Aberrant CpG Islands Methylation Of WWOX Gene In Breast Cancer And Effect Of Hydralazine On Demethylation Of WWOX Gene In Breast Cancer Cells

The WWOX(WW domain containing oxidoreductase)gene is a tumor suppressor gene that is detected in chromosome 16q,spanning the common fragile site FRA16D. Aberrations of WWOX at either genome and expression level or both were common in various neoplasms including breast,ovarian,bladder,esophageal and prostate carcinomas.WWOX proteins contain two WW domains in the NH₂-terminal region and a short chain dehydrogenase/reduetase(SDR)domain in the central region of the protein.The WW domains are involved in protein-protein interactions.While the WWOX SDR domain encode an amino acid sequence homologous to steroid oxidoreductases,WWOX expression level in normal tissue was higher in hormonally regulated tissues,such as testis,prostate,and ovary,suggesting that WWOX may play an important role in hormones regulated cancers and that WWOX protein may be an enzyme involved in steroid metabolism.Usually CpG islands are located in the promoter,exon 1 and 3' side of DNA of tumors.Expression of many tumor suppressor genes is down-regulated in cancer by epigenetic mechanisms,and CpG-rich islands of gene promoter regions are frequently methylated in cancers.A role for epigenetic mechanisms of WWOX transcriptional regulation was suggested for hemopoietic and pancreatic neoplasms, and WWOX promoter methylation status was associated with lack of expression. Studies indicate that aberrant transcriptions and expression of tumor suppress gene contribute to the carcinogenesis of breast cancer.The potential mechanisms include allelic loss and/or loss of homozygosity,dysfunction of transcription of WWOX mRNA,and hypermethylation of CpG islands DNA regulatory sites.WWOX gene maps at the location of FRA16D,one of the most active common chromosomal fragile sites.The WWOX gene is similar to the FHIT gene in the both genes encompass very active common fragile sites,both genes show frequent allelic loss and/or loss of homozygous deletion region in several human cancers,and both frequently show aberrant transcripts.A recent study showed WWOX and FHIT expression was strongly correlated,suggesting that genes at common fragile sites are likely to be coordinately inactivation in cancer,and promoter hypermthylation including the position of site-specific methylation of WWOX gene may play an impotant role in pancreatic tumor development.The renaissance of methylation studies has generated considerable interests for cancer researchers because:(1)methylation of CpG islands in gene regulatory regions,in combination with chromatin remodeling,is involved in downregulation of expression of genes,including tumor suppressor genes;(2)methylation of specific CpG islands in easily detected in tissues and body fluids of individuals with cancer, or at high risk for cancer development,so that specific gene methylation patterns can be useful in diagnosis or prognosis;and(3)epigenetic and chromatin remodeling marks can be reversed by specific agents or inhibitiors,suggesting such inhibitors as therapeutic agents.Methylation of regulatory regions of many genes has been reported in cancer cells,but which of these methylation marks will be most useful in diagnostic or prognostic clinical trials,or as surrogate markers in preclinical/clinical prevention and therapy trials of specific cancers,is only beginning to be defined.In the present study,WWOX protein expression pattern was analyzed in patients with normal breast and in those with breast carcinoma.We correlated WWOX protein expression with staging of breast carcinoma and with a few clinicopathological parameters as well.And this study aimed to determine if DNA methylation is a mechanism of WWOX(WW domain containing oxidoreductase)inactivation in breast cancer and MAB-MD-231,MCF-7 cell line.The expression of WWOX mRNA was detected with RT-PCR in breast cancer cell lines and tissues.Methylation specific PCR(MSP)was used to check whether it was methylated in the promoter and exon 1 CpG island of WWOX in MDA-MB-231,MCF-7 cell lines and tissues.Furthermore, the effect of hydralazine on demetlrylation of WWOX gene regulatory region CpG islands was evaluated,and 5-Aza-dcA was used as positive control.Part 1 Expression of WWOX protein in breast cancer and cell linesObjective To determine the expression of WWOX protein in breast cancer tissues and cells,and evaluate the association between WWOX expression and clinicopathological features.Methods Expression of WWOX was determined by immunohistochemical(IHC) staining on 155 tissues which was comprised of specimens of 56 normal breast tissurs,12 ductal carcinoma in situ(DCIS)and 87 primary invasive carcinoma. Furthermore,expression of WWOX protein was evaluated by IHC on breast cancer cell lines,MDA-MB-231 and MCF-7 cells.Results Decreased or lost WWOX expression was observed more frequently in invasive tumor(62.1%)than control(28.6%)(P=0.001).82.1%of ER negative tumors and 52.5%of ER positive tumors showed decreased or lost WWOX expression(P=0.026).22.4%of premenopausal tumors were completely negative for WWOX expression versus 51.8%for postmenopausal breast carcinomas(P=0.024). Furthermore,23.1%of Stageâ… (6/26),28.6%of Stageâ…¡(10/35)and 46.2%of Stageâ…¢(12/26)tumors showed negative WWOX expression(P=0.001). Immunohistochemical analysis showed a highly reduced WWOX staining in MDA-MB-231 cells and a moderately reduced WWOX reaction in MCF-7 cells.Conclusions Lost WWOX expression is a common event in breast cancer.The associations of WWOX expression with ER status,menopausal status and clinical stage of breast cancer respectively support the hypothesis that WWOX play a role in carcinogenesis and development of breast cancer in a pathway or pathways involved sex steroid metabolism.Part 2 Methylation of the WWOX promoter and exon 1 CpG islands in breast cancer tissue and breast cancer cellsObjective To evaluate CpG islands methylation status of WWOX gene in breast cancer and anlysis the association between methylation and WWOX mRNA expression.Methods The expression of WWOX mRNA was detected with RT-PCR in breast cancer cell lines and tissues.Methylation specific PCR(MSP)was used to check whether it was methylated in the promoter and exon 1 CpG island of WWOX in MDA-MB-231,MCF-7 cell lines and tissues,and normal breast tissues were used as control.Results The breast cancer cell lines MDA-MB-231 and specimens of breast cancers were found methylated in WWOX DNA promoter and exon 1 CpG island.In breast cancer specimens,methylation rate of promoter and exon 1 CpG island of WWOX gene was 55%and 45%respectively.Whereas,WWOX CpG islands of normal mammary tissues were completely unmethylated in all cases.CpG islands of WWOX promoter and exon 1 were methylated in MDA-MB-231 cell line but not MCF-7 cell.The methylated cell lines and breast cancer tissues expressed decreassed WWOX mRNA.DNA CpG islands methylation contributes to the tumor-specific silencing of WWOX mRNA expression.The expression of WWOX mRNA was decreased significantly in MDA-MB-231 cells and partly due to the methylation of WWOX CpG islands.Conclusions The disfigurement of WWOX gene plays an important role in the carcinogenesis of breast cancer,and this alteration is partially caused by methylation of WWOX DNA regulatery region CpG islands. Part 3 Effect of hydralazine on demethylation of WWOX gene promoter and exon 1 in breast cancer cellsObjective To evaluate the effect of hydralazien on demethylation of WWOX gene promoter and exon 1 in breast cancer cellsMethods The demethylating agent,hydralazine,was used to treat methylated breast cancer cells and then,breast cancer cell growth was measured in vivo,and 5-Aza-CdR was used as positive control.The expression of WWOX mRNA was detected with RT-PCR in breast cancer cell lines before and after demethylating treatment,and WWOX protein expression was evaluated by immunohistochemical staining. Furthermore,to study the effects of restoration of WWOX expression in breast cancer cells,cell proliferation was evaluated.Results After hydralazine treatmentm WWOX CpG islands of MDA-MB-231 cells were unmethylated.Hydralazine could reactivate the expression of WWOX mRNA in methylated MDA-MB-231 cells.Restoration of WWOX expression led to suppression of growth of Wwox-deficient breast cancer-derived cells,through activation of the intrinsic caspase pathway,but did not affect growth of Wwox-sufficient MCF7 cells.Conclusions Hydralazine could effectively cause demethylation and inhibit the growth of MDA-MB-231 cells by reactivation the gene transcription silenced by aberrant hypermethylation.There are no significant differences in the demethylation of methylated MDA-MB-231 cells,restoration of WWOX mRNA expression,and suppression of the growth of cell lines for 1.0μmol/L,2.0μmol/L,4.0μmol/L, 6.0μmol/L 5-Aza-CdR.Summary WWOX gene is a candidate tumor suppressor gene located on chromosome 16q23.3-24.1.Abnormalities of WWOX at genomic and/or expression levels have been reported in numerous neoplasias including breast cancer.Lost WWOX expression is a common event in breast cancer.The associations of WWOX expression with ER status,menopausal status and clinical stage of breast cancer respectively supported the hypothesis that WWOX played a role in carcinogenesis and development of breast cancer in a pathway or pathways involved sex steroid metabolism.The disfigurement of WWOX gene plays an important role in the development of ovarian cancer,and this alteration is partially caused by methylation of WWOX DNA.MDA-MB-231 was found methylated by MSP in WWOX DNA promoter and exon 1 CpG island and methylation rate of the promoter and exon 1 CpG islands was 55%and 45%in breast cancer specimens respectively.The methylated cell lines and breast cancer tissues expressed decreased WWOX mRNA. The expression of WWOX mRNA was decreased significantly in MDA-MB-231breast cancer cells.The WWOX mRNA decrease was partly due to the methylation of WWOX CpG islands.Hydralazine and 5-Aza-CdR could reactivate the expression of WWOX mRNA in methylated MDA-MB-231 cells.Restoration of WWOX expression led to suppression of growth of WWOX-deficient breast cancer-derived cells,through activation of the intrinsic caspase pathway,but did not affect growth of WWOX-sufficient MCF7 cells.The finding that the WWOX regulatory region was methylated in MDA-MB-231 cells suggested that WWOX might be restored in breast cancer cells by hydralazine treatment.The cardiovascular drugs have a promising tumor suppressor-reactivating action and could pontentially be used in clinic as an anticancer treatment,mostly likely to increase the efficacy of current biological or chemotherapeutic treatments.