Promoter hypermethylation of TMS1, BRCA1, ERα and PRB in serum and tumor DNA of invasive ductal breast carcinoma patients

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Promoter hypermethylation of TMS1, BRCA1, ERα and PRB in serum and tumor DNA of invasive ductal breast carcinoma patients

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  Promoter hypermethylation of   TMS1, BRCA1, ER α  and  PRB  in serum andtumor DNA of invasive ductal breast carcinoma patients Sameer Mirza  a  , Gayatri Sharma  a  , Chandra P. Prasad  a  , Rajinder Parshad  b , Anurag Srivastava  b ,Siddartha Dutta Gupta  c , Ranju Ralhan  a, ⁎ a   Department of Biochemistry, All India Institute of Medical Sciences, Ansari Nagar, New Delhi 110029, India  b  Department of Surgery, All India Institute of Medical Sciences, Ansari Nagar, New Delhi 110029, India c  Department of Pathology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi 110029, India Received 21 February 2007; accepted 14 May 2007 Abstract Breast cancer is fast emerging as the leading cancer amongst females, especially in younger age group in India; a large proportion of thesetumors are often aggressive and ER and/or PR negative. Promoter methylation of genes involved in DNA repair and hormonal regulation may, in part, account for this behavior. To test this hypothesis methylation status of tumor suppressor genes  TMS1 ,  BRCA1 ,  ER α  and  PRB  was determinedin invasive ductal carcinoma of breast and paired serum DNA. Of the 50 breast cancer patients investigated, 36/50 (72%) tumors and 32/50 (64%) paired sera showed methylation of at least one of these genes, while 17/50 (34%) tumors and 12/50 (24%) sera showed methylation of three genes.Methylation frequencies ranged from 24% for   TMS1  to 63% for   ER α . Significant association was observed between  ER α  and  PRB  methylation(  p ≤ 0.001) and there was concordance between DNA methylation in tumor and serum for each gene. Immunohistochemical analysis showed nodetectable expression of ER  α , PRB and BRCA1 in 21/36 (58%), 20/36 (56%) and 23/36 (64%) tumors respectively; significant correlation wasobserved between promoter methylation and loss of protein expression confirming our hypothesis that promoter methylation is an important mechanism for transcriptional silencing of these genes in breast cancer in this population. This study also underscores the potential utility of DNAmethylation based screening of serum (a surrogate for tumor DNA methylation) as a tool for detection of breast cancer.© 2007 Elsevier Inc. All rights reserved.  Keywords:  Methylation; ER  α ; TMS1; BRCA1; PRB Introduction Breast cancer is the third most common cancer in the world,accounting for an annual incidence of 1.05 million new cases,representing over 20% of all malignancies among females(Parkin et al., 2001). It is fast emerging as the leading cancer among females in India, especially in the younger age group(Chopra, 2001). Often, these tumors are Estrogen receptor (ER)and/or Progesterone receptor (PR) negative; their mechanistic basis remains elusive. Methylation of promoter CpG islands,CG-rich regions that coincide with the promoters of protein-coding genes, is an important mechanism of gene inactivation inhuman cancers, including breast cancer. Many cellular path-ways, such as DNA repair, are inactivated by this type of epigenetic lesion, resulting in mutator pathways (Jacinto andEsteller, 2007). The clinical relevance of promoter hypermethy-lation in diagnosis/prognosis and treatment of breast cancer isunder intense investigation. In this context, our group hasdetermined the clinical significance of promoter methylationstatus of several key genes involved in cell proliferation intumor and circulating DNA in breast cancer patients (Shuklaet al., 2006; Bagadi et al., 2006; Sharma et al., 2007).This study was designed to investigate the promoter methylation status of   BRCA1 ,  TMS1, ER α  and  PRB  genes,with the goal of determining the clinical significance of their  Life Sciences 81 (2007) 280 – 287www.elsevier.com/locate/lifescie ⁎  Corresponding author. Tel.: +91 11 2659 3478; fax: +91 11 26588663.  E-mail addresses:  ralhanr@rediffmail.com, ralhanr@hotmail.com(R. Ralhan).0024-3205/$ - see front matter © 2007 Elsevier Inc. All rights reserved.doi:10.1016/j.lfs.2007.05.012  epigenetic silencing in breast tumsrcenesis. The rationale for choice of these genes for promoter methylation analysis is basedon their importance in breast carcinogenesis.The  familial breast cancer gene 1 (BRCA1)  is an important component of the cellular DNA repair machinery; its inactiva-tion by promoter methylation is being increasingly implicated inmany human cancers, including sporadic breast cancers. The  BRCA1  gene, originally cloned as the gene responsible for familial breast cancers (Miki et al., 1994), encodes amultifunctional protein involved in DNA repair, cell cyclecheck point control, protein ubiquitinylation and chromatinremodeling (Ralhan et al., 2006).  BRCA1 -associated familial breast cancers are more likely to occur at an early age, char-acterized by poor histological differentiation, aneuploidy, highS-phase fraction, and hormone receptor negativity; though  BRCA1 hasbeensuggestedtocontributetonon-hereditaryformsofbreastcanceraswell(Arveretal.,2000).Hypermethylationof   BRCA1  promoter is one of the mechanisms for its functionalinactivation, reported to occur in 7% to 31% of sporadic breast and ovarian cancers (Catteau and Morris, 2002). Furthermore,sporadic tumors with aberrant methylation of the  BRCA1  promoter have been shown to be clustered with tumors derivedfromwomenwithinherited  BRCA1 germlinemutationsbecauseofsimilaritiesintheirglobalgeneexpressionprofiles(van'tVeer et al., 2002).Estrogen receptor   α  (ER  α ), a ligand-activated transcriptionfactor, mediates the action of estrogen and is strongly implicatedin the development and progression of breast cancer (Anderson,2002). Loss of ER- α  expression has been associated withaberrant CpG island hypermethylation in breast cancer cell linesand tumors (Leu et al., 2004; Giacinti et al., 2006) and shown tomodulate breast cancer progression (Shinozaki et al., 2005).Diverse DNA methylation and gene expression profiles have been shown to correlate with differential adaptation of breast cancer cells to the antiestrogens, tamoxifen and fulvestrant (Fanet al., 2006).Progesterone receptor (PR) is an estrogen regulated protein;its synthesis in normal and cancer cells requires estrogen andER. CpG islands within the estrogen-regulated  PR  gene are not methylated in normal breast tissue but have been shown to behypermethylated in 40% of PR-negative primary tumors(Lapidus et al., 1996). Furthermore, a large proportion of breast cancers in Indian women are ER and/or PR negative; whether this loss of ER/PR can be attributed to promoter methylationmediated gene silencing remains an enigma.Target of methylation induced silencing-1  (TMS1),  identifiedas a target of methylation-mediated silencing in breast cancer cells, is a candidate tumor suppressor gene proposed to play pivotal role in regulation of apoptosis, inflammatory signaling pathwaysandimmuneresponsepathways(BertinandDiStefano,2000; McConnell and Vertino, 2004). Its clinical significance in breast cancer needs remains elusive. In view of the importanceof hormonal regulation and DNA repair gene defects in breast tumsrcenesis, we investigated the promoter methylation statusof   BRCA1 ,  ER α  , PRB  and  TMS1  genes in tumors and serumderived circulating DNA of breast cancer patients, to determinetheconcordancebetweentumorandcirculatingDNAmethylationand correlate with clinical parameters for determining their di-agnostic significance and utility as a surrogate for tumor meth-ylation in breast cancer patients. In addition, expression of these proteins was assessed in the tumors to determine if promoter methylation correlated with loss of protein expression in breast carcinomas. Materials and methods Tissue specimens Surgically resected specimens from untreated primary breast carcinomas, matched normal tissues, trucut biopsies and peripheral blood samples were collected from 50 breast cancer  patients enrolled in the Out Patients Department of SurgicalDisciplines, All India Institute of Medical Sciences, New Delhi,India, after approval of the study by Institutional Human EthicsCommittee. Written consent was taken from all the patientsenrolled in the study. The age of the patients ranged from 30 to81 years (median age 50 years). All the patients were diagnosedwith Invasive Ductal Carcinoma, the most common type foundin Delhi (Saxena et al., 2005). 10 ml blood was collected from breast cancer patients at the time of surgery. Blood was alsocollected from 5 healthy females (age range 24 – 50 years); 2 of these women were pre-menopausal and 2 were post-menopausal;1 was nulliparous. All these five healthy females did not havehistory of any chronic disease and had no evidence of disease at the time of enrollment in the study. Serum was isolated fromclotted blood by centrifugation at 1000 ×  g   for 10 min. A part of each tumor and representative normal tissue was kept in formalinfor histopathological characterization to confirm the diagnosisand immunohistochemistry and the other part was snap frozenand stored at  − 70 °C. Cell culture Human breast cancer cell lines MCF-7, MDA-MB-231 andMDA-MB-157 were obtained from National Centre for CellScience, Pune, India and American Type Culture Collection(ATCC) respectively. Cells were grown as monolayer culturesin Dulbecco's modified Eagles Medium (DMEM) (SigmaAldrich, Bangalore, India) supplemented with 10% fetal bovineserum (Sigma Aldrich) at 37 °C in a humidified atmosphere of 95% air and 5% carbon dioxide. Sub-confluent cultures wereroutinely passaged by trypsinization. Cells in log phase wereharvested and DNA was extracted as described previously(Shukla et al., 2006).  DNA extraction DNA was extracted from breast tumor/normal tissue or  breast cancer cell lines MCF-7, MDA-MB-231, MDA-MB-157using standard technique of digestion with proteinase K in the presence of SDS at 37 °C overnight, followed by phenol/ chloroform extraction as described previously (Sambrook andRussell, 2001). Serum DNA was extracted using Qiamp DNABlood Mini Kit (Qiagen, Hilden, Germany) according to 281 S. Mirza et al. / Life Sciences 81 (2007) 280  –  287   manufacturers' instructions. The quality and integrity of DNAfrom tissues was checked by electrophoresis on 0.8% agarosegel, quantitated spectrophotometrically and stored at  − 20 °C tillfurther use. Placental DNA was treated with Sss1 methylaseusing the manufacturer's recommendations (New EnglandBiolabs, Hertfordshire, UK.). This treated DNA was used as a positive control for methylation.  Methylation-specific PCR Bisulphite modification of the DNA (up to 2  μ g) fromvarious sources was carried out as described (Frommer et al.,1992). Primers and PCR conditions used for Methylation-Specific PCR (MSP) of   ER α ,  PRB ,  TMS1  and  BRCA1 (promoter   α ) are from published studies and have beenstandardized in our laboratory (Conway et al., 2000; Lapiduset al., 1996; Sasaki et al., 2001; Esteller et al., 2000). For  positive and negative controls of the MSP, a breast cancer cellline (MCF-7, MDA-MB-231, MDA-MB-157) or tumor withknown hypermethylation as a positive control, normal lympho-cyte and normal breast tissue DNA as negative controls andwater with no DNA template as a control for contaminationwere included in each experiment. After amplification, eachPCR product was electrophoresed using a 2% agarose gel,stained with ethidium bromide, and visualized under UVillumination. Fig. 1. MSP analysis of   TMS1 ,  BRCA1 ,  ER α and  PRB  genes in breast tumors and serum DNA. Panel A.  TMS1  panel viewed from left to right shows a 50-bp ladder asmolecular weight marker, a water control for contamination in the PCR reaction, patient 1 shows presence of both methylated and unmethylated DNA in tumor andcorresponding sera, patient 2shows presence of unmethylated DNA detectedin both tumorandcorrespondingsera.Breast cancer cell line MDA-231used as a positivecontrol shows methylated DNA. MCF-7 used as a positive control for unmethylated DNA, normal breast tissue used as another negative control also showsunmethylated DNA. Panel B. BRCA1 viewed from left to right shows a 50-bp ladder as molecular weight marker, a water control for contamination in the PCR reaction, patient 1 shows presence of methylated DNA in tumor and corresponding sera, patient 2 shows presence of unmethylated DNA detected in both tumor andcorresponding sera, Sss1 treated placental DNA used as a positive control for methylated DNA. Breast cancer cell line MCF-7 DNA used as a positive control for unmethylated DNA, normal breast tissue used as another negative control also shows unmethylated DNA. Panel C.  ER α  panel viewed from left to right shows a 50-bpladder as molecular weight marker, a water control for contamination in the PCR reaction, patient 1 shows presence methylated DNA in tumor and corresponding sera, patient 2 shows presence of both methylated and unmethylated DNA detected in both tumor and corresponding sera. Breast cancer cell line MDA-231 used as a positive control shows methylated DNA. MCF-7 used as a positive control for unmethylated DNA, normal breast tissue used as another negative control also showsunmethylated DNA. Panel D.  PRB  panel viewed from left to right shows a 50-bp ladder as molecular weight marker, a water control for contamination in the PCR reaction,patient1 methylatedDNA isdetectedinbothtumorandcorresponding serapatient2showspresenceof bothmethylatedandunmethylatedDNA intumorandcorresponding sera. Breast cancer cell line MDA-231 used as a positive control shows methylated DNA. T-47D used as a positive control for unmethylated DNA,normal breast tissue used as another negative control also shows unmethylated DNA.282  S. Mirza et al. / Life Sciences 81 (2007) 280  –  287    Immunohistochemistry Monoclonal antibodies were purchased against BRCA1(OP-92) from Oncogene Research, Darmstadt, Germany; PR (A0098) from Dako Cytomation, Glostrup, Denmark and ER  α (sc-8005) from Santa Cruz Biotechnology Inc. (Santa Cruz,CA). Immunohistochemical analysis was carried out using paraffin embedded tissue sections as described by us (Sharmaet al., 2007). Briefly, tissue sections were deparaffinized inxylene, hydrated and incubated with 0.3% (v/v) H 2 0 2  inmethanol for 45 min, to inactivate the endogenous peroxidases.Antigen was retrieved by microwave treatment for 15 min in0.01 M citrate buffer (pH 6.0), sections were incubated with primary antibody (1:50 dilution) at 4 °C overnight. Thereafter,sectionswereincubatedwithbiotinylatedanti-mouseanti-serumand subsequently with horse-radish peroxidase-streptavidinconjugate (Dako Cytomation, Glostrup, Denmark), followed by color development using 3, 3 ′ -diaminobenzidine hydrochlo-ride (DAB) as chromogen, counterstained with Mayer'shematoxylin and mounted for evaluation using microscope(NIKON microphot-FXA, Japan). In the negative control, primary antibody was replaced by isotype-specific IgG. Incase of ER and PR only nuclear staining was considered asimmunopositivity and tumors showing  N 10% cells with nuclear immunopositivity were considered as ER/PR positive. For BRCA1 protein expression nuclear immunostaining wasconsidered as positive. The slides were scored following thecriteria: negative  b 10% tumor cells showing immunoreactivity,+ ≥ 10 – 30% tumor cells showing immunoreactivity, ++ ≥ 30 – 50% tumor cells showing immunoreactivity, ++ N  50% tumor cells showing immunoreactivity. Statistical analysis Statistical analysis of correlation of methylation of genes and protein expression as well as with known histopathologiccharacteristics was performed with Fisher exact test (2 sided)usingSPSSv.10.  p valuelessthan0.05wasconsideredsignificant. Results Promoter methylation analysis of   TMS1 ,  BRCA1 ,  ER α  and  PRB  was carried out in 50 invasive ductal carcinomas of the breast and paired serum DNA and of these patients. Methylationanalysis of these genes was also carried out in 5 normal seracollected from healthy females as well as in 5 adjacent normaltissues collected from breast cancer patients. Fig. 1 showsrepresentative methylation status of   TMS1 ,  BRCA1 ,  ER α  and  PRB  in invasive breast carcinomas and paired serum DNA.Overall 36/50 (72%) tumors and 32/50 (64%) paired serashowed hypermethylation of at least one of these genes(Table 1). Only 1 case showed biallelic promoter methylationof all the 4 genes; 17/50 (34%) tumors and 12/50 (24%) serashowed methylation of at least 3 of the 4 genes investigated.Immunohistochemical analysis of BRCA1, ER  α  and PR  proteins was carried out to determine the effect of promoter methylation on gene silencing and consequent loss of proteinexpression in 36 cases; 14 of 36 cases were specimens obtainedfrom Trucut biopsies and could not be used for immunohisto-chemistry due to limited amount of tissue. For a feasibility of the study, it is important that the target genetic alteration iscancer specific and is not present in normal cells. Therefore, as acontrol, we checked methylation status of these genes in five paired normal breast tissues and sera collected from healthyfemales. None of the 5 normal breast tissues analyzed showedmethylation in any of these 4 genes. Sera from normal healthyfemales also did not show methylation in any of these 4 genesanalyzed (data not shown).  Analysis of TMS1 methylation in breast cancer  Hypermethylation of   TMS1  promoter was detected in 12/50(24%) tumors; 9 tumors had both methylated alleles and these 9 patients showed methylation of   TMS1  in the paired sera as well(Table 1). Thus, concordant   TMS1  methylation was observed inall the 9 tumors showing biallelic methylation and their paired Table 1 TMS1, BRCA1, ER α  ,  and  PRB  hypermethylation in tumor and serum of breast cancer patientsGenes Methylation status Tumor   N   (%) Serum  N   (%)  ER  U 17 (34) 26 (52)U/M 21 (42) 13 (26)M 12 (24) 11 (22)  PR  U 18 (36) 27 (54)U/M 19 (38) 12 (24)M 13 (26) 11 (22)  BRCA1  U 37 (74) 39 (78)U/M 1 3M 12 (24) 8 (16) TMS1  U 38 (76) 41 (82)U/M 3 0M 9 9 Note: M, methylated DNA; U/M, heterogeneous DNA; U, unmethylated DNA; N, number of patients analysed is 50; U/M is considered as methylated (M) for the analysis.Table 2BRCA1, ER  α , and PR protein expression in invasive ductal carcinoma patientsGenes Genes methylated (  N  =36) Protein expression (  N  =36)MethylationstatusTumor   N   (%)Serum  N   (%)Proteinexpression  N   (%)Loss of proteinexpression  N   (%)  ER  U 12 (33) 20 (55) 9 3U/M 15 (42) 7 5 10 (28)M 9 9 8 1  PR  U 14 (39) 20 (55) 10 (28) 4U/M 12 (34) 7 3 9M 10 (27) 9 3 7  BRCA1  U 27 (75) 27 (75) 13 (36) 14 (39)U/M 0 3 0 0M 9 6 0 9 TMS1  U 30 (84) 32 (89) ND NDU/M 2 0 ND NDM 4 4 ND ND Note: M, methylated DNA; U/M, heterogeneous DNA; U, unmethylated DNA; N, number of patients analysed is 36; ND, not determined; U/M is considered asmethylated (M) for the analysis.283 S. Mirza et al. / Life Sciences 81 (2007) 280  –  287   sera. However, 3 of 12 tumors showed heterogeneousmethylation and the methylated allele could not be detected inthe sera of these 3 patients, probably due to low levels incirculating DNA. Ten of these 12 breast cancers withmethylated  TMS1  promoter also showed methylation of   ER α and  PRB  promoters and 7 of these were negative for ER proteinexpression.  TMS1  methylation is associated with reduction or loss of ER protein expression (  p =0.027).  TMS1  methylationwas also associated with advanced tumor stage (  p =0.044).  BRCA1 methylation and immunostaining analysis in breast cancer  BRCA1  was observed to be methylated in 13 of 50 (26%)tumors and 11 of 50 (22%) paired sera of breast cancer patients(Table 1). Immunohistochemical analysis showed loss or markedly reduced expression of BRCA1 protein in 23 of 36(64%) cases (Table 2). Of the 13 tumors showing promoter hypermethylation, 10 cases were analyzed for BRCA1 protein Fig. 2. Immunohistochemical analysis of BRCA1, ER  α , PR protein expression in breast tissues Paraffin embedded tissue sections from invasive ductal carcinomas of  breast and normal tissues were used for immunohistochemical analysis of BRCA1 proteins(A – C), ER  α  protein(D – F)and PR protein (G – I).The photomicrographshows A) normal breast tissue showing nuclear expression of BRCA1 protein; B) invasive ductal carcinoma of breast depicting nuclear expression of BRCA1 protein;C) invasive ductal carcinoma of breast illustrating no detectable BRCA1 immunopositivity; D) normal breast tissue showing nuclear expression of ER  α  protein;F) invasive ductal carcinoma of breast depicting nuclear expression of ER  α  protein; G) normal breast tissue showing nuclear expression of PR protein; H) invasiveductal carcinoma of breast patient depicting nuclear expression of PR protein; I) invasive ductal carcinoma of breast patient illustrating no detectable PR immunopositivity. A – I, srcinal magnification ×200.284  S. Mirza et al. / Life Sciences 81 (2007) 280  –  287 
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