SNP rs1049430 in the 3'-UTR of SH3GL2 regulates its expression: Clinical and prognostic implications in head and neck squamous cell carcinoma.

Single nucleotide polymorphisms (SNPs) in the 3'-UTR region are emerging cis-regulatory factors associated with the occurrences of several human diseases. SH3GL2, which is located at chromosome 9p21-22, is associated with hyperplastic/mildly dysplastic lesions of the head and neck and has a long 3'-UTR with multiple SNPs. The aim of the present study was to determine the susceptible allele(s) in the 3'-UTR SNPs of SH3GL2 in head and neck squamous cell carcinoma (HNSCC). First, we screened the genotypes of all SNPs located in the 3'-UTR of SH3GL2 in 110 controls and 147 cases in Indian populations by sequencing. A SNP (rs1049430:>G/T) that showed only heterozygosity was further confirmed by genotyping with an Illumina GoldenGate platform in 530 controls and 764 cases. Genotype-specific survival analysis of the HNSCC patients was performed. In addition, genotype-specific mRNA stability, isoform expression and protein expression were analyzed. SNP rs1049430 was not associated with disease occurrence, but it was associated with poor patient outcome. The G allele was associated with decreased SH3GL2 mRNA stability, differential splicing and low protein expression. Thus, our data demonstrate that the presence of the susceptible G allele in SNP rs1049430 is associated with the inactivation of SH3GL2 and could be used as a prognostic marker of HNSCC.

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.

Frequent alterations of the candidate genes hMLH1, ITGA9 and RBSP3 in early dysplastic lesions of head and neck: clinical and prognostic significance.

To understand the association between candidate tumor suppressor genes (TSGs) human mismatch repair protein homologue 1 (hMLH1), AP20 region gene 1 (APRG1), integrin alpha RLC (ITGA9), RB1 serine phosphates from human chromosome 3 (RBSP3) at chromosomal 3p22.3 region and development of head and neck squamous cell carcinoma (HNSCC), alterations (deletion/promoter methylation/expression) of these genes were analyzed in 65 dysplastic lesions and 84 HNSCC samples. Clinicopathological correlations were made with alterations of the genes. In HNSCC, deletion frequencies of hMLH1, ITGA9, and RBSP3 were comparatively higher than APRG1. Overall alterations (deletion/methylation) of hMLH1, ITGA9, and RBSP3 were high (45-55%) in mild dysplasia and comparable in subsequent stages of tumor progression. Quantitative RT-PCR analysis showed reduced expression of these genes in tumors concordant to their molecular alterations. An in vitro demethylation experiment by 5-aza-2'-deoxycytidine confirmed the promoter hypermethylation of RBSP3 in Hep2 and UPCI:SCC084 cell lines. Functionally less-active RBSP3A isoform was predominant in tumor tissues contrary to the adjacent normal tissue of tumors where more active RBSP3B isoform was prevalent. In immunohistochemical analysis, intense nuclear staining of hMLH1 and pRB (phosphorylated RB, the substrate of RBSP3) proteins were seen in the basal layer of normal epithelium. In tumors, concordance was seen between (i) low/intermediate level of hMLH1 expression and its molecular alterations; and (ii) intense nuclear staining of pRB and RBSP3 alterations. Poor patient outcome was seen with hMLH1 and RBSP3 alterations. Moreover, in absence of human papilloma virus (HPV) infection, tobacco-addicted patients with hMLH1, RBSP3 alterations, and nodal invasions showed poor prognosis. Thus our data suggests that dysregulation of hMLH1, ITGA9, and RBSP3 associated multiple cellular pathways are needed for the development of early dysplastic lesions of the head and neck.

Alterations of ROBO1/DUTT1 and ROBO2 loci in early dysplastic lesions of head and neck: clinical and prognostic implications.

Deletion of chromosomal 3p12.3 was suggested to be associated with dysplastic lesions of head and neck. This region harbors two candidate tumor suppressors ROBO1/DUTT1, ROBO2 and two non-coding RNAs (ncRNAs) located at intron 2 of ROBO1/DUTT1. Aim of this study is to understand the role of these genes in development of head and neck squamous cell carcinoma. A collection of 72 dysplastic lesions and 116 HNSCC samples and two oral cancer cell lines were analyzed for ROBO1/DUTT1 and ROBO2 deletion and promoter methylation. ROBO1/DUTT1, ROBO2 and two ncRNAs mRNA expression were analyzed by Q-PCR. Immunohistochemical analysis of ROBO1/DUTT1 and ROBO2 was performed. Alterations of these genes were correlated with different clinicopathological parameters. High frequency of molecular alterations (deletion/methylation) was seen in ROBO1/DUTT1 than ROBO2. In mild dysplastic lesions both of these genes showed high molecular alterations and remained more or less constant in subsequent stages. Q-PCR analysis showed reduced expression of these genes and the two ncRNAs. In vitro demethylation experiment by 5-aza-dC showed upregulation of ROBO1/DUTT1 and ROBO2 while the expression of the ncRNAs remained unchanged. Immunohistochemical analysis of ROBO1/DUTT1 and ROBO2 showed concordance with their mRNA expression and molecular alterations. Poor patients' outcome was predicted in the cases with alteration of ROBO1/DUTT1 along with tobacco addiction and nodal involvement. Our data suggests (a) ROBO1/DUTT1 and the ncRNAs are transcribed from different promoters, and (b) inactivation of ROBO1/DUTT1 could be used as molecular signature for early detection and prognosis of the head and neck cancer.

Alterations of 3p21.31 tumor suppressor genes in head and neck squamous cell carcinoma: Correlation with progression and prognosis.

The aim of our study was to analyze the alterations of some candidate tumor suppressor genes (TSGs) viz. LIMD1, LTF, CDC25A, SCOTIN, RASSF1A and CACNA2D2 located in the chromosomal region 3p21.31 associated with the development of early dysplastic lesions of head and neck. In analysis of 72 dysplastic lesions and 116 squamous cell carcinoma of head and neck, both deletion and promoter methylation have been seen in these genes except for CDC25A and SCOTIN where no methylation has been detected. The alteration of LIMD1 was highest (50%) in the mild dysplastic lesions and did not change significantly during progression of tumor indicating its association with this stage of the disease. It was evident that alterations of LTF, CDC25A and CACNA2D2 were associated with development of moderate dysplastic lesions, while alterations in RASSF1A and CACNA2D2 were needed for progression. Novel somatic mutations were seen in exon 1 of LIMD1 (7%), intron 3/exon4 splice junction of LTF (2%) and exon 7 of cdc25A (10%). Quantitative RT-PCR analysis revealed mean reduced expression of the genes in the following order: LTF (67.6 +/- 16.8) > LIMD1 (53.2 +/- 20.1) > CACNA2D2 (23.7 +/- 7.1) > RASSF1A (15.1 +/- 5.6) > CDC25A (5.3 +/- 2.3) > SCOTIN (0.58 +/- 0.54). Immunohistochemical analysis of CDC25A showed its localization both in cytoplasm and nucleus in primary lesions and oral cancer cell lines. In absence of HPV infection, LTF and RASSF1A alterations jointly have adverse impact on survival of tobacco addicted patients. Thus, our data suggested that multiple candidate TSGs in the chromosomal 3p21.31 region were differentially associated with the early dysplastic lesions of head and neck.

Remarkable difference in Al3+ and Zn2+ sensing properties of quinoline based isomers.

Two positional isomers, 4-methyl-2-((quinolin-6-ylimino)methyl)phenol (6-QMP) and 4-methyl-2-((quinolin-2-ylimino)methyl)phenol (2-QMP), have been synthesized to compare their fluorescence sensing properties. 6-QMP and 2-QMP have been synthesized by Schiff-base condensation between 2-hydroxy-5-methylbenzaldehyde and the respective amine (6-aminoquinoline for 6-QMP and 2-aminoquinoline for 2-QMP) under mild conditions. These compounds have been characterized by standard methods. 6-QMP and 2-QMP have been found to be dual fluorescence chemosensors for Al3+ and Zn2+ ions but the increment of fluorescence intensity varies. 6-QMP can detect Al3+ (emission at 543 nm) and Zn2+ (emission at 525 nm) by the enhancement of emission intensity by 97 and 79 fold, respectively, with the same excitation wavelength at 415 nm. However, 2-QMP shows two different excitation and emission wavelengths for the detection of Al3+ (emission at 376 nm; λex = 330 nm) and Zn2+ (emission at 550 nm; λex = 435 nm). The increase in emission intensity is low (4.5 fold for Al3+ and 35 fold for Zn2+) compared to that with 6-QMP. The enhancement of intensity may be explained by the PET mechanism. Both the probes form a 1 : 1 complex with both the metal ions as indicated by the elemental and different spectral analysis. 6-QMP shows better sensitivity towards both the metal ions than 2-QMP. Both the probes are able to detect Al3+ and Zn2+ ions by producing distinct color changes that can be observed by the naked eye. Some theoretical calculations have been performed to investigate spectral transitions of the probes along with their aluminum and zinc compounds. These compounds have been used for living cell imaging studies. A comparison with the recently published studies has been made.

Frequent inactivation of SLIT2 and ROBO1 signaling in head and neck lesions: clinical and prognostic implications.

OBJECTIVE: The protein SLIT2 and its receptor ROBO1 regulate different cellular processes, such as proliferation, apoptosis, and migration. In this study our aim is to understand the alterations of these genes during development of head and neck squamous cell carcinoma (HNSCC). MATERIALS AND METHODS: First, molecular alterations of the genes were analyzed in 30 dysplastic lesions, 128 primary HNSCC samples, and 1 HNSCC cell line. Then alterations were correlated with mRNA expression (n = 22) and protein expression (n = 29). Finally, the alterations were correlated with different clinicopathologic parameters and clinical outcomes of the patients. RESULTS: ROBO1 had a comparatively high frequency of deletion (28.5%-54.2%) from dysplastic lesions and subsequent clinical stages than did SLIT2 (16.6-27%). On the contrary, SLIT2 had a high frequency (56.6%-81.2%) of promoter methylation from dysplastic lesions onward compared with ROBO1 (20%-32.8%). Interestingly, alterations of SLIT2 and ROBO1 were high in dysplastic lesions (80%), followed by comparable frequencies (92.5%-95.3%) in subsequent stages of tumor. Alterations of these genes showed concordance with their mRNA/protein expression and significant association with poor patient outcome. CONCLUSIONS: Our data suggest that inactivation of SLIT2 and/or ROBO1 is one of the early events in development of dysplastic lesions of head and neck and has prognostic importance.

Comprehensive SNP scan of DNA repair and DNA damage response genes reveal multiple susceptibility loci conferring risk to tobacco associated leukoplakia and oral cancer.

Polymorphic variants of DNA repair and damage response genes play major role in carcinogenesis. These variants are suspected as predisposition factors to Oral Squamous Cell Carcinoma (OSCC). For identification of susceptible variants affecting OSCC development in Indian population, the "maximally informative" method of SNP selection from HapMap data to non-HapMap populations was applied. Three hundred twenty-five SNPs from 11 key genes involved in double strand break repair, mismatch repair and DNA damage response pathways were genotyped on a total of 373 OSCC, 253 leukoplakia and 535 unrelated control individuals. The significantly associated SNPs were validated in an additional cohort of 144 OSCC patients and 160 controls. The rs12515548 of MSH3 showed significant association with OSCC both in the discovery and validation phases (discovery P-value: 1.43E-05, replication P-value: 4.84E-03). Two SNPs (rs12360870 of MRE11A, P-value: 2.37E-07 and rs7003908 of PRKDC, P-value: 7.99E-05) were found to be significantly associated only with leukoplakia. Stratification of subjects based on amount of tobacco consumption identified SNPs that were associated with either high or low tobacco exposed group. The study reveals a synergism between associated SNPs and lifestyle factors in predisposition to OSCC and leukoplakia.

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.

Reduction of proliferation and induction of apoptosis are associated with shrinkage of head and neck squamous cell carcinoma due to neoadjuvant chemotherapy.

BACKGROUND: Neoadjuvant chemotherapy (NACT) is a treatment modality whereby chemotherapy is used as the initial treatment of HNSCC in patients presenting with advanced cancer that cannot be treated by other means. It leads to shrinkage of tumours to an operable size without significant compromise to essential oro-facial organs of the patients. The molecular mechanisms behind shrinkage due to NACT is not well elucidated. MATERIALS AND METHODS: Eleven pairs of primary HNSCCs and adjacent normal epithelium, before and after chemotherapy were screened for cell proliferation and apoptosis. This was followed by immunohistochemical analysis of some cell cycle (LIMD1, RBSP3, CDC25A, CCND1, cMYC, RB, pRB), DNA repair (MLH1, p53) and apoptosis (BAX, BCL2) associated proteins in the same set of samples. RESULTS: Significant decrease in proliferation index and increase in apoptotic index was observed in post-therapy tumors compared to pre-therapy. Increase in the RB/ pRB ratio, along with higher expression of RBSP3 and LIMD1 and lower expression of cMYC were observed in post-therapy tumours, while CCND1 and CDC25A remained unchanged. While MLH1 remained unchanged, p53 showed higher expression in post-therapy tumors, indicating inhibition of cell proliferation and induction of apoptosis. Increase in the BAX/BCL2 ratio was observed in post-therapy tumours, indicating up-regulation of apoptosis in response to therapy. CONCLUSIONS: Thus, modulation of the G1/S cell cycle regulatory proteins and apoptosis associated proteins might play an important role in tumour shrinkage due to NACT.

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.

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.