Abstract
While p73 overexpression has been associated with increased apoptosis in cancer tissues, p73 overexpressing tumors appear to be of high grade malignancy. Why this putative tumor suppressor is overexpressed in cancer cells and what the function of overexpressed p73 is in breast cancers are critical questions to be addressed. By investigating the effect of p53 inactivation on p73 expression, we found that both protein and mRNA levels of TAp73 were increased in MCF-7/p53siRNA cells, MCF-7/p53mt135 cells and HCT-116/p53−/− cells, as compared to wild type control, suggesting that p53 inactivation by various forms upregulates p73. We showed that p53 knockdown induced p73 was mainly regulated at the transcriptional level. However, although p53 has a putative binding site in the TAp73 promoter, deletion of this binding site did not affect p53 knockdown mediated activation of TAp73 promoter. Chromatin immuno-precipitation (ChIP) data demonstrated that loss of p53 results in enhanced occupancy of E2F-1 in the TAp73 promoter. The responsive sequence of p53 inactivation mediated p73 upregulation was mapped to the proximal promoter region of the TAp73 gene. To test the role of E2F-1 in p53 inactivation mediated regulation of p73 transcription, we found that p53 knockdown enhanced E2F-1 dependent p73 transcription, and mutations in E2F-1 binding sites in the TAp73 promoter abrogated p53 knockdown mediated activation of TAp73 promoter. Moreover, we demonstrated that p21 is a mediator of p53-E2F crosstalk in the regulation of p73 transcription. We concluded that p53 knockdown/inactivation may upregulate TAp73 expression through E2F-1 mediated transcriptional regulation. p53 inactivation mediated upregulation of p73 suggests an intrinsic rescuing mechanism in response to p53 mutation/inactivation. These findings support further analysis of the correlation between p53 status and p73 expression and its prognostic/predictive significance in human cancers.
Highlights
Loss of p53 function, including genetic mutation and functional inactivation, has been associated with carcinogenesis and therapeutic resistance [1,2]
By testing the activation of the TAp73 promoter in a transient transfection system, we found TAp73 promoter activity was increased in MCF-7 cells transfected with p53siRNA or mtp53 (G135A), and it was decreased in response to wtp53 transfection (Fig. 2E)
Reports from later studies showed that co-transfection of p53 did not activate the TAp73 promoter [6,15,20]. To determine whether this p53 responsive element (p53RE) plays any roles in p53 inactivation modulated p73 regulation, we examined DNA binding activity of p53 to this putative binding site using electrophoretic mobility shift assay (EMSA)
Summary
Loss of p53 function, including genetic mutation and functional inactivation, has been associated with carcinogenesis and therapeutic resistance [1,2]. P73 is able to induce apoptosis/growth arrest in cells with mutant p53 [7] These findings support p73 as a tumor suppressor and underscore the significance of p73 in p53-independent anti-tumor mechanisms. While p73 overexpression has been associated with increased apoptosis in cancer tissues, p73 overexpressing tumors appear to be of high grade malignancy [12] Why this putative tumor suppressor is overexpressed in cancer cells and what the function of overexpressed p73 is in breast cancers are critical questions to be addressed. P73 is commonly overexpressed in cancer cells and is functional in many cell lines with mutant p53 [7] This indicates that there might be additional mechanisms that coordinate p53 inactivation and p73 activation, such that p73 is upregulated/activated to rescue some, if not all, of p53 function. The underlying mechanisms involve E2F-1 mediated regulation of TAp73 transcription modulated by p21 activity
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