Reduced expression of LIMD1 in ulcerative oral epithelium associated with tobacco and areca nut

Purpose: The aim of this study was to cast light on initiating molecular events associated with the development of premalignant oral lesions induced by tobacco and/or areca nut. Method: Immunohistochemical analyses of cell cycle regulatory proteins (LIMD1, RBSP3, p16, RB, phosphorylated RB, p53), EGFR and SH3GL2 (EGFR associated protein) were performed with inflammatory/ulcerative epithelium and adjacent hyperplastic/mild dysplastic lesions. Results: No change in expression of the proteins was seen in inflammatory epithelium. Reduced nuclear expression of LIMD1 was evident in ulcerative epithelium. In hyperplastic lesions, reduced expression of RBSP3, p16, SH3GL2 and overexpression of p-RB and EGFR were apparent. Reduced nuclear expression of p53 was observed in mild dysplastic lesions. Conclusion: Our data suggest that inactivation of LIMD1 in ulcerative epithelium might predispose the tissues to alterations of other cell cycle regulatory and EGFR signaling proteins needed for the development of premalignant oral lesions

Inactivation of 9q22.3 tumor suppressor genes predict outcome for patients with head and neck squamous cell carcinoma

Aim: This study examined the prognostic significance of candidate tumor suppressor genes (TSGs) PHD finger protein-2 (PHF2), Fanconi anaemia complementation group C (FANCC) and human homologue of Drosophila patched gene (PTCH1), in head and neck squamous cell carcinoma (HNSCC) treated primarily with surgery, or surgery followed by adjuvant radiotherapy. Patients and Methods: Eighty-four patients with HNSCC were followed-up for recurrence/death for up to five years after diagnosis. Molecular alterations (deletion/methylation) of TSGs and human papilloma virus (HPV) status were determined in previous studies of our group. Statistical analyses of correlation of genetic alterations with treatment response and survival were carried out. Results: Alterations of FANCC and PTCH1 were significantly associated with locoregional recurrence/death. In the surgery with adjuvant radiotherapy-group (n=56), patients showing alterations in FANCC and in PTCH1 were seven- and six-times, respectively, more likely to have locoregional recurrence compared to those with no alterations. In addition, the presence of alterations of both FANCC and PTCH1 had remarkable prognostic significance. Conclusion: FANCC and PTCH1 alterations might be used as molecular markers for prognosis and to develop strategies for effective treatment planning

Association of FANCC and PTCH1 with the development of early dysplastic lesions of the head and neck

Background: Alteration of chromosome 9q22.3 region is an early and frequent event in head and neck squamous cell carcinoma (HNSCC). The aim of this study was to understand the association of candidate tumor suppressor genes PHF2, FANCC, PTCH1, and XPA located in this region in the development of HNSCC. Methods: The alterations (deletion, promoter methylation, mutation, expression) of these genes were analyzed in 65 dysplastic head and neck lesions and 84 primary HNSCC samples. Clinicopathologic correlations were made with alterations of the genes. Results: Overall alterations (deletion, promoter methylation) of FANCC and PTCH1 were high in mild dysplasia and comparable in subsequent stages of tumor progression. However, PHF2 alteration was low in mild dysplasia, but increased in moderate and severe dysplasias. Alterations (deletion, promoter methylation) of FANCC and PTCH1 showed association with each other. Two novel mutations in GLI binding sites of PTCH1 promoter and a novel microsatellite marker hmPTCH1 with four alleles at immediate upstream of the gene were identified. In a case-control study, the (CGG)7 allele of hmPTCH1 was found to be susceptible for HNSCC development. Concordance was seen in the expression (RNA, protein) of these genes with their molecular alterations. Conclusions: Alterations of FANCC and PTCH1 could be used as molecular marker for early diagnosis and prognosis of HNSCC

Overexpression of EGFR in head and neck squamous cell carcinoma is associated with inactivation of SH3GL2 and CDC25A genes.

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

Allelic association results among different comparison groups.

a<p>MAF: Minor Allele Frequency of reference population is listed;</p>b<p>Association tests abbreviations, CC: case (jointly oral cancer and leukoplakia) vs. control, CAC: cancer vs. control, CAL: cancer vs. leukoplakia and LC: leukoplakia vs. control;</p>c<p>P-values without any adjustment for age, sex and tobacco habits by logistic regression and without any multiple tests correction applied,</p>d<p>P-values without any adjustment for age, sex and tobacco habits by logistic regression but corrected for multiple testing by Benjamini-Hochberg False Discovery Rate method,</p>e<p>P-values after adjustment for age, sex and tobacco habits by logistic regression but no correction multiple testing was applied,</p>f<p>P-values after adjustment for age, sex and tobacco habits by logistic regression and corrected for multiple testing by Benjamini-Hochberg False Discovery Rate method.</p

Basic characteristics of case and control data in discovery phase.

<p>Abbreviation: Con: Control, Can: Oral cancer, Leu: Leukoplakia.</p>a<p>P-values from Mann-Whitney test,</p>b<p>p-values from chi-square test.</p

Allelic associations in with respect to tobacco exposure.

a<p>MAF: Minor allele frequency of the reference population is listed;</p>b<p>Association tests abbreviations, CC: case (jointly oral cancer and leukoplakia) vs. Control, CAC: cancer vs. Control, CAL: cancer vs. Leukoplakia, LC: leukoplakia vs. control, HD: High-dose and LD: Low-dose tobacco exposed group;</p>c<p>Benjamini-Hochberg False Discovery Rate corrected P-values for multiple tests.</p

Basic characteristics of cancer and control in replication phase.

<p>Abbreviation:</p>a<p>p-values from Mann-Whitney test,</p>b<p>P-values from chi-square test.</p

Overall strategy of the association study.

<p>Overall strategy of the association study.</p

Allelic association results of replication study and comparison with discovery data.

a<p>MAF: Minor allele frequency of the reference population is listed;</p>*<p>Benjamini-Hochberg False Discovery Rate corrected P-values for multiple tests;</p>#<p>Unadjusted P-values.</p