Publications

PURPOSE: We assessed the ability of different classes of histone deacetylase inhibitors to target tumor and invasive suppressor genes in a panel of bladder carcinoma cell lines using reverse phase protein arrays.

MATERIALS AND METHODS: Three poorly, moderately and highly invasive cell lines were exposed to histone deacetylase inhibitors, trichostatin A, apicidin, valproic acid (Sigma) and MS-275 (AXXORA) for 0 to 36 hours. Lysates were harvested and arrayed in a 10-fold dilution series in duplicate. Data points were collected and analyzed using a concentration interpolation methodology after normalization.

RESULTS: Protein expression profiles revealed up-regulation of gamma-catenin in highly invasive lines, and alpha-catenin in moderately and highly invasive lines after exposure to all histone deacetylase inhibitors, apicidin and MS-275, respectively. Gelsolin was up-regulated in poorly and moderately invasive lines after exposure to all histone deacetylase inhibitors. Desmoglein was down-regulated in poorly and moderately invasive cell lines by all 4 histone deacetylase inhibitors, in addition to decreased FAK (Transduction Laboratories) expression in moderately and highly invasive lines exposed to valproic acid and MS-275.

CONCLUSIONS: Different histone deacetylase inhibitor classes have the potential to modulate tumor and invasive suppressor gene expression, identifying histone deacetylase inhibitors as potential therapeutic agents for bladder cancer. Reverse phase protein arrays enable high throughput screening of multiple compounds to assess the expression profile of specific protein groups targeted for therapy.

OBJECTIVE: To identify the frequency of change in the expression and localization of p120(ctn) in bladder tumours and its association with clinical outcomes, and to investigate the potential role of p120(ctn) in the migratory and invasive behaviour of bladder carcinoma cells.

MATERIALS AND METHODS: In all, 425 superficial tumour specimens (Ta, Tis and T1) and 305 invasive (T2-T4) tumour specimens from 534 patients were assembled in 10 tissue microarrays. P120(ctn) immunostaining was scored for intensity and cellular localization and correlated with clinical variables and survival analysis. Knockdown of p120(ctn) was achieved using small-interference RNA (siRNA) followed by the assessment of migration and invasion behaviour in standard in vitro assays.

RESULTS: The expression levels of p120 catenin inversely correlated with pathological tumour stage (P < 0.001), histological grade (P < 0.001), presence of lymphovascular invasion (P = 0.02) but not lymph node (LN) involvement (P = 0.17). Non-membranous localization of p120(ctn) correlated with stage (P < 0.001), grade (P < 0.001), lymphovascular invasion (P = 0.04) and LN-positive disease (P = 0.02). A low expression level of p120(ctn) was linked to a poor outcome in cancer-specific survival analysis. Knockdown of p120(ctn) using siRNA resulted in a significant reduction in the migration and invasive potential of bladder carcinoma cells.

CONCLUSIONS: Our findings suggest that p120(ctn) acts as a prognostic factor in bladder tumours and has a primary role to play in the migratory and invasive behaviour of bladder carcinoma cells.

OBJECTIVE: To identify changes associated with P-cadherin expression in bladder cancer and evaluate the potential role of such events in determining the clinical outcome and cell behaviour, as the function of P-cadherin in normal epithelium is unknown, as is its potential role in neoplastic progression in different cancers.

MATERIALS AND METHODS: In all, 536 bladder tumour specimens from 408 patients were assembled in seven tissue microarrays. Paraffin sections from each array were processed for immunohistochemistry to assess the expression of P-cadherin. The expression of P-cadherin was forced using lipofectin, followed by an assessment of migration and invasion potential using standard in vitro assays.

RESULTS: The absence of P-cadherin staining was associated with muscle-invasive disease, grade 3 (P < 0.001) and nodal disease (P = 0.009). Similar results were obtained when considering cytoplasmic and unrestricted localization of P-cadherin (P < 0.001), except for nodal involvement. The group with cytoplasmic location of P-cadherin showed a shorter cancer-specific survival than the group with membrane location of P-cadherin (P = 0.03). Forced expression of P-cadherin in EJ and UM-UC-3 cells, that constitutively lack P-cadherin expression, resulted in modulation of catenin expression and enhanced migration of EJ and UM-UC-3/P-cadherin transfectants (>200%).

CONCLUSIONS: These results showed that loss of expression, cytoplasmic relocation or unrestricted tissue location of P-cadherin was associated with a poor clinical outcome and prognosis in bladder cancer. From the in vitro work it is evident that P-cadherin plays a role in regulating the migration potential of bladder carcinoma cells.

Green tea has been reported as potential dietary protection against numerous cancers and has been shown to have activity in bladder tumor inhibition in different animal models. The goal of this study was to examine the effects of (-)-epigallocatechin gallate (EGCG-the major phytochemical in green tea) on growth inhibition and behavior of human bladder carcinoma cells and to identify the altered signaling pathway(s) underlying the response to EGCG exposure. EGCG inhibited the in vitro growth of invasive bladder carcinoma cells with an IC(50) range of 70-87 microM. At a concentration of 20 microM, EGCG decreased the migratory potential of bladder carcinoma cells with concomitant activation of p42/44 MAPK and STAT3 and inactivation of Akt. Using biochemical inhibitors of MAPK/ERK, and siRNA to knockdown STAT3 and Akt, inhibition of migration was recorded associated with Akt but not MAPK/ERK or STAT3 signaling in bladder cells. In addition, EGCG downregulated N-cadherin in a dose-dependent manner where reduction in N-cadherin expression paralleled declining migratory potential. Continuous feeding of EGCG to mice prior to and during the establishment of bladder carcinoma xenografts in vivo revealed >50% reduction in mean final tumor volume (P </= 0.05) with no detectable toxicity. EGCG inhibited bladder carcinoma cell growth and suppressed the in vitro migration capacity of cells via downregulation of N-cadherin and inactivation of Akt signaling. Continuous administration of EGCG to mice revealed significant inhibition of tumor growth in vivo indicating a possible preventative role for green tea in bladder cancer.

PURPOSE: Epithelial to mesenchymal transition (EMT) is reportedly an important transition in cancer progression in which the underlying cellular changes have been identified mainly using in vitro models. In this study, we examined the expression pattern of EMT markers in vivo and determined the occurrence and clinical significance of these events in a series of bladder carcinomas.

EXPERIMENTAL DESIGN: Eight hundred and twenty-five tumor samples from 572 bladder cancer patients were assembled in 10 tissue microarrays. Paraffin sections from each tissue microarray were subjected to antigen retrieval and processed by immunohistochemistry for the expression of E-cadherin, plakoglobin, beta-catenin, N-cadherin, and vimentin.

RESULTS: Pathologic expression of E-cadherin, beta-catenin, plakoglobin, and vimentin were associated with the clinicopathologic variables of grade and stage with only the cytoplasmic localization of plakoglobin found associated with lymph node status. Associations between the aforementioned markers were found significant as determined by the Spearman correlation coefficient with N-cadherin showing no associations in this analysis. In univariate survival analysis involving patients who underwent cystectomy, the reduction or loss of plakoglobin significantly influenced overall survival (P = 0.02) in which the median time to death was 2 years compared with 4 years when a normal level of plakoglobin was recorded. When the analysis was done for cancer-specific survival, low levels of both plakoglobin (P = 0.02) and beta-catenin (P = 0.02) significantly influenced survival.

CONCLUSION: The putative markers of EMT defined within a panel of bladder carcinoma cell lines were recorded in vivo, frequently associated with tumors of high grade and stage. Although multivariate analysis showed no significant influence of the EMT biomarkers on survival, alterations associated with plakoglobin were identified as significant prognostic features in these tumors.

Invasive breast cancer has a high risk of recurrence to incurable disease and needs improved prognostic and therapeutic tools. Our work combines clinical and molecular analyses to show that the transcriptional repressor HBP1 may be a new target for invasive breast cancer. Previous work indicated that HBP1 regulated proliferation and senescence and inhibited Wnt signaling. Two of these functions have been associated with invasive breast cancer. In 76 breast tumors, we identified 10 HBP1 mutations/variants that were associated with fully invasive breast cancer. In a separate analysis, we found that a subset of invasive breast cancer specimens also had reduced HBP1 mRNA levels. These clinical correlations suggested that mutation or reduction of HBP1 occurs in invasive breast cancer and that HBP1 might regulate the proliferation and invasiveness of this breast cancer type. Analysis of the HBP1 mutants showed they were functionally defective for suppressing Wnt signaling. To test the consequences of reduced HBP1 levels, we used RNA interference to knock down HBP1 and observed increased Wnt signaling, tumorigenic proliferation, and invasiveness in cell and animal breast cancer models. Lastly, statistical analysis of a breast cancer patient database linked reduced HBP1 expression to breast cancer recurrence. In considering two-gene criteria for relapse potential, reduced expression of HBP1 and SFRP1, which is another Wnt inhibitor that was recently linked to invasive breast cancer, strikingly correlated with recurrence. Together, these data indicate that HBP1 may be a molecularly and clinically relevant regulator of breast cancer transitions that eventually lead to poor prognosis.

PURPOSE: We established the frequency of mutation of the epidermal growth factor receptor in bladder cancer and determined whether the activation status of epidermal growth factor receptor confers sensitivity to erlotinib.

MATERIALS AND METHODS: The identification of mutations in the kinase domain (exons 18-21) of epidermal growth factor receptor was performed using single strand conformation polymorphism. The action of erlotinib was established within a bladder carcinoma cell panel using clonogenic assays and Western blot analysis.

RESULTS: In 112 invasive bladder tumors a total of 6 mutations in 4 patients (3.6%) were identified in exon 21. Erlotinib demonstrated concentration dependent inhibition of growth where three cell lines showed high and 2 showed low sensitivity to the drug. Erlotinib inhibited activation of epidermal growth factor receptor, mitogen activated protein kinase, Akt and STAT3. However, the activation status of Akt was maintained in cell lines that were insensitive to the inhibitory action of erlotinib and were characterized as having undergone epithelial-to-mesenchymal transition.

CONCLUSIONS: Although mutations in the coding region of epidermal growth factor receptor are rare in invasive bladder tumors, differential sensitivity to erlotinib was recorded within a panel of cell lines. Maintenance of the phosphorylation status of Akt in the presence of erlotinib along with epithelial-to-mesenchymal transition correlates with insensitivity to growth inhibition in bladder carcinoma cell lines. Even in the absence of epidermal growth factor receptor mutations erlotinib shows potential as a therapeutic agent for the treatment of bladder cancer.

Genetic and biochemical de-regulation of Wnt signaling is correlated with breast and other cancers. Our goal was to identify compounds that block Wnt signaling as a first step toward investigating new strategies for suppression of invasive and other breast cancers. In a limited phytonutrient screen, EGCG ((-)-epigallocatechin 3-gallate), the major phytochemical in green tea, emerged as an intriguing candidate. Epidemiological studies have associated green tea consumption with reduced recurrence of invasive and other breast cancers. Wnt signaling was inhibited by EGCG in a dose-dependent manner in breast cancer cells. The apparent mechanism targeted the HBP1 transcriptional repressor, which we had previously characterized as a suppressor of Wnt signaling. EGCG treatment induced HBP1 transcriptional repressor levels through an increase in HBP1 mRNA stability, but not transcriptional initiation. To test functionality, DNA-based short hairpin RNA (shRNA) was used to knockdown the endogenous HBP1 gene. Consistently, the HBP1 knockdown lines had reduced sensitivity to EGCG in the suppression of Wnt signaling and of a target gene (c-MYC). Because our ongoing studies clinically link abrogation of HBP1 with invasive breast cancer, we tested if EGCG also regulated biological functions associated with de-regulated Wnt signaling and with invasive breast cancer. EGCG reduced both breast cancer cell tumorigenic proliferation and invasiveness in an HBP1-dependent manner. Together, the emerging mechanism is that EGCG blocks Wnt signaling by inducing the HBP1 transcriptional repressor and inhibits aspects of invasive breast cancer. These studies provide a framework for considering future studies in breast cancer treatment and prevention.

PURPOSE: The aim of this study was to evaluate the utility of the DNA integrity assay (DIA) as a plasma-based screening tool for the detection of prostate cancer.

EXPERIMENTAL DESIGN: Blood samples were collected from patients with biopsy-proven prostate cancer prior to prostatectomy (n = 123) and processed as two-spin plasma preparations. The three control groups included: males <40 years old with no history of cancer (group 1, n = 20); cancer-free postprostatectomy patients (group 2, n = 25), and patients with a negative prostate biopsy (group 3, n = 22). DNA in plasma preparations were isolated, hybrid-captured, and DNA fragments (200 bp, 1.3, 1.8, and 2.4 kb) were multiplexed in real-time PCR. A baseline cutoff was determined for individual fragment lengths to establish a DIA score for each patient sample.

RESULTS: Patients with prostate cancer (86 of 123; 69.9%) were determined to have a positive DIA score of >or=7. The DIA results from control groups 1, 2, and 3 showed specificities of 90%, 92%, and 68.2%, respectively. Of the patients with negative age-adjusted prostate-specific antigen (PSA) and prostate cancer, 19 of 30 (63%) had a positive DIA score. The area under the receiver operating characteristic curve for DIA was 0.788.

CONCLUSION: While detecting 69.9% of those with prostate cancer, DIA maintained an overall specificity of 68.2% to 92%, a range favorably comparable to that currently accepted for PSA (60-70%). The variability in specificity between control groups is likely explained by the established 19% to 30% detection of prostate cancer on subsequent biopsies associated with control group 3. DIA detected 63% of the prostate cancers undetected by currently accepted PSA ranges.

OBJECTIVE: To evaluate PP2 as a modulator of the cadherin/catenin complex in late-stage bladder carcinoma cells, and to assess its potential invasion-suppressor activity in this model.

MATERIALS AND METHODS: A panel of five human bladder carcinoma cells, characterizing late-stage disease, was used to determine the concentration for 50% inhibition of PP2 in cell-proliferation assays. Modulation of cadherin/catenin expression by PP2 was determined in Western blot analysis, with an assessment of the activation status of mitogen-activated protein kinase and Akt signalling pathways. Altered invasive capacity linked to these variables was determined in standard in vitro invasion assays.

RESULTS: PP2 elicited concentration-dependent growth inhibition in all bladder cell lines within the panel, with growth suppression recorded at 10-35 micromol/L PP2. Distinct morphological changes were recorded in cell lines exposed to PP2, accompanied by up-regulation of plakoglobin expression in a subset of lines. Exposure of cells to PP2 resulted in inactivation of Akt in all cells and a concomitant reduction in in vitro invasive capacity.

CONCLUSIONS: These results show that PP2 inhibits bladder carcinoma cell growth and can modulate plakoglobin expression in a subset of cell lines. In addition, PP2 can suppress the in vitro invasive capacity of bladder carcinoma cells by modulating the activation status of Akt.