Abstract C031: Keratin 17 promotes pancreatic cancer chemoresistance through mitochondrial translocation and stabilization of dihydroorotate dehydrogenase (DHODH)
Authors: Yinghuan Lyu, Monisankar Ghosh, Chun-Hao Pan, Shayan Sarkar, Girish H Rajacharya, Bo Chen, Natalia Marchenko, Pankaj K Singh, Kenneth R. Shroyer, Luisa F. Escobar-Hoyos
Journal: Cancer Research (2024)

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) has emerged as the third leading cause of cancer-related deaths in the U.S., in part, due to intrinsic resistance to main-line chemotherapeutic protocols. Our prior data showed that the upregulation of pyrimidine but not purine de novo biosynthesis is associated with shortened survival of patients whose tumors expresses high keratin 17 (K17). Furthermore, we reported that K17 expression confers chemoresistance by impacting the inner mitochondrial membrane protein, dihydroorotate dehydrogenase (DHODH), the rate-limiting enzyme in the de novo pyrimidine synthesis pathway1. Here, we explore the mechanisms through which K17 enters mitochondria, stabilizes DHODH, and thereby, enhances downstream de novo pyrimidine biosynthesis and chemoresistance. Methods: We generated a KPC K17 stably expressing cell line and a L3.6 K17 knockout cell line to assess the effects of K17 on cell viability and performed western blot, immunoprecipitation, mass spectrometry, immunofluorescence imaging, and cellular sub- fractionation. We also generated wild type K17, MLS mutant K17 and coil 2 deletion K17 overexpression in L3.6 K17 KO and KPC cell lines to study the importance of MLS and coil 2 domains. Results: Our previous studies suggested that K17 drives chemoresistance by increasing mitochondrial DHODH, elevating intracellular pyrimidines. In our current report, we identified by Mitoprot a mitochondria localization signal (MLS) of K17 with a probability of 86% and validated that it is required for K17 to enter the mitochondria and impact DHODH. Mechanistically, we found that K17 enters the mitochondria by specifically interacting with the translocase of the outer mitochondrial membrane complex (TOM20/TOM40). Additionally, we uncovered a coil 2 domain of K17 that mediates DHODH stability at the inner mitochondrial membrane. Our data showed that K17 with deletion of the coil 2 domain fails to interact with DHODH suggesting that this domain is critical for mediating interaction and DHODH stabilization. By contrast, MLS mutant K17 did not enter the mitochondria and promote DHODH stabilization, thus having no impact in altering the downstream de novo pyrimidine synthesis. Furthermore, we found that MLS mutant or coil 2 deleted K17 expressing cells have increased sensitivity towards gemcitabine, compared to wild type K17 expressing cells. Conclusions: We report for the first time that K17 encodes an MLS, enabling K17 to enter mitochondria, and that the coil 2 domain of K17 stabilizes DHODH at the inner mitochondrial membrane, thereby upregulating de novo pyrimidine biosynthesis and chemoresistance to gemcitabine. Further studies are warranted to explore whether a therapeutic strategy targeting K17 mitochondrial transport or interaction with DHODH could enhance the therapeutic efficacy of gemcitabine or other pyrimidine analog-based pharmacologic agents. 1. Pan et. al. Targeting keratin 17- mediated reprogramming of de novo pyrimidine biosynthesis to overcome chemoresistance in pancreatic cancer. bioRxiv, 2022 Citation Format: Yinghuan Lyu, Monisankar Ghosh, Chun-Hao Pan, Shayan Sarkar, Girish H Rajacharya, Bo Chen, Natalia Marchenko, Pankaj K Singh, Kenneth R. Shroyer, Luisa F. Escobar-Hoyos. Keratin 17 promotes pancreatic cancer chemoresistance through mitochondrial translocation and stabilization of dihydroorotate dehydrogenase (DHODH) [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C031.