Mediate Cell Survival Research Articles

Antiproliferative Effects of Thymoquinone in MCF-7 Breast and HepG2 Liver Cancer Cells: Possible Role of Ceramide and ER Stress

We aimed to investigate the impact of thymoquinone (TQ), on sphingolipid metabolites, ER stress and apoptotic pathways in MCF-7 and HepG2 cancer cells. Antiproliferative effect was exerted in cancer cells via TQ incubation at different doses and durations. Cell viability was measured by MTT assay. Levels of sphingosine-1-phosphate (S1P), C16-C24 sphingomyelins (SM) and C16-C24 ceramides (CER) were determined by LC-MS/MS. Neutral sphingomyelinase (N-SMase) enzyme activity was measured by colorimetric assay and ceramide-1-phosphate (C1P) levels were determined by immunoassay. Nuclear factor kappa-b subunit 1 (NFκB1) and glucose-regulated protein 78-kd (GRP78) gene expressions were evaluated by quantitative PCR analysis, while NF-κB p65, GRP 78 and cleaved caspase-3 protein levels were assesed by immunofluorescence and western blot analysis. Incubation with TQ significantly decreased cell viability, S1P, C1P, NF-κB1 mRNA and NF-κB p65 protein levels in cancer cells compared to controls. A significant increase was observed in N-SMase activity, cellular levels of C16-C24 CERs and cleaved caspase-3 levels in cancer cells treated with TQ. GRP78 mRNA and protein levels also increased in cancer cells treated with TQ. In conclusion, TQ-induced ceramide accumulation and ER stress in conjunction with decreased S1P, C1P and NF-κB mediated cell survival may promote cancer cell death by triggering apoptosis.

Role of nutrient-driven O-GlcNAc-post-translational modification in pancreatic exocrine and endocrine islet development.

Although the developing pancreas is exquisitely sensitive to nutrient supply in utero, it is not entirely clear how nutrient-driven post-translational modification of proteins impacts the pancreas during development. We hypothesized that the nutrient-sensing enzyme O-GlcNAc transferase (Ogt), which catalyzes an O-GlcNAc-modification onto key target proteins, integrates nutrient-signaling networks to regulate cell survival and development. In this study, we investigated the heretofore unknown role of Ogt in exocrine and endocrine islet development. By genetic manipulation in vivo and by using morphometric and molecular analyses, such as immunofluorescence imaging and single cell RNA sequencing, we show the first evidence that Ogt regulates pancreas development. Genetic deletion of Ogt in the pancreatic epithelium (OgtKOPanc) causes pancreatic hypoplasia, in part by increased apoptosis and reduced levels of of Pdx1 protein. Transcriptomic analysis of single cell and bulk RNA sequencing uncovered cell-type heterogeneity and predicted upstream regulator proteins that mediate cell survival, including Pdx1, Ptf1a and p53, which are putative Ogt targets. In conclusion, these findings underscore the requirement of O-GlcNAcylation during pancreas development and show that Ogt is essential for pancreatic progenitor survival, providing a novel mechanistic link between nutrients and pancreas development.

Role of Src homology/collagen adaptor protein p66Shc in Pyk2 translocation into mitochondria under Gq protein–coupled receptor stimulation

IntroductionMitochondrial Ca2+ (mtCa2+) uptake via a main mtCa2+ channel, mtCa2+ uniporter (MCU), mediates cell survival and death via its contribution of mitochondrial ATP synthesis and apoptotic signaling activation, respectively. Our group previously showed that Gq protein–coupled receptor (GqPCR) stimulation promotes activation of a tyrosine kinase named proline‐rich tyrosine kinase 2 (Pyk2), and its translocation from cytosol to the mitochondrial matrix, which leads to the tyrosine phosphorylation of MCU, and mtCa2+ overload, and activation of apoptotic signaling in the cardiomyocytes. However, it is still unclear how Pyk2 access from cytosol to mitochondrial matrix under GqPCR stimulation. The Src homology/collagen (Shc) adaptor protein, p66Shc, is also known to be capable to translocate to the mitochondria where it promotes increased ROS production and apoptosis. Importantly, p66Shc can bind to another tyrosine kinase Src, but its association to Pyk2 has not been well investigated.Hypothesisp66Shc serves as an important chaperon for Pyk2 translocation into the mitochondrial matrix.Aimto test whether p66Shc can associate with Pyk2.MethodsMammalian expression plasmids containing p66Shc or Pyk2 were transformed in to bacteria and purified. Overexpression of p66Shc and/or Pyk2 in HEK293T cells were performed by transient plasmid transfection. Whole cell lysates were collected and used for biochemical assay including Western blotting and co‐immunoprecipitation assay.ResultsWe confirmed that transient transfections of the plasmids containing His‐tagged p66Shc and GFP‐tagged Pyk2 were successfully yield overexpression of these proteins in HEK293Tcells. Co‐immunoprecipitation assay using GFP antibody showed that p66Shc and Pyk2 forms complex in the whole cell lysates from cells co‐transfected with two plasmids.Conclusionp66Shc can bind Pyk2 and may contribute to the mechanism for Pyk2 translocation to mitochondria. Pur next plan is to test whether the removal (overexpression) of p66Shc blocks (Pyk2 transportation and prevent excessive calcium uptake by the MCU under GqPCR stimulation both in heterologous expression system and primary cardiomyocytes.Support or Funding InformationApart of this research was supported by the Lillehei Heart Institute Summer Research Fellowship (to G.D.), American Heart Association (AHA) 18CDA34110091(to B.S.J), NIH/NHLBI R01HL136757 (to J.O.‐U.), AHA 16SDG27260248 (to J.O.‐U.), and American Physiological Society (APS) 2017 Shih‐Chun Wang Young Investigator Award.

Extracellular interaction between Neurotrophic factor‐α1 and HTR1E serotonin receptor promotes cell survival

Neurotrophic factor α1 (NFα1), classically known as carboxypeptidase E (CPE) has a non‐enzymatic role in the survival of neurons and cancer cells via binding to a putative receptor. We have identified 5‐Hydroxy tryptamine receptor 1E (HTR1E), a serotonin receptor with unknown function, as a binding partner that interacts with NFα1/CPE extracellularly to mediate cell survival. Co‐immunoprecipitation using a human LN18 cells and pull‐down assay with NFα1/CPE and HTR1E expressed in HEK293 cells confirmed interaction of these two molecules. 125I NFα1/CPE binding studies demonstrated saturable, high affinity binding to HTR1E. Molecular docking studies revealed the surface interaction between NFa1/CPE and HTR1E via 3 salt bridges, further stabilized by hydrogen bonding. We also found that HTR1E was able to activate ERK/CREB signaling upon treatment with NFα1/CPE. Pretreatment of HTR1E stable cells with NFα1/CPE followed by H2O2‐induced oxidative stress prevented a decrease in pro‐survival protein BCL2 and reduced cytotoxicity. SiRNA knock‐down of HTR1E in U118 glioblastoma cells resulted in significant inhibition of these cancer cells survival. Immunocytochemical analysis of human hippocampus indicated co‐expression of HTR1E with NFα1/CPE in CA1‐3 and dentate gyrus, and cellular co‐localization at the cell membrane in CA3 neurons. This study has uncovered a novel role for HTR1E as a binding partner for NFα1/CPE, and their interaction activates the ERK‐CREB‐BCL2 pathway to promote cell survival.Support or Funding InformationDivision of intramural research, NICHD, NIH, Bethesda, MD, USA

Kinetic and Mechanistic Characterization of a Potent and Selective Inhibitor for Human AXL Receptor Tyrosine Kinase

The AXL receptor tyrosine kinase (AXL) is a member of the TAM RTK family comprising TYRO3, AXL and MER. AXL signaling is activated by binding of the growth arrest‐specific protein 6 (Gas6) or vitamin K‐dependent protein S (PROS1), which subsequently induces homodimerization and autophosphorylation, thereby initiating a conventional RTK signaling pathway. Maximal stimulation requires an extracellular lipid, phosphatidylserine (PtdSer).Activation of AXL regulates an array of signal transduction pathways, including those mediating cell survival, proliferation and migration, as well as promoting an immunosuppressive phenotype within the tumor microenvironment 1, 2. AXL has been shown to be highly expressed in sarcoma, renal cell carcinoma, pancreatic adenocarcinoma and other types of tumors, correlative with poor prognosis in cancer patients. Furthermore, AXL has been shown to facilitate both intrinsic and acquired resistance to multiple anti‐cancer drugs 3, suggesting therapeutic targeting of AXL may be an effective anti‐cancer strategy.We have developed a potent, reversible and competitive small molecule AXL inhibitor. This inhibitor (Compound 1) inhibits the activity of recombinant human AXL kinase domain with nanomolar potency, as determined by detection of phosphorylated substrate using homogeneous time‐resolved fluorescence (HTRF). Compound 1 is also highly selective against TYRO3, MERTK and MET kinase domains. The compound binds to the intracellular AXL kinase domain with a nanomolar dissociation rate constant measured by monitoring displacement of a competitive fluorescent tracer using bioluminescence resonance energy transfer (BRET). In order to assess the inhibitory effect of the compound on downstream signal transduction, AXL autophosphorylation‐induced SH2 translocation was determined using an AXL PathHunter® assay and phosphorylation of AKT Ser473 induced by AXL activation was also assessed by AlphaLISA.Support or Funding Information

Calebin A Potentiates the Effect of 5-FU and TNF-β (Lymphotoxin α) against Human Colorectal Cancer Cells: Potential Role of NF-κB.

Objective: The majority of chemotherapeutic agents stimulate NF-κB signaling that mediates cell survival, proliferation and metastasis. The natural turmeric non-curcuminoid derivate Calebin A has been shown to suppress cell growth, invasion and colony formation in colorectal cancer cells (CRC) by suppression of NF-κB signaling. Therefore, we hypothesized here that Calebin A might chemosensitize the TNF-β-treated tumor cells and potentiates the effect of 5-Fluorouracil (5-FU) in advanced CRC. Materials and Methods: CRC cells (HCT116) and their clonogenic 5-FU chemoresistant counterparts (HCT116R) were cultured in monolayer or alginate-based 3D tumor environment culture and were treated with/without Calebin A, TNF-β, 5-FU, BMS-345541 and DTT (dithiothreitol). Results: The results showed that TNF-β increased proliferation, invasion and resistance to apoptosis in chemoresistant CRC cells. Pretreatment with Calebin A significantly chemosensitized HCT116R to 5-FU and inhibited the TNF-β-induced enhanced efforts for survival, invasion and anti-apoptotic effects. We found further that Calebin A significantly suppressed TNF-β-induced phosphorylation and nuclear translocation of p65-NF-κB, similar to BMS-345541 (specific IKK inhibitor) and NF-κB-induced tumor-promoting biomarkers (NF-κB, β1-Integrin, MMP-9, CXCR4, Ki67). This was associated with increased apoptosis in HCT116 and HCT116R cells. Furthermore, blocking of p65-NF-κB stimulation by Calebin A was imparted through the downmodulation of p65-NF-κB binding to the DNA and this suppression was turned by DTT. Conclusion: Our findings indicate, for the first time, that Calebin A chemosensitizes human CRC cells to chemotherapy by targeting of the p65-NF-κB signaling pathway.

Discovery of novel molecular characteristics and cellular biological properties in ameloblastoma

Ameloblastoma is a rare odontogenic benign tumor accounting for less than 1% of head and neck tumors. Advanced next generation sequencing (NGS) analyses identified high frequency of BRAF V600E and SMO L412F mutations in ameloblastoma. Despite the existence of whole genomic sequence information from patients with ameloblastoma, entire molecular signature of and the characteristics of ameloblastoma cells are still obscure. In this study, we sought to uncover the molecular basis of ameloblastoma and to determine the cellular phenotype of ameloblastoma cells with BRAF mutations. Our comparative cDNA microarray analysis and gene set enrichment analysis (GSEA) showed that ameloblastoma exhibited a distinct gene expression pattern from the normal tissues: KRAS‐responsive gene set is significantly activated in ameloblastoma. Importantly, insulin like growth factor 2 (IGF2), a member of KRAS‐responsive genes, enhances the proliferation of an ameloblastoma cell line AMU‐AM1 with BRAF mutation. In addition, Toll‐like receptor 2 (TLR2) knockdown readily inactivated KRAS‐responsive gene sets as well as increases caspase activities, suggesting that TLR2 signaling may mediate cell survival signaling in ameloblastoma cells. Collectively, the findings may help to further clarify the pathophysiology of ameloblastoma and lead to the development of precision medicine for patients with ameloblastoma.

Loss of PI3 kinase association improves the sensitivity of secondary mutation of KIT to Imatinib

BackgroundKIT mutations are the predominant driver mutations in gastrointestinal stromal tumors (GISTs), and targeted therapy against KIT has improved treatment outcome dramatically. However, gaining secondary mutation of KIT confers drug resistance of GISTs leading to treatment failure.ResultsIn this study, we found that secondary mutation of KIT dramatically increases the ligand-independent activation of the receptor and their resistance to the often used KIT inhibitor Imatinib in the treatment of GISTs. PI3 kinase plays essential roles in the cell transformation mediated by the primary mutation of KIT. We found that loss of PI3 kinase association, but not the inhibition of the lipid kinase activity of PI3 kinase, inhibits the ligand-independent activation of secondary mutations of KIT, and increases their sensitivity to Imatinib, and loss of PI3 kinase association inhibits secondary mutations of KIT mediated cell survival and proliferation in vitro. The in vivo assay further showed that the growth of tumors carrying secondary mutations of KIT is more sensitive to Imatinib when PI3 kinase association is blocked while inhibition of the lipid kinase activity of PI3 kinase cannot inhibit tumor growth, indicating that PI3 kinase is important for the drug resistance of secondary mutation of KIT independent of the lipid kinase activity of PI3 kinase.ConclusionsOur results suggested that PI3 kinase is necessary for the ligand-independent activation of secondary mutations of KIT, and loss of PI3 kinase association improves the sensitivity of secondary mutations to the targeted therapy independent of the lipid kinase activity of PI3 kinase.

Abstract B107: Metabolic reprogramming enhances the efficacy of mTOR inhibition in colorectal cancer

Abstract Background: PI3K/mTOR pathway is mutated in 10-20% of colorectal cancer (CRC) specimens and has been associated with poor survival. Phosphatidic acid (PA) is a central lipid membrane metabolite and lipid second messenger which has been shown to target mTOR. It is thought that PA lipid signaling to mTOR in part promotes mTOR mediated cancer cell growth, proliferation and survival. Diacylglycerol kinases (DGKs) are one of several mechanisms of PA generation. In this study, we found diacylglycerol kinases to be synthetically lethal in mTOR inhibitor resistant CRC. We evaluated the anti-proliferative, pharmacodynamic and metabolic effects of dual inhibition with mTOR (TAK-228) and DGK (ritanserin and R59022) inhibitors. Methods: A synthetic lethal screen was performed with two TAK-228 resistant colorectal cancer cell lines (HCT116 and SW620). Subsequent experiments were performed with one TAK-228 sensitive (DLD1) and one resistant (HCT116) CRC cell lines. Efficacy of TAK-228/ritanserin and TAK-228/R59022 combination therapy was evaluated with CellTiter-Glo cell viability and clonogenic colony formation assays. Global metabolomics profiling of DLD1 and HCT116 cells upon treatment with TAK-228, R59022, and in combination was performed using ultra high pressure liquid chromatography coupled to mass spectrometry. Pharmacologic DGK inhibition was phenocopied using lentiviral shRNA knockdown of DGKα. Immunoblotting was performed to evaluate mechanism of action of TAK-228 combination therapy. Results: TAK-228 combined with ritanserin and R59022 demonstrated decreased cell viability and colony formation as compared to either single agent. Immunoblotting confirmed TAK-228 abrogates PI3K/mTOR pathway activity. DGK inhibition alone resulted in a compensatory activation of mTOR signaling. DGK inhibition disrupted the phosphatidic acid pathway in DLD1 and HCT116 as evidenced by a decrease in PA synthesis and elevation of glycerol 3-phosphate levels, respectively: altering energy metabolism. Specifically, in HCT116, glucose utilization, glutaminolysis, and Krebs cycle anaplerosis were elevated; while one carbon metabolism was decreased. Lentiviral shRNA transduction resulted in DGKα knockdown as evaluated by RT-PCR and immunoblot. Phenocopy combination therapy with TAK-228 and DGKα knockdown resulted in an increased sensitivity to mTOR inhibition compared to mock transduced control. Conclusions: Pharmacologic and shRNA knockdown inhibition of DGK in combination with mTOR inhibition resulted in decreased cancer cell viability and decreased colony formation. Pharmacologic inhibition of mTOR and DGK, alone or in combination, alter metabolic wiring in crucial pathways such as energy metabolism, nucleotide biosynthesis, and the generation of lipid precursors. Impaired phosphatidic acid production may sensitize cells to mTOR inhibition. These results suggest a therapeutic anti-cancer advantage of simultaneously targeting lipid signaling/metabolism via diacylglycerol kinases and mTOR. Citation Format: Peter J Klauck, Hayley J Hawkins, Madison Weber, Stacey M Bagby, Christopher H Lieu, Sarah J Hartman, Betelehem W Yacob, Kelly D Sullivan, Monica Brown, Julie A Reisz, Angelo D’Alessandro, Wells A Messersmith, S Gail Eckhardt, Todd M Pitts. Metabolic reprogramming enhances the efficacy of mTOR inhibition in colorectal cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B107. doi:10.1158/1535-7163.TARG-19-B107

Bag-1 stimulates Bad phosphorylation through activation of Akt and Raf kinases to mediate cell survival in breast cancer

BackgroundBag-1 (Bcl-2-associated athanogene) is a multifunctional anti-apoptotic protein frequently overexpressed in cancer. Bag-1 interacts with a variety of cellular targets including Hsp70/Hsc70 chaperones, Bcl-2, nuclear hormone receptors, Akt and Raf kinases. In this study, we investigated in detail the effects of Bag-1 on major cell survival pathways associated with breast cancer.MethodsUsing immunoblot analysis, we examined Bag-1 expression profiles in tumor and normal tissues of breast cancer patients with different receptor status. We investigated the effects of Bag-1 on cell proliferation, apoptosis, Akt and Raf kinase pathways, and Bad phosphorylation by implementing ectopic expression or knockdown of Bag-1 in MCF-7, BT-474, MDA-MB-231 and MCF-10A breast cell lines. We also tested these in tumor and normal tissues from breast cancer patients. We investigated the interactions between Bag-1, Akt and Raf kinases in cell lines and tumor tissues by co-immunoprecipitation, and their subcellular localization by immunocytochemistry and immunohistochemistry.ResultsWe observed that Bag-1 is overexpressed in breast tumors in all molecular subtypes, i.e., regardless of their ER, PR and Her2 expression profile. Ectopic expression of Bag-1 in breast cancer cell lines results in the activation of B-Raf, C-Raf and Akt kinases, which are also upregulated in breast tumors. Bag-1 forms complexes with B-Raf, C-Raf and Akt in breast cancer cells, enhancing their phosphorylation and activation, and ultimately leading to phosphorylation of the pro-apoptotic Bad protein at Ser112 and Ser136. This causes Bad’s re-localization to the nucleus, and inhibits apoptosis in favor of cell survival.ConclusionsOverall, Bad inhibition by Bag-1 through activation of Raf and Akt kinases is an effective survival and growth strategy exploited by breast cancer cells. Therefore, targeting the molecular interactions between Bag-1 and these kinases might prove an effective anticancer therapy.

Retraction Note: CD109 Mediates Cell Survival in Hepatocellular Carcinoma Cells.

This article [1] is retracted at the request of Editor-in-Chief.

Novel ADAM-17 inhibitor ZLDI-8 inhibits the proliferation and metastasis of chemo-resistant non-small-cell lung cancer by reversing Notch and epithelial mesenchymal transition in vitro and in vivo

Acquired drug-resistant non-small cell lung cancer (NSCLC) has strong proliferation ability and is prone to epithelial-mesenchymal transition (EMT) and subsequent metastasis. Notch pathway mediates cell survival and EMT and is involved in the induction of multidrug resistance (MDR). ZLDI-8 is an inhibitor of Notch activating/cleaving enzyme ADAM-17 we found before. However, the effects of ZLDI-8 on resistant NSCLC was unclear. Here, we demonstrated for the first time that ZLDI-8 could induce apoptosis in lung cancer, especially in chemotherapy-resistant non-small cell lung cancer cells, and also inhibit migration, invasion and EMT phenotype of drug-resistant lung cancer. ZLDI-8 inhibits the Notch signaling pathway, thereby regulating the expression of survival/apoptosis and EMT-related proteins. Moreover, ZLDI-8 suppresses multidrug-resistant lung cancer xenograft growth in vivo and blocks metastasis in a tail vein injection mice model. Therefore, ZLDI-8 is expected to be an effective agent in the treatment of drug-resistant lung cancer.

Tunneling nanotubes mediate the expression of senescence markers in mesenchymal stem/stromal cell spheroids.

The therapeutic potential of mesenchymal stem/stromal cells (MSCs) is limited by acquired senescence following prolonged culture expansion and high-passage numbers. However, the degree of cell senescence is dynamic, and cell-cell communication is critical to promote cell survival. MSC spheroids exhibit improved viability compared with monodispersed cells, and actin-rich tunneling nanotubes (TNTs) may mediate cell survival and other functions through the exchange of cytoplasmic components. Building upon our previous demonstration of TNTs bridging MSCs within these cell aggregates, we hypothesized that TNTs would influence the expression of senescence markers in MSC spheroids. We confirmed the existence of functional TNTs in MSC spheroids formed from low-passage, high-passage, and mixtures of low- and high-passage cells using scanning electron microscopy, confocal microscopy, and flow cytometry. The contribution of TNTs toward the expression of senescence markers was investigated by blocking TNT formation with cytochalasin D (CytoD), an inhibitor of actin polymerization. CytoD-treated spheroids exhibited decreases in cytosol transfer. Compared with spheroids formed solely of high-passage MSCs, the addition of low-passage MSCs reduced p16 expression, a known genetic marker of senescence. We observed a significant increase in p16 expression in high-passage cells when TNT formation was inhibited, establishing the importance of TNTs in MSC spheroids. These data confirm the restorative role of TNTs within MSC spheroids formed with low- and high-passage cells and represent an exciting approach to use higher-passage cells in cell-based therapies.

Cardiomyocyte targeted overexpression of IGF1 during detraining restores compromised cardiac condition via mTORC2 mediated switching of PKCδ to PKCα

Altered cardiac adaptation of physiologically hypertrophied heart during detraining remained obscure for long time. We had previously reported the switching of protein kinase C (PKC) isoforms (-α to -δ) associated with functional deterioration of heart at detraining in mice undergone swim exercise. Here we report that, myocardium targeted overexpression of insulin-like growth factor 1 (IGF1) and knockdown of insulin-like growth factor 1 receptor (IGF1R) during detraining and exercise respectively altered the activation of PKCs and eventual cardiac condition. Moreover, downregulation of mammalian target of rapamycin complex 2 (mTORC2) was recorded in both IGF1R knockdown or detraining groups. Additionally, knocking down of mTORC2 during exercise exhibited impaired cardiac condition. Interestingly, significantly increased interactions of mTORC2 with both PKCα and δ was recorded exclusively in exercise group. This interaction resulted into hydrophobic motif phosphorylation of both PKCs (Serine657-PKCα; Serine662-PKCδ). Serine phosphorylation on one hand activated PKCα mediated cell survival and on the other hand alleviated the apoptotic activity of PKCδ during exercise. Mutation of Serine662 of PKCδ in exercised mice showed higher Tyrosine311 phosphorylation with increased apoptotic load similar to that in detrained animals. These observations confirmed that differential and conditional activation of PKCs depend upon IGF1 induced mTORC2 activation. Furthermore, blocking of PKCα resulted in activated p53 which in turn repressed IGF1 expression during swim, mimicking the condition of detrained heart. In conclusion, this is the first report to unravel the intricate molecular mechanism of switching a physiologically hypertrophied heart to a pathologically hypertrophied heart during exercise withdrawal.

FMS-like Tyrosine Kinase 3/FLT3: From Basic Science to Clinical Implications.

FMS-like tyrosine kinase 3 (FLT3) is a receptor tyrosine kinase that is expressed almost exclusively in the hematopoietic compartment. Its ligand, FLT3 ligand (FL), induces dimerization and activation of its intrinsic tyrosine kinase activity. Activation of FLT3 leads to its autophosphorylation and initiation of several signal transduction cascades. Signaling is initiated by the recruitment of signal transduction molecules to activated FLT3 through binding to specific phosphorylated tyrosine residues in the intracellular region of FLT3. Activation of FLT3 mediates cell survival, cell proliferation, and differentiation of hematopoietic progenitor cells. It acts in synergy with several other cytokines to promote its biological effects. Deregulated FLT3 activity has been implicated in several diseases, most prominently in acute myeloid leukemia where around one-third of patients carry an activating mutant of FLT3 which drives the disease and is correlated with poor prognosis. Overactivity of FLT3 has also been implicated in autoimmune diseases, such as rheumatoid arthritis. The observation that gain-of-function mutations of FLT3 can promote leukemogenesis has stimulated the development of inhibitors that target this receptor. Many of these are in clinical trials, and some have been approved for clinical use. However, problems with acquired resistance to these inhibitors are common and, furthermore, only a fraction of patients respond to these selective treatments. This review provides a summary of our current knowledge regarding structural and functional aspects of FLT3 signaling, both under normal and pathological conditions, and discusses challenges for the future regarding the use of targeted inhibition of these pathways for the treatment of patients.

TBL1XR1 is involved in c-Met-mediated tumorigenesis of human nonsmall cell lung cancer.

Nonsmall cell lung carcinoma (NSCLC) contributes to the highest number of cancer deaths globally. Metastases and chemoresistance are two major confounders to the treatment efficacy in NSCLC. Transducin (β)-like 1 X-linked receptor 1 (TBL1XR1) has been associated with high rates of metastases in breast, gastric, and stomach cancers. However, the role of TBL1XR1 in lung cancers remains underexplored. We selected matched and cancerous lung tissues to establish the upregulation of TBL1XR1. Using in vitro assays, we assessed the influence of TBL1XR1 on various cancer phenotypes, namely cell proliferation, chemoresistance, invasion, and metastases in a CRISPR-Cas9-mediated knock out model (A549 cells), and H460 cell lines overexpressing TBL1XR1. We found that TBL1XR1 is overexpressed in NSCLC tissue and patient sera in comparison to paired adjacent normal tissue. Overexpression of TBL1XR1 in NSCLC cell lines mediates cell survival, proliferation, and metastases. TBL1XR1 was found to regulate MEK and Akt pathways through their master regulator c-Met. We observed that activation of c-Met is downregulated in the absence of TBL1XR1. Our study strengthens the contention that TBL1XR1 is a biomarker for prognosis of NSCLC. It may also be considered as an adjunct or core therapeutic target to overcome cisplatin resistance in lung cancers.

Tumour Necrosis Factor Alpha in Intestinal Homeostasis and Gut Related Diseases.

The intestinal epithelium constitutes an indispensable single-layered barrier to protect the body from invading pathogens, antigens or toxins. At the same time, beneficial nutrients and water have to be absorbed by the epithelium. To prevent development of intestinal inflammation or tumour formation, intestinal homeostasis has to be tightly controlled and therefore a strict balance between cell death and proliferation has to be maintained. The proinflammatory cytokine tumour necrosis factor alpha (TNFα) was shown to play a striking role for the regulation of this balance in the gut. Depending on the cellular conditions, on the one hand TNFα is able to mediate cell survival by activating NFκB signalling. On the other hand, TNFα might trigger cell death, in particular caspase-dependent apoptosis but also caspase-independent programmed necrosis. By regulating these cell death and survival mechanisms, TNFα exerts a variety of beneficial functions in the intestine. However, TNFα signalling is also supposed to play a critical role for the pathogenesis of inflammatory bowel disease (IBD), infectious diseases, intestinal wound healing and tumour formation. Here we review the literature about the physiological and pathophysiological role of TNFα signalling for the maintenance of intestinal homeostasis and the benefits and difficulties of anti-TNFα treatment during IBD.

TRPC5‑induced autophagy promotes the TMZ‑resistance of glioma cells via the CAMMKβ/AMPKα/mTOR pathway.

Temozolomide (TMZ) is the first choice chemotherapy agent against glioblastoma, but the TMZ chemotherapy resistance has restricted the clinical application. Although autophagy is considered an adaptive response for cell survival under the pressure of chemotherapy and associated with chemotherapy resistance, its initiator and the precise molecular mechanism remains unknown. In the present study, it was determined that TMZ increases the transient receptor potential cation channel subfamily C member 5 (TRPC5) protein expression and the basal autophagy level, and the upregulation of autophagy is mediated by TRPC5 in glioma cells. Additionally, knockdown of TRPC5 upregulated the chemotherapy sensitivity in vitro and in vivo. Furthermore, TRPC5‑small interfering RNA and pharmacological inhibition indicated that the Ca2+/calmodulin dependent protein kinase β (CaMKKβ)/AMP‑activated protein kinase α (AMPKα)/mechanistic target of rapamycin kinase (mTOR) pathway mediates cell survival autophagy during TMZ treatment. In addition, TMZ‑resistant U87/TMZ cells retained a high basal autophagy level, while silence of TRPC5 expression or inhibition of autophagy reversed TMZ resistance. Thus, the present study revealed that TRPC5, an initiator of autophagy, upregulated TMZ resistance via the CaMKKβ/AMPKα/mTOR pathway and this indicated a novel therapeutic site for drug resistance in glioma chemotherapy.

Interaction between the BAG1S isoform and HSP70 mediates the stability of anti-apoptotic proteins and the survival of osteosarcoma cells expressing oncogenic MYC

BackgroundThe oncoprotein MYC has the dual capacity to drive cell cycle progression or induce apoptosis, depending on the cellular context. BAG1 was previously identified as a transcriptional target of MYC that functions as a critical determinant of this cell fate decision. The BAG1 protein is expressed as multiple isoforms, each having an array of distinct biochemical functions; however, the specific effector function of BAG1 that directs MYC-dependent cell survival has not been defined.MethodsIn our studies the human osteosarcoma line U2OS expressing a conditional MYC-ER allele was used to induce oncogenic levels of MYC. We interrogated MYC-driven survival processes by modifying BAG1 protein expression. The function of the separate BAG1 isoforms was investigated by depleting cells of endogenous BAG1 and reintroducing the distinct isoforms. Flow cytometry and immunoblot assays were performed to analyze the effect of specific BAG1 isoforms on MYC-dependent apoptosis. These experiments were repeated to determine the role of the HSP70 chaperone complex in BAG1 survival processes. Finally, a proteomic approach was used to identify a set of specific pro-survival proteins controlled by the HSP70/BAG1 complex.ResultsLoss of BAG1 resulted in robust MYC-induced apoptosis. Expression of the larger isoforms of BAG1, BAG1L and BAG1M, were insufficient to rescue survival in cells with oncogenic levels of MYC. Alternatively, reintroduction of BAG1S significantly reduced the level of apoptosis. Manipulation of the BAG1S interaction with HSP70 revealed that BAG1S provides its pro-survival function by serving as a cofactor for the HSP70 chaperone complex. Via a proteomic approach we identified and classified a set of pro-survival proteins controlled by this HSP70/BAG1 chaperone complex that contribute to the BAG1 anti-apoptotic phenotype.ConclusionsThe small isoform of BAG1, BAG1S, in cooperation with the HSP70 chaperone complex, selectively mediates cell survival in MYC overexpressing tumor cells. We identified a set of specific pro-survival clients controlled by the HSP70/BAG1S chaperone complex. These clients define new nodes that could be therapeutically targeted to disrupt the survival of tumor cells driven by MYC activation. With MYC overexpression occurring in most human cancers, this introduces new strategies for cancer treatment.

Double-Edged Sword of Tumour Suppressor Genes in Schizophrenia.

Schizophrenia (SCZ) is a common psychiatric disorder with polygenetic pathogenesis. Among the many identified candidate genes and loci, the group of tumour suppressor genes has drawn our interest. In this mini-review article, we describe evidence of a correlation between major tumour suppressor genes and SCZ development. Genetic mutations ranging from single nucleotide polymorphisms to large structural alterations have been found in tumour-related genes in patients with SCZ. Epigenetic mechanisms, including DNA methylation/acetylation and microRNA regulation of tumour suppressor genes, have also been implicated in SCZ. Beyond genetic correlations, we hope to establish causal relationships between tumour suppressor gene function and SCZ risk. Accumulating evidence shows that tumour suppressor genes may mediate cell survival and neural development, both of which contribute to SCZ aetiology. Moreover, converging intracellular signalling pathways indicate a role of tumour suppressor genes in SCZ pathogenesis. Tumour suppressor gene function may mediate a direct link between neural development and function and psychiatric disorders, including SCZ. A deeper understanding of how neural cell development is affected by tumour suppressors may lead to improved anti-psychotic drugs.