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Purpose: Recent molecular analyses of bladder cancer open the door to significant advances in targeted therapies. NOTCH has been identified as a tumor suppressor in bladder cancer, but prior reports have focused on NOTCH1. Here we hypothesized that NOTCH2 is an oncogene suitable for therapeutic targeting in bladder cancer.Experimental design: We studied genomic aberrations of NOTCH, compared survival and tumor progression according to NOTCH2 expression levels, and studied NOTCH2 function in vitro and vivo.Results: We report a high rate of NOTCH2 copy number gain in bladder cancer. High NOTCH2 expression was identified especially in the basal subtype and in mesenchymal tumors. NOTCH2 activation correlated with adverse disease parameters and worse prognosis by immunohistochemistry. Forced overexpression of the intracellular domain of NOTCH2 (N2ICD) induced cell growth and invasion by cell-cycle progression, maintenance of stemness and epithelial-to-mesenchymal transition (EMT). These effects were abrogated by silencing of CSL, indicating that the effects were mediated through the canonical NOTCH signaling pathway. In an orthotopic xenograft model, forced overexpression of N2ICD increased growth, invasion, and metastasis. To explore the potential for therapeutic targeting of NOTCH2, we first silenced the receptor with shRNA and subsequently treated with a specific inhibitory antibody. Both interventions decreased cell growth, invasion, and metastasis in vitro and in the orthotopic xenograft model.Conclusions: We have demonstrated that NOTCH2 acts as an oncogene that promotes bladder cancer growth and metastasis through EMT, cell-cycle progression, and maintenance of stemness. Inhibition of NOTCH2 is a rational novel treatment strategy for invasive bladder cancer. Clin Cancer Res; 22(12); 2981–92. ©2016 AACR.

NOTCH2-inactivating antibody inhibited tumor progression in vitro and in vivo.

NOTCH2 silencing inhibited tumor progression through decreased cell proliferation and invasion.

NOTCH2-induced cell invasion was blocked by downstream inhibition of canonical NOTCH2 signaling.

NOTCH2 overexpression promoted bladder cancer progression through increased proliferation and invasion.

The lists of antibodies, primers and patients characteristics and detailed information of methods were described in Supplementary Material and Methods.

Purpose: Recent molecular analyses of bladder cancer open the door to significant advances in targeted therapies. NOTCH has been identified as a tumor suppressor in bladder cancer, but prior reports have focused on NOTCH1. Here we hypothesized that NOTCH2 is an oncogene suitable for therapeutic targeting in bladder cancer. Experimental design: We studied genomic aberrations of NOTCH, compared survival and tumor progression according to NOTCH2 expression levels, and studied NOTCH2 function in vitro and vivo. Results: We report a high rate of NOTCH2 copy number gain in bladder cancer. High NOTCH2 expression was identified especially in the basal subtype and in mesenchymal tumors. NOTCH2 activation correlated with adverse disease parameters and worse prognosis by immunohistochemistry. Forced overexpression of the intracellular domain of NOTCH2 (N2ICD) induced cell growth and invasion by cell-cycle progression, maintenance of stemness and epithelial-to-mesenchymal transition (EMT). These effects were abrogated by silencing of CSL, indicating that the effects were mediated through the canonical NOTCH signaling pathway. In an orthotopic xenograft model, forced overexpression of N2ICD increased growth, invasion, and metastasis. To explore the potential for therapeutic targeting of NOTCH2, we first silenced the receptor with shRNA and subsequently treated with a specific inhibitory antibody. Both interventions decreased cell growth, invasion, and metastasis in vitro and in the orthotopic xenograft model. Conclusions: We have demonstrated that NOTCH2 acts as an oncogene that promotes bladder cancer growth and metastasis through EMT, cell-cycle progression, and maintenance of stemness. Inhibition of NOTCH2 is a rational novel treatment strategy for invasive bladder cancer. Clin Cancer Res; 22(12); 2981–92. ©2016 AACR.

Neuroendocrine (NE) prostate cancer (PCa) is a highly aggressive subtype of prostate cancer associated with resistance to androgen ablation therapy. In this study, we used LNCaP prostate cancer cells cultured in a serum-free medium for 6 days as a NE model of prostate cancer. Serum deprivation increased the expression of NE markers such as neuron-specific enolase (NSE) and βIII tubulin (βIII tub) and decreased the expression of the androgen receptor protein in LNCaP cells. Using cDNA microarrays, we compared gene expression profiles of NE cells and non-differentiated LNCaP cells. We identified up-regulation of 155 genes, among them LAMP2, a lysosomal membrane protein involved in lysosomal stability and autophagy. We then confirmed up-regulation of LAMP2 in NE cells by qRT-PCR, Western blot and confocal microscopy assays, showing that mRNA up-regulation correlated with increased levels of LAMP2 protein. Subsequently, we determined autophagy activity in NE cells by assessing the protein levels of SQSTM/p62 and LC3 by Western blot and LC3 and Atg5 mRNAs content by qRT-PCR. The decreased levels of SQSTM/p62 was accompanied by an enhanced expression of LC3 and ATG5, suggesting activation of autophagy in NE cells. Blockage of autophagy with 1μM AKT inhibitor IV, or by silencing Beclin 1 and Atg5, prevented NE cell differentiation, as revealed by decreased levels of the NE markers. In addition, AKT inhibitor IV as well as Beclin1 and Atg5 kwockdown attenuated LAMP2 expression in NE cells. On the other hand, LAMP2 knockdown by siRNA led to a marked blockage of autophagy, prevention of NE differentiation and decrease of cell survival. Taken together, these results suggest that LAMP2 overexpression assists NE differentiation of LNCaP cells induced by serum deprivation and facilitates autophagy activity in order to attain the NE phenotype and cell survival. LAMP2 could thus be a potential biomarker and potential target for NE prostate cancer.

migratoryand invasiveproperties tobecomemesenchymal stemcells.EMT has been implicated in the initiation of metastasis for cancer progression. METHODS: Exosomes were isolated by ultracentrifugation from T24 or UMUC3 invasive bladder cancer cell conditioned media. T24 exosomes were added to urothelial cells for 4, 6, 24 or 48 hours. RNA or protein was collected from the urothelial cells and qRT-PCR or western blotting was performed to measure expression of EMT markers. T24or UMUC3-derived exosomes were added to urothelial cells and plated in transwell inserts for migration and invasion assays. RESULTS: Urothelial cells treated with T24 or UMUC3 bladder cancer exosomes showed an increased expression in several mesenchymal markers, including a-SMA, S100A4, and Snail, as compared to PBS treated cells. Moreover, treatment of urothelial cells with T24or UMUC3-derived exosomes resulted in decreased expression of epithelial markers, including e-cadherin and b-catenin, as compared to the control, PBS treated cells. T24and UMUC3-derived exosomes also increased the migration and invasion of urothelial cells, and this was blocked by heparin pre-treatment. We further showed that exosomes isolated from patient urine and barbotage samples were able to induce the expression of several mesenchymal markers in recipient urothelial cells. CONCLUSIONS: In thisstudy,weestablished thatexosomes from invasive bladder cancer cells are able to induce EMT in recipient urothelial cells. We further showed that exosomes from invasive bladder cancer cells can promote migration and invasion of recipient urothelial cells. We also demonstrated that the effect on migration and invasion is mediated by heparin. Finally, we established that exosomes isolated from bladder cancer patients hada similar effect on the expression ofmesenchymalmarkers in urothelial cells as the exosomes isolated from bladder cancer cell lines.

Loss of tumour suppressor proteins, such as the retinoblastoma protein (Rb), results in tumour progression and metastasis. Metastasis is facilitated by low oxygen availability within the tumour that is detected by hypoxia inducible factors (HIFs). The HIF1 complex, HIF1α and its dimerization partner the aryl hydrocarbon receptor nuclear translocator (ARNT), is the master regulator of the hypoxic response. Previously, we demonstrated that Rb represses the transcriptional response to hypoxia by virtue of its association with HIF1. In this report, we further characterized the role of Rb in HIF1-regulated genetic programs by stably ablating Rb expression with retrovirally-introduced short hairpin RNA in LNCaP and 22rV1 human prostate cancer cells. DNA microarray analysis revealed that Rb regulates specific chromosomal gene clusters and loss of Rb in conjunction with hypoxia leads to dysregulation of HIF1-regulated genetic programs that promote cell invasion and neuroendocrine differentiation. Gene ontology analysis of the hypoxia-inducible genes sensitive to loss of Rb revealed that a significant portion of these genes are involved in neuroendocrine differentiation (NED), specifically ENO2, KISS1R and HTR5A. ENO2 is the bonafide marker of neuroendocrine differentiation and it's presence is a signature of late stage castrate resistant prostate cancer. Furthermore, we have functional evidence KISS1R is linked to intracellular calcium mobilization in 22RV1 cells. We have demonstrated that increased expression of HIF-regulated genes in response to loss of Rb activates Akt and ERK signaling pathways and promotes neuroendocrine differentiation and invasion. Inhibition of these signaling pathways significantly decreased actin polymerization in LNCaP cells. For the first time, we have established a direct link between hypoxic tumour environments, Rb inactivation and progression to late stage metastatic neuroendocrine prostate cancer. Understanding the molecular pathways responsible for progression of benign prostate tumours to metastasized and lethal forms will aid in the development of more effective prostate cancer therapies. Citation Format: Mark Labrecque, Mandeep Takhar, Rebecca J. Nason, Stephanie Santacruz, Kevin Tam, Shabnam Massah, Anne Haegert, Robert Bell, Manuel Altamirano-Dimas, Colin Collins, Frank Lee, Gratien Prefontaine, Michael Cox, Timothy Beischlag. Loss of retinoblastoma protein dysregulates HIF1-mediated genetic programs, and promotes tumor cell invasiveness and neuroendocrine differentiation in prostate cancer cells. [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 2922.

The retinoblastoma protein (Rb) is capable of attenuating the hypoxic response in tumor cells. This process is mediated by the hypoxia inducible factor 1α/2α (HIF1α/2α) and its dimerization partner the aryl hydrocarbon receptor nuclear translocator (ARNT/ HIF1β). Rb modulates HIF activity by virtue of its association with the thyroid hormone receptor/retinoblastoma interacting protein 230 (TRIP230), an essential cofactor of the HIF1α/ARNT transcriptional complex. We used short hairpin RNA (shRNA) technology and microarray analysis to interrogate the Rb-negative and wild-type MCF7 cell transcriptomes and generated lists of genes that were either up- or downregulated in response to both loss of Rb and hypoxia. We found that loss of Rb enhances the expression of hypoxia-regulated genes involved in invasion and epithelial-to-mesenchymal transition and significantly decreases the expression of genes involved in cell anchoring and differentiation in MCF7 and MDA-MB-231 breast cancer cells. Genes were validated using both qRT-PCR and immuno-blot analysis. Additionally, gene ontology analysis revealed that AKT and ERK1/2 are downstream effectors of hypoxic gene programs that are sensitive to loss of Rb. Furthermore, these factors regulate the acquisition of a more invasive phenotype in breast cancer cells. Finally, we found that Rb knockdown in combination with pre-treatment of cells with hypoxia increased growth of tumor foci in the lungs after i.v. injection and increased the development of spontaneous metastases from orthotopically implanted breast tumor cells in female NOD-SCID mice. Primary tumors lacking Rb demonstrated enriched protein levels of genes identified in our arrays when compared to negative control tumors. These results show that Rb is a negative modulator of hypoxia-regulated genetic programs by virtue of its direct effects on the HIF-complex. Understanding the HIF complex and the molecular mechanisms controlling the progression from benign tumors to metastasized and lethal forms will allow us to develop more specific breast cancer therapies. Citation Format: Mandeep K. Takhar, Mark P. Labrecque, Kevin J. Tam, Anne Haegert, Robert H. Bell, Manuel Altamirano-Dimas, Colin C. Collins, Gratien G. Prefontaine, Michael E. Cox, Kevin L. Bennewith, Timothy V. Beischlag. The retinoblastoma protein regulates hypoxia-inducible factor-1α-mediated transcriptional programs, tumor cell invasiveness, tumor growth and metastasis in human breast cancer cells. [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 2921.