miR-34a and IRE1A/XBP-1(S) Form a Double-Negative Feedback Loop to Regulate Hypoxia-Induced EMT, Metastasis, Chemo-Resistance and Autophagy

SIMPLE SUMMARY: The hypoxic tumor microenvironment is a key factor in the formation of metastasis and treatment resistance. We recently identified a new regulatory network consisting of the hypoxia-inducible transcription factor HIF-1A and the p53-inducible miR-34a that determines whether a tumor cell undergoes EMT (epithelial-mesenchymal transition) or MET (mesenchymal-epithelial transition) under hypoxic conditions. Here, we characterized XBP-1 and IRE1A as new miR-34a targets, which are relevant in this context. In addition, we found that the activation of the IRE1A/XBP-1 arm of the unfolded protein response (UPR) by hypoxia results in repression of miR-34a and thereby mediates hypoxia-induced EMT, migration, invasion, and chemo-resistance in p53 mutated/deficient CRC lines and ultimately contributes to lung metastasis formation. In this context, the restoration of miR-34a may be of therapeutic value in the future. ABSTRACT: Tumor-associated hypoxia, i.e., decreased availability of oxygen, results in a poor clinical outcome since it promotes EMT, metastasis, and chemotherapy-resistance. We have previously identified p53 and its target miR-34a, as critical determinants of the effect of hypoxia on colorectal cancer (CRC). Here, we aimed to characterize mechanisms that contribute to the selective advantage of cells with loss of p53/miR-34a function in a hypoxic environment. Using in silico prediction, we identified XBP-1 and IRE1A as potential miR-34a targets. IRE1A and XBP-1 are central components of the unfolded protein response that is activated by ER stress, which is also induced in tumor cells as a response to harsh conditions surrounding tumors such as hypoxia and a limited supply of nutrients. Here we characterized the XBP-1(S) transcription factor and its regulator IRE1A as direct, conserved miR-34a targets in CRC cells. After hypoxia and DNA damage, IRE1A and XBP-1 were repressed by p53 in a miR-34a-dependent manner, whereas p53-deficient cells showed induction of IRE1A and XBP-1(S). Furthermore, miR-34a expression was directly suppressed by XBP-1(S). In p53-deficient CRC cells, hypoxia-induced EMT, migration, invasion, metastases formation, and resistance to 5-FU were dependent on IRE1A/XBP-1(S) activation. Hypoxia-induced autophagy was identified as an XBP-1(S)-dependent mediator of 5-FU resistance and was reversed by ectopic miR-34a expression. The HIF1A/IRE1A/XBP-1(S)/p53/miR-34a feedback loop described here represents a central regulator of the response to hypoxia and ER stress that maintains cellular homeostasis. In tumors, the inactivation of p53 and miR-34a may result in IRE1A/XPB-1(S)-mediated EMT and autophagy, which ultimately promotes metastasis and chemoresistance.