Role of CTCF in HOX genes regulation and breast cancer tumorigenesis

Abstract

CTCF (or CCCTC-binding factor), a ubiquitous 11-zinc finger multifunctional protein has distinct molecular functions such as transcriptional activation, transcriptional repression, or enhancer blocking activity, in a locus-specific manner. Identification of somatic mutations in CTCF in different cancers and its involvement in cellular growth, differentiation and apoptosis point towards its role in cancer progression. HOX genes not only play important roles in defining body patterning during embryonic development, but also control numerous cellular events in adult cells. Deregulated HOX gene expression in different cancers including breast cancer is now increasingly being reported.

In this work we wanted to investigate that how CTCF contribute in the breast cancer cell survival directly and through modulation of HOX genes. We performed CTCF gain and loss of function studies to see its effects on HOX genes expression and cancer cell survival and identified several directly regulated HOX genes including HOXA4, HOXC8 and HOXA10. Among HOX, HOXA10 is an emerging tumor suppressor for its role in activation of p53 and in countering tumorigenesis in breast cancer. HOXA10 silencing is associated with different cancers but the underlying mechanism is still elusive. Our data defines the putative promoter region of HOXA10 5.3-6.1 kb upstream of its start codon and its negative regulation by CTCF. Analysis of histone modification reveals that the presence of CTCF is associated with decreased active histone marks H3K4me3 and increased repressive histone marks H3K27me3 on HOXA10 locus. Together with in silico analysis and specially designed in-vitro mutation assay we identified an important promoter element, flanked with CTCF core motif in HOXA10 promoter region. Based on the evidence in our study, we propose that CTCF binding site co-exist within core promoter region of HOXA10 and CTCF presence not only maintains the inactive state of local chromatin but also interfere with the transcription machinery and induce transcriptional silencing of HOXA10. Epigenetic silencing of HOXA10 by CTCF in breast cancer cells may contribute towards tumorigenesis by avoiding cell cycle arrest, decreasing apoptosis and promoting metastasis.

We observed depletion of CTCF leads to decreased cell growth and proliferation in breast cancer cell line MCF-7 than normal breast epithelial cell line MCF10A. We found that it happens due to the direct and indirect activation of p53 signaling cascade in CTCF knock down breast cancer cells. CTCF binding near the promoter region of TP53 is marked with increased H3K27me3 and decreased H3K4me3. p53 activation by CTCF knock down resulted in cell cycle arrest at G1/S check point and enhanced apoptosis due to the activation of p21 and Bax respectively. Nuclear localization of p53 is related with CTCF depletion in MCF-7 cells and it is consistent with our observation that p53 cascade is activated in the absence of CTCF. CTCF also induce transcriptional silencing of HOXA10 which is known to activate p53.We observed that HOXA10 co-localize with p53 in breast cancer cells and depletion of nuclear HOXA10 results in decreased nuclear p53. Several key components of autophagy signaling pathway e.g., ATG13, SQSTM1 (P62) and LC3-II as well as lysosome number are positively related to CTCF levels in MCF-7 cells.

Taken together we demonstrated that CTCF induce transcriptional silencing of p53 directly as well as through HOXA10 to contribute in the breast cancer survival and tumorigenesis.