MEK-ERK Signaling Research Articles

Oleanolic acid improved intestinal immune function by activating and potentiating bile acids receptor signaling in E. coli-challenged piglets

BackgroundInfection with pathogenic bacteria during nonantibiotic breeding is one of the main causes of animal intestinal diseases. Oleanolic acid (OA) is a pentacyclic triterpene that is ubiquitous in plants. Our previous work demonstrated the protective effect of OA on intestinal health, but the underlying molecular mechanisms remain unclear. This study investigated whether dietary supplementation with OA can prevent diarrhea and intestinal immune dysregulation caused by enterotoxigenic Escherichia coli (ETEC) in piglets. The key molecular role of bile acid receptor signaling in this process has also been explored.ResultsOur results demonstrated that OA supplementation alleviated the disturbance of bile acid metabolism in ETEC-infected piglets (P < 0.05). OA supplementation stabilized the composition of the bile acid pool in piglets by regulating the enterohepatic circulation of bile acids and significantly increased the contents of UDCA and CDCA in the ileum and cecum (P < 0.05). This may also explain why OA can maintain the stability of the intestinal microbiota structure in ETEC-challenged piglets. In addition, as a natural ligand of bile acid receptors, OA can reduce the severity of intestinal inflammation and enhance the strength of intestinal epithelial cell antimicrobial programs through the bile acid receptors TGR5 and FXR (P < 0.05). Specifically, OA inhibited NF-κB-mediated intestinal inflammation by directly activating TGR5 and its downstream cAMP-PKA-CREB signaling pathway (P < 0.05). Furthermore, OA enhanced CDCA-mediated MEK-ERK signaling in intestinal epithelial cells by upregulating the expression of FXR (P < 0.05), thereby upregulating the expression of endogenous defense molecules in intestinal epithelial cells.ConclusionsIn conclusion, our findings suggest that OA-mediated regulation of bile acid metabolism plays an important role in the innate immune response, which provides a new diet-based intervention for intestinal diseases caused by pathogenic bacterial infections in piglets.Graphical

Simulating BRAFV600E-MEK-ERK signalling dynamics in response to vertical inhibition treatment strategies

In vertical inhibition treatment strategies, multiple components of an intracellular pathway are simultaneously inhibited. Vertical inhibition of the BRAFV600E–MEK-ERK signalling pathway is a standard of care for treating BRAFV600E-mutated melanoma where two targeted cancer drugs, a BRAFV600E-inhibitor, and a MEK inhibitor, are administered in combination. Targeted therapies have been linked to early onsets of drug resistance, and thus treatment strategies of higher complexities and lower doses have been proposed as alternatives to current clinical strategies. However, finding optimal complex, low-dose treatment strategies is a challenge, as it is possible to design more treatment strategies than are feasibly testable in experimental settings. To quantitatively address this challenge, we develop a mathematical model of BRAFV600E–MEK-ERK signalling dynamics in response to combinations of the BRAFV600E-inhibitor dabrafenib (DBF), the MEK inhibitor trametinib (TMT), and the ERK-inhibitor SCH772984 (SCH). From a model of the BRAFV600E–MEK–ERK pathway, and a set of molecular-level drug–protein interactions, we extract a system of chemical reactions that is parameterised by in vitro data and converted to a system of ordinary differential equations (ODEs) using the law of mass action. The ODEs are solved numerically to produce simulations of how pathway-component concentrations change over time in response to different treatment strategies, i.e., inhibitor combinations and doses. The model can thus be used to limit the search space for effective treatment strategies that target the BRAFV600E–MEK–ERK pathway and warrant further experimental investigation. The results demonstrate that DBF and DBF–TMT–SCH therapies show marked sensitivity to BRAFV600E concentrations in silico, whilst TMT and SCH monotherapies do not.

Napyradiomycin B4 Suppresses RANKL-Induced Osteoclastogenesis and Prevents Alveolar Bone Destruction in Experimental Periodontitis.

The unique structure and beneficial biological properties of marine natural products have drawn interest in drug development. Here, we examined the therapeutic potential of napyradiomycin B4 isolated from marine-derived Streptomyces species for osteoclast-related skeletal diseases. Bone marrow-derived macrophages were treated with napyradiomycin B4 in an osteoclast-inducing medium, and osteoclast formation, osteoclast-specific gene expression, and nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) localization were evaluated using tartrate-resistant acid phosphatase staining, real-time PCR, and immunostaining, respectively. Phosphorylation levels of signaling proteins were assessed by immunoblot analysis to understand the molecular action of napyradiomycin B4. The in vivo efficacy of napyradiomycin B4 was examined under experimental periodontitis, and alveolar bone destruction was evaluated by microcomputed tomography (micro-CT) and histological analyses. Among the eight napyradiomycin derivatives screened, napyradiomycin B4 considerably inhibited osteoclastogenesis. Napyradiomycin B4 significantly suppressed the receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast formation and disrupted the expression of NFATc1 and its target genes. Mitogen-activated extracellular signal-regulated kinase (MEK) and extracellular signal-regulated kinase (ERK) phosphorylation levels were reduced by napyradiomycin B4 in response to RANKL. Under in vivo experimental periodontitis, napyradiomycin B4 significantly attenuated osteoclast formation and decreased the distance between the cementoenamel junction and alveolar bone crest. Our findings demonstrate the antiosteoclastogenic activity of napyradiomycin B4 by inhibiting the RANKL-induced MEK-ERK signaling pathway and its protective effect on alveolar bone destruction.

Abstract 7070: MEK-ERK signaling maintains epidermal proliferation through H2A.Z deposition

Abstract Recent studies have connected dysregulation of H2A.Z deposition to glioblastoma as well as lung, pancreatic, and metastatic breast cancers. The H2AZ1 and H2AZ2 isoforms, collectively known as H2A.Z, are histone variants that regulate gene expression, stem cell identity, and differentiation during development and in homeostatic tissue. Most tumors originate from epithelial tissues. We utilize skin epidermis, an accessible model of epithelial tissue, to understand the regulatory mechanisms controlling proliferation and differentiation. Primary human epidermal keratinocytes retain their abilities to proliferate, differentiate, and stratify in vitro. First, we observed that progenitor keratinocytes express high levels of H2A.Z mRNA and protein, and that H2A.Z levels significantly and progressively decrease during four days of calcium-induced differentiation. Using ChIP-sequencing, we find a significant, genome-wide decrease in H2A.Z chromatin occupancy in differentiated keratinocytes. Therefore, we hypothesized that H2A.Z is critical for progenitor function. Knock down of either H2AZ1 or H2AZ2 significantly impaired keratinocyte proliferation in holoclone assays. Furthermore, H2AZ1 or H2AZ2 knockdown inhibited epidermal stratification in organotypic culture. Canonically, two chromatin remodeling complexes, SRCAP and EP400, deposit H2A.Z into nucleosomes. While both SRCAP and EP400 knockdown diminish keratinocyte proliferation, only SRCAP knockdown decreases H2A.Z deposition in epidermal keratinocytes. Finally, we find that MEK and ERK signaling is required to sustain expression of SRCAP, H2AZ1, and H2AZ2. MEK or ERK inhibition is sufficient to significantly decrease H2A.Z occupancy genome-wide in progenitor keratinocytes. Citation Format: Stephenie Droll, Benny Zhang, Celia Xue, Maxwell Levine, Xiaomin Bao. MEK-ERK signaling maintains epidermal proliferation through H2A.Z deposition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7070.

O-sialoglycoprotein Endopeptidase (OSGEP) Suppresses Hepatic Ischemia-Reperfusion Injury-Induced Ferroptosis Through Modulating the MEK/ERK Signaling Pathway.

Hepatic ischemia-reperfusion injury (HIRI) was widely accepted as a critical complication of liver resection and transplantation. A growing body of evidence suggested that O-sialoglycoprotein endopeptidase (OSGEP) was involved in cell proliferation and mitochondrial metabolism. However, whether OSGEP could mediate the pathogenesis of HIRI has still remained unclarified. This study investigated whether OSGEP could be protective against HIRI and elucidated the potential mechanisms. The OSGEP expression level was detected in cases undergoing ischemia-related hepatectomy and a stable oxygen-glucose deprivation/reoxygenation (OGD/R) condition in hepG2 cells. Additionally, it was attempted to establish a mouse model of HIRI, thus, the function and mechanism of OSGEP could be analyzed. At one day after hepatectomy, the negative association of OSGEP expression level with the elevated serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) was noted. Moreover, it was attempted to carry out gain- and loss-of-function analyses of OSGEP in hepG2 cells to reveal its influences on OGD/R-induced injury and relevant signaling pathways. The findings suggested that OSGEP overexpression significantly protected hepG2 cells against ferroptotic cell death, while OSGEP consumption had opposite effects. Consistent with in vitro studies, OSGEP deficiency exacerbated liver functions and ferroptoticcell death in a mouse model of HIRI. The results also revealed that OSGEP mediated the progression of HIRI by regulating the MEK/ERK signaling pathway. Rescue experiments indicated that ERK1/2 knockdown or overexpression reversed the effects of OSGEP overexpression or knockdown on hepG2 cells under OGD/R condition. Taken together, the findings demonstrated that OSGEP could contribute to alleviate HIRI by mediating the MEK-ERK signaling pathway, which may serve as a potential prognostic marker and a therapeutic target for HIRI.

The MEK-ERK signaling pathway promotes maintenance of cardiac chamber identity.

Ventricular and atrial cardiac chambers have unique structural and contractile characteristics that underlie their distinct functions. The maintenance of chamber-specific features requires active reinforcement, even in differentiated cardiomyocytes. Previous studies in zebrafish have shown that sustained FGF signaling acts upstream of Nkx factors to maintain ventricular identity, but the rest of this maintenance pathway remains unclear. Here, we show that MEK1/2-ERK1/2 signaling acts downstream of FGF and upstream of Nkx factors to promote ventricular maintenance. Inhibition of MEK signaling, like inhibition of FGF signaling, results in ectopic atrial gene expression and reduced ventricular gene expression in ventricular cardiomyocytes. FGF and MEK signaling both influence ventricular maintenance over a similar timeframe, when phosphorylated ERK (pERK) is present in the myocardium. However, the role of FGF-MEK activity appears to be context-dependent: some ventricular regions are more sensitive than others to inhibition of FGF-MEK signaling. Additionally, in the atrium, although endogenous pERK does not induce ventricular traits, heightened MEK signaling can provoke ectopic ventricular gene expression. Together, our data reveal chamber-specific roles of MEK-ERK signaling in the maintenance of ventricular and atrial identities.

Plm-102, a Next Generation FLT3 Inhibitor, Shows Potent Anti-Leukemic Activity on Resistance to Gilteritinib in FLT3 Mutated Acute Myeloid Leukemia

Introduction: Mutations in the FMS-like tyrosine kinase 3 (FLT3) gene occur in approximately 25 ~ 30% of all acute myeloid leukemia (AML) cases, with FLT3-ITD mutations having a poor prognosis. In 2022, the FDA granted approval to Gilteritinib, a 2nd generation FLT3 inhibitor, for the treatment of relapsed or refractory AML. Although Gilteritinib has demonstrated efficacy in R/R AML patients, its response duration is limited due to the development of secondary resistance. Herein, we report a next-generation drug candidate, PLM-102, which has shown promising pre-clinical results to overcome the current unmet needs of Gilteritinib resistance, including FLT3-ITD-TKD and Ras mutations in FLT3-mutated AML. Aims: The main objective of this study is to investigate the effect of PLM-102 in in-vitro and in-vivo acquired Gilteritinib-resistant models and to elucidate the underlying signaling pathways. Methods: To establish Gilteritinib-resistant cell lines, we exposed the AML cells to FLT3 ligand at 10 ng/ml and Gilteritinib at concentrations ranging from 10 nM to 100 nM for four months. Subsequently, for the following four months, we treated the cells with Gilteritinib alone at 100 nM, omitting FLT3 ligand from the treatment. Whole Exome Sequencing (WES) and mRNA sequencing were performed on primary FLT3 mutated AML cells and Gilteritinib resistance FLT3 mutated AML cells. Subsequently, cell viability assays were carried out on both parent FLT3-mutated AML cells and Gilteritinib-resistant FLT3-mutated AML cells. To assess the activities of PLM-102 at the on-target and off-target resistant mechanisms, immunoblot studies were conducted. An in vivo study was performed using a Gilteritinib-resistant MOLM-14 AML xenograft mouse model. Results: We conducted Whole Exome Sequencing on the Gilteritinib-resistant MOLM-13 and MOLM-14 cell lines. The analysis revealed the presence of KRAS G13D and FLT3-ITD-C828G mutations in the Gilteritinib-resistant MOLM-13 cell line, and NRAS G13D and FLT3-ITD-G697R mutations in the Gilteritinib-resistant MOLM-14 cell line. In the evaluation of the efficacies of Gilteritinib and PLM-102 in Gilteritinib-resistant MOLM-14 cell lines. The GI 50 values for Gilteritinib and PLM-102 were determined to be 427 nM and 6.17 nM, respectively. In the studies with a xenograft model with Gilteritinib-resistant MOLM-14 cells (20 mg/kg, p.o.), the Tumor Growth Inhibition (TGI, %) values were determined as 71.9% vs 17.1% for PLM-102 vs Gilteritinib, respectively. These results demonstrated the superior in-vivo efficacies of PLM-102 in Gilteritinib-resistant cells. mRNA sequencing was performed on both MOLM-14 cells and Gilteritinib-resistance MOLM-14 cells, resulting in an upregulation of the cytokine-mediated signaling pathway, including chemokines, in the Gilteritinib-resistant MOLM-14 cells. Additionally, we conducted mRNA sequencing on Gilteritinib-resistant MOLM-14 cells treated with and without PLM-102 at a concentration of 10 nM. Interestingly, in the group treated with PLM-102 at 10 nM, we observed a notable decrease in the cytokine-mediated signaling pathway, including chemokines. These findings provide valuable insights into the potential mechanism by which PLM-102 exerts its anti-leukemic effect in Gilteritinib-resistant cells by modulating the cytokine-mediated signaling pathway. In Gilteritinib-resistant MOLM-14 cells harboring the NRAS G13D mutation, we confirmed hyperactivation of the MEK-ERK signaling pathway. The significantly improved effects in the combination of PLM-102 and Trametinib, a MEK inhibitor, were observed through cell viability assays and a xenograft model, further supporting their potential as a synergistic therapeutic approach. Currently, the GLP toxicological studies of PLM-102 has successfully completed, and the Investigational New Drug (IND) for human clinical studies will be submitted near future. Conclusions: Our results have confirmed that RAS-MEK-ERK signaling pathway and cytokine signaling pathway were activated by Gilteritinib resistance. Notably, PLM-102 exhibits exceptional anti-leukemic activity in Gilteritinib-resistant cell lines through the unique mechanism of inhibiting the RAS-MEK-ERK-cytokine signaling pathway. These results highlight the potential of PLM-102 as a third-generation inhibitor with favorable efficacies in patients with resistance to current FLT3 inhibitors.

Multi-omic analysis reveals metabolic pathways that characterize right-sided colon cancer liver metastasis

There are well demonstrated differences in tumor cell metabolism between right sided (RCC) and left sided (LCC) colon cancer, which could underlie the robust differences observed in their clinical behavior, particularly in metastatic disease. As such, we utilized liquid chromatography-mass spectrometry to perform an untargeted metabolomics analysis comparing frozen liver metastasis (LM) biobank samples derived from patients with RCC (N = 32) and LCC (N = 58) to further elucidate the unique biology of each. We also performed an untargeted RNA-seq and subsequent network analysis on samples derived from an overlapping subset of patients (RCC: N = 10; LCC: N = 18). Our biobank redemonstrates the inferior survival of patients with RCC-derived LM (P = 0.04), a well-established finding. Our metabolomic results demonstrate increased reactive oxygen species associated metabolites and bile acids in RCC. Conversely, carnitines, indicators of fatty acid oxidation, are relatively increased in LCC. The transcriptomic analysis implicates increased MEK-ERK, PI3K-AKT and Transcription Growth Factor Beta signaling in RCC LM. Our multi-omic analysis reveals several key differences in cellular physiology which taken together may be relevant to clinical differences in tumor behavior between RCC and LCC liver metastasis.

Butein inhibits corticosterone-induced apoptosis of Neuro2A cells by maintaining MEK-ERK signaling.

Stress-induced overactivation of glucocorticoid signaling may contribute to mental illness by inducing neuronal death and dysfunction. We previously reported that pretreatment with the plant flavonoid butein inhibits corticosterone (CORT)-induced apoptosis of Neuro2A (N2A) cells. In the current study, we examined whether MEK-ERK and PI3K-AKT signaling pathways are involved in neuroprotection by butein. N2A cells were pre-incubated with serum-free DMEM containing 0.5μM butein for 30min, and then incubated with serum-free DMEM containing 0.5µM butein, 50µM CORT, 50µM LY294002, or 50µM PD98059 as indicated for 24h. We subsequently performed the MTT assay and the western blot analysis. As expected, CORT considerably reduced N2A cell viability and increased relative expression of the apoptosis effector cleaved caspase-3, whereas pretreatment with butein blocked these cytotoxic effects. Treatment with CORT alone also decreased both AKT and ERK protein phosphorylation. Butein pretreatment had no effect on AKT phosphorylation, and only partially reversed the reduction in phosphorylated ERK. However, cotreatment with butein and the PI3K inhibitor LY294002 during CORT exposure enhanced ERK phosphorylation, whereas cotreatment with butein and the ERK phosphorylation/activation inhibitor PD98059 enhanced AKT phosphorylation, suggesting that MEK-ERK negatively regulates AKT phosphorylation. Moreover, the protective efficacy of butein was blocked by PD98059 cotreatment but not LY294002 cotreatment. These findings suggest that butein protects neurons against glucocorticoid-induced apoptosis by sustaining ERK phosphorylation and downstream signaling.

Approaches to the treatment of a multisystem form of juvenile xanthogranuloma with central nervous system lesion

Relevance. Juvenile xanthogranuloma (JXG) is the most common form of non-Langerhans cell histiocytic disorder. Cutaneous forms of the disease spontaneously regress within a few years, while systemic forms of JXG require treatment and may pose a threat to the lives of patients. Due to the lack of unified approach to the treatment of multisystem forms of JXG, the question of effective therapy tactics remains unresolved. The most common approach is to use Langerhans cell histiocytosis (LCH) treatment regimens for JXG. With the understanding of the leading role of mutations in the MEK-ERK signaling pathway in the pathogenesis of JXG, targeted therapy, BRAF- and MEK-inhibitors, are increasingly being considered in the treatment of JXG.Clinical cases. We present two cases of multisystem JXG with central nervous system (CNS) lesions. The first patient with CNS and skin lesions was treated with chemotherapy, developed for the treatment of multisystem LCH, which allowed us to obtain an effect “active disease better” (AD better). The second JXG patient with brain, lungs, bones, and adrenal gland lesions, combined targeted therapy with BRAF- and MEKinhibitors, vemurafenib and cobimetinib, resulted in a “non active disease” (NAD) effect.Conclusion. Multisystem form of JXG with CNS involvement is a rare oncological disease, the therapy of which has not been developed. With the introduction of molecular genetic profiling technology, it became possible to obtain NAD effect using targeted therapy.

Abstract 1669: In vitro and in vivo synthetic lethality: Potent tumor suppression by a novel FLT3/cKIT/CSF1R/CDK19 inhibitor

Abstract Tumor cells are characterized by aberrant signal transduction and cell cycle control pathways that lead to unchecked cell proliferation. Unchecked tumor cell proliferation, as well as macrophage proliferation, contributes to tumor growth and spread. Inhibitors of PI3K or MEK signaling pathways can initially inhibit tumor growth until resistance due to upregulation of the alternative pathway develops. We identified a novel allosteric cKIT/FLT3/CSF1R inhibitor that induces synthetic lethality in AML, lung carcinoma, pancreatic carcinoma and other tumor cells and in proliferating macrophages in vitro and in tumors in vivo by simultaneously inhibiting the MEK-ERK and PI3K-AKT pathways and activating stress activated kinase-mediated cell cycle arrest. A series of related cKIT/FLT3/CSF1R/PDGFR allosteric inhibitors suppressed receptor tyrosine phosphorylation as well as downstream MEK-ERK and PI3K-AKT signaling and mTorc1 and cMyc gene expression pathways that control cell growth. Select cKIT/FLT3/CSF1R/PDGFR inhibitors with a unique positioning of a single side chain also inhibited CDK8/19 and stimulated phosphorylation and activation of JNK and p38. JNK activation promoted sustained ATF-2 and cJun-mediated p53 and Chk1/Chk2 activation, thereby promoting cell cycle arrest in G2/M and ultimately, apoptosis. These inhibitors potently suppressed tumor growth in vivo by halting tumor cell and macrophage proliferation in G2/M. They synergized with anti-PD-1 and anti-CTLA-4 and led to tumor eradication and immunological memory formation. These studies identify novel allosteric inhibitors with potent synthetically lethal anti-tumor activity. Citation Format: Hui Chen, Marc Paradise, Ryan Shepard, Judith Varner. In vitro and in vivo synthetic lethality: Potent tumor suppression by a novel FLT3/cKIT/CSF1R/CDK19 inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1669.

Abstract 4874: Hyperforin induces apoptosis and inhibits invasion via inhibiting AKT/NF-ΚB signaling pathway in colorectal cancer cells

Abstract Recently, the incidence of colorectal cancer (CRC) has been increasing year by year and has become the first incidence cancer in Taiwan. Epidermal growth factor receptor inhibitor (EGFR inhibitor) has been recognized as an important target of CRC. Therefore, targeting EGFR and its related signaling could be a potential strategy to overcome this disease. Hyperforin is a natural prenylated phloroglucinol and has been identified as the major molecule responsible for the antidepressant effects. Previous studies indicated that hyperforin has other potentially pharmacologically meaningful biological properties, including antibacterial ability and inhibition of inflammatory mediators. Numerous studies have also pointed out the anti-tumor potential of hyperforin is associated with the induction of apoptosis signaling. However, whether hyperforin may regulate CRC progression and its underlying mechanism is unclear. In our study, we used the MTT assay to prove the cytotoxicity of CRC cells (HT29 and HCT116 cells) was induced by hyperforin. We then used the flow cytometry to confirmed that hyperforin may induce both extrinsic/intrinsic apoptotic pathways in CRC cells. We further demonstrated hyperforin-induced apoptosis pathways is associated with mitochondrial-dependent apoptosis and death receptor dependent apoptosis. Then, transwell invasion, migration, and wound healing assay results proved the inhibition effect by hyperforin in HT29 and HCT116 cells. Additionally, we suggested that hyperforin can effectively inhibit EGFR downstream genes MEK-ERK and AKT signaling, as well as inhibit the phosphorylation of NF-ΚB and the nuclear translocation in CRC cells. In sum, our results show that hyperforin can inhibit the apoptosis and metastasis ability of CRC cells by inhibiting both AKT/NF- ΚB and ERK/NF-ΚB signaling pathway. Citation Format: Ying-Tzu Chen, I-Tsang Chiang, Yuan Chang, Fei-Ting Hsu. Hyperforin induces apoptosis and inhibits invasion via inhibiting AKT/NF-ΚB signaling pathway in colorectal cancer cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4874.

Reduced FRG1 expression promotes angiogenesis via activation of the FGF2-mediated ERK/AKT pathway.

Identifying novel targets that control both tumorigenesis and angiogenesis can aid in developing a more potent anti-angiogenic therapeutic strategy. We previously reported that reduction of FRG1 is associated with increased p38-MAPK signaling in prostate cancer and with elevated MEK-ERK signaling in breast cancer. Here, we reveal the role of FRG1 in tumor angiogenesis. Our findings demonstrate that depleted FRG1 levels enhance the proliferation, migration, and tubule formation of HUVECs in a paracrine manner, and this was further substantiated in multiple animal models. Mechanistically, FRG1 depletion activated the expression of FGF2 in breast cancer cells, which triggered the ERK/AKT cascade in endothelial cells. As FRG1 affects multiple tumorigenic properties and it is upstream of FGF2, it can be explored as a therapeutic target that is less prone to resistance.

Oscillatory ERK Signaling and Morphology Determine Heterogeneity of Breast Cancer Cell Chemotaxis via MEK-ERK and p38-MAPK Signaling Pathways.

Chemotaxis, regulated by oscillatory signals, drives critical processes in cancer metastasis. Crucial chemoattractant molecules in breast cancer, CXCL12 and EGF, drive the activation of ERK and Akt. Regulated by feedback and crosstalk mechanisms, oscillatory signals in ERK and Akt control resultant changes in cell morphology and chemotaxis. While commonly studied at the population scale, metastasis arises from small numbers of cells that successfully disseminate, underscoring the need to analyze processes that cancer cells use to connect oscillatory signaling to chemotaxis at single-cell resolution. Furthermore, little is known about how to successfully target fast-migrating cells to block metastasis. We investigated to what extent oscillatory networks in single cells associate with heterogeneous chemotactic responses and how targeted inhibitors block signaling processes in chemotaxis. We integrated live, single-cell imaging with time-dependent data processing to discover oscillatory signal processes defining heterogeneous chemotactic responses. We identified that short ERK and Akt waves, regulated by MEK-ERK and p38-MAPK signaling pathways, determine the heterogeneous random migration of cancer cells. By comparison, long ERK waves and the morphological changes regulated by MEK-ERK signaling, determine heterogeneous directed motion. This study indicates that treatments against chemotaxis in consider must interrupt oscillatory signaling.

Anti-aging Effects of Alu Antisense RNA on Human Fibroblast Senescence Through the MEK-ERK Pathway Mediated by KIF15.

To investigate whether human short interspersed nuclear element antisense RNA (Alu antisense RNA; Alu asRNA) could delay human fibroblast senescence and explore the underlying mechanisms. We transfected Alu asRNA into senescent human fibroblasts and used cell counting kit-8 (CCK-8), reactive oxygen species (ROS), and senescence-associated beta-galactosidase (SA-β-gal) staining methods to analyze the anti-aging effects of Alu asRNA on the fibroblasts. We also used an RNA-sequencing (RNA-seq) method to investigate the Alu asRNA-specific mechanisms of anti-aging. We examined the effects of KIF15 on the anti-aging role induced by Alu asRNA. We also investigated the mechanisms underlying a KIF15-induced proliferation of senescent human fibroblasts. The CCK-8, ROS and SA-β-gal results showed that Alu asRNA could delay fibroblast aging. RNA-seq showed 183 differentially expressed genes (DEGs) in Alu asRNA transfected fibroblasts compared with fibroblasts transfected with the calcium phosphate transfection (CPT) reagent. The KEGG analysis showed that the cell cycle pathway was significantly enriched in the DEGs in fibroblasts transfected with Alu asRNA compared with fibroblasts transfected with the CPT reagent. Notably, Alu asRNA promoted the KIF15 expression and activated the MEK-ERK signaling pathway. Our results suggest that Alu asRNA could promote senescent fibroblast proliferation via activation of the KIF15-mediated MEK-ERK signaling pathway.

The sodium channel subunit SCNN1B suppresses colorectal cancer via suppression of active c-Raf and MAPK signaling cascade

The incidence of colorectal cancer (CRC) is rising worldwide. Here, we identified SCNN1B as an outlier down-regulated in CRC and it functions as a tumor suppressor. SCNN1B mRNA and protein expression were down-regulated in primary CRC and CRC cells. In a tissue microarray cohort (N = 153), SCNN1B protein was an independent prognostic factor for favorable outcomes in CRC. Ectopic expression of SCNN1B in CRC cell lines suppressed cell proliferation, induced apoptosis, and cell cycle arrest, and suppressed cell migration in vitro. Xenograft models validated tumor suppressive function of SCNN1B in vivo. Mechanistically, Gene Set Enrichment Analysis (GSEA) showed that SCNN1B correlates with KRAS signaling. Consistently, MAPK qPCR and kinase arrays revealed that SCNN1B suppressed MAPK signaling. In particular, SCNN1B overexpression suppressed p-MEK/p-ERK expression and SRE-mediated transcription activities, confirming blockade of Ras-Raf-MEK-ERK cascade. Mechanistically, SCNN1B did not affect KRAS activation, instead impairing activation of c-Raf by inducing its inhibitory phosphorylation and targeting active c-Raf for degradation. The ectopic expression of c-Raf fully rescued cell proliferation and colony formation in SCNN1B-overexpressing CRC cells, confirming c-Raf as the principal molecular target of SCNN1B. In summary, we identified SCNN1B as a tumor suppressor by functioning as a c-Raf antagonist, which in turn suppressed oncogenic MEK-ERK signaling.

Autoimmune activation and hypersensitization of the AT1 and ETA receptors contributes to vascular injury in scleroderma renal crisis.

Scleroderma renal crisis (SRC) is a rare vascular complication of systemic sclerosis with substantial risks for end-stage renal disease and premature death. Activating autoantibodies (Abs) targeting the angiotensin II type 1 (AT1R) and the endothelin-1 type A receptor (ETAR) have been identified as predictors for SRC. Here, we sought to determine their pathogenic significance for acute renal vascular injury potentially triggering kidney failure and malignant hypertension. IgG from patients with SRC was studied for AT1R and ETAR dependent biologic effects on isolated rat renal interlobar arteries and vascular cells including contraction, signalling and mechanisms of receptor activation. In myography experiments, patient IgG exerted vasoconstriction sensitive to inhibition of AT1R and ETAR. This relied on MEK-ERK signalling indicating functional relevance of anti-AT1R and anti-ETAR Abs. The contractile response to angiotensin II and endothelin-1 was amplified by patient IgG containing anti-AT1R and anti-ETAR Abs with substantial crosstalk between both receptors implicating autoimmune receptor hypersensitization. Co-immunoprecipitation experiments indicated heterodimerization between both receptor types which may enable the observed functional interrelation by direct structural interactions. We provide experimental evidence that agonistic Abs may contribute to SRC. This effect is presumably related to direct receptor stimulation and additional allosteric effects, at least in heterodimeric receptor constellations. Novel therapies targeted at autoimmune hyperactivation of AT1R and ETAR might improve outcomes in severe cases of SRC.

The members of the RAS–RAF–MEK–ERK signaling pathway and cancer

Cancer is one of the biggest health problems of contemporary humanity, representing the second cause of death after cardiovascular diseases, caused by many contributing factors, such as population growth, ageing, stress, pollution, unhealthy diet, tumor diversity and heterogenity, difficulties in correct and early diagnosis and inefficiency of current treatments (e.g. cytoreduction, chemotherapy, radiotherapy, etc.). This leads us to look for new ways of dealing with cancer, such as immunotherapy and personalized therapy, which consider the type of tumour, mutations and expression levels of certain genes, making it necessary to detect specific markers. A category of such specific markers is represented by the proteins involved in inter- and intra-cellular signaling which play essential roles in tumor transformation, progression and dissemination. In cancer, some canonical signaling pathways, including RAS-RAF-MEK-ERK, P13K-PKB/AKT, JAK-STAT, HIF1-VEGF, TGFB, NOTCH, RAP1, TP53, B-catenin/WNT, HIPPO, KEAP-NRF2, MYC and CDKN2 (cell cycle), may be dysregulated. In this review, we detail the roles of RAS–RAF–MEK–ERK signaling pathway in tumorigenic processes and the types of abnormalities that affect them in cancer.

Expression of active B-Raf proto-oncogene in kidney collecting ducts induces cyst formation in normal mice and accelerates cyst growth in mice with polycystic kidney disease

Polycystic kidney disease (PKD) is characterized by the formation and progressive enlargement of fluid-filled cysts due to abnormal cell proliferation. Cyclic AMP agonists, including arginine vasopressin, stimulate ERK-dependent proliferation of cystic cells, but not normal kidney cells. Previously, B-Raf proto-oncogene (BRAF), a MAPK kinase kinase that activates MEK-ERK signaling, was shown to be a central intermediate in the cAMP mitogenic response. However, the role of BRAF on cyst formation and enlargement invivo had not been demonstrated. To determine if active BRAF induces kidney cyst formation, we generated transgenic mice that conditionally express BRAFV600E, a common activating mutation, and bred them with Pkhd1-Cre mice to express active BRAF in the collecting ducts, a predominant site for cyst formation. Collecting duct expression of BRAFV600E (BRafCD) caused kidney cyst formation as early as three weeks of age. There were increased levels of phosphorylated ERK (p-ERK) and proliferating cell nuclear antigen, a marker for cell proliferation. BRafCD mice developed extensive kidney fibrosis and elevated blood urea nitrogen, indicating a decline in kidney function, by ten weeks of age. BRAFV600E transgenic mice were also bred to Pkd1RC/RC and pcy/pcy mice, well-characterized slowly progressive PKD models. Collecting duct expression of active BRAF markedly increased kidney weight/body weight, cyst number and size, and total cystic area. There were increased p-ERK levels and proliferating cells, immune cell infiltration, interstitial fibrosis, and a decline in kidney function in both these models. Thus, our findings demonstrate that active BRAF is sufficient to induce kidney cyst formation in normal mice and accelerate cystic disease in PKD mice.

The Protective Effects of Hydrogen Sulfide New Donor Methyl S-(4-Fluorobenzyl)-N-(3,4,5-Trimethoxybenzoyl)-l-Cysteinate on the Ischemic Stroke.

In this paper, we report the design, synthesis and biological evaluation of a novel S-allyl-l-cysteine (SAC) and gallic acid conjugate S-(4-fluorobenzyl)-N-(3,4,5-trimethoxybenzoyl)-l-cysteinate (MTC). We evaluate the effects on ischemia-reperfusion-induced PC12 cells, primary neurons in neonatal rats, and cerebral ischemic neuronal damage in rats, and the results showed that MTC increased SOD, CAT, GPx activity and decreased LDH release. PI3K and p-AKT protein levels were significantly increased by activating PI3K/AKT pathway. Mitochondrial pro-apoptotic proteins Bax and Bim levels were reduced while anti-apoptotic protein Bcl-2 levels were increased. The levels of cleaved caspase-9 and cleaved caspase-3 were also reduced in the plasma. The endoplasmic reticulum stress (ERS) was decreased, which in turns the survival rate of nerve cells was increased, so that the ischemic injury of neurons was protected accordingly. MTC activated the MEK-ERK signaling pathway and promoted axonal regeneration in primary neurons of the neonatal rat. The pretreatment of MEK-ERK pathway inhibitor PD98059 and PI3K/AKT pathway inhibitor LY294002 partially attenuated the protective effect of MTC. Using a MCAO rat model indicated that MTC could reduce cerebral ischemia-reperfusion injury and decrease the expression of proinflammatory factors. The neuroprotective effect of MTC may be due to inhibition of the over-activation of the TREK-1 channel and reduction of the current density of the TREK1 channel. These results suggested that MTC has a protective effect on neuronal injury induced by ischemia reperfusion, so it may have the potential to become a new type of neuro-ischemic drug candidate.