bioRxiv2025
In pancreatic ductal adenocarcinoma cancer (PDAC) drug resistance is a severe clinical problem and patients relapse within a few months after receiving the standard-of-care chemotherapy. One contributing factor to treatment resistance is the desmoplastic nature of PDAC; the tumours are surrounded by thick layers of stroma composing up to 90% of the tumour mass. This stroma, which is mostly comprised of extracellular matrix (ECM) proteins, is secreted by cancer-associated fibroblasts (CAFs) residing in the tumour microenvironment. However, the mechanistic basis by which the tumour stroma directly contributes to chemoresistance remains unclear. Here, we show that CAF-secreted ECM proteins induce chemoresistance by blunting chemotherapy-induced DNA damage. Mechanistically, we identify N-myc downstream regulated gene 1 (NDRG1) as a key protein required for stroma-induced chemoresistance that responds to signals from the ECM and adhesion receptors. We further show that NDRG1 is a novel DNA repair protein that physically interacts with replication forks, maintains DNA replication and functions to resolve stalled forks caused by chemotherapy. More specifically, NDRG1 reduces R-loops, RNA-DNA hybrids that are known to cause genomic instability. R-loops occur during replication-transcription conflicts in S-phase and after chemotherapy treatments, thus posing a major threat to normal replication fork homeostasis. We identify NDRG1 as highly expressed in PDAC tumours, and its high expression correlates with chemoresistance and poor disease-specific survival. Importantly, knock-out of NDRG1 or inhibition of its phosphorylation restores chemotherapy-induced DNA damage and resensitizes tumour cells to treatment. In conclusion, our data reveal an unexpected role for CAF-secreted ECM proteins in enhancing DNA repair via NDRG1, a novel DNA repair protein, directly linking tumour stroma to replication fork homeostasis and R-loop biology, with important therapeutic implications for restoring DNA damage response pathways in pancreatic cancer. Summary paragraphDrug resistance is a severe clinical problem in stroma-rich tumours, such as pancreatic ductal adenocarcinoma (PDAC), and patients often relapse within a few months on chemotherapy1-9. The stroma, comprised of extracellular matrix (ECM) proteins, is secreted by cancer-associated fibroblasts (CAFs) residing in the tumour microenvironment10-13. Prior work show that ECM proteins provide survival benefits to cancer cells14,15. However, the precise role of CAF-secreted ECM in resistance to DNA damaging chemotherapies remains poorly understood. Here, we link ECM proteins to chemoresistance by enhanced DNA damage repair (DDR). Mechanistically, we identify N-myc downstream-regulated gene 1 (NDRG1) as a key effector downstream of ECM and the integrin-Src-SGK1-signalling axis that mediates enhanced DDR. We show that NDRG1 loss, mutation of conserved His194, or inhibition of NDRG1 phosphorylation by SGK1 lead to replication fork stalling, increased R-loops, and higher transcription-replication conflicts, resulting in genomic instability and sensitivity to chemotherapies. Our analysis of PDAC patient cohorts16 found that high NDRG1 expression correlates with chemoresistance and poor patient survival. In conclusion, we uncover an unexpected role for CAF-secreted ECM proteins in promoting therapeutic resistance by enhancing DDR and establish NDRG1 as a novel DNA repair protein directly linking tumour stroma to DDR.