Research on uPAR promoting proliferation, migration, and chemoresistance of pancreatic cancer by inhibiting autophagy via MAPK signaling

Abstract

Background and purpose: Amplification of the urokinase plasminogen activator receptor (uPAR) gene is closely associated with poor prognosis in pancreatic cancer patients. uPAR regulates epithelial-mesenchymal transition (EMT) and chemoresistance in pancreatic cancer cells through the mitogen-activated protein kinases (MAPK) signaling pathway, though the specific mechanisms remain unclear. This study aimed to investigate the mechanism by which uPAR promotes proliferation, invasion, and chemoresistance of pancreatic cancer cells by inhibiting autophagy. Methods: Pancreatic cancer tissue samples were collected from patients who underwent surgical resection and biopsy at the Changsha Central Hospital, Affiliated to University of South China (Changsha Central Hospital), between December 2021 and Jun 2022. The study was approved by the Ethics Committee of Changsha Central Hospital (Approval No.: 2021-S0182, 2022-S0084). Patient-derived organoids (PDOs) from pancreatic cancer samples were cultured in vitro. Six pancreatic cancer cell lines (AsPC-1, PANC-1, CAPAN-1, CAPAN-2, MIA PaCa-2 and PaTu8988T) were used in this study. uPAR-deficient models were constructed using clustered regularly interspaced short palindromic repeats (CRISPR) Cas9 technology. Cell proliferation and invasion abilities were measured using confocal microscopy, Western blot, enzyme-linked immunosorbent assay (ELISA), and MTS assays. Changes in MAPK and autophagy signaling pathways and gemcitabine-induced cell death were analyzed. The synergistic effects of combined treatments were evaluated using gene silencing (siRNA) or autophagy inhibitors. Results: In AsPC-1 cells, uPAR knockout significantly reduced the proliferation and migration abilities of clone cells compared to wild-type cells, as shown by MTS assays and wound healing experiments, and decreased sensitivity to gemcitabine (P<0.05). Re-expression of uPAR restored the proliferation and invasion abilities of clone cells and partially restored sensitivity to gemcitabine (P<0.05). Confocal microscopy revealed reduced F-actin and a rounded morphology in clone cells. Western blot analysis showed increased expressions of E-cadherin and Slug, decreased expression of vimentin, and increased expressions of phospho-focal adhesion kinase (p-FAK), p-p38MAPK, and the microtubule-associated protein light chain 3B (LC3B) in clone cells compared to wild-type cells. siRNA results indicated that silencing FAK or p38MAPK or combining autophagy inhibition could resensitize clone cells to gemcitabine (P<0.05), with p38MAPK silencing reducing LC3B expression. Organoid studies showed varying responses to gemcitabine among 8 organoids, all expressing uPAR. uPAR expression levels were negatively correlated with gemcitabine IC50 (r2=0.66, P<0.05). Three organoids responded well to the combination of gemcitabine and autophagy inhibitors (P<0.05). Conclusion: uPAR promotes pancreatic cancer cell activity through the p38MAPK signaling pathway, preventing FAK-mediated resistance and cell dormancy. The study suggests that pancreatic cancer patients with high uPAR expression respond better to gemcitabine, while tumors with low uPAR and high p38MAPK expressions may benefit from combined treatment with autophagy inhibitors and cytotoxic chemotherapy.

Keywords

• |pancreatic cancer|organoid|upar|autophagy|gemcitabine|chemoresistance