Characterisation of novel protein partners of the CCCTC-binding factor in breast cancer cell lines

CTCF is an evolutionally conserved 11-zinc finger protein. It controls multiple cellular functions and is itself partly regulated via poly (ADP-ribosyl)ation (PARylation). Recent evidence suggested that the hypo PARylated isoform was exclusively expressed in breast tumours and low expression levels was associated with worse prognostic features. It also possessed proliferative activity. The exact mechanism by which CTCF exerted its effects in breast cancer is not known. In order to define the interaction between CTCF and proliferation markers, Ki67 and PCNA, colocalisation studies were performed in a panel of five breast cancer cell lines which possessed different hormone receptor / invasive phenotypes. Following co-localisation, co-immunoprecipitation assays and mass spectrometry were carried out to determine whether CTCF was physically bound to either Ki67 or PCNA. To determine whether CTCF directly regulated ERα activity, CTCF plasmid overexpression and siRNA knockdown assays were performed in the hormone receptor positive MCF7 breast cancer cell line. Changes in endogenous expression of ERα were monitored by quantitative polymerase chain reaction (QPCR). CTCF, Ki67 and PCNA were all found to be strongly expressed in breast cancer cell lines though the strength of this expression for CTCF and Ki67 was antibody dependent. CTCF and Ki67 were shown to colocalise in the nucleoli of all breast cancer cell lines while CTCF and PCNA demonstrated nucleolar colocalisation only in the weakly invasive, hormone receptor positive cell lines. Despite colocalisation, there was no physical interaction detected between CTCF and Ki67 / PCNA on coimmunoprecipitation and mass spectrometry in all the cell lines studied suggesting that the proteins did not exist as a functional complex. Three novel CTCF - interacting protein partners (general transcriptional factor 2, glucose regulated protein 78 and the huntingtin interacting protein-1 related) were however discovered. These new protein partners, known to function at least in part via epidermal growth factor receptor (EGFR) signalling in cancer formation, were discovered only in ER positive breast cancer cell lines. Further investigation did not detect a direct regulatory effect of CTCF on ERα expression suggesting that the effect of CTCF on EGFR signalling in breast cancer cell lines did not involve an indirect action on ERα expression.