Aberrant DNA methylation but not mutation of CITED4 is associated with alteration of HIF-regulated genes in breast cancer

Abstract

CBP/p300-interacting transactivator with ED-rich carboxy-terminal domain 4 (CITED4) inhibits HIF-1α transactivation by binding to CBP/p300. We hypothesised that either somatic mutation or hypermethylation of the CITED4 gene underlies CITED4 down-regulation and thus enhanced HIF-1α expression in some breast tumours. DNA sequencing was used to screen for somatic mutations. Methylation-sensitive high resolution melting was performed to identify CITED4 methylation. RT-qPCR was carried out to measure the expression of CITED4 and selected HIF downstream targets. HIF-1α and downstream gene expression was assessed with immunohistochemistry. No somatic mutations of CITED4 were identified in 10 tumour cell lines and 100 breast carcinomas. However, CITED4 promoter methylation was identified in 5/168 breast carcinomas (four infiltrating ductal carcinomas and one infiltrating lobular carcinoma) and in 3/10 breast cancer cell lines (MDA-MB-453, MDA-MB-231 and Hs578T). CITED4 mRNA expression in cell lines was inversely correlated with DNA methylation. CITED4 mRNA expression was significantly increased in all three cell lines after 5-aza-2-deoxycytidine (DAC) treatment. Treatment of the MDA-MB-231 cell line with DAC followed by hypoxia (0.1% O²) resulted in down-regulation of expression of the HIF-1α downstream genes VEGFA and SLC2A1 (P = 0.0029). HIF-1α downstream SLC2A1 was decreased (P = 0.021) after CITED4 was re-expressed under hypoxia. Loss of expression of CITED4 in breast cancer may be due to DNA methylation but is unlikely to be due to mutation. Demethylation and histone modification can potentially reactivate CITED4 gene expression in some breast cancers and lead to changes in tumour behaviour. Strategies such as HDAC inhibitors may overcome this effect.

GATA3 expression in triple‐negative breast cancers

“…A total of 150 ng of extracted RNA from 18 TN, three luminal and three HER2 FFPE breast cancers was used in cDNA synthesis, as described previously . In addition, archival RNA from cell lines MCF7, MDA‐MB 435, MDA‐MB 468 and MDA‐MB 231, as described previously, and FirstChoice Human Breast Total RNA (Ambion, Foster City, CA, USA), were converted to cDNA using Superscript III Reverse Transcriptase (Invitrogen, Life Technologies, Carlsbad, CA, USA), random hexamers (Pharmacia, Uppsala, Sweden) and RNase inhibitor (Roche, Basel, Switzerland). cDNA quality was assessed using multiplex endpoint RT–PCR and only samples with greater than 92 base pairs (bp) fragments were used for further RT–qPCR mRNA quantification …”

Knockdown of hypoxia inducible factor-2α inhibits cell invasion via the downregulation of MMP-2 expression in breast cancer cells

“…Activated HIFs exhibit a crucial role in adaptive responses of tumor cells to changes in O 2 levels via transcriptional activation of >100 downstream genes which regulate vital biological processes required for tumor survival and progression. Examples include genes involved in glucose metabolism, cell proliferation, migration and angiogenesis (29). Currently, the role of HIF-2α in certain solid tumors is unclear.…”

CITED Proteins in the Heart of Pluripotent Cells and in Heart’s Full Potential

“…Several reports have now pointed out the expression and the role of CITED proteins in stem cells (including ESC), in which they are involved in selfrenewal and cell fate decisions [38,[41][42][43][44][45][46]. Not surprisingly, due to the plethora of cellular and embryonic functions exerted by CITED1, CITED2 and CITED4, these genes were also showed to play intricate roles in tumorigenic processes [5,45,[47][48][49][50][51][52][53][54][55][56][57][58][59]. Therefore, CITED proteins are truly Regenerative Medicine Frontiers…”