Microenvironmental Changes by Stromal MMP3 Regulate Primary Tumor Growth and Lung Metastasis in Breast Cancer

Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases that regulate the tissue microenvironment. MMP3 regulates both breast development and cancer progression and localizes to both mammary epithelial and stromal cells in normal and tumor mouse mammary tissues. Epithelial MMP3 overexpression promotes mammary tumor development. However, the functions of stromal and epithelial MMP3 have not been distinguished.

I investigated the requirements for stromal MMP3 during breast cancer. We transplanted PyMT cancer cells, which express MMP3, into mammary glands of mice expressing (MMP3 WT or Het) or not expressing (MMP3 KO) MMP3. In initial experiments, tumor burden increased in MMP3 KO mice, suggesting an inhibitory role for stromal MMP3. I next analyzed proliferation, apoptosis, and immune cell infiltration by immunohistochemistry to identify stromal MMP3’s contribution to tumor progression. Tumors from KO recipient tumors had decreased cell proliferation marker phospho-histone H3 and neutrophil recruitment but increased CD4⁺ and CD8⁺ T cell infiltration compared to tumors expressing MMP3. Apoptosis was not significantly different. Subsequent bone marrow transplantation experiments confirmed an inhibitory role for a stromal MMP3. However, neither MMP3-deficient bone marrow cells, CD11b⁺ cells, nor carcinoma-associated fibroblasts (CAFs) were sufficient to increase tumor burden. In fact, co-injection of MMP3 KO CAFs with cancer cells significantly decreased the primary tumor growth in both Het and KO mouse backgrounds, suggesting that MMP3 produced by CAFs significantly promotes tumorigenicity. In addition, after backcrossing the mice for several years, the MMP3 KO mice did not have increased tumor burden, which suggests that a passenger mutation of another gene might be required for any inhibitory role of MMP3.

MMP3 exists as inactive (pro-MMP3 or inhibited by TIMPs (Tissue Inhibitors of Metalloproteases) and catalytically active MMP3. To test whether MMP3 activity is contributing to the observed phenotypes, MMP3 substrates in PyMT tumors were explored through Tandem Mass Tag-Terminal amine isotopic labelling of substrates (TMT-TAILS) degradomics. The consolidated list of in vivo candidate substrates included: Heat shock protein-90β (HSP-90β), pyruvate carboxylase, Cathepsin B, myeloperoxidase, α-2-Macroglobulin, and superoxide dismutase. In vitro cleavage assays and MALDI-TOF analysis confirmed cleavage of Cathepsin B, and myeloperoxidase and of α-2-Macroglobulin and superoxide dismutase by western analysis.

I next investigated stromal MMP3’s role during lung metastasis. In contrast to the inhibitory role for stromal MMP3 in the primary tumors, stromal MMP3 was required for lung metastasis by orthotopic transplantation of breast cancer cells. In contrast, stromal MMP3 inhibited lung metastasis by tail vein injections of breast cancer cells, suggesting that MMP3 impacts metastasis by effects at the primary tumor site.

Together, this study suggests that stromal MMP3 has both protective and tumor promoting roles during breast cancer and highlights the context-specific functions of MMP3 during breast cancer.