Figure 1.
Schematic representation of the usage of primary HNSCC tumor in different experimental analyses. N,sample number; HNSCC, head and neck squamous cell carcinoma; IHC, Immunohistochemical analysis; AMP, Gene amplification analysis; Del, deletion analysis; MSRA, Methylation Sensitive Restriction Analysis; Mut, Mutation analysis by SSCP; QRT-PCR, Real time-PCR quantification.
Table 1.
Clinicopathological feature of head and neck lesions.
Figure 2.
Molecular alterations of EGFR in HNSCC.
a) Immunohistochemical analysis of EGFR proteins in normal, dysplasia and HNSCC. Distinct cytosolic/membrane expression of EGFR in the basal lining/parabasal cells/spinous cells of normal oral epithelium, dysplasia and HNSCC samples. EGFR has high cytoplasmic expression in the basal cells followed by gradual decrease in parabasal and spinus layer of normal oral epithelium. #L50 (AMP+), #51T(AMP+) and #5999T(AMP-) showed overexpression of cytoplasmic and membrane EGFR. # 403 (AMP-) showed medium EGFR expressions. Arrows point to membrane/cytoplasmic expression. Magnification of tissue samples is 20×, and inset magnification is 40×. Scale bars in tissue sections represent 100 µm. AMP+/−, gene amplification present/absent. b) Representative gel diagram featuring amplification of EGFR locus. DDR2 locus was used as the control locus. The sample number was indicated above the figure. Arrow head indicate the amplified band in #3371T. T: Tumor DNA, N: corresponding normal DNA. c) Quantitative RT-PCR showing mRNA expression pattern of EGFR in HNSCC samples and cell lines. Bars represent the gene expression normalized to β2-microglobulin and relative to corresponding adjacent normal tissues, using 2-ddCt method. The line illustrates the mean fold of expression level. X-axis indicates the sample numbers.
Table 2.
Correlation of molecular alterations with RNA/protein expression of the genes SH3GL2 and CDC25A and the relationship between the protein expression of EGFR vs SH3GL2 and CDC25A vs p-EGFR.
Figure 3.
Molecular alterations of SH3GL2 and CDC25A.
a) Representative autoradiographs showing deletion and MA in HNSCC samples at D9S157 marker loci. (i) LOH, loss of heterozygosity, (ii) MA-I, microsatellite size alteration of one allele, (iii) LMA microsatellite size alteration of of one allele and LOH in other allele. b) Genetic alterations of CDC25A analyzed by microsatellite based deletion mapping showing deletion and MA in HNSCC at D3S3560 marker loci (i) LOH loss of heterozygosity, (ii) MA-I microsatellite size alteration of one allele. (iii) MA2 microsatellite size alteration of both alleles. Arrow heads indicate the lost allele and star indicates the allele with MA. Representative gel electrophorogram showing the methylation status of c) SH3GL2 and d) CDC25A in tumor samples and in corresponding normal sample by MSRA. SH3GL2 showed methylation in tumor sample and absence of methylation in adjacent normal samples. Promoter methylation of CDC25A was absent in both tumor and normal samples. h, amplicon obtained with primer for HpaII digested DNA; m, amplicon obtained with primer for MspI digested DNA; u, amplicon obtained with primer for undigested DNA. e) The methylation analysis by MSRA was validated by MSP after bisulphate modification of DNA. The sample #2123T showed the methylalaton specific PCR band, but #2123N and #2935TN showed unmethylation specific PCR band. U; amplicon obtained with primer for modified unmethylated DNA, M amplicon obtained with primer for modified methylated DNA, T tumor DNA, N corresponding normal tissue DNA. f) Frequency of overall alterations of the genes SH3GL2 and CDC25A observed in dysplasia and different stages of HNSCC samples. Significant increase in alteration with tumor progression is shown by asterisk. Q-RT PCR showing reduced expression of g) SH3GL2 and h) CDC25A in HNSCC tumor. Bars represented the gene expression normalized to β2-microglobulin and relative to adjacent normal tissues using the 2?-ddCt method. The line illustrates the mean decreased level of the genes. X-axis indicates samples.
Table 3.
Correlation of the two methods of promoter methylation analysis.
Figure 4.
Immunohistochemical analysis of SH3GL2, CDC25A and p-EGFR.
a) Distinct cytoplasmic/membrane expression of SH3GL2 in the basal lining/parabasal cells of normal oral epithelium and primary HNSCC samples were seen. Spinus layer showed high expression of the gene. #403T and #5999T showed low expression, #4465T and #2333T showed intermediate/high expression expression level of SH3GL2. b) Differential nuclear and cytoplasmic expression of CDC25A was seen in basal/parabasal/spinus layer cells of normal oral epithelium and tumor samples. Low cytoplasmic and nuclear expression of CDC25A was evident in basal and parabasal cells of normal epithelium, but higher expression in spinus layer. #2333T and #5999T showed low expression, #4465T and #403T showed high/intermediate nuclear and cytoplasmic expression level of CDC25A. Arrows pointed to nuclear/cytoplasmic/membrane expression. c) Distinct cytoplasmic and membrane bound expression of p-EGFR was seen in normal oral epithelium. The expression was high in basal layer but gradually decreased in parabasal and spinus layer. Arrows pointed to cytoplasmic/membrane/nucleus expression. #2333T and #5999T showed high expression, #4465T and #403 showed reduced expression of p-EGFR. Magnification of tissue samples is 20X, and for inset in tissues magnification is 40X. Scale bars in tissue sections represent 100 µm.
Figure 5.
SH3GL2 and CDC25A mediated EGFR homeostasis.
a)EGFR was degraded due to upregulation of SH3GL2 and CDC25A by 5-aza-dc treatment. Hep2 cell line was incubated with 20 µm of 5-aza-dc up to 120 h. Cells were harvested after zero hour of treatment and then every 24 h interval. Equal amounts of protein were subjected to western blotting. The amount of EGFR protein decreased gradually and degradation was maximum after 120 hour. Similarly, the expression of SH3GL2 and CDC25A was gradually increased after treatment. b) The amount of proteins (normalized band OD) was plotted as a function of time of 5-aza-dc treatment. The intensity of the bands were determined by densitometry and normalized with tubulin. c) SCC084 cell line was treated with siRNA of CDC25A and SH3GL2. Protein expression of the genes were analysed by western blot. Expression of EGFR and phosphorylated EGFR were assayed during knock down either of SH3GL2 and CDC25A or of both. Both EGFR and p-EGFR level was up regulated due to reduction of SH3GL2 and CDC25A. d) The amount of proteins (normalized band OD) was plotted. The intensity of the bands were determined by densitometry and normalized with actin. The bar diagram showing the level of EGFR and p-EGFR up regulation during siRNA treatment of SH3GL2 and CDC25A.
Figure 6.
Immunocytochemical analysis of EGFR, SH3GL2, CDC25A and p-EGFR.
over night subconfluent cover slip culture of Hep2 cell line was treated with 5-aza-dc for 72 hour and protein expression of the genes were analyzed by ICC after fixing the cells. EGFR and p-EGFR showed reduced expression in treated cells (b) compare to non treated cells (a). On the contrary, SH3GL2 and CDC25A showed upregulation of cytoplasmic/nuclear expression after aza treatment (b) compare to untreated cells (a). Scale bars in microphotograph represent 50 µm.
Figure 7.
Kaplan–Meier analysis of survival (up to 5 years) of HNSCC patients.
a) Co-alteration of CDC25A and SH3GL2 was significantly associated with poor overall survival (OS). b) The significant association with poor overall survival of patients having co-alterations of the genes was also seen in presence of HPV infection; however, co-alterations did not associate significantly with OS in absence of HPV infection (c). d) Poor survival was also seen of the patients having high EGFR expression irrespective of HPV infection. d) Similarly, reduced SH3GL2 expression and high EGFR expression was a predictor for poor OS. However f), protein expression of p-EGFR and CDC25A did not show any significant association with survival of the patients. Survival time was defined as the time from the date of surgery to the date of last follow-up, known recurrence or death (up to 5 years). n, total number of samples; C+/−, CDC25A deletion present/absent; S+/−, SH3GL2 alterations present/absent; H+/−, HPV infection present/absent; EH/ML, EGFR protein expression high/medium to low; S-L/MH, SH3GL2 protein expression low/medium to high; C-H/ML, CDC25A protein expression high/medium to low; p-EH/LM, p-EGFR protein expression high/low to medium.
Table 4.
Univariate and Multivariate analyses of genetic, clinical, and etiological parameters in predicting overall survival (OS) of 148 HNSCC patients.