title: Hypoxia-inducible factor-1alpha polymorphisms and TSC1/2 mutations are complementary in head and neck cancers.
abstract: BACKGROUND
  Polymorphisms or mutations in hypoxia inducible factor-1 alpha (HIF-1alpha) 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-1alpha. Here, we investigate the novel polymorphisms in exon 12, that approximate the oxygen-dependent degradation domain of HIF-1alpha 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-1alpha expression.
  
  
  RESULTS
  Denaturing high pressure liquid chromatography (DHPLC) analysis on PCR fragments in exon 12 of HIF-1alpha 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-1alpha were significantly greater for tumors possessing a HIF-1alpha polymorphism or mutation within exon 12, whereas tumors possessing a deletion or polymorphism in TSC1/TSC2 displayed a trend for higher levels of HIF-1alpha. Western blot analyses for HIF-1alpha, TSC1 and TSC2 in five SCC cell lines revealed high levels of HIF-1alpha in SCC cells possessing TSC1 and/or TSC2 mutations. Wild-type TSC2 cells targeted with siRNA to TSC2 exhibited increased levels of HIF-1alpha. Transfection of a HIF-1alpha mutant produced higher levels of HIF-1alpha 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-1alpha to those observed in cell lines with wild type TSC1/TSC2.
  
  
  CONCLUSIONS
  Dysregulation of the TSC1/TSC2 complex by mutation compliments HIF-1alpha polymorphisms in the expression of HIF-1alpha in SCC of the head and neck, and may provide biomarkers to predict responses to specific therapies and overall disease prognosis.
authors.affiliation: Department of Diagnostic Sciences and Pathology, University of Maryland Baltimore, Maryland 21201-1586, USA. chebert@umaryland.edu
authors.forename: Carla
authors.initials: C
authors.lastname: Hebert
authors.forename: Kathleen
authors.initials: K
authors.lastname: Norris
authors.forename: Pallavi
authors.initials: P
authors.lastname: Parashar
authors.forename: Robert A
authors.initials: RA
authors.lastname: Ord
authors.forename: Nikolaos G
authors.initials: NG
authors.lastname: Nikitakis
authors.forename: John J
authors.initials: JJ
authors.lastname: Sauk
mesh_terms: D002294:Carcinoma, Squamous Cell / Q000097:blood / Q000235:genetics* / Q000473:pathology; D045744:Cell Line, Tumor; D005260:Female; D006258:Head and Neck Neoplasms; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics* / Q000378:metabolism; D008297:Male; D009062:Mouth Neoplasms / Q000097:blood / Q000235:genetics* / Q000473:pathology; D009154:Mutation; D011110:Polymorphism, Genetic; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011494:Protein Kinases / Q000378:metabolism; D034622:RNA Interference; D020123:Sirolimus / Q000494:pharmacology; D058570:TOR Serine-Threonine Kinases; D014162:Transfection; D000077004:Tuberous Sclerosis Complex 1 Protein; D000077005:Tuberous Sclerosis Complex 2 Protein; D025521:Tumor Suppressor Proteins / Q000235:genetics* / Q000378:metabolism
chemical_list: D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D047428:Protein Kinase Inhibitors; C000624650:TSC1 protein, human; C000624653:TSC2 protein, human; D000077004:Tuberous Sclerosis Complex 1 Protein; D000077005:Tuberous Sclerosis Complex 2 Protein; D025521:Tumor Suppressor Proteins; D011494:Protein Kinases; C546842:MTOR protein, human; D058570:TOR Serine-Threonine Kinases; D020123:Sirolimus