Involvement of TSC genes and differential expression of other members of the mTOR signaling pathway in oral squamous cell carcinoma

<p>Abstract</p> <p>Background</p> <p>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, <it>TSC1 </it>and <it>TSC2</it>, and other members of this pathway in tumorigenesis of OSCC.</p> <p>Methods</p> <p>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 <it>TSC1</it>, <it>TSC2 </it>and <it>PTEN </it>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 <it>TSC2 </it>promoter in tissue samples was examined by combined bisulfite restriction analysis (COBRA).</p> <p>Results</p> <p>The semi-quantitative RT-PCR analysis showed downregulation of <it>TSC1</it>, <it>TSC2</it>, <it>EIF4EBP1 </it>and <it>PTEN</it>, and upregulation of <it>PIK3C2A</it>, <it>AKT1</it>, <it>PDPK1</it>, <it>RHEB</it>, <it>FRAP1</it>, <it>RPS6KB1</it>, <it>EIF4E </it>and <it>RPS6 </it>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 <it>TSC2 </it>gene only. In order to confirm the results in patient tumor samples, the methylation status of the <it>TSC2 </it>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.</p> <p>Conclusion</p> <p>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.</p

A) Representative RT-PCR gel pictures showing restoration or increase in the expression of TSC genes

b) Graphical representation of expression levels of and following the drug treatment. Expressions of these genes in untreated SCC 131 and HeLa cells at day 0, 2 and 5 were used as controls. '-' indicates no 5-azacytidine treatment and '+' indicates treatment with 5-azacytidine. Graphs represent mean ± SEM of two separate experiments. * indicates significant difference at p < 0.05. Although the expression of was increased in HeLa cells following the drug treatment, the relative expression levels of treated and untreated cells were not significantly different from each other. The expression of was significantly increased in both cell lines following the drug treatment.<p><b>Copyright information:</b></p><p>Taken from "Involvement of TSC genes and differential expression of other members of the mTOR signaling pathway in oral squamous cell carcinoma"</p><p>http://www.biomedcentral.com/1471-2407/8/163</p><p>BMC Cancer 2008;8():163-163.</p><p>Published online 6 Jun 2008</p><p>PMCID:PMC2430211.</p><p></p

A) Semi-quantitative RT-PCR analysis of TSC genes in 16 matched normal and tumor samples

Note, downregulation of both genes in tumor samples. Each square or triangle corresponds to data from one sample. Horizontal lines represent mean values of mRNA expression across normal or tumor samples. b) Western blot analysis of TSC1 and TSC2 in eight matched normal and tumor tissues. TSC1 and TSC2 show no expression or downregulation in tumor samples. c) Western blot analysis of TSC1 and TSC2 in three oral cancer (KB, SCC 104 and SCC 131), lung carcinoma (A549), embryonic kidney (HEK-293T), cervical carcinoma (HeLa) and hepatic carcinoma (HepG2) cell lines. Note, the expression of TSC2 is not detectable in the oral cancer cell line SCC 131.<p><b>Copyright information:</b></p><p>Taken from "Involvement of TSC genes and differential expression of other members of the mTOR signaling pathway in oral squamous cell carcinoma"</p><p>http://www.biomedcentral.com/1471-2407/8/163</p><p>BMC Cancer 2008;8():163-163.</p><p>Published online 6 Jun 2008</p><p>PMCID:PMC2430211.</p><p></p

A) Representative gel pictures showing LOH for markers from the and candidate regions

B and T denote constitutive blood and tumor DNA respectively. Arrows indicate loss or allelic imbalance of the corresponding allele in tumor DNA. b) LOH analysis of 50 matched samples at both the TSC loci. Approximate locations of microsatellite markers with respect to TSC genes are shown on the left. Tumors are grouped according to their T classification (T1–T4). Numbers represent patient numbers. Abbreviations: NI, non-informative; IN, informative; LOH, loss of heterozygosity; and MI, microsatellite instability.<p><b>Copyright information:</b></p><p>Taken from "Involvement of TSC genes and differential expression of other members of the mTOR signaling pathway in oral squamous cell carcinoma"</p><p>http://www.biomedcentral.com/1471-2407/8/163</p><p>BMC Cancer 2008;8():163-163.</p><p>Published online 6 Jun 2008</p><p>PMCID:PMC2430211.</p><p></p