A Reporter System Evaluates Tumorigenesis, Metastasis, β-catenin/MMP Regulation, and Druggability.

Abstract

Cancer invasion, metastasis, and therapy resistance are the crucial phenomena in cancer malignancy. The high expression of matrix metalloproteinase 9 (MMP9) is a biomarker as well as a causal factor of cancer invasiveness and metastatic activity. However, a regulatory mechanism underlying MMP9 expression in cancer is not clarified yet. In addition, a new strategy for anticancer drug discovery is becoming an important clue. In the present study, we aimed (i) to develop a novel reporter system evaluating tumorigenesis, invasiveness, metastasis, and druggability with a combination of three-dimensional tumoroid model and <i>Mmp9</i> promoter and (ii) to examine pharmacological actions of anticancer medications using this reporter system. High expression and genetic amplification of <i>MMP9</i> were found in colon cancer cases. We found that proximal promoter sequences of <i>MMP9</i> in murine and human contained conserved binding sites for transcription factors β-catenin/TCF/LEF, glucocorticoid receptor (GR), and nuclear factor kappa-B (NF-κB). The murine <i>Mmp9</i> promoter (-569 to +19) was markedly activated in metastatic colon cancer cells and additionally activated by tumoroid formation and by β-catenin signaling stimulator lithium chloride. The <i>Mmp9</i> promoter-driven fluorescent reporter cells enabled the monitoring of activities of MMP9/gelatinase, tumorigenesis, invasion, and metastasis in syngeneic transplantation experiments. We also demonstrated pharmacological actions as follows: dexamethasone and hydrocortisone, steroidal medications binding to GR, inhibited the <i>Mmp9</i> promoter but did not inhibit tumorigenesis. On the contrary, antimetabolite 5-fluorouracil, a gold standard for colon cancer chemotherapy, inhibited tumoroid formation but did not inhibit <i>Mmp9</i> promoter activity. Notably, antimalaria medication artesunate inhibited both tumorigenesis and the <i>Mmp9</i> promoter <i>in vitro</i>, potentially through inhibition of β-catenin/TCF/LEF signaling. Thus, this novel reporter system enabled monitoring tumorigenesis, invasiveness, metastasis, key regulatory signalings such as β-catenin/MMP9 axis, and druggability. Impact Statement Cancer invasion and metastasis have been shown to be driven by matrix metalloproteinase 9 (MMP9), whose expression mechanism is not clarified yet. In addition, a new strategy for anticancer drug discovery is becoming important. We established a novel reporter system evaluating tumorigenesis, invasiveness, metastasis, and druggability with a combination of three-dimensional (3D) tumoroid model and <i>Mmp9</i> promoter. Using this reporter system, we demonstrated pharmacological actions of anticancer medications such as antimetabolite 5-fluorouracil (5-FU) and antimalaria medication artesunate (ART), which inhibited both tumorigenesis and β-catenin/MMP regulatory signaling. Our study impacts the translational fields of oncology, drug discovery, and organoid model.