Faculty Bibliography

Background: The two major melanoma histologic subtypes, superficial spreading and nodular melanomas, are believed to differ in their speed of dermal invasion but to converge biologically once they invade and metastasize. Here, we tested the hypothesis that distinct molecular alterations arising in primary melanoma cells might persist as these tumors progress to invasion and metastasis. Materials and methods: Expression of 141 signaling proteins was evaluated by protein pathway array in 3 Radial Growth Phase (RGP)/SSM and 3 Vertical Growth Phase (VGP)/NM cell lines. The impact of p90- ribosomal-S6-kinase (RSK1) and its inhibition on proliferation, migration and invasion was assessed in SSM and NM cell lines, and confirmed using NM cells treated with a RSK inhibitor (BI-D1870) in microarray profiling studies. The effect of constitutive RSK1 activation in vivo was further studied using a zebrafish model. Results: We show that p90-ribosomal-S6-kinase (RSK1) was significantly hyper-activated in human melanoma lines and metastatic tissues derived from nodular compared with superficial spreading melanoma. RSK1 was constitutively phosphorylated at Ser-380 in nodular but not superficial spreading melanoma and was not directly correlated with BRAF or MEK activation. Nodular melanoma cells were more sensitive to RSK1 inhibition using both siRNA and pharmacological inhibitor BI-D1870 compared with superficial spreading cells. In addition, gene expression microarray analyses revealed that RSK1 orchestrates a program of gene expression that promotes cell motility and invasion. Our data also demonstrate a differential over expression of the pro- metastatic MMP-8 and TIMP-1 in metastatic nodular compared to metastatic superficial spreading melanoma. Finally, using an in vivo zebrafish model, constitutive RSK1 activation increased melanoma invasion. Conclusions: Together, our data reveal a novel role for activated RSK1 in the progression of nodular melanoma, and suggest that melanoma originating from different histological subtypes may be biologically distinct and that these differences are maintained as the tumors invade and metastasize

PURPOSE: Brain metastasis is the major cause of mortality among melanoma patients. A molecular prognostic test that can reliably stratify patients at initial melanoma diagnosis by risk of developing brain metastasis may inform the clinical management of these patients. EXPERIMENTAL DESIGN: We performed a retrospective, cohort-based study analyzing genome-wide and targeted microRNA expression profiling of primary melanoma tumors of three patient cohorts (n= 92, n= 119, n= 45) with extensive clinical follow up. We used Cox regression analysis to establish a microRNA-based signature that improves the ability of the current clinicopathologic staging system to predict the development of brain metastasis. RESULTS: Our analyses identified a 4-microRNA (miR-150-5p, miR-15b-5p, miR-16-5p, and miR-374b-3p) prognostic signature that, in combination with stage, distinguished primary melanomas that metastasized to the brain from non-recurrent and non-brain-metastatic primary tumors (training cohort: C-index=81.4%, validation cohort: C-index=67.4%, independent cohort: C-index=76.9%). Corresponding Kaplan-Meier curves of high- vs. low-risk patients displayed a clear separation in brain-metastasis-free and overall survival (training: p<0.001, p<0.001, validation: p=0.033, p=0.007, independent: p=0.021, p=0.022, respectively). Finally, of the microRNA in the prognostic model, we found that the expression of a key lymphocyte miRNA, miR-150-5p, which is less abundant in primary melanomas metastatic to brain, correlated with presence of CD45+ tumor infiltrating lymphocytes. CONCLUSIONS: A prognostic assay based on the described miRNA expression signature combined with the currently used staging criteria may improve accuracy of primary melanoma patient prognoses and aid clinical management of patients, including selection for adjuvant treatment or clinical trials of adjuvant therapies.

Histone variants are emerging as key regulatory molecules in cancer. We report a unique role for the H2A.Z isoform H2A.Z.2 as a driver of malignant melanoma. H2A.Z.2 is highly expressed in metastatic melanoma, correlates with decreased patient survival, and is required for cellular proliferation. Our integrated genomic analyses reveal that H2A.Z.2 controls the transcriptional output of E2F target genes in melanoma cells. These genes are highly expressed and display a distinct signature of H2A.Z occupancy. We identify BRD2 as an H2A.Z-interacting protein, levels of which are also elevated in melanoma. We further demonstrate that H2A.Z.2-regulated genes are bound by BRD2 and E2F1 in an H2A.Z.2-dependent manner. Importantly, H2A.Z.2 deficiency sensitizes melanoma cells to chemotherapy and targeted therapies. Collectively, our findings implicate H2A.Z.2 as a mediator of cell proliferation and drug sensitivity in malignant melanoma, holding translational potential for novel therapeutic strategies.

Heat-shock factor 1 (HSF1) orchestrates the heat-shock response in eukaryotes. Although this pathway has evolved to help cells adapt in the presence of challenging conditions, it is co-opted in cancer to support malignancy. However, the mechanisms that regulate HSF1 and thus cellular stress response are poorly understood. Here we show that the ubiquitin ligase FBXW7alpha interacts with HSF1 through a conserved motif phosphorylated by GSK3beta and ERK1. FBXW7alpha ubiquitylates HSF1 and loss of FBXW7alpha results in impaired degradation of nuclear HSF1 and defective heat-shock response attenuation. FBXW7alpha is either mutated or transcriptionally downregulated in melanoma and HSF1 nuclear stabilization correlates with increased metastatic potential and disease progression. FBXW7alpha deficiency and subsequent HSF1 accumulation activates an invasion-supportive transcriptional program and enhances the metastatic potential of human melanoma cells. These findings identify a post-translational mechanism of regulation of the HSF1 transcriptional program both in the presence of exogenous stress and in cancer.

The overexpression of microRNA cluster miR-17-92 has been implicated in development of solid tumors and hematological malignancies. The role of miR-17-92 in lymphomagenesis has been extensively investigated; however, because of the developmental defects caused by miR-17-92 dysregulation, its ability to drive tumorigenesis has remained undetermined until recently. Here we demonstrate that overexpression of miR-17-92 in a limited number of hematopoietic cells is sufficient to cause B cell malignancies. In sum, our study provides a novel and physiologically relevant model that exposes the potent ability of miR-17-92 to act as a driver of tumorigenesis.

BACKGROUND: Surgical management of primary melanoma is curative for most patients with clinically localized disease at diagnosis; however, a substantial number of patients recur and progress to advanced disease. Understanding molecular alterations that influence differential tumor progression of histopathologically similar lesions may lead to improved prognosis and therapies to slow or prevent metastasis. METHODS: We examined microRNA dysregulation by expression profiling of primary melanoma tumors from 92 patients. We screened candidate microRNAs selected by differential expression between recurrent and nonrecurrent tumors or associated with primary tumor thickness (Student's t test, Benjamini-Hochberg False Discovery Rate [FDR] < 0.05), in in vitro invasion assays. We performed in vivo metastasis assays, matrix remodeling experiments, and molecular studies to identify metastasis-regulating microRNAs and their cellular and molecular mechanisms. All statistical tests were two-sided. RESULTS: We identified two microRNAs (hsa-miR-382, hsa-miR-516b) whose expression was lower in aggressive vs nonaggressive primary tumors, which suppressed invasion in vitro and metastasis in vivo (mean metastatic foci: control: 37.9, 95% confidence interval [CI] = 25.6 to 50.2; miR-382: 19.5, 95% CI = 12.2 to 26.9, P = .009; miR-516b: 12.5, 95% CI = 7.7 to 17.4, P < .001, Student's t test). Mechanistically, miR-382 overexpression inhibits extracellular matrix degradation by melanoma cells. Moreover, we identified actin regulators CTTN, RAC1, and ARPC2 as direct targets of miR-382. Depletion of CTTN partially recapitulates miR-382 effects on matrix remodeling, invasion, and metastasis. Inhibition of miR-382 in a weakly tumorigenic melanoma cell line increased tumor progression and metastasis in vivo. CONCLUSIONS: Aberrant expression of specific microRNAs that can functionally impact progression of primary melanoma occurs as an early event of melanomagenesis.

BACKGROUND: Identification of primary melanoma patients at the highest risk of recurrence remains a critical challenge, and monitoring for recurrent disease is limited to costly imaging studies. We recently reported our array-based discovery of prognostic serum miRNAs in melanoma. In the current study, we examined the clinical utility of these serum-based miRNAs for prognosis as well as detection of melanoma recurrence. METHODS: Serum levels of 12 miRNAs were tested using qRT-PCR at diagnosis in 283 melanoma patients (training cohort, n = 201; independent validation, n = 82; median follow-up, 68.8 months). A refined miRNA signature was chosen and evaluated. We also tested the potential clinical utility of the miRNAs in early detection and monitoring of recurrence using multiple longitudinal samples (pre- and postrecurrence) in a subset of 82 patients (n = 225). In addition, we integrated our miRNA signature with publicly available Cancer Genome Atlas data to examine the relevance of these miRNAs to melanoma biology. RESULTS: Four miRNAs (miR-150, miR-30d, miR-15b, and miR-425) in combination with stage separated patients by recurrence-free survival (RFS) and overall survival (OS) and improved prediction of recurrence over stage alone in both the training and validation cohorts (training RFS and OS, P < .001; validation RFS, P < .001; OS, P = .005). Serum miR-15b levels significantly increased over time in recurrent patients (P < .001), adjusting for endogenous controls as well as age, sex, and initial stage. In nonrecurrent patients, miR-15b levels were not significantly changed with time (P =.17). CONCLUSIONS: Data demonstrate that serum miRNAs can improve melanoma patient stratification over stage and support further testing of miR-15b to guide patient surveillance. Cancer 2015;121:51-59. (c) 2014 American Cancer Society.

Malignant melanoma is the most lethal form of skin cancer with rising incidence. Once metastasis occurs, patients have a dismal prognosis, largely due to limited systemic treatment with chemotherapy and resistance to targeted therapies. Thus, effective therapies with long-term responses are currently lacking. Although much effort has focused on characterizing and targeting the genetic alterations in melanoma, the identification of epigenetic players remains poorly understood. Chromatin dynamics have recently been shown to exert a critical function in a number of cancers, including melanoma, and emerging evidence points towards a role of histone variants as key regulatory molecules in cancer. H2A.Z is a highly conserved H2A variant, harboring two different isoforms in vertebrates, H2A.Z.1 and H2A.Z.2. High levels of H2A.Z promote cell proliferation in breast, prostate and bladder cancers, however studies so far have focused primarily on H2A.Z.1 or did not clearly distinguish between the two isoforms. Here, we report a role for the unappreciated isoform H2A.Z.2 as a mediator of cell proliferation and drug sensitivity in malignant melanoma. To our knowledge, this is the first evidence to implicate a distinct role for this H2A.Z isoform in any tumor type. While both H2A.Z.1 and H2A.Z.2 are highly expressed in metastatic melanoma and correlate with decreased patient survival, only H2A.Z.2 deficiency results in impaired cellular proliferation, which occurs through a G1 to S arrest. Integrated gene expression and ChIP-seq analyses revealed that H2A.Z.2 positively regulates E2F target genes, which are highly expressed and acquire a distinct H2A.Z occupancy signature over the promoter and gene body in metastatic cells. We further identified the BET (bromodomain and extraterminal domain) family member BRD2 as an H2A.Z-interacting protein in melanoma cells, and our data suggest that H2A.Z.2 exerts its oncogenic function by maintaining the global levels of BRD2 and histone H4 acetylation. Furthermore, H2A.Z.2 depletion sensitizes melanoma cells to targeted therapies and chemotherapy. Collectively, our findings implicate H2A.Z.2 as a driver of melanoma pathogenesis. Owing to the fact that histone modification is a reversible process, H2A.Z.2 and BRD2 hold translational potential for novel therapeutic strategies

Transcription factors and chromatin-remodeling complexes are key determinants of embryonic stem cell (ESC) identity. Here, we demonstrate that BRD4, a member of the bromodomain and extraterminal domain (BET) family of epigenetic readers, regulates the self-renewal ability and pluripotency of ESCs. BRD4 inhibition resulted in induction of epithelial-to-mesenchymal transition (EMT) markers and commitment to the neuroectodermal lineage while reducing the ESC multidifferentiation capacity in teratoma assays. BRD4 maintains transcription of core stem cell genes such as OCT4 and PRDM14 by occupying their super-enhancers (SEs), large clusters of regulatory elements, and recruiting to them Mediator and CDK9, the catalytic subunit of the positive transcription elongation factor b (P-TEFb), to allow Pol-II-dependent productive elongation. Our study describes a mechanism of regulation of ESC identity that could be applied to improve the efficiency of ESC differentiation.