CPEB2 inhibit cell proliferation through upregulating p21 mRNA stability in glioma.

Glioma is the most common primary malignant brain tumor in adults and remains an incurable disease at present. Thus, there is an urgent need for progress in finding novel molecular mechanisms that control the progression of glioma which could be used as therapeutic targets for glioma patients. The RNA binding protein cytoplasmic polyadenylate element-binding protein 2 (CPEB2) is involved in the pathogenesis of several tumors. However, the role of CPEB2 in glioma progression is unknown. In this study, the functional characterization of the role and molecular mechanism of CPEB2 in glioma were examined using a series of biological and cellular approaches in vitro and in vivo. Our work shows CPEB2 is significantly downregulated in various glioma patient cohorts. Functional characterization of CPEB2 by overexpression and knockdown revealed that it inhibits glioma cell proliferation and promotes apoptosis. CPEB2 exerts an anti-tumor effect by increasing p21 mRNA stability and inducing G1 cell cycle arrest in glioma. Overall, this work stands as the first report of CPEB2 downregulation and involvement in glioma pathogenesis, and identifies CPEB2 as an important tumor suppressor gene through targeting p21 in glioma, which revealed that CPEB2 may become a promising predictive biomarker for prognosis in glioma patients.

Physical Interaction between Embryonic Stem Cell-Expressed Ras (ERas) and Arginase-1 in Quiescent Hepatic Stellate Cells.

Embryonic stem cell-expressed Ras (ERas) is an atypical constitutively active member of the Ras family and controls distinct signaling pathways, which are critical, for instance, for the maintenance of quiescent hepatic stellate cells (HSCs). Unlike classical Ras paralogs, ERas has a unique N-terminal extension (Nex) with as yet unknown function. In this study, we employed affinity pull-down and quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses and identified 76 novel binding proteins for human and rat ERas Nex peptides, localized in different subcellular compartments and involved in various cellular processes. One of the identified Nex-binding proteins is the nonmitochondrial, cytosolic arginase 1 (ARG1), a key enzyme of the urea cycle and involved in the de novo synthesis of polyamines, such as spermidine and spermine. Here, we show, for the first time, a high-affinity interaction between ERas Nex and purified ARG1 as well as their subcellular colocalization. The inhibition of ARG1 activity strikingly accelerates the activation of HSCs ex vivo, suggesting a central role of ARG1 activity in the maintenance of HSC quiescence.

Recent Developments in Targeting RAS Downstream Effectors for RAS-Driven Cancer Therapy.

Aberrant activity of oncogenic rat sarcoma virus (RAS) protein promotes tumor growth and progression. RAS-driven cancers comprise more than 30% of all human cancers and are refractory to frontline treatment strategies. Since direct targeting of RAS has proven challenging, efforts have been centered on the exploration of inhibitors for RAS downstream effector kinases. Two major RAS downstream signaling pathways, including the Raf/MEK/Erk cascade and the phosphatidylinositol-3-kinase (PI3K) pathway, have become compelling targets for RAS-driven cancer therapy. However, the main drawback in the blockade of a single RAS effector is the multiple levels of crosstalk and compensatory mechanisms between these two pathways that contribute to drug resistance against monotherapies. A growing body of evidence reveals that the sequential or synergistic inhibition of multiple RAS effectors is a more convenient route for the efficacy of cancer therapy. Herein, we revisit the recent developments and discuss the most promising modalities targeting canonical RAS downstream effectors for the treatment of RAS-driven cancers.

The seasonal profile of proliferation and apoptosis in the prostate gland of the wild ground squirrel (Spermophilus dauricus).

The seasonal cycle of growth and regression in the prostate gland of wild ground squirrel provide a unique research model to understand the morphological changes of prostate glands. Our previous studies showed that the local production of dihydrotestosterone could affect the morphology and function of the prostate gland in either an autocrine or paracrine manner. In the present study, we attempted to gain more insight into this process by investigating the expression of key factors implicated in cell proliferation, apoptosis, and the cell cycle, including mechanistic target of rapamycin (mTOR), cyclin-D2, p21, p27 and retinoblastoma 1 (pRB). Morphological and histological observations confirmed that the prostate increased significantly in both size and weight during the breeding season. Positive immunostaining for proliferating cell nuclear antigen (PCNA) was mainly localized to the prostate epithelial cells during the breeding season, which is significantly higher in the prostate gland during the breeding season (2470 ± 81/mm2) than that in the nonbreeding season (324 ± 54/mm2). However, there was no significant difference in the prostate gland when compared between the breeding and nonbreeding seasons, with regards to TUNEL staining. Moreover, cell cycle regulators were mainly localized to the epithelial cells, including mTOR, cyclin-D2, p21, p27 and pRB. the immunostaining of mTOR and cyclin D2 were stronger during the breeding season, whereas the immunostaining of p27 and pRB were stronger during the nonbreeding season. The mRNA expression levels of mTOR, cyclin D2, and PCNA, were higher during the breeding season while those of p27 and p21 were higher during the nonbreeding season. Collectively, this study profiled the distinct expression pattern of key cell cycle regulators throughout the breeding and nonbreeding seasons. Collectively, these factors may play important roles in regulating the seasonal growth and regression of the prostatic epithelium in the wild ground squirrel.

A phospho-switch controls RNF43-mediated degradation of Wnt receptors to suppress tumorigenesis.

Frequent mutation of the tumour suppressor RNF43 is observed in many cancers, particularly colon malignancies. RNF43, an E3 ubiquitin ligase, negatively regulates Wnt signalling by inducing degradation of the Wnt receptor Frizzled. In this study, we discover that RNF43 activity requires phosphorylation at a triplet of conserved serines. This phospho-regulation of RNF43 is required for zebrafish development and growth of mouse intestinal organoids. Cancer-associated mutations that abrogate RNF43 phosphorylation cooperate with active Ras to promote tumorigenesis by abolishing the inhibitory function of RNF43 in Wnt signalling while maintaining its inhibitory function in p53 signalling. Our data suggest that RNF43 mutations cooperate with KRAS mutations to promote multi-step tumorigenesis via the Wnt-Ras-p53 axis in human colon cancers. Lastly, phosphomimetic substitutions of the serine trio restored the tumour suppressive activity of extracellular oncogenic mutants. Therefore, harnessing phospho-regulation of RNF43 might be a potential therapeutic strategy for tumours with RNF43 mutations.

ERas regulates cell proliferation and epithelial-mesenchymal transition by affecting Erk/Akt signaling pathway in pancreatic cancer.

Pancreatic cancer is the fourth most common lethal malignancy with an overall 5-year survival rate of less than 5%. ERas, a novel Ras family member, was first identified in murine embryonic stem cells and is upregulated in various cancers. However, the expression and potential role of ERas in pancreatic cancer have not been investigated. In this study, we found that ERas mRNA and protein were upregulated in pancreatic cancer tissues and cells compared with controls. Knockdown of ERas in pancreatic cancer cells by siRNA significantly decreased cell proliferation, colony formation, migration, and invasion and promoted cell apoptosis in vitro. Epithelial-mesenchymal transition (EMT) is closely related to tumor progression. We observed a significant decrease in N-cadherin expression in pancreatic cancer cells in response to ERas gene silencing by immunofluorescence assay and western blot. Furthermore, tumor growth and EMT were inhibited in xenografts derived from pancreatic cancer cells with ERas downregulation. We further investigated the regulatory mechanisms of ERas in pancreatic cancer and found that ERas may activate the Erk/Akt signaling pathway. Moreover, Erk inhibitor decreased pancreatic cancer cells proliferation and colony formation activities. Our data suggest that targeting ERas and its relevant signaling pathways might represent a novel therapeutic approach for the treatment of pancreatic cancer.

Silybum marianum (milk thistle) improves vancomycin induced nephrotoxicity by downregulating apoptosis.

Increased use of vancomycin for treating infections, and the associated risk of causing nephrotoxicity lead to the present study. The antioxidant and anti-apoptotic potential of Silybum marianum is used along with vancomycin to reduce adverse effects on the kidney. Vero cells (monkey kidney cells) and mice were used to test S. marianum extract on vancomycin induced nephrotoxicity. Vero cells were treated with different concentrations of vancomycin and S. marianum for 24 h for determination of cytotoxic potential and mRNA levels of apoptotic genes p53 , p21, and cyt-c were measured. For in-vivo studies mice were divided into five groups; G1 control (untreated), G2 vehicle (olive oil), G3 vancomycin treated (300 mg/kg body weight), G4 (S. marianum; 400 mg/kg bodyweight and vancomycin 300 mg/kg bodyweight simultaneously) and G5 (S. marianum 400 mg/kg bodyweight and vancomycin 300 mg/kg bodyweight treatment started after day 4 of S. marianum treatment). After 10 days histopathological analysis of mice kidneys was performed, serum urea and creatinine were analysed and mRNA expression of p53 , p21, and cyt-c was evaluated. Expression of p53, p21, and cyt-c in Vero cells was elevated in response to vancomycin treatment, whereas after S. marianum administration expression of these genes reduced. Vancomycin showed apoptosis in cells at the concentration of 6 mg/ml (LC50). Urea and creatinine levels in mice were increased in response to vancomycin administration and kidney histology showed an abnormality in functional units. The apoptotic cells were very visible in kidney structure in vancomycin treated group. These symptoms were however relieved in groups where treatment of S. marianum extract was given. mRNA expression of p53 , p21, and cyt-c also reduced in S. marianum treated groups of mice. S. marianum extract has protective effects against renal damage from vancomycin induced oxidative stress and relieves symptoms may be by downregulating apoptotic genes.

Epithelial-mesenchymal transition suppresses ERas to activate autophagy in retinal pigment epithelial cells in proliferative vitreoretinopathy.

OBJECTIVE: Proliferative vitreoretinopathy (PVR) is a complex ocular disease that leads to detached retinas and irreversible vision loss. The epithelial-mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells plays a critical role in PVR occurrence. However, the core targets driven by the EMT process that lead to the pathogenesis of PVR remain unclear. In our study, the relationship between embryonic stem cell-expressed Ras (ERas) and EMT in RPE cells was investigated. PATIENTS AND METHODS: The subretinal and epiretinal membrane specimens of human PVR were examined for ERas and hallmarks of autophagy and EMT using Western blotting and immunofluorescence. EMT was induced by transforming growth factor (TGF)-ß1 or epidermal growth factor (EGF) in ARPE-19 cells. Autophagy was inhibited by U0126 or bafilomycin A1 in ARPE-19 cells. RESULTS: ERas was decreased and the classical autophagy biomarker microtubule associated protein 1 light chain 3 alpha (LC3) was upregulated in the subretinal and epiretinal membranes of PVR patients in vivo. Moreover, ERas was downregulated and autophagy was activated in RPE ARPE-19 cells in response to transforming growth factor (TGF)-ß1 and epidermal growth factor (EGF) induction. Finally, overexpression of ERas in RPE cells inhibited autophagy via impaired formation of autophagosomes and lysosomes. CONCLUSIONS: Our study revealed the role of ERas in the pathogenesis of PVR through EMT and provided a novel therapeutic target for PVR prevention and treatment.

Plasma membrane V-ATPase controls oncogenic RAS-induced macropinocytosis.

Oncogenic activation of RAS is associated with the acquisition of a unique set of metabolic dependencies that contribute to tumour cell fitness. Cells that express oncogenic RAS are able to internalize and degrade extracellular protein via a fluid-phase uptake mechanism termed macropinocytosis1. There is increasing recognition of the role of this RAS-dependent process in the generation of free amino acids that can be used to support tumour cell growth under nutrient-limiting conditions2. However, little is known about the molecular steps that mediate the induction of macropinocytosis by oncogenic RAS. Here we identify vacuolar ATPase (V-ATPase) as an essential regulator of RAS-induced macropinocytosis. Oncogenic RAS promotes the translocation of V-ATPase from intracellular membranes to the plasma membrane via a pathway that requires the activation of protein kinase A by a bicarbonate-dependent soluble adenylate cyclase. Accumulation of V-ATPase at the plasma membrane is necessary for the cholesterol-dependent plasma-membrane association of RAC1, a prerequisite for the stimulation of membrane ruffling and macropinocytosis. These observations establish a link between V-ATPase trafficking and nutrient supply by macropinocytosis that could be exploited to curtail the metabolic adaptation capacity of RAS-mutant tumour cells.

KRAS: A Promising Therapeutic Target for Cancer Treatment.

Kirsten rat sarcoma 2 viral oncogene homolog (KRAS) is the most commonly mutated oncogene in human cancer. The developments of many cancers depend on sustained expression and signaling of KRAS, which makes KRAS a high-priority therapeutic target. Scientists have not successfully developed drugs that target KRAS, although efforts have been made last three decades. In this review, we highlight the emerging experimental strategies of impairing KRAS membrane localization and the direct targeting of KRAS. We also conclude the combinatorial therapies and RNA interference technology for the treatment of KRAS mutant cancers. Moreover, the virtual screening approach to discover novel KRAS inhibitors and synthetic lethality interactors of KRAS are discussed in detail.

Targeting Mutant KRAS for Anticancer Therapy.

Over the past decades, designing therapeutic strategies to target KRAS-mutant cancers, which is one of the most frequent mutant oncogenes among all cancer types, have proven unsuccessful regardless of many concerted attempts. There are key challenges for KRAS-mutant anticancer therapy, as the complex cellular processes involved in KRAS signaling has present. Herein, we highlight the emerging therapeutic approaches for inhibiting KRAS signaling and blocking KRAS functions, in hope to serve as a more effective guideline for future development of therapeutics.

Recent Advances in Developing K-Ras Plasma Membrane Localization Inhibitors.

The Ras proteins play an important role in cell growth, differentiation, proliferation and survival by regulating diverse signaling pathways. Oncogenic mutant K-Ras is the most frequently mutated class of Ras superfamily that is highly prevalent in many human cancers. Despite intensive efforts to combat various K-Ras-mutant-driven cancers, no effective K-Ras-specific inhibitors have yet been approved for clinical use to date. Since K-Ras proteins must be associated to the plasma membrane for their function, targeting K-Ras plasma membrane localization represents a logical and potentially tractable therapeutic approach. Here, we summarize the recent advances in the development of K-Ras plasma membrane localization inhibitors including natural product-based inhibitors achieved from high throughput screening, fragment-based drug design, virtual screening, and drug repurposing as well as hit-to-lead optimizations.

Mitophagy mediated by BNIP3 and BNIP3L/NIX in urothelial cells of the urinary bladder of cattle harbouring bovine papillomavirus infection.

Autophagy is a powerful tool that host cells use to defend against viral infection. Mitophagy, the selective autophagic removal of dysfunctional mitochondria was upregulated in urothelial cancer cells harbouring bovine papillomavirus (BPV) infection, as detected by the expression of BPV E5 protein, the major oncoprotein of bovine Deltapapillomavirus genus. HIF-1α-induced mitophagy receptors, BNIP3 and BNIP3L/Nix, were found to be overexpressed in these cells. The BNIP3 and BNIP3L/Nix receptors were amplified, and amplicon sequencing showed homology between bovine BNPI3 and BNIP3L/Nix sequences deposited in GenBank (accession number: NM_001076366.1 and NM_001034614.2, respectively). The transcripts and protein levels of BNIP3 and BNIP3L/Nix were significantly overexpressed in hypoxic neoplastic cells relative to healthy, non-neoplastic cells. BNIP3 and BNIP3L/Nix interacted with the LC3 protein, a marker of autophagosome (mitophagosome) membrane, ERAS, a small GTPase, and p62, known to be a specific autophagy receptor protein, that plays a role in mitochondrial priming for mitophagy and subsequent elimination. ERAS also interacted with the BPV E5 oncoprotein at mitochondrial level. Furthermore, in anti-Bag3 mitochondrial immunoprecipitates, a complex composed of the Hsc70/Hsp70 chaperone, CHIP co-chaperone, Synpo2, ERAS, LC3, p62, BNPI3, and BNIP3L/Nix was also detected. Bag3 may play a role in mitophagosome formation together with the Synpo2 protein and may be involved in the degradation of Hsc70/Hsp70-bound CHIP-ubiquitinated cargo, in association with its chaperone. ERAS may be involved in mitophagosome maturation via the PI3K signalling pathway. Ultrastructural findings revealed the presence of mitochondria exhibiting severe fragmentation and loss of cristae, as well as numerous mitochondria-containing autophagosomes.

The Extract of Cordyceps Militaris Inhibited the Proliferation of Cisplatin-Resistant A549 Lung Cancer Cells by Downregulation of H-Ras.

We investigated the antitumor effect of Cordyceps militaris extract (CME) on A549 cisplatin-resistant (CR) lung cancer cells. The proliferation of A549/CR cells was suppressed by CME. Apoptosis of the cells was induced by CME. The cell cycle arrest was observed in the sub-G1 phase in the cells treated with CME. Proteomic profile analysis showed that H-Ras was downregulated in CME-treated cells and it was confirmed by western blot analysis. Collectively, these data demonstrated that CME is an alternative treatment for the anticancer effect.

Mir-455-3p-1 represses FGF7 expression to inhibit pulmonary arterial hypertension through inhibiting the RAS/ERK signaling pathway.

OBJECTIVE: To analyze the effects of miR-455-3p-1 and its possible mechanisms in pulmonary arterial hypertension (PAH). METHODS: A microarray assay was used to examine the expressed genes between normal and PAH. The expressed genes in PAH was assessed by qRT-PCR. The targeted interaction between miRNAs and FGF7 was confirmed using a dual luciferase reporter assay. A CCK-8 assay and cell count were used to analyze the pulmonary artery smooth muscle cells (PASMCs) activity and proliferation level, respectively. Apoptotic PASMCs were detected by flow cytometry. In addition, the mRNA and protein expression levels of RAS/ERK signaling pathway were determined by qRT-PCR and a Western blot assay, respectively. A PAH rat model was used to identify the effects of miR-455-3p-1 in vivo. RESULTS: FGF7 was upregulated in PAH. MiR-455-3p-1 was downregulated in PAH. MiR-455-3p-1 targeted FGF7. MiR-455-3p-1 decreased the expression of FGF7. Moreover, the effect of FGF7 on PASMCs was suppressed by miR-455-3p-1. MiR-455-3p-1 upregulation was associated with reduced mRNA and protein levels of core RAS/ERK signal genes, suggesting the inhibition of the RAS/ERK pathway. Furthermore, miR-455-3p-1 upregulation improved the RVSP, mPAP, ratio of RV/LV + S, CO and RV function of PAH rat model in vivo. CONCLUSION: Our findings illustrate a role for miR-455-3p-1 in modulating FGF7-RAS/ERK signaling and suggest that an agomir of miR-455-3p-1 could inhibit the proliferation of PASMCs and mitigate PAH in vivo.

A missense variant in PTPN12 associated with the risk of colorectal cancer by modifying Ras/MEK/ERK signaling.

BACKGROUND: The classical protein tyrosine phosphatases (PTPs) have been widely reported to be associated with various human malignancies including colorectal cancer (CRC). However, there are few comprehensive analyses of the association between the classical PTP genes and CRC risk. METHODS: First, a bioinformatics analysis was performed to identify missense variants within the classical PTP gene family. Second, exome-wide association data and an independent population study were conducted to evaluate effects of candidate variants on CRC risk. Finally, functional assays based on signaling pathways were applied to uncover the potential pathogenic mechanism. RESULTS: We identified that PTPN12 rs3750050 G allele presented a 19% increase the risk of CRC, with an OR of 1.19 (95% CI = 1.09-1.30, P = 1.015×10-4) under an additive model in the combined analysis. Furthermore, biochemical assays illustrated that rs3750050 could impair the inhibitory effect of PTPN12 on Ras/MEK/ERK signaling by impeding SHC dephosphorylation, increase the expression of cyclin D1 and ultimately lead to aberrant cell proliferation, thus contributing to CRC pathogenesis. CONCLUSION: Our study highlights that PTPN12 rs3750050 could increase CRC risk by modifying Ras/MEK/ERK signaling. This work provides a novel insight into the roles of genetic variants within PTP genes in the pathogenesis of CRC.

Structure-based development of new RAS-effector inhibitors from a combination of active and inactive RAS-binding compounds.

The RAS gene family is frequently mutated in human cancers, and the quest for compounds that bind to mutant RAS remains a major goal, as it also does for inhibitors of protein-protein interactions. We have refined crystallization conditions for KRAS169Q61H-yielding crystals suitable for soaking with compounds and exploited this to assess new RAS-binding compounds selected by screening a protein-protein interaction-focused compound library using surface plasmon resonance. Two compounds, referred to as PPIN-1 and PPIN-2, with related structures from 30 initial RAS binders showed binding to a pocket where compounds had been previously developed, including RAS effector protein-protein interaction inhibitors selected using an intracellular antibody fragment (called Abd compounds). Unlike the Abd series of RAS binders, PPIN-1 and PPIN-2 compounds were not competed by the inhibitory anti-RAS intracellular antibody fragment and did not show any RAS-effector inhibition properties. By fusing the common, anchoring part from the two new compounds with the inhibitory substituents of the Abd series, we have created a set of compounds that inhibit RAS-effector interactions with increased potency. These fused compounds add to the growing catalog of RAS protein-protein inhibitors and show that building a chemical series by crossing over two chemical series is a strategy to create RAS-binding small molecules.

Novel anti-inflammatory target of geniposide: Inhibiting Itgß1/Ras-Erk1/2 signal pathway via the miRNA-124a in rheumatoid arthritis synovial fibroblasts.

Geniposide (GE) is an active component isolated from the fruit of Gardenia jasminoides Ellis that has anti-inflammatory and other pharmacological effects; however, the underlying mechanism of GE action has not been elucidated in rheumatoid arthritis (RA). Previous studies have shown that GE plays a therapeutic role in RA via regulation of the integrin beta 1 (Itgß1)-mediated Ras-Erk1/2 signalling pathway. However, the specific mechanism of GE action on Itgß1 has not been clarified. Recent evidence indicates that microRNAs (miRNAs) are involved in the development of RA. In this study, we developed a miRNA-124a-based synoviocyte repair strategy. We demonstrated that miRNA-124a can directly inhibit the expression of the Itgß1 gene and decrease TNF-α-stimulated cell proliferation in vitro. MH7A cells were obtained from the patient with RA and treated with GE in the presence of TNF-α (10 ng/mL). Additionally, we demonstrated that the expression of miRNA-124a can be regulated by GE. GE upregulated the expression of miRNA-124a and decreased the expression of Itgß1 at the mRNA and protein levels. The results of the present study are the first to suggest that GE inhibits TNF-α-stimulated cell proliferation and blocks the activation of the Ras-Erk1/2 pathway via the upregulation of miRNA-124a expression. Our study elucidates the role of miRNA-124a as a protected miRNA in RA and may provide a novel strategy for the diagnosis and treatment of RA in the future.

Andrographolide enhances the anti-metastatic effect of radiation in Ras-transformed cells via suppression of ERK-mediated MMP-2 activity.

BACKGROUND: Activation of Ras oncogene in human tumors is associated with radiation-associated metastatic potential. Although ionizing radiation is one important method of cancer treatments, it has been shown to enhance matrix metalloproteinases (MMPs) activity and facilitates a more aggressive cancer phenotype. Our previous studies showed that andrographolide with lower dose rates of radiation could inhibit RAS-transformed cancer metastasis in vivo; however, the molecular mechanisms are not yet clear. In this study, we aimed to explore the anti-metastatic effect of andrographolide combined with radiation on Ras-transformed cells. METHODS: RAS-transformed cells were treated with andrographolide in the presence or absence of irradiation (2-4 Gy) or angiotensin II to examine cell invasion. In vivo tumorigenesis assays were also performed. The MMP-2 activity was detected by using Gelatin zymography. Signal transduction of NF-κB subunit, p65 and phosphor-ERK 1/2, were examined by using Western blotting analysis. RESULTS: Treatment with andrographolide inhibited migration of Ras-transformed cells. Andrographolide treatment with radiation significantly inhibited cancer metastasis in vivo. We found that andrographolide exhibited anti-migration and anti-invasive ability against cancer metastasis via inhibition of MMP2 activity rather than affected MMP-9 and EMT. In addition, combined andrographolide with radiation appeared to be more effective in reducing MMP-2 expression, and this effect was accompanied by suppression of ERK activation that inhibits cancer cell migration and invasion. CONCLUSIONS: These findings suggest that andrographolide enhances the anti-metastatic effect of radiation in Ras-transformed cells via suppression of ERK-mediated MMP-2 activity.