Abstract 7154: The role of TNIK and MINK1 in TNBC tumorigenesis and invasion

Abstract Introduction: TRAF2- and NCK-interacting kinase (TNIK) and Misshapen-like kinase 1 (MINK1) are implicated in MAP kinase signaling cascades, including Wnt signaling. These kinases play a role in cancer-associated processes, like migration and epithelial-to-mesenchymal transition (EMT). Migration and EMT are hallmarks associated with poorer cancer outcomes, specifically in breast cancer patients. Triple-negative breast cancer (TNBC) is more aggressive and challenging to treat, making the identification of novel targets of the utmost importance. TNIK and MINK1 are relatively understudied in TNBC; therefore, our group is studying the effect of TNIK and MINK1 knockdown on TNBC tumorigenesis, invasion, and migration. Methods: To better understand the role of TNIK and MINK1 in TNBC, an shRNA-mediated knockdown of TNIK and MINK1 was performed in a patient-derived xenograft-derived TNBC cell line: TU-BcX-4IC (4IC). Using pre-validated shRNAs that were pre-packaged in lentiviral particles and specifically target TNIK and MINK1, the expression of these genes was diminished. We confirmed knockdown at the transcript level, as well as by monitoring GFP expression via fluorescent microscopy. We then assessed the effects of knockdown on 4IC cell spheroid formation and invasion, as well as EMT marker gene expression, using a collagen-embedded sphere migration assay and quantitative real-time PCR (qRT-PCR), respectively. Results: TNIK and MINK1 expression were diminished after stable transduction. TNIK expression was reduced by 82% compared to the scrambled shRNA control. MINK1 expression was decreased by 92%. shTNIK and shMINK1 cell lines were unable to form as tightly compact tumor spheroids as their scrambled shRNA control counterparts. Upon characterizing the spheroids, the area and perimeter of the shMINK1 spheroids were significantly larger, while the circularity and solidity were diminished considerably compared to the control. There was no significant difference in these parameters for the shTNIK spheres, but a biological difference appears to exist. There was no significant difference in spheroid migration through collagen in either group, despite the spheres being much larger and less compact. Conclusions: Overall, MINK1 and TNIK knockdown have an effect on TNBC cells' spheroid-forming capacity, despite no significant difference in migration being observed compared to the scrambled control. This suggests that MINK1 and TNIK may play a role in tumorigenesis, but not in migration, from a central tumor. However, further research is needed. In the future, we plan to repeat these experiments, as well as stain sectioned spheroids for extracellular matrix (ECM) markers to assess if TNIK and MINK1 are associated with changes in the ECM that may be driving the inability to aggregate. We also plan to generate a double knockdown cell line, which could provide clearer insight into the roles of both proteins simultaneously. Citation Format: Caroline McCauley, Bruce Bunnell, . The role of TNIK and MINK1 in TNBC tumorigenesis and invasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 7154.