Extracellular Vesicles Enriched with Moonlighting Metalloproteinase Are Highly Transmissive, Pro-Tumorigenic, and Trans-Activates Cellular Communication Network Factor (CCN2/CTGF): CRISPR against Cancer

Abstract

Matrix metalloproteinase 3 (MMP3) plays multiple roles in extracellular proteolysis as well as intracellular transcription, prompting a new definition of moonlighting metalloproteinase (MMP), according to a definition of protein moonlighting (or gene sharing), a phenomenon by which a protein can perform more than one function. Indeed, connective tissue growth factor (CTGF, aka cellular communication network factor 2 (CCN2)) is transcriptionally induced as well as cleaved by MMP3. Moreover, several members of the MMP family have been found within tumor-derived extracellular vesicles (EVs). We here investigated the roles of MMP3-rich EVs in tumor progression, molecular transmission, and gene regulation. EVs derived from a rapidly metastatic cancer cell line (LuM1) were enriched in MMP3 and a C-terminal half fragment of CCN2/CTGF. MMP3-rich, LuM1-derived EVs were disseminated to multiple organs through body fluid and were pro-tumorigenic in an allograft mouse model, which prompted us to define LuM1-EVs as oncosomes in the present study. Oncosome-derived MMP3 was transferred into recipient cell nuclei and thereby trans-activated the CCN2/CTGF promoter, and induced CCN2/CTGF production in vitro. TRENDIC and other cis-elements in the CCN2/CTGF promoter were essential for the oncosomal responsivity. The CRISPR/Cas9-mediated knockout of MMP3 showed significant anti-tumor effects such as the inhibition of migration and invasion of tumor cells, and a reduction in CCN2/CTGF promoter activity and fragmentations in vitro. A high expression level of MMP3 or CCN2/CTGF mRNA was prognostic and unfavorable in particular types of cancers including head and neck, lung, pancreatic, cervical, stomach, and urothelial cancers. These data newly demonstrate that oncogenic EVs-derived MMP is a transmissive trans-activator for the cellular communication network gene and promotes tumorigenesis at distant sites.

Knockout of MMP3 Weakens Solid Tumor Organoids and Cancer Extracellular Vesicles

“…Earlier studies showed that MMPs constituted a large family of zinc/calcium-dependent endopeptidases, playing key roles in extracellular matrix (ECM) remodeling because of their ability to degrade numerous components of ECM (such as collagens and proteoglycans) and non-ECM proteins (such as adhesion molecules, cytokines, protease inhibitors, and membrane receptors) [24]. Later studies have shown that matrix metalloproteinase 3 (MMP3) was a bifunctional protein that acted as a proteolytic enzyme and a transcriptional factor, playing crucial roles in tumor progression [30][31][32]. Therefore, we defined MMP3 as a moonlighting protein, as it plays multiple roles in the cells as a proteinase as well as a transcription factor [32,33].…”

“…Later studies have shown that matrix metalloproteinase 3 (MMP3) was a bifunctional protein that acted as a proteolytic enzyme and a transcriptional factor, playing crucial roles in tumor progression [30][31][32]. Therefore, we defined MMP3 as a moonlighting protein, as it plays multiple roles in the cells as a proteinase as well as a transcription factor [32,33]. It has been shown that some members of MMPs were expressed at high levels in particular cancer types and promoted tumor progression.…”

“…It has been shown that some members of MMPs were expressed at high levels in particular cancer types and promoted tumor progression. We have compared protumorigenic phenotypes of rapidly metastatic murine cancer cell line LuM1 with a parental cancer cell line Colon26 (aka CT26) [31,32,[34][35][36] and shown that MMP3 and MMP9 were highly expressed in LuM1 cells. The RNA interference (RNAi)-mediated knockdown of MMP3 and/or MMP9 significantly attenuated tumor growth and metastasis in the tumor allograft mouse model [31].…”

“…The RNA interference (RNAi)-mediated knockdown of MMP3 and/or MMP9 significantly attenuated tumor growth and metastasis in the tumor allograft mouse model [31]. Most recently, we established MMP3-knockout (KO) cells using CRISPR/Cas9 genome editing technology on the LuM1 cells and then compared the oncogenic effects of MMP3-KO cells versus LuM1 cells [32]. This study indicated that knockout of Mmp3 results in a significant inhibition of tumor growth in vivo, cellular migration and invasion in vitro [32].…”

“…Most recently, we established MMP3-knockout (KO) cells using CRISPR/Cas9 genome editing technology on the LuM1 cells and then compared the oncogenic effects of MMP3-KO cells versus LuM1 cells [32]. This study indicated that knockout of Mmp3 results in a significant inhibition of tumor growth in vivo, cellular migration and invasion in vitro [32]. However, a mechanism of how MMP3 enriched EVs influences the characteristics of EVs and tumors has not been completed yet.…”