Faculty Bibliography

In this study, we investigated the mechanism(s) of altered expression of protooncogene SKP2 in metastatic melanoma and its clinical relevance in patients with metastatic melanoma. The genomic status of SKP2 was assessed in cell lines by sequencing, single nucleotide polymorphism array, and genomic PCR. Copy number status was then evaluated for concordance with SKP2 mRNA and protein expression. SKP2 protein was further evaluated by immunohistochemistry in 93 human metastatic tissues. No mutations were identified in SKP2. Increased copy number at the SKP2 locus was observed in 6/14 (43%) metastatic cell lines and in 9/22 (41%) human metastatic tissues which was associated with overexpression of SKP2 protein. Overexpression of SKP2 protein in human tissues was associated with worse survival in a multivariate model controlling for the site of metastasis. Copy number gain is a major contributing mechanism of SKP2 overexpression in metastatic melanoma. Results may have implications for the development of therapeutics that target SKP2

Targeting oncogenic microRNAs (miRNAs) is emerging as a promising strategy for cancer therapy. In this study, we provide proof of principle for the safety and efficacy of miRNA targeting against metastatic tumors. We tested the impact of targeting miR-182, a pro-metastatic miRNA frequently overexpressed in melanoma, the in vitro silencing of which represses invasion and induces apoptosis. Specifically, we assessed the effect of anti-miR-182 oligonucleotides synthesized with 2' sugar modifications and a phosphorothioate backbone in a mouse model of melanoma liver metastasis. Luciferase imaging showed that mice treated with anti-miR-182 had a lower burden of liver metastases compared with control. We confirmed that miR-182 levels were effectively downregulated in the tumors of anti-miR-treated mice compared with tumors of control-treated mice, both in the liver and in the spleen. This effect was accompanied by an upregulation of multiple miR-182 direct targets. Transcriptional profiling of tumors treated with anti-miR-182 or with control oligonucleotides revealed an enrichment of genes controlling survival, adhesion and migration modulated in response to anti-miR-182 treatment. These data indicate that in vivo administration of anti-miRs allows for efficient miRNA targeting and concomitant upregulation of miRNA-controlled genes. Our results demonstrate that the use of anti-miR-182 is a promising therapeutic strategy for metastatic melanoma and provide a solid basis for testing similar strategies in human metastatic tumors

Minority melanoma patients have worse survival. In this study, we evaluated the impact of socioeconomic and demographic factors on minority melanoma patients presenting to two different New York City hospitals (one public and one private) managed by the same multidisciplinary team. Sociodemographic and clinicopathologic characteristics were retrieved for melanoma patients presenting to Bellevue Hospital Center (BHC), a public hospital, and the New York University Cancer Institute (NYUCI), a private cancer center. Socioeconomic data was obtained from the United States Census Bureau database. The Kruskal-Wallis and chi-square tests were used to evaluate the associations between race/ethnicity and continuous and categorical variables (e.g. income, stage at presentation), respectively. Minorities comprised 2% (27/1296) of melanoma patients at the NYUCI compared to 42% (50/119) at BHC. Those presenting to the NYUCI were more likely to have a higher median household income (P = 0.05), a higher educational level (P = 0.04), and an earlier stage at presentation (P = 0.02) than those at BHC. NYUCI patients were predominantly covered by commercial insurance (70%), whereas Medicaid (62%) was common among BHC patients. Only 19% of Hispanic patients at BHC chose English as their preferred language. Our data demonstrate that language and health care system factors affect melanoma presentation in minorities

Several reports have demonstrated a role for aberrant NOTCH signaling in melanoma genesis and progression, prompting us to explore if targeting this pathway is a valid therapeutic approach against melanoma. We targeted NOTCH signaling using RO4929097, a novel inhibitor of gamma secretase, which is a key component of the enzymatic complex that cleaves and activates NOTCH. The effects of RO4929097 on the oncogenic and stem cell properties of a panel of melanoma cell lines were tested both in vitro and in vivo, using xenograft models. In human primary melanoma cell lines, RO4929097 decreased the levels of NOTCH transcriptional target HES1. This was accompanied by reduced proliferation and impaired ability to form colonies in soft agar and to organize in tridimensional spheres. Moreover, RO4929097 affected the growth of human primary melanoma xenograft in NOD/SCID/IL2gammaR-/- mice and inhibited subsequent tumor formation in a serial xenotransplantation model, suggesting that inhibition of NOTCH signaling suppresses the tumor initiating potential of melanoma cells. In addition, RO4929097 decreased tumor volume and blocked the invasive growth pattern of metastatic melanoma cell lines in vivo. Finally, increased gene expression of NOTCH signaling components correlated with shorter post recurrence survival in metastatic melanoma cases. Our data support NOTCH inhibition as a promising therapeutic strategy against melanoma

The incidence of melanoma has increased 3-7% per year, and is now approaching 30 per 100,000 individuals. This rate is faster than any other cancer, and is predicted to double every 10-20 years [1]. Surgery can be curative in Stage I, II, or III disease, but 75% of patients with deep primary lesions will develop extensive recurrence or distant metastases (Stage IV disease). To date, no curative treatment exists for Stage IV melanoma and these patients have a median overall survival of only 7 months [2]. A subset of patients can be salvaged with surgical resection of metastatic sites, but no adjuvant therapy has further improved the outcome [3]. The only FDA approved adjuvant therapy for Stage IV melanoma is alpha-interferon. Several trials have demonstrated an increase in relapse-free survival; however, toxicity is high and overall survival remains controversial. Several reports have demonstrated a role for aberrant Notch signaling in melanoma genesis or progression, prompting us to explore if targeting this pathway is a valid therapeutic approach against melanoma. To investigate the potential benefits of Notch inhibition in melanoma, we are using RO4929097, a novel inhibitor of gamma secretase, a key component of the enzymatic complex that cleaves and activates Notch. RO4929097 is completing Phase I dose escalation in cancer patients and a CTEP-sponsored Phase II clinical trial in melanoma is currently under review. We have generated DNA microarray data for a series of 22 melanoma cell lines at both the gene expression and DNA copy number level. Preliminary gene expression analysis has indicated that melanoma cells over express crucial components of NOTCH-pathway, such as Hey1 Hey2 and NOXA. Furthermore, integration of gene expression and copy number data for NOTCH2 suggests that the increased DNA copy number play a role in the increase in gene expression. We have also tested the efficacy of RO4929047 in human melanoma cell lines. We have observed that treatment with RO4929097 for 24h causes a!

PURPOSE: To identify a melanoma microRNA (miRNA) expression signature that is predictive of outcome and then evaluate its potential to improve risk stratification when added to the standard-of-care staging criteria. EXPERIMENTAL DESIGN: Total RNA was extracted from 59 formalin-fixed paraffin-embedded melanoma metastases and hybridized to miRNA arrays containing 911 probes. We then correlated miRNA expression with post-recurrence survival and other clinicopathologic criteria. RESULTS: We identified a signature of 18 miRNAs whose overexpression was significantly correlated with longer survival, defined as more than 18 months post-recurrence survival. Subsequent cross-validation showed that a small subset of these miRNAs can predict post-recurrence survival in metastatic melanoma with an estimated accuracy of 80.2% (95% confidence interval, 79.8-80.6%). In contrast to standard-of-care staging criteria, a six-miRNA signature significantly stratified stage III patients into 'better' and 'worse' prognostic categories, and a multivariate Cox regression analysis revealed the signature to be an independent predictor of survival. Furthermore, we showed that most miRNAs from the signature also showed differential expression between patients with better and worse prognoses in the corresponding paired primary melanoma. CONCLUSIONS: MiRNA signatures have potential as clinically relevant biomarkers of prognosis in metastatic melanoma. Our data suggest that molecularly based models of risk assessment can improve the standard staging criteria and support the incorporation of miRNAs into such models. Clin Cancer Res; 16(5); 1577-86

Background: Most skin cancer-related deaths are due to malignant melanoma. Risk assessment criteria for melanoma currently include skin phenotype, family and sun exposure history, factors that are subject to observer and recall bias. Genetic markers of susceptibility have been identified in association studies; however little progress has been made in developing them to improve screening and identification of individuals at risk of melanoma. Tyrosinase (TYR), a known susceptibility gene and a determinant of skin pigmentation, was thus investigated further to characterize its association with melanoma susceptibility and to identify markers which can be used in a risk assessment model. Methods: The cohort consisted of 326 individuals diagnosed with melanoma and 400 control subjects. TYR was interrogated using fifteen tag single nucleotide polymorphisms (SNPs) spanning the gene and statistical association tests performed. Additionally, ancestry informative markers were utilized to correct for population genetic sub-structure. Haplotype analysis was performed to determine if specific regions of the gene contributed more significantly to susceptibility. Coding regions of the gene are currently being sequenced and identified variants will be tested for impact on enzymatic function. Results: Of the 15 SNPs, 8 were associated with melanoma; 4 with decreased risk (Odds ratios 0.41-0.71) and 4 with increased risk (Odds ratios 1.43-1.96). SNPs localized to 2 regions of the gene (spanning exon 1 to intron 2 and intron 3 to 4) with markers of increased as well as decreased susceptibility present in both areas. With the exception of one coding region variant, SNPs were localized to introns. Conclusions: SNPs localized to TYR may serve as useful biomarkers for determining susceptibility to melanoma. We are currently sequencing the gene in our population in order to identify additional and potentially more potent markers of melanoma susceptibility. Coding region variants are being characterized for their effect on protein stability and enzyme activity such that functional active variants (most likely to affect susceptibility to melanoma) can be identified and assessed for their utility in melanoma risk assessment