Targeting PRPK and TOPK for skin cancer prevention and therapy.
ABSTRACT: Solar ultraviolet (sUV) irradiation is a major environmental carcinogen that can cause inflammation and skin cancer. The costs and morbidity associated with skin cancer are increasing, and therefore identifying molecules that can help prevent skin carcinogenesis is important. In this study, we identified the p53-related protein kinase (PRPK) as a novel oncogenic protein that is phosphorylated by the T-LAK cell-originated protein kinase (TOPK). Knockdown of TOPK inhibited PRPK phosphorylation and conferred resistance to solar-simulated light (SSL)-induced skin carcinogenesis in mouse models. In the clinic, acute SSL irradiation significantly increased epidermal thickness as well as total protein and phosphorylation levels of TOPK and PRPK in human skin tissues. We identified two PRPK inhibitors, FDA-approved rocuronium bromide (Zemuron®) or betamethasone 17-valerate (Betaderm®) that could attenuate TOPK-dependent PRPK signaling. Importantly, topical application of either rocuronium bromide or betamethasone decreased SSL-induced epidermal hyperplasia, neovascularization, and cutaneous squamous cell carcinoma (cSCC) development in SKH1 (Crl: SKH1-Hrhr) hairless mice by inhibiting PRPK activation, and also reduced expression of the proliferation and oncogenesis markers, COX-2, cyclin D1, and MMP-9. This study is the first to demonstrate that targeting PRPK could be useful against sUV-induced cSCC development.
Project description:Nonmelanoma skin cancer (NMSC) such as cutaneous squamous cell carcinoma (cSCC) is caused by solar ultraviolet (SUV) exposure and is the most common cancer in the United States. T-LAK cell-originated protein kinase (TOPK), a serine-threonine kinase is activated by SUV irradiation and involved in skin carcinogenesis. Strategies with research focusing on the TOPK signaling pathway and targeted therapy in skin carcinogenesis may helpful for the discovery of additional treatments against skin cancer. In this study, we found that TOPK can directly bind to and phosphorylate c-Jun (as one of the core member of AP-1) at Ser63 and Ser73 after SSL exposure in a JNKs-independent manner. TOPK knocking down, or HI-TOPK-032 (TOPK specific inhibitor) attenuated colony formation and cell proliferation of skin cancer cells. Phosphorylated levels of c-Jun were overexpressed in human AK and cSCC compared with normal skin tissues, and HI-TOPK-032 inhibited the phosphorylation of c-Jun in SCC cell line in a dose-dependent manner. Furthermore, HI-TOPK-032 decreased SSL-induced AP-1 transactivation activity. Moreover, acute SSL-induced inflammation was attenuated by the topical application of HI-TOPK-032 in SKH1 hairless mice. Importantly, HI-TOPK-032 suppressed chronic SSL-induced skin carcinogenesis and c-Jun phosphorylation levels in SKH1 hairless mice. Our results demonstrate that TOPK can phosphorylate and activate c-Jun at Ser63 and Ser73 in the process of skin carcinogenesis and HI-TOPK-032 could be used as a potential chemopreventive drug against cSCC development.
Project description:Solar ultraviolet (SUV) exposure is a major risk factor in the etiology of cutaneous squamous cell carcinoma (cSCC). People commonly use sunscreens to prevent SUV-induced skin damage and cancer. Nonetheless, the prevalence of cSCC continues to increase every year, suggesting that commercially available sunscreens might not be used appropriately or are not completely effective. In the current study, a solar simulated light (SSL)-induced cSCC mouse model was used to investigate the efficacy of eight commonly used FDA-approved sunscreen components against skin carcinogenesis. First, we tested FDA-approved sunscreen components for their ability to block UVA or UVB irradiation by using VITRO-SKIN (a model that mimics human skin properties), and then the efficacy of FDA-approved sunscreen components was investigated in an SSL-induced cSCC mouse model. Our results identified which FDA-approved sunscreen components or combinations are effective in preventing cSCC development. Not surprisingly, the results indicated that sunscreen combinations that block both UVA and UVB significantly suppressed the formation of cutaneous papillomas and cSCC development and decreased the activation of oncoproteins and the expression of COX-2, keratin 17, and EGFR in SSL-exposed SKH-1 (Crl:SKH1-Hrhr) hairless mouse skin. Notably, several sunscreen components that were individually purported to block both UVA and UVB were ineffective alone. At least one component had toxic effects that led to a high mortality rate in mice exposed to SSL. Our findings provide new insights into the development of the best sunscreen to prevent chronic SUV-induced cSCC development.
Project description:Solar ultraviolet irradiation is an environmental carcinogen that causes skin cancer. Caspase-7 is reportedly expressed at reduced levels in many cancers. The present study was designed to examine the role of caspase-7 in solar-simulated light (SSL)-induced skin cancer and to elucidate its underlying molecular mechanisms. Our study revealed that mice with genetic deficiency of caspase-7 are highly susceptible to SSL-induced skin carcinogenesis. Epidermal hyperplasia, tumor volume and the average number of tumors were significantly increased in caspase-7 knockout (KO) mice compared with SKH1 wild-type mice irradiated with SSL. The expression of cell proliferation markers, such as survivin and Ki-67, was elevated in SSL-irradiated skin of caspase-7 KO mice compared with those observed in SSL-exposed wild-type SKH1 mouse skin. Moreover, SSL-induced apoptosis was abolished in skin from caspase-7 KO mice. Two-dimensional gel electrophoresis, followed by matrix-assisted laser desorption/ionization-time-of-flight analysis of skin tissue lysates from SSL-irradiated SKH1 wild-type and caspase-7 KO mice revealed an aberrant induction of keratin-17 in caspase-7 KO mice. Immunohistochemical analysis of skin tumors also showed an increase of keratin-17 expression in caspase-7 KO mice compared with SKH1 wild-type mice. The expression of keratin-17 was also elevated in SSL-irradiated caspase-7 KO keratinocytes as well as in human basal cell carcinomas. The in vitro caspase activity assay showed keratin-17 as a substrate of caspase-7, but not caspase-3. Overall, our study demonstrates that genetic loss of caspase-7 promotes SSL-induced skin carcinogenesis by blocking caspase-7-mediated cleavage of keratin-17.
Project description:Approximately 90% of all cancer deaths arise from the metastatic dissemination of primary tumors. Metastasis is the most lethal attribute of colorectal cancer. New data regarding the molecules contributing to the metastatic phenotype, the pathways they control and the genes they regulate are very important for understanding the processes of metastasis prognosis and prevention in the clinic. The purpose of this study was to investigate the role of T-LAK cell-originated protein kinase (TOPK) in the promotion of colorectal cancer metastasis. TOPK is highly expressed in human metastatic colorectal cancer tissue compared with malignant adenocarcinoma. We identified p53-related protein kinase (PRPK) as a new substrate of TOPK. TOPK binds with and phosphorylates PRPK at Ser250 in vitro and ex vivo. This site plays a critical role in the function of PRPK. Cell lines stably expressing mutant PRPK (S250A), knockdown TOPK, knockdown PRPK or knockdown of both TOPK and PRPK significantly inhibited liver metastasis of human HCT116 colon cancer cells in a xenograft mouse model. Therefore, we conclude that TOPK directly promotes metastasis of colorectal cancer by modulating PRPK. Thus, these findings may assist in the prediction of prognosis or development of new therapeutic strategies against colon cancer.
Project description:Skin inflammation, and skin cancer induced by excessive solar ultraviolet (SUV) is a great threat to human health. SUV induced skin inflammation through activating p38 mitogen-activated protein kinase (p38) and c-Jun N-termeinal kinases (JNKs). T-LAK cell-originated protein kinase (TOPK) plays an important role in this process. Herein, the clinical data showed TOPK, phospho-p38, phospho-JNKs were highly expressed in human solar dermatitis. Ex vivo studies showed that SUV induced the phosphorylation of p38 and JNKs in HaCat and JB6 cells in a dose and time dependent manner. Molecule docking model indicated cefradine, an FDA-approved cephalosporin antibiotic, directly binds with TOPK. The result of in vitro binding assay verified cefradine can directly bind with TOPK. In vitro kinase results showed cefradine can inhibit TOPK activity. Ex vivo studies further showed cefradine inhibited SUV-induced the phosphorylation level of p38, JNKs and H2AX through inhibiting TOPK activity in a dose and time dependent manner, and cefradine inhibited the secretion of IL6 and TNF-? in HaCat and JB6 cells. In vivo studies showed that cefradine down-regulated SUV-induced the phosphorylation of p38, JNKs and H2AX and inhibited the secretion of IL6 and TNF-? in Babl/c mice. These results indicated that cefradine can inhibit SUV-induced skin inflammation by blocking TOPK signaling pathway, and TOPK is an effective target for suppressing inflammation induced by SUV irradiation.
Project description:Excessive exposure to solar UV (SUV) is related with numerous human skin disorders, such as skin inflammation, photoaging and carcinogenesis. T-LAK cell- originated protein kinase (TOPK), an upstream of p38 mitogen-activated protein kinase (p38) and c-Jun N-terminal kinases (JNKs), plays an important role in SUV -induced skin inflammation, and targeting TOPK has already been a strategy to prevent skin inflammation. In this study, we found that the expression of TOPK, phosphorylation of p38 or JNKs was increased in human solar dermatitis tissues. The level of phosphorylation of p38 or JNKs increased in a dose and time dependent manner in HaCat cells or JB6 Cl41 cells after SUV treatment. Paeonol is an active component isolated from traditional Chinese herbal medicines, and MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2H-tetrazdium) assay showed that it has no toxicity to cells. Microscale thermophoresis (MST) assay showed that paeonol can bind TOPK ex vivo. In vitro kinase assay showed paeonol can inhibit TOPK activity. Ex vivo studies further showed paeonol suppressed SUV-induced phosphorylation level of p38, JNKs, MSK1 and histone H2AX by inhibiting TOPK activity in a time and dose dependent manner. Paeonol inhibited the secretion of IL-6 and TNF-? in HaCat and JB6 cells ex vivo. In vivo studies demonstrated that paeonol inhibited SUV-induced increase of TOPK, the phosphorylation of p38, JNKs and H2AX, and the secretion of IL-6 and TNF-? in Babl/c mouse. In summary, our data indicated a protective role of paeonol against SUV-induced inflammation by targeting TOPK, and paeonol could be a promising agent for the treatment of SUV-induced skin inflammation.
Project description:<h4>Background</h4>Skin cancer is one of the most commonly diagnosed cancers worldwide. The 5-year survival rate of the most aggressive late-stage skin cancer ranges between 20 and 30%. Thus, the discovery and investigation of novel target therapeutic agents that can effectively treat skin cancer is of the utmost importance. The T-lymphokine-activated killer cell-originated protein kinase (TOPK), which belongs to the serine-threonine kinase class of the mitogen-activated protein kinase kinase (MAPKK) family, is highly expressed and activated in skin cancer. The present study investigates the role of 3-deoxysappanchalcone (3-DSC), a plant-derived functional TOPK inhibitor, in suppressing skin cancer cell growth.<h4>Purpose</h4>In the context of skin cancer prevention and therapy, we clarify the effect and mechanism of 3-DSC on different types of skin cancer and solar-simulated light (SSL)-induced skin hyperplasia.<h4>Methods</h4>In an <i>in vitro</i> study, western blotting and <i>in vitro</i> kinase assays were utilized to determine the protein expression of TOPK and its activity, respectively. Pull-down assay with 3-DSC and TOPK (wild-type and T42A/N172 mutation) was performed to confirm the direct interaction between T42A/N172 amino acid sites of TOPK and 3-DSC. Cell proliferation and anchorage-independent cell growth assays were utilized to determine the effect of 3-DSC on cell growth. In an <i>in vivo</i> study, the thickness of skin and tumor size were measured in the acute SSL-induced inflammation mouse model or SK-MEL-2 cell-derived xenografts mouse model treated with 3-DSC. Immunohistochemistry analysis of tumors isolated from SK-MEL-2 cell-derived xenografts was performed to determine whether cell-based results observed upon 3-DSC treatment could be recapitulated <i>in vivo</i>.<h4>Results</h4>3-DSC is able to inhibit cell proliferation in skin cancer cells in an anchorage-dependent and anchorage-independent manner by regulation of TOPK and its related signaling pathway <i>in vitro</i>. We also found that application of 3-DSC reduced acute SSL-induced murine skin hyperplasia. Additionally, we observed that 3-DSC decreased SK-MEL-2 cell-derived xenograft tumor growth through attenuating phosphorylation of TOPK and its downstream effectors including ERK, RSK, and c-Jun.<h4>Conclusions</h4>Our results suggest that 3-DSC may function in a chemopreventive and chemotherapeutic capacity by protecting against UV-induced skin hyperplasia and inhibiting tumor cell growth by attenuating TOPK signaling, respectively.
Project description:Cutaneous squamous cell carcinoma (cSCC) is the most common type of non-melanoma skin cancer that can metastasize. The major etiological factor associated with cSCC is Ultraviolet radiation (UVR) with a limited understanding of its molecular mechanism. It was hypothesized that there is a direct effect of UVR on modulation of microRNAs (miRNAs), a novel class of short noncoding RNAs which affects translation and stability of mRNAs. To test the hypothesis, the dorsal skin of the SKH1 mice (6-7 week old) was exposed to acute and chronic doses of UVR. In miRNA array profiling, we found differential expression (log fold change>1) of miR-25-5p between untreated and acute UVR treated (4kJ/m2) SKH1 mice skin. However, differential expression (>1 log fold) of miR-144-3p, miR-33-5p, miR-32-5p, miR-1983, miR-136-5p, miR-142-3p, miR-376a-3p, miR-142-5p, miR-3968, and miR-29b-3p was observed between untreated and chronically UVR treated mice skin. Differentially expressed selected miRNAs (miR-32-5p, miR-33-5p, miR-144-3p, and miR-376a-3p) were further validated in real time PCR using miRNA specific primers. Web based data mining, for the prediction of potential miRNA associated gene pathways in miRBase database revealed a link with important pathways (PI3K-Akt, MAPK, Wnt, transcriptional misregulation, and other oncogenic pathway) associated with cSCC. Furthermore, findings of PI3K-Akt pathway genes affected due to chronic UVR were confirmed using cDNA array.