RSK2 phosphorylates T-bet to attenuate colon cancer metastasis and growth.

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.

Ceftriaxone, an FDA-approved cephalosporin antibiotic, suppresses lung cancer growth by targeting Aurora B.

Ceftriaxone, an FDA-approved third-generation cephalosporin antibiotic, has antimicrobial activity against both gram-positive and gram-negative organisms. Generally, ceftriaxone is used for a variety of infections such as community-acquired pneumonia, meningitis and gonorrhea. Its primary molecular targets are the penicillin-binding proteins. However, other activities of ceftriaxone remain unknown. Herein, we report for the first time that ceftriaxone has antitumor activity in vitro and in vivo. Kinase profiling results predicted that Aurora B might be a potential 'off' target of ceftriaxone. Pull-down assay data confirmed that ceftriaxone could bind with Aurora B in vitro and in A549 cells. Furthermore, ceftriaxone (500 µM) suppressed anchorage-independent cell growth by targeting Aurora B in A549, H520 and H1650 lung cancer cells. Importantly, in vivo xenograft animal model results showed that ceftriaxone effectively suppressed A549 and H520 lung tumor growth by inhibiting Aurora B. These data suggest the anticancer efficacy of ceftriaxone for the treatment of lung cancers through its inhibition of Aurora B.

T-LAK cell-originated protein kinase (TOPK) phosphorylation of Prx1 at Ser-32 prevents UVB-induced apoptosis in RPMI7951 melanoma cells through the regulation of Prx1 peroxidase activity.

Protein kinases are potential targets for the prevention and control of UV-induced skin cancer. T-cell-originated protein kinase (TOPK) is highly expressed in skin cancer cells, but its specific function is still unknown. We investigated the role of TOPK in UVB-induced apoptosis in RPMI7951 human melanoma cells. Liquid chromatography-tandem mass spectrometry analysis was used to identify proteins that bind with TOPK. Immunofluorescence, Western blot, and flow cytometry were used to assess the effect of UVB on TOPK, peroxiredoxin 1 (Prx1), and apoptosis in RPMI7951 cells. TOPK binds with Prx1 and its phosphorylation of Prx1 at Ser-32 is important for regulation of H(2)O(2)-mediated signal transduction. Analysis of the CD spectra of Prx1 and mutant Prx1 (S32A) proteins showed that the secondary structure of Prx1 was significantly altered by phosphorylation of Prx1 at Ser-32. UVB irradiation induced phosphorylation of TOPK in RPMI7951 human melanoma cells and phosphorylated TOPK co-localized with Prx1 in the nucleus. UVB induced the peroxidase activity of Prx1 in vitro and ex vivo. Following treatment with UVB, H(2)O(2) levels and apoptosis were increased in RPMI7951 cells stably expressing TOPK siRNA or stably mutant Prx1 (S32A). Phosphorylation of Prx1 (Ser-32) by TOPK prevents UVB-induced apoptosis in RPMI7951 melanoma cells through regulation of Prx1 peroxidase activity and blockade of intracellular H(2)O(2) accumulation.

Suppression of the solar ultraviolet-induced skin carcinogenesis by TOPK inhibitor HI-TOPK-032.

Nonmelanoma skin cancer (NMSC) such as cutaneous squamous cell carcinoma (cSCC) is caused by solar ultraviolet (SUV) exposure and is the most common cancer in the United States. T-LAK cell-originated protein kinase (TOPK), a serine-threonine kinase is activated by SUV irradiation and involved in skin carcinogenesis. Strategies with research focusing on the TOPK signaling pathway and targeted therapy in skin carcinogenesis may helpful for the discovery of additional treatments against skin cancer. In this study, we found that TOPK can directly bind to and phosphorylate c-Jun (as one of the core member of AP-1) at Ser63 and Ser73 after SSL exposure in a JNKs-independent manner. TOPK knocking down, or HI-TOPK-032 (TOPK specific inhibitor) attenuated colony formation and cell proliferation of skin cancer cells. Phosphorylated levels of c-Jun were overexpressed in human AK and cSCC compared with normal skin tissues, and HI-TOPK-032 inhibited the phosphorylation of c-Jun in SCC cell line in a dose-dependent manner. Furthermore, HI-TOPK-032 decreased SSL-induced AP-1 transactivation activity. Moreover, acute SSL-induced inflammation was attenuated by the topical application of HI-TOPK-032 in SKH1 hairless mice. Importantly, HI-TOPK-032 suppressed chronic SSL-induced skin carcinogenesis and c-Jun phosphorylation levels in SKH1 hairless mice. Our results demonstrate that TOPK can phosphorylate and activate c-Jun at Ser63 and Ser73 in the process of skin carcinogenesis and HI-TOPK-032 could be used as a potential chemopreventive drug against cSCC development.

Resveratrol directly targets COX-2 to inhibit carcinogenesis.

Targeted molecular cancer therapies can potentially deliver treatment directly to a specific protein or gene to optimize efficacy and reduce adverse side effects often associated with traditional chemotherapy. Key oncoprotein and oncogene targets are rapidly being identified based on their expression, pathogenesis and clinical outcome. One such protein target is cyclooxygenase-2 (COX-2), which is highly expressed in various cancers. Research findings suggest that resveratrol (RSVL; 3,5,4'-trihydroxy-trans-stilbene) demonstrates nonselective COX-2 inhibition. We report herein that RSVL directly binds with COX-2 and this binding is absolutely required for RSVL's inhibition of the ability of human colon adenocarcinoma HT-29 cells to form colonies in soft agar. Binding of COX-2 with RSVL was compared with two RSVL analogues, 3,3',4',5',5-pentahydroxy-trans-stilbene (RSVL-2) or 3,4',5-trimethoxy-trans-stilbene (RSVL-3). The results indicated that COX-2 binds with RSVL-2 more strongly than with RSVL, but does not bind with RSVL-3. RSVL or RSVL-2, but not RSVL-3, inhibited COX-2-mediated PGE(2) production in vitro and ex vivo. HT-29 human colon adenocarcinoma cells express high levels of COX-2 and either RSVL or RSVL-2, but not RSVL-3, suppressed anchorage independent growth of these cells in soft agar. RSVL or RSVL-2 (not RSVL-3) suppressed growth of COX-2(+/+) cells by 60% or 80%, respectively. Notably, cells deficient in COX-2 were unresponsive to RSVL or RSVL-2. These data suggest that the anticancer effects of RSVL or RSLV-2 might be mediated directly through COX-2.

Targeting PRPK and TOPK for skin cancer prevention and therapy.

Solar ultraviolet (sUV) irradiation is a major environmental carcinogen that can cause inflammation and skin cancer. The costs and morbidity associated with skin cancer are increasing, and therefore identifying molecules that can help prevent skin carcinogenesis is important. In this study, we identified the p53-related protein kinase (PRPK) as a novel oncogenic protein that is phosphorylated by the T-LAK cell-originated protein kinase (TOPK). Knockdown of TOPK inhibited PRPK phosphorylation and conferred resistance to solar-simulated light (SSL)-induced skin carcinogenesis in mouse models. In the clinic, acute SSL irradiation significantly increased epidermal thickness as well as total protein and phosphorylation levels of TOPK and PRPK in human skin tissues. We identified two PRPK inhibitors, FDA-approved rocuronium bromide (Zemuron®) or betamethasone 17-valerate (Betaderm®) that could attenuate TOPK-dependent PRPK signaling. Importantly, topical application of either rocuronium bromide or betamethasone decreased SSL-induced epidermal hyperplasia, neovascularization, and cutaneous squamous cell carcinoma (cSCC) development in SKH1 (Crl: SKH1-Hrhr) hairless mice by inhibiting PRPK activation, and also reduced expression of the proliferation and oncogenesis markers, COX-2, cyclin D1, and MMP-9. This study is the first to demonstrate that targeting PRPK could be useful against sUV-induced cSCC development.

The Ashitaba (Angelica keiskei) Chalcones 4-hydroxyderricin and Xanthoangelol Suppress Melanomagenesis By Targeting BRAF and PI3K.

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.

Lymphokine-activated killer T-cell-originated protein kinase phosphorylation of histone H2AX prevents arsenite-induced apoptosis in RPMI7951 melanoma cells.

PURPOSE: Arsenic is a valuable therapeutic tool in cancer treatment. Lymphokine-activated killer T-cell-originated protein kinase (TOPK) is highly expressed in cancer cells, but its specific function is still unknown. We investigated the role of TOPK in arsenic-induced apoptosis in RPMI7951 human melanoma cells. EXPERIMENTAL DESIGN: Expression of TOPK was evaluated in different melanoma cell lines, and liquid chromatography-tandem mass spectrometry analysis was used to identify proteins binding with TOPK. Immunofluorescence, Western blot, and flow cytometry were used to assess the effect of arsenic on TOPK, histone H2AX, and apoptosis in RPMI7951 cells. RESULTS: Melanoma cell lines expressing high levels of TOPK were more resistant to arsenite (As(3+))-induced apoptosis. As(3+) treatment induced phosphorylation of TOPK and histone H2AX in RPMI7951 human melanoma cells. Liquid chromatography-tandem mass spectrometry results indicated that TOPK could bind with histone H2AX, and in vitro and in vivo assays confirmed that TOPK binds with and phosphorylates histone H2AX. As(3+) treatment caused phosphorylation of TOPK, which colocalized with phosphorylated histone H2AX in the nucleus. TOPK small interfering RNA cells exhibited a decreased phosphorylation of histone H2AX with As(3+) treatment. As(3+)-induced apoptosis was decreased in H2AX(-/-) cells but increased in TOPK small interfering RNA cells. CONCLUSIONS: TOPK binds with histone H2AX and inhibits As(3+)-induced apoptosis through phosphorylation of histone H2AX. Melanoma cell lines with high levels of TOPK are more resistant to As(3+)-induced apoptosis. Therefore, inhibition of TOPK activity combined with As(3+) treatment may be helpful in the treatment of melanomas.

TRAF1 Is Critical for DMBA/Solar UVR-Induced Skin Carcinogenesis.

TRAF1 is a member of the TRAF protein family, which regulates the canonical and noncanonical NF-κB signaling cascades. Although aberrant TRAF1 expression in tumors has been reported, the role of TRAF1 remains elusive. Here, we report that TRAF1 is required for solar UV-induced skin carcinogenesis. Immunohistochemical analysis showed that TRAF1 expression is up-regulated in human actinic keratosis and squamous cell carcinoma. In vivo studies indicated that TRAF1 expression levels in mouse skin are induced by short-term solar UV irradiation, and a long-term skin carcinogenesis study showed that deletion of TRAF1 in mice results in a significant inhibition of skin tumor formation. Moreover, we show that TRAF1 is required for solar UV-induced extracellular signal-regulated kinase-5 (ERK5) phosphorylation and the expression of AP-1 family members (c-Fos/c-Jun). Mechanistic studies showed that TRAF1 expression enhances the ubiquitination of ERK5 on lysine 184, which is necessary for its kinase activity and AP-1 activation. Overall, our results suggest that TRAF1 mediates ERK5 activity by regulating the upstream effectors of ERK5 and also by modulating its ubiquitination status. Targeting TRAF1 function might lead to strategies for preventing and treating skin cancer.

The T-LAK Cell-originated Protein Kinase Signal Pathway Promotes Colorectal Cancer Metastasis.

Approximately 90% of all cancer deaths arise from the metastatic dissemination of primary tumors. Metastasis is the most lethal attribute of colorectal cancer. New data regarding the molecules contributing to the metastatic phenotype, the pathways they control and the genes they regulate are very important for understanding the processes of metastasis prognosis and prevention in the clinic. The purpose of this study was to investigate the role of T-LAK cell-originated protein kinase (TOPK) in the promotion of colorectal cancer metastasis. TOPK is highly expressed in human metastatic colorectal cancer tissue compared with malignant adenocarcinoma. We identified p53-related protein kinase (PRPK) as a new substrate of TOPK. TOPK binds with and phosphorylates PRPK at Ser250 in vitro and ex vivo. This site plays a critical role in the function of PRPK. Cell lines stably expressing mutant PRPK (S250A), knockdown TOPK, knockdown PRPK or knockdown of both TOPK and PRPK significantly inhibited liver metastasis of human HCT116 colon cancer cells in a xenograft mouse model. Therefore, we conclude that TOPK directly promotes metastasis of colorectal cancer by modulating PRPK. Thus, these findings may assist in the prediction of prognosis or development of new therapeutic strategies against colon cancer.

Serine 347 Phosphorylation by JNKs Negatively Regulates OCT4 Protein Stability in Mouse Embryonic Stem Cells.

The POU transcription factor OCT4 is critical for maintaining the undifferentiated state of embryonic stem cells (ESCs) and generating induced pluripotent stem cells (iPSCs), but its precise mechanisms of action remain poorly understood. Here, we investigated the role of OCT4 phosphorylation in the biological functions of ESCs. We observed that c-Jun N-terminal kinases (JNKs) directly interacted with and phosphorylated OCT4 at serine 347, which inhibited the transcriptional activity of OCT4. Moreover, phosphorylation of OCT4 induced binding of FBXW8, which reduced OCT4 protein stability and enhanced its proteasomal degradation. We also found that the mutant OCT4 (S347A) might delay the differentiation process of mouse ESCs and enhance the efficiency of generating iPSCs. These results demonstrated that OCT4 phosphorylation on serine 347 by JNKs plays an important role in its stability, transcriptional activities, and self-renewal of mouse ESCs.

Biologically active polypeptides from the tropical sea anemone Radianthus macrodactylus.

Some biologically active polypeptides, three high and two low molecular weight cytolysins and four trypsin inhibitors were isolated from the sea anemone Radianthus macrodactylus and characterized. The purification steps involved acetone precipitation, gel filtration, ion-exchange, and affinity chromatography, and ion-exchange and reverse-phase HPLC. The relative molecular weight of high molecular weight Radianthus cytolysins named according to their N-terminal amino acids RTX-A (Ala), RTX-S (Ser) and RTX-G (Gly) was about 20,000. The isoelectric points were 9.8 for RTX-A and RTX-S, and 10.5 for RTX-G. The hemolytic activities of RTX-A, RTX-S and RTX-G were 3.5 x 10(4), 5.0 x10(4), and 1.0 x10(4)HU/mg, respectively, and were inhibited by sphingomyelin. The N-terminal amino acid sequence of RTX-A was determined as ALAGAIIAGAGLGLKILIEVLGEG-VKVKI-. Molecular weight of low molecular weight Radianthus cytolysins RmI, RmII, and of one trypsin inhibitor InI were 5100, 6100 and 7100, respectively. Isoelectric points for RmI and RmII were 9.2 and 9.3. Their hemolytic activity worked out 25 and 20 HU/mg, and was not inhibited by sphingomyelin. Toxicity of RmI and RmII was assessed by their histaminolytic activity. Amino acid composition of RmI and RmII was similar to that of tealiatoxin, histaminolytic cytolysin from the sea anemone Tealia felina.

Isolation, properties and partial amino acid sequence of a new actinoporin from the sea anemone Radianthus macrodactylus.

A new cytolytic toxin, actinoporin RTX-S II, was isolated from the sea anemone Radianthus macrodactylus with a high degree of purity by a combination of gel filtration, ion-exchange and reverse-phase chromatography. RTX-S II has molecular mass of 19,280 Da and isoelectric point of 10.0. The hemolytic activity of RTX-S II is inhibited by sphingomyelin. RTX-S II had an LD(50) of 70 mg/kg, and is lacking in phospholipase activity. The amino acid composition of this protein contains a high amount of basic and non-polar amino acids and no cysteine. The N-terminal sequence of RTX-S II was determined. The partial amino acid sequence (141 aa) of RTX-S II was deduced based on the cDNA sequence obtained with two oligonucleotides encoding the N-terminal portion of RTX-S II and the internal conserved cytolysin peptide by PCR. A comparison of the RTX-S II cDNA sequence and the rtx-s II gene obtained with the same PCR primers indicates that they are 100% identical at the nucleotide level. It shows that no introns are present in the corresponding region of the rtx-s II gene. Multiple alignments of RTX-S II with known sequences of actinoporins show that RTX-S II is highly homologous to magnificalysin II from Heteractis magnifica. The predicted secondary structure of RTX-S II is predominantly anti-parallel beta-structure, which is in good agreement with experimental data obtained from other sea anemones-actinoporins.

Epigallocatechin-gallate suppresses tumorigenesis by directly targeting Pin1.

The most active anticancer component in green tea is epigallocatechin-3-gallate (EGCG). The human peptidyl prolyl cis/trans isomerase (Pin1) plays a critical role in oncogenic signaling. Herein, we report the X-ray crystal structure of the Pin1/EGCG complex resolved at 1.9 Å resolution. Notably, the structure revealed the presence of EGCG in both the WW and PPIase domains of Pin1. The direct binding of EGCG with Pin1 was confirmed and the interaction inhibited Pin1 PPIase activity. In addition, proliferation of cells expressing Pin1 was inhibited and tumor growth in a xenograft mouse model was suppressed. The binding of EGCG with Arg17 in the WW domain prevented the binding of c-Jun, a well-known Pin1 substrate. EGCG treatment corresponded with a decreased abundance of cyclin D1 and diminution of 12-O-tetradecanoylphorbol-l3-acetate-induced AP-1 or NF-κB promoter activity in cells expressing Pin1. Overall, these results showed that EGCG directly suppresses the tumor-promoting effect of Pin1.

Phosphorylation of H2AX at Ser139 and a new phosphorylation site Ser16 by RSK2 decreases H2AX ubiquitination and inhibits cell transformation.

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.

Extracellular signal-regulated kinase 8-mediated c-Jun phosphorylation increases tumorigenesis of human colon cancer.

Extracellular signal-regulated kinase 8 (ERK8), a recently discovered member of the mitogen-activated protein kinase protein family, has been less studied than other family members, leaving its physiologic functions mostly unknown. The biological consequences of overexpression of ERK8 in JB6 Cl41 epidermal skin cells or knockdown of ERK8 in HCT15 colorectal cancer cells was studied. Kinase assays and transient transfection experiments were performed to study the signaling pathway between ERK8 and c-Jun. We found that ERK8 is relatively highly expressed in HCT15 human colorectal cancer cells and plays an important role in the promotion and progression of colorectal cancer. ERK8 promoted neoplastic transformation, and knockdown of ERK8 in HCT15 colorectal cancer cells reduced the tumorigenic properties of these cell lines. Furthermore, a direct interaction between ERK8 and c-Jun was shown. With epidermal growth factor treatment, overexpression of ERK8 in JB6 Cl41 cells caused an increased phosphorylation of c-Jun at Ser(63) and Ser(73), resulting in increased activator protein-1 transactivation. In contrast, knockdown of ERK8 in HCT15 colorectal cancer cells blocked c-Jun phosphorylation. The interaction between ERK8 and c-Jun seems to increase the tumorigenic properties of HCT15 colorectal cancer cells. Thus, ERK8-regulated signaling might serve as a potential therapeutic target in colorectal cancer.

Involvement of ERKs, RSK2 and PKR in UVA-induced signal transduction toward phosphorylation of eIF2alpha (Ser(51)).

Double-stranded RNA-dependent protein kinase R (PKR) has been implicated in anti-viral (antitumor) and apoptotic responses. PKR is activated by extracellular stresses and phosphorylates the alpha subunit of protein synthesis initiation factor eIF2, thereby inhibiting protein synthesis and impeding virus multiplication. Phosphorylation of eIF2alpha in mammalian cells has been shown to be increased after ultraviolet (UV) stress and to be required for UV-induced repression of protein translation. UVA is an important etiological factor in skin carcinogenesis and we observed that UVA induced phosphorylation of PKR (Thr(451)) and eIF2alpha (Ser(51)) in mouse skin epidermal JB6 Cl41 cells. The induction was suppressed by the MEK1 inhibitor, PD 98059. UVA stimulation of PKR and eIF2alpha phosphorylation was also inhibited by a dominant-negative mutant (DNM) of ERK2- or RSK2-deficient cells (RSK2(-)). An inhibitor of p38, SB 202190 or a DNM of p38alpha kinase (DNM-p38alpha) suppressed UVA-induced phosphorylation of eIF2alpha (Ser(51)) but had no effect on phosphorylation of PKR (Thr(451)). Our data indicated that phosphorylation of PKR at Thr(451) is mediated through ERK2 and RSK2, but not through p38 kinase, and is involved in the regulation of Ser(51) phosphorylation of eIF2alpha in UVA-irradiated JB6 cells. In vitro and in vivo kinase assays indicated that phosphorylation of eIF2alpha at Ser(51) occurred indirectly through ERK2, RSK2 or p38 kinase in the cellular response to UVA. These data may lead to the use of these signaling molecules as targets to develop more effective chemopreventive agents with fewer side effects to control UV-induced skin cancer.

Bidirectional signals transduced by TOPK-ERK interaction increase tumorigenesis of HCT116 colorectal cancer cells.

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.

The signal transduction networks required for phosphorylation of STAT1 at Ser727 in mouse epidermal JB6 cells in the UVB response and inhibitory mechanisms of tea polyphenols.

Signal transducers and activators of transcription (STATs) play a critical role in signal transduction pathways. STATs are a family of cytoplasmic proteins with roles as signal messengers and transcription factors that participate in normal cellular responses to cytokines and growth factors. Phosphorylation of STAT1 at Ser727 is essential for its activation and occurs in response to stress signals, inflammation or infection. We observed that UVB induced phosphorylation of STAT1 (Ser727) in mouse epidermal JB6 Cl41 cells. This stimulation was inhibited by PD98059 and UO126, wortmannin, LY294002, SB202190 and SP600125 and dominant negative mutants of ERK2 (DNM-ERK2), p38 (DNM-p38) and JNK1 (DNM-JNK1). The response was absent in Jnk1(-/-) or Jnk2(-/-) knockout cells, but was unaffected by a dominant negative mutant of the phosphatidylinositol-3 kinase (PI-3K) p85 subunit (DNM-Deltap85). STAT1 (Ser727) phosphorylation was also blocked in a Rsk2(-) cell line. In Pdk1(-/-) cells STAT1 was not activated by UVB stimulation compared with strong activation in Pdk1(+/+) cells. Our data indicate that phosphorylation of STAT1 (Ser727) occurs through PI-3K, ERKs, p38 kinase, JNKs, PDK1 and p90RSK2 in the cellular response to UVB. We also show an inhibitory effect of theaflavins and EGCG on UVB-induced STAT1 (Ser727), ERKs, JNKs, PDK1 and p90RSK2 phosphorylation.