The p53 tumor suppressor protein is a transcription factor that exerts its effects on the cell cycle via regulation of gene expression. Although the mechanism of p53-dependent transcriptional activation has been well-studied, the molecular basis for p53-mediated repression has been elusive. The E2F family of transcription factors has been implicated in regulation of cell cycle-related genes, with E2F6, E2F7, and E2F8 playing key roles in repression. In response to cellular DNA damage, E2F7, but not E2F6 or E2F8, is up-regulated in a p53-dependent manner, with p53 being sufficient to increase expression of E2F7. Indeed, p53 occupies the promoter of the E2F7 gene after genotoxic stress, consistent with E2F7 being a novel p53 target. Ablation of E2F7 expression abrogates p53-dependent repression of a subset of its targets, including E2F1 and DHFR, in response to DNA damage. Furthermore, E2F7 occupancy of the E2F1 and DHFR promoters is detected, and expression of E2F7 is sufficient to inhibit cell proliferation. Taken together, these results show that p53-dependent transcriptional up-regulation of its target, E2F7, leads to repression of relevant gene expression. In turn, this E2F7-dependent mechanism contributes to p53-dependent cell cycle arrest in response to DNA damage.

The p53 tumor suppressor is a transcription factor that mediates varied cellular responses. The C terminus of p53 is subjected to multiple and diverse post-translational modifications. An attractive hypothesis is that differing sets of combinatorial modifications therein determine distinct cellular outcomes. To address this in vivo, a Trp53ΔCTD/ΔCTD mouse was generated in which the endogenous p53 is targeted and replaced with a truncated mutant lacking the C-terminal 24 amino acids. These Trp53ΔCTD/ΔCTD mice die within 2 wk post-partum with hematopoietic failure and impaired cerebellar development. Intriguingly, the C terminus acts via three distinct mechanisms to control p53-dependent gene expression depending on the tissue. First, in the bone marrow and thymus, the C terminus dampens p53 activity. Increased senescence in the Trp53ΔCTD/ΔCTD bone marrow is accompanied by up-regulation of Cdkn1 (p21). In the thymus, the C-terminal domain negatively regulates p53-dependent gene expression by inhibiting promoter occupancy. Here, the hyperactive p53ΔCTD induces apoptosis via enhanced expression of the proapoptotic Bbc3 (Puma) and Pmaip1 (Noxa). In the liver, a second mechanism prevails, since p53ΔCTD has wild-type DNA binding but impaired gene expression. Thus, the C terminus of p53 is needed in liver cells at a step subsequent to DNA binding. Finally, in the spleen, the C terminus controls p53 protein levels, with the overexpressed p53ΔCTD showing hyperactivity for gene expression. Thus, the C terminus of p53 regulates gene expression via multiple mechanisms depending on the tissue and target, and this leads to specific phenotypic effects in vivo.

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Melanoma is the leading cause of skin cancer-related death. Survival rates are high if the disease is diagnosed early, but drop precipitously at later stages. Small molecule inhibitor therapy has given robust responses in the clinic, but relapse is almost certain. This relapse may be due, in part, to tumor heterogeneity. Not only are there multiple cell types within a tumor, but cancer cells themselves can exhibit various phenotypes. This can be due to genotype variation or nutrient availability, and result in populations with different proliferative and invasive capabilities. As these cells display various behaviors, they could also respond to therapies uniquely. Understanding the molecular signature influencing different sub-populations is therefore crucial to design the most effective therapeutic regimen. The fluorescence ubiquitination cell cycle indicator (FUCCI) system, which delineates phases of the cell cycle by visual means, was employed to better understand melanoma tumor heterogeneity. Using this model, it was found that tumor xenografts grown in mice produce two cohorts. One that contained distinct clusters of either arrested or proliferating cells, and another that displayed a homogenous dispersion of proliferating cells throughout the breadth of the tumor. These cohorts were subsequently discovered to display either low or high levels of microphthalmia-associated transcription factor (MITF) expression, respectively. Additionally, loss of MITF by shRNA treatment resulted in conversion of the ability of melanoma cells to give rise to a homogenous xenograft, to instead produce a clustered tumor phenotype. Furthermore, in a 3D in vitro tumor spheroid model, MITF expression was predominantly found in the periphery of the spheroid, which corresponds with the region of highly proliferative cells. Forced over-expression of MITF within these spheroids results in loss of the distinct proliferative ring, and instead a homogenous growth pattern. Not only do spheroids express MITF around the perimeter, but also markers of the Epithelial to Mesenchymal Transition (EMT). These markers, such as Vimentin and Slug, also switch to become expressed homogenously upon high MITF expression. Surprisingly, the increased levels of EMT marker expression by MITF do not correlate to increased migration, and these spheroids in fact show reduced invasion into collagen. We are currently exploring what other means of cancer cell migration could trump an enhanced EMT phenotype and slow invasion in our model. These data outline how tumor heterogeneity, including proliferative and invasive potential, is tightly intertwined with MITF expression, making it an important marker for therapy design. Citation Format: Crystal A. Tonnessen, Kimberley A. Beaumont, David S. Hill, Sheena M. Daignault, Andrea Anfosso, Russell J. Jurek, Wolfgang Weninger, Nikolas K. Haass. MITF regulates proliferative subpopulation tumor architecture and modifies invasion and characteristics of the epithelial to mesenchymal transition within melanoma. [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 1420. doi:10.1158/1538-7445.AM2015-1420

While previous studies identify gender differences in melanoma, limited research on the phenomenon exists. We aim to investigate melanoma prognostic factors amongst males versus females.In this retrospective chart review, 1,156 adults diagnosed with melanoma between 2006-2016 at the University of Colorado were included. Breslow's depth, mitotic rate, ulceration status, and location were extracted between March and August 2016. Cochran-Armitage trend tests and cumulative logistic regression were used to examine the association between gender and Breslow's depth, univariately and after adjusting for potential confounders. In univariate analysis, males were significantly more likely to present with lesions with higher Breslow's depths (p for trend¼0.005). In models adjusted for age, melanoma subtype, and location, males were marginally more likely to present with lesions with higher Breslow's depths (cumulative OR: 1.261, 95% CI: 0.988-1.611, p¼0.060). Males were also marginally more likely to present with lesions with higher mitotic rates, after further adjustments for all other prognostic factors (cumulative OR: 1.244, 95% CI: 0.979-1.580, p¼0.074). Differences in mitotic rates among melanomas in males versus females, even after adjustments for all other prognostic factors, suggests that biological differences may contribute to the female advantage.