Activation Of ERK Pathways Research Articles

Upregulation of ASIC1a channels in an in vitro model of Fabry disease

Neuropathic pain is one of the key features of the classical phenotype of Fabry disease (FD). Acid sensing ion channels (ASICs) are H+-gated cation channels, which belong to the epithelial sodium channel/DeGenerin superfamily, sensitive to the diuretic drug Amiloride. Molecular cloning has identified several distinct ASIC subunits. In particular the ASIC1a subunit has been associated to pain and its upregulation has been documented in animal models of pain. We analyzed the expression of ASIC1a channels in cellular models that mimic the accumulation of glycosphingolipids in FD (FD-GLs) like Gb3, and LysoGb3. We used mouse primary neurons from brain cortex and hippocampus -supraspinal structures that accumulate FD-GLs-, as well as HEK293 cells. Incubation with Gb3, lysoGb3 and the inhibitor (1-deoxy-galactonojirymicin, DJG) of the enzyme α-galactosidase A (Gla) lead to the upregulation of ASIC1a channels. In addition, activation of ASIC1a results in the activation of the MAPK ERK pathway, a signaling pathway associated with pain. Moreover, accumulation of glycosphingolipids results in activation of ERK, an effect that was prevented by blocking ASIC1a channels with the specific blocker Psalmotoxin. Our results suggest that FD-GLs accumulation and triggering of the ERK pathway via ASIC channels might be involved in the mechanism responsible for pain in FD, thus providing a new therapeutic target for pain relief treatment.

Abstract 1907: Src-Met signaling confers apoptosis resistance to EGFR and PI3K co-targeting independently of AKT and ERK pathway activation in head and neck cancer

Abstract Purpose: Molecularly targeted agents will play a major role in the next generation of personalized cancer therapies. However, intrinsic and acquired resistance to targeted agents poses challenges to the success of such treatments. The purpose of the current study was to investigate the intrinsic resistance of head and neck cancer (HNC) cell lines to apoptosis induced by the combination of EGFR inhibitor erlotinib and PI3K inhibitor BKM120. Methods: In a panel of 10 malignant and 1 premalignant HNC cell lines, we evaluated cell growth inhibition (by SRB assay), IC50, combination index, dose reduction index (by CalcuSyn Software), and apoptosis (by Annexin-V staining). Protein expression levels were measured by Western blotting. Small molecule inhibitors and siRNA-mediated knockdown strategies were used to inactivate and shut down the expression of the relevant proteins, respectively. Results: We have reported previously that single targeting of EGFR with erlotinib or PI3K with BKM120 (pan-PI3K inhibitor) suppressed cellular growth without inducing significant apoptosis. Co-targeting EGFR and PI3K demonstrated in vitro synergy in all except the JHU022 cell line and more effectively inhibited HNC xenograft growth in vivo. The combination of erlotinib and BKM120 induced variable apoptosis: some cell lines were very sensitive (MDA686TU, MDA686LN, 93-VU-147T), some moderately (Fadu, SqCCy1, MDA1483, UPCI:SCC090) and others were resistant (UD-SCC2, MSK-LEUK1, JHU022) to apoptosis induction. We found that the resistant cell line JHU022 expressed very high levels of p-Met (Y1234/1235). Inhibition of Met with small molecule inhibitor crizotinib or siRNA rendered this cell line highly sensitive to apoptosis induced by the combination of erlotinib and BKM120. Although inhibition of hepatocyte growth factor (HGF), the only known ligand for Met, with soluble neutralization antibody had no effect on Met phosphorylation, inhibition of Src family kinases (SFK) with dasatinib abolished Met phosphorylation at Y1234/1235. Dasatinib also made this cell line sensitive to apoptosis. Ablation of c-Src with siRNA had similar effects on p-Met (Y1234/1235) and apoptosis. Interestingly, inhibition of Met had no effects on AKT and ERK phosphorylation, two critical downstream pathways of receptor tyrosine kinases including Met. Conclusions: Our results strongly suggest that co-targeting of EGFR and PI3K has synergistic activity, is effective in vivo, and induces apoptosis of some HNC cell lines. Some resistant cell lines express a high level of p-Met as a downstream target of SFK, which confers resistance of these cells to EGFR and PI3K co-targeting. Finally, Met confers apoptosis resistance independently of AKT and ERK pathway activation. Citation Format: Abu S. Anisuzzaman, Abedul Haque, Zhuo G. Chen, Dong M. Shin, A.R.M. Ruhul Amin. Src-Met signaling confers apoptosis resistance to EGFR and PI3K co-targeting independently of AKT and ERK pathway activation in head and neck cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1907.

CircHIPK3 Promotes Metastasis of Gastric Cancer via miR-653-5p/miR-338-3p-NRP1 Axis Under a Long-Term Hypoxic Microenvironment.

As a vital feature of the microenvironment, hypoxia, especially long-term hypoxia, is known to promote metastasis and lead to poor prognosis in solid tumors. Circular RNAs (circRNAs) participate in important processes of cell proliferation and metastasis in cancers. However, the contribution of circRNAs to metastasis under long-term hypoxia is obscure. In this study, we aim to explore specific functions of circHIPK3 in long-term hypoxia-promoting metastasis of gastric cancer (GC). The hypoxic resistant gastric cancer (HRGC) cell lines we established previously, which were tolerant to 2% O2 conditions, were used as the long-term hypoxia model. We found that circHIPK3 was upregulated by HIF-2α in HRGC cells, and circHIPK3 facilitated the migration and invasion ability of HRGC cells. Further investigation proved that circHIPK3 promoted metastasis of HRGC cells directly by interacting with miR-653-5p and miR-338-3p to relieve the suppression of neuropilin 1 (NRP1), resulting in the activation of downstream ERK and AKT pathways. Our study identified oncogene functions of circHIPK3 under a long-term hypoxic microenvironment and the possibility of using circHIPK3 as a potential biomarker of long-term hypoxia in GC. In conclusion, circHIPK3 could promote GC metastasis via the miR-653-5p/miR-338-3p-NRP1 axis under a long-term hypoxic microenvironment.

Abstract 642: TRAF2 mediates sensitivity to immunomodulatory drugs via NF-κB and ERK signaling in myeloma cells

Abstract The development of novel agents including immunomodulatory drugs (IMiDs) lenalidomide (Len) and pomalidomide (Pom) has improved patient outcome in multiple myeloma (MM); however, acquired resistance to IMiDs commonly underlies relapse of disease. Previous studies show that IMiDs bind to the CRL4CRBN ubiquitin ligase cereblon (CRBN) and promote proteasomal degradation of IKZF1 and IKZF3 followed by downregulation of c-Myc and IRF4, resulting in MM cell growth inhibition. Importantly, some MM cells show resistance to IMiDs despite harboring high CRBN expression levels. Here we utilize genome-wide CRISPR-Cas9 knockout (KO) screening to identify novel mechanisms mediating resistance to IMiDs in MM. Among positively selected sgRNAs, we found three different sgRNAs targeting TRAF2 were enriched after IMiDs treatment. Individually KO of TRAF2 induced significant resistance of MM cells to Pom and Len treatment independent of CRBN-IKZF1/3 axis. TRAF2 KO cells show hyperactivation of the non-canonical NF-κB pathway and p52 KO re-sensitizes the cells to IMiDs treatment, suggesting that TRAF2 predominantly suppresses non-canonical NF-κB pathway, maintaining sensitivity to IMiDs in MM cells. We next examined the relevance of TRAF2 downregulation in the context of the bone marrow (BM) microenvironment. Co-culture of MM cells with either BM stromal cells (BMSCs) or culture supernatants (BM-CS) confers resistance to IMiDs. Importantly, TRAF2 in MM cells is downregulated by BMSCs and BM-CS co-culture. TNF-α was elevated in BM-CS, and exogenous TNF-α triggered decreased TRAF2 expression in MM cells, associated with resistance to IMiDs. Taken together, these results suggest that IMiDs resistance in the BM microenvironment may be mediated, at least in part, by TRAF2 downregulation. As observed in the TRAF2 KO, exogenous TNF-α hyper-activated the noncanonical NF-κB pathway without affecting CRBN expression, further confirming a major role of TNF-α mediating protection against IMiDs-driven cytotoxicity. Interestingly, we also found that TNF-α induces IMiDs resistance even in p52 KO cells, implicating additional mediating IMiDs resistance. Indeed, we found that ERK pathway was highly activated in TRAF2 KO MM cells, and confirmed that both BM-CS and TNF-α induced activation of ERK pathway in MM cells. Moreover, constitutive overexpression of phosphorylated-ERK-2 significantly reduced IMiDs-induced cytotoxicity. Finally, our in vitro and in vivo murine xenograft studies show that MEK inhibitor AZD6244 overcomes IMiDs resistance in TRAF2 KO MM cells or in BM-CS treated wild-type MM cells, indicating that MEK inhibitor can overcome IMiDs resistance in the BM microenvironment. Taken together, our studies therefore identify TRAF2 as a mediator of IMIDs sensitivity in MM cells independent of CRBN-IKZF1/3, and validate the therapeutic potential of MEK/ERK inhibitor to overcome IMiDs resistance. Citation Format: Jiye Liu, Teru Hideshima, Lijie Xing, Su Wang, Wenrong Zhou, Daisuke Ogiya, Kenneth Wen, Yu-Tzu Tai, Yong Cang, Kenneth C. Anderson. TRAF2 mediates sensitivity to immunomodulatory drugs via NF-κB and ERK signaling in myeloma cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 642.

Abstract 5969: Novel tumor suppressor FRG1 reduces cancerous properties of breast cancer cell lines by regulating ERK pathway, irrespective of molecular subtypes

Abstract Breast cancer is the most leading cause of cancer related deaths in women. Although combination of improved earlier detection and effective adjuvant therapies has dropped the breast cancer mortality, still different molecular subtypes, acquisition of quick drug resistance make it difficult to combat against this deadliest disease. So, an urging has to be compelled to inspect the additional players which are still unexplored in breast cancer. The focus of our lab is to find out novel molecules regulating cancer development and progression. In this process, we discovered the role of FRG1 in tumorigenesis. FRG1 (FSHD Region Gene 1) is an evolutionary conserved gene which is reported to have role in muscle development, angiogenesis, RNA biogenesis, F actin bundling. In our previous study, we first time reported the association of FRG1 with cancer. In this study, our key interest is to explore the molecular mechanism behind the differential expression of FRG1 in breast cancer. Here we started our study by exploring the expression of FRG1 in patient samples where we found reduced FRG1 expression in around 87% of breast cancer patients compared to their normal counterparts. Further, we also observed that FRG1 level was lower in triple negative breast cancer (TNBC) patients compared to Luminal A (hormone responsive) type. Similar trends of FRG1 expression was observed in Luminal A (ER+) cell line MCF7 and TNBC cell line MDAMB231. Therefore, we decided to evaluate the effect of perturbed FRG1 level in two breast cancer cell lines MCF7 (Luminal A) and MDAMB231 (TNBC). Knockdown of FRG1 in MCF7 cells led to increased cell proliferation, migration, colony formation and invasion; whereas ectopic FRG1 expression in MDAMB231 showed a reduction in these properties. Next, we investigated the signaling molecules associated with FRG1 and affected the above mentioned cellular properties. We observed low FRG1 expression in breast cancer cell line MCF7 resulted into activation of MEK/ERK pathway. Various EMT molecules like snail, slug, twist and tumor promoting cytokines such as IL1/8, growth factor like PDGFα/β was found to be up regulated in vitro due to reduction of FRG1. Similarly, FRG1 overexpression resulted into considerable downregulation of these EMT markers and cytokines. Furthermore, we found a rescue of these EMT molecules when ERK was inhibited or activated which confirmed that reduction in FRG1 in breast cancer cell lines activated ERK pathway which in turn upregulated the EMT molecules. Interestingly our data showed similar kind of trends irrespective of molecular subtypes of breast cancer cell lines. Also our study showed an activation of estrogen signaling in breast cancer due to the unliganded mechanism of estrogen receptor by FRG1. Thus, our study first time suggests that FRG1 level is important for breast cancer progression irrespective of molecular subtypes; but further understanding and characterization of signalling mechanisms and interacting partners are required to conclusively comment on its tumor suppressive role. Citation Format: Bratati Mukherjee, Ankit Tiwari, Manjusha Dixit. Novel tumor suppressor FRG1 reduces cancerous properties of breast cancer cell lines by regulating ERK pathway, irrespective of molecular subtypes [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5969.

Autocrine CCL5 Effect Mediates Trastuzumab Resistance by ERK Pathway Activation in HER2-Positive Breast Cancer.

HER2-positive breast cancer is currently managed with chemotherapy in combination with specific anti-HER2 therapies, including trastuzumab. However, a high percentage of patients with HER2-positive tumors do not respond to trastuzumab (primary resistance) or either recur (acquired resistance), mostly due to molecular alterations in the tumor that are either unknown or undetermined in clinical practice. Those alterations may cause the tumor to be refractory to treatment with trastuzumab, promoting tumor proliferation and metastasis. Using continued exposure of a HER2-positive cell line to trastuzumab, we generated a model of acquired resistance characterized by increased expression of several cytokines. Differential gene expression analysis indicated an overexpression of 15 genes, including five different chemokines, and highlighting CCL5/RANTES as the most overexpressed one. Functional studies, either by in vitro gene silencing or by in vitro and in vivo pharmacologic inhibition of the CCL5/CCR5 interaction with maraviroc, confirmed that CCL5 overexpression was implicated in acquired resistance to trastuzumab, which was mediated by ERK activation. In patient samples, increased CCL5 expression significantly correlated with lower rates of complete response after neoadjuvant therapy, confirmed by detection of high serum CCL5 levels by ELISA. Overexpression of CCL5 correlated with ERK phosphorylation in tumor cells and was statistically associated with worse disease-free survival and overall cancer survival in patients with early HER2-positive breast cancer.

Abstract 255: Cardiac Ischemia and Reperfusion Injury: The Emerging Role of Sialidase Neu3

Reperfusion strategies, together with thrombolytic therapies, represent life-saving approaches to restore the blood flow in the cardiac tissue after acute myocardial infarction (AMI). However, they inevitably induce the so-called ischemia/reperfusion injury (IRI), resulting in increased cardiomyocytes damage and heart failure. In this context, many efforts have been made to clarify the molecular mechanisms involved in IRI, and the activation of pro-survival kinases, such as Akt and Erk, as well as of the hypoxia-inducible factor (HIF) has been recognized to be critical. Along this line, we discovered a novel mechanism of HIF-1α activation mediated by sialidase Neu3, which is PHDs independent, and that it increased muscle cells resistance to hypoxic stress, through the activation of Akt and Erk pathways. Moreover, an upregulation of Neu3 expression was observed under chronic hypoxia in cyanotic congenital cardiac patients.On these premises, this study aims at investigating the role of Neu3 in protecting cardiac cells against IRI. In particular, H9C2 rat cardiomyoblasts were exposed to an IRI model in vitro revealing a marked reduction in cell proliferation. This was accompanied by the modulation of Neu3 that was characterized by its progressive down-regulation during the ischemic phase, followed by its reactivation during reperfusion. These experiments resembled Neu3 modulation we observed in an IRI mouse model, obtained by the temporary occlusion of the LAD coronary vessel. Interestingly, overexpression of Neu3 significantly increased cardiomyoblasts resistance to IRI, both in terms of cell proliferation and resistance to apoptosis, as well as promoted HIF-1α and Akt/Erk activation. Remarkably, the treatment with Akt and Erk inhibitors completely reverted the beneficial effects mediated by Neu3 upregulation. Likewise, sialidase Neu3 inhibition reduced Akt/Erk activation, resulting in the complete loss of Neu3-mediated cardioprotection. In conclusion, our results demonstrate the role of sialidase Neu3 in counteracting the detrimental effects of IRI, calling for further studies to unveil its full potential as a therapeutic target to support current strategies to manage cardiac damage and to improve patients recover after AMI.

Blocking ERK signaling pathway lowers MMP-9 expression to alleviate brain edema after traumatic brain injury in rats

To investigate the effects of blocking the activation of ERK pathway on the expression of matrix metalloproteinase-9 (MMP-9) and the formation of cerebral edema in SD rats after brain injury. Ninety SD rats were randomly divided into 3 equal groups, including a sham-operated group, modified Feeney's traumatic brain injury model group, and ERK inhibition group where the ERK inhibitor SCH772984 (500 μg/kg) was injected via the femoral vein 15 min before brain trauma. At 2 h and 2 days after brain trauma, the permeability of blood-brain barrier was assessed by Evans blue method, the water content of the brain tissue was determined, and the phosphorylation level of ERK and the expression level of MMP-9 mRNA and protein were measured by RT-PCR and Western blotting. Compared with the sham-operated group, the rats with brain trauma exhibited significantly increased level of ERK phosphorylation at 2 h and significantly increased expression of MMP-9 mRNA and protein 2 days after the injury (P < 0.01). Treatment with the ERK inhibitor significantly decreased the phosphorylation level of ERK after the injury (P < 0.01), suppressed over-expression of MMP-9 mRNA and protein 2 days after the injury (P < 0.01). The permeability of blood-brain barrier increased significantly 2 h after brain trauma (P < 0.05) and increased further at 2 days (P < 0.01); the water content of the brain did not change significantly at 2 h (P > 0.05) but increased significantly 2 d after the injury (P < 0.01). Treatment with the ERK inhibitor significantly lowered the permeability of blood-brain barrier and brain water content after brain trauma (P < 0.01). Blocking the activation of ERK pathway significantly reduced the over-expression of MMP-9 and alleviates the damage of blood-brain barrier and traumatic brain edema, suggesting that ERK signaling pathway plays an important role in traumatic brain edema by regulating the expression of MMP-9.

CB2R induces a protective response against epileptic seizures through ERK and p38 signaling pathways

Background and purpose Epilepsy is a pivotal neurological disorder characterized by the synchronous discharging of neurons to induce momentary brain dysfunction. Temporal lobe epilepsy is the most common type of epilepsy, with seizures originating from the mesial temporal lobe. The hippocampus forms part of the mesial temporal lobe and plays a significant role in epileptogenesis; it also has a vital influence on the mental development of children. In this study, we aimed to explore the effects of CB2 receptor (CB2R) activation on ERK and p38 signaling in nerve cells of a rat epilepsy model. Materials and methods We treated Sprague-Dawley rats with pilocarpine to induce an epilepsy model and treated such animals with a CB2R agonist (JWH133) alone or with a CB2R antagonist (AM630). Nissl’s stain showed the neuron conditon in different groups. Western blot analyzed the level of p-ERK and p-p38. Results JWH133 can increase the latent period of first seizure attack and decrease the Grades IV–V magnitude ratio after the termination of SE. Nissl’s stain showed JWH133 protected neurons in the hippocampus while AM630 inhibited the functioning of CB2R in neurons. Western blot analysis showed that JWH133 decreased levels of p-ERK and p-p38, which is found at increased levels in the hippocampus of our epilepsy model. In contrast, AM630 inhibited the protective function of JWH133 and also enhanced levels of p-ERK and p-p38. Conclusions CB2R activation can induce neurons proliferation and survival through activation of ERK and p38 signaling pathways.

Discovery of new benzhydrol biscarbonate esters as potent and selective apoptosis inducers of human melanomas bearing the activated ERK pathway: SAR studies on an ERK MAPK signaling modulator, ACA-28

The recent discovery that an ERK signaling modulator [ACA-28 (2a)] preferentially kills human melanoma cell lines by inducing ERK-dependent apoptosis has generated significant interest in the field of anti-cancer therapy. In the first SAR study on 2a, here, we successfully developed candidates (2b, 2c) both of which induce more potent and selective apoptosis towards ERK-active melanoma cells than 2a, thus revealing the structural basis for inducing the ERK-dependent apoptosis and proposing the therapeutic prospect of these candidates against ERK-dependent cancers represented by melanoma.

LncRNA OIP5-AS1 promotes the malignancy of pancreatic ductal adenocarcinoma via regulating miR-429/FOXD1/ERK pathway

BackgroundPancreatic ductal adenocarcinoma (PDAC), a subtype of pancreatic cancer, is a malignant tumor with unfavorable prognosis. Despite accumulating researches have made efforts on finding novel therapeutic methods for this disease, the underlying mechanism of long non-coding RNAs (lncRNAs) remains elusive. OIP5 antisense RNA 1 (OIP5-AS1) has been reported to play important role in the occurrence and development of multiple human cancers. This study was aimed at unveiling the regulatory role of OIP5-AS1 in PDAC.MethodsRT-qPCR analysis revealed the OIP5-AS1 expression in PDAC tissues and adjacent normal ones. Kaplan–Meier method was applied to analyze the overall survival of patients with high or low level of OIP5-AS1. Gain- or loss-of function assays were performed to assess the effects of OIP5-AS1 knockdown on cell functions, including proliferation, migration and EMT process. Mechanism experiments, such as luciferase reporter and RNA pull-down assays proved the interaction between OIP5-AS1 and miR-429 as well as that between miR-429 and FOXD1.ResultsOIP5-AS1 was up-regulated in PDAC tissues and cell lines, and high level of OIP5-AS1 indicated poor prognosis in PDAC patients. OIP5-AS1 knockdown hindered cell proliferation, migration and epithelial-mesenchymal transition (EMT) process, while overexpression of OIP5-AS1 caused the opposite results. OIP5-AS1 activated ERK pathway through up-regulating forkhead box D1 (FOXD1) expression by sponging miR-429. Furthermore, OIP5-AS1 facilitated cell growth in vivo.ConclusionOIP5-AS1 exerted oncogenic function in PDAC cells through targeting miR-429/FOXD1/ERK pathway.

CXCL5 promotes gastric cancer metastasis by inducing epithelial-mesenchymal transition and activating neutrophils

Deregulated expression of chemokines in tumor microenvironment contributes to tumor metastasis by targeting distinct cells. Epithelial-derived neutrophil-activating peptide-78 (ENA78/CXCL5) is upregulated in many cancers and involved in tumor progression. The role and underlying mechanism of CXCL5 in gastric cancer (GC) metastasis remain unclear. In this study, we reported that the expression of CXCL5 was elevated in tumor tissues and positively associated with lymphatic metastasis and tumor differentiation. Stimulation by recombinant human CXCL5 (rhCXCL5) induced epithelial-mesenchymal transition (EMT) in GC cells through the activation of ERK pathway, which enhanced their migration and invasion abilities. The culture supernatant from tumor tissues also enhanced the migration and invasion abilities of GC cells, however, this effect was reversed by pre-treatment with CXCL5 neutralizing antibody. Further studies showed that rhCXCL5 could induce the expression of IL-6 and IL-23 in neutrophils through the activation of ERK and p38 signaling pathways, which in turn facilitated GC cell migration and invasion. The culture supernatant from tumor tissues showed similar effects on neutrophils in a CXCL5-dependent manner. Blockade of IL-6 and IL-23 with neutralizing antibodies reversed the induction of EMT and the increased migration and invasion abilities in GC cells by CXCL5-activated neutrophils. Moreover, CXCL5 activated neutrophils could promote gastric cancer metastasis in vivo. Taken together, our results indicate that CXCL5 acts on gastric cancer cells to induce EMT and mediates pro-tumor activation of neutrophils, which synergistically promotes the metastatic ability of GC cells.

β-Sitosterol Alleviates Inflammatory Response via Inhibiting the Activation of ERK/p38 and NF-κB Pathways in LPS-Exposed BV2 Cells.

Neurodegenerative disease is a disease state in which neurons in the spinal cord and brain are lost. Studies show that sustained neuroinflammatory reactions release toxic factors, damage neurons, and lead to neurodegenerative diseases. Therefore, inhibiting neuroinflammation may be an effective measure to alleviate neurodegenerative diseases. Microglia is an important participant in the neuroinflammatory response. β-Sitosterol (BS) is widely found in various vegetable oils, nuts, and other plant seeds. Studies have found that BS has a wide range of anti-inflammatory effects in peritoneal macrophages and other peripheral tissues. However, no studies have reported the effect of BS that impacts microglia activity. Herein, we further study the effect of BS on impacts microglia activity. Firstly, BV2, a murine microglial cell, was treated with different concentrations of BS prior to stimulation of LPS, and then the inflammatory mediators and the expression of related signaling molecules were tested. The inflammatory response results illustrated that BS treatment can reduce the LPS-induced expression of inflammatory mediators (interleukin-6 (IL-6), inducible nitric oxide (iNOS), tumor necrosis factor-α (TNF-α), and cyclooxygenase-2(COX-2)). The related signaling pathway analysis demonstrated that BS treatment can inhibit the LPS-induced activation of p38, ERK, and NF-κB pathways. To sum up, the results demonstrated that BS impacts microglia activity via repressing the activation of p38, ERK, and NF-κB pathways.

IGFBP-1 Expression Promotes Tamoxifen Resistance in Breast Cancer Cells via Erk Pathway Activation.

Insulin-like growth factor (IGF) system plays a significant role in many cellular processes, including proliferation, and survival. In estrogen receptor positive breast cancer, the level of circulating IGF-1 is positively associated with the incidence and at least 50% of cases have elevated IGF-1R signaling. Tamoxifen, a selective estrogen receptor modulator and antagonist for estrogen receptor alpha (ERα) in breast tissue, is a commonly prescribed adjuvant treatment for patients presenting with ERα-positive breast cancer. Unfortunately, tamoxifen resistance is a frequent occurrence in patients receiving treatment and the molecular mechanisms that underlie tamoxifen resistance not adequately defined. It has recently been reported that the inhibition of IGF-1R activation and the proliferation of breast cancer cells upon tamoxifen treatment is mediated by the accumulation of extracellular insulin-like growth factor binding protein 1 (IGFBP-1). Elevated IGFBP-1 expression was observed in tamoxifen-resistant (TamR) MCF-7 and T-47D cells lines suggesting that the tamoxifen-resistant state is associated with IGFBP-1 accumulation. MCF-7 and T-47D breast cancer cells stably transfected with and IGFBP-1 expression vector were generated (MCF7-BP1 and T47D-BP1) to determine the impact of breast cancer cell culture in the presence of increased IGFBP-1 expression. In these cells, the expression of IGF-1R was significantly reduced compared to controls and was similar to our observations in tamoxifen-resistant MCF-7 and T-47D cells. Also similar to TamR breast cancer cells, MCF7-BP1 and T47D-BP1 were resistant to tamoxifen treatment, had elevated epidermal growth factor receptor (EGFR) expression, increased phospho-EGFR (pEGFR), and phospho-Erk (pErk). Furthermore, tamoxifen sensitivity was restored in the MCF7-BP1 and T47D-BP1 upon inhibition of Erk phosphorylation. Lastly, the transient knockdown of IGFBP-1 in MCF7-BP1 and T47D-BP1 inhibited pErk accumulation and increased tamoxifen sensitivity. Taken together, these data support the conclusion that IGFBP-1 is a key component of the development of tamoxifen resistance in breast cancer cells.

Upregulation of circ-0000745 in acute lymphoblastic leukemia enhanced cell proliferation by activating ERK pathway.

Acute lymphoblastic leukemia (ALL) is a common hematologic malignancy. Circular RNAs (circRNAs) have been reported as an important factor in regulating cellular process expressed in all hematopoietic compartment. But the molecular mechanism of circRNAs in ALL remain unclear. In this study, we observed circ-0000745 upregulated in leukemia cells (Kasumi-1 and KG-1). Upregulated the expression of circ-0000745 significantly enhanced the proliferation of Kasumi-1 and KG-1 cells, and reduced the ratio of apoptosis cells. Knock down the endogenous expression of circ-0000745 suppressed the cell proliferation and increased the ratio of apoptosis cells. Furthermore, western blot assay showed that overexpression of circ-0000745 increased the activity of ERK1/2. Altogether, these results revealed that upregulated circ-0000745 in leukemia cells could promoter cell viability by increasing the activation of ERK pathway. This study supported the important of circRNAs in the process of acute lymphoblastic leukemia, and provided a new biomarker on ALL diagnosis and therapy.

Small proline-rich repeat 3 is a novel coordinator of PDGFRβ and integrin β1 crosstalk to augment proliferation and matrix synthesis by cardiac fibroblasts.

Nearly 6 million Americans suffer from heart failure. Increased fibrosis contributes to functional decline of the heart that leads to heart failure. Previously, we identified a mechanosensitive protein, small proline‐rich repeat 3 (SPRR3), in vascular smooth muscle cells of atheromas. In this study, we demonstrate SPRR3 expression in cardiac fibroblasts which is induced in activated fibroblasts following pressure‐induced heart failure. Sprr3 deletion in mice showed preserved cardiac function and reduced interstitial fibrosis in vivo and reduced fibroblast proliferation and collagen expression in vitro. SPRR3 loss resulted in reduced activation of Akt, FAK, ERK, and p38 signaling pathways, which are coordinately regulated by integrins and growth factors. SPRR3 deletion did not impede integrin‐associated functions including cell adhesion, migration, or contraction. SPRR3 loss resulted in reduced activation of PDGFRβ in fibroblasts. This was not due to the reduced PDGFRβ expression levels or decreased binding of the PDGF ligand to PDGFRβ. SPRR3 facilitated the association of integrin β1 with PDGFRβ and subsequently fibroblast proliferation, suggesting a role in PDGFRβ‐Integrin synergy. We postulate that SPRR3 may function as a conduit for the coordinated activation of PDGFRβ by integrin β1, leading to augmentation of fibroblast proliferation and matrix synthesis downstream of biomechanical and growth factor signals.

LncRNA LINC-PINT increases SOCS1 expression by sponging miR-155-5p to inhibit the activation of ERK signaling pathway in rheumatoid arthritis synovial fibroblasts induced by TNF-α

Rheumatoid arthritis (RA) is a systemic and chronic autoimmune disease associated with altered gene expression in synovial tissue. Long intergenic non-protein encoding long-chain RNA p53-induced transcript (LncRNA LINC-PINT) has been reported to be involved in multiple physiological and pathological processes. However, the role of lncRNA LINC-PINT in RA is rarely mentioned. To investigate the mechanism of LINC-PINT in RA, we constructed a RA model using TNF-α-induced method to explore the downstream effector and signaling pathway. We found that LINC-PINT was downregulated in RA tissues and TNF-α stimulated RA cells. And overexpression of LINC-PINT could inhibit cell proliferation and invasion induced by TNF-α through downregulating the levels of IL-1β and MMPs and inhibiting the activation of ERK pathway. Using bioinformatics analysis and RNA Binding Protein Immunoprecipitation (RIP) assay, we verified that LINC-PINT directly interacted with miR-155-5p, and miR-155-5p could regulate the expression of SOCS1. Whereas, downregulation of miR-155-5p inhibited cell growth and invasion. Overexpression of miR-155-5p could reverse the inhibitory effect of LINC-PINT induced by TNF-α. Furthermore, silencing SOCS1 promoted cell proliferation and invasion, upregulated the expression of IL-1β and MMPs, and activated ERK pathway, while overexpression of LINC-PINT could reverse the control of knocking down SOCS1. In conclusion, we revealed that LINC-PINT suppressed TNF-α induced cell proliferation and invasion which might be induced through downregulating miR-155-5p, influencing the expression of SOCS1, IL-1β and MMPs and inactivating ERK signaling pathway.

Curcumin inhibits epithelial-mesenchymal transition in oral cancer cells via c-Met blockade.

Oral squamous cell carcinoma (OSCC) is the most common type of oral cancer. OSCC cells are highly invasive, a characteristic that involves epithelial-mesenchymal transition (EMT); the conversion of immotile epithelial cells into motile mesenchymal cells. EMT is involved in the progression of various types of cancer by promoting tumour cell scattering and conferring to these cells cancer stem cell (CSC)-like characteristics, such as self-renewal. Hepatocyte growth factor (HGF) signalling plays an important role in EMT induction and, therefore, contributes to cell invasion and metastasis in cancer. Due to its potential chemopreventative and anti-tumour activities, curcumin has attracted much interest and has been shown to act as a potent EMT inhibitor in various types of cancer. However, at present, the potential effects of curcumin on HGF-induced EMT in OSCC have not been investigated. Here, we demonstrated that HGF signalling could induce EMT in the HSC4 and Ca9-22 OSCC cell lines via the HGF receptor c-Met and downstream activation of the pro-survival ERK pathway. Notably, curcumin inhibited HGF-induced EMT and cell motility in HSC-4 and Ca9-22 cells via c-Met blockade. Therefore, these findings establish curcumin as a candidate drug for OSCC treatment. Furthermore, curcumin was able to effectively inhibit the HGF-induced increase in the levels of vimentin by downregulating the expression of phosphorylated c-Met, an ERK. In conclusion, the results of the present study demonstrated that curcumin was able to reverse HGF-induced EMT, possibly by inhibiting c-Met expression in oral cancer cells, providing a strong basis for the development of novel approaches for the treatment of oral cancer.

SLAMF3-Mediated Signaling via ERK Pathway Activation Promotes Aggressive Phenotypic Behaviors in Multiple Myeloma.

The signaling lymphocytic activation molecule family 3 (SLAMF3) is a member of the immunoglobulin superfamily expressed on T, B, and natural killer cells and modulates the activation and cytotoxicity of these cells. SLAMF3 is also expressed on plasma cells from patients with multiple myeloma (MM), although its role in MM pathogenesis remains unclear. This study found that SLAMF3 is highly and constitutively expressed on MM cells regardless of disease stage and that SLAMF3 knockdown/knockout suppresses proliferative potential and increases drug-induced apoptosis with decreased levels of phosphorylated ERK protein in MM cells. SLAMF3-overexpressing MM cells promote aggressive myeloma behavior in comparison with cytoplasmic domain-truncated SLAMF3 (ΔSLAMF3) cells. SLAMF3 interacts directly with adaptor proteins SH2 domain-containing phosphatase 2 (SHP2) and growth factor receptor bound 2 (GRB2), which also interact with each other. SLAMF3 knockdown, knockout, ΔSLAMF3, and SHP2 inhibitor-treated MM cells decreased phosphorylated ERK protein levels. Finally, serum soluble SLAMF3 (sSLAMF3) levels were markedly increased in advanced MM. Patients with high levels of sSLAMF3 progressed to the advanced stage significantly more often and had shorter progression-free survival times than those with low levels. This study revealed that SLAMF3 molecules consistently expressed on MM cells transmit MAPK/ERK signals mediated via the complex of SHP2 and GRB2 by self-ligand interaction between MM cells and induce a high malignant potential in MM. Furthermore, high levels of serum sSLAMF3 may reflect MM disease progression and be a useful prognostic factor. IMPLICATIONS: SLAMF3 may be a new therapeutic target for immunotherapy and novel agents such as small-molecule inhibitors.

Regulation of the small GTPase Ran by miR-802 modulates proliferation and metastasis in colorectal cancer cells

BackgroundThe small GTPase Ran is upregulated in multiple cancers and fundamental for cancer cell survival and progression, but its significance and molecular mechanisms in colorectal cancer (CRC) remain elusive.MethodsRan expression was detected in CRC cell lines and tumour tissues. In vitro and in vivo functional assays were performed to examine the effects of Ran on cell proliferation and metastasis. The pathways and effectors regulated by Ran were explored by an unbiased screening. Bioinformatics prediction and experimental validation were used to identify the miRNA regulator for Ran.ResultsRan expression was frequently increased in metastatic CRC cells and tissues, especially in metastatic tissues. The upregulation of Ran correlated with poor CRC patient prognosis. Ran silencing reduced proliferation and metastasis of CRC cells both in vitro and in vivo. Ran regulated the expression of EGFR and activation of ERK and AKT signalling pathways. miR-802 was identified as an upstream regulator of Ran and miR-802 overexpression resulted in antiproliferative and antimetastatic activities.ConclusionOur study demonstrates the oncogenic roles and underlying mechanisms of Ran in CRC and the novel miR-802/Ran/EGFR regulatory axis may provide potential biomarkers for the treatment of CRC.