Stromal MMP3 inhibits oncogenic potential during breast cancer progression

Abstract The tumor microenvironment is composed of multiple cell types and extracellular proteins that interact with breast cancer epithelium. Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases that regulate the microenvironment. MMP3 functions during both breast development and cancer progression. We assessed MMP3 protein localization by immunohistochemistry. MMP3 localized to epithelial ductal cells, adipocytes, and stroma, varying its localization and intensity according to the mouse age and developmental process (pregnancy, lactation, and involution). Roles of stromal and epithelial MMP3 have not been distinguished. To determine the requirement for MMP3 in breast cancer, we transplanted cancer cells (PyMT) into the mammary glands of MMP3 knockout (KO) and heterozygous (Het) syngeneic mice. Tumor burden increased in MMP3 KO recipient mice compared to Het, suggesting an inhibitory role for stromal MMP3. H&Es of primary tumors show similar pathologies between the MMP3 Het and KO cohorts. To identify how stromal MMP3 contributes to tumor progression, we next analyzed proliferation, apoptosis, and immune cell infiltration by immunohistochemistry. The expression of the G2/M cell proliferation marker phospho-histone H3 decreased, while Ki67 did not change in KO recipient tumors. Apoptosis was not significantly different between tumors of Het and KO recipients. Neutrophil recruitment decreased in tumors from KO recipients. In contrast, macrophages and extracellular matrix detected by trichrome staining were not different between tumor cohorts. We also investigated the role of stromal MMP3 during metastasis by analyzing the metastatic tumor burden in recipient mouse lungs. Unlike Het mice, KO recipient mice did not develop visible metastatic tumors in the lung. These data suggest that stromal MMP3 was required for visible lung metastases but does not specify the step in cancer progression where MMP3 was required. MMP3 KO mice have significantly higher levels of trichrome staining in their lungs, which suggests an MMP3-dependent alteration of the extracellular matrix at the metastatic site. To determine if stromal MMP3 plays a similar role after the cancer cells circulate through the vasculature, we injected cancer cells into tail veins of MMP3 Het and KO mice. In vivo imaging of these animals determined that lung metastasis increased in MMP3 KO compared to Het recipient mice, suggesting stromal MMP3 in the lungs inhibits metastatic tumors after tail vein injection. Together, our results suggest that stromal MMP3 primarily has a protective role during breast cancer development by inhibiting primary and metastatic tumor growth. In contrast, epithelial MMP3 promotes initial primary tumor formation. Stromal MMP3 increases early metastasis events between primary tumor invasion and extravasation but inhibits later metastasis during extravasation or colonization steps of the metastatic cascade. Citation Format: Maria Cristina Miranda Vergara, Emilia Hartland, Charley Jang, Megan McGarel, Ricardo Romero Moreno, William Kaliney, Laurie E. Littlepage. Stromal MMP3 inhibits oncogenic potential during breast cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5078.