PBRM1 Regulates Stress Response in Epithelial Cells.

Polybromo1 (PBRM1) is a chromatin remodeler subunit highly mutated in cancer, particularly clear cell renal carcinoma. PBRM1 is a member of the SWI/SNF subcomplex, PBAF (PBRM1-Brg1/Brm-associated factors), and is characterized by six tandem bromodomains. Here we establish a role for PBRM1 in epithelial cell maintenance through the expression of genes involved in cell adhesion, metabolism, stress response, and apoptosis. In support of a general role for PBRM1 in stress response and apoptosis, we observe that loss of PBRM1 results in an increase in reactive oxygen species generation and a decrease in cellular viability under stress conditions. We find that loss of PBRM1 promotes cell growth under favorable conditions but is required for cell survival under conditions of cellular stress.

PBRM1 Regulates the Expression of Genes Involved in Metabolism and Cell Adhesion in Renal Clear Cell Carcinoma.

Polybromo-1 (PBRM1) is a component of the PBAF (Polybromo-associated-BRG1- or BRM-associated factors) chromatin remodeling complex and is the second most frequently mutated gene in clear-cell renal cell Carcinoma (ccRCC). Mutation of PBRM1 is believed to be an early event in carcinogenesis, however its function as a tumor suppressor is not understood. In this study, we have employed Next Generation Sequencing to profile the differentially expressed genes upon PBRM1 re-expression in a cellular model of ccRCC. PBRM1 re-expression led to upregulation of genes involved in cellular adhesion, carbohydrate metabolism, apoptotic process and response to hypoxia, and a downregulation of genes involved in different stages of cell division. The decrease in cellular proliferation upon PBRM1 re-expression was confirmed, validating the functional role of PBRM1 as a tumor suppressor in a cell-based model. In addition, we identified a role for PBRM1 in regulating metabolic pathways known to be important for driving ccRCC, including the regulation of hypoxia response genes, PI3K signaling, glucose uptake, and cholesterol homeostasis. Of particular novelty is the identification of cell adhesion as a major downstream process uniquely regulated by PBRM1 expression. Cytoskeletal reorganization was induced upon PBRM1 reexpression as evidenced from the increase in the number of cells displaying cortical actin, a hallmark of epithelial cells. Genes involved in cell adhesion featured prominently in our transcriptional dataset and overlapped with genes uniquely regulated by PBRM1 in clinical specimens of ccRCC. Genes involved in cell adhesion serve as tumor suppressor and maybe involved in inhibiting cell migration. Here we report for the first time genes linked to cell adhesion serve as downstream targets of PBRM1, and hope to lay the foundation of future studies focusing on the role of chromatin remodelers in bringing about these alterations during malignancies.

Metronomic administration of chlorambucil for treatment of dogs with urinary bladder transitional cell carcinoma.

OBJECTIVE: To determine the antitumor effects and toxicoses of metronomic oral administration of a low dose of chlorambucil in dogs with transitional cell carcinoma (TCC). DESIGN: Prospective clinical trial. ANIMALS: 31 client-owned dogs with TCC for which prior treatments had failed or owners had declined other treatments. Procedures-Chlorambucil (4 mg/m2, PO, q 24 h) was administered to dogs. Before and at scheduled times during treatment, evaluations of dogs included physical examination, CBC, serum biochemical analyses, urinalysis, thoracic and abdominal imaging including cystosonography for measurement of TCCs, and grading of toxicoses. RESULTS: 29 of 31 dogs had failed prior TCC treatment. Of the 30 dogs with available data, 1 (3%) had partial remission (≥ 50% reduction in tumor volume), 20 (67%) had stable disease (< 50% change in tumor volume), and 9 (30%) had progressive disease (≥ 50% increase in tumor volume or development of additional tumors); 1 dog was lost to follow-up. The median progression-free interval (time from the start of chlorambucil treatment to the day progressive disease was detected) for the dogs was 119 days (range, 7 to 728 days). The median survival time of dogs from the time of the start of chlorambucil treatment was 221 days (range, 7 to 747 days). Few toxicoses were detected; chlorambucil administration was discontinued because of toxicoses in only 1 dog. CONCLUSIONS AND CLINICAL RELEVANCE: Metronomic administration of chlorambucil was well tolerated, and 70% of dogs had partial remission or stable disease. Metronomic administration of chlorambucil may be a treatment option for dogs with TCC.

Detection of herbicides in the urine of pet dogs following home lawn chemical application.

Exposure to herbicide-treated lawns has been associated with significantly higher bladder cancer risk in dogs. This work was performed to further characterize lawn chemical exposures in dogs, and to determine environmental factors associated with chemical residence time on grass. In addition to concern for canine health, a strong justification for the work was that dogs may serve as sentinels for potentially harmful environmental exposures in humans. Experimentally, herbicides [2,4-dichlorophenoxyacetic acid (2,4-D), 4-chloro-2-methylphenoxypropionic acid (MCPP), dicamba] were applied to grass plots under different conditions (e.g., green, dry brown, wet, and recently mowed grass). Chemicals in dislodgeable residues were measured by LC-MS at 0.17, 1, 24, 48, 72 h post treatment. In a separate study, 2,4-D, MCPP, and dithiopyr concentrations were measured in the urine of dogs and in dislodgeable grass residues in households that applied or did not apply chemicals in the preceding 48 h. Chemicals were measured at 0, 24, and 48 h post application in treated households and at time 0 in untreated control households. Residence times of 2,4-D, MCPP, and dicamba were significantly prolonged (P<0.05) on dry brown grass compared to green grass. Chemicals were detected in the urine of dogs in 14 of 25 households before lawn treatment, in 19 of 25 households after lawn treatment, and in 4 of 8 untreated households. Chemicals were commonly detected in grass residues from treated lawns, and from untreated lawns suggesting chemical drift from nearby treated areas. Thus dogs could be exposed to chemicals through contact with their own lawn (treated or contaminated through drift) or through contact with other grassy areas if they travel. The length of time to restrict a dog's access to treated lawns following treatment remains to be defined. Further study is indicated to assess the risks of herbicide exposure in humans and dogs.

DNMT1: an emerging target in the treatment of invasive urinary bladder cancer.

OBJECTIVES: More than 14,000 people die from invasive urothelial carcinoma (iUC) of the urinary bladder each year in the USA, and more effective therapies are needed. Naturally occurring canine iUC very closely resembles the disease in humans and serves as a highly relevant translational model for novel therapy of human iUC. Work was undertaken to identify new targets for anticancer therapy in dogs with the goal of translating successful therapeutic strategies into humans with iUC. MATERIALS AND METHODS: Microarray expression analyses were conducted on mRNA extracted from canine normal bladder (n = 4) and iUC tissues (n = 4) using Genome Array 1.0 and analyzed by GeneSpring GX 11, with the stringency of P < 0.02 and a ≥ 2-fold change. The genes thus identified were further analyzed for functional and pathway analysis using Protein ANalysis THrough Evolutionary Relationships (PANTHER) Classification System. In selecting genes for further study, consideration was given for evidence of a role of the gene in human iUC. From these analyses, DNA methyltransferase 1 (DNMT1) was selected for further study. Immunohistochemistry (IHC) of canine normal bladder and iUC tissues was performed to confirm the microarray expression analyses. The effects of targeting DNMT1 in vitro was assessed through MTT assay and Western blot of canine iUC cells treated with 5-azacitidine (5-azaC) and trichostatin A (TSA). RESULTS: DNMT1 was expressed in 0 of 6 normal canine bladder samples and in 10 of 22 (45%) canine iUC samples. The proliferation of canine iUC cells was inhibited by 5-azaC (at concentrations ≥ 5 µm) and by TSA (at concentrations ≥ 0.1 µm). Western blot results were supportive of DNMT1-related effects having a role in the antiproliferative activity. CONCLUSIONS: Microarray expression analyses on canine tissues identified DNMT1 as a potentially "targetable" gene. Expression of DNMT1 in canine iUC was confirmed by IHC, and in vitro studies confirmed that drugs that inhibit DNMT1 have antiproliferative effects. These findings are similar to those recently reported in human iUC and are also in line with results of a preclinical (prehuman) trial of 5-azaC in dogs with naturally occurring iUC. DNMT1 has excellent potential as a target for iUC therapy in humans.

Targeting folate receptors to treat invasive urinary bladder cancer.

Folate receptors (FR) may be of use for targeted delivery of cytotoxic drugs in invasive urothelial carcinoma (iUC), for which improved therapy is needed. FR expression and function in iUC were explored and the antitumor activity and toxicity of a folate-targeted vinblastine conjugate were evaluated in dogs with naturally occurring iUC, an excellent model for human iUC. FR immunohistochemistry was carried out on iUC and normal human and dog bladder tissues together with nuclear scintigraphy in dogs to monitor iUC folate uptake. Dose escalation of a folate-targeted vinblastine compound, EC0905, was conducted in dogs with biopsy-confirmed, FR-positive iUC. FRs were detected by immunohistochemistry (PU17) in most primary iUC and many nodal and lung metastases from dogs, and scintigraphy confirmed folate uptake in both primary and metastatic lesions. The maximum tolerated dose of EC0905 in dogs was 0.25 mg/kg IV weekly, with neutropenia at higher doses. Tumor responses included partial remission (≥ 50% reduction in tumor volume) in five dogs and stable disease (<50% change in tumor volume) in four dogs. Immunoreactivity to PU17 was similar in humans (78% of primary iUC, 80% of nodal metastases). Less immunoreactivity to mab343 (22% of cases) occurred. FR-ß was noted in 21% of human iUC cases. Our findings suggest folate-targeted therapy holds considerable promise for treating iUC, where FR-ß may be important in addition to FR-α.

Effects of short-term celecoxib treatment in patients with invasive transitional cell carcinoma of the urinary bladder.

High-grade invasive transitional cell carcinoma (InvTCC) kills >14,000 people yearly in the United States, and better therapy is needed. Cyclooxygenase-2 (Cox-2) is overexpressed in bladder cancer. Cox inhibitors have caused remission of InvTCC in animal studies, and cancer regression was associated with doubling of the apoptotic index in the tumor. The purpose of this study was to determine the apoptosis-inducing effects of celecoxib (a Cox-2 inhibitor) in InvTCC in humans. Patients (minimum of 10 with paired tumor samples) with InvTCC who had elected to undergo cystectomy were enrolled. The main study end point was induction of apoptosis in tumor tissues. Patients received celecoxib (400 mg twice daily p.o. for a minimum of 14 days) between the time of diagnosis [transurethral resection of bladder tumor (TURBT)] and the time of cystectomy (standard frontline treatment for InvTCC). Terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling assay and immunohistochemistry were done on TURBT and cystectomy samples. Of 13 cases treated with celecoxib, no residual invasive cancer was identified in 3 patients at the time of cystectomy (post celecoxib). Of the 10 patients with residual cancer, 7 had induction of apoptosis in their tumor. Induction of apoptosis was less frequent (3 of 13 cases; P < 0.04) in control patients not receiving a Cox inhibitor. Expression of vascular endothelial growth factor in the tumor cells decreased more frequently (P < 0.026) in the treated patients as compared with nontreated control cases. The biological effects of celecoxib treatment (increased apoptosis) justify further study of the antitumor effects of Cox-2 inhibitors in InvTCC.

Effects of cyclooxygenase inhibitor treatment on the renal toxicity of cisplatin in rats.

PURPOSE: The purpose of this study was to determine the effects of a nonselective cyclooxygenase (cox) inhibitor and of a selective cox-2 inhibitor on the renal toxicity of cisplatin. METHODS: Cisplatin with or without a cox-1 inhibitor (SC560), a cox-2 inhibitor (SC236), or a nonselective cox inhibitor (piroxicam) was administered to Sprague-Dawley rats. Renal toxicity was assessed by serum creatinine concentration (SCR), urine specific gravity (USG), and histopathologic lesion score (HLS). RESULTS: Acutely, the SCR was significantly higher in rats receiving cisplatin/SC560 (1.62+/-0.34 mg/dl) or cisplatin/piroxicam (2.0+/-0.41 mg/dl) than in rats receiving cisplatin alone (1.09+/-0.40 mg/dl). The apparent increase in SCR in the rats receiving cisplatin/SC236 (1.58+/-0.31) was not significantly different from that of rats receiving cisplatin alone (1.09+/-0.40 mg/dl). No significant differences in USG or HLSs were noted between rats receiving cisplatin alone and cisplatin combined with any cox inhibitor. In a chronic study, no differences in renal toxicity were found between rats treated with cisplatin alone and cisplatin/SC236 or cisplatin/piroxicam. CONCLUSIONS: The acute rise in SCR following cisplatin treatment can be worsened by the addition of cox inhibitors, especially those that inhibit cox-1.

Canine invasive transitional cell carcinoma cell lines: in vitro tools to complement a relevant animal model of invasive urinary bladder cancer.

OBJECTIVES: Urinary bladder cancer is the fifth most common form of cancer in humans in the United States. Urinary bladder cancer also occurs in pet dogs, and naturally-occurring bladder cancer in pet dogs very closely resembles invasive bladder cancer (intermediate to high grade invasive transitional cell carcinoma, InvTCC) in humans. Pet dogs with InvTCC offer a highly relevant resource for preclinical studies in bladder cancer. For translational research in which findings are moved from in vitro experiments through in vivo studies in dogs to human trials, access to human and canine bladder cancer cell lines is important. Cell lines derived from canine InvTCC have been lacking. Here we describe eight such cell lines. MATERIALS AND METHODS: Eight cell lines were established from canine InvTCC. Cells were characterized using immunocytochemistry, evaluated for anchorage independent growth in soft agar, and assessed for tumorigenicity in athymic mice. Western blotting was used to identify expression of proteins of interest in human InvTCC. RESULTS: The cell lines were confirmed to be of epithelial origin by their expression of cytokeratin and E-cadherin. Seven cell lines were found to be tumorigenic in athymic mice, and 4 of these cell lines grew in an anchorage independent manner. The cell lines expressed several proteins of interest associated with bladder cancer prognosis and progression in humans, including p53, cox-2, and pRb protein. CONCLUSIONS: These established cell lines can be used for comparative bladder cancer research and to evaluate new therapy approaches in vitro prior to in vivo testing.

Cyclooxygenase-2 dependent and independent antitumor effects induced by celecoxib in urinary bladder cancer cells.

Transitional cell carcinoma of the urinary bladder is the second most common genitourinary malignancy in people in the United States. Cyclooxygenase-2 (COX-2) is overexpressed in bladder cancer. COX-2 inhibitors have had antitumor activity against bladder cancer, but the mechanisms of action are unclear. Clinically relevant concentrations of COX-2 inhibitors fail to inhibit proliferation in standard in vitro assays. In pilot experiments, different culture conditions [standard monolayer, modified monolayer, soft agar, collagen, and poly(2-hydroxyethyl methacrylate)-coated plates] were assessed to determine conditions suitable for the study of COX inhibitor growth-inhibitory effects. This was followed by studies of the effects of clinically relevant concentrations of a selective COX-2 inhibitor (celecoxib) on urinary bladder cancer cell lines (HT1376, TCCSUP, and UMUC3). Celecoxib (

E-cadherin binding modulates EGF receptor activation.

We found that E-cadherin and epidermal growth factor receptor (EGFR) are associated in mammary epithelial cells and that E-cadherin engagement in these cells induces transient activation of EGFR, as previously seen in keratinocytes (37). In contrast, EGFR does not associate with and is not activated by N-cadherin. Analysis of cells expressing chimeric cadherins revealed that the extracellular domain of E-cadherin is required for interaction with and activation of EGFR. This activation results in tyrosine phosphorylation of known EGFR substrates and reduction in focal adhesions. These interactions, however, are not necessary for suppression of cell motility by E-cadherin.

Antibody targeting of the EphA2 tyrosine kinase inhibits malignant cell behavior.

EphA2 is a transmembrane receptor tyrosine kinase that is up-regulated on many aggressive carcinoma cells. Despite its overexpression, the EphA2 on malignant cells fails to bind its ligand, ephrinA1, which is anchored to the membrane of adjacent cells. Unlike other receptor kinases, EphA2 demonstrates kinase activity that is independent of ligand binding. However, ligand binding causes EphA2 to negatively regulate tumor cell growth and migration. Herein, we translate knowledge of EphA2 into strategies that selectively target malignant cells. Using a novel approach to preserve extracellular epitopes and optimize antibody diversity, we generated monoclonal antibodies that identify epitopes on the extracellular domain of EphA2. EphA2 antibodies were selected for their abilities to inhibit behaviors that are unique to metastatic cells while minimizing damage to nontransformed cells. A subset of EphA2 monoclonal antibodies were found to inhibit the soft agar colonization by MDA-MB-231 breast tumor cells but did not affect monolayer growth by nontransformed MCF-10A breast epithelial cells. These EphA2 antibodies also prevented tumor cells from forming tubular networks on reconstituted basement membranes, which is a sensitive indicator of metastatic character. Biochemical analyses showed that biologically active antibodies induced EphA2 phosphorylation and subsequent degradation. Antisense-based targeting of EphA2 similarly inhibited soft agar colonization, suggesting that the antibodies repress malignant behavior by down-regulating EphA2. These results suggest an opportunity for antibody-based targeting of the many cancers that overexpress EphA2. Our studies also emphasize how tumor-specific cellular behaviors can be exploited to identify and screen potential therapeutic targets.