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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.

Preparations of Echinacea (Asteraceae) are frequently consumed for the control and prevention of rhinovirus-induced colds and other respiratory disorders. Since it is now generally believed that the symptoms of rhinovirus colds are due to the enhanced secretion of inflammatory chemokines and cytokines, we decided to analyze the effects of rhinovirus infection and Echinacea treatment [defined extracts of E. purpurea (L.) Moench] on cytokine/chemokine gene expression and protein secretion in a line of human tracheo- bronchial epithelial cells. Among the collection of more than 50 cytokines and chemokines present in the gene arrays, 12 showed significant induction of expression by the virus (> 2-fold), some of them by more than 5-fold. However, not all of these resulted in similar changes in the corresponding proteins, presumably as a consequence of post-transcriptional changes. A total of 16 cytokines, mostly chemokines, showed substantial protein increases, including several, such as the well known pro-inf...

Echinacea extracts have traditionally been used in the treatment of many infectious and other diseases (such as rhinovirus colds), and research has revealed the presence of various bioactivities in these extracts, particularly those connected with immune responses. We examined the effects of Echinacea by using gene expression analysis in a line of human bronchial epithelial cells, with or without rhinovirus infection. More than 13 000 human genes were evaluated. From these analyses we focused primarily on immune response genes and found that both Echinacea extracts, one predominantly rich in polysaccharides and the other rich in alkylamides and caffeic acid derivatives, stimulated the expression of numerous genes. These included a number of cytokines and chemokines, although the pattern of stimulation was different. In addition, Echinacea extracts tended to neutralize the effects of the rhinovirus. When the immune response gene pathways were analyzed with the Ingenuity Pathway program, it became apparent that many of them were interconnected through a major node, the transcription factor C/EBPβ (CAAT/enhancer-binding protein β) and its related C/EBP proteins. This suggests that Echinacea can bring about important biological responses in cells by virtue of interactions between components of the extract and a small number of intracellular factors involved in multiple signaling pathways.

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.

Approximately half of prostate cancers (PCa) carry TMPRSS2-ERG translocations; however, the clinical impact of this genomic alteration remains enigmatic. Expression of v-ets erythroblastosis virus E26 oncogene like (avian) gene (ERG) promotes prostatic epithelial dysplasia in transgenic mice and acquisition of epithelial-to-mesenchymal transition (EMT) characteristics in human prostatic epithelial cells (PrECs). To explore whether ERG-induced EMT in PrECs was associated with therapeutically targetable transformation characteristics, we established stable populations of BPH-1, PNT1B and RWPE-1 immortalized human PrEC lines that constitutively express flag-tagged ERG3 (fERG). All fERG-expressing populations exhibited characteristics of in vitro and in vivo transformation. Microarray analysis revealed >2000 commonly dysregulated genes in the fERG-PrEC lines. Functional analysis revealed evidence that fERG cells underwent EMT and acquired invasive characteristics. The fERG-induced EMT transcript signature was exemplified by suppressed expression of E-cadherin and keratins 5, 8, 14 and 18; elevated expression of N-cadherin, N-cadherin 2 and vimentin, and of the EMT transcriptional regulators Snail, Zeb1 and Zeb2, and lymphoid enhancer-binding factor-1 (LEF-1). In BPH-1 and RWPE-1-fERG cells, fERG expression is correlated with increased expression of integrin-linked kinase (ILK) and its downstream effectors Snail and LEF-1. Interfering RNA suppression of ERG decreased expression of ILK, Snail and LEF-1, whereas small interfering RNA suppression of ILK did not alter fERG expression. Interfering RNA suppression of ERG or ILK impaired fERG-PrEC Matrigel invasion. Treating fERG-BPH-1 cells with the small molecule ILK inhibitor, QLT-0267, resulted in dose-dependent suppression of Snail and LEF-1 expression, Matrigel invasion and reversion of anchorage-independent growth. These results suggest that ILK is a therapeutically targetable mediator of ERG-induced EMT and transformation in PCa.

315 Background: Notch is a family of cell surface receptors that has been demonstrated in several malignancies to regulate differentiation, proliferation, and metastasis. We have investigated the role of Notch signaling in bladder cancer and report here effects of modulating Notch2 in preclinical models of bladder cancer. Methods: Notch2 staining was examined in cystectomy patients by immunohistochemistry (IHC). The high NOTCH2 expressing UM-UC3 and UM-UC13 bladder cancer cells were stably transduced with either Notch2 specific or control shRNA. The low Notch2 expressing RT4V6 and RT112 cells were transduced with NOTCH2 intracellular domain or empty vector. A Notch2 inactivating antibody, NRR2Mab, was provided by Genentech, Inc. (San Francisco, CA). The effects of NOTCH2 overexpression and inhibition were assessed for changes in proliferation under both adherent (AH) and anchorage independent conditions (AI), and invasive activities in vitro, as well as for expression of markers of epithelial to mesenchymal transition (EMT) and stem cell markers. In vivo effects were evaluated in an orthotopic bladder cancer xenograft model with bioluminescence imaging. Results: NOTCH2 overexpression in IHC correlated with higher grade and stage. Both NOTCH2 overexpressing cells showed spindle shaped morphology and increased cell growth in AH and AI and invasion compared to mock cells. This was associated with cell cycle progression and increased expression of EMT and stem cell markers. Stable NOTCH2 knockdown cells and cells treated with NRR2Mab demonstrated decreased growth in AI and invasion, and this was associated with decreased expression of EMT and stem cell markers. In vivo, RT4V6 NOTCH2 overexpression increased xenograft tumor growth and NOTCH2 overexpression increased ki67 positive cells and promoted EMT in IHC. Stable Notch2 knockdown inhibited xenograft tumor growth in UM-UC3 and UM-UC13. NRR2Mab treatment also inhibited UM-UC13 xenograft tumor growth and lymph node metastasis. Conclusions: NOTCH2 plays a crucial role in growth, self-renewal and invasion in bladder cancer. These results provide pre-clinical proof of principle that Notch2 inhibition may be a rational treatment for bladder cancer.

e15590 Background: Notch signaling guides cell fate decisions, establishment of cell lineages, stem cell maintenance and differentiation during early development. Notch is re-activated in many malignancies where it has been shown to regulate tumor growth and progression. We report effects of Notch-2 knockdown and treatment with a specific Notch-2 inhibitor, NRR2Mab, in pre-clinical models of bladder cancer. Methods: UM-UC3, UM-UC13 and UM-UC16 cells, representing invasive bladder cancer cells with high Notch-2 expression, were stably transduced with Notch-2 or non-targeting shRNA in a lentiviral vector. A Notch-2-inactivating monoclonal antibody, NRR2Mab, and an isotype-matched, non-targeting antibody were provided by Genentech, Inc. (San Francisco, CA). Cells were grown under adherent (AH) or anchorage-independent conditions (AI) and the effects of Notch-2 silencing or NRR2Mab inactivation were quantitatively assessed for changes in proliferation, migration and invasive activities using in vitro assays, and for expression of Notch-2 or other common stem cell-related genes using quantitative RT-PCR, Western blotting and immunohistochemistry. In vivo effects were evaluated in an orthotopic bladder cancer xenograft model with bioluminescence imaging. Results: Cells transduced with the Notch-2 shRNA or treated with NRR2Mab demonstrated markedly decreased Notch-2 expression. Both treatments diminished cell proliferation under AI but not AH conditions in UM-UC3 and UM-UC13. When grown in AI conditions, UM-UC3 and UM-UC13 were found to be enriched for expression of stem cell genes. Notch-2 silencing or treatment with NRR2Mab diminished the expression of these genes. Both treatments significantly inhibited cell migration and invasion of these cells. In vivo, stable Notch-2 knockdown significantly inhibited xenograft growth for all 3 cell lines. NRR2Mab treatment also inhibited UM-UC13 xenograft growth and metastasis. Conclusions: Our results provide preclinical evidence that Notch-2 may be a useful target to inhibit growth and progression of bladder cancer. Targeting Notch-2 with a specific inhibitory monoclonal antibody warrants further evaluation in this context.

The fine structure of 5 human papillary thyroid carcinomas was described and compared with previous publications on the ultrastructure of the normal gland and of diffuse hyperplastic goiter. The 5 tumors were composed of predominantly tall columnar epithelial cells. Interspersed with the neoplastic cells and inside the epithelial basement membrane were a few lymphocytes, the significance of which is undetermined. Most neoplastic cells showed numerous mitochondria and cytosomes. A well-developed granular endoplasmic reticulum and a prominent Golgi apparatus often were observed. Microvilli, cytoplasmic protrusions, apical vesicles, multivesicular bodies, microfibrils, and microtubules also were identified. Nuclei were large, were often indented, and contained prominent nucleoli and distinct granulofibrillar inclusions in two different stages of development. Their etiology is unknown. Nuclear structures containing cytoplasmic organelles were common and were caused by invagination of the nuclear envelope. Except for the intranuclear inclusions, more variation in the size and shape of the nuclei, and absence of colloid droplets, human papillary thyroid carcinoma cells closely resemble hyper-plastic follicular cells.

Alveolar soft part sarcoma is a distinct clinicopathological entity of unknown histogenesis described by Christopherson et al1in 1952. It commonly occurs in adolescents and young adults, and females are affected more often than males.2Although frequently found in association with muscles of the extremities,3-5it has also been encountered in other anatomic sites, including the orbit, where it appears to be an exceedingly rare tumor.6 The purpose of this paper is to report two cases of alveolar soft part sarcoma involving the orbit of a 10-month-old boy and a 3-year-old girl, both recently observed at the Unidad de Patologia de la Facultad de Medicina de la Universidad Nacional Autonoma de Mexico, in the Hospital General. Report of Cases Case 1. —Eight months before admission the parents of a 10-month-old boy noticed external deviation of his right eye. A few weeks later a slowly growing mass