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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-14-3-3e interactions may be an effective strategy for inducing skin cancer cell apoptosis. Co-immunoprecipitation revealed that CDC25A associated with 14-3-3e, 14-3-3γ and 14-3-3ζ in SCC cells but not normal keratinocytes. 14-3-3e and CDC25A activated Akt/BAD/Survivin pro-survival signaling. To target the interaction of 14-3-3e 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 14-3-3e binding to CDC25A. Peptides pT (IC50 = 22.1 μM), and pS (IC50 = 29 μM) induced SCC cell death and blocked 14-3-3e 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 14-3-3e-CDC25A interactions for skin cancer treatment.

More than a million cases of cutaneous squamous cell carcinoma are diagnosed in the USA each year, and its incidence is increasing. Most of these malignancies arise from premalignant lesions, providing an opportunity for intervention before malignant progression. We previously documented how cytoplasmic mislocalization of CDC25A in premalignant and malignant skin cancers confers resistance to apoptotic cell death via a mechanism that depends on its interaction with 14-3-3ε. From these data, we hypothesized that 14-3-3ε overexpression drives skin tumor development and progression, such that targeting 14-3-3ε may be a useful strategy for skin cancer treatment. Like CDC25A, 14-3-3ε was overexpressed and mislocalized to the cytoplasm of both benign and malignant human skin cancer. Skin-targeted deletion of the 14-3-3ε gene reduced skin tumor development by 75% and blocked malignant progression. 14-3-3ε suppressed apoptosis through activation of Akt, leading to inhibition of BCL2 associated agonist of cell death and upregulation of Survivin. Using virtual tetrapeptide libraries, we developed a novel peptide that specifically blocked 14-3-3ε heterodimerization and thereby prevented its interaction with CDC25A. The peptide reduced prosurvival signaling, killed skin cancer cells and reduced skin tumor growth in xenograft. Normal skin keratinocytes were unaffected by inhibition or deletion of 14-3-3ε. Thus, targeting of 14-3-3ε dimerization is a promising strategy for the treatment of premalignant skin lesions.

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.

Scavenger receptors clear pathogens, transport lipid, and mediate polyanionic ligand uptake in macrophages, but their expression and role in skin is poorly understood. Although the epidermal barrier typically excludes nucleic acid entry, topically applied, spherically-arranged oligonucleotide nanoconjugates (spherical nucleic acids or SNAs) penetrate mouse skin, 3D skin equivalents, and human skin. We explored the mechanism of SNA uptake in normal human epidermal keratinocytes (NHEKs) and 3D skin equivalents. NHEKs and 3D raft treatment with class A scavenger receptor (SR-A) inhibitors reduced SNA uptake by >80%. Human epidermis expresses SR-As scavenger receptor class A type 3 (SCARA3) and, to a lesser extent, macrophage receptor with collagenous structure (MARCO). Simultaneous lentiviral knockdown of SCARA3 and MARCO reduced SNA uptake in NHEKs and 3D rafts after topical application, affirming a role for SR-As in SNA uptake and 3D raft penetration. Incubation of NHEKs at 4°C or with sodium azide prevented SNA uptake, suggesting active endocytosis. Endocytosis inhibitors, immunofluorescence, immunoprecipitation, and knockdown studies localized functional SR-As to flotillin-1 (FLOT-1)-containing lipid rafts throughout the epidermis and caveolin-1 (CAV-1)-containing rafts only in the upper epidermis. These studies suggest a central role for SR-A complexes in epidermal lipid rafts in mediating uptake of nucleic acid-laden nanoparticles.

Despite its documented role in cell cycle regulation, over-expression of the cyclin-dependent kinase activator CDC25A does not consistently correlate with worse cancer patient outcomes or predict successful clinical response to CDC25A inhibition. The current study was undertaken to investigate CDC25A in skin cancer and understand predictors of positive response to CDC25A targeting. CDC25A was increased in human squamous cell carcinoma (SCC) associated with a shift from a primarily nuclear localization in skin to a strong cytoplasmic localization in SCC, a pattern that was reproduced in skin cancer cell lines. Surprisingly, siRNA-targeting or forced expression of CDC25A failed to alter SCC proliferation. Instead, CDC25A suppressed apoptotic cell death in a manner dependent on both its cytoplasmic localization and interaction with 14-3-3. Normal keratinocytes with nuclear localization of the phosphatase were resistant to CDC25A modulation. Additionally, the CDC25A inhibitors Vitamin K3 or NSC663284 were more toxic to SCC than normal keratinocytes, and CDC25A inhibition effectively suppressed skin cancer growth by increasing apoptosis without affecting normal skin biology. These studies provide proof-of-concept evidence for the potential of CDC25A inhibitors for skin cancer treatment and suggest that an assessment of the cytoplasmic localization of CDC25A may be a strategy for identification of skin and other cancers susceptible to CDC25A targeting.

p53 is an important tumor-suppressor protein that is mutated in more than 50% of cancers. Strategies for restoring normal p53 function are complicated by the oncogenic properties of mutant p53 and have not met with clinical success. To counteract mutant p53 activity, a variety of drugs with the potential to reconvert mutant p53 to an active wildtype form have been developed. However, these drugs are associated with various negative effects such as cellular toxicity, nonspecific binding to other proteins, and inability to induce a wildtype p53 response in cancer tissue. Here, we report on the effects of a curcumin analog, HO-3867, on p53 activity in cancer cells from different origins. We found that HO-3867 covalently binds to mutant p53, initiates a wildtype p53-like anticancer genetic response, is exclusively cytotoxic toward cancer cells, and exhibits high anticancer efficacy in tumor models. In conclusion, HO-3867 is a p53 mutant–reactivating drug with high clinical anticancer potential.

Cytoplasmic relocalization of the dual-specificity phosphatase, CDC25A, occurs during nonmelanoma skin cancer development and progression, leading to suppression of cell death. Cytoplasmic CDC25A is phosphorylated at Ser178, which allows for its association with 14-3-3ϵ. 14-3-3ϵ is a member of the 14-3-3 family of proteins that bind phosphoserine/threonine-containing proteins and are involved in various processes including scaffolding, nuclear transport, protein degradation, cell cycle regulation, apoptosis and cell signaling. We hypothesized that CDC25A's antiapoptotic function depends upon 14-3-3ϵ binding to trigger activation of prosurvival signaling pathways in SCC cells. Co-immunoprecipitation of CDC25A from the SCC cell line SCC12B.2 followed by immunoblotting for 14-3-3ϵ confirmed an association between CDC25A and14-3-3ϵ. CDC25A over-expression in SCC12B.2 cells reduced apoptosis by approximately 50%, as assessed by a caspase 3/7 glo assay. However, inhibition of 14-3-3ϵ with the peptide R18, or silencing of 14-3-3ϵ, blocked the anti-apoptotic effect of CDC25A in SCC cells. Treatment with serial dilutions of R18 increased cell death with an IC50 of 8.9 μM in SCC12B.2 cells. Interestingly, over-expression of CDC25A significantly increased P-Akt (p=0.011), P-BAD (p=0.034) and Survivin (p=0.036) when compared to control. Consistent with our hypothesis that 14-3-3ϵ facilitates CDC25A's antiapoptotic activity, silencing of 14-3-3ϵ decreased P-Akt (p=0.044), P-BAD (p=0.034) and Survivin (p=0.008). Taken together, these results indicate that cytoplasmic CDC25A and 14-3-3ϵ interact in skin carcinomas to suppress apoptosis through a mechanism involving activation of Akt/BAD/Survivin signaling, rendering SCC cells sensitive to 14-3-3ϵ or CDC25A inhibition, a mechanism that requires further study. Citation Format: Thomas R. Holmes, Jenan Al-Matouq, Brianna Hammiller, Yawah T. Nicholson, Matti J. Holmes, Laura A. Hansen. CDC25A suppression of apoptosis in cutaneous squamous cell carcinoma depends on 14-3-3ϵ [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-190.

The glmS riboswitch is unique among gene-regulating riboswitches and catalytic RNAs. This is because its own metabolite, glucosamine-6-phosphate (GlcN6P), binds to the riboswitch and catalytically participates in the RNA self-cleavage reaction, thereby providing a novel negative feedback mechanism. Given that a number of pathogens harbor the glmS riboswitch, artificial actuators of this potential RNA target are of great interest. Structural/kinetic studies point to the 2-amino and 6-phosphate ester functionalities in GlcN6P as being crucial for this actuation. As a first step toward developing artificial actuators, we have synthesized a series of nine GlcN6P analogs bearing phosphatase-inert surrogates in place of the natural phosphate ester. Self-cleavage assays with the Bacillus cereus glmS riboswitch give a broad SAR. Two analogs display significant activity, namely, the 6-deoxy-6-phosphonomethyl analog (5) and the 6-O-malonyl ether (13). Kinetic profiles show a 22-fold and a 27-fold higher catalytic efficiency, respectively, for these analogs vs glucosamine (GlcN). Given their nonhydrolyzable phosphate surrogate functionalities, these analogs are arguably the most robust artificial glmS riboswitch actuators yet reported. Interestingly, the malonyl ether (13, extra O atom) is much more effective than the simple malonate (17), and the "sterically true" phosphonate (5) is far superior to the chain-truncated (7) or chain-extended (11) analogs, suggesting that positioning via Mg coordination is important for activity. Docking results are consistent with this view. Indeed, the viability of the phosphonate and 6-O-malonyl ether mimics of GlcN6P points to a potential new strategy for artificial actuation of the glmS riboswitch in a biological setting, wherein phosphatase-resistance is paramount.

tries are currently in the process of reconstructing democracy, with a new openness to the Western world. In Central and Eastern European countries, a "partial vacuum" now exists in the health delivery system because the economic and policy-making structures have shifted so dramatically that new patterns of health-care policy and service delivery must emerge. This review of mental health systems in the Czech Republic gives mental health policy administrators and service providers a unique opportunity to investigate policy and service delivery in the 1990s. As the country moves from a state-mandated service system under a totalitarian socialist government to a mix of government-managed and free-market health-care systems in a newly

This article reports on a clinical outcome study assessing the history of childhood abuse as a predictor variable for outcome research in clinical social work practice. Although this variable is widely viewed by clinicians as important in predicting the response of clients to treatment, little research has been done to test this assumption. This study assesses the effect of short-term treatment on depression and anxiety levels of 39 clients in a family service agency. The ANCOVA results indicated that for this treatment group, a history of childhood abuse was related to clients' response to treatment. A nonrandomized pretest-posttest design was used to compare the outcomes of clients with a history of childhood abuse with those without such a history. The results showed significant difference between the two groups on outcome levels of anxiety and depression. Implications for clinical research are discussed.

A family service counseling program was studied to assess the efficacy of short-term treatment. Previous research indicating effectiveness of short-term treatment did not control for a history of childhood abuse as a factor affecting treatment outcome. This study of 39 adult outpatient clients showed that after 3 to 6 sessions of treatment, those with no history of childhood abuse showed clinically and statistically significant improvement in their anxiety and depression as measured by the Beck anxiety and depression inventories. Clients with a history of childhood abuse showed no change after the short-term intervention in either anxiety or depression.

Identifying the role orientation of recently divorced women holds promise for further understanding the divorce process and the family life cycle of nonnuclear families. The authors describe how 30 female heads of family perceived their adaptation to divorce and to the reconstruction and development of their family units. Results of this exploratory study further suggest that the meaning and value a divorced woman attaches to the mothering component of her role as well as the comfort she feels in accepting a work identity affect the ways in which she adapts to being a single parent. The consequences of this adaptation, in turn, alter the content of the family life cycle.

Amelioration de la resistance aux agents alcalins. Developpement d'un refractaire de SiC lie par un sialon

There are strong suggestions in the literature that nursing home employees may not be properly trained to deal with the behavioral problems of geriatric residents. The present results support this conclusion. Social service/activity directors and nurse aides were found to do poorly on a test (The Behavioral Knowledge Questionnaire) which purports to measure one's skills at working with this population. Their performance was found to be relatively independent of knowledge about the aging process per se. Finally, it was suggested that the instrument be utilized in in-service training with these groups.

A 66-year old obese man with asthma was given a hypocaloric (1100 Kcal) and low protein (35 g) diet for nine days. While receiving theophylline (Theodur), 200 mg bid, his morning trough theophylline concentrations rose from 3.40 micrograms/ml to 12.7 micrograms/ml by day 9 of this diet. Following discontinuation, his theophylline concentration fell to 5.95 micrograms/ml by day 6 on home diet. The patient lost 3.67 kg during the nine-day study. Thus, a brief exposure to a hypocaloric, low protein diet in this elderly patient with asthma caused a dramatic rise in trough theophylline concentrations.