Functional analysis of ANKRD11 and FBXO31: two candidate tumour suppressor genes from the 16q24.3 breast cancer loss of heterozygosity region.

Abstract

Loss of heterozygosity (LOH) on the long arm of chromosome 16 is frequently observed during the onset of breast cancer. Our laboratory has recently identified both ANKRD11 and FBXO31 as candidate tumour suppressor genes in the chromosome band 16q24.3, which is the smallest region of overlap for breast cancer LOH. This thesis focuses on the functional analysis of these two novel genes and implicates a role for them as breast cancer tumour suppressors. ANKRD11: a novel p53 coactivator involved in the rescue of mutant p53. The ability of p53 to act as a transcription factor is critical for its function as a tumour suppressor. Ankyrin repeat domain 11 (ANKRD11) was found to be a novel p53-interacting protein which enhanced the transcriptional activity of p53. ANKRD11 expression in breast cancer cell lines was shown to be down-regulated when compared to ANKRD11 expression in finite life-span HMECs and non-malignant immortalized breast epithelial cells. Restoration of ANKRD11 expression in MCF-7 (p53 wild-type) and MDA-MB-468 (p53[superscript R273H] mutant) cells suppressed the oncogenic properties of these breast cancer cell lines through enhancement of p21[superscript waf1] expression. ShRNA-mediated silencing of ANKRD11 reduced the ability of p53 to activate p21[superscript waf1] expression in response to DNA damage. ANKRD11 was shown to associate with the p53 acetyltransferase, P/CAF, and exogenous ANKRD11 expression increased the levels of acetylated p53. Exogenous ANKRD11 expression enhanced the DNA-binding properties of the p53[superscript R273H] mutant to the CDKN1A promoter, implicating a role for ANKRD11 in the restoration of mutant p53[superscript R273H] function. These findings demonstrate a role for ANKRD11 as a p53 coactivator and illustrate the potential of ANKRD11 in the restoration of mutant p53[superscript R273H] function. ANKRD11 has roles beyond that of p53 coactivation. This thesis also presents preliminary findings to suggest that ANKRD11 may be involved in the regulation of eukaryotic cell division. Furthermore, ANKRD11 was shown to function as an estrogen receptor coactivator. Taken together, these finding suggest that ANKRD11 is a multi-functional cancer-related protein. FBXO31: the 16q24.3 senescence gene. A BAC located in the 16q24.3 breast cancer loss of heterozygosity region was previously shown to restore cellular senescence when transferred into breast tumour cell lines. We have shown that FBXO31, although located just distal to this BAC, can induce cellular senescence in the breast cancer cell line MCF-7 and is the likely candidate senescence gene. Exogenous FBXO31 expression inhibited the oncogenic properties of the MCF-7 breast cancer cell line. In addition, compared to the relative expression in normal breast, levels of FBXO31 were down-regulated in breast tumour cell lines and primary tumours. FBXO31 protein levels were cell cycle regulated, with maximal expression from late G2 to early G1 phase. Ectopic expression of FBXO31 in the breast cancer cell line MDA-MB-468 resulted in the accumulation of cells at the G1 phase of the cell cycle. FBXO31 was also shown to be a component of a SCF ubiquitination complex. We propose that FBXO31 functions as a tumour suppressor by generating SCF[superscript FBXO31] complexes that target particular substrates, critical for the normal execution of the cell cycle, for ubiquitination and subsequent degradation.