Impact of hyperglycemia on the expression of GLUT1 during oral carcinogenesis in rats.

BACKGROUND: On the background of the epidemiological link between diabetes and oral cancer, the present study aimed to analyze the potential involvement of selected glucose transporters (GLUT1/GLUT3/GLUT4), if any, in such putative association. METHODS AND RESULTS: Oral carcinogenesis was induced by 4-nitroquinoline N-oxide in 10 non-diabetic and 10 diabetic rats; 8 non-diabetic and 7 diabetic rats served as controls. Expressions of selected GLUTs at mRNA and protein levels were analyzed in oral tissue (normal/lesion) by quantitative real-time PCR and immunohistochemistry respectively. Premalignant lesions (hyperplasia/dysplasia/carcinoma-in-situ) appeared on tongues of carcinogen-treated animals. Significant increase of GLUT1mRNA level was seen from normal to lesion tongues, along increasing lesion grades (from hyperplasia/mild dysplasia to moderate/severe dysplasia) and in lesions induced under hyperglycemic condition than that induced under normoglycemic one; a similar trend was found in transcript variant-1 of GLUT1, but not in variant-2. GLUT3 and GLUT4 mRNA levels were comparable among lesions irrespective of grades and glycemic status. Concordant to mRNA level, overall expression of GLUT1 protein was higher in tongue lesions in presence of hyperglycemia than in absence of such condition; non-lesion portions of tongues exposed to carcinogen showed a similar trend. Moreover in carcinogen-treated groups, non-lesion and lesion portions of tongues under hyperglycemic condition showed predominantly membranous expression for GLUT1 which was again significantly higher than equivalent portions of tongue under normoglycemic condition. CONCLUSION: Hyperglycemia seemed to favor GLUT1 over-expression and membrane localization of the protein during oral carcinogenesis. GLUT1 transcript variant-1 appeared to be more important than variant-2 in disease pathogenesis.

Deregulation of H19 is associated with cervical carcinoma.

CACX is one of the most common cancer affecting women world-wide. Here, expression microarray analysis revealed 8 over-expressed transcribed pseudogenes (GBP1P1, HLA-DRB6, HLA-H, SLC6A10P, NAPSB, KRT16P2, PTTG3P and RNF126P1), down-regulated 7 lincRNAs (H19, MIR100HG, MEG3, DIO3OS, HOXA11-AS, CD27-AS1 and EPB41L4A-AS) and 6 snoRNAs (SNORD97, SNORD3A, SNORD3C, SNORD3D, SNORA12 and SCARNA9) as DEncGs (log2 fold-change ≥ ±1.0) in CACX. Consequently, down-regulation of lincRNA MEG3 and over-expression of pseudogenes, GBP1P1 and PTTG3P in the microarray analysis were found concordant with the real-time quantitative PCR results upon validation. Then, Ingenuity® Pathway analysis (IPA®) analysis with deregulated DEncGs identified functionally important gene, H19. Further, validation (n = 52) of expression confirmed frequent downregulation of H19 with significant association with its deletion (LOH) and promoter methylation (n = 128) in CACX. Moreover, clinicopathological analysis found Indian CACX patients (n = 26) with alterations of H19 by deletion or, promoter methylation with concomitant low expression have poor prognosis.

Integrative genomics and pathway analysis identified prevalent FA-BRCA pathway alterations in arsenic-associated urinary bladder carcinoma: Chronic arsenic accumulation in cancer tissues hampers the FA-BRCA pathway.

Arsenic in drinking water is one of the major etiological factors in urinary bladder carcinoma (BlCa). Here, high-resolution CGH-SNP microarray analysis in arsenic accumulated BlCa tissues showed significant (p < 0.05) association of chromosomal alterations with high arsenic (≥112 ng/g) accumulation, further corroborated by high γH2AX nuclear expression. Cytobands 5q11-35, 9p24.3-21.5, 18q11.1-25, etc. showed deletion, whereas 12q was amplified in high arsenic samples (AsH). Consecutively, IPA® found FA-BRCA pathway to be exclusively altered in AsH group. Validation of several key regulatory genes (RAD50, BRIP1, UIMC1, FANCD2, BRCA2 and BRCA1) of the pathway, were performed in independent BlCa cases (n = 81). UIMC1, RAD50 and BRIP1 were differentially deleted and associated with poor survival of AsH samples. Moreover, reduced nuclear expression with diffused cytoplasmic expression of FANCD2 was higher in AsH samples. Collectively, frequent deregulation of RAD50, UIMC1 and BRIP1 may result in reduced nuclear translocation of FANCD2, which may cause more chromosomal aberrations among AsH samples.

Reduction of nuclear Y654-p-ß-catenin expression through SH3GL2-meditated downregulation of EGFR in chemotolerance TNBC: Clinical and prognostic importance.

Triple negative breast cancer (TNBC) originates from a less differentiated ductal cell of breast, which is less sensitive to chemotherapy. The chemotolerance mechanism of TNBC has not yet been studied in detail. For this reason, molecular profiles (expression/genetic/epigenetic) of Y654-p-ß-catenin (active) and its kinase epidermal growth factor receptor (EGFR) along with SH3GL2 (regulator of EGFR homeostasis) were compared between neoadjuvant chemotherapy treated (NACT) and pretherapeutic TNBC samples. Reduced nuclear expression of Y654-p-ß-catenin protein with low proliferation index and CD44 prevalence showed concordance with reduced expression of EGFR/Y1045-p-EGFR proteins in the NACT samples than the pretherapeutic TNBC samples. Infrequent messenger RNA expression, gene amplification (10-32.5%), and mutation (1%) of EGFR were seen in the TNBC samples irrespective of therapy, suggesting the importance of EGFR protein stabilization in this tumor. The upregulation of SH3GL2 seen in the NACT samples in contrast to the pretherapeutic samples might be due to its promoter hypomethylation, as seen in the quantitative methylation assay. A similar trend of upregulation of SH3GL2 and downregulation of EGFR, Y1045-p-EGFR, Y654-p-ß-catenin were seen in the MDA-MB-231 cell line using antharacycline antitumor drugs (doxorubicin/nogalamycin). The NACT patients with reduced expression of Y654-p-ß-catenin and/or EGFR and high expression of SH3GL2 showed comparatively better prognosis than the pretherapeutic patients. Thus, our study showed that reduced nuclear expression of Y654-p-ß-catenin in NACT samples due to downregulation of EGFR protein through promoter hypomethylation-mediated upregulation of SH3GL2, resulting in low proliferation index/CD44 prevalence with better prognosis of the NACT patients, might have an important role in the chemotolerance of TNBC.

Divergent molecular profile of PIK3CA gene in arsenic-associated bladder carcinoma.

The activation of PIK3CA in bladder carcinoma (BlCa) with its recurrent mutations in exon 9 and 20 were well reported. But the association of arsenic on the activation of the pathway is not well elucidated. Therefore, we aimed to analyse the effect of arsenic on the genetic (copy number variation/mutation) and expression profiles of PIK3CA in primary BlCa samples. Infrequent amplification (16%) of the PIK3CA locus was observed, with higher frequency among the arsenic-high (AsH) than arsenic-low (AsL) samples. Frequent (54%) tumour-specific mutations in exon 9 and 20 of PIK3CA were observed in the BlCa samples with prevalent (47%) C>T transition mutations. Exon 9 and 20 harboured 48% and 73% of the total mutations, respectively, with 37% in E542K/E545K and 25% of the mutation in H1047Y/R. Though mutation frequency in AsH and AsL was found to be comparable, we observed some arsenic-specific mutation at c.1633G>A, c.1634A>C (E545K) and c.2985C>T and c.3130G>T mutations, as well as prevalent transverse mutations of A>C and G>T in AsH group. Furthermore, 73% of the BlCa samples showed overexpression (mRNA/protein) of PIK3CA with genetic alterations (amplification/mutation), significantly (P = 0.01) higher in AsH group. However, 36% of the samples showed overexpressed PIK3CA, independent of mutation or amplification, signifying a transcriptional upregulation of PIK3CA gene. Therefore, the expression status of NFκB, a transcription factor of PIK3CA, was assessed and found to be significantly correlated with the overexpression of PIK3CA (mRNA/protein) in AsH group. Similarly, the expression pattern of pAKT1 (Thr 308) was also found to be significantly correlated with PIK3CA overexpression. Finally, AsH patients with the overexpression of PIK3CA or NFκB had the worst overall survival, signifying a strong impact of arsenic on this pathway and outcome of the patients. Thus, our study showed that the arsenic-associated differential molecular profile of PIK3CA/AKT1/NFkB in BlCa has an important role in the molecular pathogenesis of the disease.

Induction of HRR genes and inhibition of DNMT1 is associated with anthracycline anti-tumor antibiotic-tolerant breast carcinoma cells.

The aim of the study was to understand the role of homologous recombination repair (HRR) pathway genes in development of chemotolerance in breast cancer (BC). For this purpose, chemotolerant BC cells were developed in MCF-7 and MDA MB 231 cell lines after treatment with two anthracycline anti-tumor antibiotics doxorubicin and nogalamycin at different concentrations for 48 h with differential cell viability. The drugs were more effective in MCF-7 (IC50: 0.214-0.242 µM) than in MDA MB 231 (IC50: 0.346-0.37 µM) as shown by cell viability assay. The drugs could reduce the protein expression of PCNA in the cell lines. Increased mRNA/protein expression of the HRR (BRCA1, BRCA2, FANCC, FANCD2, and BRIT1) genes was seen in the cell lines in the presence of the drugs at different concentrations (lower IC50, IC50, and higher IC50) irrespective of the cell viability (68-41%). Quantitative methylation assay showed an increased percentage of hypomethylation of the promoters of these genes after drug treatment in the cell lines. Similarly, chemotolerant neoadjuvant chemotherapy (NACT) treated primary BC samples showed significantly higher frequency of hypomethylation of the genes than the pretherapeutic BC samples. The drugs in different concentrations could reduce m-RNA and protein expression of DNMT1 (DNA methyltransferase 1) in the cell lines. Similar phenomenon was also evident in the NACT samples than in the pretherapeutic BC samples. Thus, our data indicate that reduced DNMT1 expression along with promoter hypomethylation and increased expression of the HRR genes might have importance in chemotolerance in BC.

Hypomethylation of mismatch repair genes MLH1 and MSH2 is associated with chemotolerance of breast carcinoma: Clinical significance.

BACKGROUND AND OBJECTIVES: The aim of the study was to understand the importance of mismatch repair genes MLH1 and MSH2 in chemotolerance and prognosis of breast carcinoma (BC). METHODS: First, the alterations (deletion/methylation/expression) of MLH1 and MSH2 were analyzed in 45 neoadjuvant chemotherapy (NACT)-treated and 133 pretherapeutic BC samples. The chemotolerant BC cells were characterized by treating two BC cell lines MCF-7 and MDA MB 231 with two anthracycline antitumor antibiotics, doxorubicin and nogalamycin. RESULTS: The deletion frequencies were 32% to 38% in MLH1/MSH2 genes and promoter methylation frequencies were 49% to 62% in MLH1 and 41% to 51% in MSH2 in both NACT-treated and pretherapeutic samples. The overall alteration of MLH1 and MSH2 was 58% to 71% in the samples. Reduced messenger RNA (mRNA) and protein expression were found in both the genes and it showed concordance with the molecular alterations. NACT-treated patients showed better prognosis. The chemotherapeutic drug induced increased mRNA/protein expression of the genes in BC cell lines was due to their promoter hypomethylation, as analyzed by quantitative methylation assay. This phenomenon was also evident in NACT-treated BC samples. CONCLUSION: MLH1/MSH2 genes play a critical role in the development of BC. Hypomethylation of MLH1/MSH2 genes might be important in chemotolerance of the disease.

Deregulation of LIMD1-VHL-HIF-1α-VEGF pathway is associated with different stages of cervical cancer.

To understand the mechanism of cellular stress in basal-parabasal layers of normal cervical epithelium and during different stages of cervical carcinoma, we analyzed the alterations (expression/methylation/copy number variation/mutation) of HIF-1α and its associated genes LIMD1, VHL and VEGF in disease-free normal cervix (n = 9), adjacent normal cervix of tumors (n = 70), cervical intraepithelial neoplasia (CIN; n = 32), cancer of uterine cervix (CACX; n = 174) samples and two CACX cell lines. In basal-parabasal layers of normal cervical epithelium, LIMD1 showed high protein expression, while low protein expression of VHL was concordant with high expression of HIF-1α and VEGF irrespective of HPV-16 (human papillomavirus 16) infection. This was in concordance with the low promoter methylation of LIMD1 and high in VHL in the basal-parabasal layers of normal cervix. LIMD1 expression was significantly reduced while VHL expression was unchanged during different stages of cervical carcinoma. This was in concordance with their frequent methylation during different stages of this tumor. In different stages of cervical carcinoma, the expression pattern of HIF-1α and VEGF was high as seen in basal-parabasal layers and inversely correlated with the expression of LIMD1 and VHL. This was validated by demethylation experiments using 5-aza-2'-deoxycytidine in CACX cell lines. Additional deletion of LIMD1 and VHL in CIN/CACX provided an additional growth advantage during cervical carcinogenesis through reduced expression of genes and associated with poor prognosis of patients. Our data showed that overexpression of HIF-1α and its target gene VEGF in the basal-parabasal layers of normal cervix was due to frequent inactivation of VHL by its promoter methylation. This profile was maintained during different stages of cervical carcinoma with additional methylation/deletion of VHL and LIMD1.

IGF2 is Deregulated During the Development of Uterine Cervical Carcinoma in Indian Patients.

Uterine cervical carcinoma (CACX) is one of the leading causes of deaths in Indian women. Chromosomal alterations including 11p15.5 locus were reported in CACX. Consequently, we strived for the first time to understand the molecular status of the candidate gene Insulin-like growth factor 2, IGF2 (11p15.5) in Indian CACX patients (n = 128). DNA copy number (CN) analysis using CGH-SNP analysis showed no genetic alteration and it was further validated by comparison with publicly available CN datasets. But promoter hypo-methylation during the progression of CACX was observed and also found to be concordant with publicly available DNA methylation datasets. Interestingly, we found diverse expression of IGF2 transcript in both normal cervical epithelium (NCE) and CACX tumors. Similar heterogeneous expression pattern was seen in publicly available expression datasets as well. Finally, protein expression analysis in NCE showed concordance with transcript expression but tumors showed frequent low expression. Log-rank test showed a difference (p-value = 0.057) in overall survival between cases with and without alteration for IGF2 in Indian CACX patients. Collectively, our study proposes that regulation of IGF2 expression in NCE appeared to be multifaceted and deregulation during the development of CACX resulted in the differential expression.

Activation of Wnt-ß-catenin pathway in basal-parabasal layers of normal cervical epithelium comparable during development of uterine cervical carcinoma.

In this study, importance of Wnt-ß-catenin pathway in the development of uterine cervical carcinoma was evaluated. For this purpose, the profiles (expression/methylation/deletion) of ß-catenin, p-ß-catenin (Y654), Wnt3a, and APC were studied in disease free normal cervical epithelium (n = 9), adjacent normal cervical epithelium of primary tumors (n = 70), CIN (n = 28), CACX (n = 102) samples, and two CACX cell lines (HeLa and SiHa). Immunohistochemical analysis revealed high/medium (74-95%) expression of ß-catenin/p-ß-catenin (Y654) and Wnt3a and low expression (23-26%) of APC in proliferating basal-parabasal layers contrary to differentiated spinous layer in normal cervix irrespective of HPV16 infection. The expression profile of the genes in the basal-parabasal layers did not change significantly during development of CACX. High (66%) promoter methylation of APC was seen in basal-parabasal layers and the cervical lesions (42-69%), unlike in spinous layers (25%). The promoter methylation status of APC was validated by in vitro demethylation experiments using 5-aza-dC in CACX cell lines. However, additional deletion of APC was significantly increased from CIN (12%) to stage I/II (40%) and became comparable in stage III/IV (48%) of the tumor. Patients with alterations (deletion/methylation) of APC and high/medium expression of Wnt3a/ß-catenin/p-ß-catenin (Y654) showed significantly poor survival. Thus our data indicate that cumulative effect of Wnt3a overexpression and APC inactivation are needed for overexpression of ß-catenin during the development of CACX.

Frequent inactivation of MCC/CTNNBIP1 and overexpression of phospho-beta-catenin(Y654) are associated with breast carcinoma: Clinical and prognostic significance.

Transcriptional activation of ß-catenin is a hallmark of Wnt/ß-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 ß-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 ß-catenin. Then transcriptional activity of ß-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 ß-catenin/p-ß-catenin(Y654) expression. Also, a significant correlation was seen between nuclear ß-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 ß-catenin and its target genes. The expression of nuclear p-ß-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 ß-catenin mediated transcription of target genes in BC.

Integrative genomic and network analysis identified novel genes associated with the development of advanced cervical squamous cell carcinoma.

BACKGROUND: CSCC is one of the most common cancer affecting women globally. Though it is caused by the infection of hrHPV but long latency period for malignant outcome in only a subset of hrHPV infected women indicates involvement of additional alterations, primarily CNVs. Here, we showed how CNVs played a crucial role in development of advanced tumors (stage III/IV) in Indian patients. METHODS: Initially, high-resolution CGH-SNP microarray analysis pointed out frequent CNVs followed by significantly altered genes. After comparison with TCGA dataset, expressions of the genes were checked in three CSCC datasets to identify key genes followed by Ingenuity® Pathway analysis. Then node effect property analysis was applied on the constructed PPI network to rank the key proteins. Finally, validations in independent samples were performed. RESULTS: For the first time, frequent chromosomal amplifications at 3q13.13-3q29, 1p36.11-1p31.1, 1q21.1-1q44 and 5p15.33-5p12 followed by common deletions at 11q14.1-11q25, 2q34-2q37.3, 4p16.3-4p12 and 13q13.3-13q14.3 were identified in Indian CSCC patients. Integrative analysis found 78 key genes including several novel ones, which were mostly associated with 'Cancer' and may regulate DNA repair and metabolic pathways. Analysis showed PARP1 and ATR were among the top ranking protein interactors. CONCLUSIONS: Frequent amplification and over-expression of ATR and PARP1 were further confirmed in cervical lesions, indicating their association with poor prognosis of advanced CSCC patients. GENERAL SIGNIFICANCE: Our novel approach identified precise CNVs along with several novel genes within these loci and showed that PARP1 and ATR, having biologically significant interactions, may be involved in development of advanced CSCC.

Frequent alterations of homologous recombination repair pathway in primary and chemotolerant breast carcinomas: clinical importance.

AIM: To understand the importance of homologous recombination repair pathway in development of breast carcinoma (BC), alterations of some key regulatory genes like BRCA1, BRCA2, FANCC and FANCD2 were analyzed in pretherapeutic/neoadjuvant chemotherapy (NACT)-treated BC samples. MATERIALS & METHODS: Alterations (deletion/methylation/expression) of the genes were analyzed in 118 pretherapeutic and 41 NACT-treated BC samples. RESULTS: High deletion/methylation (29-68%) and 64-78% overall alterations of the genes were found in the samples. Concordance was evident between alteration and protein expression of the genes. Estrogen/progesterone receptor-negative tumors showed significantly high alterations even in NACT-treated samples having low CD44 and proliferating cell nuclear antigen expression. Pretherapeutic patients with alterations showed poor prognosis. CONCLUSION: Alterations of homologous recombination repair pathway genes are needed for the development of BC.

Association of P16-RBSP3 inactivation with phosphorylated RB1 overexpression in basal-parabasal layers of normal cervix unchanged during CACX development.

To understand the molecular mechanism of RB1 phosphorylation in basal-parabasal layers of normal cervix and during cervical cancer (CACX) development, we analyzed the alterations (expression/methylation/deletion/mutation) of RB1/phosphorylated RB1 (p-RB1) (ser807/811 and ser567) and two RB1 phosphorylation inhibitors, P16 and RBSP3, in disease-free normal cervical epithelium (n = 9), adjacent normal cervical epithelium of tumors (n = 70), cervical intraepithelial neoplasia (CIN; n = 28), CACX (n = 102) samples and two CACX cell lines. Immunohistochemical analysis revealed high/medium expression of RB1/p-RB1 (ser807/811 and ser567) and low expression of P16 and RBSP3 in proliferating basal-parabasal layers of majority of normal cervical epitheliums, irrespective of HPV16 infection. Interestingly, 35-52% samples showed high/medium expression of P16 in basal-parabasal layers of normal and had significant association with deleterious non-synonimous SNPs of P16. Methylation of P16 and RBSP3 in basal-parabasal layers of normal cervix (32 and 62%, respectively) showed concordance with their respective expressions in basal-parabasal layers. The methylation frequency of P16 and RBSP3 in basal-parabasal layers of normal did not change significantly in CIN and CACX. The deletion frequency of P16 and RB1 increased significantly with CACX progression. While, deletion of RBSP3 was high in CIN and comparable during CACX progression. P16 showed scattered and infrequent mutation in CACX. The alteration of P16 and RBSP3 was synergistic and showed association with overexpression of p-RB1 in tumors and associated with poor prognosis of patients. Thus, our data suggest that overexpression of p-RB1 in basal-parabasal layers of normal cervical epithelium was due to methylation/low functional-linked non-synonimous SNPs of P16 and RBSP3. This pattern was maintained during cervical carcinogenesis by additional deletion/mutation.

Amarogentin regulates self renewal pathways to restrict liver carcinogenesis in experimental mouse model.

Amarogentin, a secoiridoid glycoside isolated from medicinal plant Swertia chirata, was found to restrict CCl4 /N-nitrosodiethyl amine (NDEA) induced mouse liver carcinogenesis by modulating G1/S cell cycle check point and inducing apoptosis. To understand its therapeutic efficacy on stem cell self renewal pathways, prevalence of CD44 positive cancer stem cell (CSC) population, expressions (mRNA/protein) of some key regulatory genes of self renewal Wnt and Hedgehog pathways along with expressions of E-cadherin and EGFR were analyzed during the liver carcinogenesis and in liver cancer cell line HepG2. It was observed that amarogentin could significantly reduce CD44 positive CSCs in both pre and post initiation stages of carcinogenesis than carcinogen control mice. In Wnt pathway, amarogentin could inhibit expressions of ß-catenin, phospho ß-catenin (Y-654) and activate expressions of antagonists sFRP1/2 and APC in the liver lesions. In Hedgehog pathway, decreased expressions of Gli1, sonic hedgehog ligand, and SMO along with up-regulation of PTCH1 were seen in the liver lesions due to amarogentin treatment. Moreover, amarogentin could up-regulate E-cadherin expression and down-regulate expression of EGFR in the liver lesions. Similarly, amarogentin could inhibit HepG2 cell growth along with expression and prevalence of CD44 positive CSCs. Similar to in vivo analysis, amarogentin could modulate the expressions of the key regulatory genes of the Wnt and hedgehog pathways and EGFR in HepG2 cells. Thus, our data suggests that the restriction of liver carcinogenesis by amarogentin might be due to reduction of CD44 positive CSCs and modulation of the self renewal pathways. © 2015 Wiley Periodicals, Inc.

Tea polyphenols EGCG and TF restrict tongue and liver carcinogenesis simultaneously induced by N-nitrosodiethylamine in mice.

The aim of this study is to understand the molecular mechanisms of N-nitrosodiethylamine (NDEA) induced multi-organ carcinogenesis in tongue and liver of the same mouse and restriction of carcinogenesis by Epigallocatechin gallate (EGCG) and Theaflavin (TF), if any. For that purpose, cellular proliferation/apoptosis, prevalence of CD44 positive stem cell population and expressions of some key regulatory genes of self renewal Wnt and Hedgehog (Hh) pathways and some of their associated genes were analyzed in the NDEA induced tongue and liver lesions in absence or presence of EGCG/TF. Chronic NDEA exposure in oral cavity could decrease mice body weights and induce tongue and liver carcinogenesis with similar histological stages (severe dysplasia up to 30thweeks of NDEA administration). Increasing mice body weights were seen in continuous and post EGCG/TF treated groups. EGCG/TF treatment could restrict both the carcinogenesis at similar histological stages showing potential chemopreventive effect in continuous treated groups (mild dysplasia) followed by pre treatment (moderate dysplasia) and therapeutic efficacy in post treated groups (mild dysplasia) up to 30thweek. The mechanism of carcinogenesis by NDEA and restriction by the EGCG/TF in both tongue and liver were similar and found to be associated with modulation in cellular proliferation/apoptosis and prevalence of CD44 positive population. The up-regulation of self renewal Wnt/ß-catenin, Hh/Gli1 pathways and their associated genes Cyclin D1, cMyc and EGFR along with down regulation of E-cadherin seen during the carcinogenesis processes were found to be modulated during the restriction processes by EGCG/TF.

Inactivation of PTCH1 is associated with the development of cervical carcinoma: clinical and prognostic implication.

The aim of this study was to analyze the alterations of PTCH1 (deletion/promoter methylation/mutation/expression) during the development of cervical cancer (CACX). For this purpose, deletion/methylation of PTCH1 were analyzed in HPV16 positive exfoliated asymptomatic cervical swabs (n = 74), cervical intraepithelial neoplasia (CIN) (n = 32), CACX (n = 174) samples, and two CACX cell lines. The deletion of PTCH1 increased significantly from CIN (11.5%) to stage I/II (42%) and comparable in stage III/IV (46%). Low frequency (14-16%) of PTCH1 methylation was seen in the asymptomatic exfoliated cervical cells and in the normal epithelium adjacent to the tumor followed by a significant increase in CIN (31%) to stage I/II (57%) and comparable in stage III/IV (58%). The overall alterations (deletion/methylation) of PTCH1 significantly increased from CIN (34%) to stage I/II (70%) and comparable in stage III/IV (69%). Interestingly, in the normal epithelium, methylation of PTCH1 was high in basal/parabasal layers (83%), followed by decrease in the spinous layer (33 %), and showed significant inverse correlation with its expression. Reduced expression of PTCH1 seen in tumors showed a significant association with its alterations (deletion/methylation). The expression pattern of PTCH1 showed an inverse correlation with the nuclear expression of GLI1 in the normal epithelium as well as in the tumors. High nuclear expression of HPV16, E6, and E7 were seen in basal/parabasal layers of the normal epithelium and also in tumors. The PTCH1 alterations (deletion and/or methylation) in tumors and its methylation in adjacent normal epithelium were associated with poor prognosis of patients. Thus, our data suggests that activation of the Hedgehog pathway due to PTCH1 inactivation along with HPV infection is important in CACX development.

Down-regulation of FA-BRCA Pathway in Cervical Carcinoma Gradually Reversed During the Development of Chemo-tolerance: Clinical Implications.

Cervical cancer is one of the leading causes of cancer death among females, worldwide. The contributory role of different cellular pathways in the process of carcinogenesis is still poorly understood. Our study was focused here to understand the functional evaluation of key regulatory genes of FA-BRCA pathway in the development of CACX and their role in chemo-tolerance of the disease by analyzing the molecular profile of the genes both in normal and tumour tissue of our sample pool, also validated in in silico datasets. Later on, prognostic importance of the genes was further evaluated in plasma DNA and cisplatin-treated in vitro system. We found that expression profile of FA-BRCA pathway genes was gradually reduced from undifferentiated basal-parabasal layers of normal tissue towards the progression of the disease. Further analysis revealed that frequent promoter methylation [32-55%] and deletion [34-52%] events were the plausible reasons for their reduced expression in CACX. Noticeably, invasion of promoter methylation of the genes [11-17%] in plasma CTCs of CACX patients was positively correlated [p < 0.001] with poor prognosis among patients. On the other hand, functional upregulation of these genes at higher concentrations [IC50-70] of cisplatin was a predictor for the development of drug tolerance, as evaluated in our in vitro study. This finding was supported further by low prevalence of γ-H2X foci formation and reduced expression of DNMT1 at higher concentrations of cisplatin. In totality, we discovered that the FA-BRCA pathway must be inactivated for cancer formation. In contrast, elevated gene expression played a substantial role in building of chemo-tolerance and might be associated with developing increased risk of disease recurrence among patients.

Frequent alterations of MCPH1 and ATM are associated with primary breast carcinoma: clinical and prognostic implications.

BACKGROUND: MCPH1 is a proximal regulator of DNA damage response pathway that is involved in recruitment of phosphorylated ATM to double-stranded DNA breaks. METHODS: To understand the importance of MCPH1 and ATM in deregulation of DNA damage response pathway in breast carcinoma, we studied m-RNA expression and genetic/epigenetic alterations of these genes in primary breast carcinoma samples. RESULTS: Our study revealed reduced expression (mRNA/protein) and high alterations (deletion/methylation) (96 %, 121 of 126) of MCPH1 and ATM. Mutation was, however, rare in inactivation of MCPH1. In immunohistochemical analysis, reduced protein expression of MCPH1, ATM and p-ATM were concordant with their molecular alterations (P = 0.03-0.01). Alterations of MCPH1 and deletion of ATM were significantly high in estrogen/progesterone receptor-negative than estrogen/progesterone receptor-positive breast carcinoma samples compared to early or late age of onset tumors, indicating differences in pathogenesis of the molecular subtypes (P = 0.004-0.01). These genes also showed differential association with tumor stage, grade and lymph node status in different subtypes of breast carcinoma (P = 0.00001-0.01). Their coalterations showed significant association with tumor progression and prognosis (P = 0.003-0.05). Interestingly, patients with alterations of these genes or MCPH1 alone had poor outcome after treatment with DNA-interacting drugs and/or radiation (P = 0.01-0.05). CONCLUSIONS: Inactivation of MCPH1-ATM-associated DNA damage response pathway might have an important role in the development of breast carcinoma with diagnostic, prognostic and therapeutic implications.

Role of MLH1 and MSH2 deficiency in the development of tumorigenesis and chemo-tolerance of cervical Carcinoma: Clinical implications.

Cervical cancer (CACX) is one of the top causes of cancer death in women globally. The involvement of several cellular pathways in carcinogenesis is still poorly understood. Here, we focused to evaluate the contributory role of Mismatch Repair (MMR) pathway genes-MLH1 and MSH2 in CACX and their association with chemo-tolerance of the disease. For this purpose, molecular profiles (expression/promoter methylation/deletion) of the genes were analysed in both normal cervical epithelium and tumour tissue, also validated in in-silico dataset as well. Later on, prognostic importance of the genes was identified through analysis of their methylation/expression status in plasma DNA of circulating tumour cells (CTCs) and cisplatin-tolerant CACX cell lines respectively. It was found that the expression profile of MLH1 and MSH2 genes was considerably reduced from undifferentiated basal-parabasal layers of normal cervical epithelium towards progression of the disease. Further analysis showed that frequent deletion [34-48%] and promoter methylation events [28-46%] of the genes were the plausible reasons for their reduced expression during tumorigenesis. Incidentally, the prevalence of MLH1 [32%] and MSH2 [27%] promoter methylation found in CTCs of plasma of the clinically advanced CACX patients implicated their prognostic importance of the disease. In addition, the patients having high alterations of those genes resulted in poor patient outcomes even after the therapy. In in-depth analysis of this result in cisplatin-tolerant CACX cell lines, we discovered that increased promoter methylation frequency of those genes at higher concentrations of cisplatin and gradual accumulation of the cells in the G2/M phase of the cell cycle were the rational causes for their reduced expression and MMR deficiency in the system. Hence, it is possible to conclude that the gradual down-regulation of MLH1 and MSH2 proteins may be a key event for MMR pathway inactivation in CACX. This might also be associated with chemo-tolerance and overall poor survival among the patients.