title: Simultaneous CXCL12 and ESR1 CpG island hypermethylation correlates with poor prognosis in sporadic breast cancer.
abstract: BACKGROUND
  CXCL12 is a chemokine that is constitutively expressed in many organs and tissues. CXCL12 promoter hypermethylation has been detected in primary breast tumours and contributes to their metastatic potential. It has been shown that the oestrogen receptor alpha (ESR1) gene can also be silenced by DNA methylation. In this study, we used methylation-specific PCR (MSP) to analyse the methylation status in two regions of the CXCL12 promoter and ESR1 in tumour cell lines and in primary breast tumour samples, and correlated our results with clinicopathological data.
  
  
  METHODS
  First, we analysed CXCL12 expression in breast tumour cell lines by RT-PCR. We also used 5-aza-2'-deoxycytidine (5-aza-CdR) treatment and DNA bisulphite sequencing to study the promoter methylation for a specific region of CXCL12 in breast tumour cell lines. We evaluated CXCL12 and ESR1 methylation in primary tumour samples by methylation-specific PCR (MSP). Finally, promoter hypermethylation of these genes was analysed using Fisher's exact test and correlated with clinicopathological data using the Chi square test, Kaplan-Meier survival analysis and Cox regression analysis.
  
  
  RESULTS
  CXCL12 promoter hypermethylation in the first region (island 2) and second region (island 4) was correlated with lack of expression of the gene in tumour cell lines. In the primary tumours, island 2 was hypermethylated in 14.5% of the samples and island 4 was hypermethylated in 54% of the samples. The ESR1 promoter was hypermethylated in 41% of breast tumour samples. In addition, the levels of ER alpha protein expression diminished with increased frequency of ESR1 methylation (p < 0.0001). This study also demonstrated that CXCL12 island 4 and ESR1 methylation occur simultaneously at a high frequency (p = 0.0220).
  
  
  CONCLUSIONS
  This is the first study showing a simultaneous involvement of epigenetic regulation for both CXCL12 and ESR1 genes in Brazilian women. The methylation status of both genes was significantly correlated with histologically advanced disease, the presence of metastases and death. Therefore, the methylation pattern of these genes could be used as a molecular marker for the prediction of breast cancer outcome.
authors.affiliation: Department of Basic Pathology, Federal University of Parana, Curitiba, Brazil.
authors.forename: Edneia A S
authors.initials: EA
authors.lastname: Ramos
authors.forename: Anamaria A
authors.initials: AA
authors.lastname: Camargo
authors.forename: Karin
authors.initials: K
authors.lastname: Braun
authors.forename: Renata
authors.initials: R
authors.lastname: Slowik
authors.forename: Iglenir J
authors.initials: IJ
authors.lastname: Cavalli
authors.forename: Enilze M S F
authors.initials: EM
authors.lastname: Ribeiro
authors.forename: Fábio de O
authors.initials: Fde O
authors.lastname: Pedrosa
authors.forename: Emanuel M
authors.initials: EM
authors.lastname: de Souza
authors.forename: Fabrício F
authors.initials: FF
authors.lastname: Costa
authors.forename: Giseli
authors.initials: G
authors.lastname: Klassen
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D001943:Breast Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D054377:Chemokine CXCL12 / Q000096:biosynthesis / Q000235:genetics*; D018899:CpG Islands; D019175:DNA Methylation; D004252:DNA Mutational Analysis; D047628:Estrogen Receptor alpha / Q000096:biosynthesis / Q000235:genetics*; D005260:Female; D020868:Gene Silencing; D020022:Genetic Predisposition to Disease; D006801:Humans; D007150:Immunohistochemistry; D008875:Middle Aged; D011379:Prognosis; D011401:Promoter Regions, Genetic; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: C515255:CXCL12 protein, human; D054377:Chemokine CXCL12; C506487:ESR1 protein, human; D047628:Estrogen Receptor alpha