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. 2009 Jan;135(1):91-102.
doi: 10.1007/s00432-008-0435-x. Epub 2008 Aug 1.

Down-regulation of CXCL12 mRNA expression by promoter hypermethylation and its association with metastatic progression in human breast carcinomas

Wei Zhou  1 , Zheng Jiang, Ningbo Liu, Fenghua Xu, Peie Wen, Yanbing Liu, Weixia Zhong, Xianrang Song, Xiaotian Chang, Xiuli Zhang, Guangsheng Wei, Jinming Yu
Affiliations

Down-regulation of CXCL12 mRNA expression by promoter hypermethylation and its association with metastatic progression in human breast carcinomas

Wei Zhou et al. J Cancer Res Clin Oncol. 2009 Jan.

Abstract

Purpose: Not only is the expression of CXCR4 on breast cancers a key determinant of tumor metastasis, CXCL12 exhibiting peak levels of constitutive expression in organs representing the first destinations of cancer metastasis, but is proposed to be also essential for the organ-specific metastatic process.

Methods: In this study, the expressions of CXCR4 and CXCL12 were investigated using quantitative RT-PCR and immunohistochemistry in samples of 63 primary breast carcinomas and 20 normal breast tissues. Using methylation-specific PCR, we also analyzed the methylation status of CXCL12.

Results: Both up-regulation of CXCR4 and down-regulation of CXCL12 were observed in primary breast carcinomas. Over-expression of CXCR4 mRNA was significantly related to lymph node metastasis status and strong Her-2 expression, while decreased expression of CXCL12 mRNA was significantly associated with positive lymph node metastasis and estrogen receptor negativity. Methylation-specific PCR showed that 52.4% of breast tumors were hypermethylated in the CXCL12 promoter region. The expression levels of DNA methyltransferase (DNMT) 1 and DNMT3B were significantly higher in the CXCL12-methylated breast carcinomas than in the CXCL12-unmethylated ones.

Conclusions: In summary, DNA hypermethylation of CXCL12 plays an important role in the down-regulation of CXCL12 expression in breast carcinomas. Cancer cells lacking expression of CXCL12, but maintaining over-expression of CXCR4, can selectively spread to target organs in which the ligand is highly secreted.

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Figures

Fig. 1
Fig. 1
Expression and methylation analyses of CXCL12/CXCR4 in primary breast carcinomas and normal breast tissues. a, b Representative expression of CXCR4 and CXCL12 mRNA in primary breast carcinomas (T) compared to the normal breast tissues (N) by semi-quantitative RT-PCR. c Methylation analysis of CXCL12 in normal breast tissues (N) and primary breast carcinomas (T) by methylation-specific PCR. Lane U unmethylated, Lane M methylated. Data in (a–c) are representative of two independent experiments
Fig. 2
Fig. 2
Immunohistochemical analysis of CXCR4 and CXCL12 protein in primary breast carcinomas and normal breast tissues. a A normal breast sample showing negative CXCR4 expression. b A breast tumor sample showing moderate CXCR4 expression. c A breast tumor sample showing high CXCR4 expression. d A normal breast sample showing positive CXCL12 expression. e A CXCL12-unmethylated breast tumor showing positive CXCL12 expression. f A CXCL12-methylated breast tumor showing negative CXCL12 expression. Panels a–f, ×400
Fig. 3
Fig. 3
Over-expressions of DNA methyltransferase enzymes in CXCL12-methylated breast carcinomas as compared with in CXCL12-unmethylated ones. a Representative mRNA expressions of DNMT1, DNMT3A, and DNMT3B in the CXCL12-methylated (Lanes 1–6) and CXCL12-unmethylated (Lanes 7–12) breast carcinomas by semi-quantitative RT-PCR. GAPDH served as endogenous control. b Semi-quantification of expression levels of DNMT1, 3A and 3B relative to GAPDH in these 12 breast carcinoma samples. c Quantitative real-time PCR analysis of gene expression levels of DNMT1, 3A and 3B in the CXCL12-methylated and CXCL12-unmethylated breast carcinoma samples, as well as normal breast tissues. Statistically significant differences are denoted by brackets with P values. In addition, the transcript levels of DNMT3A (P = 0.025) and DNMT3B (P = 0.011) in the CXCL12-unmethylated astrocytomas were significantly higher than normal breast tissues
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