Involvement of TSC genes and differential expression of other members of the mTOR signaling pathway in oral squamous cell carcinoma.
BACKGROUND
Despite extensive research, the five-year survival rate of oral squamous cell carcinoma (OSCC) patients has not improved. Effective treatment of OSCC requires the identification of molecular targets and signaling pathways to design appropriate therapeutic strategies. Several genes from the mTOR signaling pathway are known to be dysregulated in a wide spectrum of cancers. However, not much is known about the involvement of this pathway in tumorigenesis of OSCC. We therefore investigated the role of the tumor suppressor genes, TSC1 and TSC2, and other members of this pathway in tumorigenesis of OSCC.


METHODS
Expression of genes at the RNA and protein levels was examined by semi-quantitative RT-PCR and western blot analyses, respectively. Loss of heterozygosity was studied using matched blood and tumor DNA samples and microsatellite markers from the TSC1, TSC2 and PTEN candidate regions. The effect of promoter methylation on TSC gene expression was studied by treating cells with methyltransferase inhibitor 5-azacytidine. Methylation status of the TSC2 promoter in tissue samples was examined by combined bisulfite restriction analysis (COBRA).


RESULTS
The semi-quantitative RT-PCR analysis showed downregulation of TSC1, TSC2, EIF4EBP1 and PTEN, and upregulation of PIK3C2A, AKT1, PDPK1, RHEB, FRAP1, RPS6KB1, EIF4E and RPS6 in tumors. A similar observation was made for AKT1 and RPS6KB1 expression in tumors at the protein level. Investigation of the mechanism of downregulation of TSC genes identified LOH in 36.96% and 39.13% of the tumors at the TSC1 and TSC2 loci, respectively. No mutation was found in TSC genes. A low LOH rate of 13% was observed at the PTEN locus. Treatment of an OSCC cell line with the methyltransferase inhibitor 5-azacytidine showed a significant increase in the expression of TSC genes, suggesting methylation of their promoters. However, the 5-azacytidine treatment of non-OSCC HeLa cells showed a significant increase in the expression of the TSC2 gene only. In order to confirm the results in patient tumor samples, the methylation status of the TSC2 gene promoter was examined by COBRA. The results suggested promoter hypermethylation as an important mechanism for its downregulation. No correlation was found between the presence or absence of LOH at the TSC1 and TSC2 loci in 50 primary tumors to their clinicopathological variables such as age, sex, T classification, stage, grade, histology, tobacco habits and lymph node metastasis.


CONCLUSIONS
Our study suggests the involvement of TSC genes and other members of the mTOR signaling pathway in the pathogenesis of OSCC. LOH and promoter methylation are two important mechanisms for downregulation of TSC genes. We suggest that known inhibitors of this pathway could be evaluated for the treatment of OSCC.
Department of Molecular Reproduction Development and Genetics, Indian Institute of Science, Bangalore, India. sanjuc8@gmail.com
Sanjukta
S
Chakraborty
S M Azeem
SM
Mohiyuddin
K S
KS
Gopinath
Arun
A
Kumar
D001374:Azacitidine / Q000494:pharmacology; D015153:Blotting, Western; D002294:Carcinoma, Squamous Cell / Q000188:drug therapy / Q000235:genetics* / Q000473:pathology; D019175:DNA Methylation / Q000187:drug effects; D004252:DNA Mutational Analysis; D004273:DNA, Neoplasm / Q000032:analysis; D015536:Down-Regulation; D005260:Female; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D016147:Genes, Tumor Suppressor; D006801:Humans; D008297:Male; D009062:Mouth Neoplasms / Q000188:drug therapy / Q000235:genetics* / Q000473:pathology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics*; D000077004:Tuberous Sclerosis Complex 1 Protein; D000077005:Tuberous Sclerosis Complex 2 Protein; D014407:Tumor Cells, Cultured; D025521:Tumor Suppressor Proteins / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism
D004273:DNA, Neoplasm; C000624650:TSC1 protein, human; C000624653:TSC2 protein, human; D000077004:Tuberous Sclerosis Complex 1 Protein; D000077005:Tuberous Sclerosis Complex 2 Protein; D025521:Tumor Suppressor Proteins; D001374:Azacitidine