Fhit Expression Research Articles

Expression and clinical significance of miRNAs that may be associated with the FHIT gene in breast cancer

The dysregulation of miRNA expression has frequently been observed in breast cancer. Therefore, we investigated the expression profile of miRNAs that may be associated with expression of the FHIT gene in breast cancer and assessed their clinicopathological significance. The expression levels of miR-143, miR-663a, miR-668, miR-922 and FHIT were analyzed in normal and malignant breast tissues from 65 patients with breast cancer. We studied the correlation between the expression of miR-143, miR-663a, miR-668, miR-922 and FHIT and the clinicopathological features presented by the patients. The expression levels of the miRNAs and FHIT were downregulated in breast cancer tissue. The expression levels of miR-143, miR-663a and miR-668 were significantly reduced in FHIT downregulated tumors. miR-668 expression was also significantly altered relative to FHIT down- and up- regulated tumor tissues. Reduced miR-663a expression was statistically associated with high-grade ER/PR (+) status, benign reactive hyperplasia, lymph-node metastasis, in-situ component >25% and Ki 67>15% compared with non-tumor tissues. Additionally, reduced miR-668 expression was significantly different between tumors with and without lymph-node metastasis. miR-668 may play an important role in breast cancer development and progression by regulating the expression of FHIT. Furthermore, miR-668 and miR-663a may be potential prognostic biomarkers for breast cancer.

BRAF and FHIT expression in hcv related cirrhosis and hepatocellular carcinoma and their association with prognostic anatomopathological features

BRAF and FHIT expression in hcv related cirrhosis and hepatocellular carcinoma and their association with prognostic anatomopathological features

Exome-wide single-base substitutions in tissues and derived cell lines of the constitutive Fhit knockout mouse.

Loss of expression of Fhit, a tumor suppressor and genome caretaker, occurs in preneoplastic lesions during development of many human cancers. Furthermore, Fhit‐deficient mouse models are exquisitely susceptible to carcinogen induction of cancers of the lung and forestomach. Due to absence of Fhit genome caretaker function, cultured cells and tissues of the constitutive Fhit knockout strain develop chromosome aneuploidy and allele copy number gains and losses and we hypothesized that Fhit‐deficient cells would also develop point mutations. On analysis of whole exome sequences of Fhit‐deficient tissues and cultured cells, we found 300 to >1000 single‐base substitutions associated with Fhit loss in the 2% of the genome included in exomes, relative to the C57Bl6 reference genome. The mutation signature is characterized by increased C>T and T>C mutations, similar to the “age at diagnosis” signature identified in human cancers. The Fhit‐deficiency mutation signature also resembles a C>T and T>C mutation signature reported for human papillary kidney cancers and a similar signature recently reported for esophageal and bladder cancers, cancers that are frequently Fhit deficient. The increase in T>C mutations in −/− exomes may be due to dNTP imbalance, particularly in thymidine triphosphate, resulting from decreased expression of thymidine kinase 1 in Fhit‐deficient cells. Fhit‐deficient kidney cells that survived in vitro dimethylbenz(a)anthracene treatment additionally showed increased T>A mutations, a signature generated by treatment with this carcinogen, suggesting that these T>A transversions may be evidence of carcinogen‐induced preneoplastic changes.

Association of activated Gαq to the tumor suppressor Fhit is enhanced by phospholipase Cβ.

BackgroundG proteins are known to modulate various growth signals and are implicated in the regulation of tumorigenesis. The tumor suppressor Fhit is a newly identified interaction partner of Gq proteins that typically stimulate the phospholipase C pathway. Activated Gαq subunits have been shown to interact directly with Fhit, up-regulate Fhit expression and enhance its suppressive effect on cell growth and migration. Other signaling molecules may be involved in modulating Gαq/Fhit interaction.MethodsTo test the relationship of PLCβ with the interaction between Gαq and Fhit, co-immunoprecipication assay was performed on HEK293 cells co-transfected with different combinations of Flag-Fhit, Gα16, Gα16QL, pcDNA3 vector, and PLCβ isoforms. Possible associations of Fhit with other effectors of Gαq were also demonstrated by co-immunoprecipitation. The regions of Gαq for Fhit interaction and PLCβ stimulation were further evaluated by inositol phosphates accumulation assay using a series of Gα16/z chimeras with discrete regions of Gα16 replaced by those of Gαz.ResultsPLCβ1, 2 and 3 interacted with Fhit regardless of the expression of Gαq. Expression of PLCβ increased the affinities of Fhit for both wild-type and activated Gαq. Swapping of the Fhit-interacting α2-β4 region of Gαq with Gαi eliminated the association of Gαq with Fhit without affecting the ability of the mutant to stimulate PLCβ. Other effectors of Gαq including RGS2 and p63RhoGEF were unable to interact with Fhit.ConclusionsPLCβ may participate in the regulation of Fhit by Gq in a unique way. PLCβ interacts with Fhit and increases the interaction between Gαq and Fhit. The Gαq/PLCβ/Fhit complex formation points to a novel signaling pathway that may negatively regulate tumor cell growth.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1802-z) contains supplementary material, which is available to authorized users.

Oncogenes and tumor suppressor genes in squamous cell carcinoma of the tongue in young patients

The occurrence of squamous cell carcinoma of the tongue (SCCT) of young patients increased. There are still controversies about patient prognosis. The underlying molecular mechanisms remain unclear. 276 patients (66 ≤45, 210 >45 years) with SCCT were included. Clinical parameters and survival data were assessed. Oncogenes and tumor suppressors were analyzed via immunohistochemistry (p53, CXCR4, p16, EGFR) and qPCR (CDK4, CDKN2A, TP53, MDM2, AKT1, PIK3CA, NRAS, HRAS, KRAS, HGF, MET, EGF, ATM, BRCA1, E2F1, FHIT, RUNX3, STK11, BCL2, CTNNB1). The median overall survival was 142 (≤45 years) and 34 months (>45 years) (p < 0.0001; HR [95%CI]: 0.37 [0.30-0.58]). Disease specific survival in patients ≤45 years was with 181 months significantly higher than in patients >45 years (p < 0.0001; HR [95%CI]: 0.33 [0.26-0.57]). Immunhistochemistry visualized a comparable expression of analyzed proteins. QPCR demonstrated in patients ≤45 years a higher expression of genes that are associated with carcinogenesis (CTNNB1, STK11, CDKN2A, HGF, MET) as well as tumor suppressors that constitute an enhanced radio-sensitivity (ATM, BRCA1E2F1, FHIT). Derogation of the WNT-CTNNB1-STK11 and CDKN2A-HGF-MET pathway can constitute the carcinogenesis, while the higher expression of radio-sensitizers ATM, BRCA1E2F1 and FHIT can explain the better OS/DSS in young patients.

Methylation and Hydroxymethylation Status of Tumor Suppressor Genes and Common Fragile Sites Genes in Acute Myeloid Leukemia

Background: 5-methylation (5-mC) is the predominant epigenetic mark in mammalian genomic DNA. When promoter region of certain gene is hypermethylated, the gene becomes transcription silent. Promoter of tumor suppressor genes (TSG) usually exists in CpG islands, and silencing of TSGs in cancer cells is often associated with hypermethylation. p15, CDH1 are frequently methylated in myeloid malignancies such as acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS). Common Fragile Site (CFS) is a fragile site on the chromosomes easy to produce gap and break, and it contains putative TSGs. FHIT, WWOX and PARK2 are the CFS genes known to be frequently methylated in solid tumors, but their status of hematologic malignancies has not been fully elucidated yet. 5-hydroxymethylaiton (5-hmC) is a newly discovered epigenetic modification that is presumably generated by oxidation of 5-mC by the TET family of cytosine oxygenases. Techniques identifying 5-mC cannot distinguish between 5-mC and 5-hmC, therefore 5-hmC status of the genes have not fully elucidated yet too. Recently it has been demonstrated that mutation of epigenetic modifiers (DNMT3A, TET2, IDH1/2) play important role on AML pathogenesis. We tried to clarify 5-mC and 5-hmC status of TSG p15, CDH1 and CFS genes FHIT, WWOX and PARK2 by using new techniques and the relationships with expression levels of epigenetic modifiers in AML.Methods: BM samples obtained from 74 of AML patients are subjected to the study after informed consent. This study was approved by IRB of Gunma University Hospital. DNA, RNA were extracted from BM mononuclear cells. Methylation specific PCR (MSP) was carried out to assay 5-mC of p15, CDH1, WWOX, PARK2. Quantification of 5-mC and 5-hmC (except PARK2) was carried out by methylation sensitive restriction enzyme assay (MSRE) with glucosylation and Q-PCR. Total DNA 5-mC and 5-hmC were analyzed by ELISA. The mRNA expression levels of p15, CDH1, FHIT, WWOX, PARK2, DNMT1, 3A, TET2 were quantified by Q-PCR.Results: MSP revealed that p15, CDH1, WWOX and PARK2 were methylated in 43.1%, 94.3%, 35.7% and 36.9% of AML, respectively. PARK2 methylation was not found in t(15;17) APL, but in 32% of normal karyotype AML (NK-AML), in 67% of t(8;21) CBF-AML. In contrast, the p15 methylation was found in 83.3% of APL, 45.5% of NK-AML, 50% of CBF-AML. WWOX methylation was found in 42.9% of APL, in 16% of NK-AML and 66.7% of CBF-AML. Adverse karyotype AML (adv-AML) tended to show lower % of WWOX, PARK2 and p15 methylation with 15.8%, 21.1% and 18.8% compare to good risk karyotype. The frequency of the methylation of PARK2 and WWOX were varied among karyotypes and the methylation was mutually exclusive. ELISA demonstrated that mean % of total 5-mC DNA was 1.08% and ratio of 5-hmC in 5-mC was 0.95% in AML. Interestingly, 5-hmC was 0% in adv-AML although 5-mC existed (mean: 1.05%). Locus specific MSRE-QPCR demonstrated that mean % of 5-mC of p15, CDH1, WWOX and FHIT were 6.62%, 1.25%, 8.33%, 2.88%, respectively., In adv-AML, 5-hmC of CDH1, WWOX and FHIT were not detected, although 5-mC of these genes were detected (0.41%, 9.0%, 2.14%) in accordance with whole DNA analysis. In good and intermediate AML, 5-hmC of these genes was 3.44%, 1.07%, 2.69% ,respectively. RQ-PCR demonstrated that CDH1, p15, WWOX, PARK2 and epigenetic modifier DNMT1, DNMT3A and TET2 expression were not different among various karyotype risks, but only FHIT expression significantly higher in good risk group (p=0.047). The expression levels of the genes were not significantly different between mentylated and unmethylated. The ratio of 5-hmC/5-mC of the TSGs tended to be associated with the expression levels of the corresponding genes, but the association did not reach statistical significance. DNMT3A expression in AML with 5-mC PARK2 was higher than in other AML (p=0.016). Contrary to the intuition, DNMT3A expression was positively correlated with FHIT, PARK2 expression (r=0.776, p<0.001, r=0.689, p<0.001). CDH1 expression was positively correlated with DNMT1 and negatively correlated with TET2 expression (r=0.447, p=0.009, r=-0.349, p=0.022). OS and EFS were not different among the methylation status of these genes.Conclusion: CFS genes are selectively methylated in AML. MSRE-QPCR can distinguish 5-mC and 5-hmC and quantify the ratio of them with locus specific manner. The relationship between gene expression and 5-hmC, 5-mC should be pursued. DisclosuresNo relevant conflicts of interest to declare.

FHIT loss-induced DNA damage creates optimal APOBEC substrates: Insights into APOBEC-mediated mutagenesis

APOBEC cytidine deaminase activity is a major source of hypermutation in cancer. But previous studies have shown that the TC context signature of these enzymes is not observed in sizable fractions of cancers with overexpression of APOBEC, suggesting that cooperating factors that contribute to this mutagenesis should be identified. The fragile histidine triad protein (Fhit) is a tumor suppressor and DNA caretaker that is deleted or silenced in >50% of cancers. Loss of Fhit protein activity causes replication stress through reduced Thymidine Kinase 1 expression, increased DNA breaks, and global genome instability in normal and cancer cells. Using data from The Cancer Genome Atlas (TCGA), we show that FHIT-low/APOBEC3B-high expressing lung adenocarcinomas display significantly increased numbers of APOBEC signature mutations. Tumor samples in this cohort with normal FHIT expression do not exhibit APOBEC hypermutation, despite having high APOBEC3B expression. In vitro, silencing Fhit expression elevates APOBEC3B-directed C > T mutations in the TP53 gene. Furthermore, inhibition of Fhit loss-induced DNA damage via thymidine supplementation decreases the TP53 mutation burden in FHIT-low/APOBEC3B-high cells. We conclude that APOBEC3B overexpression and Fhit-loss induced DNA damage are independent events that, when occurring together, result in a significantly increased frequency of APOBEC-induced mutations that drive cancer progression.

C-Myc suppresses microRNA-29b to promote tumor aggressiveness and poor outcomes in non-small cell lung cancer by targeting FHIT.

The dual role of the microRNA-29 (miR-29) family in tumor progression and metastasis in solid tumors has been reported. Evidence for the role of miR-29 in tumor malignancy and its prognostic value in overall survival (OS) and relapse-free survival (RFS) in non-small cell lung cancer (NSCLC) remains conflicting. Mechanistic studies presented herein demonstrated that c-Myc suppressed the expression of miR-29b, promoting soft agar growth and invasion capability in lung cancer cells. Interestingly, the decrease in the expression of miR-29b by c-Myc is responsible for soft agar growth and invasiveness mediated by FHIT loss due to promoter methylation. Among patients, low expression of miR-29b and FHIT was more common in tumors with high c-Myc expression than in tumors with low c-Myc expression. Kaplan-Meier and Cox regression analysis showed that tumors with high c-Myc, low miR-29b and low FHIT expression had shorter OS and RFS periods than their counterparts. In conclusion, the decrease in the expression of miR-29b by c-Myc may be responsible for FHIT loss-mediated tumor aggressiveness and for poor outcome in NSCLC. Therefore, we suggest that restoration of the miR-29b expression using the c-Myc inhibitor might be helpful in suppressing tumor aggressiveness mediated by FHIT loss and consequently improving outcomes in NSCLC patients with tumors with low expression of FHIT.

Mo1048 BRAF and FHIT Expression in HCV Related Cirrhosis and Hepatocellular Carcinoma and Their Association With Prognostic Anatomopathological Features

Mo1048 BRAF and FHIT Expression in HCV Related Cirrhosis and Hepatocellular Carcinoma and Their Association With Prognostic Anatomopathological Features

Fhit Regulates EMT Targets through an EGFR/Src/ERK/Slug Signaling Axis in Human Bronchial Cells

In many cancers, including lung carcinomas, Fragile histidine triad (Fhit) is frequently decreased or lost. Fhit status has recently been shown to be associated with elevated in vitro and in vivo invasiveness in lung cancer. Tumor cell invasion is facilitated by epithelial-mesenchymal transition (EMT), a process by which tumor cells lose their epithelial features to acquire a mesenchymal cell-like phenotype. In this study, the mechanism underlying Fhit-regulated EMT was deciphered. Using Slug knockdown, pharmacologic inhibitors PD98059, PP1, and gefitinib as well as an anti-EGFR antibody, it was demonstrated that Fhit silencing in bronchial cells induced overexpression of two primary EMT-associated targets, MMP-9 and vimentin, to regulate cell invasion dependent on an EGFR/Src/ERK/Slug signaling pathway. Moreover, ectopic expression of Fhit in Fhit-deficient lung cancer cells downregulated this pathway. Finally, an inverse correlation was observed between Fhit and phospho-EGFR levels in a cohort of human squamous cell lung carcinoma specimens. These results demonstrate a Fhit-dependent mechanism in the control of EMT-regulated EGFR signaling. This study adds new insight into the regulatory mechanism of EMT, a process known to increase resistance to conventional and targeted therapies in lung cancer.

Identification of a Tumor-Suppressive Human-Specific MicroRNA within theFHITTumor-Suppressor Gene

Loss or attenuated expression of the tumor-suppressor gene FHIT is associated paradoxically with poor progression of human tumors. Fhit promotes apoptosis and regulates reactive oxygen species; however, the mechanism by which Fhit inhibits tumor growth in animals remains unclear. In this study, we used a multidisciplinary approach based on bioinformatics, small RNA library screening, human tissue analysis, and a xenograft mouse model to identify a novel member of the miR-548 family in the fourth intron of the human FHIT gene. Characterization of this human-specific microRNA illustrates the importance of this class of microRNAs in tumor suppression and may influence interpretation of Fhit action in human cancer.

Reduced FHIT expression is associated with tumor progression in sporadic colon adenocarcinoma

PurposeTumor supressor gene FHIT was identified at chromosome 3p14.2 spanning the FRA3B fragile site and is very often inactivated in different types of cancer. The aim of this study was to examine the frequency of FHIT gene LOH as well as FHIT mRNA and protein expression in sporadic colon adenocarcinoma. MethodsThe results of LOH, real-time qRT-PCR and imunohistochemical analyses were correlated with clinico-pathological characteristics of patients and their tumors in order to evaluate the role of FHIT gene/protein in sporadic colon adenocarcinoma tumorigenesis. ResultsOne hundred and thirty one (96.3%) samples were informative for both markers and 33/131 (25.2%) demonstrated LOH. Expression of FHIT mRNA was significantly decreased in colon tumors relative to that in corresponding normal tissue (p=7.2×10−6). Most of the samples (54.0%) were negative for FHIT protein, 26.4% adenocarcinomas showed a weak to moderate immunostaining and 19.6% adenocarcinomas showed strong FHIT immunostaining. No correlation was found between FHIT gene LOH status, mRNA expression or FHIT protein immunostaining and clinico-pathological characteristics. Expression of FHIT mRNA was significantly decreased in FHIT LOH positive tumors (p=0.027). Patients with LOH negative tumors or FHIT protein positive tumors had longer survival but this findings were not statistically significant. ConclusionsOur overall results suggest that reduced expression of FHIT gene may be associated with the progression of these malignant tumors.

Abstract P2-06-05: Association of promoter methylation status of the FHIT gene and Fhit expression with HER2/neu status in breast cancer

Abstract Purpose: Aberrant DNA methylation has been recognized to contribute to breast carcinogenesis, and promoter hypermethylation of many tumor suppressor genes has been correlated with decreased gene expression. The fragile histidine triad (FHIT) gene is a putative tumor suppressor gene in breast and other cancers, and loss of Fhit expression has been observed in breast cancers. The aim of this study was to evaluate the association between methylation of the FHIT gene and its expression in breast cancer, and to investigate whether methylation and expression of the FHIT gene would correlate with clinicopathological characteristics in relation to human epidermal growth factor receptor 2 (HER2) status. Methods: Pyrosequencing of bisulfite treated DNA was performed to study the methylation status of the FHIT gene in 60 breast cancer samples from Korean women obtained at Daegu Catholic University Hospital. We examined the expression of FHIT using tissue microarrays by immunohistochemical staining. Association between the methylation status of the FHIT gene and its expression was analyzed, and the relationship between the FHIT expression and the clinicopathological characteristics of the patients was evaluated. Results: FHIT methylation was detected in 96.7% and positive expression rate of Fhit was 87.3% of the patients. The mean methylation level of the FHIT gene was associated with intratumoral inflammation. Methylation level of the FHIT gene had no significant differences according to molecular subtypes. Loss of Fhit expression was associated with large tumor size, basal-like subtype and positive expression of EGFR. In HER2-negative breast cancers, loss of Fhit expression was significantly associated with tumor size, estrogen receptor status and Ki-67 proliferation index. There was no significant correlation between methylation of the FHIT gene and its expression in this study. Conslusion: Our study revealed that loss of Fhit expression in breast cancer is associated with poor prognostic features, although there is no significance association between the FHIT gene methylation and Fhit expression. We found that in HER2-negatvie breast cancers, loss of Fhit expression was associated with poor prognostic features. These results support the possibility of potential complementation between HER2 and the Fhit pathway. The clinical significance of our findings needs to be further evaluated in larger cohorts with longer follow-up. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-06-05.

Fhit Deficiency-Induced Global Genome Instability Promotes Mutation and Clonal Expansion

Loss of Fhit expression, encoded at chromosome fragile site FRA3B, leads to increased replication stress, genome instability and accumulation of genetic alterations. We have proposed that Fhit is a genome ‘caretaker’ whose loss initiates genome instability in preneoplastic lesions. We have characterized allele copy number alterations and expression changes observed in Fhit-deficient cells in conjunction with alterations in cellular proliferation and exome mutations, using cells from mouse embryo fibroblasts (MEFs), mouse kidney, early and late after establishment in culture, and in response to carcinogen treatment. Fhit -/- MEFs escape senescence to become immortal more rapidly than Fhit +/+ MEFs; -/- MEFs and kidney cultures show allele losses and gains, while +/+ derived cells show few genomic alterations. Striking alterations in expression of p53, p21, Mcl1 and active caspase 3 occurred in mouse kidney -/- cells during progressive tissue culture passage. To define genomic changes associated with preneoplastic changes in vivo, exome DNAs were sequenced for +/+ and -/- liver tissue after treatment of mice with the carcinogen, 7,12-dimethylbenz[a]anthracene, and for +/+ and -/- kidney cells treated in vitro with this carcinogen. The -/- exome DNAs, in comparison with +/+ DNA, showed small insertions, deletions and point mutations in more genes, some likely related to preneoplastic changes. Thus, Fhit loss provides a ‘mutator’ phenotype, a cellular environment in which mild genome instability permits clonal expansion, through proliferative advantage and escape from apoptosis, in response to pressures to survive.

Activation of Gαq subunits up-regulates the expression of the tumor suppressor Fhit

The tumor suppressor Fhit protein is defective or absent in many tumor cells due to methylation, mutation or deletion of the FHIT gene. Despite numerous attempts to unravel the functions of Fhit, the mechanisms by which the function and expression of Fhit are regulated remain poorly understood. We have recently shown that activated Gαq subunits interact directly with Fhit and enhance its inhibitory effect on cell growth. Here we investigated the regulation of Fhit expression by Gq. Our results showed that Fhit was up-regulated specifically by activating Gα subunits of the Gq subfamily but not by those of the other G protein subfamilies. This up-regulation effect was mediated by a PKC/MEK pathway independent of Src-mediated Fhit Tyr114 phosphorylation. We further demonstrated that elevated Fhit expression was due to the specific regulation of Fhit protein synthesis in the ribosome by activated Gαq, where the regulations of cap-dependent protein synthesis were apparently not required. Moreover, we showed that activated Gαq could increase cell–cell adhesion through Fhit. These findings provide a possible handle to modulate the level of the Fhit tumor suppressor by manipulating the activity of Gq-coupled receptors.

Promoter methylation status of the FHIT gene and Fhit expression: Association with HER2/neu status in breast cancer patients

Aberrant DNA methylation has been recognized to contribute to breast carcinogenesis, and promoter hypermethylation of several tumor suppressor genes has been correlated with decreased gene expression. The fragile histidine triad (FHIT) gene is a putative tumor suppressor gene in breast and other types of cancer, and loss of Fhit expression has been observed in breast cancer. The aim of the present study was to evaluate the association between methylation of the FHIT gene and its expression in breast cancer, and to investigate whether methylation and expression of the FHIT gene correlates with clinicopathological characteristics in relation to human epidermal growth factor receptor 2 (HER2) status. Pyrosequencing of bisulfite-treated DNA was performed to study the methylation status of the FHIT gene in 60 breast cancer samples. We examined the expression of Fhit using tissue microarrays by immunohistochemical staining. FHIT methylation was detected in 96.7% and the positive expression rate of Fhit was 87.3% of the patients. The mean methylation level of the FHIT gene was associated with intratumoral inflammation. Methylation level of the FHIT gene had no significant differences according to molecular subtypes. Loss of Fhit expression was associated with large tumor size, basal-like subtype and positive expression of EGFR. In HER2-negative breast cancer, loss of Fhit expression was significantly associated with tumor size, estrogen receptor status and Ki-67 proliferation index. No significant correlation between methylation of the FHIT gene and its expression was observed in the present study. Our results suggest that loss of Fhit expression in breast cancer is associated with poor prognostic features, and it is also relevant to the results in HER2-negative breast cancer. Further studies with larger sample sizes and longer follow-up are required to clarify the predictive and prognostic value of Fhit expression and the FHIT gene methylation status in breast cancer.

P2.187 Leucine-Rich Immunoglobulin-Like Repeats (LRIG) 1, 2 and 3 in Cervical Neoplasia

Leucine-rich immunoglobulin-like repeats (LRIG) 1, 2 and 3 in cervical neoplasia Background Cervical neoplasms; invasive cancer and intraepithelial neoplasia (CIN), are sexually transmitted infections, HPV infection is the main etiological agent. Defining factors that are correlated to increased risk, diagnosis, prognosis and other clinical features are important. Methods 129 invasive cervical cancers in stages IB to IV, 47 cases of high grade CIN, 59 cases of low-grade CIN and 64 biopsies from normal epithelium were consecutively recruited. The cervical biopsies were evaluated for LRIG expression, and a total of 15 other relevant biological tissue markers (tumour markers) in invasive cancer and CIN. A structured questionnaire, and serum estradiol and progesterone were included. Results In early stages of invasive cancer LRIG 1 expression correlated to a favourable prognosis (90% vs. 64% survival), while the reverse was true for LRIG 2 expression (60% vs. 87% survival). Low expression of LRIG 1 and high for LRIG 2 indicated a very poor prognosis (26% vs. 66%). LRIG 3 expression had no impact on prognosis. Smoking and high serum progesterone correlated to absence of LRIG 1 expression. In CIN both LRIG 1 and LRIG 2 expression increased with increasing severity of the lesion. There was a correlation between LRIG 3 expression and HPV infection as well as three tumour suppressors (Rb, p53 and p16) and use of progestogenic contraceptives, whereas LRIG 2 correlated negatively to Rb. Both LRIG 1 and LRIG 2 correlated to expression of tumour suppressor FHIT. Conclusion There seems to be biological roles for LRIG 1, LRIG 2 and LRIG 3 in HPV-associated cervical neoplasia. In invasive lesions LRIG 1 is associated with suppression, LRIG 2 with progression of the tumour, while the role of LRIG 3 remains obscure. In CIN LRIG expression correlates to a number of events associated with outcome.

Expression of WWOX and FHIT is downregulated by exposure to arsenite in human uroepithelial cells

Ecological studies in Taiwan, Chile, Argentina, Bangladesh, and Mexico have confirmed significant dose-dependent associations between ingestion of arsenic-contaminated drinking water and the risk of various human malignancies. The FHIT and WWOX genes are active in common fragile sites FRA3B and FRA16D, respectively. Reduced expression of FHIT or WWOX is known to be an early indicator of carcinogen-induced cancers. However, the effect of arsenite on the expressions and molecular mechanisms of these markers is still unclear. The aims of this study were (i) to observe the expression of ATR, WWOX and FHIT proteins in urothelial carcinoma (UC) between endemic and non-endemic areas of blackfoot disease (BFD) by immunohistochemical analyses; (ii) to compare expression of these genes between arsenite-treated SV-HUC-1 human epithelial cells and rat uroepithelial cells; and (iii) to determine the role of DNMT and MEK inhibitors on expressions of WWOX and FHIT in response to arsenite in SV-HUC-1. The experiments revealed that expressions of ATR, WWOX and FHIT in UC significantly differed between BFD areas and non-BFD areas (p=0.003, 0.009 and 0.021, respectively). In fact, the results for the arsenite-treated groups showed that ATR, WWOX and FHIT are downregulated by arsenite in SV-HUC-1. However, the inhibitors suppressed the effects of arsenite on WWOX and FHIT proteins and mRNA expression. In conclusion, arsenite decreased expressions of ATR, WWOX and FHIT via ERK1/2 activation in SV-HUC-1 cells. These findings confirm that dysregulations of these markers may contribute to arsenite-induced carcinogenesis.

Chromosome Fragile Sites Signature and Genome Instability in Human Epithelial cells

Common chromosome fragile sites (CFSs) are highly sensitive to genotoxic stress and perturbation of replication, resulting in deletions within genes located at these sites. CFSs at 3p14.2, 16q23, 4q, 7q, and 6q are the most frequently expressed in lymphoblasts but CFSs have not yet been mapped in epithelial cells. We are defining CFSs in epithelial cells derived from breast tissue to determine if they represent the sites most frequently altered in cancer cells of epithelial organs from which most human cancers derive. We observed that in MCF10A noncancerous breast epithelial cells, chromosome regions 16q23, 2q, 12q, 1p most frequently exhibited chromosome breaks typical of CFS after aphidicolin treatment. This suggests that, as observed for cells of fibroblast origin, CFSs in epithelia are epigenetically determined. In parallel, we have focused on the role of Fhit protein, encoded at the CFS at 3p14.2, on chromosome stability and association with nucleotide pyrimidine pool imbalance. We investigated chromosome instability in epithelial cells deficient for Fhit expression vs Fhit‐expressing cells and observed increased aneuploidy, sister chromatid exchanges, chromosome breaks and gaps in Fhit‐deficient cells. Moreover, Fhit deficiency leads to thymidine kinase 1 down regulation, resulting in pyrimidine pool disequilibrium and accounting, in part, for genome instability.

Promoter methylation of DAPK1, FHIT, MGMT, and CDKN2A genes in cervical carcinoma

Aberrant DNA methylation contributes to the malignant phenotype in virtually all types of human cancer. This study explored the relationship between promoter methylation and inactivation of the DAPK1, FHIT, MGMT, and CDKN2A genes in cervical cancer. The promoter methylation of DAPK1, FHIT, MGMT, and CDKN2A was investigated by using a methylation-specific polymerase chain reaction in 53 specimens of cervical cancer (42 squamous cell carcinoma, 11 adenocarcinoma), 22 specimens of intraepithelial neoplasia tissues, and 24 control normal cervical tissue specimens. The correlation of promoter methylation with the clinicopathological features of cervical cancer was analyzed. The expressions of DAPK1, FHIT, MGMT, and CDKN2A were detected by measuring relative mRNA levels. The promoter methylation of DAPK1, FHIT, MGMT, and CDKN2A in cervical cancer vs. intraepithelial neoplasia vs. normal cervical tissue was 75.5 vs. 31.8 vs. 4.2 % (p < 0.0001), 66.0 vs. 59.1 vs. 25.0 % (p = 0.0033), 34.0 vs. 27.3 vs. 20.8 % (p = 0.76), and 17.0 vs. 31.8 vs. 8.3 % (p = 0.11), respectively. The methylation of the promoter region significantly decreased the expression of only DAPK1 (p = 0.03). The methylation rate of the DAPK1 gene promoter was significantly higher in cervical cancer tissues than in cervical intraepithelial neoplasia and normal cervical tissues. Promoter methylation may therefore lead to the inactivation of the DAPK1 gene, and may be related to the progression of cervical oncogenesis.