The GALNT9, BNC1 and CCDC8 genes are frequently epigenetically dysregulated in breast tumours that metastasise to the brain

Rajendra P. Pangeni, Prasanna Channathodiyil, David S. Huen, Lawrence W. Eagles, Balraj K. Johal, Dawar Pasha, Natasa Hadjistephanou, Oliver Nevell, Claire L. Davies, Ayobami I. Adewumi, Hamida Khanom, Ikroop S. Samra, Vanessa C. Buzatto, Preethi Chandrasekaran, Thoraia Shinawi, Timothy P. Dawson, Katherine M. Ashton, Charles Davis, Andrew R. Brodbelt, Michael D. Jenkinson, Ivan Bièche, Farida Latif, John L. Darling, Tracy J. Warr, Mark R. Morris

Tumour metastasis to the brain is a common and deadly development in certain cancers; 18-30 % of breast tumours metastasise to the brain. The contribution that gene silencing through epigenetic mechanisms plays in these metastatic tumours is not well understood. We have carried out a bioinformatic screen of genome-wide breast tumour methylation data available at The Cancer Genome Atlas (TCGA) and a broad literature review to identify candidate genes that may contribute to breast to brain metastasis (BBM). This analysis identified 82 candidates. We investigated the methylation status of these genes using Combined Bisulfite and Restriction Analysis (CoBRA) and identified 21 genes frequently methylated in BBM. We have identified three genes, GALNT9, CCDC8 and BNC1, that were frequently methylated (55, 73 and 71 %, respectively) and silenced in BBM and infrequently methylated in primary breast tumours. CCDC8 was commonly methylated in brain metastases and their associated primary tumours whereas GALNT9 and BNC1 were methylated and silenced only in brain metastases, but not in the associated primary breast tumours from individual patients. This suggests differing roles for these genes in the evolution of metastatic tumours; CCDC8 methylation occurs at an early stage of metastatic evolution whereas methylation of GANLT9 and BNC1 occurs at a later stage of tumour evolution. Knockdown of these genes by RNAi resulted in a significant increase in the migratory and invasive potential of breast cancer cell lines. These findings indicate that GALNT9 (an initiator of O-glycosylation), CCDC8 (a regulator of microtubule dynamics) and BNC1 (a transcription factor with a broad range of targets) may play a role in the progression of primary breast tumours to brain metastases. These genes may be useful as prognostic markers and their products may provide novel therapeutic targets.