents directly targeting MYC has been largely unsuccessful, thus emphasizing the need to indirectly target MYC activity through inhibition of downstream cellular processes it regulates. One of the main effects of MYC in cancer cells is to accelerate proliferative growth via stimulation of high levels of ribosome biogenesis. Accordingly, the control of protein synthesis rate has emerged as the “Achilles' heel” of a wide array of tumors. MYC also regulates and cooperates with PIM kinases to increase the activity of the eIF4F translation initiation complex and MYC-driven tumors are addicted to eIF4E. Here, we investigate the efficacy of a single and dual approach targeting ribosome biogenesis and function to treat prostate cancer (PC). Experimental design: We employed numerous models of PC, including a novel CRPC patient derived xenograft system, which showed the pre-clinical efficacy of therapies that combine to target MYC directed signaling to the ribosome. The inhibition of ribosomal RNA (rRNA) synthesis with CX-5461, a potent, selective and orally bioavailable inhibitor of RNA polymerase I (Pol I) transcription has been successfully exploited therapeutically, but only in models of hematological malignancy. CX-5461 and CX-6258, a pan-PIM kinase inhibitor, were tested alone and in combination in PC cell lines, in Hi-MYC and PTEN-deficient mouse models and in patient derived xenografts (PDX) of metastatic tissue obtained from a castration-resistant PC patient. Results: CX-5461 inhibited anchorage-independent growth and induced cell cycle arrest in PC cell lines at nanomolar concentrations. Oral administration of 50 mg/kg CX-5461 induced p53 expression and activity and reduced proliferation (Ki-67) and invasion (loss of ductal actin) in Hi-MYC tumors, but not in PTEN null (low MYC) tumors. While 100 mg/kg CX-6258 showed limited effect alone, its combination with CX-5461 further suppressed proliferation and dramatically reduced large invasive lesions in both models. This rational combination strategy significantly inhibited proliferation and induced cell death in PDX of PC. Conclusion: Our results demonstrate preclinical efficacy of targeting the ribosome at multiple levels and provide a new approach for the treatment of PC. In addition, a key conclusion of our study is that the androgen receptor (AR) presence or activity has no significant impact on the therapeutic activity of our novel combination therapy. Therefore, we believe our new exciting combination therapy could be used in the clinic in combination with current anti-androgens or as salvage therapy in multi-drug resistant CRPC. Citation Format: Richard J. Rebello, Eric Kusnadi, Don P. Cameron, Helen B. Pearson, Analia Lesmana, Jennifer R. Devlin, Denis Drygin, Ashlee K. Clark, Laura Porter, John Pedersen, Shahneen Sandhu, Gail P. Risbridger, Richard B. Pearson, Ross D. Hannan, Luc Furic. Inhibition of ribosomal RNA synthesis as a new therapeutic approach to treat advanced prostate cancer. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr B23.

Aberrant Ras and Wnt signaling are emerging as key events in the multistep nature of prostate tumorigenesis and progression. Here, we report the generation of a compound model of prostate cancer to define the synergism of activated K-ras (K-ras+/V12) and dominant stabilized β-catenin (Catnb+/lox(ex3)) in the murine prostate. Recombination of floxed alleles and subsequent expression of oncogenic transgenes was mediated by Cre recombinase expression governed by the composite Probasin (PB) promoter (termed PBCre). Concomitant with elevated mitogen-activated protein kinase (MAPK) signaling, PBCre+K-ras+/V12 mice developed AH at 100 days (100% incidence) and low-grade prostate intraepithelial neoplasia and adenocarcinoma (60% and 7% incidence) by 500 days. PBCre+Catnb+/lox(ex3) mice showed reduced longevity (average 428 days) and were predisposed to PIN-like keratinized squamous metaplasia at 100 days (100% incidence) and adenocarcinoma (100% incidence) at end-point. These lesions displayed elevated Wnt signaling and basal levels of MAPK signaling. Synchronous activation of K-ras and β-catenin significantly reduced survival (average 189 days), reflecting accelerated tumorigenesis and the development of invasive carcinoma that displayed activated Wnt and MAPK signaling. Notably, expression of the basal cell marker p63 negatively correlated with tumor grade, resembling human prostate adenocarcinoma. Taken together, our data show that combinatorial oncogenic mutations of K-ras and β-catenin drive rapid progression of prostate tumorigenesis to invasive carcinoma, characterized by the synergistic elevation of androgen receptor, cyclooxygenase-2, and c-Myc. [Cancer Res 2009;69(1):94–101]

Prostate cancer, a hormone-dependent disease of aging men, occurs as the relative levels of estrogen compared to testosterone are increasing. Indeed, high doses of estrogen, in combination with androgens, can initiate prostate cancer. This effect is attributed to the activity of the estrogen receptor α (ERα), but a paucity of suitable models means that the precise role of ERα in prostate cancer is still poorly understood. In this study we observed increased ERα expression in three different models of prostate cancer, PTEN null mice, Hi-Myc mice, and high grade human tumor specimens. Within the PTEN null prostate, there was a consistent pattern of ERα expression: low in benign glands, moderate in tumors within the dorsal, lateral and ventral lobes, and high in tumors within the anterior prostate. This pattern significantly correlated with the levels of the proliferative marker Ki67. There was also a significant correlation between ERα and Ki67 within individual malignant glands in the anterior prostate. Furthermore, we demonstrated that ERα sustained the in vitro proliferation of cells derived from a PTEN null tumor in 2D and 3D assays. There was a significant decrease in proliferation in cells treated with TPSF, a non-competitive ERα antagonist, or with ERα-specific shRNA. Loss of ERα caused a significant decrease in the levels of MYC and other ERα target genes. It also reduced the activity of both the PI3K and MAPK pathways as measured by decreased levels of phosphorylated erk1/2, S6 kinase and other downstream factors. This effect was reversed in rescue experiments with expression constructs for both full length ERα, capable of genomic and non-genomic actions, and membrane-only ERα, only able to trigger rapid non-genomic signalling. Collectively, these results demonstrate that ERα levels increase in prostate cancer, which promotes proliferation through classical genomic and rapid non-genomic signalling. Citation Format: Itsuhiro Takizawa, Mitchell Lawrence, Helen Pearson, John Pedersen, Normand Pouliot, Australian Prostate Cancer BioResource, Patrick Humbert, Luc Furic, Gail Risbridger. Estrogen receptor alpha drives proliferation of prostate cancer through PI3K and MAPK signaling. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2106. doi:10.1158/1538-7445.AM2014-2106

In recent years, loss of cell polarity and tissue architecture has been defined as a hallmark of epithelial cancer yet the molecular mechanisms underlying this predisposition are not fully understood. Two distinct polarity programs are required for the correct assembly and function of all mammalian epithelial tissues; apical-basal polarity and non-canonical Wnt/planar cell polarity (1). The Scribble complex functions to establish and maintain both aspects of epithelial polarity and has been shown to mediate cell adhesion, migration and asymmetric cell division (2). In human colorectal, breast, and cervical cancers, expression of the Scribble complex member SCRIB is often mislocalised and deregulated, suggesting that polarity regulators plays a role in maintaining homeostasis of epithelial tissues (1,3). Scrib null mice are neonatal lethal owing to a neural tube closure defect. To determine the tumor suppressive role of Scrib within epithelial tissues, we have employed tissue specific Cre-LoxP technology to deplete Scrib specifically within the prostate, mammary or lung. Scrib depletion in these epithelial tissues results in tumor initiation, supporting previous work in Drosophila (1). We and others have previously shown that SCRIB loss stimulates Ras/MAPK signaling in vitro, suggesting that SCRIB negatively regulates the Ras/MAPK cascade to suppress tumorigenesis (4,5). We now show elevated p-ERK1/2 expression is a common feature of Scrib deficient tumors in vivo. Accordingly, administration of the MEK inhibitor PD0325901 in Scrib+/− mice partially rescued the hyperplastic prostate phenotype. Together, these studies establish that depletion of the polarity regulator Scrib can initiate murine tumorigenesis in several epithelial tissues in a process involving deregulation of the Ras/MAPK cascade. Interestingly, tissue specific activation of oncogenic K-ras in combination with Scrib depletion accelerated tumor progression in both the prostate and lung. Thus, K-ras activation and Scrib deficiency synergise to facilitate tumor growth, demonstrating that Scrib depletion can contribute to tumor progression in the presence of an additional oncogenic mutation. The clinical relevance of this research is highlighted by our observation that SCRIB deregulation strongly correlated with PSA recurrence free survival in human prostate cancer (3). Together, our findings suggest that the polarity network may hold prognostic value or provide a novel route for therapeutic intervention. (1) Humbert PO et al (2008) Oncogene 27(55):6888-6907 (2) Kallay LM et al (2006) J Cell Biochem 99: 647-64 (3) Pearson HB et al (2011) J Clin Invest 121(11):4257-4267 (4) Dow LE et al (2008) Oncogene 27(46):5988-6001 (5) Nagasaka K et al (2010) Oncogene 29(38):5311-5321 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3291. doi:1538-7445.AM2012-3291

A subset of patients with gastric cancer have mutations in genes that participate in or regulate Wnt signaling at the level of ligand (Wnt) receptor (Fzd) binding. Moreover, increased Fzd expression is associated with poor clinical outcome. Despite these findings, there are no in vivo studies investigating the potential of targeting Wnt receptors for treating gastric cancer, and the specific Wnt receptor transmitting oncogenic Wnt signaling in gastric cancer is unknown. Here, we use inhibitors of Wnt/Fzd (OMP-18R5/vantictumab) and conditional gene deletion to test the therapeutic potential of targeting Wnt signaling in preclinical models of intestinal-type gastric cancer and ex vivo organoid cultures. Pharmacologic targeting of Fzd inhibited the growth of gastric adenomas in vivo. We identified Fzd7 to be the predominant Wnt receptor responsible for transmitting Wnt signaling in human gastric cancer cells and mouse models of gastric cancer, whereby Fzd7-deficient cells were retained in gastric adenomas but were unable to respond to Wnt signals and consequently failed to proliferate. Genetic deletion of Fzd7 or treatment with vantictumab was sufficient to inhibit the growth of gastric adenomas with or without mutations to Apc. Vantictumab is currently in phase Ib clinical trials for advanced pancreatic, lung, and breast cancer. Our data extend the scope of patients that may benefit from this therapeutic approach as we demonstrate that this drug will be effective in treating patients with gastric cancer regardless of APC mutation status. Significance: The Wnt receptor Fzd7 plays an essential role in gastric tumorigenesis irrespective of Apc mutation status, therefore targeting Wnt/Fzd7 may be of therapeutic benefit to patients with gastric cancer.