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The aim of this study is to understand the mechanism of EGFR overexpression in head and neck squamous cell carcinoma (HNSCC). For this reason, expression/mutation of EGFR were analyzed in 30 dysplastic head and neck lesions and 148 HNSCC samples of Indian patients along with 3 HNSCC cell lines. In addition, deletion/methylation/mutation/expression of SH3GL2 (associated with EGFR degradation) and CDC25A (associated with dephosphorylation of EGFR) were analyzed in the same set of samples. Our study revealed high frequency of EGFR overexpression (66–84%), low frequency of gene amplification (10–32.5%) and absence of functional mutation in the dysplastic lesions and HNSCC samples. No correlation was found between protein overexpression and mRNA expression/gene amplification status of EGFR. On the other hand, frequent alterations (deletion/methylation) of SH3GL2 (63–77%) and CDC25A (37–64%) were seen in the dysplastic and HNSCC samples. Two novel single nucleotide polymorphism (SNPs) were found in the promoter region of SH3GL2. Reduced expression of these genes showed concordance with their alterations. Overexpression of EGFR and p-EGFR were significantly associated with reduced expression and alterations of SH3GL2 and CDC25A respectively. In-vitro demethylation experiment by 5-aza-2′-deoxycytidine (5-aza-dC) showed upregulation of SH3GL2 and CDC25A and downregulation of EGFR expression in Hep2 cell line. Poor patient outcome was predicted in the cases with alterations of SH3GL2 and CDC25A in presence of human papilloma virus (HPV) infection. Also, low SH3GL2 and high EGFR expression was a predictor of poor patient survival. Thus, our data suggests that overexpression of EGFR due to its reduced degradation and dephosphorylation is needed for development of HNSCC.

ObjectivesHuman papillomavirus (HPV) causes tumors primarily Cervical cancer. Recently, inconsistent reports came up in Breast cancer (BC) too. In India, despite treatment 70,218 BC patients die each year. So, we explored the association of HPV, if any, with BC prognosis in Indian pre-therapeutic (PT) and Neo-adjuvant chemotherapy (NACT) patients with subsequent analysis of HPV profile.MethodsHPV prevalence was checked and analysis of physical status, copy number, genome variation, promoter methylation and expression (mRNA and protein) of the prevalent subtype was done.ResultsHigh prevalence of HPV was observed in both PT (64.0%) and NACT (71.0%) cases with significant association with younger (20–45 yrs) PT patients. Interestingly, HPV infection was significantly increased from adjacent normal breast (9.5%, 2/21), fibro adenomas (30%, 3/10) to tumors (64.8%, 203/313) samples. In both PT and NACT cases, HPV16 was the most prevalent subtype (69.0%) followed by HPV18 and HPV33. Survival analysis illustrated hrHPV infected PT patients had worst prognosis. So, detailed analysis of HPV16 profile was done which showed Europian-G350 as the most frequent HPV16 variant along with high rate of integration. Moreover, low copy number and hyper-methylation of P97 early promoter were concordant with low HPV16 E6 and E7 mRNA and protein expression. Notably, four novel variations (KT020838, KT020840, KT020841 and KT020839) in the LCR region and two (KT020836 and KT020837) in the E6 region were identified for the first time along with two novel E6^E7*I (KU199314) and E6^E7*II (KU199315) fusion transcript variants.ConclusionThus, significant association of hrHPV with prognosis of Indian BC patients led to additional investigation of HPV16 profile. Outcomes indicated a plausible role of HPV in Indian BC patients.

The aim of the study is to understand the importance of the Wnt ⁄ b-catenin pathway in the development of breast cancer (BC) and its association with different clinicopathological parameters. Alterations (deletion ⁄ methylation ⁄ expression) of some Wnt ⁄ b-catenin pathway antagonists like APC, SFRP1 ⁄ 2, CDH1 and activator b-catenin (CTNNB1) were analyzed in primary BC in Indian patients. High frequencies (65-70%) of overall alterations (deletion ⁄ methylation) of the antagonists were seen in the BC samples. Also, 99% (156 ⁄ 158) of the samples showed alterations in any one of the genes, indicating the importance of this pathway in the development of this tumor. Co-alterations of these genes were observed in 30% of samples, with significantly high alterations in late-onset (37%) and estrogen receptor (ER)) ⁄ progesterone receptor (PR)) (37%) BC compared with early onset (21%) and ER ⁄ PR+ (18%) BC samples, respectively. Significantly high (P-value = 0.001-0.02) alterations of APC and CDH1 genes were seen in ER) ⁄ PR) BC compared with ER ⁄ PR+ BC. Immunohistochemical analysis showed reduced expression of the Wnt antagonists in BC concordant with their molecular alterations. Nuclear localization of b-catenin showed significant association with alterations in the antagonists and was also significantly high in the ER) ⁄ PR) BC samples. Alterations of SFRP2 coupled with a late clinical stage and low ⁄ nulliparity predicted the worst prognosis in BC patients. Therefore, the present study suggests that cumulative alterations in more than one Wnt antagonist along with increased nuclear accumulation of b-catenin play an important role in the development of BC and have significant clinical as well as prognostic importance. (Cancer Sci 2012; 103: 210-220) B reast cancer (BC) is a frequently occurring cancer in women worldwide and accounts for 25% of cancer cases in the Eastern Indian population, especially in urban areas.(1) There are several known risk factors for BC.(2) In general, younger women with BC (age £ 40 years) exhibit more aggressive pathological features and lower survival rates compared with older women with BC.(3) The molecular subtypes of BC, which usually lack expression of the estrogen receptor (ER) and progesterone receptor (PR), also show the worst prognosis of BC.(4) It has been suggested that ER and PR play important roles in regulating the mammary stem cell population and its development. The stem cell population is regulated by a number of selfrenewal pathways, (6) among which the Wnt ⁄ b-catenin pathway is quite important due to its cross-talk with the ER signaling pathway (7) and its alterations in several carcinomas including BC. (8) There are several antagonists of the Wnt ⁄ b-catenin pathway like SFRP1, SFRP2, APC and CDH1.(9,10) In different ways, these antagonists regulate the cytoplasmic and nuclear accumulation of b-catenin, the signal transducer of this pathway. (8,11) Accumulation of data shows varying frequencies of deletion (23-73%) and frequent promoter methylation in the Wnt antag...

The molecular mechanisms involving CDKN2A inactivation seem to follow similar pathway in the pathogenesis of both age groups of BC while significant association of SH3GL2 with CDKN2A might play a synergistic role in the development of group A.

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Alterations of FANCC and PTCH1 could be used as molecular marker for early diagnosis and prognosis of HNSCC.

Transcriptional activation of β-catenin is a hallmark of Wnt/β-catenin pathway activation. The MCC (Mutated in colorectal cancers) and CTNNBIP1 (catenin, beta interacting protein 1) are two candidate genes which inhibit the transcriptional activity of nuclear β-catenin. The importance of MCC and CTNNBIP1 in breast cancer (BC) development has not yet been studied in detail. For this reason, in present study, the alterations (deletion/methylation/mutation/expression) of MCC and CTNNBIP1 were analyzed in BC of Indian patients (N=120) followed by expression/mutation analysis of β-catenin. Then transcriptional activity of β-catenin was checked by expression analysis of its target genes (EGFR, C-MYC and CCND1) in the same set of samples. Frequent methylation (44-45%) than deletion (20-32%) with overall alterations of 52-55% was observed in MCC/CTNNBIP1 in the BC samples. The alterations of MCC/CTNNBIP1 showed significant correlation with increased nuclear β-catenin/p-β-catenin(Y654) expression. Also, a significant correlation was seen between nuclear β-catenin expression and overexpression of its target genes like EGFR, MYC and CCND1 in the BC samples (P<0.0001). An upregulation of MCC and CTNNBIP1 expression by 5-Aza-2'-deoxycytidine treatment of MCF7 and MDA-MB-231 cell lines lead to downregulation of β-catenin and its target genes. The expression of nuclear p-β-catenin(Y654), EGFR, MYC and CCND1 were significantly high in TNBC (Triple negative BC) and Her2+ compared to Luminal A/B+ subtypes. The TNBC patients in stage III/IV having reduced expression of MCC in the tumors showed poor prognosis. Thus, our data suggests that inactivation of MCC/CTNNBIP1 could be an important event in activation of β-catenin mediated transcription of target genes in BC.