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SUV induces MAP kinase phosphorylation in JB6 P+ mouse epidermal skin cells in a time- or dose-dependent manner. (A) JB6 P+ cells were cultured, treated with SUV (60 kJ/m2) and harvested at the times indicated (0, 15, 30, 60, or 120 min). (B) JB6 P+ cells were cultured, treated with different doses of SUV (0, 5, 15, 30, 60 or 90 kJ/m2) and harvested at 15 min. Protein abundance was analyzed by Western blot. �-Actin was used to verify equal protein loading. Each assay was performed 3 times and similar results were obtained. Representative Western blots are shown.

(A) Kinase screening assays were conducted by Millipore's KinaseProfiler according to their established protocols. Scores represent the percent of control, which was derived from the following formula: % of control = [(sample − mean no enzyme)/(mean plus enzyme − mean no enzyme)] × 100. The kaempferol concentration for each specific kinase assay was set at 25 μM and ATP concentration was set at 10 μM. Data are representative of results from triplicate experiments. (B) Kaempferol can bind at the ATP binding pocket of RSK2. Enlarged view shows kaempferol binding with RSK2 at the ATP binding pocket and forming hydrogen bonds (shown as red lines). (C) Kaempferol can bind at the ATP binding pocket of MSK1. Enlarged view shows kaempferol binding with MSK1 at the ATP binding pocket and forming hydrogen bonds (shown as red lines). (D) Kaempferol does not bind with JNK2 or p38. Whole lysates from JB6 P+ cells were incubated with 30 μL of kaempferol-Sepharose 4B beads (50% slurry) for 2 h at 4{degree sign}C. Beads were washed and JNK2 or p38 binding was visualized by Western blotting.

Supplemental Figure Legends from Kaempferol Targets RSK2 and MSK1 to Suppress UV Radiation-Induced Skin Cancer

Figure S1. OPN4 expression is upregulated in human lung adenocarcinoma. Figure S2. Knockdown of OPN4 inhibits cells growth and induces cells apoptosis in H441 and A549 cells. Figure S3. Loss OPN4 suppresses urethane-induced lung carcinogenesis. Figure S4. Effects of OPN4 antagonist AE 51310 on normal lung cells growth. Figure S5. OPN4 antagonist inhibits tumor growth in lung PDX mouse model.

RSK2 and MSK1 play an important role in Ras-mediated foci formation. A focus formation assay was performed by introducing combinations of vectors as indicated. Foci were stained with 0.5% crystal violet and counted under a microscope using the Image-Pro Plus software program (vs. 6). Data are shown as means {plus minus} S.D. of values obtained from 2 independent triplicate experiments. Asterisks indicate a significant change in the number of foci (*, p < 0.05; **, p < 0.01).

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Supplementary Table 1

The key functional molecules involved in inflammatory bowel disease (IBD) and IBD-induced colorectal tumorigenesis remain unclear. In this study, we found that the apoptosis repressor with caspase recruitment domain (ARC) protein plays critical roles in IBD. ARC-deficient mice exhibited substantially higher susceptibility to dextran sulfate sodium (DSS)-induced IBD compared with wild-type mice. The inflammatory burden induced in ARC-deficient conditions was inversely correlated with CCL5 and CXCL5 levels in immune cells, especially CD4-positive T cells. Pathologically, ARC expression in immune cells was significantly decreased in clinical biopsy specimens from patients with IBD compared with normal subjects. In addition, ARC levels inversely correlated with CCL5 and CXCL5 levels in human biopsy specimens. ARC interacted with TNF receptor associated factor (TRAF) 6, regulating ubiquitination of TRAF6, which was associated with NF-κB signaling. Importantly, we identified a novel ubiquitination site at lysine 461, which was critical in the function of ARC in IBD. ARC played a critical role in IBD and IBD-associated colon cancer in a bone marrow transplantation model and azoxymethane/DSS-induced colitis cancer mouse models. Overall, these findings reveal that ARC is critically involved in the maintenance of intestinal homeostasis and protection against IBD through its ubiquitination of TRAF6 and subsequent modulation of NF-κB activation in T cells.Significance:This study uncovers a crucial role of ARC in the immune system and IBD, giving rise to a novel strategy for IBD and IBD-associated colon cancer therapy.

Supplementary Figure 1-6

Supplementary Figure 1 from Phosphorylation of H2AX at Ser139 and a New Phosphorylation Site Ser16 by RSK2 Decreases H2AX Ubiquitination and Inhibits Cell Transformation

Supplementary Methods from Transient Receptor Potential Type Vanilloid 1 Suppresses Skin Carcinogenesis

Supplementary Figure 2 from Cyclin-Dependent Kinase 3–Mediated Activating Transcription Factor 1 Phosphorylation Enhances Cell Transformation

Supplementary Methods from Phosphorylation of H2AX at Ser139 and a New Phosphorylation Site Ser16 by RSK2 Decreases H2AX Ubiquitination and Inhibits Cell Transformation

Blockade of the transient receptor potential channel vanilloid subfamily 1 (TRPV1) is suggested as a therapeutic approach to pain relief. However, TRPV1 is a widely expressed protein whose function might be critical in various nonneuronal physiologic conditions. The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase that is overexpressed in many human epithelial cancers and is a potential target for anticancer drugs. Here, we show that TRPV1 interacts with EGFR, leading to EGFR degradation. Notably, the absence of TRPV1 in mice results in a striking increase in skin carcinogenesis. The TRPV1 is the first membrane receptor shown to have a tumor-suppressing effect associated with the down-regulation of another membrane receptor. The data suggest that, although a great deal of interest has focused on TRPV1 as a target for pain relief, the chronic blockade of this pain receptor might increase the risk for cancer development. [Cancer Res 2009;69(3):905–13]

Supplementary Figure Legends 1-2 from Phosphorylation of H2AX at Ser139 and a New Phosphorylation Site Ser16 by RSK2 Decreases H2AX Ubiquitination and Inhibits Cell Transformation

Supplementary Figure 2 from Transient Receptor Potential Type Vanilloid 1 Suppresses Skin Carcinogenesis

Supplementary Figure 3 from Nuclear Factor of Activated T3 Is a Negative Regulator of Ras-JNK1/2-AP-1–Induced Cell Transformation

Supplementary Figure 3 from Transient Receptor Potential Type Vanilloid 1 Suppresses Skin Carcinogenesis

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]

Supplementary Figure 2 from Phosphorylation of H2AX at Ser139 and a New Phosphorylation Site Ser16 by RSK2 Decreases H2AX Ubiquitination and Inhibits Cell Transformation

Supplementary Figures 1-2 from Nuclear Factor of Activated T3 Is a Negative Regulator of Ras-JNK1/2-AP-1–Induced Cell Transformation

Supplementary Figure 1 from Cyclin-Dependent Kinase 3–Mediated Activating Transcription Factor 1 Phosphorylation Enhances Cell Transformation

Supplementary Figure 1 from Transient Receptor Potential Type Vanilloid 1 Suppresses Skin Carcinogenesis

The key functional molecules involved in inflammatory bowel disease (IBD) and IBD-induced colorectal tumorigenesis remain unclear. In this study, we found that the apoptosis repressor with caspase recruitment domain (ARC) protein plays critical roles in IBD. ARC-deficient mice exhibited substantially higher susceptibility to dextran sulfate sodium (DSS)-induced IBD compared with wild-type mice. The inflammatory burden induced in ARC-deficient conditions was inversely correlated with CCL5 and CXCL5 levels in immune cells, especially CD4-positive T cells. Pathologically, ARC expression in immune cells was significantly decreased in clinical biopsy specimens from patients with IBD compared with normal subjects. In addition, ARC levels inversely correlated with CCL5 and CXCL5 levels in human biopsy specimens. ARC interacted with TNF receptor associated factor (TRAF) 6, regulating ubiquitination of TRAF6, which was associated with NF-κB signaling. Importantly, we identified a novel ubiquitination site at lysine 461, which was critical in the function of ARC in IBD. ARC played a critical role in IBD and IBD-associated colon cancer in a bone marrow transplantation model and azoxymethane/DSS-induced colitis cancer mouse models. Overall, these findings reveal that ARC is critically involved in the maintenance of intestinal homeostasis and protection against IBD through its ubiquitination of TRAF6 and subsequent modulation of NF-κB activation in T cells. Significance: This study uncovers a crucial role of ARC in the immune system and IBD, giving rise to a novel strategy for IBD and IBD-associated colon cancer therapy.

Rationale: Melanoma is an aggressive tumor of the skin and drug resistance is still a major problem in melanoma therapy. Novel targets and effective agents to overcome drug resistant melanoma are urgently needed in clinical therapy. Methods: Gene Expression Omnibus (GEO) database analysis, pathway enrichment analysis, and survival rate analysis were utilized to identify a candidate target. An anchorage-independent cell growth assay, flow cytometry, Western blot, and a xenograft mouse model were used to study the function of Aurora kinase B (AURKB) in both drug-sensitive and drug-resistant melanoma. Next, HI-511, a novel dual-target inhibitor targeting both AURKB and BRAF V600E, was designed and examined by an in vitro kinase assay. Methods as indicated above in addition to a BRAF V600E/PTEN-loss melanoma mouse model were used to demonstrate the effect of HI-511 on melanoma development in vitro and in vivo. Results: AURKB is highly expressed in melanoma and especially in vemurafenib-resistant melanoma and the expression was correlated with patient survival rate. Knocking down AURKB inhibited cell growth and induced apoptosis in melanoma, which was associated with the BRAF/MEK/ERKs and PI3-K/AKT signaling pathways. Importantly, we found that HI-511, a novel dual-target inhibitor against AURKB and BRAF V600E, suppresses both vemurafenib-sensitive and vemurafenib-resistant melanoma growth in vitro and in vivo by inducing apoptosis and mediating the inhibition of the BRAF/MEK/ERKs and PI3K/AKT signaling pathways. Conclusion: AURKB is a potential target for melanoma treatment. HI-511, a novel dual-target inhibitor against both AURKB and BRAF V600E, could achieve durable suppression of melanoma growth, even drug-resistant melanoma growth.

Lung cancer is the leading cause of cancer-related death worldwide. However, promising agents for lung cancer prevention are still very limited. Identification of preventive targets and novel effective preventive agents is urgently needed for clinical applications. In this study, we found that fluvastatin targeted 3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (HMGCR), which a rate-limiting enzyme in the mevalonate pathway, and inhibited non–small cell lung cancer (NSCLC) tumorigenesis. Initially, we demonstrated that HMGCR is overexpressed in human lung adenocarcinoma tissues compared with normal tissues. Knockdown of HMGCR in NSCLC cells attenuated growth and induced apoptosis in vitro and in vivo. Furthermore, we found that fluvastatin, an inhibitor of HMGCR, suppressed NSCLC cell growth and induced apoptosis. Intriguingly, fluvastastin functions by inhibiting the HMGCR-driven Braf/MEK/ERK1/2 and Akt signaling pathways. Notably, fluvastatin attenuated tumor growth in 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung tumorigenesis and in a patient-derived xenograft lung tumor model. Overall, our findings suggest that fluvastatin might be promising chemopreventive or potential therapeutic drug against NSCLC tumorigenesis, providing hope for rapid clinical translation.

Malignant melanoma is an aggressive tumor of the skin and still lacks effective preventive and therapeutic treatments. In melanoma, both the BRAF/MEK/ERK and PI3-K/AKT signaling pathways are constitutively activated through multiple mechanisms, which result in cell-cycle progression and prevention of apoptosis. Therefore, the development of novel strategies for targeting BRAF and PI3K are of utmost importance. In this study, we found that Ashitaba (Angelica keiskei) chalcones, 4-hydroxyderricin (4HD) and xanthoangelol (XAG), suppressed melanoma development by directly targeting both BRAFV600E and PI3K, which blocked the activation of downstream signaling. This led to the induction of G1 phase cell-cycle arrest and apoptosis in melanoma cells. Importantly, 4HD or XAG dramatically attenuated tumor incidence and volume in the BRAF-activated Pten-deficient melanoma mouse model. Our findings suggest that 4HD and XAG are promising chemopreventive or potential therapeutic agents against melanomagenesis that act by targeting both BRAF and PI3K, providing hope for rapid clinical translation. Cancer Prev Res; 11(10); 607–20. ©2018 AACR.

Proper repair of damaged DNA is critical for the maintenance of genome stability. A complex composed of Integrator subunit 3 (Ints3), single-stranded DNA-binding protein 1 (SSB1), and SSB-interacting protein 1 (SSBIP1) is required for efficient homologous recombination-dependent repair of double-strand breaks (DSBs) and ataxia-telangiectasia mutated (ATM)-dependent signaling pathways. It is known that in this complex the Ints3 N-terminal domain scaffolds SSB1 and SSBIP1. However, the molecular basis for the function of the Ints3 C-terminal domain remains unclear. Here, we present the crystal structure of the Ints3 C-terminal domain, uncovering a HEAT-repeat superhelical fold. Using structure and mutation analysis, we show that the C-terminal domain exists as a stable dimer. A basic groove and a cluster of conserved residues on two opposite sides of the dimer bind single-stranded RNA/DNA (ssRNA/ssDNA) and Integrator complex subunit 6 (Ints6), respectively. Dimerization is required for nucleic acid binding, but not for Ints6 binding. Additionally, in vitro experiments using HEK 293T cells demonstrate that Ints6 interaction is critical for maintaining SSB1 protein level. Taken together, our findings establish the structural basis of a multifunctional Ints3 C-terminal module, allowing us to propose a novel mode of nucleic acid recognition by helical repeat protein and paving the way for future mechanistic studies.

Mutations of the p53-related protein kinase (PRPK) and TP53RK-binding protein (TPRKB) cause Galloway-Mowat syndrome (GAMOS) and are found in various human cancers. We have previously shown that small compounds targeting PRPK showed anti-cancer activity against colon and skin cancer. Here we present the 2.53 Å crystal structure of the human PRPK-TPRKB-AMPPNP (adenylyl-imidodiphosphate) complex. The structure reveals details in PRPK-AMPPNP coordination and PRPK-TPRKB interaction. PRPK appears in an active conformation, albeit lacking the conventional kinase activation loop. We constructed a structural model of the human EKC/KEOPS complex, composed of PRPK, TPRKB, OSGEP, LAGE3, and GON7. Disease mutations in PRPK and TPRKB are mapped into the structure, and we show that one mutation, PRPK K238Nfs*2, lost the binding to OSGEP. Our structure also makes the virtual screening possible and paves the way for more rational drug design., Jian Li and Xinli Ma et al. present a 2.53 Å crystal structure of a complex consisting of the human p53-related protein kinase (PRPK), TP53RK-binding protein, and adenylyl-imidodiphosphate. They find that one disease mutation, PRPK K238Nfs*2, is important for PRPK’s binding to O-sialoglycoprotein endopeptidase, providing insights into rational drug design.

Rationale: Melanoma is an aggressive tumor of the skin and drug resistance is still a major problem in melanoma therapy. Novel targets and effective agents to overcome drug resistant melanoma are urgently needed in clinical therapy. Methods: Gene Expression Omnibus (GEO) database analysis, pathway enrichment analysis, and survival rate analysis were utilized to identify a candidate target. An anchorage-independent cell growth assay, flow cytometry, Western blot, and a xenograft mouse model were used to study the function of Aurora kinase B (AURKB) in both drug-sensitive and drug-resistant melanoma. Next, HI-511, a novel dual-target inhibitor targeting both AURKB and BRAF V600E, was designed and examined by an in vitro kinase assay. Methods as indicated above in addition to a BRAF V600E/PTEN-loss melanoma mouse model were used to demonstrate the effect of HI-511 on melanoma development in vitro and in vivo. Results: AURKB is highly expressed in melanoma and especially in vemurafenib-resistant melanoma and the expression was correlated with patient survival rate. Knocking down AURKB inhibited cell growth and induced apoptosis in melanoma, which was associated with the BRAF/MEK/ERKs and PI3-K/AKT signaling pathways. Importantly, we found that HI-511, a novel dual-target inhibitor against AURKB and BRAF V600E, suppresses both vemurafenib-sensitive and vemurafenib-resistant melanoma growth in vitro and in vivo by inducing apoptosis and mediating the inhibition of the BRAF/MEK/ERKs and PI3K/AKT signaling pathways. Conclusion: AURKB is a potential target for melanoma treatment. HI-511, a novel dual-target inhibitor against both AURKB and BRAF V600E, could achieve durable suppression of melanoma growth, even drug-resistant melanoma growth.

The identification of oncogenic biomolecules as drug targets is an unmet need for the development of clinically effective novel anticancer therapies. In this study, we report for the first time that opsin 4/melanopsin (OPN4) plays a critical role in the pathogenesis of non–small cell lung cancer (NSCLC) and is a potential drug target. Our study has revealed that OPN4 is overexpressed in human lung cancer tissues and cells, and is inversely correlated with patient survival probability. Knocking down expression of OPN4 suppressed cells growth and induced apoptosis in lung cancer cells. We have also found that OPN4, a G protein-coupled receptor, interacted with Gα11 and triggered the PKC/BRAF/MEK/ERKs signaling pathway in lung adenocarcinoma cells. Genetic ablation of OPN4 attenuated the multiplicity and the volume of urethane-induced lung tumors in mice. Importantly, our study provides the first report of AE 51310 (1-[(2,5-dichloro-4-methoxyphenyl)sulfonyl]-3-methylpiperidine) as a small-molecule inhibitor of OPN4, suppressed the anchorage-independent growth of lung cancer cells and the growth of patient-derived xenograft tumors in mice. Implications: Overall, this study unveils the role of OPN4 in NSCLC and suggests that targeting OPN4 with small molecules, such as AE 51310 would be interesting to develop novel anticancer therapies for lung adenocarcinoma.

Tumor necrosis factor receptor (TNFR)–associated factor 1 (TRAF1) is a unique TRAF protein that can interact directly or indirectly with multiple TNFR family members, regulatory proteins, kinases, and adaptors that contribute to its diverse functions in specific tissues. However, the role of TRAF1 in non–small cell lung cancer (NSCLC) remains unknown. In this study, we report that TRAF1 is overexpressed in human lung cancer cells and tissues. TRAF1 expression level inversely correlated with patient survival probability. Loss of TRAF1 decelerated tumor invasion in a urethane-induced lung carcinogenesis mouse model. Furthermore, TRAF1 expression affected TRAF2-mediated BRAF Lys48–linked ubiquitination, which was followed by the inhibition of growth and differentiation, and the induction of death in lung cancer cells. Overall, our work suggests that TRAF1 plays a novel role in the regulation of the BRAF/MEK/ERK signaling pathway in NSCLC and offers a candidate molecular target for lung cancer prevention and therapy. Significance: These findings identify TRAF1 as a new therapeutic target for NSCLC. Cancer Res; 78(14); 3982–94. ©2018 AACR.

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.

Because the transcription factor activator protein-1 (AP-1) regulates a variety of protein-encoding genes, it is a participant in many cellular functions, including proliferation, transformation, epithelial mesenchymal transition (EMT), and apoptosis. Inhibitors targeting AP-1 have potential use in the treatment of cancer and other inflammatory diseases. Here, we identify veratramine as a potent natural modulator of AP-1, which selectively binds to a specific site (TRE 5′-TGACTCA-3′) of the AP-1 target DNA sequence and regulates AP-1-dependent gene transcription without interfering with cystosolic signaling cascades that might lead to AP-1 activation. Moreover, RNA-seq experiments demonstrate that veratramine does not act on the Hedgehog signaling pathway in contrast to its analogue, cyclopamine, and likely does not harbor the same teratogenicity and toxicity. Additionally, veratramine effectively suppresses EGF-induced AP-1 transactivation and transformation of JB6 P+ cells. Finally, we demonstrate that veratramine inhibits solar-ultraviolet-induced AP-1 activation in mice. The identification of veratramine and new findings in its specific regulation of AP-1 down stream genes pave ways to discovering and designing regulators to regulate transcription factor.

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.

Cumulative exposure to solar ultraviolet (SUV) irradiation is regarded as the major etiologic factor in the development of skin cancer. The activation of the MAPK cascades occurs rapidly and is vital in the regulation of SUV-induced cellular responses. The T-LAK cell–originated protein kinase (TOPK), an upstream activator of MAPKs, is heavily involved in inflammation, DNA damage, and tumor development. However, the chemopreventive and therapeutic effects of specific TOPK inhibitors in SUV-induced skin cancer have not yet been elucidated. In the current study, ADA-07, a novel TOPK inhibitor, was synthesized and characterized. Pull-down assay results, ATP competition, and in vitro kinase assay data revealed that ADA-07 interacted with TOPK at the ATP-binding pocket and inhibited its kinase activity. Western blot analysis showed that ADA-07 suppressed SUV-induced phosphorylation of ERK1/2, p38, and JNKs and subsequently inhibited AP-1 activity. Importantly, topical treatment with ADA-07 dramatically attenuated tumor incidence, multiplicity, and volume in SKH-1 hairless mice exposed to chronic SUV. Our findings suggest that ADA-07 is a promising chemopreventive or potential therapeutic agent against SUV-induced skin carcinogenesis that acts by specifically targeting TOPK. Mol Cancer Ther; 16(9); 1843–54. ©2017 AACR.

Background: Barrett's esophagus (BE), a complication of gastroesophageal reflux disease (GERD), predisposes patients to esophageal adenocarcinoma (EAC). Reliable biomarkers for early detection and discovery of potential drug targets are urgently needed for improved BE and EAC patient outcomes. Methods: Patient biopsy samples were evaluated for COX1, COX2, and thromboxane A2 synthase (TBXAS) expression. Circulating prostaglandins biosynthesis was determined using enzyme immunoassay kits. Anchorage-independent cell growth assay, crystal violet staining assay, and xenograft experiments were conducted to assess BE and EAC cell growth. A surgical mouse model of reflux (i.e., esophagoduodenostomy) was established and samples were analyzed using an enzyme immunoassay kit, immunohistochemistry, immunoblotting, or RT-PCR. Esophageal biopsy samples (pre- and post-intervention) were obtained from a randomized clinical trial in which participants were administered esomeprazole (40 mg) twice daily in combination with an aspirin placebo or 81 or 325 mg aspirin for 28 days. Esophageal biopsy specimens before and after the intervention period were analyzed. Findings: COX2 and TBXAS are highly expressed in BE and EAC patients accompanied by a pronounced elevation of circulating TXA2 levels. Aspirin suppressed BE and EAC growth by targeting the TXA2 pathway. Additionally, biopsies from 49 patients (with similar baseline characteristics) showed that aspirin substantially decreased serum TXA2 levels, resulting in reduced inflammation. Interpretation: This study establishes the importance of the COX1/2-driven TXA2 pathway in BE and EAC pathophysiology and lays the groundwork for introducing a TXA2-targeting strategy for EAC prevention and early detection. Funding Statement: This work was supported by the Hormel Foundation and National Institutes of Health grants CA166011, CA187027, and CA196639 (Z. Dong). Declaration of Interests: The authors declare no potential conflicts of interest. Ethics Approval Statement: All animal studies were approved by the University of Minnesota Institutional Animal Care and Use Committee (IACUC).

This study examined the effects of a 12-week physical activity intervention on the motor skill proficiency and executive function of 22 boys (aged 9.08 ± 1.75 years) with autism spectrum disorder. In Phase I of the 12 weeks, 11 boys with autism spectrum disorder (Group A) received the intervention, whereas the other 11 boys with autism spectrum disorder (Group B) did not (true control, no intervention). The arrangement was reversed in Phase II, which lasted an additional 12 weeks. The Bruininks–Oseretsky Test of Motor Proficiency, Second Edition, and the Wisconsin Card Sorting Test were conducted three times for each participant (Group A, primary grouping: baseline (T1), post-assessment (T2), and follow-up assessment (T3); Group B, control grouping: T1−T2; intervention condition, T2−T3). The main findings were that both groups of children with autism spectrum disorder significantly exhibited improvements in motor skill proficiency (the total motor composite and two motor-area composites) and executive function (three indices of the Wisconsin Card Sorting Test) after 12 weeks of physical activity intervention. In addition, the effectiveness appeared to have been sustained for at least 12 weeks in Group A. The findings provide supporting evidence that physical activity interventions involving table tennis training may be a viable therapeutic option for treating children with autism spectrum disorder.

// Yuqiao Sheng 1, 2, 3, * , Kangdong Liu 1, 2, 4, * , Qiong Wu 1, 4, * , Naomi Oi 2 , Hanyong Chen 2 , Kanamata Reddy 2 , Yanan Jiang 1 , Ke Yao 2 , Haitao Li 2 , Wei Li 2 , Yi Zhang 1, 2, 3, 4 , Mohammad Saleem 2 , Wei-Ya Ma 2 , Ann M. Bode 2 , Ziming Dong 1 , Zigang Dong 1, 2, 4 1 Basic Medical College, Zhengzhou University, Zhengzhou, Henan, China 2 The Hormel Institute, University of Minnesota, Austin, Minnesota, USA 3 The First Affiliated Hospital, Zhengzhou University, Zhengzhou, China 4 China-US (Henan) Hormel Cancer Institute, ZhengZhou, Henan, China * These authors contributed equally to this work Correspondence to: Zigang Dong, e-mail: zgdong@hi.umn.edu Ziming Dong, e-mail: Dongzm@zzu.edu.cn Keywords: tubulin-depolymerizing agent, esophageal cancer, patient-derived tumor xenograft Received: December 17, 2015 Accepted: April 10, 2016 Published: April 27, 2016 ABSTRACT Esophageal cancer is one of the least studied and deadliest cancers worldwide with a poor prognosis due to limited options for treatment. Chemotherapy agents such as the microtubule-targeting compounds are the mainstay of palliation for advanced esophageal cancer treatment. However, the toxicity and side effects of tubulin-binding agents (TBAs) have promoted the development of novel, more potent but less toxic TBAs. Herein, we identified 2-[4-(3,4-dimethoxyphenyl)-3-methyl-1H-pyrazol-5-yl]-5-[(2-methylprop-2-en-1-yl)oxy] phenol (PPMP) as a novel TBA for esophageal cancer treatment. PPMP markedly inhibited tubulin polymerization, and decreased viability and anchorage-independent growth of esophageal cancer cell lines, effects that were accompanied by G2/M arrest and apoptosis. Importantly, we produced patient-derived esophageal cancer xenografts to evaluate the therapeutic effect of PPMP in a setting that best mimics the clinical context in patients with esophageal cancer. Overall, we identified PPMP as a novel microtubule-destabilizing compound and as a new therapeutic agent against esophageal carcinoma.

Barrett's esophagus (BE), a complication of gastroesophageal reflux disease (GERD), predisposes patients to esophageal adenocarcinoma (EAC). Reliable biomarkers for early detection and discovery of potential drug targets are urgently needed for improved BE and EAC patient outcomes. In this study, COX2 and TBXAS are highly expressed in BE and EAC patients accompanied by a pronounced elevation of circulating TXA2 levels in patient biopsy samples. Acetylsalicylic acid (ASA) suppressed BE and EAC growth by targeting the TXA2 pathway in a esophagoduodenostomy mouse model of reflux. Additionally, biopsies from patients showed that ASA substantially decreased serum TXA2 levels, resulting in reduced inflammation in a window opportunity clinical trial. Overall, this study establishes the importance of the COX1/2-driven TXA2 pathway in BE and EAC pathophysiology and lays the groundwork for introducing a TXA2-targeting strategy for EAC prevention and early detection. ASA targets the TXA2 pathway and suppresses BE and EAC. Citation Format: Tianshun Zhang, Qiushi Wang, Wei-Ya Ma, Keke Wang, Xiaoyu Chang, Michele L. Johnson, Ruihua Bai, Ann M. Bode, Nathan R. Foster, Gary W. Falk, Prasad G. Iyer, Zigang Dong. Inhibition of COX1/2-driven thromboxane A2 pathway suppresses Barrett's esophagus and esophageal adenocarcinoma development [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2593.

3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (HMGCR) is a rate-limiting enzyme in the mevalonate pathway and is associated with the development of several tumor types. However, the role of HMGCR in non-small cell lung cancer (NSCLC) is still unknown. In the present study, we found that HMGCR is overexpressed in human lung adenocarcinoma tissues compared with normal tissues. Knockdown of HMGCR in NSCLC cells attenuated growth and induced apoptosis in vitro and in vivo. Furthermore, we found that fluvastatin, an inhibitor of HMGCR, suppressed NSCLC cell growth and induced apoptosis. Intriguingly, fluvastastin functions by inhibiting the HMGCR-driven Braf/MEK/ERK1/2 and Akt signaling pathways. Notably, fluvastatin attenuated tumor growth in 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung tumorigenesis and in a patient-derived xenograft (PDX) lung tumor model. Overall, our findings suggest that fluvastatin might be promising chemopreventive or potential therapeutic drug against NSCLC tumorigenesis, providing hope for rapid clinical translation. Citation Format: Qiushi Wang, Tianshun Zhang, Ruihua Bai, Keke Wang, Xiang Li, Kangdong Liu, Ting Wang, Xiaoyu Chang, Weiya Ma, Ann Bode, Qingxin Xia, Yongping Song, Zigang Dong. Inhibiting HMG-CoA reductase suppresses non-small cell lung carcinogenesis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1443.

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 epidermal growth factor receptor (EGFR) is aberrantly activated in various cancer cells and an important target for cancer treatment. Deep understanding of EGFR conformational changes between the active and inactive states is of pharmaceutical interest. Here we present a strategy combining multiply targeted molecular dynamics simulations, unbiased molecular dynamics simulations, and Bayesian clustering to investigate transition pathways during the activation/inactivation process of EGFR kinase domain. Two distinct pathways between the active and inactive forms are designed, explored, and compared. Based on Bayesian clustering and rough two-dimensional free energy surfaces, the energy-favorable pathway is recognized, though DFG-flip happens in both pathways. In addition, another pathway with different intermediate states appears in our simulations. Comparison of distinct pathways also indicates that disruption of the Lys745-Glu762 interaction is critically important in DFG-flip while movement of the A-loop significantly facilitates the conformational change. Our simulations yield new insights into EGFR conformational transitions. Moreover, our results verify that this approach is valid and efficient in sampling of protein conformational changes and comparison of distinct pathways.

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.

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 over-expression 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 pro-inflammatory 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 up-regulated 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. Citation Format: Ke Yao, Sung-Young Lee, Cong Peng, Do Young Lim, Hiroyuki Yamamoto, Joohyun Ryu, Tae-Gyu Lim, Hanyong Chen, Wei-Ya Ma, Ann M. Bode, Zigang Dong. RSK2 is required for TRAF6 phosphorylation-mediated colon inflammation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1770.

TRAF4 is an adapter protein overexpressed in certain cancers, but its contributions to tumorigenesis are unclear. In lung cancer cells and primary lung tumors, we found that TRAF4 is overexpressed. RNA interference-mediated attenuation of TRAF4 expression blunted the malignant phenotype in this setting, exerting inhibitory effects on cell proliferation, anchorage-independent growth, and tumor development in a xenograft mouse model. Unexpectedly, we discovered that TRAF4, but not Skp2, was required for activation of the pivotal cell survival kinase Akt through ubiquitination. Furthermore, TRAF4 attenuation impaired glucose metabolism by inhibiting expression of Glut1 and HK2 mediated by the Akt pathway. Overall, our work suggests that TRAF4 offers a candidate molecular target for lung cancer prevention and therapy. Cancer Res; 73(23); 6938–50. ©2013 AACR.

In recent years, PQ (power quality) problem makes quality of power supply drop, which even triggers power accident, so PQ has attracted widespread attention. To improve and increase PQ, it needs to analyze and research on PQ disturbance systematically. The paper establishes models of short-term voltage change and harmonic disturbance with the method of setting fault and changing load based on simulation platform of Matlab. The simulation of the model gets wavelet of short-term voltage change and harmonic disturbance, the waveform is analyzed through Flourier transform. The simulation results and the theoretical analysis show that the model in this paper could simulate the voltage change and harmonic disturbance well, which can provide data and basis for detection and identification of PQ and further control measures.

To against the problem of the difficult for indentifying the reflected wave of traveling wave and the need of plentiful practical experience and operation skills, this article focuses on designing and developing a digital cable insulation automatic fault locator. The instruments which can locate the fault without the data of length of the cable is based on the principle of HV bridge method and consist of bridge use for measuring cable length and bridge use for locating cable insulation fault. It can automatically improve voltage output of the high-voltage power through constant current detection circuit. To make sure the abilities of high accuracy,stability and fast for this instrument, This article introduces high-precision digital potentiometer, optical isolation and self-balancing bridge.The optical isolation between high and low pressure not only can achieve a complete electrical isolation,but also improve the anti-interference for the system. 