Abstract A046: Mechanotransduction in pancreatic ductal adenocarcinoma: the role of the hERG1/β1integrin complex

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy causing ∼7% of cancer-related deaths. Its poor treatment response is driven by a desmoplastic tumor microenvironment (TME) that renders the cancer tissue rigid, hypoxic and almost avascular. The unraveling of novel mechanisms sustaining tumor progression and therapy resistance in PDAC is urgently needed. Ion channels are novel, crucial drivers in tumor progression. Among them, the potassium channel hERG1, through its binding to β1 integrins, triggers a signaling cascade that reorganizes the cytoskeleton and activates cell migration. This project was aimed to elucidate the role of the hERG1/β1 integrin complex in mechanotransduction in PDAC malignancy. The mechanical profiles of different pancreatic cell lines HPDE (Immortal Human Pancreatic Duct Epithelial), serving as the healthy epithelial cells, PANC-1, SUIT-028, and SUIT-007, all PDAC cell lines were characterized through traction force measurements using fibronectin (FN)-coated elastic micropillar arrays at three stiffnesses: 10, 29, and 47 kPa. PDAC cells developed higher traction forces on stiffer micropillars compared to HPDE. Force generation inversely correlated with the abundance of the hERG1/β1 integrin complex, quantified through either Western blot (WB) or immunofluorescence (IF) as in Duranti, C. & Iorio, J et al., Life Science Alliance, 2024. Treating cells with a single-chain bispecific antibody in the format of a diabody (scDb) which targets and harnesses the hERG1/β1 integrin complex consistently increased traction forces in PDAC cells but had no effect on HPDE. The effects of the treatment were particularly evident in PANC-1 cells seeded onto FN-coated micropillar with a stiffness of 28 kPa, i.e. very close to the stiffness of pancreatic cancers in vivo. Key mechanotransduction effectors Yes-Associated Protein 1 (YAP1) and Activating Transcription Factor 3 (ATF3) were analyzed through IF, WB and RT-qPCR. ATF3 expression decreased with increasing substrate stiffness, in agreement with literature, while YAP1 was unaffected. Notably, ATF3 downregulation was reversed by treatment with scDb. Finally, Stochastic Optical Reconstruction Microscopy (STORM) (LENS, University of Florence) and custom code analysis was applied to PANC-1 and PANC-1 KO cells. These analyses identified distinct morphological and ultrastructural actin cytoskeleton phenotypes in PANC-1 compared to PANC-1 KO cells. Overall, our results demonstrate that the hERG1/β1 integrin complex acts as a key mediator of mechanotransduction in PDAC cells, mainly through the modulation of ATF3 transcription factor. Targeting this pathway with scDb successfully alters these cascades, offering potential promising therapeutic strategies against PDAC progression. Finally, applying STORM microscopy to oncology research allows for detecting nanoscale actin changes, providing deeper insights into tumor mechanobiology.