Gene Expression Profiling And Insights Into The Involvement Of The Insulin Signaling Pathway In Oral Cancer

Abstract

1. Despite extensive research on oral squamous cell carcinoma (OSCC), its five-year survival rate has not improved for the last two decades. Effective treatment of OSCC requires the identification of molecular targets to design appropriate therapeutic strategies. To this end, DDRT-PCR analysis was used to identify molecular markers, which could be used as therapeutic targets.

2. DDRT-PCR in combination with reverse Northern analysis identified 25 differentially expressed genes in oral tumors. Fourteen genes did not show homology to any known gene in the database and therefore may represent non-specific genomic DNA sequences or novel genes that have not yet been identified. The remaining 11 genes showed homology to known genes such as DIAPH1, NJMU-R1, RBM28, PCNA, GLTP, MTATP6, ZKSCAN1, TNKS2, PAM, TUBB2C and C14orf154. TNKS2, PAM, TUBB2C and C14orf154 showed downregulation and the remaining seven genes were upregulated in oral tumor samples.

3. To reconfirm the results of DDRT-PCR and reverse Northern blot analyses, Northern blot analysis was carried out on matched normal and tumor samples for a few genes. As expected, PCNA, NJMU-R1 and ZKSCAN1 showed upregulation, whereas TUBB2C showed downregulation in the tumor sample. PCNA was also found to be upregulated in tumor samples at the protein level.

4. The expression of eight differentially expressed genes (viz., DIAPH1, NJMU-R1, RBM28, PCNA, GLTP, TNKS2, PAM and TUBB2C) was also validated in a panel of 16 matched normal and tumor samples. The mean mRNA expression levels of GLTP, PCNA, RBM28, NJMU-R1 and DIAPH1 were significantly greater in tumor samples than in normal samples. The mean expression levels of TNKS2, PAM and TUBB2C were significantly lower in tumor samples than in normal samples.

5. As some of the genes like NJMU-R1, RBM28, GLTP and PAM are found to differentially regulated in a majority of the tumors, they could be used as potential markers in oral cancer.

6. Tuberin and hamartin have been placed as a complex in the insulin signaling pathway and are known to negatively regulate this pathway. Since overexpression of TSC2 has been previously shown to exert antitumor effect on two oral cancer cell lines, and some components of the insulin signaling pathway have already been implicated in head and neck cancers, we reasoned that both TSC genes and other key players of this pathway might be differentially regulated in oral tumors. Northern blot analysis showed downregulation of the TSC2 gene in an oral tumor sample. In order to further validate the expression pattern of the TSC2 gene, a semiquantative RT-PCR analysis was carried out in a panel of 16 matched normal and tumor samples. The mean expression level of TSC2 was significantly lower in tumor samples than in normal tissue samples. The mean expression level of its interacting partner TSC1 was also significantly lower in tumor samples than in normal tissue samples, suggesting the involvement of these genes in the etiology of oral cancer. TSC1 and TSC2 were also downregulated in eight matched normal and tumor samples at the protein level. We wanted further to determine the expression of both TSC genes in cell lines. Interestingly, TSC2 did not show a detectable level of expression in an oral cancer cell line SCC 131, whereas it was expressed in two other oral cancer cell lines KB and SCC 104 as well as in four non-oral cell lines: A549, HEK-293T, HeLa and HepG2 at the protein level. The TSC2 expression in KB was, however, lower than in other cell lines. TSC1 was expressed in all the cell lines, albeit at different levels. The TSC1 expression was lower in SCC 131 as compared to two other cell lines KB and SCC 104. 7. Given the fact that both are tumor suppressors, it was hypothesized that LOH, inactivating somatic mutations and/or promoter methylation might be playing a role for their downregulation in oral tumors. Mutation analysis of all the coding regions of both the TSC genes failed to detect any mutation in a panel of 25 tumor samples. However, seven normal population variants were identified in different patients. Our analysis of the matched peripheral blood and tumor DNA samples from 52 patients showed LOH at both the TSC loci. At the TSC1 locus, 17/48 (35.42%) tumors showed an allelic loss for one or more markers. At the TSC2 locus, LOH was found in 18/48 (37.5%) informative cases. Nine patients (9/48, 18.75%) had LOH at both the TSC loci. Since PTEN is another tumor suppressor in the insulin signaling pathway, we then sought to determine if LOH is also present in the PTEN candidate region in a panel of 50 matched samples. Microsatellite analysis using three markers showed a low LOH rate of 13% in tumor samples.

8. As the OSCC cell line SCC 131 did not show a detectable level of TSC2 expression, we treated this cell line with methylation inhibition drug 5-azacytidine. The treatment restored the expression of TSC2 and increased the expression of TSC1, suggesting that the promoter methylation and LOH are the important mechanisms for their downregulation. In order to see if the downregulation of the TSC genes is due to their promoters being methylated in tumors from the patients, we examined the methylation status of their promoters in 16 oral tumors, three normal oral tissues, two peripheral blood DNA samples from normal individuals and two cell lines HeLa and SCC 131 by COBRA. Our repeated efforts to amplify the TSC1 promoter using different DNA polymerases failed. However, we were able to successfully amplify the 571 bp long TSC2 promoter. Our analysis showed methylation of the TSC2 promoter in all tumors and two cell lines. As expected, the TSC2 promoter was not methylated in normal oral tissues and control blood DNA samples. Our bisulfite sequencing data suggested a low level and a considerable heterogeneity of methylation.

9. Using Fisher’s exact test, no correlation was found between LOH at the TSC loci and different clinical parameters such as age, sex, T classification, stage, grade, histology, tobacco habits and lymph node metastasis.

10. Using Fisher’s exact test, no correlation was found between the TSC2 promoter methylation and its downregulation in 16 tumor samples. We believe that this could be due to small sample size.

11. Since TSC1 and TSC2 are important regulators of the insulin pathway, it was hypothesized that other key players of this pathway might also be dysregulated in oral cancer. To this end, the expression pattern of some of the major regulators of the insulin pathway (viz., PI3K, AKT, PDK1, RHEB, mTOR, S6K1, S6, eIF4E, 4E-BP1, PTEN, 14-3-3ﾟ and IRS1) was investigated using semiquantative RT-PCR in a panel of 16 matched normal and tumor samples. The mean expression levels of the following genes showed significant upregulation in tumor samples: AKT, PI3K, PDK1, RHEB, mTOR, S6K1, S6 and eIF4E. On the other hand, 4E-BP1 and PTEN showed significant downregulation in tumor tissues. No significant difference in the expression was found for 14-3-3ﾟ and IRS1 between tumor and normal tissues. The expression pattern of some of these genes was also analyzed at the protein level using Western blot analysis and eight matched normal and tumor tissues. The level of total AKT was upregulated in 2/8 tumor samples only. However, phosphorylated-AKT (Thr308) showed upregulation in 6/8 samples. p70S6K1 and phosphorylated-p70S6K1 (Thr389) were upregulated in 8/8 and 6/8 tumor samples, respectively. Increase in the phosphorylated forms of both AKT and its downstream effector p70S6K1 suggested an increase in their kinase activity, indicating a constitutive activation of this pathway in oral cancer.

12. Based on our findings of mutation analysis, LOH study, 5-azacytidine treatment of an oral cancer cell line and COBRA analysis, we suggest that LOH at the TSC gene loci and promoter methylation are important mechanisms for the downregulation of the TSC genes. Loss of function of these genes may thus contribute to the constitutive activation of the insulin signaling pathway in oral cancer, leading to overall cell growth and proliferation. Our studies have shown that several key members of this pathway show aberrant expression in a subset of cancers of the oral cavity and can provide useful therapeutic targets. Several inhibitors of the insulin signaling pathway, such as rapamycin and its derivatives which inhibit mTOR and the PI3K inhibitor wortmannin, are now being actively evaluated for clinical trials for other cancers. We suggest that these inhibitors could also be evaluated for the treatment of oral cancer in future. Our differential display analysis has served to identify several genes that may be important for the onset and progression of oral cancer. Further analysis of these genes is warranted.