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The biological functions of the p53-related protein kinase (PRPK) remain unclear. We have previously demonstrated that PRPK is phosphorylated by the T-LAK cell-originated protein kinase (TOPK) and that phosphorylated PRPK (p-PRPK) promotes colon cancer metastasis. Here, we analyzed colon adenocarcinomas from 87 patients and found that higher expression levels of p-PRPK were associated with later stages of metastatic dissemination (stage III and IV) as compared with earlier stages (stages I and II). Indeed, levels of p-PRPK were higher in metastatic versus malignant human colon adenocarcinomas. Knocking down PRPK expression attenuated colorectal liver and lung metastasis of colon cancer cells in vivo. An in vitro kinase assay indicated that active PRPK does not phosphorylate p53 directly. We found that PRPK phosphorylates survivin, a regulator of colon cancer metastasis. PRPK phosphorylates survivin at Thr34, which is important for survivin stability. Taken together, our data strongly suggest that the PRPK signaling pathway promotes colon cancer metastasis by modulating survivin stability, and that PRPK could be a new prognostic marker for the survival of colon cancer patients. In addition, we identified an FDA-approved bacteriostatic antibiotic, fusidic acid sodium salt (fusidic acid or FA) as an inhibitor of PRPK, and show that FA combined with 5-fluorouracil (5-FU) inhibited PRPK activity and colon cancer metastasis to the lung in mice. We contend that the combination of FA with 5-FU could be an alternative therapeutic strategy to traditional chemotherapy for colon cancer patients with poor prognosis. Mol Cancer Ther; 17(5); 1101–13. ©2018 AACR.

Inflammation is a complex biological host reaction to tissue damage, infection and trauma. Extensive study of the inflammatory response has led to the identification of several protein kinases that are essential for signaling and could be potential therapeutic targets. The RSK family of kinases has multiple cellular functions. In our study, we found that RSK2 is a mediator for inflammation signaling and interacts with TRAF6. In vitro kinase assay results indicated that RSK2 strongly phosphorylates TRAF6 at serines 46, 47 and 48. Ectopic overexpression of TRAF6 or knocking down RSK2 expression confirmed that RSK2 is a positive regulator of TRAF6 K63 ubiquitination. TRAF6 is also required for RSK2 ubiquitination. TRAF6 bridges the TNF receptor superfamily and intracellular signaling for the induction of proinflammatory cytokines. We developed a colon inflammation model using RSK2 wild type (WT) and knockout (KO) mice. As expected, F4/80 and CD3 infiltration were significantly upregulated in WT mice compared to RSK2 KO mice. Furthermore, inflammation signaling, including Ikkα/β, p38 and JNKs, was dramatically upregulated in WT mice. Colon tissue immunoprecipitation results further confirmed that TRAF6 K63 ubiquitination was lower in RSK2 KO mice. Overall, these results indicate that phosphorylation of TRAF6 (S46, 47, 48) by RSK2 is required for TRAF6 K63 ubiquitination and inflammation signaling.

Metastasis is a major cause of cancer-related deaths. Approximately 80% of patients with colorectal cancer develop liver metastasis and 20% develop lung metastasis. We found that at different stages of colon cancer, IFNγ secretion from peripheral blood mononuclear cells was decreased compared with healthy controls. The ribosomal S6 kinase (RSK) family of kinases has multiple cellular functions, and we examined their roles in this observed IFNγ decrease. Flow cytometry analysis of wild-type (WT) and RSK2 knockout (KO) mice revealed significantly lower levels of IFNγ in the RSK2 KO mice compared with the WT mice. Since IFNγ is a component of immunity, which contributes to protection against metastatic carcinomas, we conducted a colon cancer liver metastasis experiment. We found significantly greater metastasis in RSK2 KO mice compared with WT mice. Transcription factor T-bet can directly activate Ifnγ gene transcription. In vitro kinase assay results showed that RSK2 phosphorylated T-bet at serines 498 and 502. We show that phosphorylation of T-bet by RSK2 is required for IFNγ expression, because knockdown of RSK2 expression or overexpression of mutant T-bet reduces IFNγ mRNA expression. To verify the function of the phosphorylation sites, we overexpressed a constitutively active mutant T-bet (S498E/S502E) in bone marrow. Mutant T-bet restored the IFNγ mRNA levels and dramatically reduced the metastasis rate in these mice. Overall, these results indicate that phosphorylation of T-bet is required for the inhibition of colon cancer metastasis and growth through a positive regulation of RSK2/T-bet/IFNγ signaling.

Cyclin-dependent kinase 2 (CDK2) is a known regulator in the cell cycle control of the G1/S and S/G2 transitions. However, the role of CDK2 in tumorigenesis is controversial. Evidence from knockout mice as well as colon cancer cell lines indicated that CDK2 is dispensable for cell proliferation. In this study, we found that ectopic CDK2 enhances Ras (G12V)-induced foci formation and knocking down CDK2 expression markedly decreases epidermal growth factor (EGF)-induced cell transformation mediated through the downregulation of c-fos expression. Interestingly, CDK2 directly phosphorylates ELK4 at Thr194 and Ser387 and regulates the ELK4 transcriptional activity, which serves as a mechanism to regulate c-fos expression. In addition, ELK4 is overexpressed in melanoma and knocking down the ELK4 or CDK2 expression significantly attenuated the malignant phenotype of melanoma cells. Taken together, our study reveals a novel function of CDK2 in EGF-induced cell transformation and the associated signal transduction pathways. This indicates that CDK2 is a useful molecular target for the chemoprevention and therapy against skin cancer.

The c-Jun N-terminal kinases (JNK) play an important role in many physiologic processes induced by numerous stress signals. Each JNK protein appears to have a distinct function in cancer, diabetes, or Parkinson's disease. Herein, we found that licochalcone A, a major phenolic constituent isolated from licorice root, suppressed JNK1 activity but had little effect on JNK2 in vitro activity. Although licochalcone A binds with JIP1 competitively with either JNK1 or JNK2, a computer simulation model showed that after licochalcone A binding, the ATP-binding cleft of JNK1 was distorted more substantially than that of JNK2. This could reduce the affinity of JNK1 more than JNK2 for ATP binding. Furthermore, licochalcone A inhibited JNK1-mediated, but not JNK2-mediated, c-Jun phosphorylation in both ex vivo and in vitro systems. We also observed that in colon and pancreatic cancer cell lines, JNK1 is highly expressed compared with normal cell lines. In cancer cell lines, treatment with licochalcone A or knocking down JNK1 expression suppressed colon and pancreatic cancer cell proliferation and colony formation. The inhibition resulted in G1 phase arrest and apoptosis. Moreover, an in vivo xenograft mouse study showed that licochalcone A treatment effectively suppressed the growth of HCT116 xenografts, without affecting the body weight of mice. These results show that licochalcone A is a selective JNK1 inhibitor. Therefore, we suggest that because of the critical role of JNK1 in colon cancer and pancreatic carcinogenesis, licochalcone A might have preventive or therapeutic potential against these devastating diseases. Cancer Prev Res; 7(1); 139–49. ©2013 AACR.

Histone H2AX is a histone H2A variant that is ubiquitously expressed throughout the genome. It plays a key role in the cellular response to DNA damage and has been designated as the histone guardian of the genome. Histone H2AX deficiency decreases genomic stability and increases tumor susceptibility of normal cells and tissues. However, the role of histone H2AX phosphorylation in malignant transformation and cancer development is not totally clear. Herein, we found that ribosomal S6 kinase 2 (RSK2) directly phosphorylates histone H2AX at Ser139 and also at a newly discovered site, Ser16. Epidermal growth factor (EGF)–induced phosphorylation of histone H2AX at both sites was decreased in RSK2 knockout cells. Phosphorylated RSK2 and histone H2AX colocalized in the nucleus following EGF treatment, and the phosphorylation of histone H2AX by RSK2 enhanced the stability of histone H2AX and prevented cell transformation induced by EGF. RSK2 and DNA-PK, but not ATM or ATR, are required for EGF-induced phosphorylation of H2AX at Ser139; however, only RSK2 is required for phosphorylation of H2AX at Ser16. Phosphorylation of histone H3 was suppressed in cells expressing wild-type H2AX compared with H2AX knockout (H2AX−/−) cells. EGF-associated AP-1 transactivation activity was dramatically lower in H2AX−/− cells overexpressing wild-type H2AX than H2AX−/− cells expressing mutant H2AX-AA. Thus, the RSK2/H2AX signaling pathway negatively regulates the RSK2/histone H3 pathway and therefore maintains normal cell proliferation. Cancer Res; 71(2); 393–403. ©2011 AACR.

The oncoprotein c-Jun is one of the components of the activator protein-1 (AP-1) transcription factor complex. AP-1 regulates the expression of many genes and is involved in a variety of biological functions such as cell transformation, proliferation, differentiation and apoptosis. AP-1 activates a variety of tumor-related genes and therefore promotes tumorigenesis and malignant transformation. Here, we found that epidermal growth factor (EGF) induces phosphorylation of c-Jun by P21-activated kinase (PAK) 2. Our data showed that PAK2 binds and phosphorylates c-Jun at five threonine sites (Thr2, Thr8, Thr89, Thr93 and Thr286) in vitro and ex vivo. Knockdown of PAK2 in JB6 Cl41 (P+) cells had no effect on c-Jun phosphorylation at Ser63 or Ser73 but resulted in decreases in EGF-induced anchorage-independent cell transformation, proliferation and AP-1 activity. Mutation at all five c-Jun threonine sites phosphorylated by PAK2 decreased the transforming ability of JB6 cells. Knockdown of PAK2 in SK-MEL-5 melanoma cells also decreased colony formation, proliferation and AP-1 activity. These results indicated that PAK2/c-Jun signaling plays an important role in EGF-induced cell proliferation and transformation.

Metastasis is the major cause of death for patients with solid malignancies. Approximately 80 and 20% of colorectal cancer (CRC) patients develop liver metastasis and lung metastasis, respectively. We found that at different stages of colon cancer, the patients' interferon (IFN) γ secretion from peripheral blood mononuclear cells (PBMCs), was decreased compared with healthy people. The ribosomal S6 kinase (RSK) family of kinases has multiple cellular functions, and we examined their role in this observed IFNγ decrease. Flow cytometry analysis of wild-type (WT) and RSK2 knockout (KO) mice, revealed that the level of IFNγ was significantly lower in RSK2 KO mice as compared to WT mice. Since IFNγ is a component of immunity, which contributes to protection against metastatic carcinomas, a colon cancer liver metastasis experiment was conducted. We found that metastasis was significantly increased in RSK2 KO mice, compared to WT mice. Transcription factor T-bet can directly activate Ifnγ gene transcription. In vitro kinase assay results showed that RSK2 phosphorylated T-bet at serines 498 and 502. We show that phosphorylation of T-bet by RSK2 is required for IFNγ expression, because knocking down RSK2 expression or over-expressing T-bet S498A/S502A mutants reduces IFNγ mRNA expression. To verify the function of the phosphorylation sites, a constitutively active mutant T-bet (S498E/S502E) was over-expressed in bone marrow. Mutant T-bet restored the IFNγ mRNA levels and dramatically reduced the metastasis rate in these mice. Overall, these results indicate that phosphorylation of T-bet is required for inhibiting colon cancer metastasis and growth through a positive regulation of RSK2/T-bet/IFNγ signaling. Citation Format: Ke Yao, Cong Peng, Yuwen Zhang, Tatyana A. Zykova, Mee-Hyun Lee, Hanyong Chen, Joohyun Ryu, Enyu Rao, Wei-Ya Ma, kangdong liu, Ann M. Bode, Bing Li, zigang dong. RSK2 phosphorylates T-bet to attenuate colon cancer metastasis and growth [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 2127.

The ribosomal S6 kinase 2 (RSK2) is a well-known serine/threonine kinase and a member of the p90 ribosomal S6 kinase (p90RSK) family of proteins. It is activated downstream of the MEK/ERKs cascade by mitogenic stimuli such as EGF or TPA. Here, we show that RSK2 is activated by treatment with tumor necrosis factor-α (TNF-α) and directly phosphorylates IκBα at Ser-32, leading to IκBα degradation. The phosphorylation of IκBα promotes the activation and translocation of the nuclear factor-κB (NF-κB) subunits p65 and p50 to the nucleus. The net result is an increased NF-κB activity, which serves as a mechanism for RSK2 blockade of TNF-α-induced apoptosis and enhanced cell survival.—Peng, C., Cho, Y.-Y., Zhu, F., Xu, Y.-M., Wen, W., Ma, W.-Y., Bode, A. M., Dong, Z. RSK2 mediates NF-κB activity through the phosphorylation of IκBα in the TNF-R1 pathway.

(-)-Epigallocatechin-3-gallate (EGCG) is a well-known chemoprevention factor. Recent studies have revealed that EGCG triggers cancer cells undergoing apoptosis through p53-dependent pathway. How EGCG activates p53-dependent apoptosis is not fully understood. In the present study using JB6 cell as a model system, we have shown that EGCG can negatively regulate protein serine/threonine phosphatase-2A (PP-2A) to positively regulate p53-dependent apoptosis. First, EGCG at physiologic levels down-regulates PP-2A at the protein and enzyme activity levels. Second, EGCG induces apoptosis of JB6 cells, which is associated with hyperphosphorylation of p53 and up-regulation of the proapoptotic gene, Bak. DNA sequence analysis, gel mobility shifting, chromatin immunoprecipitation, and reporter gene activity assays revealed that p53 directly controls Bak in JB6 cells. Knockdown of p53 and Bak expression with RNAi substantially inhibits EGCG-induced apoptosis. Third, PP-2A directly interacts with p53 and dephosphorylates p53 at Ser-15 in vitro and in vivo. Fourth, overexpression of the catalytic subunit for PP-2A down-regulates p53 phosphorylation at Ser15, attenuates expression of the downstream proapoptotic gene, Bak, and antagonizes EGCG-induced apoptosis. Inhibition of PP-2A activity enhances p53 phosphorylation at Ser-15 and up-regulates Bak expression to promote EGCG-induced apoptosis. Finally, in the p53−/− H1299 and p53+/+ H1080 cells, EGCG down-regulates PP-2A similarly but induces differential apoptosis. In summary, our results show that (a) PP-2A directly dephosphorylates p53 at Ser-15; (b) P53 directly controls Bak expression; and (c) EGCG negatively regulates PP-2A. Together, our results show that EGCG-mediated negative regulation of PP-2A is an important molecular event for the activation of p53-dependent apoptosis during its chemoprevention. [Cancer Res 2008;68(11):4150–62]

The c-jun-NH2-kinases (JNK) play a critical role in tumor promoter–induced cell transformation and apoptosis. Here, we showed that the nuclear factor of activated T3 (NFAT3) is phosphorylated by JNK1 or JNK2 at Ser213 and Ser217, which are located in the conserved SP motif. The transactivation domain of NFAT3 is found between amino acids (aa) 113 and 260 and includes the phosphorylation targets of JNK1 and JNK2. NFAT3 transactivation activity was suppressed in JNK1−/− or JNK2−/− mouse embryonic fibroblast (MEF) cells compared with wild-type MEF cells. Moreover, a 3xNFAT-luc reporter gene assay indicated that NFAT3 transcriptional activity was increased in a dose-dependent manner by JNK1 or JNK2. Double mutations at Ser213 and Ser217 suppressed NFAT3 transactivation activity; and SP600125, a JNK inhibitor, suppressed NFAT3-induced 3xNFAT-luciferase activity. Knockdown of JNK1 or JNK2 suppressed foci formation in NIH3T3 cells. Importantly, ectopic expression of NFAT3 inhibited AP-1 activity and suppressed foci formation. Furthermore, knockdown of NFAT3 enhanced Ras-JNK1 or JNK2-induced foci formation in NIH3T3 cells. Taken together, these results provided direct evidence for the anti-oncogenic potential of the NFAT3 transcription factor. [Cancer Res 2007;67(18):8725–35]

The oncoprotein c-Jun is a component of the activator protein-1 transcription factor complex, which is involved in cellular proliferation, transformation, and death. The stabilization of c-Jun is critically important for its function. The phosphorylation of c-Jun by c-Jun NH2-terminal kinase 1 and extracellular signal-regulated protein kinases reduces c-Jun ubiquitination resulting in increased stabilization of c-Jun. In this report, we showed that COOH-terminal Src kinase (CSK) binds with and phosphorylates c-Jun at Y26 and Y170. Phosphorylation of c-Jun by CSK, in opposition to c-Jun NH2-terminal kinase 1 and extracellular signal-regulated protein kinases, promoted c-Jun degradation and reduced stability. By promoting c-Jun degradation, CSK helps to maintain a low steady-state level of c-Jun, thereby inhibiting activator protein-1 activity and cell transformation caused by c-Jun. These results indicated that this function of CSK controls cell proliferation under normal growth conditions and may have implications for CSK loss of function in carcinogenesis. (Cancer Res 2006; 66(11): 5729-36)

Epidemiologic investigations demonstrated that arsenite exposure increases the risk of various human cancers, including skin, lung, bladder, and kidney cancers. However, oral administration of arsenite alone has failed to induce tumors in animal models, suggesting that arsenic may act to enhance mutagenicity induced by other carcinogens. Arsenite may function as a co-carcinogen, acting by inhibiting repair of carcinogen-induced DNA damage mediated by p53 and p21, a p53 target gene. To elucidate the interaction between arsenite and p53 tumor suppressor protein, we studied the effect of arsenite on ultraviolet B (UVB)-induced p53 phosphorylation, p53 DNA binding activity, and p53-induced target gene transactivation in the JB6 Cl41 mouse epidermal skin cell model. Our results indicated that arsenite suppressed UVB-induced p53 phosphorylation and p53 DNA binding activity. Arsenite also inhibited casein kinase 2 (CK2) activity and decreased p53-regulated p21 protein expression. These data suggest that the direct inhibition of p53 functional activation is one of the mechanisms through which arsenite interferes with p53 function, and thus may be a significant mechanism for the co-carcinogenic effects of arsenite.

Black tea is one of the most popular beverages worldwide and especially in Western nations. Theaflavins, a mixture of theaflavin (TF-1), theaflavin-3-gallate (TF-2a), theaflavin-3'-gallate (TF-2b), and theaflavin-3,3'-digallate (TF-3) are the major components of black tea. Among these black tea components, theaflavin is generally considered to be the more effective component for the inhibition of carcinogenesis. Recently, TF-3 has been shown to have an antiproliferative effect on tumor cells, but the mechanism is not clear. In this study, we showed that TF-3-induced internalization and downregulation of the epidermal growth factor receptor (EGFR). These results suggested that TF-3 induces EGFR endocytosis and degradation. We further showed that TF-3 stimulated EGFR ubiquitination and tyrosine kinase activation. Interestingly, TF-3-induced EGFR downregulation is inhibited by the proteasome inhibitor, MG132, but not by the EGFR-specific receptor tyrosine kinase inhibitor, AG1478. Furthermore, pretreatment with TF-3 inhibited EGF-induced EGFR autophosphorylation, ERKs phosphorylation and AP-1 activation in JB6 Cl41 cells. In addition, TF-3 inhibited EGF-induced anchorage-independent cell transformation. Overall, our results indicate that TF-3 might exert chemopreventive effects through the downregulation of the EGFR.

Inactivation of the p16(INK4a) tumor suppressor protein is critical for the development of human cancers, including human melanoma. However, the molecular basis of the protein's inhibitory effect on cancer development is not clear. Here we investigated a possible mechanism for p16(INK4a) inhibition of neoplastic transformation and UV-induced skin cancer. We show that p16(INK4a) suppresses the activity of c-Jun N-terminal kinases (JNKs) and that it binds to the glycine-rich loop of the N-terminal domain of JNK3. Although p16(INK4a) does not affect the phosphorylation of JNKs, its interaction with JNK inhibits c-Jun phosphorylation induced by UV exposure. This, in turn, interferes with cell transformation promoted by the H-Ras-JNK-c-Jun-AP-1 signaling axis.

The tumor suppressor p53 protein is one of the most highly connected nodes in cellular signal transduction pathways and acts as a central regulatory switch in networks controlling cell proliferation and apoptosis. It is involved in the activation of genes that maintain control over cellular responses to DNA errors such as DNA repair, chromosomal recombination, and chromosome segregation. Here we show that ribosomal S6 kinase 2 (RSK2) activates and phosphorylates p53 (Ser15) in vitro and in vivo and colocalizes with p53 in the nucleus. Deficiency of p53 diminishes RSK2-mediated phosphorylation of histone H3 (Ser10) and adding back p53 to p53−/− embryonic fibroblasts restored phosphorylation of histone H3 at Ser10. These results show that the p53 protein is an important substrate of RSK2 and a critical intermediary in the RSK2 and histone H3 interaction. The RSK2-p53-histone H3 complex may likely contribute to chromatin remodeling and cell cycle regulation.

Ultraviolet B (UVB) induces phosphorylation of histone H3 at serine 10, and mitogen-activated protein kinases are involved in this signal transduction pathway. Here we provide evidence that Fyn kinase, a member of the Src kinase family, is involved in the UVB-induced phosphorylation of histone H3 at serine 10. UVB distinctly increased Fyn kinase activity and phosphorylation. Fyn kinase inhibitors 4-amino-5-(4-chlorophenyl)-7(t-butyl)pyrazol(3,4-d)pyramide and leflunomide, an Src kinase inhibitor, suppressed both UVB-induced phosphorylation of histone H3 at serine 10 and Fyn kinase activity and phosphorylation. UVB-induced phosphorylation of histone H3 at serine 10 was blocked by either a dominant-negative mutant of Fyn (DNM-Fyn) kinase or small interfering RNA of Fyn kinase. UVB-induced phosphorylation and activities of ERKs and protein kinase B/Akt were markedly inhibited by DNM-Fyn kinase. However, DNM-Fyn kinase did not inhibit UVB-induced phosphorylation of p38 MAPK or c-Jun N-terminal kinases. Active Fyn kinase phosphorylated histone H3 at serine 10 in vitro, and the phosphorylated Fyn kinase could translocate into the nucleus of HaCaT cells. These results indicate that Fyn kinase plays a key role in the UVB-induced phosphorylation of histone H3 at serine 10.

金沢大学医学部附属病院薬剤部, Our previous study demonstrated that phosphatidylinositol 3-kinase (P13K) is necessary for epidermal growth factor (EGF)-induced cell transformation in mouse epidermal JB6 cells. Akt and the mammalian target of rapamycin (mTOR) are regarded as P13K downstream effectors. Therefore, in this study, we investigated the role of Akt and mTOR on EGF-induced cell transformation in JB6 cells using rapamycin, a specific mTOR inhibitor, and cells expressing dominant negative mutants of Akt1 (DNM-Akt1). We found that the treatment of cells with rapamycin inhibited EGF-induced cell transformation but only slightly inhibited JB6 cell proliferation at 72 h. Although LY294002, a P13K inhibitor, attenuated EGF-induced activator protein 1 (AP-1) activation, treatment with rapamycin did not affect AP-1 activity. Treatment with rapamycin inhibited EGF-induced phosphorylation and activation of ribosomal p70 S6 protein kinase (p70 S6K), an mTOR downstream target, but had no effect on phosphorylation and activation of Akt. Rapamycin also had no effect on EGF-induced phosphorylation of extracellular signal-regulated protein kinases (ERKs). We showed that introduction of DNM-Akt1 into JB6 mouse epidermal Cl 41 (JB6 Cl 41) cells inhibits EGF-induced cell transformation without blocking cell proliferation. The expression of DNM-Akt1 also suppressed EGF-induced p70 S6K activation as well as Akt activation. These results indicated an involvement of the Akt/mTOR pathway in EGF-induced cell transformation in JB6 cells. © 2005 Wiley-Liss, Inc.

Recently, blockage of p53-dependent transcriptional activation and apoptosis by pifithrin-alpha (PFTalpha) has been reported to be useful for reducing the side effects of cancer therapy and the compound is thus thought to be a specific inhibitor of p53 [Komarov et al., Science 1999;285:1733-1737]. Here, we found that PFTalpha did not inhibit UVB- or doxorubicin (Dox)-stimulated p53-mediated transcriptional activation and apoptosis in JB6 cells. Instead, p53-dependent activation and apoptosis were not only induced by PFTalpha itself but were also enhanced by a combination of PFTalpha with UVB or Dox. Furthermore, PFTalpha-induced apoptosis was mediated through p53-dependent and -independent signaling pathways. Extracellular signal-regulated kinases and p38 kinase, but not c-jun N-terminal kinases (JNKs), were activated, and these activations were required for phosphorylation and accumulation of p53 in the cellular apoptotic response to PFTalpha. Thus, we conclude that PFTalpha is not a specific p53 inhibitor in JB6 cells but is a potential activator of p53-mediated signaling and apoptosis.

Cyclooxygenases (COX) are rate-limiting enzymes that catalyze the conversion of arachidonic acid to prostaglandins, which are involved in many physiological and pathophysiological responses. COX-2, one of two isoforms of COX, was recently found to play an important role in carcinogenesis in many cell and tissue types. COX-2 inhibitors, which belong to the family of nonsteroidal anti-inflammatory drugs, are believed to be effective in many biological activities such as tumor chemoprevention because of their inhibition of COX-2. However, in the present study we found that both piroxicam, a general COX inhibitor, and NS-398, a COX-2 selective inhibitor, effectively suppressed the activation of transcription factor activator protein 1 (AP-1) induced by ultraviolet B (UVB) or 12-O-tetradecanoylphorbol-13-acetate in mouse epidermal JB6 cells. These COX-2 inhibitors could also inhibit 12-O-tetradecanoylphorbol-13-acetate-induced cell transformation. UVB significantly increased AP-1 activity in Cox-2(-/-) fibroblasts transfected with an AP-1 luciferase reporter gene, and this increase was blocked by NS-389 or piroxicam. In JB6, Cox-2(-/-), or wild-type Cox-2(+/+) cells, both NS-398 and piroxicam inhibited UVB-induced phosphorylation of c-Jun NH(2)-terminal kinases, the kinases that activate the AP-1/c-Jun complex. Based on our results, we propose that the inhibition of AP-1 activity by COX-2 inhibitors NS-398 or piroxicam may occur by a mechanism that is independent of COX-2.

Solar UVA, but not UVC, reaches the earth's surface and therefore is an important etiological factor for the induction of human skin cancer. ATM kinase is an important regulator of cell survival and cell cycle checkpoints. Here, we observe that UVA, unlike UVC, triggers ATM kinase activity, and the activation may occur through reactive oxygen species produced after irradiation of cells with UVA. We also show that ATM activation is involved in the apoptotic response to UVA but not UVC. Furthermore, we provide evidence that ATM-dependent p53 and c-Jun N-terminal kinase (JNK) pathways are linked to UVA-induced apoptosis. On the other hand, UVC-induced apoptosis occurs through ATR-dependent p53 phosphorylation as well as the JNK pathway. Therefore, these results suggest that ATM, like p53, is involved in the UVA-induced apoptosis to suppress carcinogenesis.

Solar UV (SUV) irradiation is a major factor in skin carcinogenesis, the most common form of cancer in the United States. The MAPK cascades are activated by SUV irradiation. The 90 kDa ribosomal S6 kinase (RSK) and mitogen and stress-activated protein kinase (MSK) proteins constitute a family of protein kinases that mediate signal transduction downstream of the MAPK cascades. In this study, phosphorylation of RSK and MSK1 was upregulated in human squamous cell carcinoma (SCC) and SUV-treated mouse skin. Kaempferol, a natural flavonol, found in tea, broccoli, grapes, apples, and other plant sources, is known to have anticancer activity, but its mechanisms and direct target(s) in cancer chemoprevention are unclear. Kinase array results revealed that kaempferol inhibited RSK2 and MSK1. Pull-down assay results, ATP competition, and in vitro kinase assay data revealed that kaempferol interacts with RSK2 and MSK1 at the ATP-binding pocket and inhibits their respective kinase activities. Mechanistic investigations showed that kaempferol suppresses RSK2 and MSK1 kinase activities to attenuate SUV-induced phosphorylation of cAMP-responsive element binding protein (CREB) and histone H3 in mouse skin cells. Kaempferol was a potent inhibitor of SUV-induced mouse skin carcinogenesis. Further analysis showed that skin from the kaempferol-treated group exhibited a substantial reduction in SUV-induced phosphorylation of CREB, c-Fos, and histone H3. Overall, our results identify kaempferol as a safe and novel chemopreventive agent against SUV-induced skin carcinogenesis that acts by targeting RSK2 and MSK1. Cancer Prev Res; 7(9); 958–67. ©2014 AACR.

The mechanism of 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced tumor promotion is still not well understood even though it is thought to be related to the protein kinase C/mitogen-activated protein kinase/AP-1 pathway. Recently, TPA was also found to induce epidermal growth factor receptor (EGFR) activity. Here, we investigated whether the EGFR is a necessary component for TPA-induced signal transduction associated with tumor promotion. We demonstrated that potent inhibitors of the EGFR, PD153035 and AG1478, blocked TPA-induced phosphorylation of extracellular signal-regulated kinases (ERKs), AP-1 activity, and cell transformation. Egfr gene deficiency blocked TPA-induced ERK activity and AP-1 binding activity. The blocking of the ectodomain of the EGFR by a monoclonal antibody depressed TPA-induced ERK activity and AP-1 DNA binding activity. The use of a neutralizing antibody for heparin-binding EGF, one of the ligands of EGFR, blocked TPA-induced phosphorylation of ERKs. BB-94, a potent inhibitor of matrix metalloproteinases, which are activators of ectodomain shedding of EGFR ligands, also blocked TPA-induced ERK activity, AP-1 DNA binding, and cell transformation but had no effect on EGF-induced signal transduction. Anti-EGFR, anti-heparin-binding EGF, and BB-94 each blocked TPA-induced EGFR phosphorylation, but only anti-EGFR could block EGF-induced EGFR phosphorylation. Based on these results, we conclude that the EGFR is required for mediating TPA-induced signal transduction. EGFR transactivation induced by TPA is a mechanism by which the EGFR mediates TPA-induced tumor promotion-related signal transduction.

The sphingomyelin-ceramide pathway is an evolutionarily conserved ubiquitous signal transduction system that regulates many cell functions including apoptosis. Sphingomyelin (SM) is hydrolyzed to ceramide by different sphingomyelinases. Ceramide serves as a second messenger in mediating cellular effects of cytokines and stress. In this study, we find that acid sphingomyelinase (SMase) activity was induced by UVA in normal JY lymphoblasts but was not detectable in MS1418 lymphoblasts from Niemann-Pick type D patients who have an inherited deficiency of acid SMase. We also provide evidence that UVA can induce apoptosis by activating acid SMase in normal JY cells. In contrast, UVA-induced apoptosis was inhibited in MS1418 cells. Exogenous SMase and its product, ceramide (10-40 micrometer), induced apoptosis in JY and MS1418 cells, but the substrate of SMase, SM (20-80 micrometer), induced apoptosis only in JY cells. These results suggest that UVA-induced apoptosis by SM is dependent on acid SMase activity. We also provide evidence that induction of apoptosis by UVA may occur through activation of JNKs via the acid SMase pathway.

Activator protein-1 (AP-1), a dimeric complex consisting of proteins encoded by the junand fos gene families, is a transcription factor induced by a variety of signals including those eliciting proliferation, differentiation, and neoplastic transformation. Although AP-1 has been widely studied in the last decade, physiological levels of AP-1 in different tissues are unclear. In the present study, we analyzed AP-1 activity in several organs (liver, kidney, brain, lung, spleen, heart, skin) of AP-1-luciferase transgenic mice of various ages. Results of these studies indicate that the level of AP-1 in young mice is much higher than that in older mice, and, second, that the skin contains considerably higher levels of AP-1 than other organs. The level of phosphorylated extracellular signal-regulated protein kinase (ERK) in skin was higher in 1- and 2-day-old mice than in mice of other ages. In addition, phosphorylated p38 kinase was high in 2-day-old and 1-wk-old mice, but phosphorylated c-Jun NH2-terminal kinase was not detected at any age. AP-1 activity and level of phosphorylated ERKs declined with maturation. These results imply that AP-1 activity mediated through an ERKs-dependent pathway may be involved in skin development.

Caffeic acid phenethyl ester (CAPE), which is derived from the propolis of honeybee hives, has been shown to block tumor promotion and to have toxic effects on several cancer cells. The mechanism of the anti-tumor promotion activity of CAPE is unclear, however. In this study, we found that CAPE suppressed 12-O-tetradecanoylphorbol-13-acetate-induced cell transformation and induced apoptosis in mouse epidermal JB6 Cl 41 cells. No difference in induction of apoptosis was observed between normal lymphoblasts and sphingomyelinase-deficient cell lines. Although CAPE treatment of two p53 mutant tumor cell lines, NCI-H358 and SK-OV-3, and p53-deficient (p53(-/-)) cells caused the cleavage of caspase-3 as well as DNA fragmentation, caspase-3 cleavage was seen early (at 6 h) only in cells expressing wild-type p53 (p53(+/+)) and Cl 41 cells. These results suggested that p53 may be involved in the early stage of CAPE-induced apoptosis. The p53-dependent transcription activation occurred 2 h after treatment with CAPE and reached a maximum at 6 h in Cl 41 p53 DNA-binding sequence stable transfectant cells. In addition, phosphorylation of p53 at serine 15 and serine 392 was induced in Cl 41 cells within 6 h after treatment with CAPE. Therefore, CAPE may induce apoptosis through p53-dependent and p53-independent pathways and its anti-tumor promotion activity may have occurred through the induction of apoptosis.

In this study, we investigated the mechanism by which UVB irradiation activates Akt (also known as protein kinase B (PKB)) in mouse epidermal JB6 cells. Treatment with a phosphatidylinositol 3-kinase inhibitor, LY 294002, or expression of a dominant negative mutant of p85 (regulatory component of phosphatidylinositol 3-kinase) inhibited UVB-induced Akt activation. Interestingly, Akt activation by UVB was attenuated by treatment with PD 98059, a specific mitogen-activated protein kinase/extracellular signal-regulated protein kinase (Erk) kinase 1 inhibitor, or SB 202190, a specific p38 kinase inhibitor. Furthermore, the expression of a dominant negative mutant of Erk2 or p38 kinase, but not that of c-Jun N-terminal kinase 1 (JNK1), blocked UVB-induced Akt activation. The expression of a dominant negative mutant of p85 or treatment with LY 294002 also inhibited UVB-induced Erk phosphorylation. The UVB-activated mitogen-activated protein kinase members, which were immunoprecipitated from cells exposed to UVB, did not phosphorylate Akt. Instead, Akt was phosphorylated at both threonine 308 and serine 473 and activated by UVB-activated mitogen- and stress-activated protein kinase 1 (Msk1). The expression of a Msk1 C-terminal kinase-dead mutant inhibited UVB-induced phosphorylation and activation of Akt. These data thus suggested that UVB-induced Akt activation was mediated through Msk1, which is a downstream kinase of the Erk and p38 kinase signaling pathways.

(-)-Epigallocatechin gallate (EGCG) and theaflavins are believed to be the key active components in tea for the chemoprevention of cancer. However, the molecular mechanisms by which EGCG and theaflavins block carcinogenesis are not clear. In the JB6 mouse epidermal cell line a tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), which causes cell transformation at high frequency, markedly induced NF-kappaB activation. We found that EGCG and theaflavins inhibited TPA-induced NF-kappaB activity in a concentration-dependent manner. These polyphenols blocked TPA-induced phosphorylation of IkappaBalpha at Ser32 in the same concentration range. Moreover, the NF-kappaB sequence-specific DNA-binding activity induced by TPA was also inhibited by these polyphenols. These results suggest that inhibition of NF-kappaB activation is also important in accounting for the anti-tumor promotion effects of EGCG and theaflavins.

Histone H3 is the core protein of the nucleosome. Phosphorylation of H3 involves immediate early gene expression, chromatin remodeling, and chromosome condensation during mitosis. Very recently, Rsk2 or MSK1 kinase-mediated phosphorylation of H3 at serine 10 was reported. In the present study, we show that both ERKs and p38 kinase may mediate ultraviolet B-induced phosphorylation of H3 at serine 10. PD 98059, a MEK1 inhibitor, and SB 202190, a p38 kinase inhibitor, efficiently inhibited ultraviolet B-induced phosphorylation of H3. Phosphorylation of H3 was also inhibited in cells expressing dominant negative mutant (DNM) ERK2 and DNM p38 kinase. In contrast, no inhibition of H3 phosphorylation in Jnk1 or Jnk2 knockout cells (Jnk1(-/-) or Jnk2(-/-)) and cells expressing DNM JNK1 was observed. More importantly, incubation of active ERK2 or p38 kinase with H3 protein resulted in phosphorylation of H3 at serine 10 in vitro. These results suggest that ERK and p38 kinase are at least two important mediators of phosphorylation of H3 at serine 10.

Mitogen activated protein (MAP) kinase belongs to a large family of serine/threonine protein kinases, including extracellular-signal-regulated protein kinases (Erks), P38 kinase and c-Jun N-terminal kinases (JNKs). Although previous work has shown that both Erks and JNKs are activated in cells in response to ultraviolet (UV) irradiation, most studies have focused only on the role of JNKs in UV-induced AP-1 activation. Hence, the role of Erks in UV-induced AP-1 activity is not well defined. We here have investigated this issue by using MAP kinase kinase (MEK1) inhibitor PD098059 and a dominant negative Erk2, as well as wild-type Erk2, in a JB6 cell model. PD098059 inhibited UVB- or UVC-induced AP-1 activity and phosphorylation of MEK1 and Erks, but not JNKs, in JB6 Cl 41 cells. Overexpression of wild-type Erk2 in Cl 30.7b cells that contain small amounts of Erks caused a 46.6- or 138.1-fold increase of AP-1 activity by UVB and UVC, respectively; introduction of a dominant negative Erk2 into Cl 41 cells significantly blocked the UV-induced Erks activation as well as the AP-1 activation. In contrast, overexpression of wild-type Erk2 in Cl 30.7b cells and dominant negative Erk2 in Cl 41 cells did not show a marked influence on the phosphorylation of JNKs. These results demonstrate that activation of Erks, in addition to the previously reported JNKs, is required for UV-induced AP-1 activation.

Aspirin, along with its analgesic-antipyretic uses, is now also being considered for prevention of cardiovascular disease, cancer, and treatment of human immunodeficiency virus infection. Although many of aspirin's pharmacological actions are related to its ability to inhibit prostaglandin biosynthesis, some of its beneficial therapeutic effects are not completely understood. Transcription factor activator protein 1 (AP-1) is critical for the induction of neoplastic transformation and induction of multiple genes involved in inflammation and infection. We have used the JB6 mouse epidermal cell lines, a system that has been used extensively as an in vitro model for the study of tumor promotion and anti-tumor promotion, to study the anti-carcinogenesis effect of aspirin at the molecular level. Aspirin and aspirin-like salicylates inhibited the activation of AP-1 in the same dose range as seen for the inhibition of tumor promoter-induced transformation. The inhibition of AP-1 and tumor promoter-induced transformation in JB6 cells occurs through a prostaglandin independent- and an Erk1- or Erk2-independent pathway. The mechanism of AP-1 and transformation inhibition in this cell culture model may involve the elevation of H+ concentration. The inhibition effects on the activation of AP-1 activity by aspirin and aspirin-like salicylates may further explain the anti-carcinogenesis mechanism of action of these drugs.

The phosphoinositide 3-kinase (PI3-K)/Akt and mTOR signaling pathway plays a critical role in cell survival and proliferation and is often aberrantly activated in many types of cancer. The mTOR kinase protein, one of the key molecules in this pathway, has been shown to be an important target for cancer therapy. In the present study, a ligand docking method was used to screen for novel scaffold mTOR inhibitors. Sixty thousand compounds in the Natural Product Database were screened against the mTOR homologous structure, and 13 commercially available compounds listed in the top-ranked 100 compounds were selected for further examination. Compound [(E)-3-(4-(benzo[d][1,3]dioxol-5-yl)-2-oxobut-3-en-1-yl)- 3-hydroxyindolin-2-one; designated herein as 3HOI-BA-01] was then selected for further study of its antitumor activity. An in vitro study has shown that 3HOI-BA-01 inhibited mTOR kinase activity in a dose-dependent manner by directly binding with mTOR. In a panel of non–small cell lung cancer cells, the compound also attenuated mTOR downstream signaling, including the phosphorylation of p70S6K, S6, and Akt, resulting in G1 cell-cycle arrest and growth inhibition. Results of an in vivo study have shown that intraperitoneal injection of 3HOI-BA-01 in A549 lung tumor–bearing mice effectively suppressed cancer growth without affecting the body weight of the mice. The expression of downstream signaling molecules in the mTOR pathway in tumor tissues was also reduced after 3HOI-BA-01 treatment. Taken together, we identified 3HOI-BA-01 as a novel and effective mTOR inhibitor. Mol Cancer Ther; 12(6); 950–8. ©2013 AACR.

The activation of cellular signal transduction pathways by solar ultraviolet (SUV) irradiation plays a vital role in skin tumorigenesis. Although many pathways have been studied using pure ultraviolet A (UVA) or ultraviolet B (UVB) irradiation, the signaling pathways induced by SUV (i.e., sunlight) are not understood well enough to permit improvements for prevention, prognosis, and treatment. Here, we report parallel protein kinase array studies aimed at determining the dominant signaling pathway involved in SUV irradiation. Our results indicated that the p38-related signal transduction pathway was dramatically affected by SUV irradiation. SUV (60 kJ UVA/m2/3.6 kJ UVB/m2) irradiation stimulates phosphorylation of p38α (MAPK14) by 5.78-fold, MSK2 (RPS6KA4) by 6.38-fold, and HSP27 (HSPB1) by 34.56-fold compared with untreated controls. By investigating the tumorigenic role of SUV-induced signal transduction in wild-type and p38 dominant-negative (p38 DN) mice, we found that p38 blockade yielded fewer and smaller tumors. These results establish that p38 signaling is critical for SUV-induced skin carcinogenesis. Cancer Res; 73(7); 2181–8. ©2013 AACR.

Solar UV radiation is a major environmental factor that causes DNA damage, inflammation, and even skin cancer. T-LAK cell-originated protein kinase (TOPK) is expressed widely in both normal and cancer cells and functions to inhibit apoptosis and promote carcinogenesis. However, its function in inflammation is not known. The p38 MAPK signaling pathway plays an important role in solar UV light-induced inflammation. In this study, we found that TOPK negatively regulated the activity of p38α by phosphorylating the p38α-specific phosphatase MKP1 and enhancing the stability of MKP1. Notably, the absence of TOPK in mice resulted in a striking increase in skin inflammation. Therefore, we conclude that TOPK has a protective function in solar UV light-induced inflammation.

The ribosomal S6 kinase 2 (RSK2) is a member of the p90 ribosomal S6 kinase (p90RSK) family of proteins and plays a critical role in proliferation, cell cycle, and cell transformation. Here, we report that RSK2 phosphorylates caspase-8, and Thr-263 was identified as a novel caspase-8 phosphorylation site. In addition, we showed that EGF induces caspase-8 ubiquitination and degradation through the proteasome pathway, and phosphorylation of Thr-263 is associated with caspase-8 stability. Finally, RSK2 blocks Fas-induced apoptosis through its phosphorylation of caspase-8. These data provide a direct link between RSK2 and caspase-8 and identify a novel molecular mechanism for caspase-8 modulation by RSK2.

MST1 (mammalian STE20-like kinase 1) is a serine/threonine kinase that is cleaved and activated by caspases during apoptosis. Overexpression of MST1 induces apoptotic morphological changes such as chromatin condensation, but the mechanism is not clear. Here we show that MST1 induces apoptotic chromatin condensation through its phosphorylation of histone H2AX at Ser-139. During etoposide-induced apoptosis in Jurkat cells, the cleavage of MST1 directly corresponded with strong H2AX phosphorylation. In vitro kinase assay results showed that MST1 strongly phosphory-lates histone H2AX. Western blot and kinase assay results with a mutant S139A H2AX confirmed that MST1 phos-phorylates H2AX at Ser-139. Direct binding of MST1 and H2AX can be detected when co-expressed in HEK293 cells and was also confirmed by an endogenous immunoprecipitation study. When overexpressed in HeLa cells, both the MST1 full-length protein and the MST1 kinase domain (MST1-NT), but not the kinase-negative mutant (MST1-NT-KN), could induce obvious endogenous histone H2AX phosphorylation. The caspase-3 inhibitor benzyloxycarbonyl-DEVD-fluoromethyl ketone (Z-DEVD-fmk) attenuates phosphorylation of H2AX by MST1 but cannot inhibit MST1-NT-induced histone H2AX phosphorylation, indicating that cleaved MST1 is responsible for H2AX phosphory-lation during apoptosis. Histone H2AX phosphorylation and DNA fragmentation were suppressed in MST1 knockdown Jurkat cells after etoposide treatment. Taken together, our data indicated that H2AX is a substrate of MST1, which functions to induce apoptotic chromatin condensation and DNA fragmentation.

The ribosomal S6 kinase 2 (RSK2) is a well-known serine/threonine kinase and a member of the p90 ribosomal S6 kinase (p90RSK) family of proteins. It is activated downstream of the MEK/ERKs cascade by mitogenic stimuli such as EGF or TPA. Here, we show that RSK2 is activated by treatment with tumor necrosis factor-α (TNF-α) and directly phosphorylates IκBα at Ser-32, leading to IκBα degradation. The phosphorylation of IκBα promotes the activation and translocation of the nuclear factor-κB (NF-κB) subunits p65 and p50 to the nucleus. The net result is an increased NF-κB activity, which serves as a mechanism for RSK2 blockade of TNF-α-induced apoptosis and enhanced cell survival.—Peng, C., Cho, Y.-Y., Zhu, F., Xu, Y.-M., Wen, W., Ma, W.-Y., Bode, A. M., Dong, Z. RSK2 mediates NF-κB activity through the phosphorylation of IκBα in the TNF-R1 pathway.

The cancer preventive action of (-)-epigallocatechin gallate (EGCG), found in green tea, is strongly supported by epidemiology and laboratory research data. However, the mechanism by which EGCG inhibits carcinogenesis and cell transformation is not clear. In this study, we report that EGCG suppressed epidermal growth factor (EGF)-induced cell transformation in JB6 cells. We also found that EGCG inhibited EGF-induced Fyn kinase activity and phosphorylation in vitro and in vivo. Fyn was implicated in the process because EGF-induced JB6 cell transformation was inhibited by small interfering RNA (siRNA)-Fyn-JB6 cells. With an in vitro protein-binding assay, we found that EGCG directly bound with the GST-Fyn-SH2 domain but not the GST-Fyn-SH3 domain. The K(d) value for EGCG binding to the Fyn SH2 domain was 0.367 +/- 0.122 microM and B(max) was 1.35 +/- 0.128 nmol/mg. Compared with control JB6 Cl41 cells, EGF-induced phosphorylation of p38 MAP kinase (p38 MAPK) (Thr180/Tyr182), ATF-2 (Thr71) and signal transducer and activator of transcription 1 (STAT1) (Thr727) was decreased in siRNA-Fyn-JB6 cells. EGCG could inhibit the phosphorylation of p38 MAPK, ATF-2, and STAT1. The DNA binding ability of AP-1, STAT1, and ATF-2 was also decreased in siRNA-Fyn-JB6 cells. Overall, these results demonstrated that EGCG interacted with Fyn and inhibited Fyn kinase activity and thereby regulated EGF-induced cell transformation. Inhibition of Fyn kinase activity is a novel and important mechanism that may be involved in EGCG-induced inhibition of cell transformation.

Background & Aims: Aberrant activation of Ras and Raf in mitogen-activated protein kinase (MAPK) signaling has been linked with cancer. However, the role of MAPK kinases (MAPKKs or MEKs) in cancer is unclear, although constitutively activated MEK1, which does not exist in nature, is "oncogenic." Herein, we found that T-cell-originated protein kinase (TOPK), a member of the MAPKK protein family, is highly expressed in human colorectal cancer tissues and cell lines and plays an important role in the transformation of colorectal cancer. Methods: The biologic consequences of overexpression or knockdown of TOPK in JB6 Cl41 and HCT116 colorectal cancer cells were studied in vitro and in vivo, respectively. Kinase assay or transient transfection experiments were performed to study the bidirectional signaling pathway between TOPK and extracellular signal-regulated kinase (ERK). Results: TOPK was shown to promote transformation in vitro and in vivo, and knockdown of TOPK in HCT116 colorectal cancer cells reduced this cell lines' tumorigenic properties in vitro and in vivo. Furthermore, a positive feedback loop between TOPK and ERK2 was identified. With epidermal growth factor treatment, knockdown of either TOPK or ERK2 in HCT116 cells resulted in a decreased phosphorylation of ERK2 or TOPK, respectively, and knockdown of TOPK in HCT116 colorectal cancer cells blocked the phosphorylation of downstream substrates of ERK2. Conclusions: The positive feedback loop between TOPK and ERK2 increases tumorigenesis properties of HCT116 colorectal cancer cells, and TOPK-regulated signaling may serve as a potential therapeutic target in colorectal cancer.

Ultraviolet (UV) irradiation is the leading factor in the development of skin cancer, which is the most common form of cancer in the United States. Discovering novel chemopreventive agents against this disease is extremely important. Kaempferol, a natural flavonol isolated from tea, broccoli, grapes, apples and other plant sources, is known to have anticancer activity, but its molecular mechanisms and direct target(s) in cancer chemoprevention are still unclear. In this study, our pull-down assay results showed that RSK2 and MSK1 directly interact with kaempferol in both ex vivo and in vitro systems. ATP competition and in vitro kinase assay data revealed that kaempferol interacts with RSK2 and MSK1 at the ATP-binding pocket and inhibits their respective kinase activities. Mechanistic investigations determined that kaempferol acts as an inhibitor of RSK2 and MSK1 kinase activities to attenuate solar UV-induced phosphorylation in mitogen-activated protein kinase signaling cascades in JB6 P+ mouse skin epidermal cells. In a mouse skin tumorigenesis study, kaempferol significantly suppressed solar UV-induced skin carcinogenesis. Further analysis showed that the kaempferol-treated group had a substantial reduction in solar UV-induced phosphorylation of CREB and c-Fos in mouse skin. Taken together, our results identify kaempferol as a safe and novel chemopreventive agent against solar UV-induced skin carcinogenesis that acts by targeting RSK2 and MSK1. Citation Format: Ke Yao, Hanyong Chen, Mee-Hyun Lee, Alyssa Langfald, Myoung Ok Kim, Dong Hoon Yu, Kangdong Liu, Wei-Ya Ma, Ann M. Bode, Ziming Dong, Zigang Dong. Kaempferol suppresses solar ultraviolet radiation-induced skin cancers by targeting RSK2 and MSK1. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1241. doi:10.1158/1538-7445.AM2014-1241

Immunofluorescence studies have revealed that H2AX is phosphorylated at the sites of DNA double-strand breaks induced by ionizing radiation and is required for recruitment of repair factors into nuclear foci after DNA damage. Therefore, the function of H2AX is believed to be associated primarily with repair of DNA damage. Here, we report a function of H2AX in cellular apoptosis. Our data showed that H2AX is phosphorylated by UVA-activated JNK. We also provided evidence showing that UVA induces caspase-3 and caspase-activated DNase (CAD) activity in both H2AX wild-type and H2AX knockout mouse embryonic fibroblasts (MEFs). However, DNA fragmentation occurred only in H2AX wild-type MEFs. Furthermore, H2AX phosphorylation was critical for DNA degradation triggered by CAD in vitro. Taken together, these data indicated that H2AX phosphorylation is required for DNA ladder formation, but not for the activation of caspase-3; and the JNK/H2AX pathway cooperates with the caspase-3/CAD pathway resulting in cellular apoptosis.

Caffeine is a key component of many popular drinks, especially tea and coffee. Previous reports have shown that caffeine may contribute to the chemopreventive effect of tea in animals. Here, we report that treatment with low concentrations of caffeine induced apoptosis in JB6 Cl41 cells. JB6 Cl41 cells were starved in 0.1% fetal bovine serum/MEM for 72 h and then treated with 50-450 microM caffeine for 24 h. Cells showed the typical DNA laddering pattern and other characteristics of apoptosis. The IC(50) of caffeine on JB6 Cl41 cells was 2.7 mM. Induction of apoptosis by caffeine appeared to be p53-dependent because cells lacking p53 (p53(-/-)) showed no signs of apoptosis after treatment with caffeine. Immunoprecipitation assays and Western blot analysis showed that caffeine induced phosphorylation of p53 at Ser(15) in JB6 Cl41 cells. The same low concentration of caffeine that was effective for inducing phosphorylation of p53 was also shown to increase p53 activation. Expression of Bax, another p53 target, distinctly increased in a time- and dose-dependent manner. Cleaved caspase 3 was also increased in a time- and dose-dependent manner. These data show that a low concentration of caffeine can induce p53-dependent apoptosis in JB6 cells through the Bax and caspase 3 pathways.

The relationship between cell transformation and p38 MAP kinase, a major mitogen-activated protein (MAP) kinase pathway converting signals of various extracellular stimuli into expression of specific target genes through activation of transcription factors, still remains unclear. The aim of the present study was to investigate the role of the p38 MAP kinase pathway in epidermal growth factor (EGF)-induced cell transformation in JB6 cells. Our data show that a dominant negative mutant of p38 MAP (DN-p38) kinase inhibits EGF-promoted JB6 Cl41 cell transformation and that SB202190, an inhibitor of p38 MAP kinase, also inhibits JB6 Cl41 cell transformation in a dose-dependent manner. Moreover, our results show that DN-p38 MAP kinase inhibits the phosphorylation of EGF-stimulated activating transcription factor-2 (ATF-2) and signal transducer and activator of transcription 1 (STAT1). Additionally, DN-p38 MAP kinase inhibits EGF-induced phosphorylation of c-Myc (Thr58/Ser62). Gel shift assays indicate that DN-p38 MAP kinase inhibits EGF-induced activator protein-1 (AP-1) DNA binding in a dose-dependent manner. These results show that p38 MAP kinase plays a key role in the regulation of EGF-induced cell transformation in JB6 cells through regulation of phosphorylation of p38 MAP kinase and activation of its target genes in phosphorylation, c-Myc cell transformation-related genes, and AP-1 binding ability.

Arsenite is known to be an environmental human carcinogen. However, the mechanism of action of this compound in skin carcinogenesis is not completely clear. Here, we provide evidence that arsenite can induce phosphorylation of histone H3 at serine 10 in a time- and dose-dependent manner in JB6 Cl 41 cells. Arsenite induces phosphorylation of Akt1 at serine 473 and increases Akt1 activity. A dominant-negative mutant of Akt1 inhibits the arsenite-induced phosphorylation of histone H3 at serine 10. Additionally, active Akt1 kinase strongly phosphorylates histone H3 at serine 10 in vitro. The arsenite-induced phosphorylation of histone H3 at serine 10 was almost completely blocked by a dominant-negative mutant of extracellular signal-regulated kinase 2 and the mitogen-activated protein kinase/extracellular signal-regulated kinase inhibitor PD98059. N- or C-terminal mutant mitogen- and stress-activated protein kinase 1 or its inhibitor H89 had no effect on arsenite-induced phosphorylation of histone H3 at serine 10 in JB6 Cl 41 cells. However, cells deficient in p90 ribosomal S6 kinase 2 (Rsk2(-/-)) totally block this phosphorylation in a dose- and time-dependent manner. Taken together, these results suggested that arsenite-induced phosphorylation of histone H3 at serine 10 is mediated by Akt1, extracellular signal-regulated kinase 2 and p90 ribosomal S6 kinase 2 but not mitogen- and stress-activated protein kinase 1.

The Bcl-2 family member Bad is a pro-apoptotic protein, and phosphorylation of Bad by cytokines and growth factors promotes cell survival in many cell types. Induction of apoptosis by UV radiation is well documented. However, little is known about UV activation of cell survival pathways. Here, we demonstrate that UVB induces Bad phosphorylation at serine 112 in JNK1, RSK2, and MSK1-dependent pathways. Inhibition of mitogen-activated protein (MAP) kinases including ERKs, JNKs, and p38 kinase by the use of their respective dominant negative mutant or a specific inhibitor for MEK1 or p38 kinase, PD98059 or SB202190, resulted in abrogation of UVB-induced phosphorylation of Bad at serine 112. Incubation of active MAP kinase members with Bad protein showed serine 112 phosphorylation of Bad by JNK1 only. However, activated RSK2 and MSK1, downstream kinases of ERKs and p38 kinase, respectively, also phosphorylated Bad at serine 112 in vitro. Cells from a Coffin-Lowry syndrome patient (deficient in RSK2) or expressing an N-terminal or C-terminal kinase-dead mutant of MSK1 were defective for UVB-induced serine 112 phosphorylation of Bad. Furthermore, MAP kinase pathway-dependent serine 112 phosphorylation was shown to be required for dissociation of Bad from Bcl-X(L). These data illustrated that UVB-induced phosphorylation of Bad at serine 112 was mediated through MAP kinase signaling pathways in which JNK1, RSK2, and MSK1 served as direct mediators.

Most of the signal pathways involved in ultraviolet (UV)-induced skin carcinogenesis are thought to originate at plasma membrane receptors. However, UVA-induced signal transduction to downstream ribosomal protein S6 kinases, p70(S6K) and p90(RSK), is not well understood. In this report, we show that UVA stimulation of the epidermal growth factor receptor (EGFR) may lead to activation of p70(S6K)/p90(RSK) through phosphatidyl isositol (PI)-3 kinase and extracellular receptor-activated kinases (ERKs). Evidence is provided that phosphorylation and activation of p70(S6K)/p90(RSK) induced by UVA were prevented in Egfr(-/-) cells and were also markedly inhibited by the EGFR-specific tyrosine kinase inhibitors AG1478 and PD153035. Furthermore, EGFR tyrosine kinase inhibitors and EGFR deficiency significantly suppressed activation of PI-3 kinase and ERKs in regulating activation of p90(RSK)/p70(S6K) but had no effect on activation of c-Jun NH(2)-terminal kinases (JNKs) and p38 kinase in response to UVA. Thus, our results suggest that UVA-induced EGFR signaling may be required for activation of p90(RSK)/p70(S6K), PI-3 kinase, and ERKs but not JNKs or p38 kinase.

In resting cells, eIF4E-binding protein 1 (4E-BP1) binds to the eukaryotic initiation factor-4E (eIF-4E), preventing formation of a functional eIF-4F complex essential for cap-dependent initiation of translation. Phosphorylation of 4E-BP1 dissociates it from eIF-4E, relieving the translation block. Studies suggested that insulin- or growth factor-induced 4E-BP1 phosphorylation is mediated by phosphatidylinositol 3-kinase (PI3-kinase) and its downstream protein kinase, Akt. In the present study we demonstrated that UVB induced 4E-BP1 phosphorylation at multiple sites, Thr-36, Thr-45, Ser-64, and Thr-69, leading to dissociation of 4E-BP1 from eIF-4E. UVB-induced phosphorylation of 4E-BP1 was blocked by p38 kinase inhibitors, PD169316 and SB202190, and MSK1 inhibitor, H89, but not by mitogen-activated protein kinase kinase inhibitors, PD98059 or U0126. The PI3-kinase inhibitor, wortmannin, did not block UVB-induced 4E-BP1 phosphorylation, but blocked both UVB- and insulin-induced activation of PI3-kinase and phosphorylation of Akt. 4E-BP1 phosphorylation was blocked in JB6 Cl 41 cells expressing a dominant negative p38 kinase or dominant negative MSK1, but not in cells expressing dominant negative ERK2, JNK1, or PI3-kinase p85 subunit. Our results suggest that UVB induces phosphorylation of 4E-BP1, leading to the functional dissociation of 4E-BP1 from eIF-4E. The p38/MSK1 pathway, but not PI3-kinase or Akt, is required for mediating the UVB-induced 4E-BP1 phosphorylation.

Phosphorylation of the p53 tumor suppressor protein is one of the key regulatory steps in its activation process. Serine 20 phosphorylation of p53 has been shown to be required for the activation of p53 following UV radiation, but the signaling pathway mediating UV-induced phosphorylation is unknown. Here, we determined the role of MAP kinases in UVB-induced phosphorylation and found that JNKs are directly involved in the phosphorylation of p53 at serine 20. In a mouse JB6 epidermal cell line, dominant negative JNK1 abrogated UVB-induced phosphorylation of p53 at serine 20, whereas dominant negative p38 kinase or its inhibitor, SB202190, partially attenuated the phosphorylation. In contrast, dominant negative ERK2 or the MEK1 inhibitor, PD98059, had no effect on p53 phosphorylation at serine 20. Importantly, UVB-activated or active recombinant JNK1/2, or the p38 kinase downstream target, MAPKAPK-2, but not ERKs or p38 kinase, phosphorylated p53 at serine 20 in vitro. Furthermore, phosphorylation of p53 at serine 20 by UVB-activated JNKs and UVB-induced p53-dependent transcriptional activity were suppressed in Jnk1 or Jnk2 knockout (Jnk1(-/-) or Jnk2(-/-)) cells. Additionally, Jnk1(-/-), Jnk2(-/-), and p53-deficient (p53(-/-)) cells, as well as re-introduction of a p53 mutant with substitution of serine 20 to alanine into p53(-/-) cells, were defective for UVB-induced apoptosis. These findings strongly suggest that JNKs are the major direct signaling mediators of UVB-induced p53 phosphorylation at serine 20.

Inositol hexaphosphate (InsP6) has an effective anticancer action in many experimental models in vivo and in vitro. Ultraviolet B (UVB) radiation is believed to be responsible for many of the carcinogenic effects related to sun exposure, and alteration in UVB-induced signal transduction is associated with UVB-induced carcinogenesis. Here we report the effects of InsP6 on UVB-induced signal transduction. InsP6 strongly blocked UVB-induced activator protein-1 (AP-1) and NF-kappaB transcriptional activities in a dose-dependent manner. InsP6 also suppressed UVB-induced AP-1 and nuclear factor kappaB (NF-kappaB) DNA binding activities and inhibited UVB-induced phosphorylation of extracellular signal-regulated protein kinases (Erks) and c-Jun NH2-terminal kinases (JNKs). Phosphorylation of p38 kinases was not affected. InsP6 also blocked UVB-induced phosphorylation of IkappaB-alpha, which is known to result in the inhibition of NF-kappaB transcriptional activity. InsP6 does not block UVB-induced phosphotidylinositol-3' (PI-3) kinase activity, suggesting that the inhibition of UVB-induced AP-1 and NF-kappaB activities by InsP6 is not mediated through PI-3 kinase. Because AP-1 and NF-kappaB are important nuclear transcription factors that are related to tumor promotion, our work suggests that InsP6 prevents UVB-induced carcinogenesis by inhibiting AP-1 and NF-kappaB transcription activities.

N-terminal tail phosphorylation of histone H3 plays an important role in gene expression, chromatin remodeling, and chromosome condensation. Phosphorylation of histone H3 at serine 10 was shown to be mediated by RSK2, mitogen- and stress-activated protein kinase-1 (MSK1), and mitogen-activated protein kinases depending on the specific stimulation or stress. Our previous study showed that mitogen-activated protein kinases MAP kinases are involved in ultraviolet B-induced phosphorylation of histone H3 at serine 28 (Zhong, S., Zhong, Z., Jansen, J., Goto, H., Inagaki, M., and Dong, Z., J. Biol. Chem. 276, 12932-12937). However, downstream effectors of MAP kinases remain to be identified. Here, we report that H89, a selective inhibitor of the nucleosomal response, totally inhibits ultraviolet B-induced phosphorylation of histone H3 at serine 28. H89 blocks MSK1 activity but does not inhibit ultraviolet B-induced activation of MAP kinases p70/85(S6K), p90(RSK), Akt, and protein kinase A. Furthermore, MSK1 markedly phosphorylated serine 28 of histone H3 and chromatin in vitro. Transfection experiments showed that an N-terminal mutant MSK1 or a C-terminal mutant MSK1 markedly blocked MSK1 activity. Compared with wild-type MSK1, cells transfected with N-terminal or C-terminal mutant MSK1 strongly blocked ultraviolet B-induced phosphorylation of histone H3 at serine 28 in vivo. These data illustrate that MSK1 mediates ultraviolet B-induced phosphorylation of histone H3 at serine 28.