Hypoxia has recently been shown to activate the endoplasmic reticulum kinase PERK, leading to phosphorylation of eIF2a and inhibition of mRNA translation initiation. Using a quantitative assay, we show that this inhibition exhibits a biphasic response mediated through two distinct pathways. The first occurs rapidly, reaching a maximum at 1-2 h and is due to phosphorylation of eIF2a. Continued hypoxic exposure activates a second, eIF2a-independent pathway that maintains repression of translation. This phase is characterized by disruption of eIF4F and sequestration of eIF4E by its inhibitor 4E-BP1 and transporter 4E-T. Quantitative RT-PCR analysis of polysomal RNA indicates that the translation efficiency of individual genes varies widely during hypoxia. Furthermore, the translation efficiency of individual genes is dynamic, changing dramatically during hypoxic exposure due to the initial phosphorylation and subsequent dephosphorylation of eIF2a. Together, our data indicate that acute and prolonged hypoxia regulates mRNA translation through distinct mechanisms, each with important contributions to hypoxic gene expression.

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Purpose: Triple-negative breast cancers (TNBC) are defined by a lack of expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (ERBB2/HER2). Although initially responsive to chemotherapy, most recurrent TNBCs develop resistance, resulting in disease progression. Autophagy is a lysosome-mediated degradation and recycling process that can function as an adaptive survival response during chemotherapy and contribute to chemoresistance. Our goal was to determine whether autophagy inhibition improves treatment efficacy in TNBC cells in tumors either sensitive or refractory to anthracyclines.Experimental Design: We used in vitro and in vivo models of TNBC using cell lines sensitive to epirubicin and other anthracyclines, as well as derivative lines, resistant to the same drugs. We assessed basal autophagy levels and the effects of chemotherapy on autophagy in parental and resistant cells. Applying various approaches to inhibit autophagy alone and in combination with chemotherapy, we assessed the effects on cell viability in vitro and tumor growth rates in vivo.Results: We demonstrated that epirubicin induced autophagic flux in TNBC cells. Epirubicin-resistant lines exhibited at least 1.5-fold increased basal autophagy levels and, when treated with autophagy inhibitors, showed a significant loss in viability, indicating dependence of resistant cells on autophagy for survival. Combination of epirubicin with the autophagy inhibitor hydroxychloroquine resulted in a significant reduction in tumor growth compared with monotherapy with epirubicin.Conclusion: Autophagy inhibition enhances therapeutic response in both anthracycline-sensitive and -resistant TNBC and may be an effective new treatment strategy for this disease.