c-Kit Activation Research Articles

Platelet Derived Growth Factor Alpha (PDGFRα) Induces the Activation of Cardiac Fibroblasts by Activating c-Kit.

BackgroundEnhanced platelet-derived growth factor receptor α (PDGFRα) signaling pathway activity leads to cardiac fibrosis. However, because of the pleiotropic effects of PDGFR signaling, its role in mediating the cardiac fibrotic response remains poorly understood. This study aimed to investigate the regulatory effect of c-Kit in cardiac fibroblasts activated by PDGFRα signaling.Material/MethodsA cardiac fibrosis mice model was induced using isoproterenol, and the heart tissues of mice were tested through western blotting and real-time quantitative PCR (RT-qPCR). The cardiac fibroblasts of neonatal mice were treated with PDGF-AA or transfected with small interfering RNAs (siRNAs) specific for the mouse c-Kit gene. The levels of collagen I, collagen III, and alpha-smooth muscle actin (α-SMA) were analyzed using western blotting and RT-qPCR.ResultsIn the heart of the cardiac fibrosis mice model, the activity of c-Kit was enhanced. PDGF-AA treatment accelerated the activity of c-Kit in cardiac fibroblasts. In addition, imatinib inhibited the activity of c-Kit in vivo and in vitro. Moreover, inhibition of c-Kit by siRNAs reduced the expression of α-SMA and collagens in the activated cardiac fibroblasts. Furthermore, PDGFRα directly bound c-Kit in cardiac fibroblasts and stimulated the expression of stem cell factor (SCF).ConclusionsOur data demonstrated that PDGF/PDGFRα induced the activation of cardiac fibroblasts by activating c-Kit. This study indicated that c-Kit could be used as a potential therapeutic target for treatment of cardiac fibrosis.

The stem cell factor (SCF)/c-KIT system in carcinogenesis of reproductive tissues: What does the hormonal regulation tell us?

The tyrosine kinase receptor c-KIT and its ligand, the stem cell factor (SCF) are expressed in several tissues of male and female reproductive tract, playing an important role in the regulation of basic biological processes. The activation of c-KIT by SCF controls, cell survival and death, cell differentiation and migration. Also, the SCF/c-KIT system has been implicated in carcinogenesis of reproductive tissues due to its altered expression pattern or overactivation in consequence of gain-of-functions mutations. Over the years, it has also been shown that hormones, the primary regulators of reproductive function and causative agents in the case of hormone-dependent cancers, are also able to control the SCF/c-KIT tissue levels. Therefore, it is liable to suppose that disturbed SCF/c-KIT expression driven by (de)regulated hormone actions can be a relevant step towards carcinogenesis. The present review describes the SCF and c-KIT expression in cancers of reproductive tissues, discussing the implications of the hormonal regulation of the SCF/c-KIT system in cancer development. Understanding the relationship between hormonal imbalance and the SCF/c-KIT expression and activity would be relevant in the context of novel therapeutic approaches in reproductive cancers.

C-Kit modifies the inflammatory status of smooth muscle cells.

Backgroundc-Kit is a receptor tyrosine kinase present in multiple cell types, including vascular smooth muscle cells (SMC). However, little is known about how c-Kit influences SMC biology and vascular pathogenesis.MethodsHigh-throughput microarray assays and in silico pathway analysis were used to identify differentially expressed genes between primary c-Kit deficient (KitW/W–v) and control (Kit+/+) SMC. Quantitative real-time RT-PCR and functional assays further confirmed the differences in gene expression and pro-inflammatory pathway regulation between both SMC populations.ResultsThe microarray analysis revealed elevated NF-κB gene expression secondary to the loss of c-Kit that affects both the canonical and alternative NF-κB pathways. Upon stimulation with an oxidized phospholipid as pro-inflammatory agent, c-Kit deficient SMC displayed enhanced NF-κB transcriptional activity, higher phosphorylated/total p65 ratio, and increased protein expression of NF-κB regulated pro-inflammatory mediators with respect to cells from control mice. The pro-inflammatory phenotype of mutant cells was ameliorated after restoring c-Kit activity using lentiviral transduction. Functional assays further demonstrated that c-Kit suppresses NF-κB activity in SMC in a TGFβ-activated kinase 1 (TAK1) and Nemo-like kinase (NLK) dependent manner.DiscussionOur study suggests a novel mechanism by which c-Kit suppresses NF-κB regulated pathways in SMC to prevent their pro-inflammatory transformation.

Orai1 mediates tumor-promoting store-operated Ca2+ entry in human gastrointestinal stromal tumors via c-KIT and the extracellular signal-regulated kinase pathway.

Gastrointestinal stromal tumors originate from interstitial cells of Cajal, the pacemaker cells of the gut. Ca2+ regulates the pacemaker activity of interstitial cells of Cajal. Store-operated Ca2+ entry mediates the majority of Ca2+ entry in most cancer cells and may be a factor in regulating intracellular Ca2+ in interstitial cells of Cajal and gastrointestinal stromal tumors. Therefore, a blockade of this mechanism may affect the progression of gastrointestinal stromal tumors. Orai1 is the pore subunit of store-operated Ca2+ channels. Here, we reported that Orai1 was overexpressed in gastrointestinal stromal tumor tissues and was positively correlated with a high-risk grade in gastrointestinal stromal tumor patients. Furthermore, upon Orai1 silencing, the functional store-operated Ca2+ entry in gastrointestinal stromal tumor cells was decreased, indicating that the function of store-operated Ca2+ entry was mediated by Orai1. Inhibition of Orai1-mediated store-operated Ca2+ entry by Orai1 silencing or store-operated Ca2+ entry blockers (SKF-96365 and 2-aminoethyl diphenylborate) induced obvious cell proliferation suppression, cell-cycle distribution, and apoptosis stimulation in GIST-T1 cells. Conversely, Orai1 overexpression increased store-operated Ca2+ entry and cell proliferation in GIST882 cells. In addition, we found that activation of c-KIT and the extracellular signal-regulated kinase pathway participated in the oncogenic functions of Orai1-mediated store-operated Ca2+ entry in gastrointestinal stromal tumor cells. These results revealed that Orai1-mediated store-operated Ca2+ entry is critical for gastrointestinal stromal tumor cell proliferation via c-KIT and ERK signaling pathway activation. Orai1-mediated store-operated Ca2+ entry plays an oncogenic role and may be a novel prognostic factor and therapeutic target for patients with gastrointestinal stromal tumors.

Retinoic acid triggers c-kit gene expression in spermatogonial stem cells through an enhanceosome constituted between transcription factor binding sites for retinoic acid response element (RARE), spleen focus forming virus proviral integration oncogene (SPFI1) (PU.1) and E26 transformation-specific (ETS).

Restricted availability of retinoic acid (RA) in the testicular milieu regulates transcriptional activity of c-kit (KIT, CD117), which aids in the determination of spermatogonial stem-cell differentiation. The effect of RA on c-kit has been reported previously, but its mode of genomic action remains unresolved. We studied the molecular machinery guiding RA responsiveness to the c-kit gene using spermatogonial stem-cell line C18-4 and primary spermatogonial cells. A novel retinoic acid response element (RARE) positioned at -989 nucleotides upstream of the transcription start site (TSS) was identified, providing a binding site for a dimeric RA receptor (i.e. retinoic acid receptor gamma (RARγ) and retinoic X receptor). RA treatment influenced c-kit promoter activity, along with endogenous c-kit expression in C18-4 cells. A comprehensive promoter deletion assay using the pGL3B reporter system characterised the region spanning -271bp and -1011bp upstream of the TSS, which function as minimal promoter and maximal promoter, respectively. In silico analysis predicted that the region -1011 to +58bp comprised the distal enhancer RARE and activators such as spleen focus forming virus proviral integration oncogene (SPFI1) (PU.1), specificity protein 1 (SP1) and four E26 transformation-specific (ETS) tandem binding sites at the proximal region. Gel retardation and chromatin immunoprecipitation (ChIP) assays showed binding for RARγ, PU.1 and SP1 to the predicted consensus binding sequences, whereas GABPα occupied only two out of four ETS binding sites within the c-kit promoter region. We propose that for RA response, an enhanceosome is orchestrated through scaffolding of a CREB-binding protein (CBP)/p300 molecule between RARE and elements in the proximal promoter region, controlling germ-line expression of the c-kit gene. This study outlines the fundamental role played by RARγ, along with other non-RAR transcription factors (PU.1, SP1 and GABPα), in the regulation of c-kit expression in spermatogonial stem cells in response to RA.

Phosphoproteomic Landscaping Unveils Constitutive cKIT Activation in Human Erythroblasts from Polycythemia Vera (PV) Patients

PV is characterized by the gain-of-function V617F mutation in JAK2, the gene encoding the first signaling element of the cytokine receptor superfamily. Progenitor cells from PV are more sensitive in vitro to Imatinib, which also inhibits the SCF receptor cKIT, than those from normal sources (Gaikwad, Exp Hemat 2007;35:931) and clinical trials with similar tyrosine kinase inhibitors have been reported to have some efficacy in PV (Nussenzveig, Int J Hematol 2009;90:58; Silver, Leuk Res 2012;36:156). These data led us to examine the mechanism by which this tyrosine kinase inhibitor affects erythropoiesis in PV.We observed that in cultures containing SCF PV progenitor cells generated similar numbers of erythroid cells (Ery) as those from adult (AB) and cord (CB) blood by day 10 [fold increase (FI) ~1-2] but by day 13 PV cells generated significantly greater numbers of Ery than AB and CB [PV FI=11±0.2, p=0.021 vs AB; CB FI=6.2±1.9, p=0.025 vs PV and 0.0055 vs AB; AB FI=2.6±0.5]. Since by day 10 progenitor cells were no longer detected, we hypothesized that increases in Ery at day 13 reflect intrinsically greater Ery proliferation potentials. To test this hypothesis, we compared the phosphoproteomic landscaping of day 10 Ery from 3 PV, 3 AB and 3 CB by Reverse Phase Protein Array (RPPA) using as target >160 signaling events (data are at http://capmm.gmu.edu/data). Overall, 40 proteins were statistically different between PV and AB and 30 proteins were statistically different between CB and AB. Pathway analyses of significant hits identified that PV and CB Ery differ from the AB ones in the activation states of 1-2 proteins involved in stemness and cell cycle control inferring that there is no major change in their cycling or differentiation state. By contrast, the 3 populations showed numerous differences in cKIT signaling. PV Ery differed from AB cells by expressing greater levels of cKITY719 and cKIT703, which were reduced to barely detectable levels by the pan-JAK inhibitor Ruxolitinib, and of elements of PI3K (eNOS/NosIII, PDK1 and PKCd) and MAPK (pMARCKS, MSK1, AMPKα1 and β1 and p38 MAPK) signaling downstream, respectively, to cKITY719 and cKIT703. PV Ery expressed also greater levels of JAK2Y1007/1008 and of its downstream target STAT3Y705. CB Ery showed lower levels of cKIT, cKITY703, cKITY719 and CD63, a member of tetraspanin superfamily that binds cKITY719 switching its intracellular fate from recycling to lysosome degradation, greater phosphorylation of proteins of MAPK (pMARCKS, MSK1, PTEN and Src) and PI3K (PKCd, mTOR, p70S6K and panPKC/βII) signaling than AB Ery. These results were stoichiometrically validated by WB and indicate that PV Ery express greater degrees of cKIT activation than AB Ery suggesting that greater response to SCF might account for their greater amplification in culture. This hypothesis was tested by RPPA analyses (and stoichiometric validation by WB) of Ery from PV, AB and CB growth factor deprived (GFD) for 4h and then stimulated with SCF for 15’ and 2h. GFD altered the activation state of 25 proteins (22 de-activated and 3 activated) in PV, of 12 proteins (10 de-activated and 2 activated) in AB and 8 proteins (4 de-activated and 4 activated) in CB. SCF altered the activation state of 36 proteins in PV (18 activated and 18 de-activated), 23 proteins in CB (all activations) and 6 proteins in AB (all activations). In PV and CB Ery, GFD decreased cKITY719 and cKITY703 and the activation state of their downstream targets JAK2Y1007/1008, MAPKs and mTOR while SCF increased the stoichiometric levels of cKITY719 and cKITY703 and the activation of mTOR. SCF also increased cKITY703 and cKITY719 but did not activate mTOR in Ery from AB. In agreement with the hypothesis that Ery from PV and CB respond more readily to SCF than those from AB, SCF induced greater cell-surface cKIT down-modulation (by flow cytometry) and lower intra-cytoplasmic cKIT/CD63 association (by confocal microscopy and WB) in PV and CB Ery than in AB Ery. Screening of 97 inhibitors against targets analysed by RPPA which are approved for clinical use by FDA revealed that growth of PV Ery was more sensitive than that of AB only to JAK and cKIT inhibitors. In addition, shRNA-CD63 reduced the growth of PV Ery (FI=0.9 vs 1.3 p=0.012) while increased by 2-fold (p=0.02) that of AB Ery. These results provide the first phosphoproteomic landscaping of cKIT signalling in Ery from PV and normal sources and confirm that cKIT is an important therapeutic target for PV. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Pim1 kinase regulates c-Kit gene translation.

BackgroundReceptor tyrosine kinase, c-Kit (CD117) plays a pivotal role in the maintenance and expansion of hematopoietic stem/progenitor cells (HSPCs). Additionally, over-expression and/or mutational activation of c-Kit have been implicated in numerous malignant diseases including acute myeloid leukemia. However, the translational regulation of c-Kit expression remains largely unknown.Methods and resultsWe demonstrated that loss of Pim1 led to specific down-regulation of c-Kit expression in HSPCs of Pim1−/− mice and Pim1−/−2−/−3−/− triple knockout (TKO) mice, and resulted in attenuated ERK and STAT3 signaling in response to stimulation with stem cell factor. Transduction of c-Kit restored the defects in colony forming capacity seen in HSPCs from Pim1−/− and TKO mice. Pharmacologic inhibition and genetic modification studies using human megakaryoblastic leukemia cells confirmed the regulation of c-Kit expression by Pim1 kinase: i.e., Pim1-specific shRNA knockdown down-regulated the expression of c-Kit whereas overexpression of Pim1 up-regulated the expression of c-Kit. Mechanistically, inhibition or knockout of Pim1 kinase did not affect the transcription of c-Kit gene. Pim1 kinase enhanced c-Kit 35S methionine labeling and increased the incorporation of c-Kit mRNAs into the polysomes and monosomes, demonstrating that Pim1 kinase regulates c-Kit expression at the translational level.ConclusionsOur study provides the first evidence that Pim1 regulates c-Kit gene translation and has important implications in hematopoietic stem cell transplantation and cancer treatment.

Mastocytose cutanée diffuse du nourrisson : à propos d’un cas

Mastocytosis is a group of diseases related to abnormal accumulation and proliferation of mast cells in one or more organs. They may be associated with an acquired point mutation and the activation of the receptor tyrosine-kinase c-KIT of CFS (mast cell growth factor). The clinical manifestations are varied and secondary to the release of mast cell mediators and/or infiltration of various organs. There are two main types of mastocytosis: pure cutaneous mastocytosis and systemic mastocytosis when more than two organs are involved in mast cell infiltration (bone marrow, gastrointestinal tract, bone, liver and spleen, lymph nodes). Mastocytosis affects children in two thirds of cases, most frequently as an isolated cutaneous form. The most common clinical form in children is urticaria pigmentosa and solitary mastocytoma; bullous diffuse mastocytosis is rare. We report the case of an 8-month-old infant who presented with a diffuse pruritic bullous eruption. The histology and immunohistochemistry results were suggestive of mastocytosis. A serum tryptase test yielded positive results. Laboratory investigations did not identify systemic involvement. The patient was given antihistamine H1 medication and local care. Advice regarding the disease was offered to the parents. The course of the disease was marked by a decrease in the number of blisters and attenuation of the pruritus at the 6-month follow-up. This observation emphasizes the importance of awareness of this rare entity, which should be considered in all cases of bullous dermatosis in children, thereby allowing for early treatment.

Abstract 3336: ST6Gal-I glycosyltransferase promotes an undifferentiated cell phenotype and enhances c-kit signaling

Abstract Acute myelogenous leukemia (AML) is characterized by an increased number of myeloid cells in the bone marrow. These cells are arrested in their maturation process, frequently resulting in hematopoietic insufficiency. Recent advancements in molecular genetics have identified new markers that may be useful in diagnosing AML, including mutant c-kit (CD117). c-kit is a receptor tyrosine kinase and activating mutations in this receptor have been identified as a functional driver of AML and other myeloproliferative diseases. c-kit also plays a major role in maintaining normal stem cell pools. Prior studies suggest that ST6Gal-I, a unique glycosyltransferase upregulated in many cancers, promotes a stem-like cell phenotype in epithelial tumor cells. ST6Gal-I is a sialyltransferase that adds a negatively charged sialic acid in an α2-6 linkage to membrane glycoproteins. In this study, we show c-kit to be a substrate of ST6Gal-I, with increased sialylation correlating to increased c-kit activation. In order to study the effects of sialylation on signaling, we forced constitutive expression of ST6Gal-I in the U937 monocytic leukemia cell line. c-kit signaling was then induced by treating U937 cells with stem cell factor (SCF), the c-kit ligand. Here we show enhanced SCF-mediated c-kit activation, indicated by increased levels of phospho-c-kit in ST6Gal-I overexpressing cells. This, in turn, is associated with increased activation of downstream effecters of c-kit signaling, phospho-Erk and phospho-Akt. We further hypothesize that ST6Gal-I might play a role in the cell differentiation process. To test the effects of sialylation on differentiation, we used a well-established model to induce monocyte cell differentiation, treatment with the phorbol ester, PMA. PMA is known to induce macrophage differentiation in U937 cells, evidenced by exit from the cell cycle and upregulation of differentiation markers, including CD11b. Macrophage differentiation induces downregulation of endogenous ST6Gal-I, thereby reducing surface sialylation of specific receptors. However, forced overexpression of ST6Gal-I inhibits PMA-induced exit from the cell cycle, suggesting that ST6Gal-I acts to maintain cells in an undifferentiated state. To further understand the effects of receptor sialylation on PMA-induced differentiation, we used flow cytometric analysis to evaluate surface markers on U937 cells with or without ST6Gal-I overexpression. We show that forced expression of ST6Gal-I inhibits PMA-induced upregulation of CD11b, whereas c-kit expression is elevated in ST6Gal-I overexpressing cells relative to parental cells. These results suggest a novel mechanism by which ST6Gal-I contributes to an undifferentiated state in part through the regulation of the c-kit receptor. Citation Format: Kaitlyn A. Dorsett, Susan L. Bellis. ST6Gal-I glycosyltransferase promotes an undifferentiated cell phenotype and enhances c-kit signaling. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3336.

RACK1 overexpression is linked to acquired imatinib resistance in gastrointestinal stromal tumor.

Although treatment with imatinib, which inhibits KIT and PDGFR, controls advanced disease in about 80% of gastrointestinal stromal tumor (GIST) patients, resistance to imatinib often develops. RACK1 (Receptor for Activated C Kinase 1) is a ribosomal protein that contributes to tumor progression by affecting proliferation, apoptosis, angiogenesis, and migration. Here, we found that c-KIT binds to RACK1 and increases proteasome-mediated RACK1 degradation. Imatinib treatment inhibits c-KIT activity and prevents RACK1 degradation, and RACK1 is upregulated in imatinib-resistant GIST cells compared to non-resistant parental cells. Moreover, Erk and Akt signaling were reactivated by imatinib in resistant GIST cells. RACK1 functioned as a scaffold protein and mediated Erk and Akt reactivation after imatinib treatment, thereby promoting GIST cell survival even in the presence of imatinib. Combined inhibition of KIT and RACK1 inhibited growth in imatinib-resistant GIST cell lines and reduced tumor relapse in GIST xenografts. These findings provide new insight into the role of RACK1 in imatinib resistance in GIST.

Murine Models Demonstrate Distinct Vasculogenic and Cardiomyogenic cKit+ Lineages in the Heart.

After 2 recent genetic studies in mice addressing the developmental origins and regenerative activity of cardiac cKit+ cells, 2 additional reports by Sultana et al and Liu et al provide further information on the expression of cKit in the embryonic and adult hearts. Here, we synthesize the findings from the 4 distinct cKit models to gain insights into the biology of this important cell type.

Abstract A42: Inhibition of the invasive component of mucinous breast cancers by targeting c-kit

Abstract Introduction: Among the molecular subtypes of breast cancer are normal-like, luminal (A or B) estrogen receptor positive (ER+), HER2+, and triple negative (basal). In addition to the molecular subtypes, there are 18 histologic breast cancer subtypes that are classified on appearance, including mucinous breast cancers (MBC), which are ER+ tumors. MBC can be classified according to their mucin composition as pure mucinous (> 90% mucin) or mixed mucinous (50-90% mucin). We have established a new model of mucinous breast cancer using breast cancer cells derived from a pleural effusion of a patient that were xenografted into an immunocompromised mouse. The resulting cell line, BCK4, was subsequently implanted into mice supplemented with either cellulose or estrogen. Pure mucinous tumors (MUCp) formed in the presence of cellulose, however, estrogen induced a histologic switch to invasive lobular carcinoma (ILC) with mucinous features (mixed mucinous). These mixed mucinous tumors contained both high mucin (MUCm) and low mucin tumor regions (ILC). The purpose of this study was to determine the molecular underpinnings of this histologic switch in BCK4 tumors. Experimental procedures: We isolated an equal number of tumor cells from pure mucinous and both the MUCm and ILC regions of the mixed mucinous BCK4 tumors using LASER capture microdissection; samples were analyzed on Affymetrix arrays to examine differential gene regulation among the tumor regions. Summary: Approximately 3000 genes were differentially regulated between the mixed and pure mucinous tumors. PAM50 subtype calls for the different tumor regions were as follows: MUCp tumors are the Normal-like subtype, MUCm tumor regions are Normal-like/Luminal A, while the ILC regions of the mixed mucinous tumors were Luminal B/Basal. Cluster analysis of the BCK4 derived tumors compared with ER+ and ER- breast cancer cell lines shows the mixed mucinous BCK4 tumors (both MUCm and ILC regions) group with ER- breast cancers, while the pure mucinous tumors were more related to ER+ breast tumor cells. To elucidate early genes regulated by estrogen in BCK4 cells, we performed expression profiling using Illumina arrays of the BCK4 cell line, treated with or without estrogen in vitro for 24 hours. A set of ~200 genes were significantly regulated by estrogen in the BCK4 cell line and the BCK4 derived tumors. Metacore Pathway Analysis suggested the c-kit pathway might be a target to reduce estrogen-induced proliferation. Subsequent studies found that inhibition of c-kit activity using Imatinib Mesylate (Gleevec®) blocked estrogen mediated stimulation of BCK4 cells as effectively as the anti-estrogen Fulvestrant. Conclusions: Targeting c-kit may represent a new type of therapy for some mucinous breast cancers. This is the first study to examine estrogen regulated genes in a model of mucinous breast cancer, allowing identification of genetic pathways that cause more indolent pure mucinous tumors to switch to aggressive mixed mucinous tumors. Citation Format: J. Chuck Harrell, Megan J. Blatner, Britta M. Jacobsen. Inhibition of the invasive component of mucinous breast cancers by targeting c-kit. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr A42.

Antagonism of stem cell factor/c-kit signaling attenuates neonatal chronic hypoxia-induced pulmonary vascular remodeling.

BackgroundAccumulating evidence suggests that c-kit positive cells are present in the remodeled pulmonary vasculature bed of patients with pulmonary hypertension (PH). Whether stem cell factor (SCF)/ c-kit regulated pathways potentiate pulmonary vascular remodeling is unknown. Here, we tested the hypothesis that attenuated c-kit signaling would decrease chronic hypoxia-induced pulmonary vascular remodeling by decreasing pulmonary vascular cell mitogenesis.MethodsNeonatal FVB/NJ mice treated with non-immune IgG (PL), or c-kit neutralizing antibody (ACK2) as well as c-kit mutant mice (WBB6F1- Kit W− v/ +) and their congenic controls, were exposed to normoxia (FiO2=0.21) or hypoxia (FiO2=0.12) for two weeks. Following this exposure, right ventricular systolic pressure (RVSP), right ventricular hypertrophy (RVH), pulmonary vascular cell proliferation and remodeling were evaluated.ResultsAs compared to chronically hypoxic controls, c-kit mutant mice had decreased RVSP, RVH, pulmonary vascular remodeling and proliferation. Consistent with these findings, administration of ACK2 to neonatal mice with chronic hypoxia-induced PH decreased RVSP, RVH, pulmonary vascular cell proliferation and remodeling. This attenuation in PH was accompanied by decreased extracellular signal-regulated protein kinase (ERK) 1/2 activation.ConclusionSCF/c-kit signaling may potentiate chronic hypoxia-induced vascular remodeling by modulating ERK activation. Inhibition of c-kit activity may be a potential strategy to alleviate PH.

Butyrate suppresses murine mast cell proliferation and cytokine production through inhibiting histone deacetylase

Beyond their nutritional impact to colonic epithelial cells, the intestinal microbiota metabolite butyrate has pleotropic effects to host cells and is known for its beneficial effects on intestinal homeostasis and metabolism. However, it remains unclear how it modulates mast cell function. Here, we demonstrate that butyrate profoundly inhibited proliferation of mouse mastocytoma P815 cells through inducing cell cycle arrest and apoptosis, as well as decreasing c-Kit activation. In addition, butyrate increased early- and late-stage apoptotic P815 cells. In murine bone marrow-derived mast cells (BMMC), butyrate-suppressed FcεRI-dependent tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6) release without affecting β-Hexosaminidase, but that was associated with decreased mitogen-activated protein kinase extracellular signal-regulated kinase 1/2, p38 and c-Jun N-terminal kinases activation. Butyrate treatment substantially enhanced histone 3 acetylation in both P815 and BMMC and decreased FcεRI-dependent mRNA expression of tnf-α and il-6 in BMMC, mimicking the effect of Trichostatin A, a known histone deacetylase inhibitor. Chromatin immunoprecipitation revealed that butyrate enhanced acetylation of the tnf-α and il-6 promoter regions but blocked RNA polymerase II binding to the promoters of tnf-α and il-6 genes, indicating suppressed transcription initiation. These phenotypes mimicked those of Trichostatin A treatment. In conclusion, butyrate inhibits cell proliferation and increases cell apoptosis in mastocytoma P815 cells and suppresses FcεRI-dependent cytokine production in murine primary BMMC, which are likely mediated by HDAC inhibition.

C-Kit Promotes Growth and Migration of Human Cardiac Progenitor Cells via the PI3K-AKT and MEK-ERK Pathways.

A recent phase I clinical trial (SCIPIO) has shown that autologous c-kit+ cardiac progenitor cells (CPCs) improve cardiac function and quality of life when transplanted into patients with ischemic heart disease. Although c-kit is widely used as a marker of resident CPCs, its role in the regulation of the cellular characteristics of CPCs remains unknown. We hypothesized that c-kit plays a role in the survival, growth, and migration of CPCs. To test this hypothesis, human CPCs were grown under stress conditions in the presence or absence of SCF, and the effects of SCF-mediated activation of c-kit on CPC survival/growth and migration were measured. SCF treatment led to a significant increase in cell survival and a reduction in cell death under serum depletion conditions. In addition, SCF significantly promoted CPC migration in vitro. Furthermore, the pro-survival and pro-migratory effects of SCF were augmented by c-kit overexpression and abrogated by c-kit inhibition with imatinib. Mechanistically, c-kit activation in CPCs led to activation of the PI3K and the MAPK pathways. With the use of specific inhibitors, we confirmed that the SCF/c-kit-dependent survival and chemotaxis of CPCs are dependent on both pathways. Taken together, our findings suggest that c-kit promotes the survival/growth and migration of human CPCs cultured ex vivo via the activation of PI3K and MAPK pathways. These results imply that the efficiency of CPC homing to the injury site as well as their survival after transplantation may be improved by modulating the activity of c-kit.

Targeting c-kit receptor in neuroblastomas and colorectal cancers using stem cell factor (SCF)-based recombinant bacterial toxins.

Autocrine activation of c-kit (KIT receptor tyrosine kinase) has been postulated to be a potent oncogenic driver in small cell lung cancer, neuroblastoma (NB), and poorly differentiated colorectal carcinoma (CRC). Although targeted therapy involving tyrosine kinase inhibitors (TKIs) such as imatinib mesylate is highly effective for gastrointestinal stromal tumor carrying V560G c-kit mutation, it does not show much potential for targeting wild-type KIT (WT-KIT). Our study demonstrates the role of stem cell factor (SCF)-based toxin conjugates for targeting WT-KIT-overexpressing malignancies such as NBs and CRCs. We constructed SCF-based recombinant bacterial toxins by genetically fusing mutated form of natural ligand SCF to receptor binding deficient forms of Diphtheria toxin (DT) or Pseudomonas exotoxin A (ETA') and evaluated their efficacy in vitro. Efficient targeting was achieved in all receptor-positive neuroblastoma (IMR-32 and SHSY5Y) and colon cancer cell lines (COLO 320DM, HCT 116, and DLD-1) but not in receptor-negative breast carcinoma cell line (MCF-7) thereby proving specificity. While dose- and time-dependent cytotoxicity was observed in both neuroblastoma cell lines, COLO 320DM and HCT 116 cells, only an anti-proliferative effect was observed in DLD-1 cells. We prove that these novel targeting agents have promising potential as KIT receptor tyrosine kinase targeting system.

The Phosphatases STS1 and STS2 Regulate Hematopoietic Stem and Progenitor Cell Fitness

SummaryFLT3 and c-KIT are crucial regulators of hematopoietic stem and progenitor cells. We investigated the role of STS1 and STS2 on FLT3 and c-KIT phosphorylation, activity, and function in normal and stress-induced hematopoiesis. STS1/STS2-deficient mice show a profound expansion of multipotent progenitor and lymphoid primed multipotent progenitor cells with elevated colony-forming capacity. Although long-term hematopoietic stem cells are not increased in numbers, lack of STS1 and STS2 significantly promotes long-term repopulation activity, demonstrating a pivotal role of STS1/STS2 in regulating hematopoietic stem and progenitor cell fitness. Biochemical analysis identified STS1/STS2 as direct phosphatases of FLT3 and c-KIT. Loss of STS1/STS2 induces hyperphosphorylation of FLT3, enhances AKT signaling, and confers a strong proliferative advantage. Therefore, our study reveals that STS1 and STS2 may serve as novel pharmaceutical targets to improve hematopoietic recovery after bone marrow transplantation.

C-kit signaling promotes proliferation and invasion of colorectal mucinous adenocarcinoma in a murine model.

It was reported that the receptor tyrosine kinase (RTK) family often highly expressed in several mucinous carcinomas. In the present study, we established a murine model of colorectal mucinous adenocardinoma (CRMAC) by treating C57 mice [both wild type (WT) and loss-of-function c-kit mutant type (Wads-/-)] with AOM+DSS for 37 weeks and found that c-kit, a member of RTK family, clearly enhanced the tumor cell proliferation by decreasing p53 and increasing cyclin D1 through AKT pathway. Significantly, c-kit strongly promoted tumor cell invasiveness by increasing ETV4, which induced MMP7 expression and epithelial-mesenchymal transition (EMT) via ERK pathway. In vitro up- or down-regulating c-kit activation in human colorectal cancer HCT-116 cells further consolidated these results. In conclusion, our data suggested that the c-kit signaling obviously promoted proliferation and invasion of CRMAC. Therefore, targeting the c-kit signaling and its downstream molecules might provide the potential strategies for treatment of patients suffering from CRMAC in the future.

Abstract 3635: Histone demethylase NDY1/KDM2B as a driver of survival and proliferation of normal and neoplastic mast cells

Abstract Proliferative disorders of mast cells exhibit a range of clinical behavior from benign cutaneous forms to aggressive systemic mastocytosis and mast cell leukemia. While these disorders are rare in people, they are among the most common malignancies in the dog, a circumstance that provides a useful model for an orphan disease. Growth of both normal and neoplastic mast cells is driven by c-kit (CD117), a tyrosine kinase receptor for stem cell factor. Ligand-independent, constitutive activation of c-kit via mutation is a common feature of mastocytic neoplasia. Therefore, tyrosine kinase inhibitors (TKIs) that target c-kit have activity in aggressive mastocytosis in people and in dogs. However, resistance to c-kit targeted TKIs is common in both species, highlighting a need for other therapeutic targets. Our data indicates that chromatin modification may be important in mast cell biology. Specifically, we have shown that the JmjC domain lysine 36 specific histone H3 demethylase, NDY1/KDM2B, supports the proliferation and immortalization of bone-marrow derived mast cells. It is well known that NDY1/KDM2B is a driver of hematopoietic transformation and is associated with malignant progression in certain solid tumors (breast, pancreatic and urothelial carcinoma). Therefore, we investigated the effects of knockdown of NDY1/KDM2B in neoplastic human and canine mast cells in vitro and in xenografts. Our data demonstrate that this knockdown inhibits proliferation and induces apoptosis in vitro and inhibits tumor growth in vivo. Therefore, targeting therapy of NDY1/KDM2B or downstream effectors in mast cell tumors could be a beneficial addition to the existing therapeutic modalities. Supported by grants from the Morris Animal Foundation (D14CA) and NIH (R01CA109747). Citation Format: Parthena Foltopoulou, Raymond Pfau, Monica Betancur-Boissel, Manar AbdelMageed, Philip N. Tsichlis, Elizabeth A. McNiel. Histone demethylase NDY1/KDM2B as a driver of survival and proliferation of normal and neoplastic mast cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3635. doi:10.1158/1538-7445.AM2015-3635

House dust mite allergens mediate the activation of c‑kit in dendritic cells via Toll‑like receptor 2.

Several studies have demonstrated that the c‑kit proto‑oncogene and its ligand, stem cell factor, are important in the development of asthma. House dust mite (HDM; Dermatophagoides pteronyssinus) allergens are a major trigger in the development and exacerbation of asthma. HDM allergens can induce the activation of c‑kit in dendritic cells (DCs), leading to the development of allergic asthma. Previous studies have demonstrated that activation of Toll‑like receptor 2 (TLR2) evokes a T helper (Th)2 immune response and promotes experimental asthma. The aim of the present study was to assess whether HDM mediates the activation of c‑kit in DCs via TLR2. Monocyte‑derived DCs were generated from C57BL/6 mice, and cultured with interleukin (IL)‑4 and granulocyte‑macrophage colony‑stimulating factor. The DCs were then sensitized with HDM (10 µg/ml) for 72 h. TLR2‑specific small interfering (si)RNA was used to silence and inhibit the expression of TLR2 in the DCs. The expression levels of c‑kit and B7 (CD80/CD86) were measured, by analyzing the DC culture supernatant for the presence of IL‑6 and IL‑12. Inhibition of TLR2 using specific siRNA downregulated the expression of c‑kit in the HDM‑activated DCs. In addition, silencing of TLR2 inhibited the expression of CD80/CD86, decreased the production of IL‑6, and increased the production of IL‑12. These results indicated that TRL2 are important in the activation of c‑kit by HDM in DCs.