Involvement of TSC genes and differential expression of other members of the mTOR signaling pathway in oral squamous cell carcinoma
© Chakraborty et al; licensee BioMed Central Ltd. 2008
Received: 10 January 2008
Accepted: 06 June 2008
Published: 06 June 2008
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.
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).
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.
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.
Oral squamous cell carcinoma (OSCC) is the sixth most common cancer in the world . In India, it is the leading cancer among males and the third most common malignancy in females . The five-year survival rate for OSCC is the lowest among all major cancers . The etiology of this cancer is multifactorial, with important risk factors being tobacco intake, alcohol consumption and human papilloma virus (HPV).
A thorough understanding of the genetic and epigenetic changes that result in the activation of signaling pathways and provide the cells with a growth advantage during oral tumorigenesis is essential for the development of novel therapeutic strategies. Agents that can inhibit or reverse these changes by targeting molecularly defined pathways should receive increased attention as novel candidates for oral cancer prevention and therapy [2, 3]. The molecular interplay between phosphoinositide-3-kinase, catalytic, alpha polypeptide (PIK3CA) and FK506 binding protein 12-rapamycin associated protein 1 (FRAP1) of the mTOR (mammalian target of rapamycin) signaling pathway in the control of cell growth and proliferation has been the subject of much interest among cell biologists . Tuberin, encoded by the tumor suppressor gene tuberous sclerosis 2 (TSC2), and its interacting partner hamartin, encoded by another tumor suppressor gene tuberous sclerosis 1 (TSC1), have been placed as a complex in the mTOR signaling pathway and negatively regulate the pathway to inhibit mTOR mediated downstream signaling . Several components of the mTOR signaling pathway are known to be dysregulated in a wide spectrum of human cancers . Although some components (PIK3C2A, AKT1, PTEN, RPS6 and EIF4E) of this pathway have been implicated in OSCC [6–9], a comprehensive analysis is lacking. Further, very little is known about the roles of TSC tumor suppressor genes in tumorigenesis of OSCC . The main aim of this study was to assess the role of TSC genes and other members of this pathway in the tumorigenesis of OSCC. The results of our study are presented here.
Clinicopathological features of patients included in the study.
No. of patients (n = 52)
50 yrs/32–70 yrs
Three oral cancer cell lines (SCC 131, SCC 104 and KB) and four other cell lines (HeLa, HepG2, A549 and HEK-293T) were used. SCC 131 and SCC 104 cell lines were a kind gift from Dr. Susanne M. Gollin (University of Pittsburgh, Pittsburgh, PA). Cell lines were maintained either in Minimum Essential Medium with Earle's salt and l-glutamine or in Dulbecco's modified Eagle's medium (Sigma-Aldrich, St. Louis, MO).
Genomic DNA isolation
Genomic DNA was isolated from peripheral blood and tumor samples using a DNA isolation kit (Roche Diagnostics™, Mannheim, Germany).
Total RNA was isolated from 16 paired normal and tumor samples using the TRI REAGENT™ (Sigma-Aldrich, St. Louis, MO). cDNA was synthesized from 1 μg total RNA from each sample using random hexamers and the Revertaid™ H Minus First Strand cDNA Synthesis Kit (MBI Fermentas, Burlington, ON, Canada). For RT-PCR, forward and reverse primers were selected from two different exons of genes to rule out the possibility of amplification of contaminating genomic DNA. Primer sequences and PCR conditions are available from the authors upon request. For each gene, the PCR protocol was optimized in order to get the amplification in a linear phase. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was amplified as a normalizing control. Images of RT-PCR ethidium bromide stained agarose gels were acquired with a Kodak CCD camera and quantification of the bands was performed by densitometric analysis using the Kodak Digital Science Image Station Imaging Software version 3.6.1. Band intensity was expressed as relative absorbance units. Data was expressed in arbitrary units (relative expression) as a ratio of normal/GAPDH and tumor/GAPDH and plotted using the GraphPad Prism software version 4.00 (GraphPad Prism Software, San Diego, CA). The significance of difference in mRNA levels between normal and tumor samples for a gene was assessed by Student's t-test and the results are expressed as mean ± SEM . A probability value of p < 0.05 was assumed to be significant. PCR amplification for each gene was repeated once. A gene was considered to be upregulated when its mean expression value across 16 tumor samples was significantly higher than the mean expression value across 16 normal tissue samples and vice versa . We defined the cutoff value for determining the upregulation or downregulation of a gene in a tumor sample as ≥ 1.8 fold difference in its expression between normal and tumor samples as described by Arora et al.  for differentially expressed genes in oral squamous cell carcinoma.
Mutation screening of the entire coding regions of TSC genes was carried out using PCR-SSCP  and DNA sequencing techniques.
LOH analysis at TSC1, TSC2 and PTEN loci
For LOH studies, matched normal and tumor DNA samples from 50 patients were genotyped using following microsatellite markers: D9S179, D9S1830 and D9S915 for the TSC1 locus; D16S3024, D16S3395 and D16S475 for the TSC2 locus; and D10S215, D10S1765 and D10S541 for the PTEN locus. Microsatellite analysis was performed as described in Kumar et al. . LOH was scored if there was a complete loss of one of the two heterozygous alleles in tumor DNA or a decrease of 50% intensity of one of the two alleles in tumor DNA as compared to the corresponding peripheral blood DNA (allelic imbalance).
Antibodies and western blot analysis
Rabbit polyclonal antibodies generated against amino acids 488–1016 of TSC1 and amino acids 155–541 of TSC2 were raised in our laboratory. Mouse monoclonal anti-β-actin antibody was purchased from Sigma-Aldrich (St. Louis, MO). Rabbit polyclonal anti-Akt 1/2, anti-p-Akt 1/2/3 (Thr 308) and anti-p-p70S6K1 (Thr 389) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit polyclonal anti-p70S6K1 antibody was obtained as a kind gift from Dr. I. Juhan-Vague (Marseille, Cedex, France).
For western blot analysis, whole cell lysates were prepared from matched normal and tumor samples as well as cell lines using a standard procedure. Equal amounts of protein (~100 μg/lane) from tumor, normal oral tissue or different cancer cell lines were resolved by SDS-polyacrylamide gel electrophoresis and transferred onto a PVDF membrane. Primary antibody was detected with either HRP-conjugated goat anti-rabbit or goat anti-mouse secondary antibodies (Bangalore Genei™, India). Immunoreactive bands were visualized using the Western Lightning Chemiluminescence Reagent kit (PerkinElmer Life Sciences, Boston, MA) and X-ray films. β-actin was used to see equal protein loading.
5-azacytidine treatment of cell lines
SCC 131 and HeLa cells were seeded at a density of 1 × 106 cells/90 mm dish. After 24 hr, freshly prepared 5-azacytidine (Sigma-Aldrich, St. Louis, MO) was added into the dish to a final concentration of 10 μM. Total RNA was isolated after 2 and 5 days from the start of the treatment. Untreated cells were used as controls. Semi-quantitative RT-PCR was used to assess the expression of TSC1 and TSC2. GAPDH was used as a normalizing control.
Combined bisulfite restriction analysis
Methylation status of the TSC2 gene promoter was examined using combined bisulfite restriction analysis (COBRA) as described by Xiong and Laird . Sodium bisulfite treated DNA was used in PCR amplification using primers designed for the bisulfite treated DNA. Primers were designed using the MethPrimer program . Promoter region of the TSC2 gene is reported by Kobayashi et al. . Sodium bisulfite treated DNA was amplified with following TSC2 promoter primers: F-5'gggattttagtttgtagtttttattt-3' and R-5'-ccataacttaaaactaaaaaatact-3'. Primers were designed to exclude binding to any CpG dinucleotide to ensure amplification of both methylated and unmethylated forms of DNA. PCR conditions for primers were as follows: an initial denaturation at 95°C for 3 min was followed by 35 cycles of 94°C for 30 sec, 60°C for 45 sec and 68°C for 45 sec with a final extension at 68°C for 5 min. TSC2 primer set generated a 571 bp amplicon. A second PCR was carried out using the product of the first amplification as a template to get enough DNA for COBRA. Approximately 500–600 ng of pooled and gel purified PCR product was digested with Aci I at 37°C for 6 hr. Digests were resolved in a 2.5% agarose gel and visualized by ethidium bromide staining. There are 18 Aci I sites and 65 CpGs in the TSC2 promoter. The restriction enzyme Aci I recognizes the sequence 5'-GCGG-3'. The cleavage of this sequence will occur only when the C residue in the recognition sequence is methylated. Promoter of TSC1 has been reported by Ali et al. . Primers were also designed for the TSC1 promoter. However, despite repeated efforts using DNA polymerases from several vendors, we were not successful in amplifying the TSC1 promoter after bisulfite treatment.
Results and discussion
Downregulation of TSC genes
Mechanisms of downregulation of TSC genes
Clinicopathological characteristics, LOH and gene expression variation in folds* for 16 tumor samples.
Sample (Patient) no.
The variation in the degree of digestion by Aci I in different tumor samples might be accounted for by the fact that all surgical samples are likely to contain a heterogenous mix of normal and tumor cells as microdissection was not performed on tumor samples and also considerable heterogeneity of methylation might exist among tumor samples. An MSP (methylation-specific PCR) assay could not be designed in this study because the relevant methylated region was limited. Taken together, our 5-azacytidine and COBRA data suggested that TSC genes are targets of epigenetic inactivation in oral cancer (Figures 3 and 4).
Aberrant expression of genes from the mTOR signaling pathway
Upregulation of PIK3C2A has been reported in several cancers such as cervical, colon, breast, liver, stomach and lung cancers . Expression of this gene has been investigated in oral tongue carcinoma, and head and neck cancer cell lines where the mean expression level in tumor samples was found to be significantly higher than in normal samples . Our results have also shown that PIK3C2A is upregulated in 10/16 oral tumors (Figure 5a, Table 2).
AKT1 (AKT) is a downstream effecter of PIK3C2A (PI3K). It has emerged as a central player controlling several signal transduction pathways that are activated in response to growth factors or insulin. Activation of AKT1 has been shown to be a frequent event in breast, colorectal, ovarian, pancreatic, and head and neck cancers . One of the best characterized regulators of the mTOR signaling pathway is PTEN. The lipid phosphatase activity of PTEN acts as a negative regulator for PIK3C2A induced signaling as it dephosphorylates PIP3 . PIP3 is a potent second messenger that recruits certain kinases to the plasma membrane including the Protein kinase B/Akt family of kinases and PDPK1. On membrane localization, AKT1 is activated in part through phosphorylation by PDPK1 and elicits several downstream cellular functions. Genetic inactivation of PTEN leads to constitutive activation of the mTOR pathway . Our study has shown that AKT1 and PDPK1 show upregulation in 10/16 and 6/16 tumors respectively (Figure 5a, Table 2). PTEN on the other hand showed downregulation in 7/16 tumors (Figure 5a, Table 2). Mavros et al.  identified a low LOH rate of 12% in 50 samples of OSCC. They however did not find any mutation in the coding region of PTEN and concluded that the PTEN gene alterations do not play a key role in tumorigenesis of oral squamous cell cancers. We have also found a low LOH rate of 13% (6/46 informative cases) at the PTEN locus in the same panel of 50 paired blood and tumor DNA samples (data not shown). The frequency of LOH was 4.5% (2/44 informative cases) and 10% (3/30 informative cases) at D10S1765 and D10S541 respectively (data not shown). It is possible that the downregulation of this gene in oral tumors examined in this study is due to inactivating somatic mutations or its promoter methylation. However, these possibilities need to be investigated in the future.
FRAP1 has a central role in controlling cell cycle progression and cell growth. It has emerged as a major cancer therapeutic target . FRAP1 exerts its effect by phosphorylating EIF4EBP1 (4E-BP1) which binds to and inactivates EIF4E, thus inhibiting 5'-cap-dependent mRNA translation. Phosphorylation of EIF4EBP1 releases EIF4E and allows initiation of translation. Regulation of EIF4E mediated translation is an important target for therapeutic intervention in light of the fact that EIF4E has been shown to be overexpressed in several cancers and that overexpression can cause malignant transformation of rodent fibroblasts . In our study, EIF4EBP1 showed downregulation in 10/16 tumors (Figure 5a, Table 2). Upregulation of EIF4E in 10/16 tumors (Figure 5a, Table 2) potentiates its role in the increase of translation leading to overall cell growth and proliferation. FRAP1 also regulates translation via phosphorylation of a serine/threonine kinase p70S6K1 (RPS6KB1). Upon phosphorylation, p70S6K1 promotes translation of mRNAs containing a 5' terminal oligopyrimidine (5' TOP) by phosphorylating the ribosomal subunit S6. Since ribosomal proteins and translation elongation factors are encoded by 5' TOP mRNAs, signaling along the p70S6K1 pathway promotes ribosome biogenesis and overall protein biosynthetic capacity . Our study provides the evidence that FRAP1 is upregulated in 8/16 tumors, as also are RPS6KB1 (9/16) and RPS6 (10/16) (Figure 5a, Table 2). Our western blot results also indicated that the AKT1/RPS6KB1 pathway is active in oral tumors, as phosphorylated forms of both proteins show increased levels in tumor samples (Figure 5b).
A novel positive regulator of FRAP1 is the small GTPase RHEB . Tuberin/hamartin complex acts as a negative regulator of this pathway by C-terminal GAP activity of tuberin towards RHEB. When stimulated by growth factors, AKT1 relieves this inhibition by phosphorylation of tuberin, which dissociates the tuberin/hamartin complex . Phosphorylated tuberin binds to the 14-3-3 family of proteins which control various cellular functions . We found upregulation of RHEB in 9/16 tumors (Figure 5a, Table 2), whereas we did not find any significant difference in the expressions of the tuberin interacting protein YWHAB (14-3-3β) and IRS1 across the samples analyzed (data not shown). Interestingly, 14-3-3zeta, another tuberin interacting protein , was recently found to be upregulated in OSCC . Further, the expression of 14-3-3sigma, which also interacts with tuberin , was reduced or absent in OSCC . This suggested that different isoforms of the 14-3-3 family behave differently in OSCC.
Clinicopathological characteristics of patients with LOH at TSC1 and TSC2 loci
We correlated 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. Fisher's exact test (two-sided) was carried out and a p < 0.05 was considered to be significant. Using the above criteria, none of the parameters examined demonstrated a significant correlation with LOH at either of the TSC loci (data not shown).
Collectively, the detection of LOH in a proportion of OSCC samples coupled with reduced gene expression both at the RNA and protein levels indicates a loss of function of TSC genes, implicating their role as tumor suppressors in oral cancer for the first time. Loss of function of these genes may thus contribute to the constitutive activation of the mTOR signaling pathway leading to overall cell growth and proliferation. Our studies have also shown for the first time that several key members of this pathway show aberrant expression in oral cancer and can provide useful therapeutic targets. Several inhibitors of this pathway, such as rapamycin and its derivatives which inhibit mTOR (FRAP1) and the PI3K (PIK3C2A) inhibitor wortmannin, are in fact now being actively evaluated in clinical trials for other cancers [31, 34]. Further, rapamycin and its derivative CCI-779 have been shown to reduce OSCC tumor size in nude mice [8, 39]. Thus, these inhibitors could also be evaluated for the treatment of oral cancer.
This work was supported by a research grant from the Department of Biotechnology, New Delhi to AK and KSG, and a Council of Scientific and Industrial Research fellowship to SC. We are grateful to patients and their families for their involvement in the study. We thank Dr. Sonal Khare, Mr. Santosh Gupta and Ms. C. Yeshodari for technical help. We also thank the reviewers, Drs. Eloiza Helena Tajara and Hideki Tanzawa, for their valuable suggestions to improve the manuscript.
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