The p53 tumor suppressor is a critical regulator of genomic stability. P53 is known as “the guardian of the genome” because it plays a crucial role in inducing apoptosis and cell cycle arrest in response to DNA damage. P53 is mutated in approximately 50% of all human tumors and is downregulated in a large majority of the remainder. An important regulator of p53 is the E3 ubiquitin ligase, MDM2, which acts to ubiquitinate p53 and target it for degradation through the proteasome. There is currently a large effort in the field directed toward developing targeted therapies that inhibit MDM2′s ability to interact with p53, thus allowing p53 to be stabilized. One such drug that is already undergoing clinical trials is Nutlin-3a. Our preliminary data demonstrate that the homeobox containing transcription factor, Six1, decreases the p53 response to DNA damage in a proteasome independent manner, suggesting that it may downregulate the p53 response even in the presence of Nutlins. Six1, is an important developmental regulator as it increases cell migration, invasion, and proliferation, however, it is not expressed in most normal adult tissues. It is, however, re-expressed in cancerous cells, including human breast lesions. As we are currently developing drugs to target Six1, it is essential to understand mechanistically how Six1 downregulates p53 and whether or not inhibition of Six1 may re-sensitize cells to Nutlin therapies.

Here we show that Six1 downregulates p53, not on the mRNA level, but on the protein level, and that this downregulation leads to a decrease in p53 function. We have shown that Six1 can target p53 in a variety of different cell culture systems, including both breast and colon cancer cell lines, as well as normal breast and embryonic kidney cell lines. We have begun to examine the mechanism by which Six1 downregulates p53, and have shown that it is able to target p53 independent of MDM2, as siRNA knock down of MDM2 is not able to rescue p53 levels in the presence of Six1. Furthermore, Six1 does not affect the half life of p53, and congruently Six1 is able to downregulte p53 even in the presence of a proteosome inhibitor. Together, these results suggest that MDM2’s ability to target p53 for proteasomal degradation is dispensable for the ability of Six1 to decrease p53 levels. Furthermore, our preliminary data suggests that Six1 regulates p53 in a post-transcriptional mechanism, potentially through the use of the p53 3’UTR region. Six1 overexpressing lines are more resistant to Nutlin therapies in-vitro, demonstrating a significant increase in viability in the presence of Nutlins when compared to control cell lines. Thus, high levels of Six1 may correlate with decreased response to Nutlins clinically, and targeting of Six1 may be a novel approach to re-sensitize tumors expressing high levels of Six1 to Nutlin therapy.

Citation Format: Christina N. Garlington, Douglas S. Micalizzi, Heide L. Ford. The Six1 homeoprotein downregulates p53, via a novel, MDM2 independent mechanism. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 782. doi:10.1158/1538-7445.AM2013-782