C-kit Receptor Tyrosine Kinase Research Articles

Delayed gastric emptying and disruption of the interstitial cells of Cajal network after gastric ischaemia and reperfusion

Gastrointestinal tract is one of the most susceptible organ systems to ischaemia. Not only mucosal injury but also alterations of the intestinal motility and loss of interstitial cells of Cajal (ICC) have been reported in response to ischaemia and reperfusion (I/R). However, there are few reports on the changes in the gastric motility after gastric I/R. The present study was designed to investigate the alterations in gastric emptying, the ICC and enteric nerves that regulate smooth muscle function in response to gastric I/R. Seven-week-old male Wistar rats were exposed to gastric I/R, and the gastric emptying rates at 12 and 48 h after I/R were evaluated by the phenol red method. Expressions of gene product of c-kit receptor tyrosine kinase (c-Kit), a marker of ICC, and of neuronal proteins were also examined. Gastric emptying was transiently delayed at 12 h after I/R, but returned to normal by 48 h. Expression of c-Kit protein as assessed by Western blotting and immunofluorescent staining of the smooth muscle layer, as well as expression of the mRNA of stem cell factor, the ligand for c-Kit, were reduced at both 12 and 48 h after I/R. The expression of neuronal nitric oxide synthase (nNOS) protein as assessed by Western blotting and immunofluorescent staining was also decreased at 12 h after I/R, but was restored to normal by 48 h. Gastric I/R evokes transient gastroparesis with delayed gastric emptying, associated with disruption of the ICC network and nNOS-positive neurons.

Preliminary Results of a Phase II Trial with the Multikinase Inhibitor Sorafenib in Heavily Pretreated Patients with Relapsed/Refractory Non-Hodgkin Lymphoma (NHL).

Abstract 1658Poster Board I-684 IntroductionNHL patients with refractory disease or relapsing after autologous or allogeneic stem cell transplant (SCT) have very poor prognosis with currently available salvage chemotherapy. Sorafenib (Nexavar, BAY43-9006, Bayer) is an oral multikinase inhibitor approved by FDA for the treatment of renal cell carcinoma and hepatocellular carcinoma. Sorafenib exerts a broad range of antiproliferative, antiangiogenic, and proapoptotic effects against a variety of nonhematological tumors through the inhibition of the RAF/MEK/ERK pathway, the receptor tyrosine kinases c-kit, Flt3, RET, as well as the proangiogenic vascular endothelial growth factor receptors (VEGFRs), and platelet-derived growth factor receptor-β (PDGFR-β). Several lines of evidence suggest that Sorafenib might have a significant clinical impact in the treatment of malignant lymphomas by overcoming the cytoprotective effects of Bcl-XL, ERK, and Mcl-1 and eventually targeting additional signalling pathways relevant to lymphomagenesis. Our preclinical data demonstrating a marked cytotoxic activity of Sorafenib against NHL cell lines in vitro and in vivo in xenograft models, established the rationale for this currently ongoing phase II study aimed to determine safety and activity of Sorafenib in relapsed/refractory NHL. MethodsBetween March 2008 and May 2009, 21 patients (16 males and 5 females; median age, 65 years; range, 29-74 years) with relapsed/refractory diffuse large B cell lymphoma (DLBCL, n = 11), follicular lymphoma (FL, n =4), mantle cell lymphoma (MCL, n =2), lymphoplasmacitoid lymphoma (LPL, n =1), chronic lymphocytic leukemia (CLL, n =2), and peripheral T-cell lymphoma (PTCL, n =1) who have failed second- or subsequent-line salvage chemo-radiotherapy were enrolled in this phase II trial. Prior to study entry, patients received a median of 4 (range 2 - 7) lines of treatment, including autologous SCT in 15 (71%) and an additional allogeneic SCT in 5 (24%) cases. At study entry, 7 (33%) patients had relapsed and 14 (67%) refractory disease. Eligibility criteria included absence of any available treatment options of proven efficacy, at least one target lesion ≥2 cm, ECOG performance status of 0-1, and adequate bone marrow, liver and renal functions. Sorafenib (400 mg BID, per os) was administered continuously until disease progression or appearance of clinical significant toxicity probably related to study drug. Tumor responses were assessed according to the revised response criteria for malignant lymphoma of the International Working Group. NCI CTCAE v3.0 was used for toxicity assessment. ResultsTo date, 21 patients received a median of 3 months (range, 1 – 11) of Sorafenib therapy. All patients are evaluable for toxicity and response, and 1 patient is still on therapy. Overall, therapy was well tolerated without significant adverse events. The most common drug-related non-hematological toxicities were grade 1-2 mucositis (14%), diarrhea (24%), hand-foot syndrome (24%), anorexia (29%), and fatigue (29%). Grade 3-4 hand-foot syndrome occurred in 19% of patients. Hematological toxicities included grade 1-2 neutropenia (10%) and thrombocytopenia (24%). Grade 3-4 neutropenia and thrombocytopenia were observed in 14%, and 24% of patients, respectively. Best response to Sorafenib included 1 (5%) complete remission (CR) occurring in the patient with LPL, and 1 (5%) partial remission (PR) in a patient with cutaneous DLBCL, for an overall response rate (ORR) of 10%. In both patients, response duration was 6 months. In addition, 9 (42%) patients achieved stable disease (SD) for a median of 3 months (range, 2 – 10), with 3 (14%) patients achieving SD for ≥6 months, while 10 (48%) patients progressed. Upon Sorafenib treatment, an extensive necrosis involving the central area of the tumor associated with a nearly complete disappearance of tumor vascularization was documented by computed tomography and contrast-enhanced ultrasound in two DLBCL and one FL patients bearing latero-cervical or abdominal lymphoid masses. ConclusionsSorafenib as a single agent was well tolerated. Despite limited clinical activity (10% ORR), disease stabilization was experienced by 42% of patients. The potent antiangiogenic activity of Sorafenib in NHL patients bearing highly vascularized lymphoid masses suggests that further research should focus on combinations of Sorafenib with molecularly targeted agents eventually exerting antivascular activities. DisclosuresOff Label Use: The multikinase inhibitor Sorafenib has been used in a phase II trial in patients with relapsed/refractory NHL.

The p110α Catalytic Isoform of PI3 Kinase Is Important for Erythropoiesis, but Has a Minimal Role in Hematopoietic Stem Cell Self-Renewal.

Abstract 3620Poster Board III-556PIK3CA, which encodes the p110α catalytic isoform of PI3 kinase (PI3K), is mutated in many human cancers, and is an attractive therapeutic target. However, PI3K may also be important during hematopoiesis, as it is activated by hematopoietic growth factor receptors which control hematopoietic stem cell (HSC) and progenitor proliferation, differentiation, and self-renewal, such as erythropoietin receptor (epoR), c-kit receptor, and fms-like tyrosine kinase 3 (FLT3). In hematopoietic cells, receptor tyrosine kinases signal through the catalytic p110 subunit of PI3K, which has 3 isoforms (α, β, δ). However, the roles of PI3K and its specific catalytic isoforms in normal HSC function are poorly understood. We hypothesized that signaling through the p110α isoform is important for hematopoiesis and HSC self-renewal. We have used the Cre-loxP system to delete p110α in the HSCs of adult mice by breeding p110αF/F mice to Mx1-Cre transgenic mice. p110αF/F;Mx1-Cre+ (Cre+) mice and their p110αF/F (Cre-) littermates were injected with PolyI-PolyC (pIpC) at 4-6 weeks of age to induce Cre-mediated excision at the PIK3CA locus specifically in hematopoietic cells. Deletion of p110α in the bone marrow (BM) was verified by PCR and by immunoblot. We observed that, by four weeks after pIpC treatment, Cre+ mice developed microcytic anemia compared with Cre- littermates, characterized by a decreased mean hemoglobin (p<0.0001) and decreased mean corpuscular volume, while white blood cell counts and platelet counts were unaffected. Cre+ mice also had significantly decreased spleen, liver, and thymus weights. Flow cytometry analyses of bone marrow and spleen cells revealed a relative block in erythropoiesis in the spleens of Cre+ mice, with expansion of the basophilic erythroblast population, and a decrease in the most mature nucleated erythroblast population. Colony assays of splenocytes in erythropoietin-containing methylcellulose medium revealed a 52% decrease in BFU-E colony formation by p110α-deleted cells in response to erythropoietin, suggesting that loss of p110α may lead to blunted epoR signaling (p=0.009). Multiparameter flow cytometry revealed that the overall number of Lin-Sca1+ckit+ (LSK) cells, which contains the HSC population, was increased two-fold in the BM of Cre+ mice compared with Cre- littermates (p=0.01). To determine whether loss of p110α affects long-term HSC self-renewal in vivo, we performed competitive repopulation assays, in which CD45.2+ BM cells from PIPC-treated Cre+ mice or Cre- controls were transplanted together with CD45.1+CD45.2+ competitor BM cells into lethally irradiated CD45.1+ recipient mice. Donor BM chimerism (%CD45.2+ cells) at 16 weeks was mildly reduced in the absence of p110α, but Cre+ cells were still capable of long-term reconstitution. In summary, we have found that the p110α catalytic isoform is specifically required for erythropoiesis, but has only a small role in HSC homeostasis and in differentiation of the other hematopoietic lineages. This suggests that pharmacologic targeting of p110α in cancer therapy may result in mild anemia, but should otherwise have minimal hematologic toxicity. Disclosures:Gilliland:Merck Research Laboratories: Employment. Roberts:Novartis Pharmaceuticals, Inc.: Consultancy. Zhao:Novartis Pharmaceuticals, Inc.: Consultancy.

Phenotypic Plasticity of Mouse Spermatogonial Stem Cells

BackgroundSpermatogonial stem cells (SSCs) continuously undergo self-renewal division to support spermatogenesis. SSCs are thought to have a fixed phenotype, and development of a germ cell transplantation technique facilitated their characterization and prospective isolation in a deterministic manner; however, our in vitro SSC culture experiments indicated heterogeneity of cultured cells and suggested that they might not follow deterministic fate commitment in vitro.Methodology and Principal FindingsIn this study, we report phenotypic plasticity of SSCs. Although c-kit tyrosine kinase receptor (Kit) is not expressed in SSCs in vivo, it was upregulated when SSCs were cultured on laminin in vitro. Both Kit− and Kit+ cells in culture showed comparable levels of SSC activity after germ cell transplantation. Unlike differentiating spermatogonia that depend on Kit for survival and proliferation, Kit expressed on SSCs did not play any role in SSC self-renewal. Moreover, Kit expression on SSCs changed dynamically once proliferation began after germ cell transplantation in vivo.Conclusions/SignificanceThese results indicate that SSCs can change their phenotype according to their microenvironment and stochastically express Kit. Our results also suggest that activated and non-activated SSCs show distinct phenotypes.

Disappearance of Lentigines in a Patient Receiving Imatinib Treatment for Familial Gastrointestinal Stromal Tumor Syndrome

Gastrointestinal stromal tumors (GISTs) harbor gain-of-function mutations of the c-kit tyrosine kinase receptor. Imatinib mesylate is an inhibitor of c-kit and is indicated in the treatment of chronic myeloid leukemia and GISTs. Reported adverse effects of imatinib include hypopigmentation, depigmentation, and hyperpigmentation. Although the exact mechanism by which these occur is unclear, it is likely that inhibition of c-kit leads to downstream inhibition of the tyrosinase gene promoter and thus to inhibition of pigment production. A 45-year-old woman with a history of multiple dysplastic nevi and lentigines was diagnosed as having familial GIST syndrome. Treatment with imatinib mesylate was started in an attempt to decrease the tumor load. Three months after treatment initiation, the patient noted a decrease in the number of pigmented lesions, lightening of the skin in her genital area, and graying of her terminal hair. The potential association between a specific genetic mutation and pigmentation changes secondary to imatinib therapy may account for the variety in presentation of this potential side effect. Further genetic studies paired with melanocyte-specific or c-kit-specific stains of affected tissue are warranted to better understand the relationship between the genetic mutation and the effect of imatinib on pigmentation.

Comparative analysis of c-kit gene expression and c-Kit immunoreactivity in horses with and without obstructive intestinal disease

Previous immunohistochemical studies targeting the receptor tyrosine kinase (c-Kit) have demonstrated an apparent reduction in the number of gastrointestinal pacemaker cells--the interstitial cells of Cajal (ICC)--in horses with intestinal motility disorders. This study compared the level of transcription of the c-kit gene encoding this receptor in horses with and without such motility disorders. Transcription levels of this gene were also compared to the density of ICC immunohistochemically positive for the c-Kit antigen. Intestinal samples were collected from 18 horses with intestinal disease and from 15 control animals. Following gene extraction and identification, real-time quantitative analysis of c-kit and a control gene, ACTB (β-actin), was carried out on all samples and the density of the c-Kit-positive ICC compared. There was a significant reduction in c-Kit immunoreactivity in the ICC of horses with large intestinal obstructive disorders relative to controls but no significant difference in the transcription of the c-kit gene between normal and affected animals. Further studies will be required to elucidate the mechanisms regulating c-Kit expression and to assess the pathophysiological significance of these findings.

Aberrant luminal progenitors as the candidate target population for basal tumor development in BRCA1 mutation carriers

Basal-like breast cancers arising in women carrying mutations in the BRCA1 gene, encoding the tumor suppressor protein BRCA1, are thought to develop from the mammary stem cell. To explore early cellular changes that occur in BRCA1 mutation carriers, we have prospectively isolated distinct epithelial subpopulations from normal mammary tissue and preneoplastic specimens from individuals heterozygous for a BRCA1 mutation. We describe three epithelial subsets including basal stem/progenitor, luminal progenitor and mature luminal cells. Unexpectedly, we found that breast tissue from BRCA1 mutation carriers harbors an expanded luminal progenitor population that shows factor-independent growth in vitro. Moreover, gene expression profiling revealed that breast tissue heterozygous for a BRCA1 mutation and basal breast tumors were more similar to normal luminal progenitor cells than any other subset, including the stem cell-enriched population. The c-KIT tyrosine kinase receptor (encoded by KIT) emerged as a key marker of luminal progenitor cells and was more highly expressed in BRCA1-associated preneoplastic tissue and tumors. Our findings suggest that an aberrant luminal progenitor population is a target for transformation in BRCA1-associated basal tumors .

Reply to Dr. Peparini’s Letter to the Editor regarding the publication titled “Immunohistochemical detection of receptor tyrosine kinases c-kit, EGF-R and PDGF-R in colorectal adenocarcinomas”

Dear Editor, We enjoyed reading the comments of Peparini et al. [1] in response to our recent publication entitled “Immunohistochemical detection of receptor tyrosine kinases c-kit, EGFR and PDGF-R in colorectal adenocarcinomas” [2] as it leads interest to the combination of two promising directions of cancer research. It is pointed out by Peparini and colleagues that epidermal growth factor receptor (EGF-R) is able to induce epithelial–mesenchymal transition (EMT) as shown for breast cancer [3] and that this mechanism may play a crucial role in tumor progression and formation of metastases. Emerging evidence suggests an important participation of EMT as regarding to tumor invasiveness which is underlined by a recent series of reviews published on this topic [4, 5]. However, signaling pathways in EMT and the relationship to tyrosine kinases are still poorly understood. In our study, out of 257 samples of colorectal carcinoma, only 39 (15.2%) stained positive for the expression of EGF-R, showing no significant correlation with pathological parameters which is not concordant with many studies. The controversy of differing detection rates has been discussed in our original article. A possible explanation not mentioned in our article is the heterogeneity of tumors with varying expression patterns in the bulk tumor, lymph nodes, metastases, and the tumor invasion front, representing regions of interest for EMT. For our study, we used tissue arrays of representative areas of the primary colorectal tumor. We did not perform specific counterstaining of the core needle biopsy, e.g., for EMT markers, but focusing expression patterns on different topographic sites of the same carcinoma, as proposed in the Letter to the Editor, might clarify the relationship of EMT with the expression of EGF-R and explain the variety of published expression patterns for colorectal carcinomas. In our study, we reported a detection rate of c-kit in only 1.9% of all 263 tumor samples, confirming the rare expression of c-kit in colorectal carcinomas described in literature. The discussed possibility of a different c-kitpositive tumor entity or the recently described lack of specificity of several kinase inhibitors leading to therapeutic benefit in c-kit-negative colorectal carcinomas [6] are just two interesting theories. The expression of c-kit at the tumor invasion front and the relationship to EMT or the reverting mesenchymal–epithelial transition, a suggested by Peparini and colleagues, definitively represent possible explanations and need further studies for clarification. In summary, we completely agree with Peparini and coworkers that research in this direction might lead to a better understanding of neoplastic progression and novel therapeutic strategies.

Expression of receptors tyrosine kinase c-kit and EGF-R in colorectal adenocarcinomas: is there a relationship with epithelial–mesenchymal transition during tumor progression?

To the Editor:We read with great interest the contribution by Friederichsand colleagues regarding immunohistochemical detectionof the receptor tyrosine kinases c-kit, EGF-R, and PDGF-Rin colorectal adenocarcinomas [1]. The authors showedEGF-R expression in only 15.3% of cases; emphasized thatactivity of tyrosine kinase inhibitors does not necessarilycorrelate with the tyrosine kinase expression of the tumors;showed that there is no correlation between EGF-R incolorectal cancer, clinical response to anti-EGF-R anti-bodies, and outcome; and that pretreatment selection ofpatients cannot be based on immunohistochemical expres-sion of EGF-R.EGF has recently been identified as a novel inducer ofepithelial–mesenchymal transition (EMT) in human breastcancer [2]. EMT is a process of plasticity by whichepithelial cells lose contact with adjacent cells andbasement membranes, switch patterns of extracellularmatrix protein and intermediate filament expression, andtransdifferentiate into mesenchymal cells. EMT duringtumor progression allows tumor cells to infiltrate surround-ing tissue and to metastatize to distant sites. A key event inEMT is reduction of cell–cell adhesion by transcriptionalrepression of E-cadherin. Loss of E-cadherin expression hasbeen reported in several cancers, including advancedcolorectal carcinomas. EMT hallmarks include reductionof E-cadherin with concomitant production of N-cadherinand acquisition of mesenchymal markers such as vimentinand fibronectin [3, 4]. EMT may influence the response ofcertain tumors to EGF-R-targeted therapies. In nonsmallcell lung cancer cell lines, sensitivity to an EGF-R-targetedmonoclonal antibody (erlotinib) did not correlate with EGF-R levels, but was dependent on EMT status: non-responding nonsmall cell lung carcinomas showed lowlevels of E-cadherin and higher levels of mesenchymalmarkers [5, 6].In the study by Friederichs et al., positive EGF-Rstaining did not show significant correlation with patho-logical parameters, but higher expression of EGF-R inadvanced and metastatic stages in colorectal adenocarcino-mas was reported in other studies.Friederichs et al. concluded that c-kit expression is rarein colorectal carcinomas, and because of the small numberof cases, it is uncertain if c-kit-positive carcinomasrepresent a specific entity or in which way these carcinomasdiffer. The authors suggested that in colorectal cancerwithout c-kit kinase activation, treatment with recentlydeveloped and less selective tyrosine kinase inhibitors mayresult in inhibition of alternative or related tyrosine kinase

Wsh/ Wsh c-Kit mutant mice possess interstitial cells of Cajal in the deep muscular plexus layer of the small intestine

The c-Kit receptor tyrosine kinase regulates the development and differentiation of various progenitor cells. W mutant mice with spontaneous mutations in the c-kit gene show various phenotypes such as anemia, infertility, loss of coat color and mast cells. c-Kit also regulates the development of the interstitial cells of Cajal (ICC) that are responsible for the motility regulation of the gastrointestinal musculature. W sh / W sh mice possess an inversion mutation upstream of the c-kit promoter region; this mutation is responsible for reducing c-Kit activity, leading to a decrease in the number of mast cells, melanocytes, and ICC. We extensively examined the small intestine of W sh / W sh mice by using immunohistochemistry and electron microscopy. Although the musculature of the W sh / W sh mice did not show any c-Kit immunoreactivity, there were neurokinin 1 receptor (NK1R)-immunopositive cells that were associated with the nerve fibers in the deep muscular plexus (DMP) region. These NK1R-immunopositive cells showed a bipolar shape with long processes and were identified as ICC in the DMP layer (ICC-DMP). Electron microscopic analysis revealed that ICC-DMP had numerous mitochondria, caveolae, and gap junctions and were closely associated with nerve terminals. In contrast, ICC were not observed at the myenteric layer. In the small intestine of the W sh / W sh mice, we detected ICC-DMP that showed NK1R immunoreactivity and ultrastructural characters. This type of ICC may develop and maturate structurally without c-Kit expression and regulate gastrointestinal motility.

Dimerization of Kit‐ligand and efficient cell‐surface presentation requires a conserved Ser‐Gly‐Gly‐Tyr motif in its transmembrane domain

Kit-ligand (Kitl), also known as stem cell factor, is a membrane-anchored, noncovalently bound dimer signaling via the c-kit receptor tyrosine kinase, required for migration, survival, and proliferation of hematopoietic stem and germ cells, melanocytes, and mastocytes. Despite its fundamental role in morphogenesis and stem cell biology, the mechanisms that regulate Kitl dimerization are not well understood. By employing cell-permeable cross-linker and quantitative bimolecular fluorescence complementation of wild-type and truncated forms of Kitl, we determined that Kitl dimerization is initiated in the endoplasmic reticulum and mediated to similar levels by the transmembrane and the extracellular growth factor domain. Further biochemical and mutational analysis revealed a conserved Ser-Gly-Gly-Tyr-containing motif that is required for transmembrane domain dimerization and efficient cell-surface expression of Kitl. A novel intracellular capture assay with the Kitl transmembrane domain as bait revealed specific interactions with Kitl, but not with unrelated transmembrane proteins. During evolution, the transmembrane dimerization motif appeared in Kitl at the transition from teleosts to tetrapods, which correlates with the emergence of Kitl as a supporter of stem cell populations. Thus, transmembrane-mediated association of membrane-anchored growth factors consists of a novel mechanism to improve paracrine signaling and morphogenesis.

The D816V Mutation of c-Kit Circumvents a Requirement for Src Family Kinases in c-Kit Signal Transduction

The receptor tyrosine kinase c-Kit plays a critical role in hematopoiesis, and gain-of-function mutations of the receptor are frequently seen in several malignancies, including acute myeloid leukemia, gastrointestinal stromal tumors, and testicular carcinoma. The most common mutation of c-Kit in these disorders is a substitution of the aspartic acid residue in position 816 to a valine (D816V), leading to constitutive activation of the receptor. In this study, we aimed to investigate the role of Src family kinases in c-Kit/D816V signaling. Src family kinases are necessary for the phosphorylation of wild-type c-Kit as well as of activation of downstream signaling pathways including receptor ubiquitination and the Ras/Mek/Erk pathway. Our data demonstrate that, unlike wild-type c-Kit, the phosphorylation of c-Kit/D816V is not dependent on Src family kinases. In addition, we found that neither receptor ubiquitination nor Erk activation by c-Kit/D816V required activation of Src family kinases. In vitro kinase assay using synthetic peptides revealed that c-Kit/D816V had an altered substrate specificity resembling Src and Abl tyrosine kinases. We further present evidence that, in contrast to wild-type c-Kit, Src family kinases are dispensable for c-Kit/D816V cell survival, proliferation, and colony formation. Taken together, we demonstrate that the signal transduction pathways mediated by c-Kit/D816V are markedly different from those activated by wild-type c-Kit and that altered substrate specificity of c-Kit circumvents a need for Src family kinases in signaling of growth and survival, thereby contributing to the transforming potential of c-Kit/D816V.

Immunohistochemical detection of receptor tyrosine kinases c-kit, EGF-R, and PDGF-R in colorectal adenocarcinomas

The selective inhibition of tyrosine kinases is a promising strategy in the treatment of several human malignancies. This study aimed to clarify expression patterns of therapeutically addressable receptor tyrosine kinases in colorectal cancer. In this study, we used tissue arrays to analyze 263 specimen of colorectal carcinoma for the expression of the tyrosine kinases c-kit (CD117), epidermal growth factor receptor (EGF-R), and platelet-derived growth factor receptor (PDGF-R). Staining patterns were then correlated with tumor stage and survival. Five tumors (1.9%) showed a strong expression of c-kit (CD117), while in 40 samples (15.2%), a weak/intermediate expression was observed. Positive staining did not correlate with histopathological parameters although a trend toward a better survival of c-kit-positive patients was observed. No positivity for PDGF-R was observed in 263 samples of colorectal carcinomas. Positive EGF-R expression was identified in 39 cases (15.2%), whereas 218 samples (84.8%) stained negative. Our study confirms that expression of the tyrosine kinases c-kit and PDGF-R are rare in colorectal carcinomas and do not correlate with tumor stage.

C-Kit-negative fibroblast-like cells express platelet-derived growth factor receptor α in the murine gastrointestinal musculature

Platelet-derived growth factor receptors (PDGFRs) belong to the same kinase group as c-Kit receptor tyrosine kinase that is specifically expressed in the interstitial cells of Cajal (ICC) in the gastrointestinal tract. In this study, we examined PDGFRalpha immunoreactivity in the murine gastrointestinal tract. PDGFRalpha-immunopositive (PDGFRalpha-ip) cells were observed in the musculature in all parts of the gastrointestinal tract. Although PDGFRalpha-ip cells were distinct from ICC and neurons, these cells were closely associated with intramuscular ICC and enteric nerve fibers. In the myenteric layer, PDGFRalpha-ip cells formed a cellular network with their ramified processes and encompassed myenteric ganglia. Numerous PDGFRalpha-ip cells were observed in the subserosal plane and showed a multipolar shape. The distribution pattern of the PDGFRalpha-ip cells in the ICC-deficient W(v)/W(v) mutant mice was the same as that in normal mice. PDGFRalpha-ip cells that showed intense immunoreactivity of SK3 potassium channel were considered to correspond to fibroblast-like cells or non-Cajal interstitial cells. Our observations suggest that PDGFRalpha-ip cells are basic cellular elements throughout the gastrointestinal musculature and are involved in the gastrointestinal functions.

Apoptotic cell death of human interstitial cells of Cajal

Interstitial cells of Cajal (ICC) are specialized mesenchyme-derived cells that regulate contractility and excitability of many smooth muscles with loss of ICC seen in a variety of gut motility disorders. Maintenance of ICC numbers is tightly regulated, with several factors known to regulate proliferation. In contrast, the fate of ICC is not established. The aim of this study was to investigate whether apoptosis plays a role in the regulation of ICC numbers in the normal colon. ICC were identified by immunolabelling for the c-Kit receptor tyrosine kinase and by electron microscopy. Apoptosis was detected in colon tissue by immunolabelling for activated caspase-3, terminal dUTP nucleotide end labelling and by ultrastructural changes in the cells. Apoptotic ICC were identified and counted in double-labelled tissue sections. They were identified in all layers of the colonic muscle. In the muscularis propria 1.5 +/- 0.2% of ICC were positive for activated caspase-3 and in the circular muscle layer 2.1 +/- 0.9% of ICC were positive for TUNEL. Apoptotic ICC were identified by electron microscopy. Apoptotic cell death is a continuing process in ICC. The level of apoptosis in ICC in healthy colon indicates that these cells must be continually regenerated to maintain intact networks.

A Phase II Study of Imatinib Mesylate and Capecitabine in Metastatic Breast Cancer: Southwest Oncology Group Study 0338

Background Imatinib mesylate is a potent inhibitor of the Bcr-Abl, c-Kit, and platelet-derived growth factor receptor (PDGFR) tyrosine kinases. On the basis of variable expression of c-Kit and PDGFR in breast cancer and of in vitro data supporting synergy between imatinib and capecitabine, the Southwest Oncology Group conducted a phase II trial of the combination in metastatic breast cancer. Patients and Methods Eligible patients had progressive, measurable metastatic breast cancer and received ≤ 2 previous chemotherapy regimens for metastatic disease. Previous 5-fluorouracil or capecitabine for metastatic disease was not allowed. Patients were accrued on a 2-stage design and received imatinib mesylate 400 mg by mouth daily and capecitabine at 1000 mg/m 2 by mouth twice daily for 14 days of a 21-day cycle. The primary endpoint was the confirmed response rate (RR). Tumors were evaluated for c-Kit, PDGFR-β, and hormone receptor expression. Results Nineteen fully evaluable patients were enrolled, with a confirmed RR of 11% (95% CI, 1%-33%). Eleven percent had unconfirmed partial responses, and 42% had stable disease. The trial did not accrue to the second stage. The estimated 6-month progression-free survival was 16% (95% CI, 0%-32%), and the median overall survival was 14 months (95% CI, 7-15 months). The combination was well tolerated. Of 8 available tumor samples, 2 stained for c-Kit, and all had stromal staining for PDGFR-β. Conclusion In unselected patients, the combination of imatinib mesylate and capecitabine was well tolerated but did not result in improved RRs compared to those reported with capecitabine alone.

Gastrointestinal stromal tumors - A model for understanding solid tumor biology and development of targeted therapies, or just another low-hanging fruit?

Gastrointestinal stromal tumors - A model for understanding solid tumor biology and development of targeted therapies, or just another low-hanging fruit?

The D816V Mutation of C-Kit Circumvents a Requirement for Src Family Kinases in C-Kit Mediated Signal Tranduction and Transformation

The receptor tyrosine kinase c-Kit plays a critical role in hematopoiesis and gain-of-function mutations of the receptor are frequently seen in several malignancies, including acute myeloid leukemia (AML), mastocytoma and sinonasal NK/T cell lymphomas. The most common mutation of c-Kit in these disorders is a substitution of the aspartic acid residue in position 816 to a valine (D816V), leading to constitutive activation of the receptor. In this study we aimed to investigate the role of Src family kinases in c-Kit/D816V signaling. Src family kinases are necessary for the phosphorylation of wild-type c-Kit as well as for activation of downstream signaling pathways including receptor ubiquitination and the Ras/MEK/Erk pathway. Tyrosine 568 of c-Kit, that is important for Src activation by wild-type c-Kit, was mutated to phenylalanine in both wild-type c-Kit and c-Kit/D816V and stably transfected into the hematopoietic cell line Ba/F3. Our data demonstrate that, unlike wild-type c-Kit, the phosphorylation of c-Kit/D816V is not dependent on Src family kinases. In addition we found that neither receptor ubiquitination nor Erk activation by c-Kit/D816V required activation of Src family kinases. In vitro kinase assay using synthetic peptides revealed that c-Kit/D816V had an altered substrate specificity resembling Src and Abl tyrosine kinases. The serine/threonine kinases Akt and Erk play important roles in cell survival and proliferation mediated by receptor tyrosine kinases. We could show constitutive activation of both PI3-kinase pathway and Erk in Ba/F3 cells expressing c-Kit/D816V, although ligand stimulation induced even stronger activation. We further present evidence that, in contrast to wild-type c-Kit, Src family kinases are dispensible for c-Kit/D816V cell survival. Taken together, we demonstrate that the signal transduction pathways mediated by c-Kit/D816V are markedly different from those activated by wild-type c-Kit and that altered substrate substrate specificity of c-Kit circumvents a need for Src family kinases in signaling of growth and survival, thereby contributing to the transforming potential of c-Kit/D816V.

Wild-Type and Mutant C-Kit Activation of Stat3α Contribute to Leukemogenesis through Distinct Effects on Myeloid Cell Proliferation and Resistance to Apoptosis

Expression of a constitutively active mutant of the receptor tyrosine kinase c-Kit in patients with acute myeloid leukemia (AML) is associated with poor survival. Most mutations occur in the activation loop, at Asp 816 or Asn 822, resulting in ligand-independent tyrosine phosphorylation and resistance to many of the tyrosine kinase inhibitors currently in use. Signal transducer and activator of transcription 3 (Stat3) also is upregulated and/or constitutively activated in many malignancies, including AML. Stat3 is activated by both wild-type c-Kit, which is expressed on most AML blasts, and by mutant c-Kit. Like mutant c-Kit, increased Stat3 activity has been associated with poor outcome in patients with AML. Stat3 exists in two isoforms – full length Stat3α (p92) and C-terminally truncated Stat3β (p83) – and the contribution of each isoform to c-Kitmediated leukemogenesis is not known. To begin to address this issue, we generated IL-3-dependent 32D murine myeloid cells that inducibly overexpress Stat3α when treated with dexamethasone and doxycycline (induced: Stat3αHI; uninduced: Stat3αLO). We further transfected these cells with wild-type c-Kit (c-KitWT), c-KitD816H or c-KitN822K. First, we established by immunoblotting that c-Kit and Stat3α were tyrosine-phosphorylated only after stem cell factor (SCF) stimulation in cells expressing c-KitWT, but were constitutively phosphorylated in c-KitD816H and c-KitN822K cells. To test the effects of increased Stat3α on survival and proliferation, we withdrew cells from IL-3 and stimulated with SCF (100 ng/ml) for 3 days; control cells remained unstimulated. We measured the proportion of cells undergoing apoptosis at day 3 by FACS using annexin V-PE labeling. In cells expressing c-KitWT, apoptosis was significantly reduced in SCF-stimulated Stat3αHI cells compared to SCF-stimulated Stat3αLO cells (8.4 ± 5.0% v. 40.8 ± 19.9%; p<0.05). In cells expressing c-KitD816H or c-KitN822K, apoptosis was significantly reduced in Stat3αHI cells compared to Stat3αLO cells (c-KitD816H: 30.2 ± 10.2% v. 66.7 ± 8.1%, p<0.05; c-KitN822K: 25.1 ± 17.4% v. 61.4 ± 19.8%, p<0.05). SCF treatment had no detectable effect on apoptosis in cells expressing mutant c-Kit. Next, we measured the rate of proliferation using the membrane tracking dye PKH26. Fluorescence histograms, with gating on live cells, were modeled using ModFit LT, and the proliferation index (PI; total number of cells/calculated number of cells in parental generation) at day 3 was calculated. Cells expressing c-KitWT did not proliferate well in the absence of SCF-stimulation, with PIs 1.6 ± 0.1 and 2.1 ± 0.3 for Stat3αLO and Stat3αHI, respectively. The PI for SCF-stimulated Stat3αHI cells was significantly higher than the PI for SCF-stimulated Stat3αLO cells (5.4 ± 0.7 v. 3.7 ± 1.1, p<0.05). Surprisingly, cells expressing mutant c-Kit showed only low levels of proliferation, regardless of Stat3α level or SCF stimulation. The PI was 2.0 ± 0.5 in unstimulated Stat3αHI c-KitD816H cells and 2.4 ± 0.3 in Stat3αHI c-KitN822K cells. Thus, both wild-type and mutant c-Kit activate Stat3α, which results in marked resistance to apoptosis. Wild-type c-Kit also activates a distinct signaling pathway that drives proliferation, and this process is augmented by Stat3α activation. These findings suggest that the critical role for Stat3α in myeloid leukemogenesis is apoptosis prevention and that targeting Stat3α is likely to be an effective therapeutic approach for leukemias with wild-type or mutant c-Kit.

Changes in the Structure and Function of ICC Networks in ICC Hyperplasia and Gastrointestinal Stromal Tumors

Gastrointestinal stromal tumors (GISTs) express the receptor tyrosine kinase c-kit. Approximately 90% of GISTs have gain-of-function mutations in the Kit gene, which leads to its constitutive activation and drives malignant behavior of GISTs. Interstitial cells of Cajal (ICC) express c-kit; however, it is unknown whether uncontrolled hyperplasia of ICC is responsible for GISTs. Here, we sought to determine whether gain-of-function mutations in Kit lead to hyperplasia of all classes of ICC, whether ICC hyperplasia begins before birth, and whether functional defects occur in ICC hyperplasia or the development of GISTs. Heterozygous mutant Kit(V558Delta)/+ mice that develop symptoms of human familial GISTs and prematurely die from pathology of the gastrointestinal tract were utilized and compared with wild-type controls. C-kit-immunohistochemistry and intracellular electrical recording of spontaneous and nerve-evoked activity were applied to examine the density and functionality of ICC in these mutants. There was considerable hyperplasia in all classes of ICC throughout the GI tract of Kit(V558Delta)/+ mice, except for ICC in the deep muscular plexus of the intestine. Spontaneous electrical activity and postjunctional neural responses in hyperplastic ICC tissues appeared normal but were up-regulated in the cecum, where GISTs were commonly found. Kit gain-of-function leads to hyperplasia of most classes of ICC throughout the GI tract. ICC retain normal pacemaker function and enteric neural responses well after development of hyperplasia.