In hypoxic breast cancer cells BNIP mediated autophagy activ

In hypoxic breast cancer cells, BNIP3-mediated autophagy activation and elevation of amino TP-0903 levels were notable. BNIP3, a BCL-2 family protein, is known to be transcriptionally regulated by HIF-1α and to be involved in cancer cell death in hypoxic conditions [51]. As a link to autophagy, hypoxia-induced Bnip3 was initially identified to be critical in autophagic cell death in murine embryonic fibroblasts (MEFs) [36]. Then, it was shown that BNIP3 is essential for hypoxia-induced autophagy in cancer cells and disruption of BNIP3 triggered cell death, which suggests a positive role of HIF-1α, BNIP3, and autophagy in tumor survival and progression [52]. Of particular note, this study shows that the BH3 domain of BNIP3 disrupts the Bcl2-Beclin complex, which results in the stabilization of the BNIP3/Bcl2 complex and subsequently releases Beclin to activate the pro-survival autophagy pathway. Now, it is generally accepted that hypoxia-induced autophagy plays a crucial role in cancer cell survival by removing damaged cellular components and supplying a source of nutrients under conditions of hypoxia without nutrient deficits, whereas severe stress conditions with hypoxia and nutrient limitation causes autophagic cell death [53]. Our study shows that hypoxia-induced autophagy activation is mediated by HIF-1α/BNIP3 signaling and NRF2-silencing inhibited the HIF-1α-mediated autophagy activation and amino acid elevation. Similarly to HIF-1α-mediated metabolic changes, miR-181c was found to be involved in autophagy activation; miR-181c expression suppressed BNIP3-mediated autophagy activation and hypoxia-inducible amino acid elevation. Additionally, the inhibition of miR-181c in NRF2-silenced cancer cells restored BNIP3-mediated autophagy activation. A protective role of HIF-1α/BNIP3/autophagy from hypoxic cell death was verified by using an autophagy inhibitor; chloroquine treatment substantially reduced cell viability in hypoxic control cells, whereas viability of NRF2-silenced cells, which was significantly lower than control cells, was not affected by chloroquine treatment. These results suggest that the hypoxia-induced autophagy is impaired by NRF2-silencing and provides further evidence for the role of HIF-1α/BNIP3-mediated autophagy in adaptive survival of cancer cells. Taken together, our results demonstrate that hypoxia-induced metabolic changes to glycolysis, PPP, and autophagy are suppressed by NRF2 inhibition through the blockage of HIF-1α accumulation and, further, suggest that elevation of miR-181c levels is a molecular link to connect NRF2 to HIF-1α dysregulation (Fig. 7). Thus, NRF2 and miR-181c could be effective targets to counteract HIF-1α-orchestrated metabolic adaptation of hypoxic cancer cells.
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Introduction A number of epidemiological studies showed that disturbances of the circadian rhythm strongly correlate with carcinogenesis and tumor progression in humans (for review see Fu and Lee, 2003, Filipski and Levi, 2009, Sahar and Sassone-Corsi, 2009, Rana and Mahmood, 2010). Accordingly, the International Agency for Research on Cancer classified shift work as a group 2A carcinogenic factor (Straif et al., 2007). The circadian rhythm in mammals is maintained by an integrated network of the central (neural or brain) and peripheral (tissue-specific) clocks. The central clock is located in the suprachiasmatic nucleus of the brain, receives light cues to keep in phase with the light-dark cycle, and synchronizes the peripheral clocks in various tissues through a variety of electrical, endocrine, and metabolic signaling pathways (Albrecht, 2012, Richards and Gumz, 2012). At the genetic level, both the central and the peripheral clocks are regulated by an interplay of positive and negative feedback loops involving the same set of clock genes (Yagita et al., 2001). Thereby, the bHLH-PAS transcription factors CLOCK (circadian locomotor output cycles kaput) and BMAL1 (brain-muscle Arnt-like protein 1) represent the major components of the core clock's positive limb. They induce, among others, the expression of the proteins PER (period 1,2) and CRY (cryptochrome 1,2), which constitute the major arm of the negative limb. The induced PER and CRY proteins then form a complex with each other as well as with modulator proteins such as CK1ε, CKII, or FBXL3 and act as repressors of CLOCK/BMAL heterodimers. Subsequently, they inhibit their own expression as well as those of other CLOCK/BMAL-regulated output genes (Griffin et al., 1999, Kume et al., 1999, Yoo et al., 2005, Sato et al., 2006). The core loop is interconnected to several other feedback loops, including those of REV-ERBs or PPARα/RORs, which repress or activate BMAL1 expression, respectively (Albrecht, 2012, Ko and Takahashi, 2006, Asher and Schibler, 2011). Overall, the continuous interplay between the core CLOCK/BMAL1 positive limb and the PER/CRY negative limb in concert with post-translational modifications and interconnected loops results in the oscillation of gene expression in a circadian manner.