Abstract
Immune therapies have had limited success in the treatment of metastatic cancers despite the accumulation of intra-tumoral antigen-specific T cells. The tumor microenvironment (TME) is partly responsible for these failures through active blockade of local antitumor immune responses and facilitation of immune escape. Extracellular proteinases, such as metalloproteinases (MMPs), are key components in promoting tumor progression through modulation of the TME. MMP-2 (gelatinase A or collagenase IV) cleaves many components of the extracellular matrix and its active form is over-expressed in several cancers including melanoma as compared to normal tissue. High MMP-2 levels in cancer and/or stromal cells are often associated with later tumor stages, increased dissemination and poorer survival/prognosis. The main purpose of this study is to characterize the mechanisms underlying the influence of MMP-2, derived from tumor stroma or malignant cells, on the tumor infiltrating immune cells so as to develop effective cancer therapies.
Our lab has previously demonstrated that upon antigenic stimulation, MMP-2-specific CD4+ T cells derived from patients with metastatic melanoma secrete inflammatory TH2 cytokines, i.e. TNFα, IL-4 and IL-13 but no or little IFNγ and IL-2. We subsequently showed that active MMP-2 drives the differentiation of TH2 responses by inhibiting IL-12p70 production and up-regulating OX40L expression on dendritic cells (DCs). OX40L is a key co-stimulatory molecule for the priming of TH2 cells, especially in the absence of IL-12. Recently, we published our novel discovery identifying both enzymatically active and inactive MMP-2 as ligands for TLR2 on DCs, and showed that MMP-2 mediated TLR2 stimulation lead to up-regulation OX40L on DCs (Cell Reports, 2014).
Here we aim to identify a novel link between MMP-2-TLR-2 cross talk in human melanoma progression and survival both in vitro and in vivo. We have screened several human melanoma cell lines for expression of Toll Like Receptors (TLRs) and responsiveness to TLR ligands. Moreover these cell lines have been tested for expression and secretion of pro-MMP-2 and active MMP-2 using techniques such as RT-PCR, Western blotting, flowcytometry ELISA, zymography and CBA analysis. Over all, specific cell lines have been identified that express high levels of both MMP-2 and TLR2 or express none/low levels of MMP-2 and TLR2. These cell lines will be used for performing in vitro tumor invasion assays in the presence and absence of TLR2 neutralizing antibodies to determine relative importance of autocrine MMP-2-TLR2 signaling for melanoma invasiveness. Thereafter CRISPR/Cas9 technology will be utilized for stably knocking down MMP-2, TLR2 or both in select lines and tested for invasiveness in vitro. Finally, these cells lines will be transferred into immuno-compromised NOD/SCID/IL-2gR-/- mice to determine if presence or absence of melanoma derived MMP-2 and/or TLR2 signaling could physiologically alter tumor progression.
Our previous research and current data indicate that MMP-2 acts simultaneously as an endogenous TH2 “conditioner” and tumor-associated antigen. Therefore delving into both enzymatic and non-enzymatic MMP-2 signaling mechanisms in the TME holds a strong potential for discovering novel therapeutic options for treating melanoma. Furthermore, results from this study stand to provide crucial information that will expand our global understanding of tumorigenesis.
Citation Format: Mansi Saxena, Luciana R. Muniz, Nina Bhardwaj. Matrix metalloproteinase-2 modulates inflammation and melanoma progression via TLR2. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B160.
