Hypoxia causes downregulation of the c-Myc signaling pathway through proteasomal degradation of c-Myc and the transcriptional upregulation of Mad1 and Mxi1, repressors of c-Myc

1443 As a physiologic stimulus, hypoxia is associated with profound changes in protein expression, cellular proliferation, and energy metabolism. Using different human cancer cell lines, we have observed that c-Myc protein levels decrease during hypoxia in a time-dependent manner. In contrast, c-Myc levels remain stable during hypoxia in cells treated with a proteasome inhibitor, suggesting that hypoxic downregulation of c-Myc involves the ubiquitin-proteasome pathway. Utilizing an oligonucleotide microarray approach to characterize gene expression changes that occur in hypoxia, we identified Mxi1, a c-Myc antagonist, as a hypoxia-inducible target gene. Mxi1 is induced over a range of hypoxia levels and also following treatment with hypoxia-mimetic agents such as cobalt chloride. Interestingly, Mxi1 was not induced in a cell line deficient in ARNT (Hif1β), the dimerization partner of the hypoxia-inducible factor 1α transcription factor (Hif1α), suggesting that Mxi1 is a direct transcriptional target of the HIF-1 complex. Hypoxia also resulted in the induction of Mad1, another c-Myc antagonist, although in a HIF-independent manner. Co-immunoprecipitation experiments demonstrated that under hypoxic conditions there is a cellular switch from Myc:Max to Mad:Max heterocomplexes. Analysis of downstream transcriptional targets of c-Myc during hypoxia revealed a heterogeneous response pattern. While some c-Myc targets, such as ornithine decarboxylase (ODC), were downregulated during hypoxia as predicted, others, such as lactate dehydrogenase A (LDH-A) were upregulated. Hypoxic induction of LDH-A was dependent on Hif1α, consistent with its previously known role as a transcriptional target of the HIF-1 complex. These results suggest a complex mechanism for opposing the c-Myc pathway during hypoxia that is mediated by proteasomal degradation of c-Myc, the induction of Mxi1 and Mad1, and a dominant effect of HIF-1 transcriptional activity. Present studies are examining the contribution of altered c-Myc function to tumor growth and therapeutic response under hypoxic conditions.