Identification of Molecular Targets of Dietary Grape-Mediated Chemoprevention of Ultraviolet B Skin Carcinogenesis: A Comparative Quantitative Proteomics Analysis.

We recently showed that dietary grape powder (GP) imparts considerable protection against ultraviolet B (UVB)-mediated skin carcinogenesis in SKH-1 mice. To determine molecular mechanisms of this response, we employed tandem mass tag (TMT) quantitative global proteomics approach on skin tumors from mice exposed to 180 mJ/cm2 UVB twice per week and fed control or 5% GP diet. We found 2629 proteins modulated by GP feeding, with 34 identified using stringent cutoffs (false discovery rate (FDR) q-value ≤ 0.1, fold change ≥ 1.2, p-value ≤ 0.05, ≥ 3 unique peptides). Ingenuity Pathway Analysis helped identify seven proteins involved in protein ubiquitination, including the deubiquitinase UCHL5 and 6 subunits of the 20S proteasome (PSMA1,3,4,6 and PSMB4,7). A second data set without the FDR q-value identified 239 modulated proteins, seven of which are involved in protein ubiquitination. Further, 14 proteins involved in acute phase response signaling were modulated >1.5-fold, including acute phase proteins APCS, FGA, FGB, HP, HPX, and RBP1. Evaluation of upstream regulators found inhibition of ERK1/2 phosphorylation and NF-κB p65, and an increase in IκBα in GP-treated tumors. Overall, our data suggested that GP consumption may mitigate tumorigenesis by enhancing protein ubiquitination and degradation caused by oxidative stress, and manipulates an otherwise tumor-promoting anti-inflammatory environment.

Sirtuins in Skin and Skin Cancers.

The sirtuins are a family of proteins that comprise class III of the histone deacetylases. These NAD+-dependent proteins have been found to be intricately involved in a variety of important and skin-relevant cellular functions and processes, including aging, UV damage response, oxidative stress, and wound repair. In addition, recent research is unraveling the role of sirtuins in a variety of skin diseases, including melanoma and nonmelanoma skin cancers. In this review, we provide a discussion on the potential roles and implications of different sirtuins in skin-specific cellular processes, which may have relevance to skin health and skin diseases. Based on the available literature, the sirtuins appear to be important targets in the management of a variety of skin diseases from cosmetic (e.g., skin aging) to fatal conditions (e.g., melanoma).

Resveratrol and cancer: Challenges for clinical translation.

Significant work has been done towards identifying the health-beneficial effects of the grape antioxidant resveratrol in a variety of bioassay- and disease- models, with much research being focused on its possible application to cancer management. Despite the large number of preclinical studies dealing with different aspects of the biological effects of resveratrol, its translation to clinics is far from reality due to a variety of challenges. In this review, we discuss the issues and questions associated with resveratrol becoming an effective in vivo anticancer drug, from basic metabolic issues to the problems faced by incomplete understanding of the mechanism(s) of action in the body. We also explore efforts taken by researchers, both public and private, to contend with some of these issues. By examining the published data and previous clinical trials, we have attempted to identify the problems and issues that hinder the clinical translation of resveratrol for cancer management. This article is part of a Special Issue entitled: Resveratrol: Challenges in translating pre-clinical findings to improved patient outcomes.

Role of PLK1/NUMB/NOTCH in epithelial-mesenchymal transition in human melanoma.

Polo-like kinase 1 (PLK1), a serine/threonine kinase, is overexpressed in melanoma and its expression has been associated with poor disease prognosis. PLK1 has been shown to interact with NUMB, a NOTCH antagonist. However, the exact role of PLK1, NUMB, and NOTCH signaling in epithelial-mesenchymal transition (EMT) in melanoma progression is unclear. In this study, Affymetrix microarray analysis was performed to determine differentially expressed genes following shRNA-mediated knockdown of PLK1 in human melanoma cells that showed significant modulations in EMT and metastasis-related genes. Using multiple PLK1-modulated melanoma cell lines, we found that PLK1 is involved in the regulation of cell migration, invasion, and EMT via its kinase activity and NOTCH activation. In vitro kinase assay and mass spectrometry analysis demonstrated a previously unknown PLK1 phosphorylation site (Ser413) on NUMB. Overexpression of non-phosphorylatable (S413A) and phosphomimetic (S413D) mutants of NUMB in melanoma cells implicated the involvement of NUMB-S413 phosphorylation in cell migration and invasion, which was independent of NOTCH activation. To determine the clinical relevance of these findings, immunohistochemistry was performed using melanoma tissue microarray, which indicated a strong positive correlation between PLK1 and N-cadherin, a protein required for successful EMT. These findings were supported by TCGA analysis, where expression of high PLK1 with low NUMB or high NOTCH or N-cadherin showed a significant decrease in survival of melanoma patients. Overall, these results suggest a potential role of PLK1 in EMT, migration, and invasion of melanoma cells. Our findings support the therapeutic targeting of PLK1, NUMB, and NOTCH for melanoma management.

Potential Tumor Suppressor Role of Polo-like Kinase 5 in Cancer.

The polo-like kinase (PLK) family of serine/threonine kinases contains five members (PLK1-5). Most PLKs are involved in cell cycle regulation and DNA damage response. However, PLK5 is different as it lacks a functional kinase domain and is not involved in cell cycle control. PLK5 remains the least-studied family member, and its role in oncogenesis remains enigmatic. Here, we identified tissues with high PLK5 expression by leveraging the Protein Atlas and GTEx databases with relevant literature and selected ovarian, lung, testis, endometrium, cervix, and fallopian tube tissues as candidates for further investigation. Subsequently, we performed immunohistochemical staining for PLK5 on multiple tissue microarrays followed by Vectra scanning and quantitative inForm analysis. This revealed consistently downregulated PLK5 expression in these cancers compared to normal tissues. To validate and extend our findings, we performed pan-cancer analysis of PLK5 expression using public RNAseq databases (TCGA and GTEx). We found PLK5 is downregulated in 18 cancer types, including our selected candidates. Interestingly, we also observed PLK5 expression remains consistently low in later stages of cancer, suggesting PLK5 may have a greater role in tumor initiation than cancer progression. Overall, our study demonstrates PLK5 downregulation in multiple cancers, highlighting its role as a tumor suppressor.

Antimelanoma Effects of Concomitant Inhibition of SIRT1 and SIRT3 in BrafV600E/PtenNULL Mice.

Novel therapeutic strategies are required for the effective and lasting treatment of metastatic melanoma, one of the deadliest skin malignancies. In this study, we determined the antimelanoma efficacy of 4'-bromo-resveratrol (4'-BR), which is a small-molecule dual inhibitor of SIRT1 and SIRT3, in a BrafV600E/PtenNULL mouse model that recapitulates human disease, including metastases. Tumors were induced by topical application of 4-hydroxy-tamoxifen on shaved backs of mice aged 10 weeks, and the effects of 4'-BR (5‒30 mg/kg of body weight, intraperitoneally, 3 days per week for 5 weeks) were assessed on melanoma development and progression. We found that 4'-BR at a dose of 30 mg/kg significantly reduced the size and volume of primary melanoma tumors as well as lung metastasis with no adverse effects. Furthermore, mechanistic studies on tumors showed significant modulation in the markers of proliferation, survival, and melanoma progression. Because SIRT1 and SIRT3 are linked to immunomodulation, we performed differential gene expression analysis using a PanCancer Immune Profiling Panel (770 genes). Our data showed that 4'-BR significantly downregulated the genes related to metastasis promotion, chemokine/cytokine regulation, and innate/adaptive immune functions. Overall, inhibition of SIRT1 and SIRT3 by 4'-BR is a promising antimelanoma therapy with antimetastatic and immunomodulatory activities warranting further detailed studies, including clinical investigations.

PLK1 inhibition-based combination therapies for cancer management.

Polo-like kinase I (PLK1), a cell cycle regulating kinase, has been shown to have oncogenic function in several cancers. Although PLK1 inhibitors, such as BI2536, BI6727 (volasertib) and NMS-1286937 (onvansertib) are generally well-tolerated with a favorable pharmacokinetic profile, clinical successes are limited due to partial responses in cancer patients, especially those in advanced stages. Recently, combination therapies targeting multiple pathways are being tested for cancer management. In this review, we first discuss structure and function of PLK1, role of PLK1 in cancers, PLK1 specific inhibitors, and advantages of using combination therapy versus monotherapy followed by a critical account on PLK1-based combination therapies in cancer treatments, especially highlighting recent advancements and challenges. PLK1 inhibitors in combination with chemotherapy drugs and targeted small molecules have shown superior effects against cancer both in vitro and in vivo. PLK1-based combination therapies have shown increased apoptosis, disrupted cell cycle, and potential to overcome resistance in cancer cells/tissues over monotherapies. Further, with successes in preclinical experiments, researchers are validating such approaches in clinical trials. Although PLK1-based combination therapies have achieved initial success in clinical studies, there are examples where they have failed to improve patient survival. Therefore, further research is needed to identify and validate novel biologically informed co-targets for PLK1-based combinatorial therapies. Employing a network-based analysis, we identified potential PLK1 co-targets that could be examined further. In addition, understanding the mechanisms of synergism between PLK1 inhibitors and other agents may lead to a better approach on which agents to pair with PLK1 inhibition for optimum cancer treatment.

Protective effects of dietary grape against atopic dermatitis-like skin lesions in NC/NgaTndCrlj mice.

Atopic dermatitis (AD) is a chronic inflammatory skin disease with significant health/economic burdens. Existing therapies are not fully effective, necessitating development of new approaches for AD management. Here, we report that dietary grape powder (GP) mitigates AD-like symptoms in 2,4-dinitrofluorobenzene (DNFB)-induced AD in NC/NgaTndCrlj mice. Using prevention and intervention protocols, we tested the efficacy of 3% and 5% GP-fortified diet in a 13-weeks study. We found that GP feeding markedly inhibited development and progression of AD-like skin lesions, and caused reduction in i) epidermal thickness, mast cell infiltration, ulceration, excoriation and acanthosis in dorsal skin, ii) spleen weight, extramedullary hematopoiesis and lymph nodes sizes, and iii) ear weight and IgE levels. We also found significant modulations in 15 AD-associated serum cytokines/chemokines. Next, using quantitative global proteomics, we identified 714 proteins. Of these, 68 (normal control) and 21 (5% GP-prevention) were significantly modulated (≥2-fold) vs AD control (DNFB-treated) group, with many GP-modulated proteins reverting to normal levels. Ingenuity pathway analysis of GP-modulated proteins followed by validation using ProteinSimple identified changes in acute phase response signaling (FGA, FGB, FGG, HP, HPX, LRG1). Overall, GP supplementation inhibited DNFB-induced AD in NC/NgaTndCrlj mice in both prevention and intervention trials, and should be explored further.

EPAC Regulates Melanoma Growth by Stimulating mTORC1 Signaling and Loss of EPAC Signaling Dependence Correlates with Melanoma Progression.

Exchange proteins directly activated by cAMP (EPAC) belong to a family of RAP guanine nucleotide exchange factors (RAPGEF). EPAC1/2 (RAPGEF3/4) activates RAP1 and the alternative cAMP signaling pathway. We previously showed that the differential growth response of primary and metastatic melanoma cells to cAMP is mediated by EPAC. However, the mechanisms responsible for this differential response to EPAC signaling are not understood. In this study, we show that pharmacologic inhibition or siRNA-mediated knockdown of EPAC selectively inhibits the growth and survival of primary melanoma cells by downregulation of cell-cycle proteins and inhibiting the cell-cycle progression independent of ERK1/2 phosphorylation. EPAC inhibition results in upregulation of AKT phosphorylation but a downregulation of mTORC1 activity and its downstream effectors. We also show that EPAC regulates both glycolysis and oxidative phosphorylation, and production of mitochondrial reactive oxygen species, preferentially in primary melanoma cells. Employing a series of genetically matched primary and lymph node metastatic (LNM) melanoma cells, and distant organ metastatic melanoma cells, we show that the LNM and metastatic melanoma cells become progressively less responsive and refractory to EPAC inhibition suggesting loss of dependency on EPAC signaling correlates with melanoma progression. Analysis of The Cancer Genome Atlas dataset showed that lower RAPGEF3, RAPGEF4 mRNA expression in primary tumor is a predictor of better disease-free survival of patients diagnosed with primary melanoma suggesting that EPAC signaling facilitates tumor progression and EPAC is a useful prognostic marker. These data highlight EPAC signaling as a potential target for prevention of melanoma progression. IMPLICATIONS: This study establishes loss of dependency on EPAC-mTORC1 signaling as hallmark of primary melanoma evolution and targeting this escape mechanism is a promising strategy for metastatic melanoma.

A preclinical and clinical study of lithium in low-grade neuroendocrine tumors.

BACKGROUND: Low-grade neuroendocrine tumors (NETs) respond poorly to chemotherapy; effective, less toxic therapies are needed. Glycogen synthase kinase (GSK)-3ß has been shown to regulate growth and hormone production in NETs. Use of lithium chloride in murine models suppressed carcinoid cell growth, reduced GSK-3ß levels, and reduced expression of chromogranin A. This study assessed the efficacy of lithium chloride in patients with NETs. DESIGN: Eligible patients had low-grade NETs. A single-arm, open-label phase II design was used. Lithium was dosed at 300 mg orally three times daily, titrated to serum levels of 0.8-1.0 mmol/L. The primary endpoint was objective tumor response by the Response Evaluation Criteria in Solid Tumors. Secondary endpoints included overall survival, progression-free survival, GSK-3ß phosphorylation, and toxicity. RESULTS: Fifteen patients were enrolled between October 3, 2007 and July 17, 2008, six men and nine women. The median age was 58 years. Patient diagnoses were carcinoid tumor for eight patients, islet cell tumor for five patients, and two unknown primary sites. Eastern Cooperative Oncology Group performance status scores were 0 or 1. Two patients came off study because of side effects. The median progression-free survival interval was 4.50 months. There were no radiographic responses. Because of an early stopping rule requiring at least one objective response in the first 13 evaluable patients, the study was closed to further accrual. Patients had pre- and post-therapy biopsies. CONCLUSIONS: Lithium chloride was ineffective at obtaining radiographic responses in our 13 patients who were treated as part of this study. Based on the pre- and post-treatment tumor biopsies, lithium did not potently inhibit GSK-3ß at serum levels used to treat bipolar disorders.

The grape antioxidant resveratrol for skin disorders: promise, prospects, and challenges.

Resveratrol, a phytoalexin antioxidant found in red grapes, has been shown to have both chemopreventive and therapeutic effects against many diseases and disorders, including those of the skin. Studies have shown protective effects of resveratrol against ultraviolet radiation-mediated oxidative stress and cutaneous damages including skin cancer. Because many of the skin conditions stem from ultraviolet radiation and oxidative stress, this antioxidant appears to have promise and prospects against a wide range of cutaneous disorders including skin aging and skin cancers. However, there are a few roadblocks in the way of this promising agent regarding its translation from the bench to the bedside. This review discusses the promise and prospects of resveratrol in the management of skin disorders and the associated challenges.

Role of Polo-Like Kinase 4 (PLK4) in Epithelial Cancers and Recent Progress in its Small Molecule Targeting for Cancer Management.

The polo-like kinases (PLKs) are a family of serine/threonine kinases traditionally linked to cell-cycle regulation. A structurally unique member of this family, PLK4, has been shown to regulate centriole duplication during the cell cycle via interactions with a variety of centrosomal proteins. Recent findings suggest that PLK4 is overexpressed in various human cancers and associated with poor cancer prognosis. Although several studies have shown that PLK4 inhibition may lead to cancer cell death, the underlying mechanisms are largely unknown. In this review, we discuss the structure, localization, and function of PLK4, along with the functional significance of PLK4 in epithelial cancers and some preliminary work suggesting a role for PLK4 in the key cancer progression process epithelial-mesenchymal transition. We also discuss the potential of PLK4 as a druggable target for anticancer drug development based on critical analysis of the available data of PLK4 inhibitors in preclinical development and clinical trials. Overall, the emerging data suggest that PLK4 plays an essential role in epithelial cancers and should be further explored as a potential biomarker and/or therapeutic target. Continued detailed exploration of available and next-generation PLK4 inhibitors may provide a new dimension for novel cancer therapeutics following successful clinical trials.

PLK1 and NOTCH Positively Correlate in Melanoma and Their Combined Inhibition Results in Synergistic Modulations of Key Melanoma Pathways.

Melanoma is one of the most serious forms of skin cancer, and its increasing incidence coupled with nonlasting therapeutic options for metastatic disease highlights the need for additional novel approaches for its management. In this study, we determined the potential interactions between polo-like kinase 1 (PLK1, a serine/threonine kinase involved in mitotic regulation) and NOTCH1 (a type I transmembrane protein deciding cell fate during development) in melanoma. Employing an in-house human melanoma tissue microarray (TMA) containing multiple cases of melanomas and benign nevi, coupled with high-throughput, multispectral quantitative fluorescence imaging analysis, we found a positive correlation between PLK1 and NOTCH1 in melanoma. Furthermore, The Cancer Genome Atlas database analysis of patients with melanoma showed an association of higher mRNA levels of PLK1 and NOTCH1 with poor overall, as well as disease-free, survival. Next, utilizing small-molecule inhibitors of PLK1 and NOTCH (BI 6727 and MK-0752, respectively), we found a synergistic antiproliferative response of combined treatment in multiple human melanoma cells. To determine the molecular targets of the overall and synergistic responses of combined PLK1 and NOTCH inhibition, we conducted RNA-sequencing analysis employing a unique regression model with interaction terms. We identified the modulations of several key genes relevant to melanoma progression/metastasis, including MAPK, PI3K, and RAS, as well as some new genes such as Apobec3G, BTK, and FCER1G, which have not been well studied in melanoma. In conclusion, our study demonstrated a synergistic antiproliferative response of concomitant targeting of PLK1 and NOTCH in melanoma, unraveling a potential novel therapeutic approach for detailed preclinical/clinical evaluation.

Mitochondrial Sirtuins in Skin and Skin Cancers.

Mammalian sirtuins (SIRTs 1-7) are a family of NAD+-dependent deacetylases with distinct subcellular localization and biological functions that regulate various important cellular processes. Among these, SIRTs -3, -4 and -5 are located in the mitochondria and have been implicated in caloric restriction, oxidative stress, aging and various human diseases. Emerging evidence has found dysregulation of mitochondrial sirtuins in multiple dermatological conditions, including responses to ultraviolet radiation (UVR), suggesting their importance in maintaining skin health. In this review, we discuss the roles and implications of mitochondrial sirtuins in cutaneous cellular processes, and their emerging potential as a target for the management of skin diseases, including skin cancer. Among mitochondrial sirtuins, SIRT3 is the most studied and linked to multiple skin conditions and diseases (keratinocyte differentiation, wound healing, chronological aging, UVR and ozone response, systemic sclerosis, melanoma, basal cell carcinoma (BCC) and squamous cell carcinoma (SCC)). SIRT4 has been connected to keratinocyte differentiation, chronological aging, UVR response, alopecia, BCC and SCC. Further, SIRT5 has been associated with keratinocyte differentiation, melanoma, BCC and SCC. Overall, while there is compelling evidence for the involvement of mitochondrial sirtuins in skin, additional detailed studies are needed to understand their exact roles in skin and skin cancers.

Quercetin-Resveratrol Combination for Prostate Cancer Management in TRAMP Mice.

Prostate Cancer (PCa) is a leading cause of cancer-related morbidity and mortality in men. Therefore, novel mechanistically-driven approaches are needed for PCa management. Here, we determined the effects of grape antioxidants quercetin and/or resveratrol (60 and 600 mg/kg, respectively, in diet) against PCa in Transgenic Adenocarcinoma of Mouse Prostate (TRAMP)-model in prevention and intervention settings. We found resveratrol alone and in combination significantly inhibited prostate tumorigenesis in prevention setting, while the same was seen only in combination after intervention. The observed effects were associated with marked inhibition in proliferation, oxidative stress, and tumor survival markers, and induced apoptosis markers. Utilizing PCa PCR array analysis with prevention tumor tissues, we identified that quercetin-resveratrol modulates genes involved in promoter methylation, cell cycle, apoptosis, fatty acid metabolism, transcription factors, androgen response, PI3K/AKT and PTEN signaling. Ingenuity Pathway Analysis (IPA) identified IGF1 and BCL2 as central players in two gene networks. Functional annotation predicted increased apoptosis and inhibited cell viability/proliferation, hyperplasia, vasculogenesis, and angiogenesis with dual treatment. Furthermore, IPA predicted upstream inhibition of major PCa signaling VEGF, Ca2+, PI3K, CSF2, PTH). Based on PCR array, we identified decreased levels of EGFR, EGR3, and IL6, and increased levels of IGFBP7 and NKX3.1, overall supporting anti-PCa effects of quercetin-resveratrol.

Protective Effects of Dietary Grape on UVB-Mediated Cutaneous Damages and Skin Tumorigenesis in SKH-1 Mice.

Non-melanoma skin cancers (NMSCs) are the most diagnosed cancers in the US and occur more frequently in males. We previously demonstrated chemoprotective effects of dietary grape powder (GP) against UVB-mediated skin tumorigenesis in female SKH-1 mice. To expand on this, here, we determined the effects of GP in a short-term UVB exposure protocol (0 or 5% GP, followed by UVB every other day for 2 weeks) in male and female SKH-1 mice, as well as explored any sex-related differences in UVB carcinogenesis via male SKH-1 mice (0, 3, or 5% GP; UVB twice weekly for 28 weeks). In the short-term study, we found that GP protects against early-stage epithelial hyperplasia and mast cell infiltration in both sexes. In the long term, GP markedly reduced tumor counts and malignant conversion, along with significant decreases in mast cell infiltration, serum IgE and Eotaxin. We also found inhibition of P38 phosphorylation and reduced PCNA, Ki67 and BCL2 levels, suggesting that the anti-inflammatory effects of GP inhibits P38, acting as an upstream regulator to inhibit proliferation and reduce tumor cell survival. Together, GP appears to protect against UVB-mediated skin damage and carcinogenesis in SKH-1 mice and should be explored further as a supplement for NMSC prevention.

CRISPR/Cas9-mediated Knockout of SIRT6 Imparts Remarkable Antiproliferative Response in Human Melanoma Cells in vitro and in vivo.

Melanoma is one of the most aggressive, potentially fatal forms of skin cancer and has been shown to be associated with solar ultraviolet radiation-dependent initiation and progression. Despite remarkable recent advances with targeted and immune therapeutics, lasting and recurrence-free survival remain significant concerns. Therefore, additional novel mechanism-based approaches are needed for effective melanoma management. The sirtuin SIRT6 appears to have a pro-proliferative function in melanocytic cells. In this study, we determined the effects of genetic manipulation of SIRT6 in human melanoma cells, in vitro and in vivo. Our data demonstrated that CRISPR/Cas9-mediated knockout (KO) of SIRT6 in A375 melanoma cells resulted in a significant (1) decrease in growth, viability and clonogenic survival and (2) induction of G1-phase cell cycle arrest. Further, employing a RT2 Profiler PCR array containing 84 key transformation and tumorigenesis genes, we found that SIRT6 KO resulted in modulation of genes involved in angiogenesis, apoptosis, cellular senescence, epithelial-to-mesenchymal transition, hypoxia signaling and telomere maintenance. Finally, we found significantly decreased tumorigenicity of SIRT6 KO A375 cells in athymic nude mice. Our data provide strong evidence that SIRT6 promotes melanoma cell survival, both in vitro and in vivo, and could be exploited as a target for melanoma management.

Chemoprotective Effects of Dietary Grape Powder on UVB Radiation-Mediated Skin Carcinogenesis in SKH-1 Hairless Mice.

Skin cancer is the most frequently diagnosed cancer in the United States, and solar UVR is an established causative factor for approximately 90% of these cases. Despite efforts aimed at UV protection, including use of sunscreen and clothing, annual cases of skin cancer continue to rise. Here, we report that dietary grape powder mitigates UVB-mediated skin carcinogenesis in an SKH-1 hairless mouse model. Using a UVB initiation-promotion protocol, whereby mice were exposed to 180 mJ/cm2 UVB two times per week for 28 weeks, we determined the effects of a grape powder-fortified diet (3% or 5%) on skin carcinogenesis. Grape powder consumption at both doses resulted in marked inhibition in tumor incidence, as well as a delay in onset of tumorigenesis. Molecular analyses of skin and tumor tissue showed that grape powder-mediated protective response against UVB-induced skin cancer was accompanied by enhanced DNA damage repair, reduced proliferation, increased apoptosis, and modulations in several oxidative stress markers specifically related to inhibition of oxidative stress and increased reactive oxygen species metabolism. NRF2, an activator of cellular antioxidant response, was decreased by grape powder feeding, suggesting a supportive role in tumor cell survival. Overall, our study suggested that dietary grape, containing several antioxidants in natural amalgamation, may protect against UVB-mediated skin carcinogenesis.

Inactivation of glycogen synthase kinase-3beta, a downstream target of the raf-1 pathway, is associated with growth suppression in medullary thyroid cancer cells.

Glycogen synthase kinase-3beta (GSK-3beta) is an important regulator of cell proliferation and survival. Conflicting observations have been reported regarding the regulation of GSK-3beta and extracellular signal-regulated kinase (ERK1/2) in cancer cells. In this study, we found that raf-1 activation in human medullary thyroid cancer cells, TT cells, resulted in phosphorylation of GSK-3beta. Inactivation of GSK-3beta in TT cells with well-known GSK-3beta inhibitors such as lithium chloride (LiCl) and SB216763 is associated with both growth suppression and a significant decrease in neuroendocrine markers such as human achaete-scute complex-like 1 and chromogranin A. Growth inhibition by GSK-3beta inactivation was found to be associated with cell cycle arrest due to an increase in the levels of cyclin-dependent kinase inhibitors such as p21, p27, and p15. Additionally, LiCl-treated TT xenograft mice had a significant reduction in tumor volume compared with those treated with control. For the first time, we show that GSK-3beta is a key downstream target of the raf-1 pathway in TT cells. Also, our results show that inactivation of GSK-3beta alone is sufficient to inhibit the growth of TT cells both in vitro and in vivo.

RNA interference-mediated knockdown of SIRT1 and/or SIRT2 in melanoma: Identification of downstream targets by large-scale proteomics analysis.

Melanoma is the most notorious and fatal of all skin cancers and the existing treatment options have not been proven to effectively manage this neoplasm, especially the metastatic disease. Sirtuin (SIRT) proteins have been shown to be differentially expressed in melanoma. We have shown that SIRTs 1 and 2 were overexpressed in melanoma and inhibition of SIRT1 imparts anti-proliferative responses in human melanoma cells. To elucidate the impact of SIRT 1 and/or 2 in melanoma, we created stable knockdowns of SIRTs 1, 2, and their combination using shRNA mediated RNA interference in A375 human melanoma cells. We found that SIRT1 and SIRT1&2 combination knockdown caused a decreased cellular proliferation in melanoma cells. Further, the knockdown of SIRT 1 and/or 2 resulted in a decreased colony formation in melanoma cells. To explore the downstream targets of SIRTs 1 and/or 2, we employed a label-free quantitative nano-LC-MS/MS proteomics analysis using the stable lines. We found aberrant levels of proteins involved in many vital cellular processes, including cytoskeletal organization, ribosomal activity, oxidative stress response, and angiogenesis. These findings provide clear evidence of cellular systems undergoing alterations in response to sirtuin inhibition, and have unveiled several excellent candidates for future study. SIGNIFICANCE: Melanoma is the deadliest form of skin cancer, due to its aggressive nature, metastatic potential, and a lack of sufficient treatment options for advanced disease. Therefore, detailed investigations into the molecular mechanisms of melanoma growth and progression are needed. In the search for candidate genes to serve as therapeutic targets, the sirtuins show promise as they have been found to be upregulated in melanoma and they regulate a large number of proteins involved in cellular processes known to affect tumor growth, such as DNA damage repair, cell cycle arrest, and apoptosis. In this study, we used a large-scale label-free comparative proteomics system to identify novel protein targets that are affected following knockdown of SIRT1 and/or 2 in A375 metastatic melanoma cell line. Our study offers important insight into the potential downstream targets of SIRTs 1 and/or 2. This may unravel new potential areas of exploration in melanoma research.