Abstract LB-340: Early alterations of microRNA expression predict and functionally impact melanoma metastasis
Authors: Hanniford Doug , Shang Shulian , Segura Miguel , Tu Ting , Ma Michelle , Greenwald Holly , Pavlick Anna C. , Shapiro Richard L. , Berman Russell S. , Shao Yongzhao , Osman Iman , Hernando Eva 
Published: July 2012

Introduction

Early-stage, localized melanoma is well treatable by surgical resection yielding 5-year survival rates of 98%. Patients with advanced melanoma, however, have progressively worse 5-year survival rates of 62% and 16% in those with regional lymph node involvement and distal metastasis, respectively. Understanding molecular events that occur early in melanoma development may inform clinical decision-making and uncover novel therapeutic targets.

Experimental Procedures

We analyzed microRNA (miR) expression levels by Exiqon LNA arrays of RNA isolated from formalin-fixed paraffin embedded primary melanomas (n=92). We compared miR expression levels between primary tumors that did and did not metastasize (2.5-year minimum follow-up) to derive a predictive signature. Further, we used this analysis and a comparison between thick (&gt;2mM) and thin (&lt;2mM) lesions to identify candidate miRs most closely associated with a metastatic phenotype. We selected 40 candidate miRs from these analyses based on strength of significance and expression level for screening in a fluorescence-based in vitro invasion assay to test their functional relevance in a characteristic metastasis assay. Active candidates were further characterized in invasion assays across a larger panel of melanoma cell lines as well as by proliferation assays to distinguish between proliferative and invasive effects.

Results

The expression of 26 miRs was significantly altered (t-tests, FDR&lt;.01) in primary tumors that metastasized vs. those that did not. We built a logistic regression model using these 26 miRs to test their ability to classify tumors (metastatic vs. non-metastatic), and selected 6 significant miRs (p-values&lt;0.04) as a signature that has robust predictive power with an AUC of 93% of the ROC curve. We are currently validating our results in a second, large cohort of primary melanomas (n=114). Further analysis revealed that 197 miRs were significantly (p&lt;.01) altered in thick v. thin lesions. Moreover, of the 40 candidates selected from these analyses for invasion assay screening, we identified 5 miRs (miR-215, miR-374b*, miR-382, miR-516b, and miR-7) that robustly inhibit in vitro invasion in a panel of 5 melanoma cell lines, supporting the functional relevance of the miR profiling. Of those, miR-215, miR-382, and miR-516b over-expression yields cell-type specific growth inhibition, while miR-7 and miR-374b* impact invasion only. miR target identification is ongoing.

Conclusions

MicroRNA expression alterations occur at early stages of melanoma development. This study furthers our understanding of the molecular defects that occur during melanomagenesis and how they may impact the progression from a treatable primary tumor to an invasive and ultimately metastatic disease. These results suggest that miRNA expression changes may be predictive of melanoma metastasis and/or functionally impact melanoma progression.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-340. doi:10.1158/1538-7445.AM2011-LB-340