Faculty Bibliography

PURPOSE: Extracellular matrix remodeling during tumor growth plays an important role in angiogenesis. Our preclinical data suggest that a newly identified cryptic epitope (HU177) within collagen type IV regulates endothelial and melanoma cell adhesion in vitro and angiogenesis in vivo. In this study, we investigated the clinical relevance of HUI77 shedding in melanoma patient sera. EXPERIMENTAL DESIGN: Serum samples from 291 melanoma patients prospectively enrolled at the New York University Medical Center and 106 control subjects were analyzed for HU177 epitope concentration by a newly developed sandwich ELISA assay. HU177 serum levels were then correlated with clinical and pathologic parameters. RESULTS: Mean HU177 epitope concentration was 5.8 ng/mL (range, 0-139.8 ng/mL). A significant correlation was observed between HU177 concentration and nodular melanoma histologic subtype [nodular, 10.3 +/- 1.6 ng/mL (mean +/- SE); superficial spreading melanoma, 4.5 +/- 1.1 ng/mL; all others, 6.1 +/- 2.1 ng/mL; P = 0.01 by ANOVA test]. Increased HU177 shedding also correlated with tumor thickness (< or =1.00 mm, 3.8 +/- 1.1 ng/mL; 1.01-3.99 mm, 8.7 +/- 1.3 ng/mL; > or =4.00 mm, 10.3 +/- 2.4 ng/mL; P = 0.003 by ANOVA). After multivariate analysis controlling for thickness, the correlation between higher HU177 concentration and nodular subtype remained significant (P = 0.03). The mean HU177 epitope concentration in control subjects was 2.4 ng/mL. CONCLUSIONS: We report that primary melanoma can induce detectable changes in systemic levels of cryptic epitope shedding. Our data also support that nodular melanoma might be biologically distinct compared with superficial spreading type melanoma. As targeted interventions against cryptic collagen epitopes are currently undergoing phase I clinical trial testing, these findings indicate that patients with nodular melanoma may be more susceptible to such targeted therapies

OBJECTIVES: The role of socioeconomic factors in the worse outcome of black men with prostate cancer remains unclear. To determine whether socioeconomic factors affect prostate cancer outcomes, we studied a cohort of only black patients to minimize known confounding factors. METHODS: We studied black men treated with radical prostatectomy at New York Veterans Administration Medical Center and Memorial Sloan-Kettering Cancer Center between 1990 and 2005. A centralized pathology review process determined the Gleason score of all cases. Prostate-specific antigen (PSA) recurrence at both sites was defined as PSA of 0.2 or greater with a confirmatory rise. By matching patients' home zip codes to the U.S. Census Bureau database, we obtained corresponding socioeconomic data regarding median household income (income) and percentage of population with a high school (degree). We analyzed income, education, and clinical and pathological parameters for the whole cohort. RESULTS: We studied 430 black patients. They resided in neighborhoods where median household income was $41,498.10 and mean percentage of high school graduates was 73.4%. A total of 88 patients (20.9%) had PSA recurrence. Median follow-up for survivors was 37 months. Neither income nor education evaluated as continuous or categorical variables were predictors of PSA recurrence. When evaluated as composite categorical variable, the combination of greater income and education did not predict disease-free survival. CONCLUSIONS: Data suggest that socioeconomic factors have limited impact on PSA recurrence in black men treated with radical prostatectomy. Thus, biologic factors might have a role in the poor outcomes in this population

Most metastatic melanoma patients fail to respond to available therapy, underscoring the need for novel approaches to identify new effective treatments. In this study, we screened 2,000 compounds from the Spectrum Library at a concentration of 1 micromol/L using two chemoresistant melanoma cell lines (M-14 and SK-Mel-19) and a spontaneously immortalized, nontumorigenic melanocyte cell line (melan-a). We identified 10 compounds that inhibited the growth of the melanoma cells yet were largely nontoxic to melanocytes. Strikingly, 4 of the 10 compounds (mebendazole, albendazole, fenbendazole, and oxybendazole) are benzimidazoles, a class of structurally related, tubulin-disrupting drugs. Mebendazole was prioritized to further characterize its mechanism of melanoma growth inhibition based on its favorable pharmacokinetic profile. Our data reveal that mebendazole inhibits melanoma growth with an average IC(50) of 0.32 micromol/L and preferentially induces apoptosis in melanoma cells compared with melanocytes. The intrinsic apoptotic response is mediated through phosphorylation of Bcl-2, which occurs rapidly after treatment with mebendazole in melanoma cells but not in melanocytes. Phosphorylation of Bcl-2 in melanoma cells prevents its interaction with proapoptotic Bax, thereby promoting apoptosis. We further show that mebendazole-resistant melanocytes can be sensitized through reduction of Bcl-2 protein levels, showing the essential role of Bcl-2 in the cellular response to mebendazole-mediated tubulin disruption. Our results suggest that this screening approach is useful for identifying agents that show promise in the treatment of even chemoresistant melanoma and identifies mebendazole as a potent, melanoma-specific cytotoxic agent

BACKGROUND: The tumor suppressors p14(ARF) (ARF) and p16(INK4A) (p16) are encoded by overlapping reading frames at the CDKN2A/INK4A locus on chromosome 9p21. In human melanoma, the accumulated evidence has suggested that the predominant tumor suppressor at 9p21 is p16, not ARF. However, recent observations from melanoma-prone families and murine melanoma models suggest a p16-independent tumor suppressor role for ARF. We analyzed a group of melanoma metastases and cell lines to investigate directly whether somatic alterations to the ARF gene support its role as a p16-independent tumor suppressor in human melanoma, assuming that two alterations (genetic and/or epigenetic) would be required to inactivate a gene. METHODS: We examined the p16/ARF locus in 60 melanoma metastases from 58 patients and in 9 human melanoma cell lines using multiplex ligation-dependent probe amplification and multiplex polymerase chain reaction (PCR) to detect deletions, methylation-specific PCR to detect promoter methylation, direct sequencing to detect mutations affecting ARF and p16, and, in a subset of 20 tumors, immunohistochemistry to determine the effect of these alterations on p16 protein expression. All statistical tests were two-sided. RESULTS: We observed two or more alterations to the ARF gene in 26/60 (43%) metastases. The p16 gene sustained two or more alterations in 13/60 (22%) metastases (P = .03). Inactivation of ARF in the presence of wild-type p16 was seen in 18/60 (30%) metastases. CONCLUSION: Genetic and epigenetic analyses of the human 9p21 locus indicate that modifications of ARF occur independently of p16 inactivation in human melanoma and suggest that ARF is more frequently inactivated than p16

Background: Although remission rates for metastatic melanoma are generally very poor, some patients can survive for prolonged periods following metastasis. Here we use gene expression profiling of metastatic melanoma samples to search for a molecular basis for this observation. Methods: We analyzed gene expression profiles of 44 metastatic melanoma specimens collected from 38 patients who were followed clinically for a median of 20 months after surgery. RNA was processed using standard methods and hybridized to Affymetrix Human Genome HU133 Plus 2.0 arrays. Data were analyzed using GeneSpring and PathwayAssist software, normalizing using a PLIER algorithm and utilizing ANOVA statistical tests. Results: Unsupervised clustering yielded 4 distinct groups of subjects, 2 of which had large differences in overall survival. We then used supervised clustering to compare patients whose post-surgery survival was less then 1.5 years (11 subjects, 9.5 month median survival) to those who survived greater than 1.5 years (23 subjects, 26 month median survival, 17 alive at last follow-up). Only genes with changes in expression of > 2 with a p value of < 0.05 were accepted, resulting in a group of approximately 200 genes. Genes associated with immune response (TNFa, IRF1, Granzyme B, CD8a, CXCL11, IL27AR, GBP1, CXCL10, CCBP2, GBP2, CCR9 and IFIT4) or with cell proliferation (FGFR1, MET, MDM2, CDC25A, RFC2, SOS1, HOXA3, MCM4, MCM7, ORC5L, KIF23 and VAV3) were highly represented. Prolonged survival was associated with elevated expression of immune response genes and decreased expression of genes associated with cell proliferation. Conclusions: Gene expression profiling of metastatic melanoma samples identified a set of genes associated with patient survival, and suggests that immune surveillance is a mechanism for prolonged survival in these patients

While epidermal growth factor receptor (EGFR) dysregulation is known to play a critical role in prostate carcinogenesis, there has been no direct evidence indicating EGFR mutations induce tumorigenesis in prostate cancer. We previously identified four novel EGFR somatic mutations in the EGFR tyrosine kinase domain of prostate cancer patients: G735S, G796S, E804G and R841K. In this study, we investigated the oncogenic potential of these somatic mutations by establishing stable clonal NIH3T3 cells expressing these four mutations and WT EGFR to determine their ability to increase cell proliferation and invasion. In the absence of the EGF ligand, cell proliferation was readily increased in G735S, G796S and E804G mutants compared to WT EGFR. The addition of EGF ligand greatly increased cell growth and transforming ability of these same EGFR mutants. Matrigel invasion assays showed enhanced invasion with G735S, G796S and E804G mutants. Western blot analysis showed that these EGFR mutations enhanced cell growth and invasion via constitutive and hyperactive tyrosine phosphorylation and led to the activation of mitogen-activated protein kinase (MAPK), signal transducer and activator of transcription 3 (STAT3) and Akt pathways. Our findings demonstrate the oncogenic activation of three novel EGFR somatic missense mutations in prostate cancer. Molecules that regulate the mechanisms of their oncogenic activation represent novel targets for limiting tumor cell progression, and further elucidation of these mutations will have utility in prostate cancer treatment.Oncogene advance online publication, 14 January 2008; doi:10.1038/sj.onc.1210983

Despite the increasing use of gene transfer strategies in the study of cellular and molecular biology, melanoma cells have remained difficult to transfect in a safe, efficient, and reproducible manner. In the present study, we report the successful use of nucleofector technology to transfect human melanoma cell lines. This technology uses an empirically derived combination of cell line-specific solutions and nucleofector programmes to electroporate nucleic acid substrates directly into the cell nucleus. Using a colorimetric beta-galactosidase assay, we optimized nucleofection parameters for 13 melanoma cell lines, leading to maximum transfection efficiency and cell survival. The combinations of cell solutions NHEM or T and nucleofector programmes A-24 or U-20 produced the best results. We compared nucleofection with two commercially available lipid-based gene transfer systems, effectene and lipofectamine 2000 using a green fluorescent protein reporter vector. Nucleofection demonstrated a 3- to 40-fold improvement in transfection efficiency when compared with the lipid-based counterparts. Nucleofection was also superior in transfecting small-interfering RNA (siRNA) as determined by Western blot analysis. Lastly, we applied nucleofection to the simultaneous transfection of a p53-dependent luciferase plasmid and p53-siRNA. Experiments using dual transfection showed knockdown of p53 expression and silencing of the reporter plasmid. In conclusion, nucleofection is highly effective for the transfer of nucleic acid substrates, singly or in combination, into human melanoma cell lines

OBJECTIVE: To determine the utility of the current diameter criterion of larger than 6 mm of the ABCDE acronym for the early diagnosis of cutaneous melanoma. DESIGN: Cohort study. SETTING: Dermatology hospital-based clinics and community practice offices. Patients A total of 1323 patients undergoing skin biopsies of 1657 pigmented lesions suggestive of melanoma. MAIN OUTCOME MEASURE: The maximum lesion dimension (diameter) of each skin lesion was calculated before biopsy using a novel computerized skin imaging system. RESULTS: Of 1657 biopsied lesions, 853 (51.5%) were 6 mm or smaller in diameter. Invasive melanomas were diagnosed in 13 of 853 lesions (1.5%) that were 6 mm or smaller in diameter and in 41 of 804 lesions (5.1%) that were larger than 6 mm in diameter. In situ melanomas were diagnosed in 22 of 853 lesions (2.6%) that were 6 mm or smaller in diameter and in 62 of 804 lesions (7.7%) that were larger than 6 mm in diameter. Conclusion The diameter guideline of larger than 6 mm provides a useful parameter for physicians and should continue to be used in combination with the A, B, C, and E criteria previously established in the selection of atypical lesions for skin biopsy

ABSTRACT: BACKGROUND: Treatment options for patients with recurrent superficial bladder cancer are limited, necessitating aggressive exploration of new treatment strategies that effectively prevent recurrence and progression to invasive disease. We assessed the effects of belinostat (previously PXD101), a novel histone deacetylase inhibitor, on a panel of human bladder cancer cell lines representing superficial and invasive disease, and on a transgenic mouse model of superficial bladder cancer. METHODS: Growth inhibition and cell cycle distribution effect of belinostat on 5637, T24, J82, and RT4 urothelial lines were assessed. Ha-ras transgenic mice with established superficial bladder cancer were randomized to receive either belinostat or vehicle alone, and assessed for bladder weight, hematuria, gene expression profiling, and immunohistochemistry (IHC). RESULTS: Belinostat had a significant linear dose-dependent growth inhibition on all cell lines (IC50 range of 1.0-10.0 micromolar). The 5637 cell line, which was derived from a superficial papillary tumor, was the most sensitive to treatment. Belinostat (100 mg/kg, intraperitoneal, 5 days each week for 3 weeks) treated mice had less bladder weight (p<0.05), and no hematuria compared with 6/10 control mice that developed at least one episode. IHC of bladder tumors showed less cell proliferation and a higher expression of p21WAF1 in the belinostat-treated mice. Gene expression profile analysis revealed 56 genes significantly different in the treated group; these included the upregulation of p21WAF1, induction of core histone deacetylase (HDAC), and cell communication genes. CONCLUSION: Our data demonstrate that belinostat inhibits bladder cancer and supports the clinical evaluation of belinostat for the treatment of patients with superficial bladder cancer