Preclinical Evaluation of the Novel Small-Molecule MSI-N1014 for Treating Drug-Resistant Colon Cancer via the LGR5/β-catenin/miR-142-3p Network and Reducing Cancer-Associated Fibroblast Transformation

Abstract

Colorectal cancer represents one of the most prevalent malignancies globally, with an estimated 140,000 new cases in the United States alone in 2019. Despite advancements in interventions, drug resistance occurs in virtually all patients diagnosed with late stages of colon cancer. Amplified epidermal growth factor receptor (EGFR) signaling is one of the most prevalent oncogenic drivers in patients and induces increased Janus kinase (JAK)/signal transduction and activator of transcription (STAT) and β-catenin functions, all of which facilitate disease progression. Equally important, cancer-associated fibroblasts (CAFs) transformed by cancer cells within the tumor microenvironment (TME) further facilitate malignancy by secreting interleukin (IL)-6 and augmenting STAT3 signaling in colon cancer cells and promoting the generation of cancer stem-like cells (CSCs). Based on these premises, single-targeted therapeutics have proven ineffective for treating malignant colon cancer, and alternative multiple-targeting agents should be explored. Herein, we synthesized a tetracyclic heterocyclic azathioxanthone, MSI-N1014, and demonstrated its therapeutic potential both in vitro and in vivo. First, we used a co-culture system to demonstrate that colon cancer cells co-cultured with CAFs resulted in heightened 5-fluorouracil (5-FU) resistance and tumor sphere-forming ability and increased side populations, accompanied by elevated expression of cluster of differentiation 44 (CD44), β-catenin, leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5), and ATP-binding cassette super-family G member 2 (ABCG2). MSI-N1014 suppressed cell viability, colony formation, and migration in both DLD1 and HCT116 cells. MSI-N1014 treatment led to decreased expressions of oncogenic markers, including mammalian target of rapamycin (mTOR), EGFR, and IL-6 and stemness markers such as CD44, β-catenin, and LGR5. More importantly, MSI-N1014 treatment suppressed the transformation of CAFs, and was associated with decreased secretion of IL-6 and vascular endothelial growth factor (VEGF) by CAFs. Furthermore, MSI-N1014 treatment resulted in significantly reduced oncogenic properties, namely the migratory ability, tumor-sphere generation, and resistance against 5-FU. Notably, an increased level of the tumor suppressor, miR-142-3p, whose targets include LGR5, IL-6, and ABCG2, was detected in association with MSI-N1014 treatment. Finally, we demonstrated the therapeutic potential of MSI-N1014 in vivo, where combined treatment with MSI-N1014 and 5-FU led to the lowest tumor growth, followed by MSI-N1014 only, 5-FU, and the vehicle control. Tumor samples from the MSI-N1014 group showed markedly reduced expressions of LGR5, β-catenin, IL-6, and mTOR, but increased expression of the tumor suppressor, miR-142-3p, according to qRT-PCR analysis. Collectively, we present preclinical support for the application of MSI-N1014 in treating 5-FU-resistant colon cancer cells. Further investigation is warranted to translate these findings into clinical settings.