Invasive Behavior of Tumor Cells Producing Collagenase-1

Dartmouth College, Hanover NH

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Abstract

DESCRIPTION (provided by applicant): Destruction of the extracellular matrix in tumor invasion and metastasis is mediated primarily by Matrix Metalloproteinases (MMPs), a family of enzymes that are over-expressed in many cancers. Degradation of type IV collagen in basement membrane by MMP-2 and MMP-9 is an essential step, but degradation of the interstitial collagens (types I and III) is also required. This is accomplished by the collagenases, of which MMP-1 is the most ubiquitously expressed. Malignant melanoma is an aggressive cancer where MMP-1 contributes to an invasive phenotype and is associated with poor outcome. Melanoma is the most common fatal skin cancer, with approximately 59,000 new cases diagnosed and 8,000 patient deaths in 2007. Melanoma progression from benign to metastatic tumor is classified histologically, and early stage melanoma is defined as radial growth phase (RGP), which is confined to the epidermis. In contrast, later stage vertical growth phase (VGP) is characterized by invasion of melanoma cells into the dermis, and VGP frequently progresses to metastatic melanoma. However, mechanisms mediating the RGP to VGP transition are not known. Increasing evidence points to Protease Activated Receptor-1 (PAR-1), a G- coupled protein receptor, as critical in the progression to VGP. Based on our Preliminary Data, we hypothesize that an autocrine MMP-1/PAR-1 axis in RGP melanoma initiates signaling cascades that facilitate the RGP to VGP transition, and that this autocrine signaling axis then contributes to the metastatic behavior of VGP melanomas. We also hypothesize a paracrine signaling axis in which melanoma-derived MMP-1 activates PAR-1 on endothelial cells to mediate angiogenesis and melanoma progression. To determine how MMP-1 contributes to melanoma progression, we inhibited MMP-1 production in VGP melanoma cells with RNA interference (RNAi) technology. Cells stably expressing MMP-1 shRNAs had > 85% knock-down of MMP-1 compared to MAMMX control shRNAs, and when injected interdermally into nude mice, MMP-1 shRNAs and shMAMMX cells formed primary tumors with similar growth rates. However, shMAMMX tumors metastasized to the lung, while tumors from MMP-1 shRNA cells did not. Further, melanoma-produced MMP-1 mediated invasion through the dermis, and the MMP-/PAR-1 paracrine signaling axis enhanced angiogenesis. Thus, based on these data, we now hypothesize that tumor-produced MMP-1 mediates melanoma growth and metastasis via novel autocrine and paracrine activation of PAR-1 signaling, initiation of pathways transduction pathways and changes in gene expression. We will test this hypothesis by investigating: (1) the autocrine MMP-1/PAR-1 signaling axis in the RGP to VGP transition and in the metastatic behavior of VGP melanoma; (2) the MMP-1/PAR-1 paracrine axis in endothelial cell behavior; and (3) using siRNAs, the mechanisms by which MMP-1 facilitates tumor growth at metastatic sites. Our studies will define novel roles for MMP-1 in melanoma progression.