Activation Of ERK Signaling Research Articles

Targeting ARF1-IQGAP1 interaction to suppress colorectal cancer metastasis and vemurafenib resistance

IntroductionAcquired resistance to BRAF inhibitor vemurafenib is frequently observed in metastatic colorectal cancer (CRC), and it is a thorny issue that results in treatment failure. As adaptive responses for vemurafenib treatment, a series of cellular bypasses are response for the adaptive feedback reactivation of ERK signaling, which warrant further investigation. ObjectivesWe identified ARF1 (ADP-ribosylation factor 1) as a novel regulator of both vemurafenib resistance and cancer metastasis, its molecular mechanism and potential inhibitor were investigated in this study. MethodsDIA-based quantitative proteomics and RNA-seq were performed to systematic analyze the profiling of vemurafenib-resistant RKO cells (RKO-VR) and highly invasive RKO cells (RKO-I8), respectively. Co‑immunoprecipitation assay was performed to detect the interaction of ARF1 and IQGAP1 (IQ-domain GTPase activating protein 1). An ELISA-based drug screen system on FDA-approved drug library was established to screen the compounds against the interaction of ARF1-IQGAP1.The biological functions of ARF1 and LY2835219 were determined by transwell, western blotting, Annexin V-FITC/PI staining and in vivo experimental metastasis assays. ResultsWe found that ARF1 strongly interacted with IQGAP1 to activate ERK signaling in VR and I8 CRC cells. Deletion of IQGAP1 or inactivation of ARF1 (ARF-T48S) restored the invasive ability induced by ARF1. As ARF1-IQGAP1 interaction is essential for ERK activation, we screened LY2835219 as novel inhibitor of ARF1-IQGAP1 interaction, which inactivated ERK signaling and suppressed CRC metastasis and vemurafenib-resistance in vitro and in vivo with no observed side effect. Furthermore, LY2835219 in combined treatment with vemurafenib exerted significantly inhibitory effect on ARF1-mediated cancer metastasis than used independently. ConclusionThis study uncovers that ARF1-IQGAP1 interaction-mediated ERK signaling reactivation is critical for vemurafenib resistance and cancer metastasis, and that LY2835219 is a promising therapeutic agent for CRC both as a single agent and in combination with vemurafenib.

Inhibition of Angiogenesis by MiR-524-5p through Suppression of AKT and ERK Activation by Targeting CXCR7 in Colon Cancer Cells.

Increasing evidence shows that alterations in microRNA (miRNA) expression are involved in the occurrence and development of various malignant tumors, including colon cancer. MiRNA-524-5p has been reported to have anticancer activity in colon cancer. This study explored the influence of the miRNA-524-5p/CXCR7 axis on angiogenesis using colon cancer cells and further studied the mechanisms involved. We found that changing the expression of miRNA-524-5p can affect colonic proliferation, migration, and angiogenesis. Furthermore, angiogenesis induced by miRNA-524-5p overexpression was reversed by overexpression of CXCR7 in HT-29 cells, while the opposite was observed in Caco-2 cells. Furthermore, miRNA-524-5p inhibited the activation of AKT and ERK signaling by targeting CXCR7. Overall, our results indicated that the miRNA-524-5p/CXCR7 axis regulated angiogenesis in colon cancer cells through the AKT and ERK pathways.

R-spondin 3 deletion induces Erk phosphorylation to enhance Wnt signaling and promote bone formation in the appendicular skeleton.

Activation of Wnt signaling leads to high bone density. The R-spondin family of four secreted glycoproteins (Rspo1-4) amplifies Wnt signaling. In humans, RSPO3 variants are strongly associated with bone density. Here, we investigated the role of Rspo3 in skeletal homeostasis in mice. Using a comprehensive set of mouse genetic and mechanistic studies, we show that in the appendicular skeleton, Rspo3 haplo-insufficiency and Rspo3 targeted deletion in Runx2+ osteoprogenitors lead to an increase in trabecular bone mass, with increased number of osteoblasts and bone formation. In contrast and highlighting the complexity of Wnt signaling in the regulation of skeletal homeostasis, we show that Rspo3 deletion in osteoprogenitors results in the opposite phenotype in the axial skeleton, i.e., low vertebral trabecular bone mass. Mechanistically, Rspo3 deficiency impairs the inhibitory effect of Dkk1 on Wnt signaling activation and bone mass. We demonstrate that Rspo3 deficiency leads to activation of Erk signaling which in turn, stabilizes β-catenin and Wnt signaling activation. Our data demonstrate that Rspo3 haplo-insufficiency/deficiency boosts canonical Wnt signaling by activating Erk signaling, to favor osteoblastogenesis, bone formation, and bone mass.

Contributions of extracellular-signal regulated kinase 1/2 activity to the memory trace.

The ability to learn from experience and consequently adapt our behavior is one of the most fundamental capacities enabled by complex and plastic nervous systems. Next to cellular and systems-level changes, learning and memory formation crucially depends on molecular signaling mechanisms. In particular, the extracellular-signal regulated kinase 1/2 (ERK), historically studied in the context of tumor growth and proliferation, has been shown to affect synaptic transmission, regulation of neuronal gene expression and protein synthesis leading to structural synaptic changes. However, to what extent the effects of ERK are specifically related to memory formation and stabilization, or merely the result of general neuronal activation, remains unknown. Here, we review the signals leading to ERK activation in the nervous system, the subcellular ERK targets associated with learning-related plasticity, and how neurons with activated ERK signaling may contribute to the formation of the memory trace.

LPAR5 confers radioresistance to cancer cells associated with EMT activation via the ERK/Snail pathway

BackgroundEpithelial-to-mesenchymal transition (EMT) is a critical event contributing to more aggressive phenotypes in cancer cells. EMT is frequently activated in radiation-targeted cells during the course of radiotherapy, which often endows cancers with acquired radioresistance. However, the upstream molecules driving the signaling pathways of radiation-induced EMT have not been fully delineated.MethodsIn this study, RNA-seq-based transcriptome analysis was performed to identify the early responsive genes of HeLa cells to γ-ray irradiation. EMT-associated genes were knocked down by siRNA technology or overexpressed in HeLa cells and A549 cells, and the resulting changes in phenotypes of EMT and radiosensitivity were assessed using qPCR and Western blotting analyses, migration assays, colony-forming ability and apoptosis of flow cytometer assays. ResultsThrough RNA-seq-based transcriptome analysis, we found that LPAR5 is downregulated in the early response of HeLa cells to γ-ray irradiation. Radiation-induced alterations in LPAR5 expression were further revealed to be a bidirectional dynamic process in HeLa and A549 cells, i.e., the early downregulating phase at 2 ~ 4 h and the late upregulating phase at 24 h post-irradiation. Overexpression of LPAR5 prompts EMT programing and migration of cancer cells. Moreover, increased expression of LPAR5 is significantly associated with IR-induced EMT and confers radioresistance to cancer cells. Knockdown of LPAR5 suppressed IR-induced EMT by attenuating the activation of ERK signaling and downstream Snail, MMP1, and MMP9 expression.ConclusionsLPAR5 is an important upstream regulator of IR-induced EMT that modulates the ERK/Snail pathway. This study provides further insights into understanding the mechanism of radiation-induced EMT and identifies promising targets for improving the effectiveness of cancer radiation therapy.

PD-L1 induction via the MEK-JNK-AP1 axis by a neddylation inhibitor promotes cancer-associated immunosuppression

MLN4924 is a first-in-class small molecule inhibitor of NEDD8-activating enzyme (NAE), which is currently in several clinical trials for anti-cancer applications. However, MLN4924 also showed some off-target effects with potential to promote the growth of cancer cells which counteracts its anticancer activity. In this study, we found that MLN4924 increases the levels of PD-L1 mRNA and protein in dose- and time-dependent manners. Mechanistic study showed that this MLN4924 effect is largely independent of neddylation inactivation, but is due to activation of both ERK and JNK signals, leading to AP-1 activation, which is blocked by the small molecule inhibitors of MEK and JNK, respectively. Biologically, MLN4924 attenuates T cell killing in a co-culture model due to PD-L1 upregulation, which can be, at least in part, abrogated by either MEK inhibitor or anti-PD-L1 antibody. In an in vivo BALB/c mouse xenograft tumor model, while MLN4924 alone had no effect, combination with either MEK inhibitor or anti-PD-L1 antibody enhanced the suppression of tumor growth. Taken together, our study provides a sound rationale for effective anticancer therapy in combination of anti-PD-L1 antibody or MEK inhibitor with MLN4924 to overcome the side-effect of immunosuppression by MLN4924 via PD-L1 induction.

A functional role for Serum Amyloid A in the molecular regulation of autophagy in breast cancer

It has been established that the acute phase protein, Serum amyloid A (SAA), which is usually synthesized by the liver, is also synthesized by cancer cells and cancer-associated cells in the tumor microenvironment. SAA also activates modulators of autophagy, such as the PI3K/Akt and MAPK signaling pathways. However, the role of SAA in autophagy in breast cancer still remains to be elucidated. The aim of this study was to investigate the role of SAA in the regulation of signaling pathways and autophagy in in vitro and in vivo models of breast cancer. The MDA-MB-231 and MCF7 cell lines were transiently transfected to overexpress SAA1. A tumor-bearing SAA1/2 knockout mouse model was also utilized in this study. SAA1 overexpression activated ERK signaling in the MDA-MB-231 cells, downregulated the PI3K pathway protein, PKB/Akt, in the MCF7 cell line, while SAA1/2 knockout also inhibited Akt. Furthermore, SAA1 overexpression in vitro downregulated autophagy, while the expression of SQSTM1/p62 was increased in the MCF7 cells, and SAA1/2 knockout induced autophagy in vivo. SAA overexpression in the MDA-MB-231 and MCF7 cells resulted in an increase in cell viability and increased the expression of the proliferation marker, MCM2, in the MCF7 cells. Furthermore, knockout of SAA1/2 resulted in an altered inflammatory profile, evident in the decrease of plasma IL-1β, IL-6 and IL-10, while increasing the plasma levels of MCP-1 and TNF-α. Lastly, SAA1/2 knockout promoted resistance to apoptosis and necrosis through the regulation of autophagy. SAA thus regulates autophagy in breast cancer cells to promote tumorigenesis.

Molecular mechanism of aquaporin 3 (AQP3) regulating by LMP2A and its crosstalk with 4E-BP1 via ERK signaling pathway in EBV-associated gastric cancer

Aquaporin 3(AQP3) is involved in epithelial-mesenchymal transformation of tumor cells and is closely related to the occurrence and development of tumors. However, the regulatory mechanism and function of AQP3 in EBV-associated gastric cancer (EBVaGC) are still poorly understood. This study aims to explore the regulatory effect of EBV on AQP3 and the cross talk of AQP3 with EIF4E-binding proteins 1(4E-BP1) in EBVaGC. The effect of LMP2A on the expression of AQP3 and 4E-BP1 was analyzed using real-time PCR and western blotting. The biological functions of AQP3 and 4E-BP1 in gastric cancer cells were detected by cell biological experiments. In addition, we examined the role of mTOR and ERK signaling pathways in the LMP2A/AQP3/4E-BP1 regulatory axis. We found that LMP2A could down-regulate AQP3 expression by inhibiting the activation of mTOR signaling pathway, and further promote autophagy and migration of gastric cancer cells. AQP3 up-regulated the expression of 4E-BP1 and its phosphorylated protein by activating ERK signaling pathway, thus promoting the autophagy and proliferation of gastric cancer cells. In conclusion, EBV-encoded LMP2A inhibits AQP3 expression, and further participates in cell proliferation, migration and autophagy through the mTOR/AQP3/ERK/4E-BP1 axis.

Eupalinolide A induces autophagy via the ROS/ERK signaling pathway in hepatocellular carcinoma cells invitro and invivo.

Hepatocellular carcinoma is the most common primary malignancy of the liver. The current systemic drugs used to treat hepatocellular carcinoma result in low overall survival time. It has therefore been suggested that new small-molecule drugs should be developed for treating hepatocellular carcinoma. Eupatorium lindleyanum DC. (EL) has been used to treat numerous diseases, particularly respiratory diseases; however, to the best of our knowledge, studies have not yet fully elucidated the effect of EL on hepatocellular carcinoma. In the present study, the effect of eupalinolide A (EA), one of the extracts of EL, was evaluated on tumor growth in a xenograft model of human hepatocellular carcinoma cells, and on the proliferation and migration of hepatocellular carcinoma cell lines. Cell cycle progression and the type of cell death were then evaluated using the Cell Counting Kit 8 assay, flow cytometry, electron microscopy and western blotting. EA significantly inhibited cell proliferation and migration by arresting the cell cycle at the G1 phase and inducing autophagy in hepatocellular carcinoma cells. EA-induced autophagy was mediated by reactive oxygen species (ROS) and ERK signaling activation. Specific inhibitors of ROS, autophagy and ERK inhibited EA-induced cell death and migration. In conclusion, the present study revealed that EA may inhibit the proliferation and migration of hepatocellular carcinoma cells, highlighting its potential as a promising antitumor compound for treating hepatocellular carcinoma.

Abstract A020: Combined inhibition of SHP2 and CDK4/6 is active in preclinical models of NF1-associated malignant peripheral nerve sheath tumor

Abstract Background: NF1 is an essential negative regulator of RAS activity and its function is lost in nearly 90% of malignant peripheral nerve sheath tumors (MPNST). Additional recurrent molecular changes include loss of function alterations in CDKN2A, TP53, EED and SUZ12, but molecular targeting of these genomic events represents a unique challenge. We previously reported that the efficacy of MEK inhibitor is limited by adaptive activation of receptor tyrosine kinases and the adaptor protein SHP2, and that combined inhibition of MEK and SHP2 is effective in MPNST. However, the clinical potential of combination MEK and SHP2 inhibitors may be limited by the overlapping toxicity induced by ERK pathway inhibition. Loss of CDKN2A, inactivation of RB1, and hyperactivation of cyclin dependent kinases (CDK) in MPNST also suggest that small-molecule CDK4/6 inhibitors (CDK4/6i) may be a potential therapeutic strategy, but monotherapy with CDK4/6i also demonstrates limited activity. We hypothesize that the anti-tumor response of SHP2i may be potentiated by agents targeting the cell cycle in combination. Methods: The effects of shRNA-mediated inducible SHP2 knockdown on RAS signaling, short-term and long-term cell growth, and response to CDK4/6i were examined using immunoblotting, high throughput proliferation assays, and colony formation assays. Combined effects of SHP2i plus CDK4/6i on signaling, cell cycle, apoptosis, cell and in vivo tumor growth were assessed. Pharmacodynamic (PD) assays were performed on tumors extracted following drug treatment in patient-derived xenograft (PDX) models of MPNST. Results: Despite a modest effect of SHP2 knockdown on ERK signaling, shPTPN11 reduced MPNST cell growth. SHP2 knockdown or SHP2i treatment alleviated activation of ERK signaling and cyclin D1 expression induced by CDK4/6i, and enhanced the sensitivity to CDK4/6i. Combination benefit was observed in in vitro cell growth and in vivo PDX. Although some PDX models demonstrated similar responses to SHP2i alone or SHP2i + CDK4/6i during the initial four weeks on treatment, we found sustained tumor growth inhibition exerted by the combination on longer therapy. PD studies demonstrated a decrease in p-ERK levels in tumors treated with either SHP2i alone or the SHP2i/CDK4/6i combination, as well as a synergistic suppression of cell cycle regulators by the combination. Conclusions: Our preliminary data demonstrate that the combined inhibition of SHP2 and CDK4/6 is active and produces deep and durable response in models of NF1-associated MPNST. This combination strategy may represent a novel treatment approach for patients with MPNST. Citation Format: Jiawan Wang, Ana Calizo, Kai Pollard, Lindy Zhang, John M. Gross, Nicolas Llosa, Angela C. Hirbe, Christine A. Pratilas. Combined inhibition of SHP2 and CDK4/6 is active in preclinical models of NF1-associated malignant peripheral nerve sheath tumor [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr A020.

Nonylphenol Promoted Epithelial-Mesenchymal Transition in Colorectal Cancer Cells by Upregulating the Expression of Regulator of Cell Cycle.

Nonylphenol (NP) is a widely used chemical, which has been considered a kind of endocrine-disrupting chemical and is involved in the occurrence and development of many types of cancers. Our recent studies demonstrated that NP exposure is related to colorectal cancer (CRC) progression. In this study, we also found epithelial-mesenchymal transition (EMT) promoted by NP treatment in CRC cells. However, the mechanism of NP on tumor metastasis is still unclear. In this study, we focused on the effect of the regulator of cell cycle (RGCC) induced by NP treatment. The cancer genome atlas (TCGA) analysis suggested that the expression of RGCC increased in CRC tissues, and our clinical samples showed that the expression of RGCC in tumor tissues is positively correlated with the serum level of NP in CRC patients. Further studies revealed that overexpression of RGCC could enhance the NP-induced EMT process in CRC cells and activate ERK signaling pathways. Inhibiting ERK signaling by ERK inhibitors or the knockdown of RGCC could attenuate the NP-induced EMT process. In addition, both RGCC overexpression and NP treatment could activate ERK pathways and attenuate the effect of ERK inhibitors on the EMT process in CRC cells. Altogether, this study demonstrated that NP could induce cell invasion and migration by increasing the expression of RGCC to enhance the EMT process, which might be through the activation of ERK signaling pathways. This finding supported a potential target for studying NP exposure-related colorectal cancers.

A NANOG‐pERK reciprocal regulatory circuit regulates Nanog autoregulation and ERK signaling dynamics

The self‐renewal and differentiation potential of embryonic stem cells (ESCs) is maintained by the regulated expression of core pluripotency factors. Expression levels of the core pluripotency factor Nanog are tightly regulated by a negative feedback autorepression loop. However, it remains unclear how ESCs perceive NANOG levels and execute autorepression. Here, we show that a dose‐dependent induction of Fgfbp1 and Fgfr2 by NANOG activates autocrine‐mediated ERK signaling in Nanog‐high cells to trigger autorepression. pERK recruits NONO to the Nanog locus to repress transcription by preventing POL2 loading. This Nanog autorepression process establishes a self‐perpetuating reciprocal NANOG‐pERK regulatory circuit. We further demonstrate that this reciprocal regulatory circuit induces pERK heterogeneity and ERK signaling dynamics in pluripotent stem cells. Collectively our data suggest that NANOG induces Fgfr2 and Fgfbp1 to activate ERK signaling in Nanog‐high cells to establish a NANOG‐pERK reciprocal regulatory circuit. This circuit regulates ERK signaling dynamics and Nanog autoregulation in pluripotent cells.

Lymphatic endothelial sphingosine 1-phosphate receptor 1 enhances macrophage clearance via lymphatic system following myocardial infarction.

Lymphatic endothelial cell homeostasis plays important roles in normal physiological cardiac functions, and its dysfunction significantly influences pathological cardiac remodeling after myocardial infarction (MI). Our results revealed that sphingosine 1-phosphate receptor 1 (S1pr1) expression in cardiac lymphatic endothelial cells (LECs) was sharply changed after MI. It has been shown that S1pr1 tightly controlled LEC functions and homeostasis. We thus hypothesized that lymphatic endothelial S1pr1 might be involved in post-MI cardiac remodeling. We generated LEC-conditional S1pr1 transgenic mice, in which S1pr1 expression was reduced in cardiac LECs. We performed the left anterior descending coronary artery (LAD) ligation operation to induce MI in these mice. Cardiac functions and remodeling were examined by echocardiography analysis and serial histological analysis. Meanwhile, we performed adoptive cell transfer experiments to monitor macrophage trafficking in post-MI myocardium and their draining lymphatic system. Furthermore, in vitro cell culture experiments and mechanism studies were undertaken to uncover the molecular mechanism by which LEC-S1pr1 regulated cardiac inflammation and remodeling after MI. Our results showed that S1pr1 expression significantly decreased in cardiac LECs after MI. Our in vivo experiments showed that the reduced expression of LEC-S1pr1 deteriorated cardiac function and worsened pathological cardiac remodeling after MI. Our further results demonstrated that the reduced expression of LEC-S1pr1 did not influence macrophage infiltration in an early inflammatory phase of MI, but significantly affected macrophages clearance in the later phase of MI via afferent cardiac lymphatics, and thus influenced inflammatory responses and cardiac outcome after MI. Further study showed that S1P/S1pr1 activated ERK signaling pathway and enhanced CCL2 expression, which promoted macrophage trafficking in a paracrine manner. This study reveals that cardiac lymphatic endothelial cells tightly control macrophage trafficking via lymphatic vessels in injured hearts via S1P/S1pr1/ERK/CCL2 pathway and thus regulate post-MI immune modulation and heart repair. This study highlights the importance of cardiac lymphatic vessel system in orchestrating post-MI immune responses and cardiac remodeling by regulating macrophage transit in injured hearts. Our finding implies that a feasible modulation of S1pr1 signaling in LECs might provide a promising target to resolve excessive inflammation and to ameliorate adverse cardiac remodeling after MI.

Phikud Navakot extract attenuates lipopolysaccharide-induced inflammatory responses through inhibition of ERK1/2 phosphorylation in a coculture system of microglia and neuronal cells

Ethnopharmacological relevancePhikud Navakot (PN), a mixture of nine herbal plants, is an ancient Thai traditional medicine used for relieving circulatory disorders and dizziness. PN has also shown anti-inflammatory effects in rats with acute myocardial infarction. Moreover, phytochemical-inhibiting neuroinflammation, including gallic acid, vanillic acid, ferulic acid, and rutin were detected in PN extract; however, the anti-neuroinflammatory activity of PN extract and its components in a coculture system of microglia and neuronal cells is limited. ObjectiveTo investigate the anti-neuroinflammatory activities of PN on lipopolysaccharide (LPS)-induced inflammation in a coculture system of microglia and neuronal cells. MethodsELISA and qRT-PCR were used to assess cytokine expression. The phosphorylation of mitogen-activated protein kinases (MAPKs) was determined by Western blotting. Microglia-mediated neuroinflammation was evaluated using a BV-2 microglia-N2a neuron transwell co-culture. ResultsPN extract and its component, gallic acid, decreased LPS-induced the mRNA expression of interleukin-6 (IL-6) and inducible nitric oxide synthase (iNOS), as well as IL-6 protein levels in both microglial monoculture and coculture systems. This was accompanied by a reduction in neurodegeneration triggered by microglia in N2a neurons with increased neuronal integrity markers (βIII tubulin and tyrosine hydroxylase (TH)). These effects were caused by the ability of PN extract to inhibit extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) activation. ConclusionThis is the first study to show that PN extract inhibits neurodegeneration in LPS-activated BV-2 microglia by targeting ERK signaling activity.

Functional analysis reveals that Tinagl1 is required for normal muscle development in mice through the activation of ERK signaling

Tinagl1 (tubulointerstitial nephritis antigen-like 1) is a matricellular protein involved in female infertility and breast cancer tumorigenesis. In this study, we analyzed the function of Tinagl1 in skeletal muscle using knockout mice and cell experiments. Although primary myoblasts isolated from Tinagl1-decifient (Tinagl1−/−) mice differentiated into normal myotubes, and treatment with recombinant Tinagl1 did not affect the proliferation or differentiation of C2C12 myoblasts, Tinagl1−/− mice exhibited reduced body mass and calf muscle weights compared to the control group (Tinagl1flox/flox). Furthermore, Tinagl1−/− mice showed myofibers with centrally located nuclei, which is a morphological marker of regenerating muscle or myopathy. In addition, the capillary density in the soleus muscle of Tinagl1−/− mice showed a decreasing trend compared to that of the control group. Importantly, si-RNA-mediated knockdown of TINAGL1 resulted in reduced tube formation in human umbilical vein endothelial cells (HUVECs), whereas treatment with Tinagl1 promoted tube formation. Immunoblot analysis revealed that Tinagl1 activates ERK signaling in both HUVECs and C2C12 myoblasts and myotubes, which are involved in the regulation of myogenic differentiation, proliferation, metabolism, and angiogenesis. Our results demonstrate that Tinagl1 may be required for normal muscle and capillary development through the activation of ERK signaling.

Inhibition of FGF receptor blocks adaptive resistance to RET inhibition in CCDC6-RET-rearranged thyroid cancer.

Genetic alterations in RET lead to activation of ERK and AKT signaling and are associated with hereditary and sporadic thyroid cancer and lung cancer. Highly selective RET inhibitors have recently entered clinical use after demonstrating efficacy in treating patients with diverse tumor types harboring RET gene rearrangements or activating mutations. In order to understand resistance mechanisms arising after treatment with RET inhibitors, we performed a comprehensive molecular and genomic analysis of a patient with RET-rearranged thyroid cancer. Using a combination of drug screening and proteomic and biochemical profiling, we identified an adaptive resistance to RET inhibitors that reactivates ERK signaling within hours of drug exposure. We found that activation of FGFR signaling is a mechanism of adaptive resistance to RET inhibitors that activates ERK signaling. Combined inhibition of FGFR and RET prevented the development of adaptive resistance to RET inhibitors, reduced cell viability, and decreased tumor growth in cellular and animal models of CCDC6-RET–rearranged thyroid cancer.

Egfl6 promotes ovarian cancer progression by inducing the immunosuppressive functions of tumor-infiltrating myeloid cells.

Abstract Myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs) are critical negative regulators of immunity in cancer. Understanding factors which regulate these cells could result in the identification of new approaches to enhance anti-tumor immunotherapy. One such factor is epidermal growth factor-like 6 (EGFL6), a secreted factor known to promote cancer stem like cell migration and regulate cancer cell differentiation. We found that mice which overexpress Egfl6 have an increased numbers of granulocytes and monocytes in both the bone marrow and spleen. In vitro and ex-vivo analysis indicated that EGFL6, via binding with beta integrins and activation of SYK and ERK signaling, (i) acts as a chemotactic factor for myeloid cells migration and (ii) promotes their differentiation toward a suppressive state. Suggesting an important role in promoting an immunosuppressive tumor microenvironment (TME), using two syngeneic mouse models of ovarian cancer, we found that expression of Egfl6 in tumor cells resulted in increased accumulation of intra-tumoral MDSCs and TAMs and fewer cytotoxic CD8+ T cells. This was associated with increased tumor growth and shortened animal survival. Gene expression profiling of tumor infiltrating myeloid cells indicated that Egfl6 induced the expression of immunosuppressive factors, including CXCL2, IL-10 and PD-L1. Moreover, in an immune ‘hot’ tumor model, EGFL6 completely inhibited response to a-PD-L-1 therapy. Combined our data show that EGFL6 induces the recruitment of myeloid cells into the ovarian TME and subsequently promotes their immunosuppressive functions. This suggest EGFL6 is a potential novel therapeutic target to enhance response to immune therapy in OvCa patients. Supported by Ovarian Cancer Research Alliance

VPO1/HOCl/ERK pathway mediates the right ventricular remodeling in rats with hypoxic pulmonary hypertension

Right ventricular (RV) remodeling is a major feature of pulmonary arterial hypertension (PAH). Vascular peroxidase 1 (VPO1) is reported to participate in the process of PAH. This study aims to explore whether VPO1 contributes to hypoxia-induced cardiac hypertrophy and the underlying mechanisms. SD rats were exposure to continuous hypoxia (10% O2) for 3 weeks, which showed RV hypertrophy (increases in the ratio of RV weight to tibia length, cardiac cell size and hypertrophic markers), concomitant with upregulation of VPO1, elevation in hypochlorous acid (HOCl) production and ERK phosphorylation. In hypoxia (3% O2)-induced hypertrophic H9c2 cells, similar characteristics of cardiac hypertrophy to that of hypoxia-treated rats were observed. Administration of VPO1 siRNA or NaHS (the HOCl inhibitor) suppressed HOCl production, ERK phosphorylation, and cardiac hypertrophy. Replacement of hypoxia with NaClO (exogenous HOCl) could also induce cardiac cell hypertrophy and activate ERK signaling pathway. In addition, hypoxia-induced cardiac hypertrophy could be blocked by PD98059 (the ERK-specific inhibitor). Based on these observations, we conclude that VPO1 promotes RV remodeling in PAH rats through catalyzing HOCl production, leading to the activation of ERK signaling. Thus, VPO1 may have the potential as a therapeutic target for PAH.

Suppression of LMCD1 ameliorates renal fibrosis by blocking the activation of ERK pathway.

Tubulointerstitial fibrosis is a common pathway of chronic kidney disease (CKD) and is closely related to the progression of CKD. LMCD1, acting as an intermediary, has been reported to play a role in cardiac fibrosis. However, its role in renal fibrosis is yet to be deciphered. Based on the GEO database, we found the expression of LMCD1 is increased in kidney tissues of CKD patients and in human proximal tubular epithelial (HK-2) cells treated with transforming growth factor-β1 (TGF-β1), suggesting that LMCD1 may be involved in tubulointerstitial fibrosis. Herein, we investigated the role of LMCD1 in mice with unilateral ureteral obstruction (UUO) and in TGF-β1-stimulated HK-2 cells. In the UUO model, the expression of LMCD1 was upregulated. UUO-induced renal histopathological changes were mitigated by knockdown of LMCD1. LMCD1 silence alleviated renal interstitial fibrosis in UUO mice by decreasing the expression of TGF-β1, fibronectin, collagen I, and collagen III. LMCD1 deficiency suppressed cell apoptosis in kidney to prevent UUO-triggered renal injury. Furthermore, LMCD1 deficiency blocked the activation of ERK signaling in UUO mice. In vitro, LMCD1 was upregulated in HK-2 cells after TGF-β1 stimulation. LMCD1 silence abrogated TGF-β1-mediated upregulation of fibrotic genes. Treatment of HK-2 cells with ERK-specific inhibitor SCH772984 and agonist TPA validated LMCD1 exerted its function via activating ERK signaling. Together, our findings suggest that inhibition of LMCD1 protects against renal interstitial fibrosis by impeding ERK activation.

Characterization of Cisplatin Effects in Lenvatinib-resistant Hepatocellular Carcinoma Cells.

Drug resistance to molecular targeted agents, such as lenvatinib, is an important issue. The aim of this study was to explore the mechanism of lenvatinib resistance and to investigate potential drugs that may improve the treatment of lenvatinib-resistant (LR) hepatocellular carcinoma (HCC). LR cells were developed by long-term culture under lenvatinib exposure. We analyzed the biological characteristics of LR cells in vitro, and investigated the antitumor effects and endogenous mechanisms of cisplatin in LR cells. The proliferative potential of LR cells was enhanced by activation of ERK signaling and changes in several miRNAs. Cisplatin inhibited cell proliferation of LR cells and induced G2/M cell cycle arrest. Furthermore, cisplatin triggered the DNA damage response, via the ATM/ATR-Chk1/Chk2 signaling pathway. Proliferation of LR cells was induced upon ERK signaling activation. Cisplatin exerted antitumor effects in LR cells and was involved in the regulation of miRNAs associated with drug resistance.