Interstitial cells of Cajal in Wsh/Wsh c-kit mutant mice.

The c-Kit receptor tyrosine kinase regulates the development and differentiation of several progenitor cells. In the gastrointestinal (GI) tract, the c-Kit regulates the development of the interstitial cells of Cajal (ICC) that are responsible for motility regulation of the GI musculature. W-sash (Wsh) is an inversion mutation upstream of the c-kit promoter region that affects a key regulatory element, resulting in cell-type-specific altered gene expression, leading to a decrease in the number of mast cells, melanocytes, and ICC. We extensively examined the GI tract of Wsh/Wsh mice using immunohistochemistry and electron microscopy. Although the musculature of the Wsh/Wsh mice did not show any c-Kit immunoreactivity, we detected intensive immunoreactivity for transmembrane member 16A (TMEM16A, anoctamin-1), another ICC marker. TMEM16A immunopositive cells were observed as ICC-MY in the gastric corpus-antrum and the large intestine, ICC-DMP in the small intestine, and ICC-SM in the colon. Electron microscopic analysis revealed these cells as ICC from their ultrastructural features, such as numerous mitochondria and caveolae, and their close contact with nerve terminals. In the developmental period, we examined 14.5 and 18.5 day embryos but did not observe c-Kit immunoreactivity in the Wsh/Wsh small intestine. From this study, ICC subtypes developed and maturated structurally without c-Kit expression. Wsh/Wsh mice are a new model to investigate the effects of c-Kit and unknown signaling on ICC development and function.

KIT ligand protects against both light-induced and genetic photoreceptor degeneration.

Photoreceptor degeneration is a major cause of blindness and a considerable health burden during aging but effective therapeutic or preventive strategies have not so far become readily available. Here, we show in mouse models that signaling through the tyrosine kinase receptor KIT protects photoreceptor cells against both light-induced and inherited retinal degeneration. Upon light damage, photoreceptor cells upregulate Kit ligand (KITL) and activate KIT signaling, which in turn induces nuclear accumulation of the transcription factor NRF2 and stimulates the expression of the antioxidant gene Hmox1. Conversely, a viable Kit mutation promotes light-induced photoreceptor damage, which is reversed by experimental expression of Hmox1. Furthermore, overexpression of KITL from a viral AAV8 vector prevents photoreceptor cell death and partially restores retinal function after light damage or in genetic models of human retinitis pigmentosa. Hence, application of KITL may provide a novel therapeutic avenue for prevention or treatment of retinal degenerative diseases.

Nuclear KIT induces a NFKBIB-RELA-KIT autoregulatory loop in imatinib-resistant gastrointestinal stromal tumors.

Gastrointestinal stromal tumors (GISTs) are frequently driven by auto-activated, mutant KIT and have durable response to KIT tyrosine kinase inhibitor. However, acquired resistance is an increasing clinical issue in GIST patients receiving front-line imatinib therapy. Our previous studies showed the colocalization of KIT with DAPI-stained nuclei in GIST cells without knowing the role of nuclear KIT in GIST tumorigenesis. In this article, we first identified the binding of nuclear KIT to the promoter of NFKB inhibitor beta (NFKBIB) by chromatin immunoprecipitation (ChIP) sequencing and ChIP assays, which was accompanied with enhanced NFKBIB protein expression in GIST cells. Clinically, high NCCN risk GISTs had significantly higher mean expression levels of nuclear phospho-KIT and NFKBIB as compared with those of intermediate or low/very low-risk GISTs. Conversely, downregulation of NFKBIB by siRNA led to RELA nuclear translocation that could bind to the KIT promoter region and subsequently reduced KIT transcription/expression and the viability of GIST cells. These findings were further confirmed by either RELA overexpression or NFKB/RELA inducer, valproic acid, treatment to result in reduced KIT expression and relative cell viability of imatinib-resistant GIST cells. Combining valproic acid with imatinib showed significantly better growth inhibitory effects on imatinib-resistant GIST48 and GIST430 cells in vitro, and in the GIST430 animal xenograft model. Taken together, these results demonstrate the existence of a nuclear KIT-driven NFKBIB-RELA-KIT autoregulatory loop in GIST tumorigenesis, which are potential targets for developing combination therapy to overcome imatinib-resistant of KIT-expressing GISTs.

Immunohistochemical and ultrastructural evidence for telocytes in the different physiological stages of the female rat mammary gland.

INTRODUCTION: Telocytes (TCs) are recently described to integrate a variety of different cells. AIM OF THE WORK: The aim was to investigate the presence of TCs in the rat mammary gland at its different physiological stages. MATERIAL AND METHODS: Twenty four adult female albino rats were classified into 4 groups: resting, mid-pregnancy, lactating, and involution groups. Inguinal mammary glands were processed for immunohistochemical and transmission electron microscopic (TEM) examination. RESULTS: TCs were immune-positive for c-kit and CD34 and showed significant differences in the different studied groups indicating variable roles at the different stages. TEM results characterized TCs by its shape and the long slender and moniliform telopodes linking the cells into stromal networks. The extracellular exosomes, homo-cellular synapsis and hetero-cellular synapsis were observed. CONCLUSION: Our study provides evidence for the presence of TCs in all stages of the gland; not only in the resting stage as proved by other studies, but with immune-labeling differences suggesting different structural and physiological roles of TCs according to the stage requirements. These functions might via controlling the proliferation during pregnancy and lactation and the involution of the gland after weaning. Thus, more future functional studies of TCs will be important to help understanding the mechanism by which TCs contribute to tissue homeostasis concerning the role of the stromal/epithelial interactions in mammary gland biology and pathology including breast cancer which would be revolutionary for future therapeutic applications.

The Proto-oncogene c-Kit Inhibits Tumor Growth by Behaving as a Dependence Receptor.

c-Kit is a classic proto-oncogene either mutated or upregulated in cancer cells, and this leads to its constitutive kinase activation and, thus, to uncontrolled proliferation. Although the pro-oncogenic role of c-Kit is of no doubt, some observations do not fit well with c-Kit solely as a tumor-promoting moiety. We show here that c-Kit actively triggers cell death in various cancer cell lines unless engaged by its ligand stem cell factor (SCF). This pro-death activity is enhanced when the kinase activation of c-Kit is silenced and is due to c-Kit intracellular cleavage by caspase-like protease at D816. Moreover, in vivo, overexpression of a c-Kit kinase-dead mutant inhibits tumor growth, and this intrinsic c-Kit tumor-suppressive activity is dependent on the D816 cleavage. Thus, c-Kit acts both as a proto-oncogene via its kinase activity and as a tumor suppressor via its dependence receptor activity.

Preinduction with bone morphogenetic protein-2 enhances cardiomyogenic differentiation of c-kit+ mesenchymal stem cells and repair of infarcted myocardium.

BACKGROUND: Preclinical and clinical trails show that c-kit+ cardiac stem cells can differentiate towards cardiovascular cells and improve cardiac function after myocardial infarction (MI). However, survival and differentiation of the engrafted stem cells within ischemic and inflammatory microenvironment are poor. METHODS: c-Kit+ cells were isolated from mesenchymal stem cells (MSCs) of rat bone marrow. Reliability of preinduction with bone morphogenetic protein-2 (BMP-2) in promotion of survival and differentiation of c-kit+ MSCs was assessed in vitro and after transplantation. RESULTS: c-Kit+ MSCs have a potential to differentiate towards cardiomyocytes. BMP-2 promotes proliferation, migration and paracrine of the cells, and protects the cells to survive in the hypoxic condition. After induction with 10 ng/mL BMP-2 for 24 h, the cells can differentiate into cardiomyocytes at four weeks. The electrophysiological characteristics of the differentiated cells are same as adult ventricular cardiomyocytes. In rat MI models, cardiac function was improved, the size of scar tissue was reduced, and regeneration of the myocardium and microvessels was enhanced significantly at four weeks after transplantation of BMP-2-preinduced cells. The survived cells and cardiomyocytes differentiated from the engrafted cells were increased greatly. CONCLUSION: The results suggest that transient treatment with BMP-2 can induce c-kit+ MSCs to differentiate into functional cardiomyocytes. Preinduction with BMP-2 enhances survival and differentiation of the cells. BMP-2-primed cells promote repair of the infarcted myocardium and improvement of cardiac function. Transplantation of BMP-2-preinduced c-kit+ MSCs is a feasible strategy for MI therapy.

SRC-like adaptor protein 2 (SLAP2) is a negative regulator of KIT-D816V-mediated oncogenic transformation.

KIT is a receptor tyrosine kinase (RTK) involved in several cellular processes such as regulation of proliferation, survival and differentiation of early hematopoietic cells, germ cells and melanocytes. Activation of KIT results in phosphorylation of tyrosine residues in the receptor, and recruitment of proteins that mediate downstream signaling and also modulate receptor signaling. Here we show that the SRC-like adaptor protein 2 (SLAP2) binds to wild-type KIT in a ligand-dependent manner and is furthermore found constitutively associated with the oncogenic mutant KIT-D816V. Peptide fishing analysis mapped pY568 and pY570 as potential SLAP2 association sites in KIT, which overlaps with the SRC binding sites in KIT. Expression of SLAP2 in cells expressing the transforming mutant KIT-D816V led to reduced cell viability and reduced colony formation. SLAP2 also partially blocked phosphorylation of several signal transduction molecules downstream of KIT such as AKT, ERK, p38 and STAT3. Finally, SLAP2 expression enhanced ubiquitination of KIT and its subsequent degradation. Taken together, our data demonstrate that SLAP2 negatively modulates KIT-D816V-mediated transformation by enhancing degradation of the receptor.

Cardiac c-Kit Biology Revealed by Inducible Transgenesis.

RATIONALE: Biological significance of c-Kit as a cardiac stem cell marker and role(s) of c-Kit+ cells in myocardial development or response to pathological injury remain unresolved because of varied and discrepant findings. Alternative experimental models are required to contextualize and reconcile discordant published observations of cardiac c-Kit myocardial biology and provide meaningful insights regarding clinical relevance of c-Kit signaling for translational cell therapy. OBJECTIVE: The main objectives of this study are as follows: demonstrating c-Kit myocardial biology through combined studies of both human and murine cardiac cells; advancing understanding of c-Kit myocardial biology through creation and characterization of a novel, inducible transgenic c-Kit reporter mouse model that overcomes limitations inherent to knock-in reporter models; and providing perspective to reconcile disparate viewpoints on c-Kit biology in the myocardium. METHODS AND RESULTS: In vitro studies confirm a critical role for c-Kit signaling in both cardiomyocytes and cardiac stem cells. Activation of c-Kit receptor promotes cell survival and proliferation in stem cells and cardiomyocytes of either human or murine origin. For creation of the mouse model, the cloned mouse c-Kit promoter drives Histone2B-EGFP (enhanced green fluorescent protein; H2BEGFP) expression in a doxycycline-inducible transgenic reporter line. The combination of c-Kit transgenesis coupled to H2BEGFP readout provides sensitive, specific, inducible, and persistent tracking of c-Kit promoter activation. Tagging efficiency for EGFP+/c-Kit+ cells is similar between our transgenic versus a c-Kit knock-in mouse line, but frequency of c-Kit+ cells in cardiac tissue from the knock-in model is 55% lower than that from our transgenic line. The c-Kit transgenic reporter model reveals intimate association of c-Kit expression with adult myocardial biology. Both cardiac stem cells and a subpopulation of cardiomyocytes express c-Kit in uninjured adult heart, upregulating c-Kit expression in response to pathological stress. CONCLUSIONS: c-Kit myocardial biology is more complex and varied than previously appreciated or documented, demonstrating validity in multiple points of coexisting yet heretofore seemingly irreconcilable published findings.

Chasing c-Kit through the heart: Taking a broader view.

Stem cell mediated cardiac repair is an exciting and controversial area of cardiovascular research that holds the potential to produce novel, revolutionary therapies for the treatment of heart disease. Extensive investigation to define cell types contributing to cardiac formation, homeostasis and regeneration has produced several candidates, including adult cardiac c-Kit+ expressing stem and progenitor cells that have even been employed in a Phase I clinical trial demonstrating safety and feasibility of this therapeutic approach. However, the field of cardiac cell based therapy remains deeply divided due to strong disagreement among researchers and clinicians over which cell types, if any, are the best candidates for these applications. Research models that identify and define specific cardiac cells that effectively contribute to heart repair are urgently needed to resolve this debate. In this review, current c-Kit reporter models are discussed with respect to myocardial c-Kit cell biology and function, and future designs imagined to better represent endogenous myocardial c-Kit expression.

Induced haploinsufficiency of Kit receptor tyrosine kinase impairs brain development.

Kit receptor tyrosine kinase is highly expressed in the developing mammalian brain, yet little is known about its contribution to neural cell development and function. Here we introduced a brain-specific conditional Kit loss-of-function mutation in mice and observed severe hypoplasia of the central nervous system. This was accompanied by an increase in apoptotic cell death in the early embryonic brain and the gradual loss of the self-renewal capacity of neuronal stem/precursor cells. A single copy of the brain-specific conditional Kit loss-of-function allele resulted in the observed phenotype, including impaired in vitro differentiation of neural cells from Kit-haploinsufficient embryonic stem (ES) cells. Our findings demonstrate that Kit signaling is required for the early development of neural cells. This potentially novel Kit-haploinsufficient lethal phenotype may represent an embryonic lethal phenomenon previously unobserved because of its dominantly acting nature.

A regulatory circuit composed of DNA methyltransferases and receptor tyrosine kinases controls lung cancer cell aggressiveness.

Overexpression of DNMT1 and KIT is prevalent in lung cancer, yet the underlying molecular mechanisms are poorly understood. While the deregulated activation of DNMT1 or KIT has been implicated in lung cancer pathogenesis, whether and how DNMT1 and KIT orchestrate lung tumorigenesis are unclear. Here, using human lung cancer tissue microarrays and fresh frozen tissues, we found that the overexpression of DNMT1 is positively correlated with the upregulation of KIT in tumor tissues. We demonstrated that DNMT1 and KIT form a positive regulatory loop, in which ectopic DNMT1 expression increases, whereas targeted DNMT1 depletion abrogates KIT signaling cascade through Sp1/miR-29b network. Conversely, an increase of KIT levels augments, but a reduction of KIT expression ablates DNMT1 transcription by STAT3 pathway leading to in-parallel modification of the DNA methylation profiles. We provided evidence that KIT inactivation induces global DNA hypomethylation, restores the expression of tumor suppressor p15INK4B through promoter demethylation; in turn, DNMT1 dysfunction impairs KIT kinase signaling. Functionally, KIT and DNMT1 co-expression promotes, whereas dual inactivation of them suppresses, lung cancer cell proliferation and metastatic growth in vitro and in vivo, in a synergistic manner. These findings demonstrate the regulatory and functional interplay between DNA methylation and tyrosine kinase signaling in propelling tumorigenesis, providing a widely applicable approach for targeting lung cancer.

Review of various molecular targets on mast cells and its relation to obesity: A future perspective.

Mast cells are stimulatory factors in prognosis of various immunogenic and allergic diseases in human body. These cells play an important role in various immunological and metabolic diseases. The aim of present article is to explore the molecular targets to suppress the over expression of mast cells in obesity. The last 20 years literature were searched by various bibliographic data bases like Pubmed, google Scholar, Scopus and web of Science. The data were collected by keywords like "Mast Cell" "obesity" and "role of mast cell or role in obesity". Articles and their abstract were reviewed with a counting of 827 publications, in which 87 publications were considered for study and remaining was excluded because of its specificity to the subject. This review explains the characteristics, molecular targets and role of mast cells in obesity and existing research with mast cells to the area of metabolic diseases.

KIT signaling is dispensable for human mast cell progenitor development.

Human hematopoietic progenitors are generally assumed to require stem cell factor (SCF) and KIT signaling during differentiation for the formation of mast cells. Imatinib treatment, which inhibits KIT signaling, depletes mast cells in vivo. Furthermore, the absence of SCF or imatinib treatment prevents progenitors from developing into mast cells in vitro. However, these observations do not mean that mast cell progenitors require SCF and KIT signaling throughout differentiation. Here, we demonstrate that circulating mast cell progenitors are present in patients undergoing imatinib treatment. In addition, we show that mast cell progenitors from peripheral blood survive, mature, and proliferate without SCF and KIT signaling in vitro. Contrary to the prevailing consensus, our results show that SCF and KIT signaling are dispensable for early mast cell development.

[Stem cell factor enhances invasive ability of BxPC-3 cells via hypoxia-inducible factor 1α (HIF-1α)].

Objective To investigate the effect of stem cell factor (SCF)/c-KIT system on the invasion of BxPC-3 pancreatic cancer cells and the role of hypoxia-inducible factor 1 alpha (HIF-1α) in this effect. Methods BxPC-3 cells were cultured in normoxia and hypoxia. The cells cultured in normoxia were treated with SCF. Real-time quantitative PCR and Western blotting were used to investigate the expressions of HIF-1α mRNA and protein, respectively. Small interference RNA of HIF-1α (siHIF-1α) was designed and synthesized, and then transfected into BxPC-3 cells. The expressions of matrix metalloproteinase-2 (MMP2), MMP9, and urokinase type plasminogen activator (uPA) were detected by real-time quantitative PCR and Western blotting in BxPC-3 cells after treated with SCF or siHIF-1α. The effects of SCF/c-KIT and HIF-1α knockdown on BxPC-3 cell invasion were examined by TranswellTM assay. Results Either SCF or hypoxia could induce the protein expression of HIF-1α. The siHIF-1α down-regulated the expression of HIF-1α specifically in BxPC-3 cells. SCF up-regulated the mRNA and protein expressions of MMP2, MMP9 and uPA, while siHIF-1α down-regulated the expressions of these genes. The invasive ability of BxPC-3 cells were enhanced by SCF, while siHIF-1α played an opposite role. Conclusion SCF/c-KIT system up-regulates the mRNA and protein expressions of MMP2, MMP9 and uPA through HIF-1α, which enhances the invasive ability of pancreatic cancer cells. And siHIF-1α can effectively inhibit these effects.

Hyperglycemia Increases Interstitial Cells of Cajal via MAPK1 and MAPK3 Signaling to ETV1 and KIT, Leading to Rapid Gastric Emptying.

BACKGROUND & AIMS: Depletion of interstitial cells of Cajal (ICCs) is common in diabetic gastroparesis. However, in approximately 20% of patients with diabetes, gastric emptying (GE) is accelerated. GE also occurs faster in obese individuals, and is associated with increased blood levels of glucose in patients with type 2 diabetes. To understand the fate of ICCs in hyperinsulinemic, hyperglycemic states characterized by rapid GE, we studied mice with mutation of the leptin receptor (Leprdb/db), which in our colony had accelerated GE. We also investigated hyperglycemia-induced signaling in the ICC lineage and ICC dependence on glucose oxidative metabolism in mice with disruption of the succinate dehydrogenase complex, subunit C gene (Sdhc). METHODS: Mice were given breath tests to analyze GE of solids. ICCs were studied by flow cytometry, intracellular electrophysiology, isometric contractility measurement, reverse-transcription polymerase chain reaction, immunoblot, immunohistochemistry, enzyme-linked immunosorbent assays, and metabolite assays; cells and tissues were manipulated pharmacologically and by RNA interference. Viable cell counts, proliferation, and apoptosis were determined by methyltetrazolium, Ki-67, proliferating cell nuclear antigen, bromodeoxyuridine, and caspase-Glo 3/7 assays. Sdhc was disrupted in 2 different strains of mice via cre recombinase. RESULTS: In obese, hyperglycemic, hyperinsulinemic female Leprdb/db mice, GE was accelerated and gastric ICC and phasic cholinergic responses were increased. Female KitK641E/+ mice, which have genetically induced hyperplasia of ICCs, also had accelerated GE. In isolated cells of the ICC lineage and gastric organotypic cultures, hyperglycemia stimulated proliferation by mitogen-activated protein kinase 1 (MAPK1)- and MAPK3-dependent stabilization of ets variant 1-a master transcription factor for ICCs-and consequent up-regulation of v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog (KIT) receptor tyrosine kinase. Opposite changes occurred in mice with disruption of Sdhc. CONCLUSIONS: Hyperglycemia increases ICCs via oxidative metabolism-dependent, MAPK1- and MAPK3-mediated stabilization of ets variant 1 and increased expression of KIT, causing rapid GE. Increases in ICCs might contribute to the acceleration in GE observed in some patients with diabetes.

ANO1 in intramuscular interstitial cells of Cajal plays a key role in the generation of slow waves and tone in the internal anal sphincter.

KEY POINTS: The internal anal sphincter develops tone important for maintaining high anal pressure and continence. Controversy exists regarding the mechanisms underlying tone development. We examined the hypothesis that tone depends upon electrical slow waves (SWs) initiated in intramuscular interstitial cells of Cajal (ICC-IM) by activation of Ca2+ -activated Cl- channels (ANO1, encoded by Ano1) and voltage-dependent L-type Ca2+ channels (CavL , encoded by Cacna1c). Measurement of membrane potential and contraction indicated that ANO1 and CavL have a central role in SW generation, phasic contractions and tone, independent of stretch. ANO1 expression was examined in wildtype and Ano1/+egfp mice with immunohistochemical techniques. Ano1 and Cacna1c expression levels were examined by quantitative PCR in fluorescence-activated cell sorting. ICC-IM were the predominant cell type expressing ANO1 and the most likely candidate for SW generation. SWs in ICC-IM are proposed to conduct to smooth muscle where Ca2+ entry via CavL results in phasic activity that sums to produce tone. ABSTRACT: The mechanism underlying tone generation in the internal anal sphincter (IAS) is controversial. We examined the hypothesis that tone depends upon generation of electrical slow waves (SWs) initiated in intramuscular interstitial cells of Cajal (ICC-IM) by activation of Ca2+ -activated Cl- channels (encoded by Ano1) and voltage-dependent L-type Ca2+ channels (encoded by Cacna1c). Phasic contractions and tone in the IAS were nearly abolished by ANO1 and CavL antagonists. ANO1 antagonists also abolished SWs as well as transient depolarizations that persisted after addition of CavL antagonists. Tone development in the IAS did not require stretch of muscles, and the sensitivity of contraction to ANO1 antagonists was the same in stretched versus un-stretched muscles. ANO1 expression was examined in wildtype and Ano1/+egfp mice with immunohistochemical techniques. Dual labelling revealed that ANO1 expression could be resolved in ICC but not smooth muscle cells (SMCs) in the IAS and rectum. Ano1, Cacna1c and Kit gene expression were the same in extracts of IAS and rectum muscles. In IAS cells isolated with fluorescence-activated cell sorting, Ano1 expression was 26.5-fold greater in ICC than in SMCs while Cacna1c expression was only 2-fold greater in SMCs than in ICC. These data support a central role for ANO1 and CavL in the generation of SWs and tone in the IAS. ICC-IM are the probable cellular candidate for ANO1 currents and SW generation. We propose that ANO1 and CavL collaborate to generate SWs in ICC-IM followed by conduction to adjacent SMCs where phasic calcium entry through CavL sums to produce tone.

Aberrant expressions of stem cell factor/c-KIT in rat testis with varicocele.

BACKGROUND/PURPOSE: Varicocele (VC) is considered by the World Health Organization as the main cause of male infertility. Studies have shown that VC can affect spermatogenesis and then result in male infertility. But the exact mechanism by which VC affects spermatogenesis is still unclear. Stem cell factor (SCF) and c-KIT receptor are crucial molecules during spermatogenesis in testis. This study aims to investigate whether SCF/c-KIT signaling is involved in the pathophysiology of VC on spermatogenesis. METHODS: Rat models of VC were built (n = 13), and sham-operated rats were used as controls (n = 8). The seminiferous tubules of the testis were observed with hematoxylin and eosin staining, expression of SCF was analyzed via enzyme-linked immunosorbent assay and Western blot, and expression of c-KIT was assessed with Western blot and immunofluorescence. RESULTS: Compared with controls, the seminiferous epithelium was disorganized and had significantly fewer cells in the testes of rats with VC. Expression of SCF increased in testes of VC rats, while expression of c-KIT was decreased. CONCLUSION: These results suggest that sperm counts in seminiferous epithelium are affected by VC, and the SCF/c-KIT system is aberrantly expressed in VC testis, which could be involved in male infertility caused by VC.

Distinct cellular properties of oncogenic KIT receptor tyrosine kinase mutants enable alternative courses of cancer cell inhibition.

Large genomic sequencing analysis as part of precision medicine efforts revealed numerous activating mutations in receptor tyrosine kinases, including KIT. Unfortunately, a single approach is not effective for inhibiting cancer cells or treating cancers driven by all known oncogenic KIT mutants. Here, we show that each of the six major KIT oncogenic mutants exhibits different enzymatic, cellular, and dynamic properties and responds distinctly to different KIT inhibitors. One class of KIT mutants responded well to anti-KIT antibody treatment alone or in combination with a low dose of tyrosine kinase inhibitors (TKIs). A second class of KIT mutants, including a mutant resistant to imatinib treatment, responded well to a combination of TKI with anti-KIT antibodies or to anti-KIT toxin conjugates, respectively. We conclude that the preferred choice of precision medicine treatments for cancers driven by activated KIT and other RTKs may rely on clear understanding of the dynamic properties of oncogenic mutants.

SCF/c-kit transactivates CXCR4-serine 339 phosphorylation through G protein-coupled receptor kinase 6 and regulates cardiac stem cell migration.

C-kit positive cardiac stem cells (CSCs) have been shown to contribute to myocardial regeneration after infarction. Previously, we have shown that the c-kit ligand stem cell factor (SCF) can induce CSC migration into the infarcted area during myocardial infarction (MI). However, the precise mechanism involved is not fully understood. In this study, we found that CSCs also express C-X-C chemokine receptor type 4 (CXCR4), which is a typical member of the seven transmembrane-spanning G protein-coupled receptor (GPCR). In vitro, activation of c-kit signalling by SCF promotes migration of CSCs with increased phosphorylation of CXCR4-serine 339, p38 mitogen-activated protein kinase (p38 MAPK) and extracellular regulated protein kinases 1/2 (ERK1/2). Knockdown of CXCR4 expression by siRNA reduces SCF/c-kit-induced migration and downstream signalling. As previously reported, CXCR4-serine 339 phosphorylation is mainly regulated by GPCR kinase 6 (GRK6); thus, silencing of GRK6 expression by siRNA impairs CXCR4-serine 339 phosphorylation and migration of CSCs caused by SCF. In vivo, knockdown of GRK6 impairs the ability of CSCs to migrate into peri-infarcted areas. These results demonstrate that SCF-induced CSC migration is regulated by the transactivation of CXCR4-serine 339 phosphorylation, which is mediated by GRK6.

Cancer cell motility is affected through 3D cell culturing and SCF/c-Kit pathway but not by X-irradiation.

BACKGROUND AND PURPOSE: Success of radiotherapy is often limited by therapy resistance and metastasis resulting from cancer cell motility. It was tested in vitro whether this cancer cell motility is affected by growth condition, active SCF/c-Kit pathway or X-irradiation. MATERIALS AND METHODS: Cell motility was measured with BioCoat™ Matrigel™ invasion chamber using four different cancer cell lines (NSCLC: H23, H520, H226 and PrCa: DU145). Cells were grown in 2D or 3D, SCF was knocked down by siRNA and cells were irradiated with 2 or 6Gy. RESULTS: All cell lines except H520 showed a 2-3-fold increase in cell motility when grown in 3D. This effect was considered to result from the EMT-like change seen when cells were grown in 3D as indicated by the enhanced expression of vimentin and N-cadherin and reduction of E-cadherin. Just the opposite trends were found for H520 cells. Knockdown of SCF was found to result in reduced cell motility for both 2D and 3D. In contrast, X-irradiation did not modulate cell motility neither under 2D nor 3D. In line with this, X-irradiation did neither induce the expression of EMT-associated genes nor SCF. CONCLUSION: X-irradiation affects neither the expression of important EMT genes such as vimentin, E-cadherin and N-cadherin nor SCF/c-Kit signaling and, as a consequence, does not alter cell motility.