ABSTRACT
PURPOSE: The extracellular matrix (ECM) is an intriguing, yet understudied component of therapy resistance. Here, we investigated the role of ECM remodeling by the collagenase, MT1-MMP, in conferring resistance of v-Raf murine sarcoma viral oncogene homolog B1 (BRAF)-mutant melanoma to BRAF inhibitor (BRAFi) therapy. EXPERIMENTAL DESIGN: Publicly available RNA-sequencing data and reverse phase protein array were used to determine the relevance of MT1-MMP upregulation in BRAFi-resistant melanoma in patients, patient-derived xenografts, and cell line-derived tumors. Short hairpin RNA (shRNA)-mediated knockdown of MT1-MMP, inhibition via the selective MT1-MMP/MMP2 inhibitor, ND322, or overexpression of MT1-MMP was used to assess the role of MT1-MMP in mediating resistance to BRAFi. RESULTS: MT1-MMP was consistently upregulated in posttreatment tumor samples derived from patients upon disease progression and in melanoma xenografts and cell lines that acquired resistance to BRAFi. shRNA- or ND322-mediated inhibition of MT1-MMP synergized with BRAFi leading to resensitization of resistant cells and tumors to BRAFi. The resistant phenotype depends on the ability of cells to cleave the ECM. Resistant cells seeded in MT1-MMP uncleavable matrixes were resensitized to BRAFi similarly to MT1-MMP inhibition. This is due to the inability of cells to activate integrinß1 (ITGB1)/FAK signaling, as restoration of ITGB1 activity is sufficient to maintain resistance to BRAFi in the context of MT1-MMP inhibition. Finally, the increase in MT1-MMP in BRAFi-resistant cells is TGFß dependent, as inhibition of TGFß receptors I/II dampens MT1-MMP overexpression and restores sensitivity to BRAF inhibition. CONCLUSIONS: BRAF inhibition results in a selective pressure toward higher expression of MT1-MMP. MT1-MMP is pivotal to an ECM-based signaling pathway that confers resistance to BRAFi therapy.
Subject(s)
Melanoma/drug therapy , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins B-raf/genetics , Vemurafenib/pharmacology , Animals , Arginine/analogs & derivatives , Arginine/pharmacology , Cell Line, Tumor , Drug Resistance, Neoplasm/drug effects , Focal Adhesion Kinase 1/genetics , Gene Expression Regulation, Neoplastic/drug effects , Heterografts , Humans , Integrin beta1/genetics , Matrix Metalloproteinase 14/genetics , Melanoma/genetics , Melanoma/pathology , Mice , Proto-Oncogene Proteins B-raf/antagonists & inhibitors , Signal Transduction/drug effects , Sulfones/pharmacology , Transforming Growth Factor beta/geneticsABSTRACT
MT1-MMP and MMP2 have been implicated as pro-tumorigenic and pro-metastatic factors in a wide variety of cancers including melanoma. We have previously demonstrated that MT1-MMP is highly expressed in melanoma where it promotes melanoma cell invasion and metastasis in part through the activation of its target MMP2. Given the accessibility of MMPs, as they are either secreted (e.g. MMP2) or membrane-tethered (e.g. MT1-MMP), they represent ideal targets for specific inhibition via small molecules. Here we show that the novel small-molecule inhibitor ND-322 with high selectivity for MT1-MMP and MMP2, effectively inhibits MT1-MMP and MMP2 activity resulting in reduced in vitro melanoma cell growth, migration and invasion. Importantly, these inhibitory effects lead to significant reduction of melanoma tumor growth and metastasis. We further show that while cell migration and invasion could be similarly hampered by specific inhibition of either MT1-MMP or MMP2 via shRNAs, the growth inhibitory activity of ND-322 could only be mirrored by specific inhibition of MT1-MMP. These data support ND-322 as a novel effective inhibitor capable of counteracting both MT1-MMP and MMP2, two key proteases involved in melanoma growth and metastasis. ND-322 may therefore represent a new inhibitor in the repertoire of treatments against melanoma.
