Binding Of Stem Cell Factor Research Articles

Cryo-EM analyses of KIT and oncogenic mutants reveal structural oncogenic plasticity and a target for therapeutic intervention.

The receptor tyrosine kinase KIT and its ligand stem cell factor (SCF) are required for the development of hematopoietic stem cells, germ cells, and other cells. A variety of human cancers, such as acute myeloid leukemia, gastrointestinal stromal tumor, and mast cell leukemia, are driven by somatic gain-of-function KIT mutations. Here, we report cryo electron microscopy (cryo-EM) structural analyses of full-length wild-type and two oncogenic KIT mutants, which show that the overall symmetric arrangement of the extracellular domain of ligand-occupied KIT dimers contains asymmetric D5 homotypic contacts juxtaposing the plasma membrane. Mutational analysis of KIT reveals in D5 region an "Achilles heel" for therapeutic intervention. A ligand-sensitized oncogenic KIT mutant exhibits a more comprehensive and stableD5 asymmetric conformation. A constitutively active ligand-independent oncogenic KIT mutant adopts a V-shaped conformation solely held by D5-mediated contacts. Binding of SCF to this mutant fully restores the conformation of wild-type KIT dimers, including the formation of salt bridges responsible for D4 homotypic contacts and other hallmarks of SCF-induced KIT dimerization. These experiments reveal an unexpected structural plasticity of oncogenic KIT mutants and a therapeutic target in D5.

A fully human anti-c-Kit monoclonal antibody 2G4 inhibits proliferation and degranulation of human mast cells

Given that mast cells are pivotal contributors to allergic diseases, various allergy treatments have been developed to inhibit them. Omalizumab, an anti-immunoglobulin E antibody, is a representative therapy that can alleviate allergy symptoms by inhibiting mast cell degranulation. However, omalizumab cannot reduce the proliferation and accumulation of mast cells, which is a fundamental cause of allergic diseases. c-Kit is essential for the proliferation, survival, and differentiation of mast cells. Excessive c-Kit activation triggers various mast cell diseases, such as asthma, chronic spontaneous urticaria, and mastocytosis. Herein, we generated 2G4, an anti-c-Kit antibody, to develop a therapeutic agent for mast cell diseases. The therapeutic efficacy of 2G4 antibody was evaluated in LAD2, a human mast cell line. 2G4 antibody completely inhibited c-Kit signaling by blocking the binding of stem cell factor, known as the c-Kit ligand. Inhibition of c-Kit signaling led to the suppression of proliferation, migration, and degranulation in LAD2 cells. Moreover, 2G4 antibody suppressed the secretion of pro-inflammatory cytokines, including granulocyte–macrophage colony-stimulating factor, vascular endothelial growth factor, C–C motif chemokine ligand 2, brain-derived neurotrophic factor, and complement component C5/C5a, which can exacerbate allergy symptoms. Taken together, these results suggest that 2G4 antibody has potential as a novel therapeutic agent for mast cell diseases.

Role and significance of c-KIT receptor tyrosine kinase in cancer: A review.

c-kit is a classical proto-oncogene that encodes a receptor tyrosine kinase (RTK) that responds to stem cell factor (SCF). C-KIT signaling is a critical regulator of cell proliferation, survival, and migration and is implicated in several physiological processes, including pigmentation, hematopoiesis, and gut movement. Accumulating evidence suggests that dysregulated c-KIT function, caused by either overexpression or mutations in c-kit, promotes tumor development and progression in various human cancers. In this review, we discuss the most important structural and biological features of c-KIT, as well as insights into the activation of intracellular signaling pathways following SCF binding to this RTK. We then illustrate how different c-kit alterations are associated with specific human cancers and describe recent studies that highlight the contribution of c-KIT to cancer stemness, epithelial-mesenchymal transition and progression to metastatic disease in different experimental models. The impact of tyrosine kinase inhibitors in treating c-KIT-positive tumors and limitations due to their propensity to develop drug resistance are summarized. Finally, we appraise the potential of novel therapeutic approaches targeting c-KIT more selectively while minimizing toxicity to normal tissue.

Stem cell factor and NSC87877 synergism enhances c-Kit mediated proliferation of human erythroid cells

The biological mechanisms underlying the effects of stem cell factor (SCF) and an inhibitor, NSC87877 (N) of the c-Kit negative regulator (SHP-1 and SHP-2) on cell proliferation are different. Therefore, we compared the cell's response to these two either alone or in combination in K562 cells. Binding of SCF (S) to c-Kit induces dimerization that activates its kinase activity. The activated c-Kit undergoes autophosphorylation at tyrosine residues that serve as a docking site for signal transduction molecules containing SH2 domains. Predominantly, the phosphotyrosine 568 (pY568) in Juxtamembrane (JM) region of c-Kit interacts with adaptor protein APS, Src family kinase, and SHP-2, while phosphotyrosine 570 (pY570) interacts with the SHP-1 and the adaptor protein Shc. The dephosphorylation of phosphotyrosine residues by SHP-1/SHP-2 leads to inhibition of c-Kit proliferative signaling. A chemical molecule, N is reported to inhibit the enzymatic activity of SHP-1/SHP-2, but its effect on c-Kit-mediated proliferation has not been studied yet. Thus, this work aims at examining the effect of the combination of S and N on cells growth as compared to individual treatment. The present study is performed with erythroleukemic K562 cells, chosen for its mRNA expression concerning the c-Kit, and SHP-1/SHP-2. Interestingly, proliferation assay showed that combination significantly increased proliferation when G1 sorted K562 cells were used. These changes were significantly higher when K562 cells were initially treated with N followed by S treatment. Collectively, these results give mechanistic insight into the proliferation enhancement of bone marrow transplantation through the synergistic effect of S and N by inhibiting SHP-1/SHP-2. The study gives solid evidence that S and N combination can be used to enhance cell proliferation/growth.

Silencing Stem Cell Factor Gene in Fibroblasts to Regulate Paracrine Factor Productions and Enhance c-Kit Expression in Melanocytes on Melanogenesis.

Melanogenesis is a complex physiological mechanism involving various paracrine factors. Skin cells such as keratinocytes, fibroblasts, and melanocytes communicate with one another through secreted regulators, thereby regulating the melanocytes’ bio-functions. The stem cell factor (SCF) is a paracrine factor produced by fibroblasts, and its receptor, c-kit, is expressed on melanocytes. Binding of SCF to c-kit activates autophosphorylation and tyrosine kinase to switch on its signal transmission. SCF inhibition does not suppress fibroblast proliferation in MTT assay, and SCF silencing induced mRNA expressions of paracrine factor genes, HGF, NRG-1, and CRH in qPCR results. Following UVB stimulation, gene expressions of HGF, NRG, and CRH were higher than homeostasis; in particular, HGF exhibited the highest correlation with SCF variations. We detected fibroblasts regulated SCF in an autocrine-dependent manner, and the conditioned medium obtained from fibroblast culture was applied to treat melanocytes. Melanogenesis-related genes, tyrosinase and pmel17, were upregulated under conditioned mediums with SCF silencing and exposed to UVB treatments. Melanin quantities in the melanocytes had clearly increased in the pigment content assay. In conclusion, SCF silencing causes variations in both fibroblast paracrine factors and melanocyte melanogenesis, and the differences in gene expressions were observed following UVB exposure.

MP31-07 URINARY STEM CELL FACTOR MAY BECOME THE DIAGNOSABLE BIOMARKER OF OVERACTIVE BLADDER IN WOMEN

You have accessJournal of UrologyUrodynamics/Lower Urinary Tract Dysfunction/Female Pelvic Medicine: Non-neurogenic Voiding Dysfunction I1 Apr 2017MP31-07 URINARY STEM CELL FACTOR MAY BECOME THE DIAGNOSABLE BIOMARKER OF OVERACTIVE BLADDER IN WOMEN Takashi Hamakawa, Yasue Kubota, Yuya Ota, Naoko Unno, Rika Banno, Masa Takada, Shoichi Sasaki, Kenjiro Kohri, and Takahiro Yasui Takashi HamakawaTakashi Hamakawa More articles by this author , Yasue KubotaYasue Kubota More articles by this author , Yuya OtaYuya Ota More articles by this author , Naoko UnnoNaoko Unno More articles by this author , Rika BannoRika Banno More articles by this author , Masa TakadaMasa Takada More articles by this author , Shoichi SasakiShoichi Sasaki More articles by this author , Kenjiro KohriKenjiro Kohri More articles by this author , and Takahiro YasuiTakahiro Yasui More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.961AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES C-kit, as known a receptor tyrosine kinase protein and a receptor of stem cell factor (SCF), not only acts as a marker of interstitial cells of Cajal, but also plays a significant role in the control of bladder spontaneous activity. And it could be an interesting target for the clinical treatment of overactive bladder (OAB). Although SCF binding to c-kit is associated with various biologic phases, the distribution and role of SCF in the urinary bladder remain unknown. Thus, we speculated that c-kit and its ligand SCF could play an important role in the control of bladder function. The objective of this study was to investigate whether SCF affects the biological behaviour of OAB. METHODS Differentiation between OAB and control was based on symptoms and a questionnaire of Overactive Bladder Symptom Score (OABSS). Urinary SCF levels were measured in patients with OAB and in control subjects by enzyme-linked immunosorbent assay (ELISA). The urinary SCF levels were compared among controls and OAB groups, and also between OAB patients ?75 years and <75 years. RESULTS A total of 93 women with OAB and 71 controls were enrolled. The mean age was 74.1± 13.0 years for the OAB groups and 67.1± 15.6 years for the control group. The average urinary SCF/creatinine levels in OAB patients was 1.589 ± 2.837, and in the control group was 0.558 ± 0.773 (p<0.001) (Fig.1). Analysis of urinary SCF/Cr levels among OAB group and controls by age showed no significant differences. CONCLUSIONS Urinary SCF levels were significantly higher in women with OAB. The urinary SCF level was not associated with ageing in OAB patients and controls. These results suggested that the SCF/C-kit pathway has a potential of contribution to the onset of OAB, and urinary stem cell factor might become the evaluable biomarker of OAB in women, objectively. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e401 Advertisement Copyright & Permissions© 2017MetricsAuthor Information Takashi Hamakawa More articles by this author Yasue Kubota More articles by this author Yuya Ota More articles by this author Naoko Unno More articles by this author Rika Banno More articles by this author Masa Takada More articles by this author Shoichi Sasaki More articles by this author Kenjiro Kohri More articles by this author Takahiro Yasui More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Identifying Residues that Determine SCF Molecular-Level Interactions through a Combination of Experimental and In silico Analyses

The stem cell factor (SCF)/c-Kit receptor tyrosine kinase complex—with its significant roles in hematopoiesis and angiogenesis—is an attractive target for rational drug design. There is thus a need to map, in detail, the SCF/c-Kit interaction sites and the mechanisms that modulate this interaction. While most residues in the direct SCF/c-Kit binding interface can be identified from the existing crystal structure of the complex, other residues that affect binding through protein unfolding, intermolecular interactions, allosteric or long-distance electrostatic effects cannot be directly inferred. Here, we describe an efficient method for protein-wide epitope mapping using yeast surface display. A library of single SCF mutants that span the SCF sequence was screened for decreased affinity to soluble c-Kit. Sequencing of selected clones allowed the identification of mutations that reduce SCF binding affinity to c-Kit. Moreover, the screening of these SCF clones for binding to a structural antibody helped identify mutations that result in small or large conformational changes in SCF. Computational modeling of the experimentally identified mutations showed that these mutations reduced the binding affinity through one of the three scenarios: through SCF destabilization, through elimination of favorable SCF/c-Kit intermolecular interactions, or through allosteric changes. Eight SCF variants were expressed and purified. Experimentally measured in vitro binding affinities of these mutants to c-Kit confirmed both the yeast surface display selection results and the computational predictions. This study has thus identified the residues crucial for c-Kit/SCF binding and has demonstrated the advantages of using a combination of computational and combinatorial methods for epitope mapping.

Expression of Epidermal c-Kit+ of Vitiligo Lesions Is Related to Responses to Excimer Laser.

BackgroundThe survival and growth of melanocytes are controlled by the binding of stem cell factor to its cell surface receptor c-kit+ (CD117). We have observed that c-kit+ melanocytes existed in some lesions of vitiligo, while Melan A+ cells were absent.ObjectiveTo verify possible relation between c-kit+ expression and treatment response in non-segmental vitiligo lesionsMethodsSkin biopsies were done from the center of the 47 lesions from the 47 patients with non-segmental vitiligo. Expression of c-kit+ and Melan A, and amounts of melanin in the epidermis were assessed in each lesion, and treatment responses to excimer laser were evaluated.ResultsThirty-five of the 47 lesions (74.5%) had c-kit+ phenotypes. There was significant difference of c-kit staining value between good responders in 3 months of excimer laser treatment (average of 24 sessions) and the others.Conclusionc-Kit expression in vitiliginous epidermis may be related to better treatment responses to excimer laser.

Withaferin A abolishes the stem cell factor-stimulated pigmentation of human epidermal equivalents by interrupting the auto-phosphorylation of c-KIT in human melanocytes.

We characterized the mechanism(s) underlying the abrogating effect of withaferin A (WFA) on the stem cell factor (SCF)-stimulated pigmentation of human epidermal equivalents (HEEs). Increased gene and protein expression levels of tyrosinase, tyrosinase-related protein1, dopachrome tautomerase, PMEL17, c-KIT and their targeted transcription factor, microphthalmia-associated transcription factor (MITF) were significantly reversed at days 7 and 10, respectively, by treatment with WFA. In WFA-treated normal human melanocytes (NHMs), there was a marked deficiency in the SCF-stimulated series of phosphorylations of c-KIT, Shc, Raf-1, MEK, ERK, MITF and CREB. Treatment with dithiothreitol (DTT) distinctly abolished the suppressive effect of WFA on the SCF-stimulated phosphorylation of c-KIT in NHMs. On the other hand, even after incubation at 4 °C for 2 h with 5 nM SCF, followed by the removal of unbound SCF by washing and then raising the temperature to 37 °C to start the signaling reaction, c-KIT was distinctly phosphorylated to a similar extent by incubation for 15 min with SCF only or with SCF + WFA. These findings indicate that WFA attenuates the SCF-induced activation of c-KIT in NHMs by interrupting the auto-phosphorylation of c-KIT through DTT-suppressible Michael addition thioalkylation reactions without interrupting the binding of SCF to the c-KIT receptor.

Abstract A227: The involvement of wild-type KIT overexpression in colon cancers.

Abstract Abnormal signaling through receptor tyrosine kinase (RTK) moieties is crucial in tumor progression and drug targeting of colorectal cancers. Wild-type KIT (WT-KIT), a RTK that is activated upon binding with stem cell factor (SCF), is highly expressed in some colon cancers; however, the functional role of SCF-dependent KIT activation is barely understood in colon cancer pathogenesis. In this study, we aimed to determine the conditions and roles of WT-KIT activation in colon cancer tumorigenesis. We found that WT-KIT was expressed in a subset of colon cancer cell lines and was activated by SCF treatment, leading to activation of downstream AKT and extracellular signal-regulated kinase (ERK) signaling pathways. We also demonstrated that KIT expression gradually decreased, after prolonged SCF stimulation, due to lysosomal degradation. Degradation of WT-KIT after SCF binding was significantly rescued when PKC-δ was activated. We also showed that activated PKC-δ directly binds to SCF bound WT-KIT, facilitating the recycling of WT-KIT. We further showed that a subset of colorectal cancers exhibit expressions of both WT-KIT and activated PKC-δ and that expression of KIT is correlated with poor patient survival (P = 0.004). We herein for the first time demonstrated that continuous downstream signal activation after KIT-SCF binding is accomplished through PKC-δ-mediated recycling of KIT. This sustained KIT activation may contribute to tumor progression in a subset of colon cancers with KIT expression and might provide the rationale for a therapeutic approach targeting KIT. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A227. Citation Format: Wonkyu Kim, Misun Park, Hogeun Kim. The involvement of wild-type KIT overexpression in colon cancers. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A227.

PI3K/mTOR pathway inhibitors sensitize chronic myeloid leukemia stem cells to nilotinib and restore the response of progenitors to nilotinib in the presence of stem cell factor

Nilotinib is a second-generation tyrosine kinase inhibitor, designed to specifically inhibit break-point cluster region (BCR)-Abelson (ABL) and developed to treat chronic myeloid leukemia (CML) in patients showing a resistance to imatinib. We previously demonstrated that nilotinib-induced apoptosis was reduced by stem cell factor (SCF) addition. Here, the SCF-activated survival pathway was investigated. BCR-ABL expression was accompanied by the activation of the SCF receptor: c-KIT. Nilotinib inhibited this activation that was restored by SCF binding. Parallel variations were observed for mammaliam target of rapamycin (mTOR) kinase and mTOR complex 1 substrate S6K. The inhibition of mTORC1 restored the response of BCR-ABL cell lines to nilotinib in the presence of SCF. PI3K inhibition restored nilotinib-induced apoptosis. On hematopoietic progenitors from CML patient's bone marrows, mTORC1 inhibition also restored nilotinib sensitivity in the presence of SCF, confirming its involvement in SCF-activated survival pathway. However, this pathway seems not to be involved in the nilotinib-induced resistance of the CML stem cell population. Conversely, PI3K inhibition sensitized both CML progenitors and stem cells to nilotinib, suggesting that, downstream PI3K, two different kinase pathways are activated in CML progenitor and stem cell populations.

Protein Kinase C-δ–Mediated Recycling of Active KIT in Colon Cancer

Abnormal signaling through receptor tyrosine kinase (RTK) moieties is important in tumorigenesis and drug targeting of colorectal cancers. Wild-type KIT (WT-KIT), a RTK that is activated upon binding with stem cell factor (SCF), is highly expressed in some colon cancers; however, little is known about the functional role of SCF-dependent KIT activation in colon cancer pathogenesis. We aimed to elucidate the conditions and roles of WT-KIT activation in colon cancer tumorigenesis. Colorectal cancers with KIT expression were characterized by immunoblotting and immunohistochemistry. The biologic alterations after KIT-SCF binding were analyzed with or without protein kinase C (PKC) activation. We found that WT-KIT was expressed in a subset of colon cancer cell lines and was activated by SCF, leading to activation of downstream AKT and extracellular signal-regulated kinase (ERK) signaling pathways. We also showed that KIT expression gradually decreased, after prolonged SCF stimulation, due to lysosomal degradation. Degradation of WT-KIT after SCF binding was significantly rescued when PKC was activated. We also showed the involvement of activated PKC-δ in the recycling of WT-KIT. We further showed that a subset of colorectal cancers exhibit expressions of both WT-KIT and activated PKC-δ and that expression of KIT is correlated with poor patient survival (P = 0.004). Continuous downstream signal activation after KIT-SCF binding is accomplished through PKC-δ-mediated recycling of KIT. This sustained KIT activation may contribute to tumor progression in a subset of colon cancers with KIT expression and might provide the rationale for a therapeutic approach targeting KIT.

In vivo knockdown of ckit impairs neuronal migration and axonal extension in the cerebral cortex

The tyrosine kinase receptor cKit and its ligand stem cell factor (SCF) are well known mediators in proliferation, survival, and positive chemotaxis of different cell types in the hematopoietic system. However, and in spite of previous reports showing robust expression of cKit and SCF in the brain during development, their possible function in the cerebral cortex has not been clarified. In this study, embryonic knockdown expression of cKit in the rat cortex by in utero electroporation of specific RNAi resulted in delayed radial migration of cortical neurons. In conditional Nestin-cKit KO homozygous mutants, radial migration in the cortex was also delayed. The opposite phenotype was observed after overexpressing cKit in the cortex: radial migration was accelerated. Callosal fibers electroporated with cKit RNAi were also delayed in their extension within the contralateral cortex and eventually failed to innervate their target area. In vitro experiments showed that, whereas SCF was able to promote migration of cortical neurons, it had no effect on cortical neurite outgrowth. In summary, our results demonstrate that (1) cKit is necessary for radial migration of cortical neurons, probably through SCF binding and (2) cKit is necessary for the correct formation of the callosal projection, most likely by a mechanism not involving SCF.

Abstract 5271: Nicotinic acetylcholine receptors and EGF induce c-Kit ligand/Stem Cell Factor (SCF) in a β-arrestin-1 and E2F1 dependent manner in NSCLC .

Abstract β-arrestin-1 (ARRB1), a scaffolding protein involved in the termination or desensitization of signals arising from activated G-protein-coupled receptors (GPCRs) has been shown to play a role in invasion and proliferation of many cancers, including nicotine-induced proliferation of human non-small cell lung cancers (NSCLCs). In this study, we carried out microarray analysis of cells lacking β-arrestin-1 which have been rendered quiescent and subsequently stimulated with nicotine or EGF. Nicotine induced and β-arrestin-1 dependent genes from the microarray data were analyzed. We identified 296 genes that were upregulated and 208 that were downregulated by nicotine in a β-arrestin-1 dependent fashion. The functional pathway analysis tool, MetaCoreTM (Genego, MI, USA) was used to obtain curated molecular interactions related to the above selected genes. We selected top 10 genes from both up and down regulated list for prognosis prediction. Prognostic prediction was carried out using a subset of NCI Director's Challenge Set. Kaplan-Meier analyses for 5 year as well as overall survival showed significance for 4 genes namely COL4A4, NFASC, SCF and ZNF137 by log-rank test. We also examined whether the expression of these gens correlated with smoking; it was found that SCF strongly differentiated smokers from non-smokers implying an important role of this gene in lung carcinogenesis induced by smoking. Stem cell factor (SCF) is the ligand of the c-Kit proto-oncogene product. It is a major human cytokine for the self-renewal, proliferation and differentiation of numerous embryonic, adult hematopoietic and primordial stem cells. Earlier reports show that uncontrolled activity of c-Kit contributes to formation of an array of human tumors. This unregulated activity of c-Kit may be due to overexpression or mutational activation suggesting that SCF-c-Kit signaling can be a potential target for cancer therapy. We elucidate the molecular mechanisms by which nicotine as well as EGF induces the expression of SCF in lung adenocarcinoma cell lines A549 and H1650. ChIP assays and transient transfection experiments showed that transcription factor E2F1 can positively regulate SCF expression at the transcriptional level; depletion of E2F1 or β-arrestin-1 prevented the nicotine-mediated induction of SCF. Given that the binding of SCF to c-Kit leads to activation of multiple downstream signaling pathways including Src, PI3-kinase and MAP/ERK pathways, our data suggest that the SCF/c-Kit pathway plays a central role in lung carcinogenesis, and may be a potential therapeutic target for combating NSCLC. Citation Format: Deepak Perumal, Smitha Pillai, Srikumar Chellappan. Nicotinic acetylcholine receptors and EGF induce c-Kit ligand/Stem Cell Factor (SCF) in a β-arrestin-1 and E2F1 dependent manner in NSCLC . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5271. doi:10.1158/1538-7445.AM2013-5271

Abstract PL04-03: Cell signaling by receptor tyrosine kinases: From basic principles to cancer therapy

Abstract Receptor tyrosine kinases (RTKs) comprise a family of cell surface receptors that control many critical cellular processes. Various human diseases are caused by dysfunction in RTKs or in their intracellular signaling pathways. Stem Cell Factor (SCF) initiates its multiple cellular responses by binding to the extracellular region of the RTK KIT resulting in receptor dimerization and tyrosine kinase activation. We have determined the crystal structure of the entire extracellular region of KIT before and after SCF stimulation. The structures show that KIT dimerization is driven by SCF binding whose sole role is to bring two KIT molecules together. We have used X-ray crystallography, electron microscopy (EM) and biochemical experiments to determine the three dimensional structure of intact, SCF-stimulated KIT dimers. Several forms of dimeric KIT molecules with different asymmetric arrangements of the two tyrosine kinase domains were identified. These asymmetric contacts may represent specific interactions occurring between two KIT tyrosine kinase domains poised towards trans autophosphorylation. We propose that cooperative interactions mediated by multiple weak homotypic contacts between the extracellular, transmembrane and cytoplasmic regions of KIT are responsible for tyrosine kinase activation and cell signaling in normal and transformed cells. Sutent is a drug that blocks the tyrosine kinase activities of several RTKs including KIT and VEGFR2. Sutent has been approved by the FDA for the treatment of advanced kidney cancers, gastrointestinal tumors and for endocrine pancreatic cancers. A scaffold-based drug discovery approach was developed enabling the development of new families of inhibitors for protein kinases and other enzymes that play a role in cell signaling. V600E B-RAF mutant is an oncogenic mutation identified in approximately 50% of melanomas and in 4% of all solid tumors. By using a scaffold-based drug discovery approach, a potent inhibitor of V600E B-RAF mutant has been discovered designated PLX 4032 (Vemurafenib). Phase-I and-II clinical trials in multiple medical centers revealed 80% responses such as tumor shrinkage, delay in tumor progression and survival of melanoma patients harboring the B-RAF mutation. Primary endpoints of phase-III clinical trials in multiple medical centers were achieved to support Vemurafenib registration. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr PL04-03. doi:1538-7445.AM2012-PL04-03

Citreorosein inhibits degranulation and leukotriene C4 generation through suppression of Syk pathway in mast cells

The aim of this study was to evaluate whether citreorosein (CIT), a naturally occurring anthraquinone isolated from Polygoni cuspidati (P. cuspidati) radix, modulates degranulation and 5-lipoxygenase (5-LO)-dependent leukotriene C(4) (LTC(4)) generation in mast cells. Cit suppresses both degranulation and the generation of LTC(4) in a dose-dependent manner in stem cell factor (SCF)-mediated mouse bone marrow-derived mast cells (BMMCs). With regard to its molecular mechanism of action, we investigated the effects of CIT on intracellular signaling and mast cell activation employing BMMCs. Binding of SCF to c-Kit on mast cell membranes induced increases in intrinsic tyrosine kinase Syk activity and activation of multiple downstream events including phosphorylation of phospholipase Cγ (PLCγ), mobilization of intracellular Ca(2+), phosphatidylinositol 3-kinase (PI3K), Akt, MAP kinases (MAPKs), translocation of phospho-phospholipase A(2) (PLA(2)) and 5-LO. The results from the biochemical analysis demonstrate that CIT attenuates degranulation and LTC(4) generation through the suppression of multiple step signaling and would be beneficial for the prevention of allergic inflammation.

C-kit binding properties of hesperidin (a major component of KMP6) as a potential anti-allergic agent.

Accumulation of mast cells can be causally related to several allergic inflammations. Stem cell factor (SCF) as a mast cell chemotaxin induces mast cell migration. To clarify a new effect of Pyeongwee-San extract (KMP6, a drug for indigestion) for the treatment of allergy, we investigated the effects of KMP6 on SCF-induced migration of rat peritoneal mast cells (RPMCs). A molecular docking simulation showed that hesperidin, a major component of KMP6, controls the SCF and c-kit binding by interaction with the active site of the c-kit. KMP6 and hesperidin significantly inhibited SCF-induced migration of RPMCs (P<0.05). The ability of the SCF to enhance morphological alteration and F-actin formation was also abolished by treatment with KMP6 or hesperidin. KMP6 and hesperidin inhibited SCF-induced p38 MAPK activation. In addition, SCF-induced inflammatory cytokine production was significantly inhibited by treatment with KMP6 or hesperidin (P<0.05). Our results show for the first time that KMP6 potently regulates SCF-induced migration, p38 MAPK activation and inflammatory cytokines production through hindrance of SCF and c-kit binding in RPMCs. Such modulation may have functional consequences during KMP6 treatment, especially mast cell-mediated allergic inflammation disorders.

Gastrointestinal Stromal Tumors: Disease and Treatment Update

Gastrointestinal Stromal Tumors: Disease and Treatment Update

Microarray profiling of gene expression in human keratinocytes suggests a new protective activity against UV-induced DNA damage for a compound previously known to interact with SCF-KIT signalling pathway

The stem cell factor (SCF) and its protein-tyrosine kinase receptor KIT are together implicated in the regulation of diverse biological processes and particularly in melanogenesis. Indeed, this signalling pathway controls melanoblast migration from the neural crest during embryogenesis and allows the communication between keratinocytes and melanocytes in the adult. In melanocytes, the binding of SCF to its transmembrane receptor leads to the activation of signalling pathways implicating protein kinases which finally control the expression of pigmentation-related genes. We have developed a biological compound called IV09.007, which we previously described as a modulator of the SCF/KIT signalling pathway with a pro-pigmenting effect. In the present work, we have studied the expression and localization of both SCF and KIT mRNAs and proteins in the skin or skin-derived cell lines. Then, we explored with a microarray approach the ability of IV09.007 to modulate the expression of genes in human keratinocytes and melanocytes in culture. Thereby, we observed the regulation of genes implicated in DNA repair, mainly related to base/nucleotides excision pathways. A modulated transcriptional response was also observed for some genes implicated in the response against oxidative stress, in apoptosis inhibition and in lowering inflammatory immune response. These microarray results predicted a conferred protective effect of IV09.007 and we verified this hypothesis by performing comet assays on UVB-irradiated keratinocytes or melanocytes, to demonstrate the efficacy of IV09.007 on preventing DNA damage.

I787 provides signals for c-Kit receptor internalization and functionality that control mast cell survival and development

AbstractMast cell (MC) differentiation, survival, and activation are controlled by the membrane tyrosine kinase c-Kit upon interaction with stem cell factor (SCF). Here we describe a single point mutation induced by N-ethyl-N-nitrosurea (ENU) mutagenesis in C57BL/6J mice—an A to T transversion at position 2388 (exon 17) of the c-Kit gene, resulting in the isoleucine 787 substitution by phenylalanine (787F), and analyze the consequences of this mutation for ligand binding, signaling, and MC development. The Kit787F/787F mice carrying the single amino acid exchange of c-Kit lacks both mucosal and connective tissue-type MCs. In bone marrow-derived mast cells (BMMCs), the 787F mutation does not affect SCF binding and c-Kit receptor shedding, but strongly impairs SCF-induced cytokine production, degranulation enhancement, and apoptosis rescue. Interestingly, c-Kit downstream signaling in 787F BMMCs is normally initiated (Erk1/2 and p38 activation as well as c-Kit autophosphorylation) but fails to be sustained thereafter. In addition, 787F c-Kit does not efficiently mediate Cbl activation, leading to the absence of subsequent receptor ubiquitination and impaired c-Kit internalization. Thus, I787 provides nonredundant signals for c-Kit internalization and functionality.