Investigating the Anticancer Potential of Novel Therapeutics Using 3D Model Systems of Colon Cancer

Abstract

Title: Investigating the Anticancer Potential of Novel Therapeutics Using 3D Model Systems of Colon Cancer Background: Chemotherapy for colorectal cancer (CRC), the second leading disease of cancer-related mortality, has so far revealed partial success. Cancer stem cells (CSCs) in CRC, which are spared by many chemotherapeutics, have tumorigenic capacity and are believed to be the reason behind cancer relapse. The inadequate response to 5-fluorouracil (5FU), the first-line therapy for advanced CRC, might be caused by surviving CSCs. So far, there have been no effective drugs to target colon CSCs. The identification of novel therapeutics that simultaneously target CSCs and chemo-resistant cells is a major challenge and is of high importance for successful cancer treatment. Quinones and imipridone have shown promising effect on targeting different types of cancer. However, research on the effects of DIQ, ONC201 and ONC206 to target CSCs in CRC is very limited. Objective: The overall aim of this thesis is to investigate the anticancer activities and targeting mechanism(s) of three novel therapeutics, diiminoquinone DIQ, and the imipridone DRD2 antagonists ONC201 and ONC206, against human colon cancer cells with stem-like properties both in 2D and in 3D using colonosphere cultures and patientderived organoids. Our first aim was to assess the toxicity of DIQ, ONC201 and ONC206 to non-tumorigenic colon FHS cells and their ability to target colon cancer stem cells in HCT116 and HT29 cells. In this aim, we used 3D sphere-formation assays to enrich cancer stem cells (CSCs) in the human colorectal cancer cell lines and determine mechanism(s) of DIQ and ONC206 for targeting colon cancer self-renewal capacity. The second aim was to establish three-dimensional patient-derived colon cancer organoid cultures and assess the effect of DIQ, ONC201 and 206 on them. Methods: We first assessed the safety of DIQ, ONC201 and ONC206 on nontumorigenic FHS74Int cells in comparison to their anticancer activity against colon cancer HCT116 and HT29 cells. Cell cycle analysis and reactive oxygen species (ROS) production in response to DIQ, ONC201 and ONC206 were investigated using propidium iodide and dihydroethidium staining, respectively. Invasion and migration ability of DIQ, ONC201 and ONC206 were assessed using wound healing and transwell invasion assays, respectively. Then, we tested their efficacy on sphere formation, sphere 4 size, and self-renewal capacity of spheres derived from colon cancer cell lines grown in 3D setting for up to 5 generations. Immunofluorescent analysis and western blot were used to determine the mechanism of action. For the second aim, we established colon cancer patient derived organoids from fresh tissue samples from consented patients with different stages of CRC undergoing colectomy at the American University of Beirut Medical Center (AUBMC, Beirut, Lebanon) according to appropriate Institutional Review Board (IRB) approval guidelines. Patient organoid model was used to assess DIQ, ONC201 and ONC206 response in comparison to 5FU. The effects of DIQ, ONC201 and ONC206 on organoids growth were evaluated by quantifying the number of organoids formed (OFC) and calculating the average size (diameters). Colon patientspecific organoids were characterized using immunofluorescent staining. Statistical analysis was performed using Graphpad prism 7. Results: Our results showed that DIQ, ONC201 and ONC206 significantly inhibited cell proliferation, migration, and invasion in HCT116 and HT29 cell lines. DIQ, ONC201 and ONC206 treatments induced apoptosis along with an accumulation of HCT116 and HT29 cancer cells in the sub-G1 region and an increase in ROS in both CRC cell lines. DIQ, ONC201 and ONC206 significantly reduced sphere-forming and self-renewal ability of colon cancer HCT116 and HT29 stem/progenitor cells by eradication of the propagated spheres at sub-toxic doses up to generation 5 (G5) . Mechanistically, DIQ and ONC206 targeted CSCs by reducing the proliferation marker Ki67 and CRC stem cell markers CD44, CD133 and CK19, as well as inducing DNA damage through decreasing gamma-H2AX (γ-H2AX) expression and downregulating the main components of stem cell-related β-catenin, AKT and ERK oncogenic signaling pathways. Potently, DIQ, ONC201, and ONC206 displayed a highly significant decrease in both the count and the size of the organoids derived from colon cancer patients as compared to control and 5FU conditions. Conclusion: This study represents the first documentation of the molecular mechanism of the novel anticancer therapeutics DIQ, ONC201 and ONC206 via targeting CSCs, findings that will certainly have therapeutic implications for colon cancer patients.