Janos Nadas

ERK1 and ERK2 regulate embryonic stem cell self-renewal through phosphorylation of Klf4

Understanding and controlling the mechanism by which stem cells balance self-renewal versus differentiation is of great importance for stem cell therapeutics. Klf4 promotes the self-renewal of embryonic stem cells, but the precise mechanism regulating this role of Klf4 is unclear. We found that ERK1 or ERK2 binds the activation domain of Klf4 and directly phosphorylates Klf4 at Ser123. This phosphorylation suppresses Klf4 activity, inducing embryonic stem cell differentiation. Conversely, inhibition of Klf4 phosphorylation enhances Klf4 activity and suppresses embryonic stem cell differentiation. Notably, phosphorylation of Klf4 by ERKs causes recruitment and binding of the F-box proteins βTrCP1 or βTrCP2 (components of an ubiquitin E3 ligase) to the Klf4 N-terminal domain, which results in Klf4 ubiquitination and degradation. Overall, our data provide a molecular basis for the role of ERK1 and ERK2 in regulating Klf4-mediated mouse embryonic stem cell self-renewal.

Eriodictyol inhibits RSK2-ATF1 signaling and suppresses EGF-induced neoplastic cell transformation

RSK2 is a widely expressed serine/threonine kinase, and its activation enhances cell proliferation. Here, we report that ATF1 is a novel substrate of RSK2 and that RSK2-ATF1 signaling plays an important role in EGF-induced neoplastic cell transformation. RSK2 phosphorylated ATF1 at Ser-63 and enhanced ATF1 transcriptional activity. Docking experiments using the crystal structure of the RSK2 N-terminal kinase domain combined with in vitro pulldown assays demonstrated that eriodictyol, a flavanone found in fruits, bound with the N-terminal kinase domain of RSK2 to inhibit RSK2 N-terminal kinase activity. In cells, eriodictyol inhibited phosphorylation of ATF1 but had no effect on the phosphorylation of RSK, MEK1/2, ERK1/2, p38 or JNKs, indicating that eriodictyol specifically suppresses RSK2 signaling. Furthermore, eriodictyol inhibited RSK2-mediated ATF1 transactivation and tumor promoter-induced transformation of JB6 Cl41 cells. Eriodictyol or knockdown of RSK2 or ATF1 also suppressed Ras-mediated focus formation. Overall, these results indicate that RSK2-ATF1 signaling plays an important role in neoplastic cell transformation and that eriodictyol is a novel natural compound for suppressing RSK2 kinase activity.

Prediction of molecular targets of cancer preventing flavonoid compounds using computational methods.

Plant-based polyphenols (i.e., phytochemicals) have been used as treatments for human ailments for centuries. The mechanisms of action of these plant-derived compounds are now a major area of investigation. Thousands of phytochemicals have been isolated, and a large number of them have shown protective activities or effects in different disease models. Using conventional approaches to select the best single or group of best chemicals for studying the effectiveness in treating or preventing disease is extremely challenging. We have developed and used computational-based methodologies that provide efficient and inexpensive tools to gain further understanding of the anticancer and therapeutic effects exerted by phytochemicals. Computational methods involving virtual screening, shape and pharmacophore analysis and molecular docking have been used to select chemicals that target a particular protein or enzyme and to determine potential protein targets for well-characterized as well as for novel phytochemicals.

(3-Chloroacetyl)-indole, a Novel Allosteric AKT Inhibitor, Suppresses Colon Cancer Growth In Vitro and In Vivo

Indole-3-carbinol (I3C) is produced in Brassica vegetables such as broccoli and cabbage and has been shown to inhibit proliferation and induce apoptosis in various cancer cells, including breast, prostate, colon, and leukemia. However, only high doses of I3C were shown to inhibit cell proliferation (IC50 = 200–300 μmol/L). Our goal here was to develop a more potent antitumor agent by modifying the structure of I3C. We created I3C derivatives and found that (3-chloroacetyl)-indole (3CAI) more strongly inhibited colon cancer cell growth than I3C. In addition, by screening 85 kinases in a competitive kinase assay, we found that 3CAI was a specific AKT inhibitor. AKT is a serine/threonine kinase that plays a pivotal role in promoting transformation and chemoresistance by inducing proliferation and inhibiting apoptosis. Therefore, AKT is regarded as a critical target for cancer therapy. 3ICA, a derivative of I3C, is a potent and specific AKT inhibitor. This compound showed significant inhibition of AKT in an in vitro kinase assay and suppressed expression of AKT direct downstream targets such as mTOR and GSK3β as well as induced growth inhibition and apoptosis in colon cancer cells. In addition, oral administration of this potent AKT inhibitor suppressed cancer cell growth in an in vivo xenograft mouse model. Cancer Prev Res; 4(11); 1842–51. ©2011 AACR.

15-Deoxy-Δ12,14-prostaglandin J2 stabilizes hypoxia inducible factor-1α through induction of heme oxygenase-1 and direct modification ofprolyl-4-hydroxylase 2

Abstract 15-Deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2), a representative J-series cyclopentenone prostaglandin, has biphasic roles in cell proliferation and apoptosis. Hypoxia inducible factor-1 (HIF-1) regulates expression of various genes involved in tumor growth and angiogenesis. In the present study, treatment of human breast cancer (MCF-7) cells with 15d-PGJ2 resulted in the accumulation of the α-subunit of HIF-1. Pretreatment with zinc protoporphyrin IX, a pharmacological inhibitor of heme oxygenase-1 (HO-1), as well as siRNA knockdown of HO-1 gene in MCF-7 cells attenuated 15d-PGJ2-mediated HIF-1α accumulation. 15d-PGJ2 treatment increased intracellular production of reactive oxygen species (ROS), which was mediated by HO-1 induction. Preincubation of MCF-7 cells with trolox, a water-soluble form of vitamin E, attenuated 15d-PGJ2-induced HIF-1α expression although HO-1 expression was unchanged. This finding suggests that ROS accumulated as a consequence of HO-1 up-regulation can enhance HIF-1α expression in MCF-7 cells treated with 15d-PGJ2. Alternatively, 15d-PGJ2 was found to covalently bind to HIF-1α prolyl-4-hydroxylase 2 (PHD2) in MCF-7 cells, which hampers the proline hydroxylation of HIF-1α, thereby disrupting ubiquitin-dependent proteasomal degradation of this transcription factor. Pretreatment with thiol reducing agents blunted 15d-PGJ2-induced HIF-1α stabilization, indicative of a cysteine residue as a direct target of 15d-PGJ2. Molecular docking analysis suggests that 15d-PGJ2 preferentially binds to PHD2 in the vicinity of the Cys201 residue based on binding energies and carbon–sulfur distances. In summary, 15d-PGJ2 stabilizes HIF-1α in MCF-7 cells through HO-1 induction with subsequent ROS generation and also through direct modification of PHD2.

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.

Targeting PRPK Function Blocks Colon Cancer Metastasis

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.

Abstract 4777: Computational strategies for finding protein targets of flavonoids

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Plant-based polyphenols (i.e., phytochemicals) have been used as treatments for human ailments for centuries. The mechanisms of action of these plant-derived medicines are a now major topic of investigation. Thousands of phytochemicals have been isolated, and a certain number of them have shown activities or effects on different disease models. Using conventional approaches to select the best or group of the best chemicals for studying the effectiveness in treating or preventing disease is extremely challenging. We have developed and used computational-based methodologies that provide an efficient and inexpensive tools to gain further understanding of the anticancer and therapeutic effects exerted by polyphenols. Computational strategies involving virtual screening, shape and pharmacophore analysis and molecular docking have been used to select chemicals that target a particular protein or enzyme and to determine potential protein targets for well-characterized as well as for novel polyphenols. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4777. doi:1538-7445.AM2012-4777

Abstract 2884: Resveratrol, a red wine polyphenol, suppresses pancreatic cancer by inhibiting leukotriene A4 hydrolase

Considerable attention has focused on the anticancer effects of red wine. Resveratrol (3,5,4’-trihydroxy-trans-stilbene) is a well-known polyphenolic compound found in red wine and recognized as a key substance for cancer chemoprevention. Leukotriene A4 hydrolase (LTA4H) is a bi-functional zinc enzyme that is regarded as a relevant target in certain types of human cancers. Our data indicate that knockdown of LTA4H decreased production of leukotriene B4 (LTB4), the enzymatic product of LTA4H, and suppressed anchorage-independent growth of MIA PaCa-2 pancreatic cancer cells. An in silico prediction using a shape-similarity approach suggested that LTA4H might be a potential target of resveratrol. Results showed that resveratrol directly bound to LTA4H in vitro and ex vivo and suppressed proliferation and anchorage-independent growth by inhibiting LTA4H activity in MIA PaCa-2 cells. Importantly, resveratrol exerted stronger inhibitory effects compared to bestatin, a well-known inhibitor of LTA4H activity. The inhibitory effects of resveratrol were reduced in MIA PaCa-2 cells transfected with sh-LTA4H compared with sh-mock transfected cells. Notably, in an in vivo xenograft mouse model, resveratrol suppressed MIA PaCa-2 tumor formation by inhibiting LTA4H activity. These findings suggest that LTA4H is a novel target for the preventive and/or therapeutic effects of resveratrol on pancreatic cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2884. doi:10.1158/1538-7445.AM2011-2884