MEK1 Expression Research Articles

MiR-16 Inhibits Extracellular Signal-Regulated Kinases (ERK) Mitogen-Activated Protein Kinases (MAPK) Signaling to Affect Epithelial-Mesenchymal Transition (EMT) and Invasion of Glioma Cells

Abnormal MEK1 expression is associated with tumor cell EMT, invasion and metastasis. Decreased miR-16 level is associated with glioma. Bioinformatics analysis showed a relationship between miR-16 and MEK1. This study assessed whether miR-16 regulates MEK1 expression and affects glioma cell EMT and invasion. The tumor tissues and adjacent glioma tissues were collected to measure miR-16 and MEK1 mRNA. The dual luciferase assay validated the relation of miR-16 with MEK1. U251 cells were cultured and assigned into NC group and mimic group, followed by analysis of cell biological behaviors, and MEK1, p-ERK1/2, E-cadherin, N-Cadherin expression. Compared with adjacent tissues, miR-16 expression was significantly decreased and MEK1 was elevated in glioma tissues. Compared with HEB, miR-16 in glioma U251 and SHG44 cells was decreased and MEK1 was increased. Dual luciferase reporter gene experiments confirmed the relation of miR-16 with MEK1. Transfection of miR-16 mimic significantly down-regulated MEK1, p-ERK1/2 and N-cadherin in U251 cells, upregulated E-cadherin, inhibited cell proliferation, promoted apoptosis, and attenuated EMT and invasion of glioma cells. In conclusion, decreased miR-16 expression and increased MEK1 expression is related to glioma pathogenesis. Overexpression of miR-16 can inhibit MEK1 expression, ERK/MAPK signaling, glioma cell proliferation, promote apoptosis, and attenuate EMT and invasion.

MOB3A Bypasses BRAF and RAS Oncogene-Induced Senescence by Engaging the Hippo Pathway.

These results suggest that MOB3 targeting to re-engage the Hippo pathway, or direct targeting of YAP/TAZ, may be viable therapeutic strategies potential for RAS-pathway driven tumours.

Inflammatory conversion of quiescent osteoblasts by metastatic breast cancer cells through pERK1/2 aggravates cancer-induced bone destruction

Disruption of bone homeostasis caused by metastatic osteolytic breast cancer cells increases inflammatory osteolysis and decreases bone formation, thereby predisposing patients to pathological fracture and cancer growth. Alteration of osteoblast function induces skeletal diseases due to the disruption of bone homeostasis. We observed increased activation of pERK1/2 in osteolytic breast cancer cells and osteoblasts in human pathological specimens with aggressive osteolytic breast cancer metastases. We confirmed that osteolytic breast cancers with high expression of pERK1/2 disrupt bone homeostasis via osteoblastic ERK1/2 activation at the bone-breast cancer interface. The process of inflammatory osteolysis modulates ERK1/2 activation in osteoblasts and breast cancer cells through dominant-negative MEK1 expression and constitutively active MEK1 expression to promote cancer growth within bone. Trametinib, an FDA-approved MEK inhibitor, not only reduced breast cancer-induced bone destruction but also dramatically reduced cancer growth in bone by inhibiting the inflammatory skeletal microenvironment. Taken together, these findings suggest that ERK1/2 activation in both breast cancer cells and osteoblasts is required for osteolytic breast cancer-induced inflammatory osteolysis and that ERK1/2 pathway inhibitors may represent a promising adjuvant therapy for patients with aggressive osteolytic breast cancers by altering the shared cancer and bone microenvironment.

Protective effects of dietary icariin on lipopolysaccharide-induced acute oxidative stress and hepatopancreas injury in Chinese mitten crab, Eriocheir sinensis

To investigate the effects of dietary icariin (ICA) supplementation on acute oxidative stress and hepatopancreatic injury induced by lipopolysaccharide (LPS) injection in Eriocheir sinensis, an 8-week feeding trial of crabs was conducted using 4 diets with different supplementation levels of ICA (0, 50, 100, and 200 mg/kg diet weight, respectively), and then challenged with LPS of 400 μg/kg body weight for 6 h. Results showed that 100 mg/kg ICA supplementation increased the antioxidant capacity, reduced the stress-related indicators in haemolymph, strengthen the mitochondrial membrane potential, and reduce apoptosis compared to the single LPS-treated crabs. The expressions of apoptosis-related genes and proteins were also evaluated to further understand the effects of dietary ICA pretreatment on LPS-induced cell apoptosis. As a result, dietary 100 mg/kg diet weight ICA pre-addition significantly down-regulated the expression of HSP60, HSP70, Caspase 3c, Caspase 8, Caspase 3, Caspase 9, P38, and Bax (P < 0.05), and alleviated the suppressed expression of PI3K, AKT, MEK, and Bcl-2 (P < 0.05) in crabs challenged with LPS. Overall, this research reveals that ICA supplementation of 100 mg/kg diet weight could enhance the resistance to oxidative damage and apoptosis in E. sinensis facing LPS challenge.

Tribbles pseudokinase 3 (TRIB3) contributes to the progression of hepatocellular carcinoma by activating the mitogen-activated protein kinase pathway.

BackgroundTribble pseudokinase 3 (TRIB3) plays a key role in regulating the malignancy of many tumors. This study examined its function in cancer cells and explored the potential mechanisms of action.MethodsThe expression of TRIB3 was examined in hepatocellular carcinomas (HCCs) using The Cancer Genome Atlas (TCGA) database. A TRIB3 lentivirus with a flag label was constructed and transfected into Huh7 and Hep3B human hepatoma cell lines to generate cells that stably overexpress TRIB3. A small interfering RNA (siRNA) was designed to knockdown TRIB3 mRNA in HepG2 and Huh7. Cell viability and cell colony formation assays were conducted. Flow cytometry was performed to assess the cell cycle in cells overexpressing TRIB3. Western blotting were performed to examine the expression of (Mitogen-activated protein kinase, MAPKK) (MEK), phosphorylated-MEK (p-MEK), extracellular signal-regulated kinase (ERK), and p-MEK in cells with TRIB3 knockdown. The correlation between TRIB3 and SMARCD3 was assessed using co-immunoprecipitation assays and immunofluorescence.ResultsTRIB3 was significantly overexpressed in advanced grade HCC tissues and was closely correlated with poor prognosis. TRIB3 overexpression promoted the cell growth and cell cycle but had little effect on migration capabilities in Huh7 and Hep3B cells. Conversely, knockdown of TRIB3 had slow down the cell growth in Huh7 and HepG2 cells detected by CCK8 and colony formation assay. The expression of MEK and ERK at both the protein and mRNA levels were downregulated when TRIB3 was knocked down. The protein expression of p-ERK and p-MEK were also downregulated upon TRIB3 silencing. SMARCD3 is a transcript factor that is belongs to the SWI/SNF complex and has been shown to regulate many genes. Indeed, co-immunoprecipitation assays demonstrated that TRIB3 interacts with SMARCD3 in the nucleus, suggesting that it may regulate TRIB3 in HCCs.ConclusionsThis study demonstrated that TRIB3 promotes the malignancy of HCC cells and its expression may be a potential diagnostic biomarker for HCC progression.

The newly identified MEK1 tyrosine phosphorylation target MACC1 is druggable by approved MEK1 inhibitors to restrict colorectal cancer metastasis

Cancer metastasis causes >90% of cancer deaths and remains a major treatment challenge. Here we deciphered the impact of tyrosine phosphorylation of MACC1, a causative driver for cancer metastasis, for cancer cell signaling and novel interventions to restrict cancer metastasis. We identified MACC1 as new MEK1 substrate. MEK1 directly phosphorylates MACC1, leading to accelerated and increased ERK1 activation. Mutating in silico predicted hierarchical MACC1 tyrosine phosphorylation sites abrogates MACC1-induced migration, invasion, and MET expression, a transcriptional MACC1 target. Targeting MEK1 by RNAi or clinically applicable MEK1 inhibitors AZD6244 and GSK1120212 reduces MACC1 tyrosine phosphorylation and restricts MACC1-induced metastasis formation in mice. Although MEK1 levels, contrary to MACC1, are not of prognostic relevance for CRC patients, MEK1 expression was found indispensable for MACC1-induced metastasis. This study identifies MACC1 as new MEK1 substrate for tyrosine phosphorylation decisively impacting cell motility, tumor growth, and metastasis. Thus, MAP kinase signaling is not linear leading to ERK activation, but branches at the level of MEK1. This fundamental finding opens new therapeutic options for targeting the MEK1/MACC1 axis as novel vulnerability in patients at high risk for metastasis. This might be extended from CRC to further solid tumor entities.

Abstract 1429: Characterization of trametinib combined with venetoclax in acute myeloid leukemia (AML) treatment

Abstract Acute myeloid leukemia (AML) is a type of hematologic malignancy with rapid progression and high genetic heterogeneity. Activating RAS pathway mutations are found in approximately 10 - 25% of AML patients and have been implicated to targeted drug resistance and disease relapse suggesting a potential benefit of MAPK pathway inhibitors in AML therapy. In patients with RAS-mutated myeloid malignancies, an inhibitor targeting MEK1/2, trametinib, demonstrated benefit in a phase 1/2 clinical study. Effective combinations of trametinib with other drugs has remained elusive. To identify potential combinatorial strategies, we generated AML cell lines resistant to MEKi. Whole exome sequencing revealed MEK1 variants in these cells. Drug sensitivity assays performed on parental AML cells and cells harboring wild type and the MEK1 variants validated their ability to confer trametinib resistance. BCL2 inhibitors, including venetoclax, were able to re-sensitize the resistant cells to trametinib, suggesting potential efficacy of combining trametinib with venetoclax. Indeed, this drug combination revealed synergistic efficacy in AML cell lines with diverse genetic backgrounds and in the resistant cells.To further investigate combination of trametinib and venetoclax in AML, we examined ~200 primary AML patient specimens using an ex vivo drug sensitivity assay, whole exome, and RNA sequencing data. We confirmed the enhanced efficacy of this combination relative to single agents. Patient samples resistant to trametinib showed increased sensitivity to venetoclax. Since, hyper-activation of MAPK signaling has been correlated with venetoclax resistance, we analyzed venetoclax resistant specimens and observed increased sensitivity to MEK and ERK inhibitors. We also observed correlation of MEK1 but not MEK2 RNA levels with trametinib sensitivity and negative correlation of MEK1 RNA levels with that of BCL2. AML samples with FLT3-ITD, NPM1, WT1, FLT3, CEBPA, DNMT3A, IDH1, IDH2, SRSF2, and TP53 mutations exhibited increased sensitivity to the combination. Samples with NRAS, KRAS, and PTPN11; although, highly sensitive to trametinib, did not benefit from this combination. Consistent with previous studies, we observed that venetoclax sensitivity was correlated with higher blast percentage. We also observed correlation of higher percentage of bone marrow blast and sensitivity to the combination. Moreover, while monocytic leukemias (AML-M4/M5) showed reduced sensitivity to venetoclax, they were sensitive to trametinib and showed increased expression of MEK1. Furthermore, we demonstrated that while venetoclax treatment increased protein levels of MCL1 in MOLM13 AML cells, combining trametinib with venetoclax reduced MCL1 levels. In summary, we demonstrate efficacy of trametinib and venetoclax combination in AML and propose mechanisms underlying the synergistic relationship of this drug combination. Citation Format: Alisa Damnernsawad, Christopher A. Eide, Sofia Beer, Haijiao Zhang, Daniel Bottomly, Stephen E. Kurtz, Jeffrey W. Tyner. Characterization of trametinib combined with venetoclax in acute myeloid leukemia (AML) treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1429.

Differential expression of phosphorylated MEK and ERK correlates with aggressive BCC subtypes.

Basal cell carcinoma (BCC) is associated with aberrant Hedgehog (HH) signalling through mutational inactivation of PTCH1; however, there is conflicting data regarding MEK/ERK signalling in BCC and the signalling pathway interactions in these carcinomas. To address this, expression of active phospho (p) MEK and ERK was examined in a panel of 15 non-aggressive and 14 aggressive BCCs. Although not uniformly expressed, both phospho-proteins were detected in the nuclei and/or cytoplasm of normal and tumour-associated epidermal cells however, whereas phospho-MEK (pMEK) was present in all non-aggressive BCCs (14/14), phospho-ERK (pERK) was rarely expressed (2/14). In contrast pERK expression was more prevalent in aggressive tumours (11/14). Interestingly, pMEK was only localized to the tumour mass whereas pERK was expressed in tumours and stroma of aggressive BCCs. Similarly, pERK (but not pMEK) was absent in mouse BCC-like tumours derived from X-ray irradiated Ptch1+/- mice with stromal pERK observed in myofibroblasts of the aggressive variant as well as in the tumour mass. RNA sequencing analysis of tumour epithelium and stroma of aggressive and non-aggressive BCC revealed the upregulation of epidermal growth factor receptor- and ERK-related pathways. Angiogenesis and immune response pathways were also upregulated in the stroma compared with the tumour. PTCH1 suppressed NEB1 immortalized keratinocytes (shPTCH1) display upregulated pERK that can be independent of MEK expression. Furthermore, epidermal growth factor pathway inhibitors affect the HH pathway by suppressing GLI1. These studies reveal differential expression of pERK between human BCC subtypes that maybe active by a pathway independent of MEK.

MTOR signaling regulates gastric epithelial progenitor homeostasis and gastric tumorigenesis via MEK1-ERKs and BMP-Smad1 pathways.

mTOR, the sensor of nutrients and growth factors, has important roles in tissue homeostasis and tumorigenesis. However, how mTOR controls gastric epithelial cell turnover and gastric cancer development, a leading malignancy, remains poorly understood. Here, we provide genetic evidence that mTOR activation promotes proliferation and inhibits differentiation of Lgr5+ gastric epithelial progenitors (GEPs) in gastric homeostasis and tumorigenesis. mTOR signaling increases MEK1 and Smad1 expression and enhances activation of MEK1-ERKs and BMP-Smad1 pathways, respectively, in GEPs and gastric tumors. Mek1 deletion or inhibition rescues hyperproliferation, whereas Bmpr1a ablation or inhibition rescues differentiation defects of Tsc1-/- GEPs. Tsc1 deficiency in Lgr5+ GEPs accelerates gastric tumor initiation and development, which require MEK1-ERKs for hyperplasia and BMP-Smad1 for differentiation suppression. These findings reveal how mTOR signaling controls Lgr5+ GEP homeostasis and cancerization and suggest that ERKs and Smad1 signaling can be safely targeted to substitute mTOR inhibitors in gastric cancer therapy.

Long non‑coding RNA BANCR promotes proliferation, invasion and migration in esophageal squamous cell carcinoma cells via the Raf/MEK/ERK signaling pathway.

Esophageal squamous cell carcinoma (ESCC) is a major histological type of esophageal cancer, identified as a leading cause of tumor-associated death worldwide. In addition, long non-coding RNA (lncRNA) BRAF-activated non-coding RNA (BANCR) expression is increased in the plasma of patients with ESCC, which can be reversed by tumor resection. Thus, the aim of the present study was to investigate the underlying mechanism of BANCR in ESCC progression. The relative mRNA expression of BANCR was determined via reverse transcription-quantitative PCR. The cell behaviors of Eca-109 cells were detected using Cell Counting Kit-8, colony formation, wound healing and Transwell chamber assays. Finally, the expression levels of proteins involved in the Raf/MEK/ERK signaling pathway and cell metastasis were analyzed with western blotting. The results revealed that lncRNA BANCR was highly expressed in ESCC cells compared with in normal esophageal cells. BANCR overexpression enhanced proliferation, migration and invasion of ESCC cells, and BANCR silencing exerted opposite effects. Moreover, BANCR overexpression induced activation of the Raf/MEK/ERK signaling pathway in ESCC cells. Notably, U0126, a specific MEK inhibitor, decreased MEK and ERK expression, and blocked the promotive effects of BANCR overexpression on the proliferation, migration and invasion of ESCC cells. Overall, lncRNA BANCR facilitated the proliferation, migration and invasion of ESCC cells via the Raf/MEK/ERK signaling pathway. Thus, lncRNA BANCR may be a promising target for inhibiting ESCC growth and metastasis.

MTOR Activation Initiates Renal Cell Carcinoma Development by Coordinating ERK and p38MAPK.

Renal cell carcinoma (RCC) mainly originates from renal proximal tubules. Intriguingly, disruption of genes frequently mutated in human RCC samples thus far has only generated RCC originated from other renal tubule parts in mouse models. This hampers our understanding of the pathogenesis of RCC. Here we show that mTOR signaling, often activated in RCC samples, initiates RCC development from renal proximal tubules. Ablation of Tsc1, encoding an mTOR suppressor, in proximal tubule cells led to multiple precancerous renal cysts. mTOR activation increased MEK1 expression and ERK activation, and Mek1 ablation or inhibition diminished cyst formation in Tsc1-deficient mice. mTOR activation also increased MKK6 expression and p38MAPK activation, and ablation of the p38α-encoding gene further enhanced cyst formation and led to RCC with clear cell RCC features. Mechanistically, Tsc1 deletion induced p53 and p16 expression in a p38MAPK-dependent manner, and deleting Tsc1 and Trp53 or Cdkn2a (encoding p16) enhanced renal cell carcinogenesis. Thus, mTOR activation in combination with inactivation of the p38MAPK-p53/p16 pathway drives RCC development from renal proximal tubules. Moreover, this study uncovers previously unidentified mechanisms by which mTOR controls cell proliferation and suggests the MEK-ERK axis to be a potential target for treatment of RCC. SIGNIFICANCE: Mouse modeling studies show that mTOR activation in combination with inactivation of the p38MAPK-p53/p16 axis initiates renal cell carcinoma that mimics human disease, identifying potential therapeutic targets for RCC treatment.

Multiple bioanalytical methods reveal a thyroid-disrupting mechanism related to the membrane receptor integrin αvβ3.

Di(2-ethylhexyl) phthalate (DEHP), a manufactured chemical, is suitable for large-scale production and has extensive applications. Although restricted for use, DEHP is still ubiquitous in the environment and shows potential to disrupt the structure or function of the thyroid system. However, its toxic mechanism is complex and not clearly understood. In this study, a battery of methods was employed to investigate DEHP-induced thyroid-disrupting effects and their mechanism of action, focusing on a newly discovered membrane receptor-mediated mechanism. The results showed that DEHP promoted rat pituitary tumor (GH3) cell proliferation and c-fos gene expression at environment level concentrations (2 and 5μmol/L) in a manner similar to that of the natural thyroid hormone 3,3',5-triiodo-L-thyronine (T3). The macromolecule DEHP-BSA cannot pass through the cell membrane to interact with nuclear receptors but upregulated the c-fos gene expression when administered at concentrations comparable to DEHP concentrations; molecular docking demonstrated that DEHP has affinity for the membrane receptor integrin αvβ3; DEHP at 2μmol/L upregulated the β3 gene expression in GH3 cells; after the addition of integrin αvβ3-inhibiting RGD peptide, DEHP-induced c-fos gene upregulation decreased. All of these findings support the supposition that DEHP-induced thyroid-disrupting effects might be mediated by the membrane receptor integrin αvβ3. Moreover, DEHP activated the downstream extracellular regulated protein kinase (ERK1/2) pathway, upregulating the gene expression of raf-1, MEK-1 and MAPK1 and increasing the protein levels of p-ERK; interestingly, ERK1/2 activation and c-fos upregulation induced by DEHP were attenuated by PD98059 (an ERK1/2 inhibitor). Taken together, the data suggest that the membrane receptor integrin αvβ3 and the downstream ERK1/2 pathway might be involved in DEHP-induced thyroid-disrupting effects. This study provides new insight into the thyroid-disrupting effect and the underlying mechanism and will advance the effort to construct adverse outcome pathways of DEHP and other thyroid hormone disrupting chemicals.

Constitutive activation of mitogen-activated protein kinase kinase (MEK1) in ileal enterocytes leads to dysplasia and a predisposition to cancer.

Activation of mitogen-activated protein kinases (MAPKs) is a key factor in the pathogenesis of cancer, although the specific role of mitogen-activated protein kinase kinase (MEK1) is not well understood. Villin promoter-driven Cre expression was used to excise a floxed stop cassette from a phosphomimetically constitutively activated MEK1 (caMEK1) expression construct in the intestine of C57BL/6 mice. Zygosity status of caMEK1 afforded assessment of the dose dependence of the effect. The expected mendelian distribution of genotypes and sex was observed in 443 progenies. Between 21 and 63 days of life, caMEK1 had no effect on body weight in male mice, but reduced body weight in female mice homozygous for caMEK1. At 10 wk of age, the ileum of caMEK1-expressing mice was characterized by the finding of dysplasia and profound changes in overall architecture. Paneth cells were nearly absent in caMEK1 homozygotes. Targeted proteomic profiling via reverse phase protein array analyses with confirmatory Western blotting revealed significant changes in protein and phosphoprotein expression, including upregulation of proteins downstream of MEK1, associated with enhanced markers of proliferation, diminished apoptosis, alterations in cell-fate determination, cell-cell interactions, and tight junctions. Long-term viability of caMEK1 homozygous mice was reduced with no survival beyond 1 yr. Invasive adenocarcinoma developed in three of ten older mice [15 wk (homozygous), 26 wk (homozygous), and 35 wk (heterozygous) of age]. Expression of caMEK1 in enterocytes leads to marked derangements in the intestinal epithelium, which is associated with a predisposition to the development of invasive cancer.NEW & NOTEWORTHY The ileum of mice with constitutive expression of activated MEK1 (via phosphomimetic changes) in enterocytes is markedly abnormal with architectural distortion and cytologic atypia, which evolves into an adenoma invasive carcinoma sequence. Phosphoproteomic analysis reveals upregulation of proteins downstream of MEK1, associated with enhanced markers of proliferation, diminished apoptosis, alterations in cell-fate determination, cell-cell interactions, and tight junctions. This novel model provides new insights into intestinal homeostasis and carcinogenesis.

LncRNA LIFR‑AS1 inhibits gastric carcinoma cell proliferation, migration and invasion by sponging miR‑4698.

The vital functions of long non-coding (lnc)RNAs have been verified in gastric carcinoma (GC). However, as a novel cancer-related lncRNA, the influence of leukemia inhibitory factor receptor antisense RNA 1 (LIFR-AS1) in GC cell biological behaviors remains unreported. The present study explored the biological effects of lncRNA LIFR-AS1 on GC progression. Reverse transcription-quantitative PCR was performed to examine lncRNA LIFR-AS1 expression in GC tissues and cells. Cell Counting Kit-8, 5-ethynyl-2′-deoxyuridine incorporation, cell wound healing and Transwell invasion assays were used to assess the functions of lncRNA LIFR-AS1 in GC cell proliferation, migration and invasion. Additionally, associations among lncRNA LIFR-AS1, microRNA (miR)-4698 and microtubule-associated tumor suppressor 1 (MTUS1) were investigated via bioinformatics software and a luciferase reporter system. In addition, western blotting was used to examine the expression of MEK and ERK. Decreased lncRNA LIFR-AS1 expression was observed in GC tissues and cells. Upregulated lncRNA LIFR-AS1 inhibited GC cell proliferation, migration and invasion. Upregulated miR-4698 and downregulated MTUS1 were identified in GC tissues and cells. The inhibitory interaction between lncRNA LIFR-AS1 and miR-4698 was confirmed. Additionally, MTUS1 was predicted as a target gene of miR-4698 positively regulated by lncRNA LIFR-AS1. The MEK/ERK pathway was inhibited by lncRNA LIFR-AS1 via regulating MTUS1. These findings revealed the inhibitory functions of lncRNA LIFR-AS1 in GC cell proliferation, migration and invasion. The process was mediated via miR-4698, MTUS1 and the MEK/ERK pathway.

Circular RNA DHX33 promotes malignant behavior in ccRCC by targeting miR-489-3p/MEK1 axis.

Mounting evidence indicates that circular RNAs modulate the initiation of clear cell renal cell carcinoma (ccRCC). However, their specific roles in the malignancy of ccRCC is understudied. Here, we present a novel circular RNA, circDHX33, that is up-regulated in ccRCC cell lines and tissues. Upregulated circDHX33 in ccRCC patients significantly correlates with advanced TNM stage and metastasis. Suppressing circDHX33 expression inhibits the proliferation and invasion of cultured cells, and suppresses tumor growth in vivo. Mechanistically, we show that circDHX33 promotes ccRCC progression by sponging miR-489-3p and modulating MEK1 expression. In conclusion, our findings suggest that circDHX33 plays a role in promoting ccRCC via the miR-489-3p/MEK1 axis and may serve as a novel therapeutic target for the treatment of ccRCC patients

POU4F1 promotes the resistance of melanoma to BRAF inhibitors through MEK/ERK pathway activation and MITF up-regulation

BRAF inhibitors (BRAFi) have shown remarkable clinical efficacy in the treatment of melanoma with BRAF mutation. Nevertheless, most patients end up with the development of BRAFi resistance, which strongly limits the clinical application of these agents. POU4F1 is a stem cell-associated transcriptional factor that is highly expressed in melanoma cells and contributes to BRAF-activated malignant transformation. However, whether POU4F1 contributes to the resistance of melanoma to BRAFi remains poorly understood. Here, we report that over-expressed POU4F1 contributed to the acquired resistance of melanoma cells to Vemurafenib. Furthermore, POU4F1 promoted the activation of ERK signaling pathway via transcriptional regulation on MEK expression. In addition, POU4F1 could increase the expression of MITF to retain the resistance of melanoma cells to BRAFi. Collectively, our findings reveal that POU4F1 re-activates the MAPK pathway by transcriptional regulation on MEK expression and promotes MITF expression, which ultimately results in the resistance to BRAFi in melanoma. Our study supports that POU4F1 is a potential combined therapeutic target with BRAFi therapy for melanoma.

MiR-195 Enhances the Sensitivity of Oral Cancer Cells to Cisplatin (DDP) by Down-Regulating the Expression of MAPK Kinase 1 (MEK1)

MAPK kinase 1 (MEK1) is involved in the development and cisplatin (DDP) resistance of various tumors. Abnormal miR-195 expression is associated with oral cancer. Bioinformatics analysis revealed a relationship between miR-195 and MEK1. This study investigated whether miR-195 affects oral cancer cells through MEK1. The DDP-resistant cell OSCC-15/DDP was separated into miR-NC group and miR-195 mimic group followed by analysis of the expression of MEK1, p-MEK1 and p-ERK1/2 protein by Western blot, cell apoptosis and proliferation by flow cytometry. MEK1 mRNA is the target gene of miR-195. miR-195 was significantly lower in OSCC-15/DDP cells than OSCC-15 cells and human normal oral epithelial cells (HOEC) cells. MEK1 was significantly upreg-ulated in OSCC-15/DDP cells compared to OSCC-15 cells and HOEC cells. miR-195 mimic significantly elevated miR-195 in OSC-15/DDP cells, downregulated MEK1, p-MEK1 and p-ERK1/2 protein, promoted apoptosis and inhibited cell proliferative ability. miR-195 is related to oral cancer cell DDP resistance and upregulation of miR-195 down-regulates MEK1 expression and reduces DDP resistance.

RAAV9-Mediated MEK1 Gene Expression Restores Post-conditioning Protection Against Ischemia Injury in Hypertrophic Myocardium.

We investigated whether increased expression of activated mitogen-activated protein kinase (MAPK) kinases 1 (MEK1) restores ischemic post-conditioning (IPostC) protection in hypertrophic myocardium following ischemia/reperfusion (I/R) injury. C57Bl/6 mice received recombinant adeno-associated virus type 9 (rAAV9)-mediated activated MEK1 gene delivery systemically, then following the induction of cardiac hypertrophy via transverse aortic constriction for 4weeks. In a Langendorff model, hypertrophic hearts were subjected to 40min/60min I/R or with IPostC intervention consisting of 6cycles of 10s reperfusion and 10s no-flow before a 60-min reperfusion. Hemodynamics, infarct size (IS), myocyte apoptosis and changes in expression of reperfusion injury salvage kinase (RISK) pathway were examined. rAAV9-MEK1 gene delivery led to a 4.3-fold and 2.7-fold increase in MEK1 mRNA and protein expression in the heart versus their control values. I/R resulted in a larger IS in hypertrophic than in non-hypertrophic hearts (52.3 ± 4.7% vs. 40.0 ± 2.5%, P < 0.05). IPostC mediated IS reduction in non-hypertrophic hearts (27.6 ± 2.6%, P < 0.05), while it had no significant effect in hypertrophic hearts (46.5 ± 3.1%, P=NS) compared with the IS in non-hypertrophic or hypertrophic hearts subjected to I/R injury only, respectively. Hemodynamic decline induced by I/R was preserved by IPostC in non-hypertrophic hearts but not in hypertrophic hearts. rAAV9-MEK1 gene delivery restored IPostC protection in hypertrophic hearts evidenced by reduced IS (32.0 ± 2.8% vs. 46.5 ± 3.1%) and cardiac cell apoptosis and largely preserved hemodynamic parameters. These protective effects were associated with significantly increased phosphorylation of ERK1/2 and ribosomal protein S6 kinases (p70S6K), but it had no influence on Akt and glycogen synthase kinase-3β. These results demonstrated that rAAV9-mediated activated MEK1 expression restores IPostC protection in the hypertrophic heart against I/R injury through the activation of ERK pathway.

Lipid Catabolism in Starved Yak Is Inhibited by Intravenous Infusion of β-Hydroxybutyrate.

Simple SummaryYak, which is the predominant and semi-domesticated livestock on the Qinghai-Tibet Plateau, suffers severe starvation and body weight reduction in the cold season because of the harsh highland environment. Lipids are important energy sources to starvation animals. β-hydroxybutyrate (BHBA) that is derived from lipid decomposition as the primary ketone body is with the function not only to provide energy for animals as energy materials, but also regulate lipid metabolism as signaling molecular. However, the effects of starvation and BHBA on lipid metabolism and its mechanism are still unclear for ruminant animals. Herein, we investigated the effects of starvation and intravenous infusion of BHBA solution on Yak growth, serum biochemistry, hormones, subcutaneous adipocyte morphology, fatty acid composition, activity of enzymes related to lipid metabolism, and signal pathway. The results showed that starvation promoted lipid catabolism and BHBA infusion up-regulated the mRNA expression of receptor GPR109A in subcutaneous adipose tissue, inhibited the Cyclic adenosine monophosphate(cAMP)/Protein kinase A (PKA)/cAMP-responsive element binding protein (CREB) signaling pathway, and inhibited lipolysis. Our study was beneficial for enriching the nutrition regulation theory of yaks and improving their growth potential.Lipid is the chief energy source for starved animals. β-hydroxybutyrate (BHBA) is the main ketone body produced by lipid decomposition. In Chinese hamster ovary (CHO) cell experiment, it was found that BHBA could be used not only as an energy substance, but also as a ligand of GPR109A for regulating lipid metabolism. However, whether BHBA can regulate lipid metabolism of yaks, and its effective concentration and signal pathway are not clear. This study investigated the effects and mechanism of starvation and BHBA on the lipid metabolism of yak. Eighteen male Jiulong yaks were selected and then randomly divided into three groups: normal feeding group (NG), starvation group (SG), and starvation with BHBA infusion group (SBG). The yaks in the NG group were freely fed during the trial, while the yaks in the SG and SBG groups fasted; from 7th to 9th days of the experiment, the NG and SG were infused continuous with 0.9% normal saline and SBG was infused 1.7 mmol/L BHBA solution respectively. The blood samples were collected on the 0th, 1st, 3rd, 5th, 7th, and 9th day of experiment. The subcutaneous adipose tissue of all the yaks in this study were taken from live bodies after infusion. Serum glucose, lipid metabolites, hormone concentrations, and mRNA and protein expressions of key factors of lipid metabolism and signaling pathway in subcutaneous adipose tissue were measured. The results showed that, as compared with NG, starvation significantly reduced the body weight of yak in SG, and significantly increased the concentration of BHBA in serum and the mRNA expression of PKA and CREB1 in subcutaneous adipose tissue, while the mRNA expression of MEK, PKC, ERK1/2, the area of adipocytes, and the proportion of saturated fatty acid were decreased. Whereas, further increase of BHBA concentration through infusion promoted the mRNA expression of GPR109A receptor in the subcutaneous adipose tissue of SBG, inhibited the mRNA expression of AC and PKA, and decreased the phosphorylation protein abundance of CREB1, and significantly increased the diameter and area of adipocytes. These findings suggest that starvation led to enhanced lipid catabolism in yaks. An increasing BHBA concentration could increase the mRNA expression of GPR109A receptor in subcutaneous adipose tissue and inhibit the cAMP/PKA/CREB signaling pathway and lipid decomposition.

Avermectin inhibits neutrophil extracellular traps release by activating PTEN demethylation to negatively regulate the PI3K-ERK pathway and reducing respiratory burst in carp

Excessive residual avermectin (AVM) in the environment can have toxic effects on non-target organisms. AVM can exert immunotoxicity by inducing genomic demethylation, but its effect on neutrophil extracellular traps (NETs) release in carp is unclear. In this study, carp neutrophils were pretreated with 5 μg/L AVM or 4 μM DNA demethylation inhibitor (aurintricarboxylic acid, ATA), alone or in combination, and then treated with 4 μM phorbol 12-myristate 13-acetate (PMA) to stimulate NETs release. The results showed that exposure of carp neutrophils to AVM significantly suppressed NETs release and MPO expression, increased ROS production, and dramatically reduced PMA-induced cellular respiratory burst. In addition, AVM could bind to the MBD2 molecule, markedly upregulate MBD2 expression to cause demethylation, and clearly activate PTEN expression, thereby inhibiting the expression of PI3K, AKT, Raf, MEK, and ERK. However, these effects were alleviated by ATA. In conclusion, our study showed that AVM could inhibit NETs release in carp by inducing demethylation of PTEN to negatively regulate NETs synthesis pathways and reducing respiratory burst level. Our findings clarify the mechanism of AVM immunotoxicity to fish and are of great significance for efforts to protect the ecological environment and human health.