Abstract 2190: Epigenetic mechanisms of transformation for the NSD3 oncogene in human breast cancer cells with the 8p11-p12 amplicon

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL An amplified region of chromosome 8, the 8p11-p12 amplicon, is observed in 12-15% of breast cancers. This region contains 55 genes, of which 22 have been identified as candidate oncogenes based on the presence of higher than normal copy number and associated elevated expression levels in breast tumor cells compared to normal breast epithelial cells. Our lab previously identified the short isoform of one amplicon gene, nuclear SET domain-containing protein 3 (NSD3s), to be the most potent inducer of transformation to a neoplastic phenotype of all genes on the 8p11-p12 amplicon when over expressed in MCF10A breast epithelial cells in vitro. NSD3 is a member of the NSD family of methyltransferases, and has been shown to methylate lysine 4, 27, and 36 on the H3 subunit of histones, however the potently transforming short isoform lacks the catalytic SET domain. Our lab has shown that over expression of NSD3s in the immortalized human breast epithelial cell line MCF10A causes a significant increase in proliferation in insulin-free culture conditions, and the ability to form colonies in soft agar. In SUM44 cells, a breast cancer cell line with the 8p11-p12 amplicon, shRNA knockdown of NSD3s resulted in decreased proliferation. Immunoblotting with anti-H3K4me2 antibody shows MCF10A cells have higher global levels of H3K4me2 than SUM 44 cells, and over expression of NSD3s in MCF10A cells reduces global H3K4me2 levels. Other NSD family members have been shown to act as oncogenes in certain leukemias by blocking differentiation in developing lymphocytes through H3K36me3-mediated inhibition of the polycomb repressor complex, resulting in constitutive activation of the HOX gene family. This epigenetic dysregulation of HOX through the NSD1 oncogene results in acquisition of a stem-like phenotype in these cells that is essential for the development of these leukemias. We predict a similar mechanism in breast cancers that over express NSD3. Genome-wide expression analysis has shown that IRX3, a gene expressed in cells of the ectoderm lineage during differentiation, is upregulated in cells over expressing NSD3s. Based on previous findings with other NSD family genes we predict that genes involved in pluripotency or stemness will be targets of NSD3s activity in breast cancers with the 8p11-p12 amplicon. To investigate whether over expression of NSD3s induces a stem-like phenotype in MCF10A cells, we have measured the expression level of breast cancer stem cell surface markers CD44 and CD24 by flow cytometry, and found that NSD3s over expression results in a sub-population of cells that has higher CD44 expression than parental MCF10A, while maintaining low CD24 expression. These data suggest that NSD3s may act as a driving oncogene in breast cancer by altering histone methylation patterns in a way that promotes the expression of genes that result in the acquisition of stem-like phenotypes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2190. doi:1538-7445.AM2012-2190