Endoplasmic reticulum stress signaling actively contributes to therapy resistance in colorectal cancer

Abstract Purpose We investigated the involvement of endoplasmic reticulum (ER) stress signaling in cancer cell responses to chemo- and radiotherapy, focusing on three main ER stress mediators, the transcription factors ATF4, XBP1 and ATF6. Methods Public cancer genome datasets were assessed for alterations in ER stress mediators. Surgically resected colorectal cancer tissues were tested by flow cytometry and used to generate patient-derived organoids. Human cell lines and organoids were characterized under oxaliplatin treatment, alone or combined with pharmacological inhibitors of the three ER stress branches, or X-ray irradiation, for cytotoxicity, activation of ER stress and proteome changes. To monitor ER stress in real time, stable HEK293 kidney epithelial cell lines were established expressing ATF4, XBP1, or ATF6, fused with a fluorophore. Results Genomic amplification of ATF6, but not ATF4 or XBP1, was frequent in solid tumor entities like breast, lung and colorectal cancer and significantly associated with reduced disease-free survival. In colorectal cancer, increased ATF6 was associated with genetic instability. Basal ER stress mediator expression was correlated to chemoresistance in colorectal cancer cell lines, and generally high in cancer cells compared to HEK293 cells. With proteomics and live HEK293-based reporter lines, we noted that oxaliplatin treatment induced ER stress in a remarkably different way from the canonical ER stress inducer thapsigargin. Moreover, modulation of ER stress signaling by exogenous expression of the stress mediators positively affects chemoresistance, and pharmacological inhibition of ATF6 sensitizes ER-stressed HCT116 colorectal cancer cells to chemotherapy. Of note, cellular stress responses was strongly dependent on the individual transcription factor: XBP1-driven response appeared multi-functional, involved in ribosome biogenesis stress and associated with oxaliplatin resistance. ATF6-dependent stress signaling was involved in DNA damage repair, and was essential for radioresistance. Moreover, chemoresistance in HCT116 cancer cells was impaired by pharmacological ATF6 inhibition. Conclusion Activation of the ER stress signaling may be critically involved in acquired chemo- and radioresistance. Due to their apparent cytoprotective roles, ATF6 and XBP1 could be attractive predictive biomarkers and putative therapeutic targets. SUMMARY To address their roles in the clinical context, genomic alterations of ATF4, XBP1 and/or ATF6 in human solid tumors were assessed with respect to prognosis and genomic instability. Moreover, surgically resected CRC patient tissues were tested for expression of ER stress markers by flow cytometry and associated with clinical characteristics. In addition, a panel of human cell lines and patient-derived colon organoids were characterized under therapeutic conditions for expression and activation of ER stress proteins, and resulting cytotoxicity was determined. To monitor and modulate ER stress activation in live cells with subcellular resolution, stable reporter cell lines expressing ATF4, sXBP1 or ATF6 proteins fused with a fluorophore were established. These lines were tested for gene or protein expression and cytotoxicity assays to analyze how activation or inhibition of ER stress proteins affects the cellular responses to oxaliplatin treatment or X-ray irradiation. Finally, mass spectrometric proteome analysis was performed to obtain an unbiased readout on the cellular responses to chemotherapy driven by the activation of the ER stress proteins.