Breast cancer cells respond to glutamine deprivation by activation of NF-kappa B: Mechanism and implications for breast cancer progression

Vascular endothelial growth factor (VEGF) and interleukin-8/CXCL8 (IL-8) are prominent pro-angiogenic and pro-metastatic cytokines up-regulated in many types of cancer mainly by hypoxia. It was hypothesized that lack of nutrients other than oxygen could stimulate the expression of these factors. The effect of glutamine availability on VEGF and IL-8 expression by TSE human breast carcinoma cells as well as the role of NF-kappaB and AP-1 transcription factors in this response were examined. VEGF and IL-8 secretion and mRNA levels were dramatically induced by glutamine deprivation. mRNA stabilization contributed to this response. Glutamine deprivation increased NF-kappaB (p65/p50) and AP-1 (Fra-1/c-Jun+JunD) DNA binding activities. Blocking NF-kappaB and AP-1 activation with curcumin and expression of dominant inhibitors, inhibitory (I)kappaB super repressor (IkappaBM) and a mutant form of c-Fos (A-Fos), demonstrated the involvement of NF-kappaB and AP-1 transcription factors in the induction of IL-8 but not VEGF expression. A macro-array containing 111 NF-kappaB-target genes identified a total of 17 that were up-regulated 2-fold or more by glutamine deprivation, including pro-metastatic neutrophil chemoattractant, growth regulated oncogene alpha (GROalpha).; We next examined the mechanism of NF-kappaB activation by glutamine deprivation of TSE cells. Inhibition of Src tyrosine kinase activity with PP1 and by expression of dominant negative Src mutant diminished NF-kappaB activation and IL-8 expression. Glutamine deprivation increased PI3-kinase activity. PI3-kinase inhibitor LY294002 blocked NF-kappaB activation and IL-8 expression. Both responses were impaired by intracellular calcium chelator, BAPTA-AM, inhibitor of calcium release from the intracellular stores, TMB-8, inhibitor of inositol 1,4,5-triphosphate (IP3) receptors, 2-APB, and extracellular calcium chelator, EGTA. Thus, store-operated calcium entry was necessary for NF-kappaB activation. Inhibition of phospholipase C (PLC) activity with U73122 or ET-18-OCH3 blocked NF-kappaB activation indicating IP3 production was necessary for this response. Increase in NF-kappaB DNA binding activity and nuclear p65 protein levels did not coincide with IkappaBalpha degradation or IkappaBalpha translation inhibition. Finally, a uniquely modified isoform of IkappaBalpha protein was observed in TSE cells. Glutamine deprivation caused loss of this modification.; These results suggest that NF-kappaB activation in response to glutamine deprivation may promote angiogenesis, survival, and motility of cancer cells within a nutrient-poor solid tumor microenvironment.