No abstract

Non-melanoma skin cancer is the most common form of cancer worldwide. We previously documented an anti-apoptotic role for CDC25A in cutaneous squamous cell carcinoma (SCC), an activity dependent on its association with 14-3-3 proteins. We hypothesized that targeting CDC25A-14-3-3ε interactions may be an effective strategy for inducing skin cancer cell apoptosis. Co-immunoprecipitation revealed that CDC25A associated with 14-3-3ε, 14-3-3γ and 14-3-3ζ in SCC cells but not normal keratinocytes. 14-3-3ε and CDC25A activated Akt/BAD/Survivin pro-survival signaling. To target the interaction of 14-3-3ε with CDC25A for cancer therapy, we developed two novel phospho-peptides, pS and pT, corresponding to each of the 14-3-3 binding sites of CDC25A, to specifically interfere with 14-3-3ε binding to CDC25A. Peptides pT (IC 50 = 22.1 μM), and pS (IC 50 = 29 μM) induced SCC cell death and blocked 14-3-3ε binding to CDC25A. pS or pT treatment of SCC xenografts increased apoptotic cell death and decreased pro-survival P-Akt (S473) and Survivin, demonstrating the effectiveness of the peptides in vivo. These findings lay a framework for the further development of peptides to target 14-3-3ε-CDC25A interactions for skin cancer treatment.

Non‐melanoma skin cancer frequently results from chronic exposure to ultraviolet (UV) irradiation. UV‐induced DNA damage activates cell cycle arrest checkpoints through degradation of the cyclin‐dependent kinase activators, the cell division cycle 25 (CDC25) phosphatases. We previously reported increased CDC25A in nonmelanoma skin cancer, but CDC25B and CDC25C had not been previously examined. Consequently, we hypothesized that increased expression of CDC25B and CDC25C increases tumor cell proliferation and skin tumor growth. We found that CDC25B and CDC25C were increased in mouse and human skin cancers. CDC25B was primarily cytoplasmic in skin and skin tumors and was significantly increased in the squamous cell carcinoma (SCC), while CDC25C was mostly nuclear in the skin, with an increased cytoplasmic signal in the premalignant and malignant tumors. Surprisingly, forced expression of CDC25B or CDC25C in cultured SCC cells did not affect proliferation, but instead suppressed apoptosis, while CDC25C silencing increased apoptosis without impacting proliferation. Targeting CDC25C to the nucleus via mutation of its nuclear export sequence, however, increased proliferation in SCC cells. Overexpression of CDC25C in the nuclear compartment did not hinder the ability of CDC25C to suppress apoptosis, neither did mutation of sites necessary for its interaction with 14‐3‐3 proteins. Analysis of apoptotic signaling pathways revealed that CDC25C increased activating phosphorylation of Akt on Ser473, increased inhibitory phosphorylation of proapoptotic BAD on Ser136, and increased the survival protein Survivin. Silencing of CDC25C significantly reduced Survivin levels. Taken together, these data suggest that increased expression of CDC25B or CDC25C are mechanisms by which skin cancers evade apoptotic cell death.

Cell division cycle 25A (CDC25A) is a dual-specificity phosphatase that removes inhibitory phosphates from cyclin-dependent kinases, allowing cell-cycle progression. Activation of cell-cycle checkpoints following DNA damage results in the degradation of CDC25A, leading to cell-cycle arrest. Ultraviolet (UV) irradiation, which causes most skin cancer, results in both DNA damage and CDC25A degradation. We hypothesized that ablation of CDC25A in the skin would increase cell-cycle arrest following UV irradiation, allowing for improved repair of DNA damage and decreased tumorigenesis. Cdc25a(fl/fl) /Krt14-Cre recombinase mice, with decreased CDC25A in the epithelium of the skin, were generated and exposed to UV. UV-induced DNA damage, in the form of cyclopyrimidine dimers and 8-oxo-deoxyguanosine adducts, was eliminated earlier from CDC25A-deficient epidermis. Surprisingly, loss of CDC25A did not alter epidermal proliferation or cell cycle after UV exposure. However, the UV-induced apoptotic response was prolonged in CDC25A-deficient skin. Double labeling of cleaved caspase-3 and the DNA damage marker γH2A.X revealed many of the apoptotic cells in UV-exposed Cdc25a mutant skin had high levels of DNA damage. Induction of skin tumors by UV irradiation of Cdc25a mutant and control mice on a skin tumor susceptible to v-ras(Ha) Tg.AC mouse background revealed UV-induced papillomas in Cdc25a mutants were significantly smaller than in controls in the first 6 weeks following UV exposure, although there was no difference in tumor multiplicity or incidence. Thus, deletion of Cdc25a increased apoptosis and accelerated the elimination of DNA damage following UV but did not substantially alter cell-cycle regulation or tumorigenesis.

Amplification of specific cancer genes leads to their over-expression contributing to tumor growth, spread, and drug resistance. Little is known about the ability of these amplified oncogenes to augment the expression of cancer genes through post-transcriptional control. The AU-rich elements (ARE)-mediated mRNA decay is compromised for many key cancer genes leading to their increased abundance and effects. Here, we performed a post-transcriptional screen for frequently amplified cancer genes demonstrating that ERBB2/Her2 overexpression was able to augment the post-transcriptional effects. The ERBB1/2 inhibitor, lapatinib, led to the reversal of the aberrant ARE-mediated process in ERBB2-amplified breast cancer cells. The intersection of overexpressed genes associated with ERBB2 amplification in TCGA datasets with ARE database (ARED) identified ERBB2-associated gene cluster. Many of these genes were over-expressed in the ERBB2-positive SKBR3 cells compared to MCF10A normal-like cells, and were under-expressed due to ERBB2 siRNA treatment. Lapatinib accelerated the ARE-mRNA decay for several ERBB2-regulated genes. The ERBB2 inhibitor decreased both the abundance and stability of the phosphorylated inactive form of the mRNA decay-promoting protein, tristetraprolin (ZFP36/TTP). The ERBB2 siRNA was also able to reduce the phosphorylated ZFP36/TTP form. In contrast, ectopic expression of ERBB2 in MCF10A or HEK293 cells led to increased abundance of the phosphorylated ZFP36/TTP. The effect of ERBB2 on TTP phosphorylation appeared to be mediated via the MAPK-MK2 pathway. Screening for the impact of other amplified cancer genes in HEK293 cells also demonstrated that EGFR, AKT2, CCND1, CCNE1, SKP2, and FGFR3 caused both increased abundance of phosphorylated ZFP36/TTP and ARE-post-transcriptional reporter activity. Thus, specific amplified oncogenes dysregulate post-transcriptional ARE-mediated effects, and targeting the ARE-mediated pathway itself may provide alternative therapeutic approaches.