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.

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.

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.

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.

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.

ADAM15 Is Functionally Associated with the Metastatic Progression of Human Bladder Cancer.

ADAM15 is a member of a family of catalytically active disintegrin membrane metalloproteinases that function as molecular signaling switches, shed membrane bound growth factors and/or cleave and inactivate cell adhesion molecules. Aberrant metalloproteinase function of ADAM15 may contribute to tumor progression through the release of growth factors or disruption of cell adhesion. In this study, we utilized human bladder cancer tissues and cell lines to evaluate the expression and function of ADAM15 in the progression of human bladder cancer. Examination of genome and transcriptome databases revealed that ADAM15 ranked in the top 5% of amplified genes and its mRNA was significantly overexpressed in invasive and metastatic bladder cancer compared to noninvasive disease. Immunostaining of a bladder tumor tissue array designed to evaluate disease progression revealed increased ADAM15 immunoreactivity associated with increasing cancer stage and exhibited significantly stronger staining in metastatic samples. About half of the invasive tumors and the majority of the metastatic cases exhibited high ADAM15 staining index, while all low grade and noninvasive cases exhibited negative or low staining. The knockdown of ADAM15 mRNA expression significantly inhibited bladder tumor cell migration and reduced the invasive capacity of bladder tumor cells through MatrigelTM and monolayers of vascular endothelium. The knockdown of ADAM15 in a human xenograft model of bladder cancer inhibited tumor growth by 45% compared to controls. Structural modeling of the catalytic domain led to the design of a novel ADAM15-specific sulfonamide inhibitor that demonstrated bioactivity and significantly reduced the viability of bladder cancer cells in vitro and in human bladder cancer xenografts. Taken together, the results revealed an undescribed role of ADAM15 in the invasion of human bladder cancer and suggested that the ADAM15 catalytic domain may represent a viable therapeutic target in patients with advanced disease.

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.

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.

HER2 drives luminal breast cancer stem cells in the absence of HER2 amplification: implications for efficacy of adjuvant trastuzumab.

Although current breast cancer treatment guidelines limit the use of HER2-blocking agents to tumors with HER2 gene amplification, recent retrospective analyses suggest that a wider group of patients may benefit from this therapy. Using breast cancer cell lines, mouse xenograft models and matched human primary and metastatic tissues, we show that HER2 is selectively expressed in and regulates self-renewal of the cancer stem cell (CSC) population in estrogen receptor-positive (ER(+)), HER2(-) luminal breast cancers. Although trastuzumab had no effects on the growth of established luminal breast cancer mouse xenografts, administration after tumor inoculation blocked subsequent tumor growth. HER2 expression is increased in luminal tumors grown in mouse bone xenografts, as well as in bone metastases from patients with breast cancer as compared with matched primary tumors. Furthermore, this increase in HER2 protein expression was not due to gene amplification but rather was mediated by receptor activator of NF-κB (RANK)-ligand in the bone microenvironment. These studies suggest that the clinical efficacy of adjuvant trastuzumab may relate to the ability of this agent to target the CSC population in a process that does not require HER2 gene amplification. Furthermore, these studies support a CSC model in which maximal clinical benefit is achieved when CSC targeting agents are administered in the adjuvant setting. Cancer Res; 73(5); 1635-46. ©2012 AACR.

The ectodomain shedding of E-cadherin by ADAM15 supports ErbB receptor activation.

The zinc-dependent disintegrin metalloproteinases (a disintegrin and metalloproteinases (ADAMs) have been implicated in several disease processes, including human cancer. Previously, we demonstrated that the expression of a catalytically active member of the ADAM family, ADAM15, is associated with the progression of prostate and breast cancer. The accumulation of the soluble ectodomain of E-cadherin in human serum has also been associated with the progression of prostate and breast cancer and is thought to be mediated by metalloproteinase shedding. Utilizing two complementary models, overexpression and stable short hairpin RNA-mediated knockdown of ADAM15 in breast cancer cells, we demonstrated that ADAM15 cleaves E-cadherin in response to growth factor deprivation. We also demonstrated that the extracellular shedding of E-cadherin was abrogated by a metalloproteinase inhibitor and through the introduction of a catalytically inactive mutation in ADAM15. We have made the novel observation that this soluble E-cadherin fragment was found in complex with the HER2 and HER3 receptors in breast cancer cells. These interactions appeared to stabilize HER2 heterodimerization with HER3 and induced receptor activation and signaling through the Erk pathway, supporting both cell migration and proliferation. In this study, we provide evidence that ADAM15 catalyzes the cleavage of E-cadherin to generate a soluble fragment that in turn binds to and stimulates ErbB receptor signaling.

ADAM15 supports prostate cancer metastasis by modulating tumor cell-endothelial cell interaction.

Using human tumor and cDNA microarray technology, we have recently shown that the ADAM15 disintegrin is significantly overexpressed during the metastatic progression of human prostate cancer. In the current study, we used lentiviral-based short hairpin RNA (shRNA) technology to down-regulate ADAM15 in the metastatic prostate cancer cell line, PC-3. ADAM15 down-regulation dramatically attenuated many of the malignant characteristics of PC-3 cells in vitro and prevented the s.c. growth of PC-3 cells in severe combined immunodeficient (SCID) mice. By inhibiting the expression of ADAM15 in PC-3 cells, we showed decreased cell migration and adhesion to specific extracellular matrix proteins. This was accompanied by a reduction in the cleavage of N-cadherin by ADAM15 at the cell surface. Fluorescence-activated cell sorting analysis revealed reduced cell surface expression of the metastasis-associated proteins alpha(v) integrin and CD44. Furthermore, matrix metalloproteinase 9 secretion and activity were abrogated in response to ADAM15 reduction. In an in vitro model of vascular invasion, loss of ADAM15 reduced PC-3 adhesion to, and migration through, vascular endothelial cell monolayers. Using an SCID mouse model of human prostate cancer metastasis, we found that the loss of ADAM15 significantly attenuated the metastatic spread of PC-3 cells to bone. Taken together, these data strongly support a functional role for ADAM15 in prostate tumor cell interaction with vascular endothelium and the metastatic progression of human prostate cancer.

Indocyanine-green-embedded PEBBLEs as a contrast agent for photoacoustic imaging.

Nanoparticles 100 nm in diameter containing indocyanine green (ICG) have been developed as a contrast agent for photoacoustic (PA) imaging based on (photonic explorers for biomedical use by biologically localized embedding PEBBLE) technology using organically modified silicate (ormosil) as a matrix. ICG is an FDA-approved dye with strong optical absorption in the near-infrared (NIR) region, where light can penetrate deepest into biological tissue. A photoacoustic imaging system was used to study image contrast as a function of PEBBLE concentration in phantom objects. ICG-embedded ormosil PEBBLEs showed improved stability in aqueous solution compared with free ICG dye. The particles were conjugated with HER-2 antibody for breast cancer and prostate cancer cell targeting. Initial in vitro characterization shows high contrast and high efficiency for binding to prostate cancer cells. ICG can also be used as a photosensitizer (generating toxic oxygen by illumination) for photodynamic therapy. We have measured the photosensitization capability of ICG-embedded ormosil nanoparticles. This feature can be utilized to combine detection and therapeutic functions in a single agent.

ADAM15 disintegrin is associated with aggressive prostate and breast cancer disease.

The aim of the current study was to evaluate the expression of ADAM15 disintegrin (ADAM15) in a broad spectrum of human tumors. The transcript for ADAM15 was found to be highly upregulated in a variety of tumor cDNA expression arrays. ADAM15 protein expression was examined in tissue microarrays (TMAs) consisting of 638 tissue cores. TMA analysis revealed that ADAM15 protein was significantly increased in multiple types of adenocarcinoma, specifically in prostate and breast cancer specimens. Statistical association was observed with disease progression within clinical parameters of predictive outcome for both prostate and breast cancers, pertaining to Gleason sum and angioinvasion, respectively. In this report, we also present data from a cDNA microarray of prostate cancer (PCa), where we compared transfected LNCaP cells that overexpress ADAM15 to vector control cells. In these experiments, we found that ADAM15 expression was associated with the induction of specific proteases and protease inhibitors, particularly tissue inhibitor of metalloproteinase 2, as validated in a separate PCa TMA. These results suggest that ADAM15 is generally overexpressed in adenocarcinoma and is highly associated with metastatic progression of prostate and breast cancers.

The role of calpain in the proteolytic cleavage of E-cadherin in prostate and mammary epithelial cells.

The E-cadherin protein mediates Ca(2+)-dependent interepithelial adhesion. Association of E-cadherin with the catenin family of proteins is critical for the maintenance of a functional adhesive complex. We have identified a novel truncated E-cadherin species of 100-kDa (E-cad(100)) in prostate and mammary epithelial cells. E-cad(100) was generated by treatment of cells with ionomycin or TPA. Cell-permeable calpain inhibitors prevented E-cad(100) induction by ionomycin. Immunoblotting for spectrin and mu-calpain confirmed calpain activation in response to ionomycin treatment. Both the mu- and m-isoforms of calpain efficiently generated E-cad(100) in vitro. The E-cad(100) fragment was unable to bind to beta-catenin, gamma-catenin, and p120, suggesting that this cleavage event would disrupt the E-cadherin adhesion complex. Mutational analysis localized the calpain cleavage site to the cytosolic domain upstream of the beta- and gamma-catenin binding motifs of E-cadherin. Because E-cadherin is inactivated in many adenocarcinomas we hypothesized that calpain may play a role in prostate tumorigenesis. A prostate cDNA microarray data base was analyzed for calpain expression in which it was found that m-calpain was up-regulated in localized prostate cancer, and to an even higher degree in metastatic prostate cancer compared with normal prostate tissue. Furthermore, we examined the cleavage of E-cadherin in prostate cancer specimens and found that E-cad(100) accumulated in both localized and metastatic prostate tumors, supporting the cDNA microarray data. These findings demonstrate a novel mechanism by which E-cadherin is functionally inactivated through calpain-mediated proteolysis and suggests that E-cadherin is targeted by calpain during the tumorigenic progression of prostate cancer.

Rescue of embryonic epithelium reveals that the homozygous deletion of the retinoblastoma gene confers growth factor independence and immortality but does not influence epithelial differentiation or tissue morphogenesis.

The ability to rescue viable prostate precursor tissue from retinoblastoma-deficient (Rb-/-) fetal mice has allowed for the isolation and characterization of the first Rb-/- prostate epithelial cell line. This cell line, designated Rb-/-PrE, was utilized for experiments examining the consequences of Rb loss on an epithelial population. These findings demonstrated that Rb deletion has no discernible effect on prostatic histodifferentiation in Rb-/-PrE cultures. When Rb-/-PrE cells were recombined with embryonic rat urogenital mesenchyme and implanted into athymic male, nude mouse hosts, the recombinants developed into fully differentiated and morphologically normal prostate tissue. The Rb-/-PrE phenotype was characterized by serum independence in culture and immortality in vivo, when compared with wild type controls. Cell cycle analysis revealed elevated S phase DNA content accompanied by increased expression of cyclin E1 and proliferating cell nuclear antigen. Rb-/-PrE cultures also exhibited a diminished ability to growth arrest under high density culture conditions. We believe that the development of Rb-/- prostate tissue and cell lines has provided a unique experimental platform with which to investigate the consequences of Rb deletion in epithelial cells under various physiological conditions. Additionally, the development of this technology will allow similar studies in other tissues and cell populations rescued from Rb-/- fetuses.