Abstract 3775: p53 is differentially regulated in proliferative and invasive melanoma cells
Authors: Marie R. Webster, Subhasree Basu, Amanpreet Kaur, Jessica Appleton, Maureen E. Murphy, Ashani T. Weeraratna
Journal: Cancer Research (2015)

Abstract Melanoma is one of the few cancer types where p53 is not often mutated. Cancers which have not mutated TP53, often attenuate p53 activity by affecting upstream regulators or downstream effectors of p53. As melanoma progresses, it by passes senescence, proliferates, and switches from a proliferative phenotype to a more invasive phenotype. We have found that invasive melanomas, characterized by high levels of the pro-invasive Wnt molecule, Wnt5A, express active p53. Conversely, melanomas with a more proliferative phenotype, characterized by increased MITF signaling and low Wnt5A expression, do not express active p53. We show that the expression of p53 appears to be controlled by Wnt5A, and this may occur via CAMKII. By Western blot, we have observed that more proliferative melanoma cells express a high molecular weight p53 (∼250 kDa). This high molecular weight p53 does not break down on an SDS-PAGE gel. Knocking down p53 in proliferative and invasive melanoma cells, using siRNA, decreases the high molecular weight band in the proliferative cells, and the 53 kDa band in the more invasive cells. When we over express HA-tagged p53 in proliferative cells, we see multiple bands for p53 by Western blot, including the high molecular weight bands, suggesting that p53 is being post-translationally modified in these cells. We confirmed that ubiquitination is involved in the formation of the high molecular weight p53, by treating proliferative cells with SMER3, an E3 ligase inhibitor. Following two hours of treatment, the high molecular weight p53 band decreased and this decrease persists for 16 h. Next, we transfected an E6 ligase into invasive melanoma cells, which express active p53. Expression of the E6 ligase increased the expression of the high molecular weight p53 band (250 kDa), but these cells also continued to express active p53 (53 kDa). To determine if ubiquitination is targeting p53 for degradation in proliferative cells, we treated invasive and proliferative cells with MG132, a proteasome inhibitor. Following treatment with MG132, p53 (53 kDa) increased in invasive cells, suggesting that p53 is degraded via the proteasome in these cells. However, when we treated proliferative cells with MG132 we did not see an increase in the high molecular weight band, suggesting that p53 is not being degraded via the proteasome in these cells. By immunofluorescence, we found that p53 is in the cytoplasm of proliferative cells, suggesting that ubiquitination may be playing a role in sequestration of p53 in proliferative cells. In invasive cells, p53 remains largely nuclear, and we demonstrate that this plays critical roles in response to therapy, counter-intuitively driving resistance, by allowing cells to undergo an adaptive stress response that we term “pseudosenescence” Citation Format: Marie R. Webster, Subhasree Basu, Amanpreet Kaur, Jessica Appleton, Maureen E. Murphy, Ashani T. Weeraratna. p53 is differentially regulated in proliferative and invasive melanoma cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3775. doi:10.1158/1538-7445.AM2015-3775