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Distinct epigenetic changes in the stromal cells of breast cancers

Abstract

Increasing evidence suggests that changes in the cellular microenvironment contribute to tumorigenesis, but the molecular basis of these alterations is not well understood. Although epigenetic modifications of the neoplastic cells in tumors have been firmly implicated in tumorigenesis1, it is not known whether epigenetic modifications occur in the non-neoplastic stromal cells. To address this question in an unbiased and genome-wide manner, we developed a new method, methylation-specific digital karyotyping, and applied it to epithelial and myoepithelial cells, stromal fibroblasts from normal breast tissue, and in situ and invasive breast carcinomas. Our analyses showed that distinct epigenetic alterations occur in all three cell types during breast tumorigenesis in a tumor stage– and cell type–specific manner, suggesting that epigenetic changes have a role in the maintenance of the abnormal cellular microenvironment in breast cancer.

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Figure 1: Outline of MSDK protocol and its validation in HCT116 wild-type and DKO cells.
Figure 2: Graphs depicting the ratio and location of tags with unique match to the genome whose presence in MSDK libraries generated from different cell types isolated from normal and tumor tissue was significantly different (P < 0.05).
Figure 3: Genome-wide view of methylation and gene-expression patterns in breast cancer.
Figure 4: Results of sequence analysis of selected genes using bisulfite-treated genomic DNA.
Figure 5: qMSP analysis of selected differentially methylated genes.
Figure 6: qMSP analysis of PRDM14 and RT-PCR analysis of selected differentially methylated genes.

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Acknowledgements

We thank A. Richardson, G. Lodeiro and R. Gomes for their help with the acquisition of human tissue samples; R. Gelman for help with statistical analysis; B. Vogelstein and K. Kinzler for providing genomic DNA from HCT116 wild-type and DKO cells and their continuous support and encouragement; and B. Vogelstein, I. Krop and other members of the laboratory of K.P. for critical reading of the manuscript and their constructive criticism throughout the execution of this project. This work was supported by the National Cancer Institute Specialized Program in Research Excellence in Breast Cancer at Dana-Farber/Harvard Cancer Center, by Department of Defense Breast Cancer Center of Excellence grants awarded to K.P. and by a Department of Defense Postdoctoral Fellowship grant awarded to J.Y.

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Correspondence to Kornelia Polyak.

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M.H. and K.P. submitted a patent application on the MSDK method and the findings described in this manuscript, in accordance with the policies of the Dana Farber Cancer Institute.

Supplementary information

Supplementary Fig. 1

SNP array analysis of genomic DNA samples used for MSDK analysis. (PDF 139 kb)

Supplementary Fig. 2

Genome-wide overview of the location of CpG islands, predicted AscI sites overlayed with methylation (MDSK), and gene expression (SAGE) patterns. (PDF 196 kb)

Supplementary Table 1

Statistical analysis of MSDK libraries generated from HCT116 WT and DKO cells. (PDF 143 kb)

Supplementary Table 2

List of breast tissue samples used for methylation assays. (PDF 81 kb)

Supplementary Table 3

Statistical analysis of MSDK libraries generated from normal (N-EPI-7) and tumor (I-EPI-7) breast epithelial cells. (PDF 128 kb)

Supplementary Table 4

Statistical analysis of MSDK libraries generated from normal (N-STR-I7) and tumor (I-STR-7) breast stromal cells. (PDF 142 kb)

Supplementary Table 5

Statistical analysis of MSDK libraries generated from normal (N-MYOEP-4) and DCIS tumor (D-MYOEP-6) breast myoepithelial cells. (PDF 134 kb)

Supplementary Table 6

Statistical analysis of MSDK libraries generated from normal breast myoepithelial (N-MYOEP-4) and epithelial (N-EPI-I7) cells. (PDF 136 kb)

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Hu, M., Yao, J., Cai, L. et al. Distinct epigenetic changes in the stromal cells of breast cancers. Nat Genet 37, 899–905 (2005). https://doi.org/10.1038/ng1596

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