A randomized phase 2 trial of gemcitabine/cisplatin with or without cetuximab in patients with advanced urothelial carcinoma.

BACKGROUND: Epidermal growth factor receptor overexpression is associated with poor outcomes in urothelial carcinoma (UC). Cetuximab (CTX) exhibited an antitumor effect in in vivo UC models. The efficacy of gemcitabine/cisplatin (GC) with or without CTX in patients with advanced UC was evaluated. METHODS: Patients with advanced UC, measurable disease, and adequate organ function were randomized 1:2 to cisplatin (70 mg/m(2) ) on day 1 plus gemcitabine (1000 mg/m(2) ) on days 1, 8, and 15 (arm A) or GC plus CTX (500 mg/m(2) ) on days 1 and 15 (arm B). The primary endpoint was the overall response rate. The secondary endpoints were the response duration, safety, progression-free survival, overall survival, determination of whether or not CTX sensitized nonresponders to GC, and exploratory biomarker analysis. The accrual targets were 27 and 54 patients for the 2 arms, respectively. The overall response rate was reported by arm with binomial confidence intervals (CIs). Kaplan-Meier methods were used for time-to-event endpoints. RESULTS: Eighty-eight eligible patients were randomized; 87 were toxicity-evaluable, and 85 were response-evaluable. The overall response rates were 57.1% for arm A (95% CI = 37%-76%) and 61.4% for arm B (95% CI = 48%-74%). The median progression-free survival times were 8.5 months for arm A (95% CI = 5.7-10.4 months) and 7.6 months for arm B (95% CI = 6.1-8.7 months). The median overall survival times were 17.4 months for arm A (95% CI = 12.8 months to unreached) and 14.3 months for arm B (95% CI = 11.6-22.2 months). The most common grade 3/grade 4 adverse events in both arms were myelosuppression and nausea. Thromboembolism, acneiform rash, fatigue, pain, hypersensitivity reactions, elevated transaminases, hyponatremia, and hypomagnesemia were more common in arm B; 3 grade 5 adverse events occurred in arm B. The presence of primary disease significantly correlated with thromboembolism. An increased soluble E-cadherin level after cycle 2 correlated with a higher risk of death. CONCLUSIONS: GC plus CTX was feasible but was associated with more adverse events and no improvements in outcomes.

Characterization of circulating tumor cells in patients with metastatic bladder cancer utilizing functionalized microfluidics.

Assessing the molecular profiles of bladder cancer (BC) from patients with locally advanced or metastatic disease provides valuable insights, such as identification of invasive markers, to guide personalized treatment. Currently, most molecular profiling of BC is based on highly invasive biopsy or transurethral tumor resection. Liquid biopsy takes advantage of less-invasive procedures to longitudinally profile disease. Circulating tumor cells (CTCs) isolated from blood are one of the key analytes of liquid biopsy. In this study, we developed a protein and mRNA co-analysis workflow for BC CTCs utilizing the graphene oxide (GO) microfluidic chip. The GO chip was conjugated with antibodies against both EpCAM and EGFR to isolate CTCs from 1 mL of blood drawn from BC patients. Following CTC capture, protein and mRNA were analyzed using immunofluorescent staining and ion-torrent-based whole transcriptome sequencing, respectively. Elevated CTC counts were significantly associated with patient disease status at the time of blood draw. We found a count greater than 2.5 CTCs per mL was associated with shorter overall survival. The invasive markers EGFR, HER2, CD31, and ADAM15 were detected in CTC subpopulations. Whole transcriptome sequencing showed distinct RNA expression profiles from patients with or without tumor burden at the time of blood draw. In patients with advanced metastatic disease, we found significant upregulation of metastasis-related and chemotherapy-resistant genes. This methodology demonstrates the capability of GO chip-based assays to identify tumor-related RNA signatures, highlighting the prognostic potential of CTCs in metastatic BC patients.

Integrative Drug Screening and Multiomic Characterization of Patient-derived Bladder Cancer Organoids Reveal Novel Molecular Correlates of Gemcitabine Response.

BACKGROUND AND OBJECTIVE: Predicting response to therapy for each patient's tumor is critical to improving long-term outcomes for muscle-invasive bladder cancer. This study aims to establish ex vivo bladder cancer patient-derived organoid (PDO) models that are representative of patients' tumors and determine the potential efficacy of standard of care and curated experimental therapies. METHODS: Tumor material was collected prospectively from consented bladder cancer patients to generate short-term PDO models, which were screened against a panel of clinically relevant drugs in ex vivo three-dimensional culture. Multiomic profiling was utilized to validate the PDO models, establish the molecular characteristics of each tumor, and identify potential biomarkers of drug response. Gene expression (GEX) patterns between paired primary tissue and PDO samples were assessed using Spearman's rank correlation coefficients. Molecular correlates of therapy response were identified using Pearson correlation coefficients and Kruskal-Wallis tests with Dunn's post hoc pairwise comparison testing. KEY FINDINGS AND LIMITATIONS: A total of 106 tumors were collected from 97 patients, with 65 samples yielding sufficient material for complete multiomic molecular characterization and PDO screening with six to 32 drugs/combinations. Short-term PDOs faithfully represent the tumor molecular characteristics, maintain diverse cell types, and avoid shifts in GEX-based subtyping that accompany long-term PDO cultures. Utilizing an integrative approach, novel correlations between ex vivo drug responses and genomic alterations, GEX, and protein expression were identified, including a multiomic signature of gemcitabine response. The positive predictive value of ex vivo drug responses and the novel multiomic gemcitabine response signature need to be validated in future studies. CONCLUSIONS AND CLINICAL IMPLICATIONS: Short-term PDO cultures retain the molecular characteristics of tumor tissue and avoid shifts in expression-based subtyping that have plagued long-term cultures. Integration of multiomic profiling and ex vivo drug screening data identifies potential predictive biomarkers, including a novel signature of gemcitabine response. PATIENT SUMMARY: Better models are needed to predict patient response to therapy in bladder cancer. We developed a platform that uses short-term culture to best mimic each patient's tumor and assess potential sensitivity to therapeutics.

The E2F1/DNMT1 axis is associated with the development of AR negative castration resistant prostate cancer.

BACKGROUND: Research on castration resistant prostate cancer (CRPC) has focused primarily on functional alterations of the androgen receptor (AR). However, little is known about the loss of AR gene expression itself and the possible contribution of AR negative cells to CRPC. METHODS: Human and murine prostate cancer tissue microarrays (TMAs) were evaluated with antibodies specific for E2F1, DNA methyltransferase 1 or AR. The human prostate cancer TMA consisted of clinical samples ranging from normal tissue to samples of metastatic disease. The murine TMA was comprised of benign, localized or metastatic prostate cancer acquired from TRAMP mice treated with castration and/or 5'-Aza-2'-deoxycytidine (5Aza). RESULTS: Immunohistochemical analysis revealed increased nuclear DNMT1 staining in localized PCa (P < 0.0001) and metastatic PCa (P < 0.0001) compared to normal tissue. Examination of specific diagnoses revealed that Gleason seven tumors exhibited greater nuclear DNMT1 staining than Gleason six tumors (P < 0.05) and that metastatic tissue exhibited greater levels of nuclear DNMT1 than Gleason seven tumors (P < 0.01). Evaluation of the murine tissue cores revealed that 8.2% and 8.1% of benign tissue cores stained positive for E2F1 and DNMT1 respectively, while 97.0% were AR positive. Conversely, 81% and 100% of tumors were positive for E2F1 and DNMT1 respectively. This was in stark contrast to only 18% of tumors positive for AR. Treatment of mice with 5Aza reduced DNMT1 staining by 30%, while AR increased by 27%. CONCLUSIONS: These findings demonstrate that the E2F1/DNMT1 inhibitory axis of AR transcription is activated during the emergence of CRPC.

Evaluation of the antitumor activity of dacomitinib in models of human bladder cancer.

Members of the human epidermal growth factor receptor (HER) family play a significant role in bladder cancer progression and may underlie the development of chemotherapy resistance. Dacomitinib is an irreversible tyrosine kinase inhibitor with structural specificity for the catalytic domains of epidermal growth factor receptor (EGFR), HER2 and HER4 that has exhibited vigorous efficacy against other solid tumors. We evaluated the antitumor activity of dacomitinib in human bladder cancer cell lines expressing varying levels of HER family receptors. These cell lines also were established as bladder cancer xenografts in nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice to assess dacomitinib activity in vivo. Significant cytotoxic and cytostatic effects were noted in cells expressing elevated levels of the dacomitinib target receptors with apoptosis and cell cycle arrest being the predominant mechanisms of antitumor activity. Cells expressing lower levels of HER receptors were much less sensitive to dacomitinib. Interestingly, dacomitinib was more active than either trastuzumab or cetuximab in vitro, and exhibited increased growth inhibition of bladder tumor xenografts compared with lapatinib. Pharmacodynamic effects of dacomitinib included decreased E-cadherin (E-cad) expression, reduction of EGFR and extracellular signal-regulated kinase (ERK) phosphorylation and reduced mitotic count. Dacomitinib also inhibited tumor growth in a chemotherapy-resistant xenograft and, when combined with chemotherapy in a sensitive xenograft, exhibited superior antitumor effects compared with individual treatments. Evaluation in xenograft-bearing mice revealed that this combination was broadly feasible and well tolerated. In conclusion, dacomitinib exhibited pronounced activity both as a single agent and when combined with chemotherapy in human bladder cancer models. Further investigation of dacomitinib in the preclinical and clinical trial settings is being pursued.

MEK is a promising target in the basal subtype of bladder cancer.

While many resources exist for the drug screening of bladder cancer cell lines in 2D culture, it is widely recognized that screening in 3D culture is more representative of in vivo response. Importantly, signaling changes between 2D and 3D culture can result in changes to drug response. To address the need for 3D drug screening of bladder cancer cell lines, we screened 17 bladder cancer cell lines using a library of 652 investigational small-molecules and 3 clinically relevant drug combinations in 3D cell culture. Our goal was to identify compounds and classes of compounds with efficacy in bladder cancer. Utilizing established genomic and transcriptomic data for these bladder cancer cell lines, we correlated the genomic molecular parameters with drug response, to identify potentially novel groups of tumors that are vulnerable to specific drugs or classes of drugs. Importantly, we demonstrate that MEK inhibitors are a promising targeted therapy for the basal subtype of bladder cancer, and our data indicate that drug screening of 3D cultures provides an important resource for hypothesis generation.

Multiparameter urine analysis for quantitative bladder cancer surveillance of orthotopic xenografted mice.

The human-derived orthotopic xenograft mouse model is an effective platform for performing in vivo bladder cancer studies to examine tumor development, metastasis, and therapeutic effects of drugs. To date, the surveillance of tumor progression in real time for orthotopic bladder xenografts is highly dependent on semi-quantitative in vivo imaging technologies such as bioluminescence. While these imaging technologies can estimate tumor progression, they are burdened with requirements such as anesthetics, specialized equipment, and genetic modification of the injected cell line. Thus, a convenient and non-invasive technology to quantitatively monitor the growth of bladder cancer in orthotopic xenografts is highly desired. In this work, using a microfluidic chemiluminescent ELISA platform, we have successfully developed a rapid, multiparameter urine-based and non-invasive biomolecular prognostic technology for orthotopic bladder cancer xenografts. This method consists of two steps. First, the concentrations of a panel of four urinary biomarkers are quantified from the urine of mice bearing orthotopic bladder xenografts. Second, machine learning and principal component analysis (PCA) algorithms are applied to analyze the urinary biomarkers, and subsequently, a score is assigned to indicate the tumor growth. With this methodology, we have quantitatively monitored the orthotopic growth of human bladder cancer that was inoculated with low, medium, and high cancer cell numbers. We also employed this method and performed a proof of principle experiment to examine the in vivo therapeutic efficacy of the EGFR inhibitor, dacomitinib.

The role of the disintegrin metalloproteinase ADAM15 in prostate cancer progression.

The metalloproteinase ADAM15 is a multi-domain disintegrin protease that is upregulated in a variety of human cancers. ADAM15 mRNA and protein levels are increased in prostate cancer and its expression is significantly increased during metastatic progression. It is likely that ADAM15 supports disease progression differentially through the action of its various functional domains. ADAM15 may downregulate adhesion of tumor cells to the extracellular matrix, reduce cell-cell adhesion, and promote metastasis through the activity of its disintegrin and metalloproteinase domains. Additionally, ADAM15 can influence cell signaling by shedding membrane-bound growth factors and other proteins that interact with receptor tyrosine kinases, leading to receptor activation. There is also evidence supporting a role for ADAM15 in angiogenesis and angioinvasion of tumor cells, which are critical for unrestrained tumor growth and metastatic spread. Given its diverse functions, ADAM15 may represent a pivotal regulatory component of tumor progression, an important target for therapeutic intervention, or emerge as a biomarker of disease progression.

ATDC mediates a TP63-regulated basal cancer invasive program.

Basal subtype cancers are deadly malignancies but the molecular events driving tumor lethality are not completely understood. Ataxia-telangiectasia group D complementing gene (ATDC, also known as TRIM29), is highly expressed and drives tumor formation and invasion in human bladder cancers but the factor(s) regulating its expression in bladder cancer are unknown. Molecular subtyping of bladder cancer has identified an aggressive basal subtype, which shares molecular features of basal/squamous tumors arising in other organs and is defined by activation of a TP63-driven gene program. Here, we demonstrate that ATDC is linked with expression of TP63 and highly expressed in basal bladder cancers. We find that TP63 binds to transcriptional regulatory regions of ATDC and KRT14 directly, increasing their expression, and that ATDC and KRT14 execute a TP63-driven invasive program. In vivo, ATDC is required for TP63-induced bladder tumor invasion and metastasis. These results link TP63 and the basal gene expression program to ATDC and to aggressive tumor behavior. Defining ATDC as a molecular determinant of aggressive, basal cancers may lead to improved biomarkers and therapeutic approaches.

A surgical orthotopic approach for studying the invasive progression of human bladder cancer.

The invasion of bladder cancer into the sub-urothelial muscle and vasculature are key determinants leading to lethal metastatic progression. However, the molecular basis is poorly understood, partly because of the lack of uncomplicated and reliable models that recapitulate the biology of locally invasive disease. We developed a surgical grafting technique, characterized by a simple, rapid, reproducible and high-efficiency approach, to recapitulate the pathobiological events of human bladder cancer invasion in mice. This technique consists of a small laparotomy and direct implantation of human cancer cells into the bladder lumen. Unlike other protocols, it does not require debriding of the urothelial lining, injection into the bladder wall, specialized imaging equipment, bladder catheterization or costly surgical equipment. With minimal practice, the procedure can be executed in <10 min. Tumors develop with a high take rate, and most cell lines exhibit local invasion within 4 weeks of implantation.

5-aza-2'-deoxycytidine delays androgen-independent disease and improves survival in the transgenic adenocarcinoma of the mouse prostate mouse model of prostate cancer.

PURPOSE: We have previously shown that 5-aza-2'-deoxycytidine (5-aza) is an effective chemopreventive agent capable of preventing early disease progression in the transgenic adenocarcinoma of the mouse prostate (TRAMP) model. The purpose of this study was to determine the effect of 5-aza on preexisting TRAMP prostate cancers and prevention of androgen-independent prostate cancer. EXPERIMENTAL DESIGN: TRAMP mice with established prostate cancers were treated with 5-aza, castration, castration + 5-aza, or vehicle control (PBS). One cohort of 22 mice per treatment was euthanized after 10 weeks of treatment, whereas a second cohort of 14 mice per group was followed until death to determine survival. Histologic sections of prostate, pelvic lymph nodes, lung, and liver were blinded and analyzed by a certified genitourinary pathologist (K.J.W.). RESULTS: Combined treatment (castration + 5-aza) provided significant survival benefits over either single treatment (combined versus castration P = 0.029, combined versus 5-aza P = 0.036). At 24 weeks of age, 86% of mice within the PBS cohort exhibited histologic evidence of prostate cancer, whereas only 47% of the combined cohort exhibited malignant disease (P < 0.0001). Additionally, whereas 43% of the PBS treatment group exhibited lymph node metastases, these were only observed in 21% of the combined treatment mice. CONCLUSIONS: This is the first study to examine the effect of 5-aza and combined castration + 5-aza on preexisting prostate cancer in an animal model. Based on these preclinical findings, we suggest that 5-aza treatment may prolong the time to an androgen-independent status and thus survival in a hormone-deprived setting in prostate cancer.

Elevated E2F1 inhibits transcription of the androgen receptor in metastatic hormone-resistant prostate cancer.

Activation of E2F transcription factors, through disruption of the retinoblastoma (Rb) tumor-suppressor gene, is a key event in the development of many human cancers. Previously, we showed that homozygous deletion of Rb in a prostate tissue recombination model exhibits increased E2F activity, activation of E2F-target genes, and increased susceptibility to hormonal carcinogenesis. In this study, we examined the expression of E2F1 in 667 prostate tissue cores and compared it with the expression of the androgen receptor (AR), a marker of prostate epithelial differentiation, using tissue microarray analysis. We show that E2F1 expression is low in benign and localized prostate cancer, modestly elevated in metastatic lymph nodes from hormone-naïve patients, and significantly elevated in metastatic tissues from hormone-resistant prostate cancer patients (P = 0.0006). In contrast, strong AR expression was detected in benign prostate (83%), localized prostate cancer (100%), and lymph node metastasis (80%), but decreased to 40% in metastatic hormone-resistant prostate cancer (P = 0.004). Semiquantitative reverse transcription-PCR analysis showed elevated E2F1 mRNA levels and increased levels of the E2F-target genes dihyrofolate reductase and proliferating cell nuclear antigen in metastatic hormone-independent prostate cancer cases compared with benign tissues. To identify a role of E2F1 in hormone-independent prostate cancer, we examined whether E2F1 can regulate AR expression. We show that exogenous expression of E2F1 significantly inhibited AR mRNA and AR protein levels in prostate epithelial cells. E2F1 also inhibited an AR promoter-luciferase construct that was dependent on the transactivation domain of E2F1. Furthermore, using chromatin immunoprecipitation assays, we show that E2F1 and the pocket protein family members p107 and p130 bind to the AR promoter in vivo. Taken together, these results show that elevated E2F1, through its ability to repress AR transcription, may contribute to the progression of hormone-independent prostate cancer.

Inhibition of DNA methyltransferase activity prevents tumorigenesis in a mouse model of prostate cancer.

Transcriptional silencing of tumor suppressor genes by DNA methylation plays an important role in tumorigenesis. These aberrant epigenetic modifications may be mediated in part by elevated DNA methyltransferase levels. DNA methyltransferase 1 (DNMT1), in particular, is overexpressed in many tumor types. Recently, we showed that Dnmt1 is transcriptionally regulated by E2F transcription factors and that retinoblastoma protein (pRb) inactivation induces Dnmt1. Based on these observations, we investigated regulation of Dnmt1 by polyomavirus oncogenes, which potently inhibit the pRb pocket protein family. Infection of primary human prostate epithelial cells with BK polyomavirus dramatically induced Dnmt1 transcription following large T antigen (TAg) translation and E2F activation. For in vivo study of Dnmt1 regulation, we used the transgenic adenocarcinoma of the mouse prostate (TRAMP) model, which expresses the SV40 polyomavirus early region, including TAg, under control of a prostate-specific promoter. Analysis of TRAMP prostate lesions revealed greatly elevated Dnmt1 mRNA and protein levels beginning in prostatic intraepithelial neoplasia and continuing through advanced prostate cancer and metastasis. Interestingly, when TRAMP mice were treated in a chemopreventive manner with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-aza), 0 of 14 mice developed prostate cancer at 24 weeks of age, whereas 7 of 13 (54%) control-treated mice developed poorly differentiated prostate cancer. Treatment with 5-aza also prevented the development of lymph node metastases and dramatically extended survival compared with control-treated mice. Taken together, these data suggest that Dnmt1 is rapidly activated by pRb pathway inactivation, and that DNA methyltransferase activity is required for malignant transformation and tumorigenesis.

3-D Cell Culture System for Studying Invasion and Evaluating Therapeutics in Bladder Cancer.

Bladder cancer is a significant health problem. It is estimated that more than 16,000 people will die this year in the United States from bladder cancer. While 75% of bladder cancers are non-invasive and unlikely to metastasize, about 25% progress to an invasive growth pattern. Up to half of the patients with invasive cancers will develop lethal metastatic relapse. Thus, understanding the mechanism of invasive progression in bladder cancer is crucial to predict patient outcomes and prevent lethal metastases. In this article, we present a three-dimensional cancer invasion model which allows incorporation of tumor cells and stromal components to mimic in vivo conditions occurring in the bladder tumor microenvironment. This model provides the opportunity to observe the invasive process in real time using time-lapse imaging, interrogate the molecular pathways involved using confocal immunofluorescent imaging and screen compounds with the potential to block invasion. While this protocol focuses on bladder cancer, it is likely that similar methods could be used to examine invasion and motility in other tumor types as well.

Molecular Correlates of In Vitro Responses to Dacomitinib and Afatinib in Bladder Cancer.

BACKGROUND: The HER family of proteins (EGFR, HER2, HER3 and HER4) have long been thought to be therapeutic targets for bladder cancer, but previous clinical trials targeting these proteins have been disappointing. Second generation agents may be more effective. OBJECTIVE: The aim of this study was to evaluate responses to two second-generation irreversible tyrosine kinase inhibitors, dacomitinib and afatinib, in bladder cancer cell lines. METHODS: Cell lines were characterized by targeted next generation DNA sequencing, RNA sequencing, western blotting and flow cytometry. Cell survival responses to dacomitinib or afatinib were determined using (3-[4,5-dimethylthioazol-2-yl]-2,5-diphenyl tetrazolium bromide) (MTT) or [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) and phenazine methosylfate (PMS) cell survival assays. RESULTS: Only two cell lines of 12 tested were sensitive to afatinib. Sensitivity to afatinib was significantly associated with mutation in either HER2 or HER3 (p < 0.05). The two cell lines sensitive to afatinib were also responsive to dacomitinib ralong with an additional 4 other cell lines out of 16 tested. No characteristic was associated with dacomitinib sensitivity. Molecular profiling demonstrated that only two genes were high in both afatinib and dacomitinib sensitive cells. Further rhigher expression of RAS pathway genes was noted for dacomitinib responsive cells. CONCLUSIONS: This study confirms that cell line screening can be useful in pre-clinical evaluation of targeted small molecule inhibitors and suggests that compounds with similar structure(s) and target(s) may have distinct sensitivity profiles. Further rcombinational targeting of additional molecularly relevant pathways may be important in enhancing responses to HER targeted agents in bladder cancer.

Targeted DNA and RNA Sequencing of Paired Urothelial and Squamous Bladder Cancers Reveals Discordant Genomic and Transcriptomic Events and Unique Therapeutic Implications.

BACKGROUND: Integrated molecular profiling has identified intrinsic expression-based bladder cancer molecular subtypes. Despite frequent histological diversity, robustness of subtypes in paired conventional (urothelial) and squamous components of the same bladder tumor has not been reported. OBJECTIVE: To assess the impact of histological heterogeneity on expression-based bladder cancer subtypes. DESIGN, SETTING, AND PARTICIPANTS: We performed clinically applicable, targeted DNA and/or RNA sequencing (multiplexed DNA and RNA sequencing [mxDNAseq and mxRNAseq, respectively]) on 112 formalin-fixed paraffin-embedded (FFPE) bladder cancer samples, including 12 cases with paired urothelial/squamous components and 21 bladder cancer cell lines. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Unsupervised hierarchical and consensus clustering of target gene expression enabled derivation of basal/luminal molecular subtyping. RESULTS AND LIMITATION: Across 21 bladder cancer cell lines, our custom mxRNAseq panel was highly concordant with whole transcriptome sequencing, and assessed targets robustly determined expression-based basal/luminal subtypes from The Cancer Genome Atlas data (in silico) and internally sequenced FFPE tissues. Frequent deleterious TP53 (56%) and activating hotspot PIK3CA (30%) somatic mutations were seen across 69 high-quality tissue samples. Potentially targetable focal ERBB2 (6%) or EGFR (6%) amplifications were also identified, and a novel subgene copy-number detection approach is described. Combined DNA/RNA analysis showed that focally amplified samples exhibit outlier EGFR and ERBB2 expression distinct from subtype-intrinsic profiles. Critically, paired urothelial and squamous components showed divergent basal/luminal status in three of 12 cases (25%), despite identical putatively clonal prioritized somatic genomic alterations. Limitations include lack of profiled paired normal tissues for formal somatic alteration determination, and the need for formal analytical and clinical validation. CONCLUSIONS: Our results support the feasibility of clinically relevant integrative bladder cancer profiling and challenge the intrinsic nature of expression subtypes in histologically diverse bladder cancers. PATIENT SUMMARY: A targeted RNA sequencing assay is capable of assessing gene expression-based subtypes in individual components of clinical bladder cancer tissue specimens. Different histological components of the same tumor may yield divergent expression profiles, suggesting that expression-based subtypes should be interpreted with caution in heterogeneous cancers.

Regulation of DNA methyltransferase 1 by the pRb/E2F1 pathway.

Tumor suppressor gene silencing by DNA hypermethylation contributes to tumorigenesis in many tumor types. This aberrant methylation may be due to increased expression and activity of DNA methyltransferases, which catalyze the transfer of methyl groups from S-adenosylmethionine to cytosines in CpG dinucleotides. Elevated expression of the maintenance DNA methyltransferase, DNA methyltransferase 1 (DNMT-1), has been shown in carcinomas of the colon, lung, liver, and prostate. Based on the nearly ubiquitous alterations of both DNA methylation and the retinoblastoma protein (pRb) pathway found in human cancer, we investigated a potential regulatory pathway linking the two alterations in murine and human prostate epithelial cells. Analysis of DNA methyltransferase levels in Rb-/- murine prostate epithelial cell lines revealed elevated Dnmt-1 levels. Genomic DNA sequence analysis identified conserved E2F consensus binding sites in proximity to the transcription initiation points of murine and human Dnmt-1. Furthermore, the Dnmt-1 promoter was shown to be regulated by the pRb/E2F pathway in murine and human cell lines of epithelial and fibroblast origin. In the absence of pRb, Dnmt-1 transcripts exhibited aberrant cell cycle regulation and Rb-/- cells showed aberrant methylation of the paternally expressed gene 3 (Peg3) tumor suppressor gene. These findings show a link between inactivation of the pRb pathway and induction of DNA hypermethylation of CpG island-containing genes in tumorigenesis.

Disruption of Rb/E2F pathway results in increased cyclooxygenase-2 expression and activity in prostate epithelial cells.

The loss of the retinoblastoma tumor suppressor gene (RB) is common in many human cancers, including prostate. We previously reported that engineered deletion of RB in prostate epithelial cells results in sustained cell growth in serum-free media, a predisposition to develop hyperplasia and dysplasia in prostate tissue recombinant grafts, and sensitization to hormonal carcinogenesis. Examining the molecular consequence of RB loss in this system, we show that cyclooxygenase-2 (COX-2) is significantly up-regulated following RB deletion in prostate tissue recombinants. To study the effect of RB deletion on COX-2 regulation, we generated wild-type (PrE) and Rb-/- (Rb-/-PrE) prostate epithelial cell lines rescued by tissue recombination. We show elevated COX-2 mRNA and protein expression in Rb-/-PrE cell lines with increased prostaglandin synthesis. We also find that loss of Rb leads to deregulated E2F activity, with increased expression of E2F target genes, and that exogenous expression of E2F1 results in elevated COX-2 mRNA and protein levels. COX-2 promoter studies reveal that E2F1 transcriptionally activates COX-2, which is dependent on the transactivation and DNA-binding domains of E2F1. Further analysis revealed that the E2F1 target gene, c-myb, is elevated in Rb-/-PrE cells and E2F1-overexpressing cells, whereas ectopic overexpression of c-myb activates the COX-2 promoter in prostate epithelial cells. Additionally, cotransfection with E2F1 and a dominant-negative c-myb inhibited E2F1 activation of the COX-2 promoter. Taken together, these results suggest activation of a transcriptional cascade by which E2F1 regulates COX-2 expression through the c-myb oncogene. This study reports a novel finding describing that deregulation of the Rb/E2F complex results in increased COX-2 expression and activity.

HER2 and EGFR Overexpression Support Metastatic Progression of Prostate Cancer to Bone.

Activation of the EGF receptors EGFR (ErbB1) and HER2 (ErbB2) drives the progression of multiple cancer types through complex mechanisms that are still not fully understood. In this study, we report that HER2 expression is elevated in bone metastases of prostate cancer independently of gene amplification. An examination of HER2 and NF-κB receptor (RANK) coexpression revealed increased levels of both proteins in aggressive prostate tumors and metastatic deposits. Inhibiting HER2 expression in bone tumor xenografts reduced proliferation and RANK expression while maintaining EGFR expression. In examining the role of EGFR in tumor-initiating cells (TIC), we found that EGFR expression was required for primary and secondary sphere formation of prostate cancer cells. EGFR expression was also observed in circulating tumor cells (CTC) during prostate cancer metastasis. Dual inhibition of HER2 and EGFR resulted in significant inhibition of tumor xenograft growth, further supporting the significance of these receptors in prostate cancer progression. Overall, our results indicate that EGFR promotes survival of prostate TIC and CTC that metastasize to bone, whereas HER2 supports the growth of prostate cancer cells once they are established at metastatic sites. Cancer Res; 77(1); 74-85. ©2016 AACR.

Cleavage of beta-catenin by calpain in prostate and mammary tumor cells.

Mutations in the NH(2)-terminal regulatory domain of the beta-catenin gene lead to aberrant stabilization and accumulation of the protein and increased TCF/LEF-dependent transcription. Although these mutations are common in some cancers, they are infrequent in prostate and breast cancer. We have found that metastatic prostate cancer specimens, obtained through a rapid autopsy tissue procurement program, expressed a novel M(r) 75,000 proteolytic fragment of beta-catenin (beta-cat(75)). beta-Cat(75) was also expressed in multiple prostate and breast cancer cell lines and was closely associated with the activity of the calcium-dependent protease, calpain. In a prostate cancer cDNA microarray, m-calpain RNA levels were found to be significantly increased in metastatic disease compared with normal prostate. We showed calpain-dependent generation of beta-cat(75) in cell culture and in vitro. Molecular mapping revealed that calpain cleavage removed the NH(2)-terminal regulatory domain of the beta-catenin protein. Treatment of MCF-7 cells with ionomycin led to increased accumulation of beta-cat(75) in the nucleus and TCF-dependent transcriptional activity. Overexpression of a similar beta-catenin fragment that lacks the NH(2)-terminal 132 amino acids and has transforming potential activated TCF-dependent transcription. Given the low frequency of mutation-induced activation of beta-catenin in prostate and breast cancers, proteolytic cleavage of beta-catenin by calpain may represent a novel mechanism by which the protein is activated during tumorigenesis.