ERK Phosphorylation Expression Research Articles

Combined Targeting of SYK and BCL-2 in Acute Myeloid Leukaemia and Mechanism Study

Acute myeloid leukemia (AML) is a highly heterogeneous haematological malignancy and is the most common acute leukaemia in adults, with a median age of onset of 68 years and a 5-year survival rate of only 30%. Only 60-80% of adult AML patients can achieve complete remission with the first induction chemotherapy, and 50-80% of AML patients ultimately relapse with the “3+7” first-line treatment regimen based on anthracycline and cytarabine (Ara-C), so there is an urgent need to find safer and more effective targeted therapies. There is an urgent need to find safer and more effective targeted therapies. Venetoclax (ABT-199) is an FDA-approved oral selective Bcl-2 inhibitor with limited anti-leukaemia activity as a single agent, and mechanisms of resistance include up-regulation of anti-apoptotic proteins, compensation of multiple metabolic pathways such as OXPHOS glycolysis, and mutations in BCL-2 and BAX.In 2018, the FDA approved the use of Venetoclax in combination with demethylating drugs (azacitidine ( AZA) or low-dose azacitidine AraC (LDAC)) in combination for newly diagnosed AML in older adults aged 75 years and older, and while this combination greatly improves remission rates, most patients relapse within 18 months. It has been suggested that direct or indirect inhibition of oxidative phosphorylation (OXPHOS) may be able to circumvent adaptive resistance of AML to Venetoclax+ AraC combination therapy. Therefore, an ideal candidate therapy in combination with Venetoclax should be able to antagonise its resistance mechanisms from metabolic pathways and down-regulation of MCL-1. Purpose To investigate the efficacy and related mechanisms of co-targeting SYK with BCL-2 for the treatment of acute myeloid leukaemia (AML). Experimental Design AML cell lines and primary patient samples were collected for cell proliferation assays. Firstly, cell activity was detected by MTS colorimetric assay, by Compusyn calculation software, the joint index CI value was calculated, apoptosis was detected by flow cytometry, mitochondrial membrane potential was altered, gene expression was detected by transcriptome sequencing, and the signalling pathways were analysed, followed by apoptosis-related proteins, BCL-2-related proteins by Western Blot, and glucose was detected by metabolism kit. sugar consumption,ATP and lactate content. The in vivo efficacy of the combination of Venetoclax and Entospletinib was assessed using an MV4-11-derived xenograft mouse model. Results SYK is highly expressed in AML and is associated with poor prognosis. ...The combination of Venetoclax and Entospletinib in AML cell lines and AML primary patient cells had significant synergistic growth inhibitory and apoptosis-inducing effects...Venetoclax in combination with Entospletinib down-regulated the expression of anti-apoptotic proteins, MCL-1, and BCL-XL, and down-regulated phosphorylated ERK expression, thereby affecting MCL1 post-translational protein stability rather than transcriptional levels.... Venetoclax in combination with Entospletinib downregulated the expression of anti-apoptotic proteins MCL-1, BCL-XL and phosphorylated ERK, thereby affecting the post-translational protein stability of MCL1, but not the transcriptional level. Conclusions The combination of Venetoclax and Entospletinib had significant synergistic killing effects in both AML cell lines and AML primary patient cells.Venetoclax in combination with Entospletinib exerts synergistic therapeutic effects on AML through down-regulation of ERK phosphorylation affecting the expression of anti-apoptotic proteins MCL-1, BCL-XL and reprogramming of glucose metabolism.

Silencing of lncRNA LOC105376794 promotes migration, invasion, and Gefitinib resistance of lung adenocarcinoma cells with EGFR 19Del mutation by ATF4/CHOP axis and ERK phosphorylation.

Epidermal growth factor receptor (EGFR) gene exon 19 in-frame deletion (19del) and exon 21 L858R point mutation (21L858R mutation) are prevalent mutations in lung adenocarcinoma. Lung adenocarcinoma patients with 19del presented with a better prognosis than the 21L858R mutation under the same epidermal growth factor receptor tyrosine kinase inhibitor treatment. Our study aimed to uncover the expression of long non-coding RNA LOC105376794 between 19del and 21L858R mutation, and explore the mechanism that regulates cells' biological behavior and gefitinib sensitivity in lung adenocarcinoma cells with 19del. Transcriptome sequencing was conducted to identify differentially expressed lncRNAs between EGFR 19del and 21L858R mutation in serum through the DNBSEQ Platform. Protein-protein interaction network and Kyoto Encyclopedia of Genes and Genomes pathway were conducted to analyze the relationship between lncRNAs and mRNAs through STRING and Dr. TOM. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to measure the expression of lncRNA LOC105376794 in serum and cells. Loss-of-function experiments were used to validate the biological function and gefitinib sensitivity of LOC105376794 in lung adenocarcinoma cells. Protein levels were detected by western blotting. Through transcriptome resequencing and RT-qPCR, we found the expression levels of LOC105376794 in serum were increased in the 19del group compared with the 21L858R mutation group. Inhibition of LOC105376794 promoted proliferation, migration and invasion, and reduced apoptosis of HCC827 and PC-9 cells. The low expression of LOC105376794 reduced gefitinib sensitivity in PC-9 cells. Mechanistically, we found that the knockdown of LOC105376794 suppressed activating transcription factor 4 (ATF4)/C/EBP homologous protein (CHOP) signaling pathway and facilitated the expression of extracellular signal-regulated kinase 1/2 (ERK) phosphorylation. LOC105376794 altered cell biological behavior and gefitinib sensitivity of lung adenocarcinoma cells with 19del through the ATF4/CHOP signaling pathway and the expression of ERK phosphorylation. The results further illustrated the fact that lung adenocarcinoma patients with 19del presented with a more favorable clinical outcome and provided a theoretical basis for treatment strategy for lung adenocarcinoma patients with 19del.

The Effects of Injectable Platelet-Rich Fibrin and Advanced-Platelet Rich Fibrin on Gingival Fibroblast Cell Vitality, Proliferation, Differentiation.

Injectable Platelet Rich Fibrin (I-PRF) and Advanced-Platelet Rich Fibrin (A-PRF) are autologous materials derived from patients' blood and employed in periodontal regenerative surgery. Although I-PRF and A-PRF have different characteristics, their biological effects on gingival tissue fibroblasts remain unclear. This research aims to compare the in vitro capacity in inducing gene expression and proliferation of human gingival fibroblasts between A-PRF and I-PRF. Human donors undergoing dental implant surgery were sampled for normal human gingival fibroblasts (NHGFCs), followed by preparing A-PRF and I-PRF membranes. Enzyme-linked immunosorbent assay (ELISA) kit was used to assess the release of platelet-derived growth factor-AA (PDGF-AA), transforming growth factor-beta1 (TGF- β1), and insulin growth factor-1 (IGF-1) at different periods. Cell viability and proliferation of A-PRF and I-PRF were compared using CCK-8 assay. The impacts of platelet concentration on human gingival fibroblast cells (HGFCs) were evaluated by quantifying the level or amount of phosphorylated extracellular signal-regulated protein kinase (p-ERK), and Matrix metalloproteinases (MMPs), MMP-1 and MMP-3. The effects of PRF on aged human gingival fibroblast cells were examined retrospectively. Overall, A-PRF demonstrated a higher release of TGF-B1 and PDGF-AA, while I-PRF reflected higher levels of IGF-1. A significantly higher level of cell proliferation was induced by higher cell proliferation by A-PRF and I-PRF. Additionally, in comparison to I-PRF, the expression of ERK phosphorylation and MMP-1 &MMP-3 in HGFCs was demonstrated by I-PRF and A-PRF. The increase in A-PRF was time-dependent (p < 0.05). Both I-PRF and A-PRF induced a stimulatory biological impact on the proliferation of human gingiva fibroblasts, with the latter demonstrating better capacity in facilitating the release of different growth factors. A-PRF also induced higher gene expression of p-ERK, MMP-1 &MMP-3, and the proliferation of fibroblasts.

Methoxylated Cinnamic Esters with Antiproliferative and Antimetastatic Effects on Human Lung Adenocarcinoma Cells.

Lung cancer is the leading cause of cancer mortality worldwide, and malignant melanomas are highly lethal owing to their elevated metastatic potential. Despite improvements in therapeutic approaches, cancer treatments are not completely effective. Thus, new drug candidates are continuously sought. We synthesized mono- and di-methoxylated cinnamic acid esters and investigated their antitumor potential. A cell viability assay was performed to identify promising substances against A549 (non-small-cell lung cancer) and SK-MEL-147 (melanoma) cells. (E)-2,5-dimethoxybenzyl 3-(4-methoxyphenyl)acrylate (4m), a monomethoxylated cinnamic acid derivative, was identified as the lead antitumor compound, and its antitumor potential was deeply investigated. Various approaches were employed to investigate the antiproliferative (clonogenic assay and cell cycle analysis), proapoptotic (annexin V assay), and antimigratory (wound-healing and adhesion assays) activities of 4m on A549 cells. In addition, western blotting was performed to explore its mechanism of action. We demonstrated that 4m inhibits the proliferation of A549 by promoting cyclin B downregulation and cell cycle arrest at G2/M. Antimigratory and proapoptotic activities of 4m on A549 were also observed. The antitumor potential of 4m involved its ability to modulate the mitogen-activated protein kinases/extracellular signal-regulated kinase (MAPK/ERK) signaling pathway once phosphorylated-ERK expression was considerably reduced in response to treatment. Our findings demonstrate that 4m is a promising anticancer drug candidate.

Unsaturated oxidated fatty acid 12(S)-HETE attenuates TNF-α expression in TNF-α/IFN-γ-stimulated human keratinocytes

Chronic skin inflammatory diseases are associated with abnormal immune responses characterized by skin barrier dysfunction. Keratinocytes participate in immune homeostasis regulated by immune cells. Immune homeostasis dysfunction contributes to the pathogenesis of skin diseases mediated by pro-inflammatory cytokines and chemokines, such as tumor necrosis factor (TNF)-α, which are produced by activated keratinocytes. 12(S)-Hydroxy eicosatetraenoic acid [12(S)-HETE], an arachidonic acid metabolite, has anti-inflammatory properties. However, the role of 12(S)-HETE in chronic skin inflammatory diseases has not been elucidated yet. In this study, we investigated the effect of 12(S)-HETE on TNF-α/interferon (IFN)-γ-induced pro-inflammatory cytokine and chemokine expression. Our data showed that 12(S)-HETE modulates TNF-α mRNA and protein expression in TNF-α-/IFN-γ-treated human keratinocytes. Molecular docking analyses demonstrated that 12(S)-HETE bound to extracellular signal-regulated kinase (ERK)1/2, thus preventing ERK activation and downregulating phosphorylated ERK expression. We also demonstrated that 12(S)-HETE treatment inhibited IκB and ERK phosphorylation and nuclear factor (NF)-κB, p65/p50, and CCAAT/enhancerbindingproteinβ (C/EBPβ) translocation. Overall, our results showed that 12(S)-HETE attenuated TNF-α expression and secretion by inhibiting the mitogen-activated protein kinase ERK/NF-κB and C/EBPβ signaling pathways. Overall, these results suggest that 12(S)-HETE effectively resolved TNF-α-induced inflammation.

SCH 23390 inhibits the acquisition of nitrous oxide-induced conditioned place preference and the changes in ERK phosphorylation expression in nucleus accumbens of mice

Nitrous oxide (N2O) has a long history of abuse, but its abuse mechanism has not been clear yet. This research aimed at the possibility of mesolimbic dopaminergic system (MLDS) involved in the rewarding effect of N2O. In this work, the rewarding behavior of N2O in mice was evaluated using a typical gas-administered conditioned place preference (CPP) procedure. SCH 23390, a Dopamine D1 receptor (D1R) antagonist, and Haloperidol, a Dopamine D2 receptor (D2R) antagonist were administered during CPP to evaluate the role of dopamine receptors in the N2O-induced CPP. The accompanying changes in phosphorylation of extracellular signal-regulated kinase (ERK) in MLDS related brain regions, including the ventral tegmental area (VTA), caudate putamen (CPu), prefrontal cortex (PFC), and nucleus accumbens (NAc) were measured to assess the neural plasticity changes in the CPP mice by Western blot analysis. Results revealed that 60% N2O induced CPP in the gas-administered mice and promoted the ERK phosphorylation (p-ERK) in the NAc and CPu during the test session of the CPP test. Pretreatment of SCH 23390 (0.5 mg/kg) inhibited the acquisition of N2O-induced CPP and the enhanced p-ERK in NAc. It suggested that Dopamine D1 receptor may play an important role in the acquisition of N2O-induced CPP and the accompanied ERK activation in the NAc, which provide insight into the molecular mechanism in the rewarding properties of nitrous oxide.

Resistance to mutant KRASV12-induced senescence in an hTERT/Cdk4-immortalized normal human bronchial epithelial cell line

Mutant KRAS, the most frequently occurring (∼30%) driver oncogene in lung adenocarcinoma, induces normal epithelial cells to undergo senescence. This phenomenon, called “oncogene-induced senescence (OIS)”, prevents mutant KRAS-induced malignant transformation. We have previously reported that mutant KRASV12 induces OIS in a subset of normal human bronchial epithelial cell line immortalized with hTERT and Cdk4. Understanding the mechanism and efficacy of this important cancer prevention mechanism is a key knowledge gap. Therefore, this study investigates mutant KRASV12-induced OIS in upregulated telomerase combined with the p16/RB pathway inactivation in normal bronchial epithelial cells. The normal (non-transformed and non-tumorigenic) human bronchial epithelial cell line HBEC3 (also called “HBEC3KT”), immortalized with hTERT (“T”) and Cdk4 (“K”), was used in this study. HBEC3 that expressed mutant KRASV12 in a doxycycline-regulated manner was established (designated as HBEC3-RIN2). Controlled induction of mutant KRASV12 expression induced partial epithelial-to-mesenchymal transition in HBEC3-RIN2 cells, which was associated with upregulated expression of ZEB1 and SNAIL. Mutant KRASV12 caused the majority of HBEC3-RIN2 to undergo morphological changes; suggestive of senescence, which was associated with enhanced autophagic flux. Upon mutant KRASV12 expression, only a small HBEC3-RIN2 cell subset underwent senescence, as assessed by a senescence-associated β-galactosidase staining (SA-βG) method. Furthermore, mutant KRASV12 enhanced cell growth, evaluated by colorimetric proliferation assay, and liquid and soft agar colony formation assays, partially through increased phosphorylated AKT and ERK expression but did not affect cell division, or cell cycle status. Intriguingly, mutant KRASV12 reduced p53 protein expression but increased p21 protein expression by prolonging its half-life. These results indicate that an hTERT/Cdk4 -immortalized normal bronchial epithelial cell line is partially resistant to mutant KRASV12-induced senescence. This suggests that OIS does not efficiently suppress KRASV12-induced transformation in the context of the simultaneous occurrence of telomerase upregulation and inactivation of the p16/Rb pathway.

Andrographolide regulates phosphorylated p38 mitogen-activated protein kinases and phosphorylated ERK expression in spinal cord and reduces postoperative pain in rats

Andrographolide (Andro) is one of the medically important phytocompounds that has multiple biological properties such as anti-biotic, analgesic, anti-diabetic, anti-viral activities. Recently, andro has gained interest of various clinicians in management of physiological and neuropathic pain mechanisms. In the current study, we analyzed the effect of andro in downregulation of phosphorylated MAPK (p38) and ERK that help in relieving the post-operative pain in rats. Open incisional wound was made in the hind paw of Sprague-Dawley rats and were segregated as control animals (saline) and andro injected animals (0.5 mg/kg of bodyweight). Spinal cord sample was collected 4 days after surgery to study the p38 & pERK expressions by immunofluorescence. Reduction of p38 (∼50%) & pERK (∼30%) expressions was observed in the dorsal funiculus and dorsal horn region of spinal cord, respectively, obtained from animals injected with andro compared to control rats. This suggested that, post-operative pain was relieved in the andro injected animals compared to control rats injected with saline, possibly through down regulated expression of these kinases at the respective regions in spinal cord. In conclusion, andro has shown the potential to relieve the surgical pain by reduction of p38 & pERK expression which could be supportive for patients in pre- and post-operative care.

Sustained O‐GlcNAcylation causes ERK Signal and APP Amplification

Activation of ERK (Extra-cellular Regulated Kinase) signal plays a major role in metabolic diseases such as cancer and Alzheimer's. Ingenuity Pathway Analysis (IPA) of RNA-Seq data from long-term O-GlcNAcase (OGA) enzyme inhibitor- Thiamet-G (TMG) treated SH-SY5Y (neuroblastoma) cells, which elevates O-GlcNAc levels, indicated ERK Signaling as a top upregulated pathway. O-GlcNAcylation consists of the addition of a single N-acetyl-glucosamine residue (GlcNAc) to specific serine/threonine residues of proteins by the enzyme OGT (O-GlcNAc Transferase) and the removal of O-GlcNAc is catalyzed by the enzyme OGA. O-GlcNAc is highly abundant and is known to modulate kinase activity. Thus, to further investigate the mechanism by which O-GlcNAcylation activates ERK, we did a serum reactivation time-course and found that there is an amplification in ERK phosphorylation after long-term TMG treatment in HeLa and SH-SY5Y cells. Next, TGF-β stimulation that specifically activates ERK signaling also indicated an increase in ERK phosphorylation with long-term TMG treatment, confirming a robust ERK activation with TMG treatment. ERK phosphorylation oscillates with time after mitogen activation; therefore, we wanted to see if TMG treatment influenced ERK oscillation for a period of 24 hours. Serum reactivation revealed an oscillation in phosphorylated-ERK expression within a 24-hour period, where it reaches a maximum at 4 hours and slowly decreases at 8,12 and 24 hours upon long-term TMG treatment. Next, we wanted to further evaluate the mechanism by which TMG treatment increases ERK phosphorylation. Of note, we did not measure any O-GlcNAcylation on ERK; hence, we probed the expression of activated mitogen activated protein kinase-kinase (phosphorylated MEK) which phosphorylates ERK and Dual specificity phosphatases (DUSPs), which dephosphorylate ERK. Interestingly, serum reactivation time course showed an increase in both phosphorylated-MEK and total DUSP4 with TMG treatment. In addition to the use of pharmacological OGA inhibitor, we measured the effect of OGT knock-down (KD) and OGA KD on ERK signaling in SH-SY5Y and HeLa cells. There was amplification in ERK phosphorylation even after OGT knock-down (OGT KD) which lowers the level of O-GlcNAcylation, in contrast to long-term TMG treatment which increases O-GlcNAcylation. These data suggest that the cycling of the modification on and off substrates is key to ERK pathway regulation. Recent research also shows APOE4 stimulates the transcription of amyloid precursor protein (APP) via a non-canonical ERK signaling pathway, leading to increased amyloid-beta secretion in Alzheimer's disease pathogenesis. Therefore, we wanted to see if there is an increase in the expression of APP in the long-term TMG treated SH-SY5Y cells after serum reactivation, and we saw an increase in APP expression correlating with amplification of ERK signaling. Further evaluation of these molecular mechanisms to elucidate how O-GlcNAcylation amplifies ERK signaling and understand if O-GlcNAcylation and ERK activation work together to increase the transcription of APP are necessary.

ERK-Directed Phosphorylation of mGlu5 Gates Methamphetamine Reward and Reinforcement in Mouse.

Methamphetamine (MA) is a highly addictive psychomotor stimulant drug. In recent years, MA use has increased exponentially on a global scale, with the number of MA-involved deaths reaching epidemic proportions. There is no approved pharmacotherapy for treating MA use disorder, and we know relatively little regarding the neurobiological determinants of vulnerability to this disease. Extracellular signal-regulated kinase (ERK) is an important signaling molecule implicated in the long-lasting neuroadaptations purported to underlie the development of substance use disorders, but the role for this kinase in the propensity to develop addiction, particularly MA use disorder, is uncharacterized. In a previous MA-induced place-conditioning study of C57BL/6J mice, we characterized mice as MA-preferring, -neutral, or -avoiding and collected tissue from the medial prefrontal cortex (mPFC). Using immunoblotting, we determined that elevated phosphorylated ERK expression within the medial prefrontal cortex (mPFC) is a biochemical correlate of the affective valence of MA in a population of C57BL/6J mice. We confirmed the functional relevance for mPFC ERK activation for MA-induced place-preference via site-directed infusion of the MEK inhibitor U0126. By contrast, ERK inhibition did not have any effect upon MA-induced locomotion or its sensitization upon repeated MA treatment. Through studies of transgenic mice with alanine point mutations on T1123/S1126 of mGlu5 that disrupt ERK-dependent phosphorylation of the receptor, we discovered that ERK-dependent mGlu5 phosphorylation normally suppresses MA-induced conditioned place-preference (MA-CPP), but is necessary for this drug’s reinforcing properties. If relevant to humans, the present results implicate individual differences in the capacity of MA-associated cues/contexts to hyper-activate ERK signaling within mPFC in MA Use Disorder vulnerability and pose mGlu5 as one ERK-directed target contributing to the propensity to seek out and take MA.

Analgesic Efficacy of a Combination of Fentanyl and a Japanese Herbal Medicine "Yokukansan" in Rats with Acute Inflammatory Pain.

Background: Fentanyl can induce acute opioid tolerance and postoperative hyperalgesia when administered at a single high dose; thus, this study examined the analgesic efficacy of a combination of fentanyl and Yokukansan (YKS). Methods: Rats were divided into control, formalin-injected (FOR), YKS-treated+FOR (YKS), fentanyl-treated+FOR (FEN), and YKS+FEN+FOR (YKS+FEN) groups. Acute pain was induced via subcutaneous injection of formalin into the paw. The time engaged in pain-related behavior was measured. Results: In the early (0–10 min) and intermediate (10–20 min) phases, pain-related behavior in the YKS+FEN group was significantly inhibited compared with the FOR group. In the late phase (20–60 min), pain-related behavior in the FEN group was the longest and significantly increased compared with the YKS group. We explored the influence on the extracellular signal-regulated kinase (ERK) pathway in the spinal cord, and YKS suppressed the phosphorylated ERK expression, which may be related to the analgesic effect of YKS in the late phase. Conclusions: These findings suggest that YKS could reduce the use of fentanyl and combined use of YKS and fentanyl is considered clinically useful.

Effects of caffeine on ethanol-elicited place preference, place aversion and ERK phosphorylation in CD-1 mice.

Epidemiological studies indicate a rise in the combined consumption of caffeinated and alcoholic beverages, which can lead to increased risk of alcoholic-beverage overconsumption. However, the effects of the combination of caffeine and ethanol in animal models related to aspects of drug addiction are still underexplored. To characterize the pharmacological interaction between caffeine and ethanol and establish if caffeine can affect the ability of ethanol (2 g/kg) to elicit conditioned place preference and conditioned place aversion, we administered caffeine (3 or 15 mg/kg) to male CD-1 mice before saline or ethanol. Moreover, we determined if these doses of caffeine could affect ethanol (2 g/kg) elicited extracellular signal-regulated kinase phosphorylation in brain areas, nucleus accumbens, bed nucleus of stria terminalis, central nucleus of the amygdala, and basolateral amygdala, previously associated with this type of associative learning. In the place-conditioning paradigm, caffeine did not have an effect on its own, whereas ethanol elicited significant conditioned-place preference and conditioned-place aversion. Caffeine (15 mg/kg) significantly prevented the acquisition of ethanol-elicited conditioned-place preference and, at both doses, also prevented the acquisition of ethanol-elicited conditioned-place aversion. Moreover, both doses of caffeine also prevented ethanol-elicited extracellular signal-regulated kinase phosphorylation expression in all brain areas examined. The present data indicate a functional antagonistic action of caffeine and ethanol on associative learning and extracellular signal-regulated kinase phosphorylation after an acute interaction. These results could provide exciting grounds for further studies, also in a translational perspective, of their pharmacological interaction modulating other processes involved in drug consumption and addiction.

SOS1 Gain-of-Function Variants in Dilated Cardiomyopathy.

Dilated cardiomyopathy (DCM) is a genetically heterogeneous cardiac disease characterized by progressive ventricular enlargement and reduced systolic function. Here, we report genetic and functional analyses implicating the rat sarcoma signaling protein, SOS1 (Son of sevenless homolog 1), in DCM pathogenesis. Exome sequencing was performed on 412 probands and family members from our DCM cohort, identifying several SOS1 variants with potential disease involvement. As several lines of evidence have implicated dysregulated rat sarcoma signaling in the pathogenesis of DCM, we assessed functional impact of each variant on the activation of ERK (extracellular signal-regulated kinase), AKT (protein kinase B), and JNK (c-Jun N-terminal kinase) pathways. Relative expression levels were determined by Western blot in HEK293T cells transfected with variant or wild-type human SOS1 expression constructs. A rare SOS1 variant [c.571G>A, p.(Glu191Lys)] was found to segregate alongside an A-band TTN truncating variant in a pedigree with aggressive, early-onset DCM. Reduced disease severity in the absence of the SOS1 variant suggested its potential involvement as a genetic risk factor for DCM in this family. Exome sequencing identified 5 additional SOS1 variants with potential disease involvement in 4 other families [c.1820T>C, p.(Ile607Thr); c.2156G>C, p.(Gly719Ala); c.2230A>G, p.(Arg744Gly); c.2728G>C, p.(Asp910His); c.3601C>T, p.(Arg1201Trp)]. Impacted amino acids occupied a number of functional domains relevant to SOS1 activity, including the N-terminal histone fold, as well as the C-terminal REM (rat sarcoma exchange motif), CDC25 (cell division cycle 25), and PR (proline-rich) tail domains. Increased phosphorylated ERK expression relative to wild-type levels was seen for all 6 SOS1 variants, paralleling known disease-relevant SOS1 signaling profiles. These data support gain-of-function variation in SOS1 as a contributing factor to isolated DCM.

NCTD elicits proapoptotic and antiglycolytic effects on colorectal cancer cells via modulation of Fam46c expression and inhibition of ERK1/2 signaling.

Colorectal cancer is a digestive tract malignancy and the third leading cause of cancer-related mortality worldwide. Norcantharidin (NCTD), the demethylated form of cantharidin, has been reported to possess anticancer properties. Family-with-sequence-similarity-46c (Fam46c), a non-canonical poly(A) polymerase, has been reported to be critical in NCTD-mediated effects in numerous types of cancer, including hepatoma. In the current study, it was found that Fam46c expression was reduced in colorectal cancer tissues and cells. Treatment with NCTD was observed to significantly enhance apoptosis and inhibit glycolysis in colorectal cancer cells. In addition, Fam46c and cleaved caspase 3 expression levels were found to be increased in response to NCTD treatment, in contrast to tumor-specific pyruvate kinase M2 and phosphorylated ERK expression, which was reduced. Importantly, overexpression of Fam46c exerted similar effects as NCTD treatment on the apoptosis and glycolysis of colorectal cancer cells, whereas Fam46c knockdown strongly attenuated the effect of NCTD. Moreover, epidermal growth factor, which acts as an agonist of ERK1/2 signaling, weakened the effects of NCTD on colorectal cancer cells. Taken together, the results indicated that NCTD promotes apoptosis and suppresses glycolysis in colorectal cancer cells by possibly targeting Fam46c and inhibiting ERK1/2 signaling, hence suggesting that Fam46c may act as a tumor suppressor in colorectal cancer. Thus, the present study identified a novel therapeutic target of NCTD in the clinical treatment of colorectal cancer.

HECTD1 regulates the expression of SNAIL: Implications for epithelial‑mesenchymal transition.

As a transcription factor, SNAIL plays a crucial role in embryonic development and cancer progression by mediating epithelial-mesenchymal transition (EMT); however, post-translational modifications, such as ubiquitination, which control the degradation of SNAIL have been observed to affect its functional role in EMT. In a previous study by the authors, it was demonstrated that the HECT domain E3 ubiquitin ligase 1 (HECTD1) regulated the dynamic nature of adhesive structures. In the present study, HECTD1 was observed to interact with SNAIL and regulate its stability through ubiquitination, and the knockdown of HECTD1 increased the expression levels of SNAIL. HECTD1 was discovered to contain putative nuclear localization and export signals that facilitated its translocation between the cytoplasm and nucleus, a process regulated by epidermal growth factor (EGF). Treatment with leptomycin B resulted in the nuclear retention of HECTD1, which was associated with the loss of SNAIL expression. The knockdown of HECTD1 in HeLa cells increased cell migration and induced a mesenchymal phenotype, in addition to through increased phosphorylated ERK expression levels. Under hypoxic conditions, HECTD1 expression levels were decreased by microRNA (miRNA or miR)-210. Upon the observation of genetic abnormalities in the HECTD1 gene in cervical cancer specimens, it was observed that the decreased expression levels of HECTD1 were significantly associated with a poor patient survival. Thus, it was hypothesized that HECTD1 may regulate EMT through the hypoxia/hypoxia inducible factor 1α/miR-210/HECTD1/SNAIL signaling pathway and the EGF/EGF receptor/HECTD1/ERK/SNAIL signaling pathway in cervical cancer. On the whole, the data of the present study indicated that HECTD1 serves as an E3 ubiquitin ligase to mediate the stability of SNAIL proteins.

Vascular endothelial growth factor enhances profibrotic activities through modulation of calcium homeostasis in human atrial fibroblasts

Vascular endothelial growth factor (VEGF), a pivotal activator of angiogenesis and calcium (Ca2+) signaling in endothelial cells, was shown to increase collagen production in atrial fibroblasts. In this study, we evaluated whether VEGF may regulate Ca2+ homeostasis in atrial fibroblasts and contribute to its profibrogenesis. Migration, and proliferation analyses, patch-clamp assay, Ca2+ fluorescence imaging, and western blotting were performed using VEGF-treated (300 pg/mL or 1000 pg/mL) human atrial fibroblasts with or without coadministration of Ethylene glycol tetra-acetic acid (EGTA, 1 mmol/L), or KN93 (a Ca2+/calmodulin-dependent protein kinase II [CaMKII] inhibitor, 10 μmol/L). VEGF (1000 pg/mL) increased migration, myofibroblast differentiation, pro-collagen type I, pro-collagen type III production, and phosphorylated VEGF receptor 1 expression of fibroblasts. VEGF (1000 pg/mL) increased the nonselective cation current (INSC) of transient receptor potential (TRP) channels and potassium current of intermediate-conductance Ca2+-activated K+ (KCa3.1) channels thereby upregulating Ca2+ entry. VEGF upregulated phosphorylated ERK expression. An ERK inhibitor (PD98059, 50 μmol/L) attenuated VEGF-activated INSC of TRP channels. The presence of EGTA attenuated the profibrotic effects of VEGF on pro-collagen type I, pro-collagen type III production, myofibroblast differentiation, and migratory capabilities of fibroblasts. VEGF upregulated the expression of phosphorylated CaMKII in fibroblasts, which was attenuated by EGTA. In addition, KN93 reduced VEGF-increased pro-collagen type I, pro-collagen type III production, myofibroblast differentiation, and the migratory capabilities of fibroblasts. In conclusion, we found that VEGF increases atrial fibroblast activity through CaMKII signaling by enhancing Ca2+ entry. Our findings provide benchside evidence leading to a potential novel strategy targeting atrial myopathy and arrhythmofibrosis.

Inhibition of Bcl-2 Synergistically Enhances the Antileukemic Activity of Midostaurin and Gilteritinib in Preclinical Models of FLT3-Mutated Acute Myeloid Leukemia.

To investigate the efficacy of the combination of the FLT3 inhibitors midostaurin or gilteritinib with the Bcl-2 inhibitor venetoclax in FLT3-internal tandem duplication (ITD) acute myeloid leukemia (AML) and the underlying molecular mechanism. Using both FLT3-ITD cell lines and primary patient samples, Annexin V-FITC/propidium iodide staining and flow cytometry analysis were used to quantify cell death induced by midostaurin or gilteritinib, alone or in combination with venetoclax. Western blot analysis was performed to assess changes in protein expression levels of members of the JAK/STAT, MAPK/ERK, and PI3K/AKT pathways, and members of the Bcl-2 family of proteins. The MV4-11-derived xenograft mouse model was used to assess in vivo efficacy of the combination of gilteritinib and venetoclax. Lentiviral overexpression of Mcl-1 was used to confirm its role in cell death induced by midostaurin or gilteritinib with venetoclax. Changes of Mcl-1 transcript levels were assessed by RT-PCR. The combination of midostaurin or gilteritinib with venetoclax potently and synergistically induces apoptosis in FLT3-ITD AML cell lines and primary patient samples. The FLT3 inhibitors induced downregulation of Mcl-1, enhancing venetoclax activity. Phosphorylated-ERK expression is induced by venetoclax but abolished by the combination of venetoclax with midostaurin or gilteritinib. Simultaneous downregulation of Mcl-1 by midostaurin or gilteritinib and inhibition of Bcl-2 by venetoclax results in "free" Bim, leading to synergistic induction of apoptosis. In vivo results show that gilteritinib in combination with venetoclax has therapeutic potential. Inhibition of Bcl-2 via venetoclax synergistically enhances the efficacy of midostaurin and gilteritinib in FLT3-mutated AML.See related commentary by Perl, p. 6567.

Abstract GMM-017: UPREGULATION OF MIR-328 CONTRIBUTES TO OVARIAN CANCER STEM CELL MAINTENANCE BY DOWNREGULATING DDB2

Abstract Cancer stem cells (CSCs) are a particular subpopulation of cells that are characterized by self renewal, differentiation and enhanced tumorigenicity. They are responsible for tumor metastasis, relapse and development of drug resistance. Thus, eradication of CSCs is essential for improved patient prognosis. Micro RNAs are a group of small non-coding, endogenous RNAs that are found to regulate cancer stem cell characteristics by binding to mRNA in a sequence specific manner. In ovarian cancers, a wide array of Micro RNAs have been found to show differential expression of which miR328-3p deserves special mention. In this study, a Micro RNA Nanostring profile analysis reveals a significant upregulation of miR-328-3p in ovarian cancer stem cells isolated from both ovarian cancer cell lines and primary ovarian tumors as compared to their corresponding bulk cells. Moreover, it was found that inhibition of miR-328 limited the CSC population in ovarian cancer cells whereas overexpression of miR-328 enriched the CSC population, thus accounting for miR-328 as an onco-miRNA. The upregulation of miR-328 not only increased the percentage of ALDH+ cells in ovarian cancer bulk cells, but also increased the tumorigenicity and sphere formation ability. This was supported by the orthotopic ovarian xenograft assay. Further investigation revealed that reduced phosphorylation of Erk in ovarian cancer stem cells owing to reduced levels of Reactive Oxygen species (ROS) could be a prospective mechanism behind elevated miR328 expression and maintenance of CSC characteristics. Inhibition of phosphorylated Erk expression in ovarian cancer bulk cells by use of commercially available Erk inhibitor, U0126, led to a significant increase in miR328 expression. Simultaneously, upregulation of phosphorylated Erk in ovarian cancer stem cells not only reduced miR328 expression, but also displayed a significant reduction in expression of cancer stem cell markers (Oct4, Sox2, Nanog), sphere formation ability and tumorigenesis. We obtained a similar trend of results on regulating the expression of pErk by use of Reactive Oxygen Species to ovarian cancer cells. These data further helped us confirm our speculation that reduced ROS promotes the maintenance of CSCs characteristics through inactivation of Erk signalling pathway. Besides, we also identified DDB2 as a direct target of miR328. Our previous findings demonstrate that DDB2 is able to limit ovarian CSC population by disrupting their self renewal capacity. Thus, we conclude that elevated miR328 in ovarian CSCs, resulting from inactivated Erk1/2 activity, is responsible for maintenance of stemness by inhibition of DDB2 expression. Targeting miR-328 could therefore be a novel therapeutic strategy to eradicate CSCs in ovarian cancer. Citation Format: Amit Kumar Srivastava, Tiantian Cui, Ananya Banerjee, Chunhua Han, Shurui Cai, Lu Liu, Dayong Wu1, Ri Cui, Zaibo Li, Xiaoli Zhang, Guozhen Xie, Selvendiran Karuppalyah, Adam Karpf, Jinsong Liu, David Cohn, Qi-En Wang. UPREGULATION OF MIR-328 CONTRIBUTES TO OVARIAN CANCER STEM CELL MAINTENANCE BY DOWNREGULATING DDB2 [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr GMM-017.

15-Lipoxygenase 1 in nasal polyps promotes CCL26/eotaxin 3 expression through extracellular signal-regulated kinase activation

15-Lipoxygenase 1 (15LO1) is expressed in airway epithelial cells in patients with type 2-high asthma in association with eosinophilia. Chronic rhinosinusitis with nasal polyps (CRSwNP) is also associated with type 2 inflammation and eosinophilia. CCL26/eotaxin 3 has been reported to be regulated by 15LO1 in lower airway epithelial cells. However, its relation to 15LO1 in patients with CRSwNP or mechanisms for its activation are unclear. We sought to evaluate 15LO1 and CCL26 expression in nasal epithelial cells (NECs) from patients with CRSwNP and healthy control subjects (HCs) and determine whether 15LO1 regulates CCL26 in NECs through extracellular signal-regulated kinase (ERK) activation. 15LO1, CCL26, and phosphorylated ERK were evaluated in NECs from patients with CRSwNP and HCs. 15LO1/CCL26 and CCL26/cytokeratin 5 were colocalized by means of immunofluorescence. IL-13-stimulated NECs were cultured at an air-liquid interface with or without 15-lipoxygenase 1 gene (ALOX15) Dicer-substrate short interfering RNAs (DsiRNA)transfection, a specific 15LO1 enzymatic inhibitor, and 2 ERK inhibitors. Expression of 15LO1 and CCL26 mRNA and protein was analyzed by using quantitative RT-PCR, Western blotting, and ELISA. 15LO1 expression was increased in nasal polyp (NP) epithelial cells compared with middle turbinate epithelial cells from patients with CRSwNP and HCs. 15LO1 expression correlated with CCL26 expression and colocalized with CCL26 expression in basal cells of the middle turbinate and NPs from patients with CRSwNP. In primary NECs invitro, IL-13 induced 15LO1 and CCL26 expression. 15LO1 knockdown and inhibition decreased IL-13-induced ERK phosphorylation and CCL26 expression. ERK inhibition (alone) similarly decreased IL-13-induced CCL26. Phosphorylated ERK expression was increased in NECs from CRSwNP subjects and positively correlated with both 15LO1 and CCL26 expression. 15LO1 expression is increased in NP epithelial cells and contributes to CCL26 expression through ERK activation. 15LO1 could be considered a novel therapeutictarget for CRSwNP.

Effect of receptor for hyaluronan-mediated motility inhibition on radiosensitivity of lung adenocarcinoma A549 cells.

BackgroundReceptor for hyaluronan-mediated motility (RHAMM), one of the major hyaluronic acid (HA) receptors, is upregulated in several forms of cancer and is a poor prognostic factor for non-small cell lung cancer (NSCLC) adenocarcinoma. RHAMM is also a potential therapeutic target for inhibition of tumor metastasis in NSCLC. However, its role in the radiosensitivity of NSCLC has yet to be determined. The aim of this study was to examine the inhibitory effect of RHAMM on the radiosensitivity of lung adenocarcinoma cell line A549 and its potential mechanism.MethodsExpression of the RHAMM gene in NSCLC cell lines A549 and H460 was detected using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Colony formation assays were used to analyze radiosensitivity of the two cell lines. Transfection with small interfering (si) RNA was used to inhibit expression of the RHAMM gene in the A549 cell line. A cell counting kit assay was used to analyze the cell proliferation rate; flow cytometry was used to evaluate the cell apoptosis and cell cycle; and Western blot was used to investigate the expression of phosphorylated-extracellular signal-regulated kinase 1/2 (p-ERK1/2) and ERK1/2 proteins.ResultsExpression of the RHAMM gene was negatively correlated with the radiosensitivity of NSCLC cell lines; inhibition of RHAMM gene expression enhanced the radiosensitivity of A549 cells. The mechanism for this inhibition was associated with reduced proliferation, increased apoptosis induced by radiotherapy, reduced ERK1/2 phosphorylation, and regulation of the ERK1/2 signaling pathway. Inhibition was not associated with increased G2/M phase arrest.ConclusionsRHAMM is a potential target for radiosensitization of lung adenocarcinomas.