Ribosomal S6 kinase 2-forkhead box protein O4 signaling pathway plays an essential role in melanogenesis.

Although previous studies have examined the signaling pathway involved in melanogenesis through which ultraviolet (UV) or α-melanocyte-stimulating hormones (α-MSH) stimuli act as key inducers to produce melanin at the stratum basal layer of the epidermis, the signaling pathway regulating melanogenesis is still controversial. This study reports that α-MSH, not UVA and UVB, acted as a major stimulus of melanogenesis in B16F10 melanoma cells. Signaling pathway analysis using gene knockdown technology and chemical inhibitors, the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK)/p90 ribosomal S6 kinase 2 (RSK2) played an important role in melanogenesis. Unexpectedly, LY294002, a PI3K inhibitor, increased melanogenesis without UV or α-MSH stimulation, suggesting that the PI3K/AKT signaling pathway may not be a major signaling pathway for melanogenesis. Chemical inhibition of the MEKs/ERKs/RSK2 signaling pathway using U0126 or BI-D1870 suppressed melanogenesis by stimulation of UVA or α-MSH stimulation, or both. In particular, the genetic depletion of RSK2 or constitutive active (CA)-RSK2 overexpression showed that RSK2 plays a key role in melanogenesis. Interestingly, forkhead box protein O4 (FOXO4) was phosphorylated by RSK2, resulting in the increase of FOXO4's transactivation activity. Notably, the FOXO4 mutant harboring serine-to-alanine replacement at the phosphorylation sites totally abrogated the transactivation activity and reduced melanin production, indicating that RSK2-mediated FOXO4 activity plays a key role in melanogenesis. Furthermore, kaempferol, a flavonoid inhibiting the RSK2 activity, suppressed melanogenesis. In addition, FOXO4-wt overexpression showed that FOXO4 enhance melanin synthesis. Overall, the RSK2-FOXO4 signaling pathway plays a key role in modulating melanogenesis.

ELK3 destabilization by speckle-type POZ protein suppresses prostate cancer progression and docetaxel resistance.

Accumulating evidence demonstrates that the activity regulation of ELK3, a member of the E26 transformation-specific oncogene family, is critical to regulating cell proliferation, migration, and survival in human cancers. However, the molecular mechanisms of how ELK3 induces chemoresistance in prostate cancer (PCa) have not been elucidated. In this study, we found that SPOP and ELK3 are an interacting partner. The interaction between SPOP and ELK3 resulted in increased ELK3 ubiquitination and destruction, assisted by checkpoint kinase-mediated ELK3 phosphorylation. Notably, the modulation of SPOP-mediated ELK3 protein stability affected the c-Fos-induced cell proliferation and invasion of PCa cells. The clinical involvement of the SPOP-ELK3 axis in PCa development was confirmed by an immunohistochemical assay on 123 PCa tissues, with an inverse correlation between increased ELK3 and decreased SPOP being present in ~80% of the specimens. This observation was supported by immunohistochemistry analysis using a SPOP-mutant PCa specimen. Finally, docetaxel treatment induced cell death by activating checkpoint kinase- and SPOP-mediated ELK3 degradation, while SPOP-depleted or SPOP-mutated PCa cells showed cell death resistance. Notably, this observation was correlated with the protein levels of ELK3. Taken together, our study reveals the precise mechanism of SPOP-mediated degradation of ELK3 and provides evidence that SPOP mutations contribute to docetaxel resistance in PCa.

Dysregulated CREB3 cleavage at the nuclear membrane induces karyoptosis-mediated cell death.

Cancer cells often exhibit resistance to apoptotic cell death, but they may be vulnerable to other types of cell death. Elucidating additional mechanisms that govern cancer cell death is crucial for developing new therapies. Our research identified cyclic AMP-responsive element-binding protein 3 (CREB3) as a crucial regulator and initiator of a unique cell death mechanism known as karyoptosis. This process is characterized by nuclear shrinkage, deformation, and the loss of nuclear components following nuclear membrane rupture. We found that the N-terminal domain (aa 1-230) of full-length CREB3 (CREB3-FL), which is anchored to the nuclear inner membrane (INM), interacts with lamins and chromatin DNA. This interaction maintains a balance between the outward force exerted by tightly packed DNA and the inward constraining force, thereby preserving INM integrity. Under endoplasmic reticulum (ER) stress, aberrant cleavage of CREB3-FL at the INM leads to abnormal accumulation of the cleaved form of CREB3 (CREB3-CF). This accumulation disrupts the attachment of CREB3-FL to the INM, resulting in sudden rupture of the nuclear membrane and the onset of karyoptosis. Proteomic studies revealed that CREB3-CF overexpression induces a DNA damage response akin to that caused by UVB irradiation, which is associated with cellular senescence in cancer cells. These findings demonstrated that the dysregulation of CREB3-FL cleavage is a key factor in karyoptotic cell death. Consequently, these findings suggest new therapeutic strategies in cancer treatment that exploit the process of karyoptosis.

Induction of Autophagy by Extract from Corydalis heterocarpa for Skin Anti-Aging.

The extracts of Corydalis heterocarpa, a salt-tolerant plant, exhibit diverse physiological properties, including anti-inflammatory, anticancer, and antiadipogenic effects. However, the anti-aging effects of C. heterocarpa extract (CHE) on human skin cells have not yet been investigated. In the present study, we determined that CHE inhibited senescence-associated ß-galactosidase (SA-ß-gal)-stained senescent human dermal fibroblasts (HDFs). Furthermore, CHE markedly suppressed the expression of major regulatory proteins involved in senescence, including p53, p21, and caveolin-1. Interestingly, CHE promoted autophagic flux, as confirmed by the formation of microtubule-associated protein 1 light chain 3B (LC3B) puncta and lysosomal activity. Notably, using RNA sequencing (RNA-seq), we showed that CHE selectively regulated the gene expression of leucine-rich repeat and sterile alpha motif-containing 1 (LRSAM1), an important regulator of autophagy. The adenosine-monophosphate activated protein kinase/mammalian target of rapamycin (AMPK/mTOR) pathway, which is essential for autophagy regulation, was also modulated by CHE. LRSAM1 depletion not only inhibited LC3B expression but also decreased the autophagy flux induced by CHE. Moreover, the knockdown of LRSAM1 suppressed the reversal of CHE-induced senescence in old HDFs. Collectively, our study has revealed the rejuvenating effects and molecular mechanisms of CHE, suggesting that CHE may be a promising anti-aging agent.

Repurposing Synthetic Congeners of a Natural Product Aurone Unveils a Lead Antitumor Agent Inhibiting Folded P-Loop Conformation of MET Receptor Tyrosine Kinase.

A library of 24 congeners of the natural product sulfuretin were evaluated against nine panels representing nine cancer diseases. While sulfuretin elicited very weak activities at 10 µM concentration, congener 1t was identified as a potential compound triggering growth inhibition of diverse cell lines. Mechanistic studies in HCT116 colon cancer cells revealed that congener 1t dose-dependently increased levels of cleaved-caspases 8 and 9 and cleaved-PARP, while it concentration-dependently decreased levels of CDK4, CDK6, Cdc25A, and Cyclin D and E resulting in induction of cell cycle arrest and apoptosis in colon cancer HCT116 cells. Mechanistic study also presented MET receptor tyrosine kinase as the molecular target mediating the anticancer activity of compound 1t in HCT116 cells. In silico study predicted folded p-loop conformation as the form of MET receptor tyrosine kinase responsible for binding of compound 1t. Together, the current study presents compound 1t as an interesting anticancer lead for further development.

Licochalcone D Inhibits Skin Epidermal Cells Transformation through the Regulation of AKT Signaling Pathways.

Cell transformation induced by epidermal growth factor (EGF) and 12-O-tetradecanoylphorbol-13-acetate (TPA) is a critical event in cancer initiation and progression, and understanding the underlying mechanisms is essential for the development of new therapeutic strategies. Licorice extract contains various bioactive compounds, which have been reported to have anticancer and anti-inflammatory effects. This study investigated the cancer preventive efficacy of licochalcone D (LicoD), a chalcone derivative in licorice extract, in EGF and TPA-induced transformed skin keratinocyte cells. LicoD effectively suppressed EGF-induced cell proliferation and anchorage-independent colony growth. EGF and TPA promoted the S phase of cell cycle, while LicoD treatment caused G1 phase arrest and down-regulated cyclin D1 and up-regulated p21 expression associated with the G1 phase. LicoD also induced apoptosis and increased apoptosis-related proteins such as cleaved-caspase-3, cleaved-caspase-7, and Bax (Bcl-2-associated X protein). We further investigated the effect of LicoD on the AKT signaling pathway involved in various cellular processes and found decreased p-AKT, p-GSK3ß, and p-NFκB expression. Treatment with MK-2206, an AKT pharmacological inhibitor, suppressed EGF-induced cell proliferation and transformed colony growth. In conclusion, this study demonstrated the potential of LicoD as a preventive agent for skin carcinogenesis.

Ginsenoside Rb2 suppresses cellular senescence of human dermal fibroblasts by inducing autophagy.

Background: Ginsenoside Rb2, a major active component of Panax ginseng, has various physiological activities, including anticancer and anti-inflammatory effects. However, the mechanisms underlying the rejuvenation effect of Rb2 in human skin cells have not been elucidated. Methods: We performed a senescence-associated ß-galactosidase staining assay to confirm cellular senescence in human dermal fibroblasts (HDFs). The regulatory effects of Rb2 on autophagy were evaluated by analyzing the expression of autophagy marker proteins, such as microtubule-associated protein 1A/1B-light chain (LC) 3 and p62, using immunoblotting. Autophagosome and autolysosome formation was monitored using transmission electron microscopy. Autophagic flux was analyzed using tandem-labeled GFP-RFP-LC3, and lysosomal function was assessed with Lysotracker. We performed RNA sequencing to identify potential target genes related to HDF rejuvenation mediated by Rb2. To verify the functions of the target genes, we silenced them using shRNAs. Results: Rb2 decreased ß-galactosidase activity and altered the expression of cell cycle regulatory proteins in senescent HDFs. Rb2 markedly induced the conversion of LC3-Ⅰ to LC3-Ⅱ and LC3 puncta. Moreover, Rb2 increased lysosomal function and red puncta in tandem-labeled GFP-RFP-LC3, which indicate that Rb2 promoted autophagic flux. RNA sequencing data showed that the expression of DNA damage-regulated autophagy modulator 2 (DRAM2) was induced by Rb2. In autophagy signaling, Rb2 activated the AMPK-ULK1 pathway and inactivated mTOR. DRAM2 knockdown inhibited autophagy and Rb2-restored cellular senescence. Conclusion: Rb2 reverses cellular senescence by activating autophagy via the AMPK-mTOR pathway and induction of DRAM2, suggesting that Rb2 might have potential value as an antiaging agent.

MEKs/ERKs-mediated FBXO1/E2Fs interaction interference modulates G1/S cell cycle transition and cancer cell proliferation.

E2F 1, 2, and 3a, (refer to as E2Fs) are a subfamily of E2F transcription factor family that play essential roles in cell-cycle progression, DNA replication, DNA repair, apoptosis, and differentiation. Although the transcriptional regulation of E2Fs has focused on pocket protein retinoblastoma protein complex, recent studies indicate that post-translational modification and stability regulation of E2Fs play key roles in diverse cellular processes. In this study, we found that FBXO1, a component of S-phase kinase-associated protein 1 (SKP1)-cullin 1-F-box protein (SCF) complex, is an E2Fs binding partner. Furthermore, FBXO1 to E2Fs binding induced K48 ubiquitination and subsequent proteasomal degradation of E2Fs. Binding domain analysis indicated that the Arg (R)/Ile (I) and R/Val (V) motifs, which are located in the dimerization domain of E2Fs, of E2F 1 and 3a and E2F2, respectively, acted as degron motifs (DMs) for FBXO1. Notably, RI/AA or RV/AA mutation in the DMs reduced FBXO1-mediated ubiquitination and prolonged the half-lives of E2Fs. Importantly, the stabilities of E2Fs were affected by phosphorylation of threonine residues located near RI and RV residues of DMs. Phosphorylation prediction database analysis and specific inhibitor analysis revealed that MEK/ERK signaling molecules play key roles in FBXO1/E2Fs' interaction and modulate E2F protein turnover. Moreover, both elevated E2Fs protein levels by knockdown of FBXO1 and decreased E2Fs protein levels by sh-E2F3a delayed G1/S cell cycle transition, resulting in inhibition of cancer cell proliferation. These results demonstrated that FBXO1-E2Fs axis-mediated precise E2Fs stability regulation plays a key role in cell proliferation via G1/S cell cycle transition.

Clinical value of urinary cytokines/chemokines as prognostic markers in patients with crescentic glomerulonephritis.

Crescentic glomerulonephritis (CrGN) usually requires urgent immunosuppressive treatment. However, aggressive immunosuppressive treatment is often difficult because of the patients' medical conditions or comorbidities. Prognostic markers including urinary cytokines/chemokines as noninvasive biomarkers were explored in CrGN patients. This prospective cohort study included 82 patients with biopsy-confirmed CrGN from 2002 to 2015 who were followed up for 5 years. Urine and serum cytokines/chemokines on the day of kidney biopsy were analyzed in 36 patients. The median age was 65 years and 47.6% were male. Baseline estimated glomerular filtration rate (eGFR) and interstitial fibrosis and tubular atrophy (IFTA) scores were identified as significant prognostic factors. Among patients with cytokines/chemokines measurement, increased IL-10 level was identified as an independent predictor of good prognosis, and increased levels of urinary MCP-1 and fractalkine tended to be associated with good prognosis after adjusting for baseline eGFR and IFTA score. However, semiquantitative analysis of intrarenal leukocytes did not show prognostic value predicting renal outcome or correlation with urinary cytokines/chemokines. This study supports the clinical importance of baseline eGFR and IFTA scores and suggests potential usefulness of urinary IL-10, MCP-1, and fractalkine as prognostic markers for predicting renal outcomes in patients with CrGN.

Positional scanning of natural product hispidol's ring-B: discovery of highly selective human monoamine oxidase-B inhibitor analogues downregulating neuroinflammation for management of neurodegenerative diseases.

Multifunctional molecules might offer better treatment of complex multifactorial neurological diseases. Monoaminergic pathways dysregulation and neuroinflammation are common convergence points in diverse neurodegenerative and neuropsychiatric disorders. Aiming to target these diseases, polypharmacological agents modulating both monoaminergic pathways and neuroinflammatory were addressed. A library of analogues of the natural product hispidol was prepared and evaluated for inhibition of monoamine oxidases (MAOs) isoforms. Several molecules emerged as selective potential MAO B inhibitors. The most promising compounds were further evaluated in vitro for their impact on microglia viability, induced production of proinflammatory mediators and MAO-B inhibition mechanism. Amongst tested compounds, 1p was a safe potent competitive reversible MAO-B inhibitor and inhibitor of microglial production of neuroinflammatory mediators; NO and PGE2. In-silico study provided insights into molecular basis of the observed selective MAO B inhibition. This study presents compound 1p as a promising lead compound for management of neurodegenerative disease.

FBXW7-mediated ERK3 degradation regulates the proliferation of lung cancer cells.

Extracellular signal-regulated kinase 3 (ERK3) is an atypical member of the mitogen-activated protein kinase (MAPK) family, members of which play essential roles in diverse cellular processes during carcinogenesis, including cell proliferation, differentiation, migration, and invasion. Unlike other MAPKs, ERK3 is an unstable protein with a short half-life. Although deubiquitination of ERK3 has been suggested to regulate the activity, its ubiquitination has not been described in the literature. Here, we report that FBXW7 (F-box and WD repeat domain-containing 7) acts as a ubiquitination E3 ligase for ERK3. Mammalian two-hybrid assay and immunoprecipitation results demonstrated that ERK3 is a novel binding partner of FBXW7. Furthermore, complex formation between ERK3 and the S-phase kinase-associated protein 1 (SKP1)-cullin 1-F-box protein (SCF) E3 ligase resulted in the destabilization of ERK3 via a ubiquitination-mediated proteasomal degradation pathway, and FBXW7 depletion restored ERK3 protein levels by inhibiting this ubiquitination. The interaction between ERK3 and FBXW7 was driven by binding between the C34D of ERK3, especially at Thr417 and Thr421, and the WD40 domain of FBXW7. A double mutant of ERK3 (Thr417 and Thr421 to alanine) abrogated FBXW7-mediated ubiquitination. Importantly, ERK3 knockdown inhibited the proliferation of lung cancer cells by regulating the G1/S-phase transition of the cell cycle. These results show that FBXW7-mediated ERK3 destabilization suppresses lung cancer cell proliferation in vitro.

Kaempferol sensitizes cell proliferation inhibition in oxaliplatin-resistant colon cancer cells.

Resolution to chemoresistance is a major challenge in patients with advanced-stage malignancies. Thus, identification of action points and elucidation of molecular mechanisms for chemoresist human cancer are necessary to overcome this challenge. In this study, we provide important evidence that kaempferol targeting RSKs might be a strategy to reduce the oxaliplatin-resistant colon cancer cells. We found that MAPK and PI3K-AKT signaling were increased in oxaliplatin (Ox)-resistant HCT116 (HCT116-OxR) cells compared to Ox-sensitive HCT116 (HCT116-OxS) cells. Comparison of cell sensitivities using SP600125 (JNK inhibitor), SB206580 (p38 kinase inhibitor), or MK-2206 (AKT inhibitor) revealed that cell proliferation inhibition was strongly observed in HT29 cells compared to that in HCT116 cells in both OxS and OxR cells. Interestingly, SP600125, SB206580, and MK-2206 treatment showed higher cell proliferation inhibition in OxS cells than that in OxR cells in both HCT116 and HT29 cells, except following treatments with 10 µM of SP600125, and 30 µM of SB206580. In comparison to magnolin and aschantin, kaempferol showed the strongest inhibitory effect on cell proliferation in both HCT116 and HT29 cells. Importantly, HCT116- and HT29-OxR cells showed higher sensitivities to cell proliferation inhibition than those of HCT116- and HT29-OxS cells, resulting in the accumulation of cells at the G2/M-phases of the cell cycle. Finally, we showed that AP-1 transactivation activity was markedly decreased by kaempferol in HCT116- and HT29-OxR cells compared to the activity levels in HCT116- and HT29-OxS cells. Taken together, the results demonstrate that kaempferol-mediated AP-1 inhibition might be an important signaling mechanism to resolve the chemoresistance of Ox-resistant colon cancer cells.

F-box Protein ßTrCP1 Is a Substrate of Extracellular Signal-regulated Kinase 2.

F-box proteins, consisting of 69 members which are organized into the three subclasses FBXW, FBXL, and FBXO, are the substrate specific recognition subunits of the SKP1-Cullin 1-F-box protein E3 ligase complex. Although ßTrCP 1 and 2, members of the FBXW subfamily, are known to regulate some protein stability, molecular mechanisms by which these proteins can recognize proper substrates are unknown. In this study, it was found that ßTrCP1 showed strong interaction with members of mitogen-activated protein kinases. Although extracellular signal-regulated kinase (ERK) 3, p38ß, and p38δ showed weak interactions, ERK2 specifically interacted with ßTrCP1 as assessed by immunoprecipitation. In interaction domain determination experiments, we found that ERK2 interacted with two independent ERK docking sites located in the F-box domain and linker domain, but not the WD40 domain, of ßTrCP1. Notably, mutations of ßTrCP1 at the ERK docking sites abolished the interaction with ERK2. ßTrCP1 underwent phosphorylation by EGF stimulation, while the presence of the mitogen-activated protein kinase kinases inhibitor U0126, genetic silencing by sh-ERK2, and mutation of the ERK docking site of ßTrCP1 inhibited phosphorylation. This inhibition of ßTrCP1 phosphorylation resulted in a shortened half-life and low protein levels. These results suggest that ERK2-mediated ßTrCP1 phosphorylation may induce the destabilization of ßTrCP1.

R (-)-methoxetamine exerts rapid and sustained antidepressant effects and fewer behavioral side effects relative to S (+)-methoxetamine.

The newfound antidepressant efficacy of ketamine has provided opportunities for the development of new-generation, rapid-acting, glutamate-based antidepressants. We previously identified that methoxetamine (MXE), a ketamine analog, and an N-Methyl-d-aspartate (NMDA) receptor antagonist, produced rapid and sustained antidepressant effects in mice. MXE (R, S (±)-MXE) is a racemic mixture containing equal parts of S (+)-MXE and R (-)-MXE. However, studies have yet to investigate the antidepressant effects of its enantiomers. Here, we examined the potential antidepressant properties and behavioral side effects of S- and R-MXE in mice. Both S- and R-MXE showed significant NMDA receptor affinity and appreciable inhibitory activity on serotonin transporter. Also, S- and R-MXE (10 mg kg-1) exerted antidepressant effects and increased gamma waves (electroencephalography) but were inhibited by NBQX (an AMPA receptor antagonist). Subsequently, they increased mammalian target of rapamycin phosphorylation and AMPA receptor subunits GluA1 and GluA2 protein levels in the hippocampus or prefrontal cortex. Furthermore, they increased 5HT2a and 5HT2c receptor mRNA levels in the prefrontal cortex, with their antidepressant effects inhibited by ketanserin (a 5HT2a/c receptor antagonist). Taken together, S-MXE and R-MXE elicit antidepressant effects that are probably mediated via glutamatergic and serotonergic mechanisms. Unlike S-MXE, R-MXE did not induce prepulse inhibition deficits, hyperlocomotion, conditioned place preference, and locomotor sensitization, although it acutely altered motor coordination. This suggests that R-MXE induces fewer behavioral side effects and is a safer antidepressant than S-MXE. Overall, this study provides significant implications for future research on the next generation of rapid-acting, glutamate-based antidepressant drugs.

Fargesin Inhibits EGF-Induced Cell Transformation and Colon Cancer Cell Growth by Suppression of CDK2/Cyclin E Signaling Pathway.

Although the lignan compound fargesin is a major ingredient in Shin-Yi, the roles of fargesin in carcinogenesis and cancer cell growth have not been elucidated. In this study, we observed that fargesin inhibited cell proliferation and transformation by suppression of epidermal growth factor (EGF)-stimulated G1/S-phase cell cycle transition in premalignant JB6 Cl41 and HaCaT cells. Unexpectedly, we found that signaling pathway analyses showed different regulation patterns in which fargesin inhibited phosphatidylinositol 3-kinase/AKT signaling without an alteration of or increase in mitogen activated protein kinase (MAPK) in JB6 Cl41 and HaCaT cells, while both signaling pathways were abrogated by fargesin treatment in colon cancer cells. We further found that fargesin-induced colony growth inhibition of colon cancer cells was mediated by suppression of the cyclin dependent kinase 2 (CDK2)/cyclin E signaling axis by upregulation of p21WAF1/Cip1, resulting in G1-phase cell cycle accumulation in a dose-dependent manner. Simultaneously, the suppression of CDK2/cyclin E and induction of p21WAF1/Cip1 were correlated with Rb phosphorylation and c-Myc suppression. Taken together, we conclude that fargesin-mediated c-Myc suppression inhibits EGF-induced cell transformation and colon cancer cell colony growth by the suppression of retinoblastoma (Rb)-E2F and CDK/cyclin signaling pathways, which are mainly regulated by MAPK and PKB signaling pathways.

Stat2 stability regulation: an intersection between immunity and carcinogenesis.

Signal transducer and activator of transcription (STAT2) is a member of the STAT family that plays an essential role in immune responses to extracellular and intracellular stimuli, including inflammatory reactions, invasion of foreign materials, and cancer initiation. Although the majority of STAT2 studies in the last few decades have focused on interferon (IFN)-α/ß (IFNα/ß) signaling pathway-mediated host defense against viral infections, recent studies have revealed that STAT2 also plays an important role in human cancer development. Notably, strategic research on STAT2 function has provided evidence that transient regulatory activity by homo- or heterodimerization induces its nuclear localization where it to forms a ternary IFN-stimulated gene factor 3 (ISGF3) complex, which is composed of STAT1 and/or STAT2 and IFN regulatory factor 9 (IEF9). The molecular mechanisms of ISGF3-mediated ISG gene expression provide the basic foundation for the regulation of STAT2 protein activity but not protein quality control. Recently, previously unknown molecular mechanisms of STAT2-mediated cell proliferation via STAT2 protein quality control were elucidated. In this review, we briefly summarize the role of STAT2 in immune responses and carcinogenesis with respect to the molecular mechanisms of STAT2 stability regulation via the proteasomal degradation pathway.

Overexpression of TC-PTP in murine epidermis attenuates skin tumor formation.

T-cell protein tyrosine phosphatase (TC-PTP), encoded by Ptpn2, has been shown to function as a tumor suppressor during skin carcinogenesis. In the current study, we generated a novel epidermal-specific TC-PTP-overexpressing (K5HA.Ptpn2) mouse model to show that TC-PTP contributes to the attenuation of chemically induced skin carcinogenesis through the synergistic regulation of STAT1, STAT3, STAT5, and PI3K/AKT signaling. We found overexpression of TC-PTP increased epidermal sensitivity to DMBA-induced apoptosis and it decreased TPA-mediated hyperproliferation, coinciding with reduced epidermal thickness. Inhibition of STAT1, STAT3, STAT5, or AKT reversed the effects of TC-PTP overexpression on epidermal survival and proliferation. Mice overexpressing TC-PTP in the epidermis developed significantly reduced numbers of tumors during skin carcinogenesis and presented a prolonged latency of tumor initiation. Examination of human papillomas and squamous cell carcinomas (SCCs) revealed that TC-PTP expression was significantly reduced and TC-PTP expression was inversely correlated with the increased grade of SCCs. Our findings demonstrate that TC-PTP is a potential therapeutic target for the prevention of human skin cancer given that it is a major negative regulator of oncogenic signaling.

FBXW7-mediated stability regulation of signal transducer and activator of transcription 2 in melanoma formation.

In this study, we provide critical evidence that STAT2 stability regulation plays an essential role in melanoma cell proliferation and colony growth. We found that the interaction of FBXW7 and STAT2 induced STAT2 destabilization via a ubiquitination-mediated proteasomal degradation pathway. Notably, GSK3ß-mediated STAT2 phosphorylation facilitated STAT2-FBXW7 interactions via the DNA binding domain of STAT2 and domains 1, 2, 6, and 7 of FBXW7 WD40. Importantly, the inverse correlation between protein levels of STAT2 and FBXW7 were observed not only in human melanoma cells but also in a human skin cancer tissue array. The relationship between protein levels of STAT2 and FBXW7, cell proliferation, and colony growth were similarly observed in the melanoma cell lines SK-MEL-2, -5, and -28. Moreover, STAT2 knockdown in melanoma cells suppressed melanoma cell proliferation and colony formation. These data demonstrated that FBXW7-mediated STAT2 stability regulation plays an essential role in melanoma cell proliferation and cancer growth.

RSK2-Mediated ELK3 Activation Enhances Cell Transformation and Breast Cancer Cell Growth by Regulation of c-fos Promoter Activity.

Ribosomal S6 kinase 2 (RSK2), regulated by Ras/Raf/MEKs/ERKs, transmits upstream activation signals to downstream substrates including kinases and transcription and epigenetic factors. We observed that ELK members, including ELK1, 3, and 4, highly interacted with RSK2. We further observed that the RSK2-ELK3 interaction was mediated by N-terminal kinase and linker domains of RSK2, and the D and C domains of ELK3, resulting in the phosphorylation of ELK3. Importantly, RSK2-mediated ELK3 enhanced c-fos promoter activity. Notably, chemical inhibition of RSK2 signaling using kaempferol (a RSK2 inhibitor) or U0126 (a selective MEK inhibitor) suppressed EGF-induced c-fos promoter activity. Moreover, functional deletion of RSK2 by knockdown or knockout showed that RSK2 deficiency suppressed EGF-induced c-fos promoter activity, resulting in inhibition of AP-1 transactivation activity and Ras-mediated foci formation in NIH3T3 cells. Immunocytofluorescence assay demonstrated that RSK2 deficiency reduced ELK3 localization in the nucleus. In MDA-MB-231 breast cancer cells, knockdown of RSK2 or ELK3 suppressed cell proliferation with accumulation at the G1 cell cycle phase, resulting in inhibition of foci formation and anchorage-independent cancer colony growth in soft agar. Taken together, these results indicate that a novel RSK2/ELK3 signaling axis, by enhancing c-Fos-mediated AP-1 transactivation activity, has an essential role in cancer cell proliferation and colony growth.

Epimagnolin targeting on an active pocket of mammalian target of rapamycin suppressed cell transformation and colony growth of lung cancer cells.

Mammalian target of rapamycin (mTOR) has a pivotal role in carcinogenesis and cancer cell proliferation in diverse human cancers. In this study, we observed that epimagnolin, a natural compound abundantly found in Shin-Yi, suppressed cell proliferation by inhibition of epidermal growth factor (EGF)-induced G1/S cell-cycle phase transition in JB6 Cl41 cells. Interestingly, epimagnolin suppressed EGF-induced Akt phosphorylation strongly at Ser473 and weakly at Thr308 without alteration of phosphorylation of MAPK/ERK kinases (MEKs), extracellular signal-regulated kinase (ERKs), and RSK1, resulting in abrogation of the phosphorylation of GSK3ß at Ser9 and p70S6K at Thr389. Moreover, we found that epimagnolin suppressed c-Jun phosphorylation at Ser63/73, resulting in the inhibition of activator protein 1 (AP-1) transactivation activity. Computational docking indicated that epimagnolin targeted an active pocket of the mTOR kinase domain by forming three hydrogen bonds and three hydrophobic interactions. The prediction was confirmed by using in vitro kinase and adenosine triphosphate-bead competition assays. The inhibition of mTOR kinase activity resulted in the suppression of anchorage-independent cell transformation. Importantly, epimagnolin efficiently suppressed cell proliferation and anchorage-independent colony growth of H1650 rather than H460 lung cancer cells with dependency of total and phosphorylated protein levels of mTOR and Akt. Inhibitory signaling of epimagnolin on cell proliferation of lung cancer cells was observed mainly in mTOR-Akt-p70S6K and mTOR-Akt-GSK3ß-AP-1, which was similar to that shown in JB6 Cl41 cells. Taken together, our results indicate that epimagnolin potentiates as chemopreventive or therapeutic agents by direct active pocket targeting of mTOR kinase, resulting in sensitizing cancer cells harboring enhanced phosphorylation of the mTORC2-Akt-p70S6k signaling pathway.