Downstream Signaling Molecules Research Articles

Latrophilin-2 Deletion in Cardiomyocyte Disrupts Cell Junction, Leading to D-CMP.

Latrophilin-2 (Lphn2), an adhesive GPCR (G protein-coupled receptor), was found to be a specific marker of cardiac progenitors during the differentiation of pluripotent stem cells into cardiomyocytes or during embryonic heart development in our previous studies. Its role in adult heart physiology, however, remains unclear. The embryonic lethality resulting from Lphn2 deletion necessitates the establishment of cardiomyocyte-specific, tamoxifen-inducible Lphn2 knockout mice, which was achieved by crossing Lphn2flox/flox mice with mice having MerCreMer (tamoxifen-inducible Cre recombinase) under the α-myosin heavy chain promoter. Tamoxifen treatment for several days completely suppressed Lphn2 expression, specifically in the myocardium, and induced the dilated cardiomyopathy (D-CMP) phenotype with serious arrhythmia and sudden death in a short period of time. Transmission electron microscopy showed mitochondrial abnormalities, blurred Z-discs, and dehiscent myofibrils. The D-CMP phenotype, or heart failure, worsened during myocardial infarction. In a mechanistic study of D-CMP, Lphn2 knockout suppressed PGC-1α and mitochondrial dysfunction, leading to the accumulation of reactive oxygen species and the global suppression of junctional molecules, such as N-cadherin (adherens junction), DSC-2 (desmocollin-2; desmosome), and connexin-43 (gap junction), leading to the dehiscence of cardiac myofibers and serious arrhythmia. In an experimental therapeutic trial, activators of p38-MAPK, which is a downstream signaling molecule of Lphn2, remarkably rescued the D-CMP phenotype of Lphn2 knockout in the heart by restoring PGC-1α and mitochondrial function and recovering global junctional proteins. Lphn2 is a critical regulator of heart integrity by controlling mitochondrial functions and cell-to-cell junctions in cardiomyocytes. Its deficiency leads to D-CMP, which can be rescued by activators of the p38-MAPK pathway.

Therapeutically targeting proinflammatory type I interferons in systemic lupus erythematosus: efficacy and insufficiency with a specific focus on lupus nephritis.

Type I interferons (IFN-Is) are important players in the immunopathogenesis of systemic lupus erythematosus (SLE). Pathogenic events in patients with SLE are potent triggers of IFN-I induction, yet IFN-I may induce or initiate the immunopathogenesis leading to these events. Because blocking IFN-I is effective in some clinical manifestations of SLE patients, concerns about the efficacy of anti-IFN-I therapy in patients with lupus nephritis remain. Tissues from kidney biopsies of patients with lupus nephritis revealed infiltration of various immune cells and activation of inflammatory signals; however, their correlation with renal damage is not clear, which raises serious concerns about how critical the role of IFN-I is among the potential contributors to the pathogenesis of lupus nephritis. This review addresses several issues related to the roles of IFN-I in SLE, especially in lupus nephritis, including (1) the contribution of IFN-I to the development and immunopathogenesis of SLE; (2) evidence supporting the association of IFN-I with lupus nephritis; (3) therapies targeting IFN-I and IFN-I downstream signaling molecules in SLE and lupus nephritis; (4) findings challenging the therapeutic benefits of anti-IFN-I in lupus nephritis; and (5) a perspective associated with anti-IFN-I biologics for lupus nephritis treatment. In addition to providing clear pictures of the roles of IFN-I in SLE, especially in lupus nephritis, this review addresses the lately published observations and clinical trials on this topic.

RIPK2 is crucial for the microglial inflammatory response to bacterial muramyl dipeptide but not to lipopolysaccharide.

Receptor-interacting serine/threonine protein kinase 2 (RIPK2) is a kinase that plays an essential role in the modulation of innate and adaptive immune responses. As a downstream signaling molecule for nucleotide-binding oligomerization domain 1 (NOD1), NOD2, and Toll-like receptors (TLRs), it is implicated in the signaling triggered by recognition of microbe-associated molecular patterns by NOD1/2 and TLRs. Upon activation of these innate immune receptors, RIPK2 mediates the release of pro-inflammatory factors by activating mitogen-activated protein kinases (MAPKs) and nuclear factor-kappa B (NF-κB). However, whether RIPK2 is essential for downstream inflammatory signaling following the activation of NOD1/2, TLRs, or both remains controversial. In this study, we examined the role of RIPK2 in NOD2- and TLR4-dependent signaling cascades following stimulation of microglial cells with bacterial muramyl dipeptide (MDP), a NOD2 agonist, or lipopolysaccharide (LPS), a TLR4 agonist. We utilized a highly specific proteolysis targeting chimera (PROTAC) molecule, GSK3728857A, and found dramatic degradation of RIPK2 in a concentration- and time-dependent manner. Importantly, the PROTAC completely abolished MDP-induced increases in iNOS and COX-2 protein levels and pro-inflammatory gene transcription of Nos2 , Ptgs2 , Il-1β , Tnfα , Il6 , Ccl2 , and Mmp9 . However, increases in iNOS and COX-2 proteins and pro-inflammatory gene transcription induced by the TLR4 agonist, LPS, were only slightly attenuated with the GSK3728857A pretreatment. Further findings revealed that the RIPK2 PROTAC completely blocked the phosphorylation and activation of p65 NF-κB and p38 MAPK induced by MDP, but it had no effects on the phosphorylation of these two mediators triggered by LPS. Collectively, our findings strongly suggest that RIPK2 plays an essential role in the inflammatory responses of microglia to bacterial MDP but not to LPS.

Fibroblast Growth Factor Receptor 1-Specific Dehydrogelation to Release Its Inhibitor for Enhanced Lung Tumor Therapy.

Fibroblast growth factor receptor 1 (FGFR1) is emerging as a promising molecular target of lung cancer, and various FGFR1 inhibitors have exhibited significant therapeutic effects on lung cancer in preclinical research. Due to their low targeting ability or bioavailability, direct administration of these inhibitors may cause side effects. Herein, a hydrogelator, Nap-Phe-Phe-Phe-Glu-Thr-Glu-Leu-Tyr-OH (Nap-Y), was rationally designed to coassemble with an FGFR1 inhibitor nintedanib (Nin) to form a peptide hydrogel Gel Y/Nin for localized administration and FGFR1-triggered release of Nin. Upon specific phosphorylation by FGFR1 overexpressed on lung cancer cells, Nap-Y in Gel Y/Nin is converted to the hydrophilic product Nap-Phe-Phe-Phe-Glu-Thr-Glu-Leu-Tyr(H2PO3)-OH (Nap-Yp), leading to dehydrogelation of the gel and subsequent Nin release. In vitro experiments demonstrate that the release of Nin in a sustained manner from Gel Y/Nin significantly suppresses the survival, migration, and invasion of A549 cells by inhibiting FGFR1 expression and its phosphorylation function on downstream signaling molecules. Nude mouse studies show that Gel Y/Nin exhibits enhanced therapeutic efficacy on lung tumor than free Nin. We anticipate that Gel Y/Nin will be utilized for lung cancer treatment in clinical settings in the near future.

Functional activation of the AKT-mTOR signalling axis in a real-world metastatic breast cancer cohort.

Mutations of the PIK3CA/AKT/mTOR axis are common events in metastatic breast cancers (MBCs). This study was designed to evaluate the extent to which genetic alterations of the PIK3CA/AKT/mTOR can predict protein activation of this signalling axis in MBCs. Molecular profiles were generated by CLIA-certified laboratories from a real-world evidence cohort of 171 MBC patients. Genetic alterations of the PIK3CA pathway were measured using next-generation sequencing. Activation levels of AKT and downstream signalling molecules were quantified using two orthogonal proteomic methods. Protein activity was correlated with underlying genomic profiles and response to CDK4/6 inhibition in combination with endocrine treatment (ET). Oncogenic alterations of the PIK3CA/AKT/PTEN pathway were identified in 49.7% of cases. Genomic profiles emerged as poor predictors of protein activity (AUC:0.69), and AKT phosphorylation levels mimicked those of mutant lesions in 76.9% of wild-type tumours. High phosphorylation levels of the PI3K/AKT/mTOR downstream target p70S6 Kinase (T389) were associated with shorter PFS in patients treated with CDK4/6 inhibitors in combination with ET (HR:4.18 95%CI:1.19-14.63); this association was not seen when patients were classified by mutational status. Phosphoprotein-based measurements of drug targets and downstream substrates should be captured along with genomic information to identify MBCs driven by the PI3K/AKT/mTOR signalling.

Chlorpyrifos and cypermethrin combination disrupts sonic hedgehog signaling and associated regulatory molecules, leading to congenital eye defects in chick embryos

Pesticides are reported to be teratogenic for non-target species. Our studies have unraveled the teratogenicity of 50 % Chlorpyrifos & 5 % Cypermethrin combination (Ci) in developing chick embryos. A sub-lethal dose of this combination when administered to chick embryos, caused several developmental anomalies, with defects in eye development being frequent. Eye development begins at an early embryonic stage, with Sonic hedgehog (Shh) serving as a crucial signaling molecule. Shh plays a pivotal role in the early development of multiple organs, including the eye, by interacting with Pax6 and other regulatory molecules to guide the proper patterning of the eye. Thus, we hypothesized that Ci administration may lead to alteration in Shh expression which subsequently hampers downstream signaling molecules potentially contributing to congenital eye defects. Morphological, anatomical, histological, transcriptional and protein level analyses at various stages (Days 1,2,4 and 10) were carried out to evaluate the hypothesis. The results revealed a remarkable alteration of key regulators in treated embryos compared to control, providing insights into plausible causative mechanisms underlying Ci-induced congenital eye defects.

Insights into molecular characterization of NOD1-RIPK interaction and transcriptional modulation in response to LPS in spotted snakehead, Channa punctata (Bloch, 1793)

NOD1, plays a pivotal role in immune responses against bacterial as well as viral invasions. While the downstream signaling pathway of NOD1 is well understood in mammals, its characterization in lower vertebrates remains elusive. In present study, an effort was made to identify and characterize downstream signaling cascade of NOD1 in response to LPS, a potential ligand of NOD1 in teleosts, in spotted snakehead. In addition, the temporal effect of LPS on transcriptional modulation of NOD1 and its downstream signaling molecule RIPK2 was investigated. Docking studies revealed well conserved leucine rich domains of NOD1 that could bind with LPS. Further, NACHT-ATP interactions revealed differences in ATP binding motifs within the NACHT domain in spotted snakehead compared to those reported in other fish species and mammals pointing towards species-specific nature of ATP interactions within the NACHT domain. Further, it was revealed that the ssNOD1-CARD domain interacts with the CARD domain of downstream signaling molecule ssRIPK. Interestingly, LPS treatment modulated the expression of both, ssNOD1 and ssRIPK2 in a time-dependent manner.

Abstract C061: Loss of CaMK2B accelerates tumor formation and enhances metastatic competency in genetically engineered mouse models of PDAC

Abstract Background Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer- related deaths in the United States and is often not diagnosed until it has already metastasized. Our prior work established that calcium signaling pathways facilitate a pro-metastatic phenotype through the induction of an epithelial-to-mesenchymal transition (EMT) program in PDAC tumor cells, but the downstream signaling molecules mediating this phenotype have remained elusive. MethodsUsing a novel genetically engineered model of pancreatic cancer, we demonstrate that loss of Calcium/calmodulin-dependent kinase-II B (CaMK2B), a downstream effector kinase activated by calcium, enhances cellular plasticity, and increases metastasis. To this end, we generated a genetically engineered mouse model where Camk2b was knocked out in tumor cells in a lineage-labeled Kras- and p53-driven PDAC mouse model (herein KPCY-Camk2bcKO). Results Genetic deletion of Camk2b in the KPCY genetically engineered model accelerated tumor formation and dramatically reduced the overall median survival of KPCY- Camk2b cKO mice (78 days) relative to KPCY controls (136 days). Pathological and immunofluorescence analysis of KPCY-Camk2b cKO animals showed an increased number of poorly differentiated tumor regions and a higher metastatic burden, with >85% of KPCY- Camk2b cKO animals presenting with multi-focal metastases. Further analysis of tumor cells in KPCY-Camk2b cKO mice showed enhanced expression of markers of the highly aggressive squamous PDAC subtypes. Strikingly, Camk2b deletion combined with oncogenic Kras G12D (in the absence of p53 alterations), was sufficient to form lethal metastatic PDAC tumors, while age-matched control Kras G12D tumors with intact Camk2b only progressed to the acinar-to-ductal metaplasia (ADM) or Pancreatic Intraepithelial Neoplasia (PanIN) stage. Collectively, these results establish Camk2b loss as a new oncogenic and pro-metastatic mechanism in PDAC Conclusion This work demonstrates that Camk2b loss enhances tumor cell evolution and plasticity within PDAC to promote metastasis. In this context, we speculate that loss of Camk2b rewires calcium signaling to drive an aggressive subtype of PDAC with increased metastasis. Citation Format: Jessica Peura, Nikita Bhalerao, Calvin Johnson, Yamini Ogoti, Faith Keller, Emma Watson, Zhen Zhao, Jason Pitarresi. Loss of CaMK2B accelerates tumor formation and enhances metastatic competency in genetically engineered mouse models of PDAC [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C061.

Development of a novel humanized anti-TSLP monoclonal antibody, QX008N, and exploration of combination therapy of anti-TSLP antibody and anti-IL-4R antibody

BackgroundSevere asthma is a complex and chronic respiratory disease, and current conventional treatments are not effective in controlling the patients’ condition. Thymic stromal lymphopoietin (TSLP) is a key regulatory factor in the initiation and maintenance of asthma. Thus, blocking TSLP during allergic inflammation emerges as a promising therapeutic approach; however, novel anti-TSLP therapies remain to be developed. Furthermore, the importance of other signaling molecules, such as IL-4 and IL-13, should be considered. Moreover, to the best of our knowledge, the inhibitory effect of binding upstream and downstream signaling molecules has not been assessed. PurposeThis study aimed to develop a novel, humanized anti-TSLP antibody and explore the enhancement in its efficacy when combined with anti-IL-4R antibodies to treat asthma. ResultsQX008N, derived from a rabbit antibody platform, exhibits a high affinity for TSLP and superior efficacy in blocking TSLP-induced signaling pathways and inflammation in vitro compared with Tezepelumab. In a cynomolgus monkey asthma model, QX008N ameliorated lung function and reduced the levels of eosinophils and IgE. Moreover, the coadministration of QX008N with anti-IL-4R antibodies enhanced the inhibition of inflammatory mediator production triggered via costimulation in vitro. In mouse asthma models, the simultaneous blockade of TSLP and IL-4R using anti-TL4R and anti-TSLP surrogates surpassed the efficacy of monotherapy. To the best of our knowledge, the therapeutic effect of a combination of anti-TSLP and IL-4R antibodies in an asthma model has not yet been reported. ConclusionThese results furnish comprehensive preclinical evidence for QX008N as an innovative anti-TSLP therapeutic agent and provide a preliminary rationale for the development of combination therapies that simultaneously target the TSLP and IL-4R signaling pathways.

Activated THP-1 Macrophage-Derived Factors Increase the Cytokine, Fractalkine, and EGF Secretions, the Invasion-Related MMP Production, and Antioxidant Activity of HEC-1A Endometrium Cells.

Endometrium receptivity is a multifactor-regulated process involving progesterone receptor-regulated signaling, cytokines and chemokines, and additional growth regulatory factors. In the female reproductive system, macrophages have distinct roles in the regulation of receptivity, embryo implantation, immune tolerance, and angiogenesis or oxidative stress. In the present study, we investigated the effects of PMA-activated THP-1 macrophages on the receptivity-related genes, cytokines and chemokines, growth regulators, and oxidative stress-related molecules of HEC-1A endometrium cells. We established a non-contact co-culture in which the culture medium of the PMA-activated macrophages exhibiting the pro-inflammatory phenotype was used for the treatment of the endometrial cells. In the endometrium cells, the expression of the growth-related factors activin and bone morphogenetic protein 2, the growth hormone EGF, and the activation of the downstream signaling molecules pERK1/2 and pAkt were analyzed by ELISA and Western blot. The secretions of cytokines and chemokines, which are involved in the establishment of endometrial receptivity, and the expression of matrix metalloproteinases implicated in invasion were also determined. Based on the results, the PMA-activated THP-1 macrophages exhibiting a pro-inflammatory phenotype may play a role in the regulation of HEC-1A endometrium cells. They alter the secretion of cytokines and chemokines, as well as the protein level of MMPs of HEC-1A cells. Moreover, activated THP-1 macrophages may elevate oxidative stress protection of HEC-1A endometrium cells. All these suggest that pro-inflammatory macrophages have a special role in the regulation of receptivity-related and implantation-related factors of HEC-1A cells.

Insight into the regulatory mechanism of β-arrestin2 and its emerging role in diseases.

β-arrestin2, a member of the arrestin family, mediates the desensitization and internalization of most G protein-coupled receptors (GPCRs) and functions as a scaffold protein in signalling pathways. Previous studies have demonstrated that β-arrestin2 expression is dysregulated in malignant tumours, fibrotic diseases, cardiovascular diseases and metabolic diseases, suggesting its pathological roles. Transcription and post-transcriptional modifications can affect the expression of β-arrestin2. Furthermore, post-translational modifications, such as phosphorylation, ubiquitination, SUMOylation and S-nitrosylation affect the cellular localization of β-arrestin2 and its interaction with downstream signalling molecules, which further regulate the activity of β-arrestin2. This review summarizes the structure and function of β-arrestin2 and reveals the mechanisms involved in the regulation of β-arrestin2 at multiple levels. Additionally, recent studies on the role of β-arrestin2 in some major diseases and its therapeutic prospects have been discussed to provide a reference for the development of drugs targeting β-arrestin2.

Salusin‑α alleviates lipid metabolism disorders via regulation of the downstream lipogenesis genes through the LKB1/AMPK pathway.

Lipid metabolism disorders are a major cause of several chronic metabolic diseases which seriously affect public health. Salusin‑α, a vasoactive peptide, has been shown to attenuate lipid metabolism disorders, although its mechanism of action has not been reported. To investigate the effects and potential mechanisms of Salusin‑α on lipid metabolism, Salusin‑α was overexpressed or knocked down using lentiviral vectors. Hepatocyte steatosis was induced by free fatty acid (FFA) after lentiviral transfection into HepG2 cells. The degree of lipid accumulation was assessed using Oil Red O staining and by measuring several biochemical indices. Subsequently, bioinformatics was used to analyze the signaling pathways that may have been involved in lipid metabolism disorders. Finally, semi‑quantitative PCR and western blotting were used to verify the involvement of the liver kinase B1 (LKB1)/AMPK pathway. Compound C, an inhibitor of AMPK, was used to confirm this mechanism's involvement further. The results showed that Salusin‑α significantly attenuated lipid accumulation, inflammation and oxidative stress. In addition, Salusin‑α increased the levels of LKB1 and AMPK, which inhibited the expression of sterol regulatory element binding protein‑1c, fatty acid synthase and acetyl‑CoA carboxylase. The addition of Compound C abrogated the Salusin‑α‑mediated regulation of AMPK on downstream signaling molecules. In summary, overexpression of Salusin‑α activated the LKB1/AMPK pathway, which in turn inhibited lipid accumulation in HepG2 cells. This provides insights into the potential mechanism underlying the mechanism by which Salusin‑α ameliorates lipid metabolism disorders while identifying a potential therapeutic target.

Unraveling the unphosphorylated STAT3-unphosphorylated NF-κB pathway in loss of function STAT3 Hyper IgE syndrome.

Patients with loss of function signal transducer and activator of transcription 3-related Hyper IgE Syndrome (LOF STAT3 HIES) present with recurrent staphylococcal skin and pulmonary infections along with the elevated serum IgE levels, eczematous rashes, and skeletal and facial abnormalities. Defective STAT3 signaling results in reduced Th17 cells and an impaired IL-17/IL-22 response primarily due to a compromised canonical Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway that involves STAT3 phosphorylation, dimerization, nuclear translocation, and gene transcription. The non-canonical pathway involving unphosphorylated STAT3 and its role in disease pathogenesis, however, is unexplored in HIES. This study aims to elucidate the role of unphosphorylated STAT3-unphosphorylated NF-κB (uSTAT3-uNF-κB) activation pathway in LOF STAT3 HIES patients. The mRNA expression of downstream molecules of unphosphorylated STAT3-unphosphorylated NF-κB pathway was studied in five LOF STAT3 HIES patients and transfected STAT3 mutants post-IL-6 stimulation. Immunoprecipitation assays were performed to assess the binding of STAT3 and NF-κB to RANTES promoter. A reduced expression of the downstream signaling molecules of the uSTAT3-uNF-κB complex pathway, viz., RANTES, STAT3, IL-6, IL-8, ICAM1, IL-8, ZFP36L2, CSF1, MRAS, and SOCS3, in LOF STAT3 HIES patients as well as the different STAT3 mutant plasmids was observed. Immunoprecipitation studies showed a reduced interaction of STAT3 and NF-κB to RANTES in HIES patients. The reduced expression of downstream signaling molecules, specially RANTES and STAT3, confirmed the impaired uSTAT3-uNF-κB pathway in STAT3 LOF HIES. Decreased levels of RANTES and STAT3 could be a significant component in the disease pathogenesis of Hyper IgE Syndrome.

Expression of Toll-like receptors in Haemonchus Contortus resistant sheep: An innate immune parameter for host defense against gastrointestinal nematode infection

Innate immune parameters, a first line of defense against invading pathogens like bacteria, parasites, fungi, etc, play a significant role in the prevention and elimination of aetiological agents primarily by recognition of invading pathogen-specific molecules by different pattern recognition receptors. Toll-like receptors (TLRs), a type-I transmembrane glycoprotein, cause innate immune responses mainly by produing inflammatory cytokines, chemokines and interferons. The objective of present study was to determine the role of TLRs in parasite resistance in Malpura sheep. In the current study, transcript variation of TLRs and its downstream signalling molecules namely MyD88, TRIF, IRF-3, TRAF, TGF-β, NFκB, and CD14 were ascertained by real-time PCR in Haemonchus contortus resistant (R) and susceptible (S) Malpura sheep. Results have shown significantly (P<0.05) up-regulated expression of TLR-2, TLR-4, TLR-5, TLR-8 and TLR-10 in July however down-regulated patterns were observed in August and September in R-line sheep compared to S-line sheep. This indicates that at more or less equal parasite load, the TLR genes in R sheep produce more transcripts, but after parasite loads have increased hugely in the S line, they easily surpass the levels seen in the S line. Result suggests that transcriptional activity of the TLR genes was related to parasite load and there were differences between the lines at different infection intensities. Three-point transcript expression observation of the signalling molecules namely TRIF, IRF-3, TRAF, a similar pattern was observed in R sheep compared with S sheep.

Abstract We102: Synthetic N-Acylurea Derivative Reduces Angiogenesis in Human Endothelial Cells by Talin Modulation

Talin is a focal adhesion protein that activates integrins and recruits other focal adhesion proteins. Talin regulates the interactions between integrins and the extracellular matrix, which are critical for endothelial cells during angiogenesis. In this study, we successfully synthesized a novel talin modulator, N-((2-(1H-indol-3-yl)ethyl)carbamoyl)-2-(benzo[d][1,3]dioxol-5-yloxy)acetamide, referred to as KCH-1521. KCH-1521 was determined to bind talin and modulate downstream signaling molecules of talin. After 24 hrs of treatment, KCH-1521 changed the cell morphology of HUVECs and reduced focal adhesion protein expression including vinculin and paxillin. Talin downstream signaling is regulated via FAK, AKT, and ERK pathways, however, treatment with KCH-1521 decreased phosphorylation of FAK, AKT, and ERK, leading to a reduction of cell proliferation, survival, and angiogenesis. Interestingly, the expression of various angiogenic genes was significantly decreased after treatment with KCH-1521. Also, in vitro tube forming assay revealed that KCH-1521 reduced angiogenic networks in a time-dependent manner. To investigate the reversibility of its effects, KCH-1521 was removed after treatment. HUVECs recovered their morphology through rearrangement of the cytoskeleton and the expression of angiogenic genes was also recovered. By further optimization and in vivo studies of KCH-1521, a novel drug of talin modulation could be used to achieve therapeutic anti-angiogenesis for vascular diseases and cancers.

Immuno-targeting the ectopic phosphorylation sites of PDGFRA generated by MAN2A1-FER fusion in HCC.

HCC is one of the most lethal cancers for humans. Mannosidase alpha class 2A member 1 (MAN2A1)-FER is one of the most frequent oncogenic fusion genes in HCC. In this report, we showed that MAN2A1-FER ectopically phosphorylated the extracellular domains of PDGFRA, MET, AXL, and N-cadherin. The ectopic phosphorylation of these transmembrane proteins led to the activation of their kinase activities and initiated the activation cascades of their downstream signaling molecules. A panel of mouse monoclonal antibodies was developed to recognize the ectopic phosphorylation sites of PDGFRA. The analyses showed that these antibodies bound to the specific phosphotyrosine epitopes in the extracellular domain of PDGFRA with high affinity and specificity. The treatment of MAN2A1-FER-positive cancer HUH7 with one of the antibodies called 2-3B-G8 led to the deactivation of cell growth signaling pathways and cell growth arrest while having minimal impact on HUH7ko cells where MAN2A1-FER expression was disrupted. The treatment of 2-3B-G8 antibody also led to a large number of cell deaths of MAN2A1-FER-positive cancer cells such as HUH7, HEPG2, SNU449, etc., while the same treatment had no impact on HUH7ko cells. When severe combined immunodeficiency mice xenografted with HEPG2 or HUH7 were treated with monomethyl auristatin E-conjugated 2-3B-G8 antibody, it slowed the progression of tumor growth, eliminated the metastasis, and reduced the mortality, in comparison with the controls. Targeting the cancer-specific ectopic phosphorylation sites of PDGFRA induced by MAN2A1-FER may hold promise as an effective treatment for liver cancer.

Signaling dynamics in coexisting monoclonal cell subpopulations unveil mechanisms of resistance to anti-cancer compounds

BackgroundTumor heterogeneity is a main contributor of resistance to anti-cancer targeted agents though it has proven difficult to study. Unfortunately, model systems to functionally characterize and mechanistically study dynamic responses to treatment across coexisting subpopulations of cancer cells remain a missing need in oncology.MethodsUsing single cell cloning and expansion techniques, we established monoclonal cell subpopulations (MCPs) from a commercially available epidermal growth factor receptor (EGFR)-mutant non-small cell lung cancer cell line. We then used this model sensitivity to the EGFR inhibitor osimertinib across coexisting cell populations within the same tumor. Pathway-centered signaling dynamics associated with response to treatment and morphological characteristics of the MCPs were assessed using Reverse Phase Protein Microarray. Signaling nodes differentially activated in MCPs less sensitive to treatment were then pharmacologically inhibited to identify target signaling proteins putatively implicated in promoting drug resistance.ResultsMCPs demonstrated highly heterogeneous sensitivities to osimertinib. Cell viability after treatment increased > 20% compared to the parental line in selected MCPs, whereas viability decreased by 75% in other MCPs. Reduced treatment response was detected in MCPs with higher proliferation rates, EGFR L858R expression, activation of EGFR binding partners and downstream signaling molecules, and expression of epithelial-to-mesenchymal transition markers. Levels of activation of EGFR binding partners and MCPs’ proliferation rates were also associated with response to c-MET and IGFR inhibitors.ConclusionsMCPs represent a suitable model system to characterize heterogeneous biomolecular behaviors in preclinical studies and identify and functionally test biological mechanisms associated with resistance to targeted therapeutics.

A TNIP1-driven systemic autoimmune disorder with elevated IgG4

Whole-exome sequencing of two unrelated kindreds with systemic autoimmune disease featuring antinuclear antibodies with IgG4 elevation uncovered an identical ultrarare heterozygous TNIP1Q333P variant segregating with disease. Mice with the orthologous Q346P variant developed antinuclear autoantibodies, salivary gland inflammation, elevated IgG2c, spontaneous germinal centers and expansion of age-associated B cells, plasma cells and follicular and extrafollicular helper T cells. B cell phenotypes were cell-autonomous and rescued by ablation of Toll-like receptor 7 (TLR7) or MyD88. The variant increased interferon-β without altering nuclear factor kappa-light-chain-enhancer of activated B cells signaling, and impaired MyD88 and IRAK1 recruitment to autophagosomes. Additionally, the Q333P variant impaired TNIP1 localization to damaged mitochondria and mitophagosome formation. Damaged mitochondria were abundant in the salivary epithelial cells of Tnip1Q346P mice. These findings suggest that TNIP1-mediated autoimmunity may be a consequence of increased TLR7 signaling due to impaired recruitment of downstream signaling molecules and damaged mitochondria to autophagosomes and may thus respond to TLR7-targeted therapeutics.

The Oncolytic Avian Reovirus p17 Protein Inhibits Invadopodia Formation in Murine Melanoma Cancer Cells by Suppressing the FAK/Src Pathway and the Formation of theTKs5/NCK1 Complex.

To explore whether the p17 protein of oncolytic avian reovirus (ARV) mediates cell migration and invadopodia formation, we applied several molecular biological approaches for studying the involved cellular factors and signal pathways. We found that ARV p17 activates the p53/phosphatase and tensin homolog (PTEN) pathway to suppress the focal adhesion kinase (FAK)/Src signaling and downstream signal molecules, thus inhibiting cell migration and the formation of invadopodia in murine melanoma cancer cell line (B16-F10). Importantly, p17-induced formation of invadopodia could be reversed in cells transfected with the mutant PTENC124A. p17 protein was found to significantly reduce the expression levels of tyrosine kinase substrate 5 (TKs5), Rab40b, non-catalytic region of tyrosine kinase adaptor protein 1 (NCK1), and matrix metalloproteinases (MMP9), suggesting that TKs5 and Rab40b were transcriptionally downregulated by p17. Furthermore, we found that p17 suppresses the formation of the TKs5/NCK1 complex. Coexpression of TKs5 and Rab40b in B16-F10 cancer cells reversed p17-modulated suppression of the formation of invadopodia. This work provides new insights into p17-modulated suppression of invadopodia formation by activating the p53/PTEN pathway, suppressing the FAK/Src pathway, and inhibiting the formation of the TKs5/NCK1 complex.

Overexpression of Nrf2 in bone marrow mesenchymal stem cells promotes B-cell acute lymphoblastic leukemia cells invasion and extramedullary organ infiltration through stimulation of the SDF-1/CXCR4 axis.

Tumor microenvironment (TME) represents the key factor inducing leukemia development. As stromal cells within the leukemia microenvironment, Bone Marrow Mesenchymal Stem Cells (BM-MSCs) can trigger leukemia progression under certain conditions. As a critical transcription factor, nuclear factor erythroid related factor 2 (Nrf2) can modulate antioxidant response and antioxidant enzyme gene expression, and prevent various oxidative changes. We previously identified a novel mechanism by which Nrf2 promotes leukemia resistance, providing a potential therapeutic target for the treatment of drug-resistant/refractory leukemias. However, the role of Nrf2 in BM-MSCs from B-cell acute lymphoblastic leukemia (B-ALL) patients has not been clearly reported. The present work focused on investigating the effect of Nrf2 overexpression within MSCs on leukemia cell invasion, extramedullary infiltration and proliferation as well as its downstream pathway. Through clinical sample detection, in vitro cell experiments and in vivo animal experiments, the role of Nrf2 within MSCs within adult B-ALL cell migration and invasion and its potential molecular mechanism was explored through transcriptome sequencing analysis, RT-PCR, Western blot, cell migration, cell invasion, lentivirus transfection and other experiments. Nrf2 was highly expressed in BM-MSCs from patients with B-ALL as well as in BM-MSCs co-cultured with leukemia cells. Overexpression of Nrf2 within MSCs significantly promoted leukemia cell migration, invasion and proliferation. The extramedullary organ infiltration rate in B-ALL model mice receiving the combined infusion of both cell types dramatically increased relative to that of leukemia cells alone, accompanied by the significantly shortened survival time. Mechanism study found that Nrf2 overexpression within MSCs promoted PI3K-AKT/ERK1/2 phosphorylation in the downstream pathway by activating SDF-1/CXCR4 axis, ultimately leading to extramedullary infiltration of leukemia cells. High Nrf2 expression with in MSCs enhances leukemia cell invasion and migration, which then accelerates infiltration in leukemic extramedullary organs. Targeting Nrf2 or inhibiting its downstream signal molecules may be the effective interventions for B-ALL patients treatment.