Raf-1 Proto-oncogene Research Articles

Bone and periosteum protein analysis via tandem mass tag quantitative proteomics in pediatric patients with osteomyelitis.

Bone healing is crucial in managing osteomyelitis after fracture fixation. Understanding the mechanism of extensive callus formation in pediatric osteomyelitis is highly important. This study aims to analyze bone and periosteum samples from pediatric patients to elucidate the essential processes involved in callus formation during osteomyelitis. The study included eight patients from our hospital: four with positive microbial culture who underwent osteomyelitis debridement and four who had osteotomy surgery as contral. We used tandem mass tag quantitative proteomics to investigate proteomic changes in bone and periosteum tissues obtained from these patients. Differential expression proteins were analyzed for their pathways through Gene Ontology (GO) annotation, GO enrichment analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, and protein-protein interaction networks. A total of 4737 proteins were successfully identified. About 2224 differentially expressed proteins were detected in the bone tissues group and periosteum tissues group. Among the differentially expressed proteins, 10 protein genes in the bone group were associated with inflammation and osteogenesis, while in the periosteum group were nine. Cytochrome b-245, beta polypeptide (CYBB), nicotinamide phosphoribosyltransferase (NAMPT), tissue inhibitor of metalloproteinases 1 (TIMP-1), Raf-1 proto-oncogene, serine/threonine kinase (RAF-1), RELA proto-oncogene, NF-KB subunit (RELA), and sphingomyelin synthase 2 (SGMS2) may play an important role in callus formation in patients with osteomyelitis. This study provides novel clues for understanding callus formation in pediatric patients with osteomyelitis.

Mechanism of gabapentinoid potentiation of opioid effects on cyclic AMP signaling in neuropathic pain

Over half of spinal cord injury (SCI) patients develop opioid-resistant chronic neuropathic pain. Safer alternatives to opioids for treatment of neuropathic pain are gabapentinoids (e.g., pregabalin and gabapentin). Clinically, gabapentinoids appear to amplify opioid effects, increasing analgesia and overdose-related adverse outcomes, but in vitro proof of this amplification and its mechanism are lacking. We previously showed that after SCI, sensitivity to opioids is reduced by fourfold to sixfold in rat sensory neurons. Here, we demonstrate that after injury, gabapentinoids restore normal sensitivity of opioid inhibition of cyclic AMP (cAMP) generation, while reducing nociceptor hyperexcitability by inhibiting voltage-gated calcium channels (VGCCs). Increasing intracellular Ca2+ or activation of L-type VGCCs (L-VGCCs) suffices to mimic SCI effects on opioid sensitivity, in a manner dependent on the activity of the Raf1 proto-oncogene, serine/threonine-protein kinase C-Raf, but independent of neuronal depolarization. Together, our results provide a mechanism for potentiation of opioid effects by gabapentinoids after injury, via reduction of calcium influx through L-VGCCs, and suggest that other inhibitors targeting these channels may similarly enhance opioid treatment of neuropathic pain.

Design, Synthesis, and Anti-Leukemic Evaluation of a Series of Dianilinopyrimidines by Regulating the Ras/Raf/MEK/ERK and STAT3/c-Myc Pathways.

Although the long-term survival rate for leukemia has made significant progress over the years with the development of chemotherapeutics, patients still suffer from relapse, leading to an unsatisfactory outcome. To discover the new effective anti-leukemia compounds, we synthesized a series of dianilinopyrimidines and evaluated the anti-leukemia activities of those compounds by using leukemia cell lines (HEL, Jurkat, and K562). The results showed that the dianilinopyrimidine analog H-120 predominantly displayed the highest cytotoxic potential in HEL cells. It remarkably induced apoptosis of HEL cells by activating the apoptosis-related proteins (cleaved caspase-3, cleaved caspase-9 and cleaved poly ADP-ribose polymerase (PARP)), increasing apoptosis protein Bad expression, and decreasing the expression of anti-apoptotic proteins (Bcl-2 and Bcl-xL). Furthermore, it induced cell cycle arrest in G2/M; concomitantly, we observed the activation of p53 and a reduction in phosphorylated cell division cycle 25C (p-CDC25C) / Cyclin B1 levels in treated cells. Additionally, the mechanism study revealed that H-120 decreased these phosphorylated signal transducers and activators of transcription 3, rat sarcoma, phosphorylated cellular RAF proto-oncogene serine / threonine kinase, phosphorylated mitogen-activated protein kinase kinase, phosphorylated extracellular signal-regulated kinase, and cellular myelocytomatosis oncogene (p-STAT3, Ras, p-C-Raf, p-MEK, p-MRK, and c-Myc) protein levels in HEL cells. Using the cytoplasmic and nuclear proteins isolation assay, we found for the first time that H-120 can inhibit the activation of STAT3 and c-Myc and block STAT3 phosphorylation and dimerization. Moreover, H-120 treatment effectively inhibited the disease progression of erythroleukemia mice by promoting erythroid differentiation into the maturation of erythrocytes and activating the immune cells. Significantly, H-120 also improved liver function in erythroleukemia mice. Therefore, H-120 may be a potential chemotherapeutic drug for leukemia patients.

Mechanism of Lingbao Huxin Dan in the treatment of bradyarrhythmia complicated with coronary heart disease: a network pharmacology analysis.

To investigate the mechanism of action of the Lingbao Huxin Dan in treating bradycardia arrhythmia with coronary heart disease (BA-CHD) by network pharmacology. The active ingredients of the Lingbao Huxin Dan were screened on the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform and Bioinformatics tools designed for the analysis of molecular mechanisms of Chinese medicine platform; target prediction was conducted with the SwissTargetPrediction database, and Cytoscape 3.8 was used to construct a drug ingredient-target network. The Genecards, Online Mendelian Inheritance in Man, and DrugBank databases were searched for disease targets. Venn plots were used to display the common targets of BA-CHD and active ingredients. The STRING platform was used to construct a protein-protein interaction network. The Metascape data platform was used for Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis to construct a signaling pathway network of the active ingredients of the Lingbao Huxin Dan. There were 121 active ingredients, 899 related targets, 39 targets important in BA-CHD and 14 targets which intersected between the active ingredients and BA-CHD. There were 27 core therapeutic ingredients, 153 biological processes, 18 cell ingredients and 20 molecular functions obtained by GO enrichment analysis. The KEGG pathway analysis yielded 19 signaling pathways. RBA-CHD may treat BA-CHD by regulating adrenergic receptor beta-1, alpha 1-α adrenergic receptor, calcium voltage-gated channel subunit alpha1 C, alpha-1β-adrenergic receptor, nitric oxide synthase 2, beta-2 adrenergic receptor, voltage-dependent calcium channel subunit alpha-2/delta-1, an- giotensin-converting enzyme, Raf-1 proto-oncogene serine/threonine-protein kinase, and other targets, potentially by affecting adrenergic receptor binding and calcium channel opening, to regulate the activity of cardiomyocytes.

3-Hydroxybutyrate ameliorates insulin resistance by inhibiting PPARγ Ser273 phosphorylation in type 2 diabetic mice

3-Hydroxybutyrate (3HB) is a small ketone body molecule produced endogenously by the body in the liver. Previous studies have shown that 3HB can reduce blood glucose level in type 2 diabetic (T2D) patients. However, there is no systematic study and clear mechanism to evaluate and explain the hypoglycemic effect of 3HB. Here we demonstrate that 3HB reduces fasting blood glucose level, improves glucose tolerance, and ameliorates insulin resistance in type 2 diabetic mice through hydroxycarboxylic acid receptor 2 (HCAR2). Mechanistically, 3HB increases intracellular calcium ion (Ca2+) levels by activating HCAR2, thereby stimulating adenylate cyclase (AC) to increase cyclic adenosine monophosphate (cAMP) concentration, and then activating protein kinase A (PKA). Activated PKA inhibits Raf1 proto-oncogene serine/threonine-protein kinase (Raf1) activity, resulting in a decrease in extracellular signal-regulated kinases 1/2 (ERK1/2) activity and ultimately inhibiting peroxisome proliferator-activated receptor γ (PPARγ) Ser273 phosphorylation in adipocytes. Inhibition of PPARγ Ser273 phosphorylation by 3HB altered the expression of PPARγ regulated genes and reduced insulin resistance. Collectively, 3HB ameliorates insulin resistance in type 2 diabetic mice through a pathway of HCAR2/Ca2+/cAMP/PKA/Raf1/ERK1/2/PPARγ.

Discovering potential inhibitors of Raf proto-oncogene serine/threonine kinase 1: a virtual screening approach towards anticancer drug development

Raf proto-oncogene serine/threonine kinase 1 (RAF1 or c-Raf) is a serine/threonine protein kinase crucial in regulating cell growth, differentiation, and survival. Any disruption or overexpression of RAF1 can result in neoplastic transformation and other disorders such as cardiomyopathy, Noonan syndrome, leopard syndrome, etc. RAF1 has been identified as a potential therapeutic target in drug development against various complex diseases, including cancer, due to its remarkable role in disease progression. Here, we carried out a multitier virtual screening study involving different in-silico approaches to discover potential inhibitors of RAF1. After applying the Lipinski rule of five, we retrieved all phytocompounds from the IMPPAT database based on their physicochemical properties. We performed a molecular docking-based virtual screening and got top hits with the best binding affinity and ligand efficiency. Then we screened out the selected hits using the PAINS filter, ADMET properties, and other druglike features. Eventually, PASS evaluation identifies two phytocompounds, Moracin C and Tectochrysin, with appreciable anti-cancerous properties. Finally, all-atom molecular dynamics simulation (MDS) followed by interaction analysis was performed on the elucidated compounds in complex with RAF1 for 200 ns to investigate their time-evolution dynamics and interaction mechanism. Molecular mechanics Poisson–Boltzmann surface area (MM-PBSA) and Dynamical Cross-Correlation Matrix (DCCM) analyses then followed these results from the simulated trajectories. According to the results, the elucidated compounds stabilize the RAF1 structure and lead to fewer conformational alterations. The results of the current study indicated that Moracin C and Tectochrysin could serve as potential inhibitors of RAF1 after required validation. Communicated by Ramaswamy H. Sarma

Tumor-associated nonmyelinating Schwann cell-expressed PVT1 promotes pancreatic cancer kynurenine pathway and tumor immune exclusion.

One of the major obstacles to treating pancreatic ductal adenocarcinoma (PDAC) is its immunoresistant microenvironment. The functional importance and molecular mechanisms of Schwann cells in PDAC remains largely elusive. We characterized the gene signature of tumor-associated nonmyelinating Schwann cells (TASc) in PDAC and indicated that the abundance of TASc was correlated with immune suppressive tumor microenvironment and the unfavorable outcome of patients with PDAC. Depletion of pancreatic-specific TASc promoted the tumorigenesis of PDAC tumors. TASc-expressed long noncoding RNA (lncRNA) plasmacytoma variant translocation 1 (PVT1) was triggered by the tumor cell-produced interleukin-6. Mechanistically, PVT1 modulated RAF proto-oncogene serine/threonine protein kinase-mediated phosphorylation of tryptophan 2,3-dioxygenase in TASc, facilitating its enzymatic activities in catalysis of tryptophan to kynurenine. Depletion of TASc-expressed PVT1 suppressed PDAC tumor growth. Furthermore, depletion of TASc using a small-molecule inhibitor effectively sensitized PDAC to immunotherapy, signifying the important roles of TASc in PDAC immune resistance.

CCT196969 effectively inhibits growth and survival of melanoma brain metastasis cells

Melanomas frequently metastasize to the brain. Despite recent progress in the treatment of melanoma brain metastasis, therapy resistance and relapse of disease remain unsolved challenges. CCT196969 is a SRC family kinase (SFK) and Raf proto-oncogene, serine/threonine kinase (RAF) inhibitor with documented effects in primary melanoma cell lines in vitro and in vivo. Using in vitro cell line assays, we studied the effects of CCT196969 in multiple melanoma brain metastasis cell lines. The drug effectively inhibited proliferation, migration, and survival in all examined cell lines, with viability IC50 doses in the range of 0.18–2.6 μM. Western blot analysis showed decreased expression of p-ERK, p-MEK, p-STAT3 and STAT3 upon CCT196969 treatment. Furthermore, CCT196969 inhibited viability in two B-Raf Proto-Oncogene (BRAF) inhibitor resistant metastatic melanoma cell lines. Further in vivo studies should be performed to determine the treatment potential of CCT196969 in patients with treatment-naïve and resistant melanoma brain metastasis.

Research progress of the CXCR4 mechanism in Alzheimer's disease.

Alzheimer's disease (AD) is a degenerative brain disease with complex clinical manifestations and pathogeneses such as abnormal deposition of beta-amyloid protein and inflammation caused by the excessive activation of microglia. CXC motif chemokine receptor type 4 (CXCR4) is a type of G protein-coupled receptor that binds to CXC motif ligand 12 (CXCL12) to activate downstream signaling pathways, such as the Janus kinase/signal transducer and activator of transcriptionand the renin-angiotensin system (Ras)/RAF proto-oncogene serine (Raf)/mitogen-activated protein kinase/extracellular-regulated protein kinase; most of these signaling pathways are involved in inflammatory responses. CXCR4 is highly expressed in the microglia and astrocytes; this might be one of the important causes of inflammation caused by microglia and astrocytes. In this review, we summarize the mechanism and therapeutics of AD, the structures of CXCR4 and the CXCL12 ligand, and the mechanisms of CXCR4/CXCL12 that are involved in the occurrence and development of AD. The possible treatment of AD through microglia and astrocytes is also discussed, with the aim of providing a new method for the treatment of AD.

Equine early pregnancy endocrine profiles and ipsilateral endometrial immune cell, gene expression and protein localisation response.

We investigated the early effects of the equine embryo on maternal serum concentrations of insulin-like growth factor 1 (IGF1), leptin and adiponectin, uterine immune cells and genes and proteins related to embryo development and the maintenance of pregnancy. Ipsilateral endometrial expression was assessed on Days 7 and 13 after ovulation for the following transcripts: oestrogen receptor ERα (ESR1), progesterone receptor (PGR), progestin and adipoQ receptor family member 5 (PAQR5), oxytocin receptor (OXTR), prostaglandin-endoperoxide synthase 2 (PTGS2), raf-1 proto-oncogene serine/threonine kinase (RAF1), p21-activated kinase 6 (PAK6), fibroblast growth factor family member 9 (FGF9), IGF1 and its receptor (IGF1R), mucin 1 (MUC1), osteopontin (OPN), leptin receptor (LEPR) and adiponectin receptors 1 and 2 (ADIPOR1 and ADIPOR2). Ipsilateral endometrial immunological cell infiltration and immunohistochemical protein localisation were evaluated on Days 7, 10 and 13 after ovulation for ERα, PGR, OXTR, PTGS2, IGF1, IGF1R, IGF2 and MUC1. Serum hormone concentrations were not affected by reproductive status. Pregnancy downregulated ESR1 and PGR mRNA levels, upregulated the expression of all other genes and affected the expression of all genes, except PGR, on Day 7 (compared with eight genes affected at Day 13). Proteins were affected by pregnancy or by its interaction with other variables (day of extraction and endometrial compartment). Pregnant mares had a higher lymphocyte count, which decreased towards Day 13. The effect of pregnancy on leucocytes and proteins was more evident in superficial endometrial compartments. The results of this study suggest that the equine embryo exerts prompt paracrine regulation of critical biological processes.

Cisplatin and Pemetrexed Have Distinctive Growth-inhibitory Effects in Monotherapy and Combination Therapy onKRAS-dependent A549 Lung Cancer Cells

Cisplatin combined with pemetrexed disodium heptahydrate (pemetrexed) is considered the standard treatment for patients with advanced, non-squamous, non-small-cell lung cancer. However, its growth-inhibitory effects on KRAS-dependent lung cancer as monotherapy and combination therapy are not well understood. The aim of this study was to compare the effects of cisplatin and pemetrexed on A549 cells as mono- and combination therapies and elucidate the underlying mechanisms. For in vitro studies, A549 cells were exposed to cisplatin with/without pemetrexed for 72 h. The results were then evaluated by cell viability, apoptosis, reactive oxygen species, terminal deoxynucleotidyl transferase dUTP nick-end labeling, and western blotting assays. Our results revealed that cisplatin monotherapy was most cytotoxic to A549 cells, while cisplatin plus pemetrexed combination had an intermediate effect, and pemetrexed monotherapy induced a minimal cytotoxic effect on A549 cells. This effect was evidenced by cell viability results, inhibition of KRAS proto-oncogene, GTPase (KRAS)/Raf proto-oncogene, serine/threonine kinase/mitogen-activated protein kinase kinase/extracellular signal-regulated kinase pathways and apoptosis induction triggered by reactive oxygen species-mediated DNA damage. The immunoblotting result of conversion of microtubule-associated protein 1 light chain 3 alpha (LC3)-I to -II indicated that the greatest inducer of autophagy was combined treatment with cisplatin plus pemetrexed, while pemetrexed monotherapy had the lowest effect on autophagy induction, with cisplatin monotherapy having an intermediate effect. We found that the AKT serine/threonine kinase 1/mechanistic target of rapamycin kinase (mTOR) and AMP-activated protein kinase/mTOR signaling pathways were associated with autophagy activation. Interestingly, combination therapy with cisplatin plus pemetrexed was the primary eliminator of cellular senescence; cisplatin monotherapy had an intermediate effect, while pemetrexed monotherapy increased cellular senescence of A549 cells, as assessed by the expression of β-galactosidase protein. Cisplatin monotherapy may be more effective than pemetrexed monotherapy or cisplatin plus pemetrexed combination therapy against KRAS-dependent lung cancer.

CircRNA CDR1as/miR-1287/Raf1 Axis Modulates Hepatocellular Carcinoma Progression Through MEK/ERK Pathway.

BackgroundHepatocellular carcinoma (HCC) is a common lethal malignant tumor worldwide. Circular RNAs (circRNAs) have been reported to affect the development of human cancers, including HCC. In this project, we aim to clarify the functional effect of circular CDR1as (circ_CDR1as) on HCC progression.MethodsQuantitative real-time polymerase chain reaction (qRT-PCR) or Western blot is implemented to detect the expression of circ_CDR1as, microRNA (miR)-1287 and Raf-1 proto-oncogene, serine/threonine kinase (Raf1). Cell proliferation is assessed via colony formation and 3-(4, 5)-dimethylthiazole-2-y1)-2, 5-biphenyl tetrazolium bromide (MTT) assays. Cell migration and invasion are measured by Transwell assay. The target relationship between miR-1287 and circ_CDR1as or Raf1 is validated through dual-luciferase reporter assay. The levels of epithelia–mesenchymal transition (EMT) markers and the MEK/ERK signal pathway-related proteins are examined by Western blot. Model in nude mice is constructed to determine the role of circ_CDR1as in vivo.ResultsExpression of circ_CDR1as and Raf1 is elevated, while miR-1287 expression is decreased in HCC. Depletion of circ_CDR1as or Raf1 could inhibit proliferation and metastasis of HCC cells. Besides, circ_CDR1as regulates Raf1 expression by targeting miR-1287. MiR-1287 upregulation or Raf1 depletion could partially counterbalance circ_CDR1as depletion-mediated inhibitory effects on HCC cell behaviors. Moreover, circ_CDR1as depletion represses HCC progression through inactivating MEK/ERK pathway. In addition, circ_CDR1as depletion suppresses tumor growth in vivo via regulating miR-1287/Raf1 pathway.ConclusionCirc_CDR1as depletion inhibits HCC cell proliferation and metastasis by miR-1287/Raf1 and MEK/ERK pathways, highlighting a promising molecular target for HCC treatment.

RAS internal tandem duplication disrupts GTPase-activating protein (GAP) binding to activate oncogenic signaling

The oncogene RAS is one of the most widely studied proteins in cancer biology, and mutant active RAS is a driver in many types of solid tumors and hematological malignancies. Yet the biological effects of different RAS mutations and the tissue-specific clinical implications are complex and nuanced. Here, we identified an internal tandem duplication (ITD) in the switch II domain of NRAS from a patient with extremely aggressive colorectal carcinoma. Results of whole-exome DNA sequencing of primary and metastatic tumors indicated that this mutation was present in all analyzed metastases and excluded the presence of any other clear oncogenic driver mutations. Biochemical analysis revealed increased interaction of the RAS ITD with Raf proto-oncogene Ser/Thr kinase (RAF), leading to increased phosphorylation of downstream MAPK/ERK kinase (MEK)/extracellular signal-regulated kinase (ERK). The ITD prevented interaction with neurofibromin 1 (NF1)-GTPase-activating protein (GAP), providing a mechanism for sustained activity of the RAS ITD protein. We present the first crystal structures of NRAS and KRAS ITD at 1.65-1.75 Å resolution, respectively, providing insight into the physical interactions of this class of RAS variants with its regulatory and effector proteins. Our in-depth bedside-to-bench analysis uncovers the molecular mechanism underlying a case of highly aggressive colorectal cancer and illustrates the importance of robust biochemical and biophysical approaches in the implementation of individualized medicine.

The protein kinase MAP3K19 phosphorylates MAP2Ks and thereby activates ERK and JNK kinases and increases viability of KRAS-mutant lung cancer cells

Identifying additional mitogen-activated protein kinase (MAPK) pathway regulators is invaluable in aiding our understanding of the complex signaling networks that regulate cellular processes, including cell proliferation and survival. Here, using in vitro kinase assays and by expressing WT or kinase-dead MAPK kinase kinase 19 (MAP3K19) in the HEK293T cell line and assessing activation of the extracellular signal-regulated kinase (ERK) and JUN N-terminal kinase (JNK) signaling pathways, we defined MAP3K19 as a novel regulator of MAPK signaling. We also observed that overexpression of WT MAP3K19 activates both the ERK and JNK pathways in a panel of cancer cell lines. Furthermore, MAP3K19 sustained ERK pathway activation in the presence of inhibitors targeting the RAF proto-oncogene Ser/Thr protein kinase (RAF) and MAPK/ERK kinase, indicating that MAP3K19 activates ERK via a RAF-independent mechanism. Findings from in vitro and in-cell kinase assays demonstrate that MAP3K19 is a kinase that directly phosphorylates both MAPK/ERK kinase (MEK) and MAPK kinase 7 (MKK7). Results from an short-hairpin RNA screen indicated that MAP3K19 is essential for maintaining survival in KRAS-mutant cancers; therefore, we depleted or inhibited MAP3K19 in KRAS-mutant cancer cell lines and observed that this reduces viability and decreases ERK and JNK pathway activation. In summary, our results reveal that MAP3K19 directly activates the ERK and JNK cascades and highlight a role for this kinase in maintaining survival of KRAS-mutant lung cancer cells.

The evolutionarily conserved MAPK/Erk signaling promotes ancestral T-cell immunity in fish via c-Myc–mediated glycolysis

The mitogen-activated protein kinase (MAPK) cascade is an ancient and evolutionarily conserved signaling pathway involved in numerous physiological processes. Despite great advances in understanding MAPK-mediated regulation of adaptive immune responses in mammals, its contribution to T-cell immunity in early vertebrates remains unclear. Herein, we used Nile tilapia (Oreochromis niloticus) to investigate the regulatory roles of MAPK/extracellular signal-regulated kinase (Erk) signaling in ancestral T-cell immunity of jawed fish. We found that Nile tilapia possesses an evolutionarily conserved MAPK/Erk axis that is activated through a classical three-tier kinase cascade, involving sequential phosphorylation of RAF proto-oncogene serine/threonine-protein kinase (Raf), MAPK/Erk kinase 1/2 (Mek1/2), and Erk1/2. In Nile tilapia, MAPK/Erk signaling participates in adaptive immune responses during bacterial infection. Upon T-cell activation, the MAPK/Erk axis is robustly activated, and MAPK/Erk blockade by specific inhibitors severely impairs T-cell activation. Furthermore, signals from MAPK/Erk were indispensable for primordial T cells to proliferate and exert their effector functions. Mechanistically, activation of the MAPK/Erk axis promoted glycolysis via induction of the transcriptional regulator proto-oncogene c-Myc (c-Myc), to ensure the proper activation and proliferation of fish T cells. Our results reveal the regulatory mechanisms of MAPK/Erk signaling in T-cell immunity in fish and highlight a close link between immune signals and metabolic programs. We propose that regulation of T-cell immunity by MAPK/Erk is a basic and sophisticated strategy that evolved before the emergence of the tetrapod lineage. These findings shed light on the evolution of the adaptive immune system.

MiR-4510 blocks hepatocellular carcinoma development through RAF1 targeting and RAS/RAF/MEK/ERK signalling inactivation.

Therapeutic outcomes using the multikinase inhibitors, sorafenib and regorafenib, remain unsatisfactory for patients with advanced hepatocellular carcinoma (HCC). Thus, new drug modalities are needed. We recently reported the remarkable capacity of miR-4510 to impede the growth of HCC and hepatoblastoma through Glypican-3 (GPC3) targeting and Wnt pathway inactivation. To identify new targets of miR-4510, we used a label-free proteomic approach and reported down-regulation of RAF proto-oncogene serine/threonine-protein kinase (RAF1) by miR-4510. Because the tumourigenic role of RAF1 in HCC is controversial, we further studied RAF1:miR-4510 interactions using cellular, molecular as well as functional approaches and a chicken chorioallantoic membrane (CAM) xenograft model. We found an increase in RAF1 protein in 59.3% of HCC patients and a specific up-regulation of its transcript in proliferative tumours. We showed that miR-4510 inactivates the RAS/RAF/MEK/ERK pathway and reduces the expression of downstream targets (ie c-Fos proto-oncogene [FOS]) through RAF1 direct targeting. At a cellular level, miR-4510 inhibited HCC cell proliferation and migration and induced senescence in part by lowering RAF1 messenger RNA (mRNA) and protein expression. Finally, we confirmed the pro-tumoural function of RAF1 protein in HCC cells and its ability to sustain HCC tumour progression in vitro and in vivo. In this work, we confirm that RAF1 acts as an oncogene in HCC and further demonstrate that miR-4510 acts as a strong tumour suppressor in the liver by targeting many proto-oncogenes, including GPC3 and RAF1, and subsequently controlling key biological and signalling pathways among which Wnt and RAS/RAF/MEK/ERK signals.

Litchi (Litchi chinensis Sonn.) flower proanthocyanidin fraction exhibited protective efficacy to suppress nickel-induced expression for vascular endothelial growth factor in HepG2 cells.

The protective efficacy of litchi (Litchi chinensis Sonn.) flower proanthocyanidin fraction (LFPF) composed of (-)-epicatechin and proanthocyanidin A2 against vascular endothelial growth factor (VEGF) generation induced by nickel (Ni) in hepatocellular carcinoma (Hep G2) cells was studied. VEGF is an angiogenic inducer, which promotes tumor angiogenesis, leading to rapid tumor growth and metastasis. VEGF could be substantially induced in the Ni-mediated Hep G2 cells. Through LFPF treatment, the Ni-induced VEGF generation could be suppressed significantly. The inhibition of HIF-1α expression by blocking phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) pathways, and the suppression of Janus kinase 2 (JAK2)/signal transducers and activators of transcription 3 (STAT 3), and Raf-1 proto-oncogene, serine/threonine kinase (RAF1)/mitogen-activated protein kinase (MEK1/2)/extracellular-signal-regulated kinase (ERK1/2) pathways are important molecular mechanisms for the LFPF action. LFPF should probably reduce the risk of liver cancer in Ni-contaminated environments by inhibiting VEGF expression. PRACTICAL APPLICATIONS: LFPF mainly contained (-)-epicatechin and proanthocyanidin A2. Our results demonstrated that LFPF considerably suppressed the Ni-induced VEGF expression through inhibition of JAK2/STAT 3 and RAF1/MEK1/2/ERK1/2 pathways and prohibited HIF-1α expression through blocking PI3K/AKT/mTOR pathway. Litchi flowers might have the potential to diminish the liver cancer risk in a Ni-contaminated environment through suitable treatment.

Epidermal growth factor receptor controls glycogen phosphorylase in T cells through small GTPases of the RAS family

We recently uncovered a regulatory pathway of the muscle isoform of glycogen phosphorylase (PYGM) that plays an important role in regulating immune function in T cells. Here, using various enzymatic, pulldown, and immunoprecipitation assays, we describe signaling cross-talk between the small GTPases RAS and RAP1A, member of RAS oncogene family (RAP1) in human Kit 225 lymphoid cells, which, in turn, is regulated by the epidermal growth factor receptor (EGFR). We found that this communication bridge is essential for glycogen phosphorylase (PYG) activation through the canonical pathway because this enzyme is inactive in the absence of adenylyl cyclase type 6 (ADCY6). PYG activation required stimulation of both exchange protein directly activated by cAMP 2 (EPAC2) and RAP1 via RAS and ADCY6 phosphorylation, with the latter being mediated by Raf-1 proto-oncogene, Ser/Thr kinase (RAF1). Consistent with this model, PYG activation was EGFR-dependent and may be initiated by the constitutively active form of RAS. Consequently, PYG activation in Kit 225 T cells could be blocked with specific inhibitors of RAS, EPAC, RAP1, RAF1, ADCY6, and cAMP-dependent protein kinase. Our results establish a new paradigm for the mechanism of PYG activation, which depends on the type of receptor involved.

Trace amine–associated receptor 1 (TAAR1) promotes anti-diabetic signaling in insulin-secreting cells

Pancreatic β-cell failure in type 2 diabetes mellitus is a serious challenge that results in an inability of the pancreas to produce sufficient insulin to properly regulate blood glucose levels. Trace amine-associated receptor 1 (TAAR1) is a G protein-coupled receptor expressed by β-cells that has recently been proposed as a potential target for improving glycemic control and suppressing binge eating behaviors. We discovered that TAAR1 is coupled to Gαs-signaling pathways in insulin-secreting β-cells to cause protein kinase A (PKA)/exchange protein activated by cAMP (Epac)-dependent release of insulin, activation of RAF proto-oncogene, Ser/Thr kinase (Raf)-mitogen-activated protein kinase (MAPK) signaling, induction of cAMP response element-binding protein (CREB)-insulin receptor substrate 2 (Irs-2), and increased β-cell proliferation. Interestingly, TAAR1 triggered cAMP-mediated calcium influx and release from internal stores, both of which were required for activation of a MAPK cascade utilizing calmodulin-dependent protein kinase II (CaMKII), Raf, and MAPK/ERK kinase 1/2 (MEK1/2). Together, these data identify TAAR1/Gαs-mediated signaling pathways that promote insulin secretion, improved β-cell function and proliferation, and highlight TAAR1 as a promising new target for improving β-cell health in type 2 diabetes mellitus.

Quantitative biophysical analysis defines key components modulating recruitment of the GTPase KRAS to the plasma membrane

The gene encoding the GTPase KRAS is frequently mutated in pancreatic, lung, and colorectal cancers. The KRAS fraction in the plasma membrane (PM) correlates with activation of the mitogen-activated protein kinase (MAPK) pathway and subsequent cellular proliferation. Understanding KRAS's interaction with the PM is challenging given the complexity of the cellular environment. To gain insight into key components necessary for KRAS signal transduction at the PM, we used synthetic membranes such as liposomes and giant unilamellar vesicles. Using surface plasmon resonance (SPR) spectroscopy, we demonstrated that KRAS and Raf-1 proto-oncogene Ser/Thr kinase (RAF1) domains interact with these membranes primarily through electrostatic interactions with negatively charged lipids reinforced by additional interactions involving phosphatidyl ethanolamine and cholesterol. We found that the RAF1 region spanning RBD through CRD (RBDCRD) interacts with the membrane significantly more strongly than the isolated RBD or CRD domains and synergizes KRAS partitioning to the membrane. We also found that calmodulin and phosphodiesterase 6 delta (PDE6δ), but not galectin3 previously proposed to directly interact with KRAS, passively sequester KRAS and prevent it from partitioning into the PM. RAF1 RBDCRD interacted with membranes preferentially at nonraft lipid domains. Moreover, a C-terminal O-methylation was crucial for KRAS membrane localization. These results contribute to a better understanding of how the KRAS-membrane interaction is tuned by multiple factors whose identification could inform drug discovery efforts to disrupt this critical interaction in diseases such as cancer.