Hypoxia-inducible factor-1α polymorphisms and TSC1/2 mutations are complementary in head and neck cancers

Abstract

Background: Polymorphisms or mutations in hypoxia inducible factor-1 alpha (HIF-1 alpha) that increases its activity and stability under normoxia have recently been identified. Likewise, disruption of the TSC1/ TSC2 complex through loss of TSC1 or TSC2 has been shown to result in abnormal accumulation of HIF-1 α. Here, we investigate the novel polymorphisms in exon 12, that approximate the oxygen-dependent degradation domain of HIF-1 alpha in five cell lines and 28 patients with oral squamous carcinomas. Moreover, we assess for the presence of polymorphisms and mutations in TSC1 and TSC2, to ascertain if dysregulation of such might complement HIF-1 alpha expression. Results: Denaturing high pressure liquid chromatography (DHPLC) analysis on PCR fragments in exon 12 of HIF-1 alpha from 28 patients with OSCC revealed that 6 of 28 patients had mismatched heteroduplex patterns. Genomic DNA was extracted from peripheral blood leukocytes and direct sequencing showed that in 5 of the six cases these changes represented polymorphisms while, one case was a somatic mutation. Analyses of TSC1 and TSC2 revealed heteroduplexes in exons: TSC1 exon 17; TSC2 exons 36,40, and 41. The relative levels of HIF-1 alpha were significantly greater for tumors possessing a HIF-1 alpha polymorphism or mutation within exon 12, whereas tumors possessing a deletion or polymorphism in TSC1/TSC2 displayed a trend for higher levels of HIF-1 alpha. Western blot analyses for HIF-1 alpha, TSC1 and TSC2 in five SCC cell lines revealed high levels of HIF-1 alpha in SCC cells possessing TSC1 and/or TSC2 mutations. Wild-type TSC2 cells targeted with siRNA to TSC2 exhibited increased levels of HIF-1 alpha. Transfection of a HIF-1 alpha mutant produced higher levels of HIF-1 alpha in TSC1/TSC2 mutant cell lines than in wild type cells. TSC1/TSC2 mutant cell lines administered Rapamycin blocked S6 phorphorylation and diminished the levels of HIF-1 alpha to those observed in cell lines with wild type TSC1/TSC2. Conclusion: Dysregulation of the TSC1/TSC2 complex by mutation compliments HIF-1α polymorphisms in the expression of HIF-1 alpha in SCC of the head and neck, and may provide biomarkers to predict responses to specific therapies and overall disease prognosis. © 2006 Hebert et al; licensee BioMed Central Ltd.