KIT Expression Research Articles

Enlarged rete pegs with excessive accumulation of melanosomes leading to darker aging spots revealed by histomorphological measurements of internal structures of the epidermis.

Various histological studies of facial pigmented spot sites such as solar lentigo have been reported, but few studies have used quantitative indices by histomorphometric analysis of the internal structure of pigmented spot sites using non-invasive methods. In the present study, to quantitatively elucidate morphological changes in the epidermis in male, darker-pigmented spots and female, light-pigmented spots, indices that characterize the internal structure of the epidermis in pigmented spot sites were measured using in vivo confocal laser scanning microscopy (CLSM). The darkness of pigmented spots on the cheeks of 69 women and 43 men was analysed using image analysis software. The L* value was calculated from RGB values obtained from facial images. The internal structures of pigmented spots on the cheeks of 13 subjects were observed by CLSM. Various parameters were measured using CLSM images from the surface of the stratum corneum to the bottom of the dermal papillae, including the thickness of the epidermis, melanosome content, and shape of the dermal papillae. Mean ΔL* values between pigmented spots and non-pigmented areas of male subjects were significantly increased in the 40s and 50s compared with those of female subjects. Conspicuous pigmented spots increased in the 40s in male subjects and the 50s in female subjects. In CLSM observations, significant increases in the thickness of the epidermis and melanosome content were confirmed in pigmented spots compared with surrounding non-pigmented areas. In particular, melanosome content in the male subject group with dark-coloured pigmented spots increased significantly to about eight times that of non-pigmented areas, and more than double that of the male subject group with light-coloured pigmented spots. From the measurements of quantitative parameters, morphological changes in the epidermis were clearly related to the surface colour tone of pigmented spots. Darker pigmented spot sites tended to show longer rete pegs in the epidermis. Accumulation of melanosomes in epidermal basal cells could be considered to increase with the degree of elongation of rete pegs at pigmented spot sites and, thus, induce darker pigmented spots.

Sodium butyrate supresses malignant human mast cell proliferation, downregulates expression of KIT and promotes differentiation.

Sodium butyrate (NaBu) is a class I histone deacetylase inhibitor (HDACi) that can impede the proliferation of transformed cells. Although some HDACi downregulate the expression of the stem cell factor receptor (KIT/CD117), the effect of NaBu on KIT expression and human mast cell proliferation requires further elucidation. In this study, we examined the effects of NaBu on three transformed human mast cell lines, HMC-1.1, HMC-1.2 and LAD2. NaBu (100 µM) inhibited the proliferation and metabolic activity of all three cell lines without significantly affecting their viability, suggesting that although the cells had ceased to divide, they were not yet undergoing apoptosis. Cell cycle analysis using the cell-permeant dye, propidium iodide, indicated that NaBu significantly blocked the cell cycle progression of HMC-1.1 and HMC-1.2 from G1 to G2/M phases. Furthermore, NaBu downregulated the expression of C-KIT mRNA and KIT protein expression in all three cell lines, but this effect was most significant in the HMC-1.1 and HMC-1.2, both of which harbour activating mutations in KIT, which proliferate more rapidly than LAD2. These data support earlier observations showing that human mast cell lines are sensitive to histone deacetylase inhibition. However, our data presents the novel observation that inhibition of cell proliferation by NaBu was not associated with a loss in cell viability but rather an arrest of the cell cycle. Higher concentrations of NaBu led to modest increases in histamine content, tryptase expression, and granularity. In conclusion, NaBu treatment of human mast cell lines led to a modest enhancement of the hallmarks of mature mast cells.

Carcinoid-Like Cell Arrangements in Basal Cell Carcinoma: A Study of 11 Cases.

Background. Carcinoid-like pattern in basal cell carcinoma is extremely rare and has only been documented in a textbook. The aim of this study is to reveal the characteristics of the carcinoid-like pattern in basal cell carcinoma as well as to establish the concept of the tumor pattern. Methods. Of 355 basal cell carcinoma cases (355 lesions of 308 patients), 11 tumors with histopathological features of carcinoid-like pattern were retrieved. Results. The patients included 5 males and 6 females with a median age of 73 years (range 38-86 years). Of the 11 lesions, 10 were observed on the head. Histopathologically, a carcinoid-like pattern with branching and anastomosing of trabecular structures was observed in 30% to 95% of the tumor area. No differentiation to sebocytes was observed in any of the tumors. The tumors were well-circumscribed, although 3 lesions contained an infiltrative-type component as well. The carcinoid-like pattern areas in all 11 lesions exhibited diffuse BerEP4 immunoexpression but no KIT, synaptophysin, or vimentin expression. Keratin 20-positive Merkel cells were not observed in the tumor areas. Conclusions. Basal cell carcinoma can show a carcinoid-like pattern cell arrangement. Based on the histopathological and immunohistochemical findings from our study, we collectively conclude that the carcinoid-like pattern in basal cell carcinoma does not seem to represent sebaceous mantle differentiation. Carcinoid-like pattern basal cell carcinoma can be distinguished from other tumors by the favorite site of the head, the co-existence of conventional basal cell carcinoma components, lack of sebaceous differentiation, and diffuse BerEP4 immunoexpression.

MITF Downregulation Induces Death in Human Mast Cell Leukemia Cells and Impairs IgE-Dependent Degranulation.

Activating mutations in KIT (CD117) have been associated with several diseases, including gastrointestinal stromal tumors and mastocytosis. Rapidly progressing pathologies or drug resistance highlight the need for alternative treatment strategies. Previously, we reported that the adaptor molecule SH3 binding protein 2 (SH3BP2 or 3BP2) regulates KIT expression at the transcriptional level and microphthalmia-associated transcription factor (MITF) expression at the post-transcriptional level in human mast cells and gastrointestinal stromal tumor (GIST) cell lines. Lately, we have found that the SH3BP2 pathway regulates MITF through miR-1246 and miR-5100 in GIST. In this study, miR-1246 and miR-5100 were validated by qPCR in the SH3BP2-silenced human mast cell leukemia cell line (HMC-1). MiRNA overexpression reduces MITF and MITF-dependent target expression in HMC-1. The same pattern was observed after MITF silencing. In addition, MITF inhibitor ML329 treatment reduces MITF expression and affects the viability and cell cycle progression in HMC-1. We also examine whether MITF downregulation affected IgE-dependent mast cell degranulation. MiRNA overexpression, MITF silencing, and ML329 treatment reduced IgE-dependent degranulation in LAD2- and CD34+-derived mast cells. These findings suggest MITF may be a potential therapeutic target for allergic reactions and deregulated KIT mast-cell-mediated disorders.

Immunohistochemical analysis of expression of VEGFR2, KIT, PDGFR-β, and CDK4 in canine urothelial carcinoma.

Urothelial carcinomas (UCs), also known as transitional cell carcinomas, are the most common canine urinary tract neoplasms. Tyrosine kinases (TKs) are enzymes that tightly regulate cell growth and differentiation through phosphorylation. Receptor TK (RTK) inhibitors are currently used to treat UCs. Toceranib phosphate (Palladia; Pfizer) is an RTK inhibitor that blocks the activity of vascular endothelial growth factor receptor 2 (VEGFR2), platelet-derived growth factor receptor-alpha and -beta (PDGFR-α, -β), FMS-like tyrosine kinase 3, stem cell factor receptor (KIT, kinase inhibitor targeting), and colony stimulating factor receptor. To better understand UCs and validate treatment targets, we performed immunohistochemical staining for RTKs, as well as a novel target, cyclin-dependent kinase 4 (CDK4, a central regulator of the mammalian cell cycle), on formalin-fixed, paraffin-embedded tissues from bladder biopsies from 17 dogs with UCs, 17 dogs with cystitis (diseased controls), and 8 normal dogs (negative controls). Although immunohistochemical scores could not be extrapolated to prognostic value, response to treatment, and outcome of patients with UC, we demonstrated expression of PDGFR-β and VEGFR2 in UCs; all UC samples staining positively for VEGFR2. Minimal positive staining for KIT was noted in the tumor samples. CDK4 staining intensity was significantly weaker in UCs compared with normal and cystitis bladder samples. The intense staining of VEGFR2 in UC cells suggested that VEGFR2 may be of prognostic and/or therapeutic value in dogs with UC. Overexpression of VEGFR2 in UC cells validates this receptor as a treatment target in UC.

Preliminary Assessment of Tumor-Associated Tissue Eosinophilia (TATE) in Canine Mast Cell Tumors: Prevalence and Prognostic Relevance and Its Association with Neoangiogenesis.

Mast cell tumor (MCT) is the most common malignant skin tumor in dogs. In order to gain more information on the prognostic markers in MCT, the role of the eosinophil granulocytes infiltrates was investigated and assessed by the evaluation of tumor-associated tissue eosinophilia (TATE) in 87 canine cutaneous MCTs. In human medicine, high TATE are often described in highly angiogenic tumors: we therefore assessed the vascular endothelial growth factor (VEGF) expression in neoplastic mast cells. TATE and VEGF expression were compared between themselves, with histological grading, immunohistochemical expression of KIT and Ki-67, and with the recurrence. We found a statistically significant correlation between TATE and Patnaik grading (p = 0.041), Kiupel grading (p = 0.022), immunohistochemical KIT expression (p = 0.015), and tumor recurrence (p = 0.000). No associations were observed with Ki-67 and VEGF expression. This is the first evaluation of TATE and its prognostic value in canine MCTs in veterinary oncology. This study suggest that this investigation could be an important source of information for this tumor and for other neoplasms.

Comparação dos padrões de KIT e infiltração de eosinófilos em mastocitoma canino

ABSTRACT KIT protein is associated with the etiology of canine mast cell tumors (MCT); however, the expression patterns of KIT are highly variable. The aim of this study was to determine if KIT patterns are related with eosinophil count in MCT. Hematoxylin eosin and May Grünwald-Giemsa stain techniques were applied, histological grading and eosinophil counting were performed in 48 MCT samples. Immunohistochemical evaluation was performed with IL-5, VEGFr, and c-KIT antibodies. The percentage of immunolabeling with IL-5 and VEGFr was determined, and the samples incubated with c-KIT were graded according to the immunolabeling pattern. Comparison of the mean eosinophil count between the histological grades and the different KIT expression patterns demonstrated a significant difference between KIT pattern 1 and KIT pattern 3, KIT pattern 3 showed a higher mean of eosinophil count. There was no significant correlation between eosinophil count and KIT patterns (p = 0.2648). However, a positive correlation was observed between the KIT patterns and Patnaik and Kiupel grades (p = 0.0006 and p = 0.0267, respectively). There was no significant correlation between eosinophil count, IL-5, or VEGFr. Further studies should determine whether eosinophil counts are an independent predictor of clinical outcome or simply correlated with already known predictors.

611 PLASMA EXOSOMES REFLECT MYOCARDIAL INJURY DETECTED BY CARDIAC MAGNETIC RESONANCE IN STEMI PATIENTS

Abstract Background CMR is the gold standard technique to detect microvascular obstruction (MVO) and myocardial salvage index (MSI) in patients with ST-segment elevation myocardial infarction (STEMI) treated by primary percutaneous coronary intervention (pPCI). Exosomes are lipid vesicles released by all cells that reflect the metabolic status of the releasing cells, thus representing potential biomarkers of disease. Objectives To evaluate the correlation between exosome profile and myocardial infarction characteristics as detected by CMR in patients after STEMI. Methods exosomes were isolated from plasma of 42 patients by different commercial kits, their concentration and size distribution determined by Nanoparticle Tracking Analysis, exosomal surface epitopes by the MACSPlex Exosome Kit and GPIIb-IIIa expression by ELISA kit. Data obtained were correlated with parameters of myocardial damage (STEMI location, culprit lesion, time to revascularization, MVO and MSI). Results Levels of circulating plasma exosomes were higher in patients with anterior STEMI (p = 0.0002) and when the culprit lesion was located in LAD (p = 0.045). In addition, a high number of exosomes was measured in the subset of patients who underwent a late revascularization (> 3 h from onset of symptoms; p = 0.038). Patients with MVO and low MSI (< 0.5) have smaller exosome (p = 0.0015 and p = 0.014, respectively). By analysing the expression of 37 surface protein markers, the strongest signals are platelet specific markers (CD41b and CD42a) followed by endothelial and immune cells markers. Interestingly, a lower expression of platelet marker GPIIb-IIIa was detected in both patients with MVO and anterior STEMI (p = 0.039 and p = 0.0038, respectively). Univariate analysis demonstrated that exosome size and GPIIb-IIIa were independent predictors of MVO/MSI and ROC analysis demonstrated that dimension and GPIIb-IIIa can identify patients with MVO/MSI (AUC 0.75 and 0.71, respectively). Conclusions The plasma exosome profile well reflects CMR-assessed myocardial injury after STEMI. In particular, the exosome dimension and the expression of platelet marker GPIIb-IIIa are independently associated with MVO. Future studies with larger populations are required to confirm the role of platelet-derived exosomes in risk stratification after STEMI.

Artesunate delays the dysfunction of age-related intestinal epithelial barrier by mitigating endoplasmic reticulum stress/unfolded protein response

The impairment of the intestinal epithelial barrier and subsequent bacterial translocation are common in aging individuals, contributory to several local and systematic disorders. However, the underlying mechanism of the age-related degeneration has not been fully understood. In this study, we demonstrated that the intestinal KIT signaling declined and de-activated with aging, parallel with epithelial barrier dysfunction. Endoplasmic reticulum stress (ERS)/unfolded protein response (UPR) was obviously increased during aging. The ERS and its downstream IRE1α were highly activated in the aging colonic epithelium. Furthermore, by the use of Tunicamycin (Tm)-induced ERS mouse and cell models, we uncovered that the activity of the ERS/IRE1α accelerated the protein degradation of KIT via ubiquitin-proteasome pathway. The deficiency of KIT signaling further reduced the transcription of the tight junction protein Claudin-3. Of significance, Artesunate (ART) could be capable of ameliorating the detrimental effect of ERS/IRE1α, indicated by the re-gained KIT and Claudin-3 expressions and the restoration of the intestinal epithelial barrier. In conclusion, our present study provided novel evidence elucidating the ERS/IRE1α-induced loss of KIT and Claudin-3 in the aging colonic epithelium and also shed light on the protective effect of Artesunate on the intestinal epithelial barrier by blocking ERS/IRE1α activity during aging.

SRSF10 is essential for progenitor spermatogonia expansion by regulating alternative splicing.

Alternative splicing expands the transcriptome and proteome complexity and plays essential roles in tissue development and human diseases. However, how alternative splicing regulates spermatogenesis remains largely unknown. Here, using a germ cell-specific knockout mouse model, we demonstrated that the splicing factor Srsf10 is essential for spermatogenesis and male fertility. In the absence of SRSF10, spermatogonial stem cells can be formed, but the expansion of Promyelocytic Leukemia Zinc Finger (PLZF)-positive undifferentiated progenitors was impaired, followed by the failure of spermatogonia differentiation (marked by KIT expression) and meiosis initiation. This was further evidenced by the decreased expression of progenitor cell markers in bulk RNA-seq, and much less progenitor and differentiating spermatogonia in single-cell RNA-seq data. Notably, SRSF10 directly binds thousands of genes in isolated THY+ spermatogonia, and Srsf10 depletion disturbed the alternative splicing of genes that are preferentially associated with germ cell development, cell cycle, and chromosome segregation, including Nasp, Bclaf1, Rif1, Dazl, Kit, Ret, and Sycp1. These data suggest that SRSF10 is critical for the expansion of undifferentiated progenitors by regulating alternative splicing, expanding our understanding of the mechanism underlying spermatogenesis.

KIT as a therapeutic target in neuroendocrine prostate cancer.

Understanding lineage plasticity in prostate cancer cells is crucial to overcoming disease progression and therapeutic resistance. In this issue of Cancer Cell, Han etal. demonstrate that Foxa2 promotes luminal-to-neuroendocrine differentiation and that it also upregulates Kit expression in neuroendocrine cells. KIT signaling inhibition represents a promising therapeutic strategy in neuroendocrine prostate cancer.

The microphthalmia-associated transcription factor is involved in gastrointestinal stromal tumor growth.

Gastrointestinal stromal tumors (GISTs) are the most common neoplasms of mesenchymal origin, and most of them emerge due to the oncogenic activation of KIT or PDGFRA receptors. Despite their relevance in GIST oncogenesis, critical intermediates mediating the KIT/PDGFRA transforming program remain mostly unknown. Previously, we found that the adaptor molecule SH3BP2 was involved in GIST cell survival, likely due to the co-regulation of the expression of KIT and Microphthalmia-associated transcription factor (MITF). Remarkably, MITF reconstitution restored KIT expression levels in SH3BP2 silenced cells and restored cell viability. This study aimed to analyze MITF as a novel driver of KIT transforming program in GIST. Firstly, MITF isoforms were characterized in GIST cell lines and GIST patients' samples. MITF silencing decreases cell viability and increases apoptosis in GIST cell lines irrespective of the type of KIT primary or secondary mutation. Additionally, MITF silencing leads to cell cycle arrest and impaired tumor growth in vivo. Interestingly, MITF silencing also affects ETV1 expression, a linage survival factor in GIST that promotes tumorigenesis and is directly regulated by KIT signaling. Altogether, these results point to MITF as a key target of KIT/PDGFRA oncogenic signaling for GIST survival and tumor growth.

AML-060 A Truncated Nucleus Isoform of FGFR1 Drives the Transformation of Leukemia Stem Cells in Leukemogenesis

Myeloid and lymphoid malignancies associated with chimeric FGFR1 kinases are the hallmark of stem cell leukemia and lymphoma syndrome (SCLL). In all cases, FGFR1 kinase is constitutively phosphoactivated in the cytoplasm as a result of chromosome translocations. Recently, we demonstrated that these chimeric kinases could be cleaved by granzyme B to generate a truncated derivative, tnFGFR1, which localized exclusively into the nucleus and was not phosphorylated. In this study, we used the mouse bone marrow transduction and transplantation model to demonstrate the leukemic transformation ability of this tnFGFR1 and identified its critical downstream targets, FLT3 and KIT, in mouse and acute myeloid leukemia (AML) patient samples. Stem cell transduction and transplantation in syngeneic mice was used to assess the transforming ability of tnFGFR1 in bone marrow stem cells, and RPPA and RNA-Seq were used to examine the related signaling pathways and regulated target genes. We now show that tnFGFR1 can independently lead to oncogenic transformation of hematopoietic stem cells. These leukemia cells show a mixed immunophenotype with a B-cell B220+Igm- profile in the majority of cells and Kit+ in virtually all cells, suggesting a stem cell disease. tnFGFR1 induces a distinct profile of altered gene expression with significant upregulation of transmembrane signaling receptors including FLT3 and KIT. De novo human AML also expresses tnFGFR1, which correlates with upregulation of FLT3 and KIT as in mouse leukemia cells. ChIP analysis demonstrates tnFGFR1 occupancy at the Flt3 and Kit promoters, suggesting a direct transcriptional regulation. Cells transformed with tnFGFR1 are insensitive to FGFR1 inhibitors but sensitive to the FLT3 inhibitor quizartinib (AC220), suggesting an alternative approach for the treatment of SCLL. This study demonstrates a novel model for the transformation of hematopoietic stem cells by chimeric FGFR1 kinases with the combined effects of direct protein activation by the full-length kinases and transcriptional regulation by tnFGFR1. In human AML, tnFGFR1 activation leads to increased FLT3 and KIT expression, and higher FLT3 and GZMB expression levels are associated with inferior prognosis. These observations provide insights into the relative therapeutic value of targeting FGFR1 and FLT3 in treating AML with this characteristic gene expression profile.

RNA-seq characterization of histamine-releasing mast cells as potential therapeutic target of osteoarthritis

ObjectiveMast cells in the osteoarthritis (OA) synovium correlate with disease severity. This study aimed to further elucidate the role of mast cells in OA by RNA-Seq analysis and pharmacological blockade of the activity of histamine, a key mast cell mediator, in murine OA. MethodsWe examined OA synovial tissues and fluids by flow cytometry, immunostaining, single-cell and bulk RNA-Seq, qPCR, and ELISA. Cetirizine, a histamine H1 receptor (H1R) antagonist, was used to treat the destabilization of the medial meniscus (DMM) mouse model of OA. ResultsFlow cytometry and immunohistology analysis of OA synovial cells revealed KIT+ FcεRI+ and TPSAB1+ mast cells. Single-cell RNA-Seq of OA synovial cells identified the expression of prototypical mast cell markers KIT, TPSAB1, CPA3 and HDC, as well as distinctive markers HPGD, CAVIN2, IL1RL1, PRG2, and CKLF, confirmed by bulk RNA-Seq and qPCR. A mast cell prototypical marker expression score classified 40 OA patients into three synovial pathotypes: mast cell-high, −medium, and -low. Additionally, we detected mast cell mediators including histamine, tryptase AB1, CPA3, PRG2, CAVIN2, and CKLF in OA synovial fluids. Elevated H1R expression was detected in human OA synovium, and treatment of mice with the H1 receptor antagonist cetirizine reduced the severity and OA-related mediators in DMM. ConclusionBased on differential expression of prototypical and distinct mast cell markers, human OA joints can be stratified into mast cell-high, −medium, and -low synovial tissue pathotypes. Pharmacologic blockade of histamine activity holds the potential to improve OA disease outcome.

Can transcription factors in the intervertebral disc of lopinavir/ritonavir prevent degeneration in the nucleus pulposus by mediating the regulation of inflammation through signaling pathways?

This study was conducted to examine whether lopinavir/ritonavir (Lop/r), an HIV protease inhibitor, can improve disc physiology and slow down intervertebral disc (IVD) degeneration through in vitro experimental methods, as well as whether it can suppress inflammation with interleukin-1 beta (IL-1β) and sex-determining region Y (SRY) protein-related high-mobility group box genes-9 (SOX9) through hypoxia-inducible factor 1-alpha (HIF-1α) and the nuclear factor kappa B (NF-κB) signaling pathway. The aim was to investigate whether Lop/r application is toxic to IVD cells and the microenvironment simultaneously. Human primary cell cultures were prepared using herniated IVD tissues obtained from patients with lumbar disc hernia who were unresponsive toconservative and medical treatment, and thereby, were operated on. The untreated culture samples served as control group, and the samples treated with Lop/r served as study group. Microscopic evaluations were performed simultaneously using fluorescent and supravital dyes in all groups. In addition to cell viability, toxicity, and proliferation analysis through a commercial kit, IL-1β, SOX9, HIF-1α, and NF-κB protein expressions were evaluated using Western blotting. In the statistical comparison of the obtained data, an alpha value less than 0.05 was considered significant. Cell proliferation decreased in the Lop/r group, but no cell death was observed (p < 0.05). Moreover, at the end of 72 hours after Lop/r application, IL-1β and NF-kB protein expressions decreased by 40% and 52%, respectively, while HIF-1α and SOX9 protein expressions increased by 4% and 59%, respectively (p< 0.05). Although these data were obtained from an in vitro experimental study, it is believed that these findings could make significant contributions to the pharmaco-regenerative treatment modalities of IVD degeneration. Lop/r suppresses the IL-1β and NF-κB and induces SOX9 and HIF-1α, since these signaling pathways may be related to human IVD degeneration.

Hsa_circ_0044907 promotes acute myeloid leukemia progression through upregulating oncogene KIT via sequestering miR-186-5p

ABSTRACT Background: It has been reported that circular RNA hsa_circ_0044907 (circ_0044907) expression is overtly elevated in acute myeloid leukemia (AML) patient-derived BMMCs. However, the effect of circ_0044907 on AML progression remains un-clarified. Methods: Expression of circ_0044907 in BM and AML cells were detected with real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR). Cell viability, proliferation, apoptosis, and cycle progression were determined by 3-(4,5-Dimethylthiazol-2-yl)−2,5-Diphenyltetrazolium Bromide (MTT), 5-ethynyl-2’-deoxyuridine (EDU), and flow cytometry assays. The regulatory mechanism of circ_0044907 was predicted by bioinformatics analysis and validated by dual-luciferase reporter, RNA pull-down, and RNA immunoprecipitation (RIP) assays. In vivo experiments were carried out to verify the function of circ_0044907. Results: Circ_0044907 was overexpressed in AML patient-derived BM and AML cells. Furthermore, circ_0044907 could distinguish AML patients from healthy controls, and high circ_0044907 expression in BM had a poor prognosis for AML patients, implying that circ_0044907 served as a diagnostic and prognostic indicator for AML. Functionally, circ_0044907 silencing reduced cell viability, restrained cell proliferation, arrested cell cycle progression, and induced cell apoptosis in AML cells in vitro. Furthermore, circ_0044907 knockdown decreased AML cell growth in xenograft mouse models. Mechanically, circ_0044907 sponged miR-186-5p to block the inhibiting effect of miR-186-5p on KIT. Silenced miR-186-5p expression weakened circ_0044907 knockdown mediated suppression on AML cell viability, proliferation, and cycle progression. Also, forced KIT expression weakened miR-186-5p upregulation mediated inhibition on AML cell viability, proliferation, and cycle progression. Conclusion: Circ_0044907 absorbed miR-186-5p to block the inhibiting impact of miR-186-5p on KIT, thus promoting AML progression.

A truncated derivative of FGFR1 kinase cooperates with FLT3 and KIT to transform hematopoietic stem cells in syndromic and de novo AML

BackgroundMyeloid and lymphoid malignancies associated with chimeric FGFR1 kinases are the hallmark of stem cell leukemia and lymphoma syndrome (SCLL). In all cases, FGFR1 kinase is constitutively phosphoactivated as a result of chromosome translocations, which lead to acquisition of dimerization motifs in the chimeric proteins. Recently, we demonstrated that these chimeric kinases could be cleaved by granzyme B to generate a truncated derivative, tnFGFR1, which localized exclusively into the nucleus and was not phosphorylated.MethodsStem cell transduction and transplantation in syngeneic mice was used to assess the transforming ability of tnFGFR1 in bone marrow stem cells, and RPPA and RNA-Seq was used to examine the related signaling pathways and regulated target genes.ResultsFor the first time, we show that this non-classical truncated form of FGFR1 can independently lead to oncogenic transformation of hematopoietic stem cells in an animal model in vivo. These leukemia cells show a mixed immunophenotype with a B-cell B220 + Igm- profile in the majority of cells and Kit+ in virtually all cells, suggesting a stem cell disease. tnFGFR1, however, does not activate classic FGFR1 downstream signaling pathways but induces a distinct profile of altered gene expression with significant upregulation of transmembrane signaling receptors including FLT3 and KIT. We further show that de novo human AML also express tnFGFR1 which correlates with upregulation of FLT3 and KIT as in mouse leukemia cells. ChIP analysis demonstrates tnFGFR1 occupancy at the Flt3 and Kit promoters, suggesting a direct transcriptional regulation. Cells transformed with tnFGFR1 are insensitive to FGFR1 inhibitors but treatment of these cells with the Quizartinib (AC220) FLT3 inhibitor, suppresses in vitro growth and development of leukemia in vivo. Combined treatment with FGFR1 and FLT3 inhibitors provides increased survival compared to FGFR1 inhibition alone.ConclusionsThis study demonstrates a novel model for transformation of hematopoietic stem cells by chimeric FGFR1 kinases with the combined effects of direct protein activation by the full-length kinases and transcriptional regulation by the truncated nuclear tnFGFR1 derivative, which is associated with GZMB expression levels. Genes significantly upregulated by tnFGFR1 include Flt3 and Kit which promote a leukemia stem cell phenotype. In human AML, tnFGFR1 activation leads to increased FLT3 and KIT expression, and higher FLT3 and GZMB expression levels are associated with an inferior prognosis. These observations provide insights into the relative therapeutic value of targeting FGFR1 and FLT3 in treating AML with this characteristic gene expression profile.

Subtype of Neuroblastoma Cells with High KIT Expression Are Dependent on KIT and Its Knockdown Induces Compensatory Activation of Pro-Survival Signaling.

Neuroblastoma (NB) is a pediatric cancer with high clinical and molecular heterogeneity, and patients with high-risk tumors have limited treatment options. Receptor tyrosine kinase KIT has been identified as a potential marker of high-risk NB and a promising target for NB treatment. We investigated 19,145 tumor RNA expression and molecular pathway activation profiles for 20 cancer types and detected relatively high levels of KIT expression in NB. Increased KIT expression was associated with activation of cell survival pathways, downregulated apoptosis induction, and cell cycle checkpoint control pathways. KIT knockdown with shRNA encoded by lentiviral vectors in SH-SY5Y cells led to reduced cell proliferation and apoptosis induction up to 50%. Our data suggest that apoptosis induction was caused by mitotic catastrophe, and there was a 2-fold decrease in percentage of G2-M cell cycle phase after KIT knockdown. We found that KIT knockdown in NB cells leads to strong upregulation of other pro-survival growth factor signaling cascades such as EPO, NGF, IL-6, and IGF-1 pathways. NGF, IGF-1 and EPO were able to increase cell proliferation in KIT-depleted cells in an ERK1/2-dependent manner. Overall, we show that KIT is a promising therapeutic target in NB, although such therapy efficiency could be impeded by growth factor signaling activation.

KIT promotes tumor stroma formation and counteracts tumor-suppressive TGFβ signaling in colorectal cancer

Expression profiling has identified four consensus molecular subtypes (CMS1-4) in colorectal cancer (CRC). The receptor tyrosine kinase KIT has been associated with the most aggressive subtype, CMS4. However, it is unclear whether, and how, KIT contributes to the aggressive features of CMS4 CRC. Here, we employed genome-editing technologies in patient-derived organoids (PDOs) to study KIT function in CRC in vitro and in vivo. CRISPR-Cas9-mediated deletion of the KIT gene caused a partial mesenchymal-to-epithelial phenotype switch and a strong reduction of intra-tumor stromal content. Vice versa, overexpression of KIT caused a partial epithelial-to-mesenchymal phenotype switch, a strong increase of intra-tumor stromal content, and high expression of TGFβ1. Surprisingly, the levels of phosphorylated SMAD2 were significantly lower in KIT-expressing versus KIT-deficient tumor cells. In vitro analyses showed that TGFβ signaling in PDOs limits their regenerative capacity. Overexpression of KIT prevented tumor-suppressive TGFβ signaling, while KIT deletion sensitized PDOs to TGFβ-mediated growth inhibition. Mechanistically, we found that KIT expression caused a strong reduction in the expression of SMAD2, a central mediator of canonical TGFβ signaling. We propose that KIT induces a pro-fibrotic tumor microenvironment by stimulating TGFβ expression, and protects the tumor cells from tumor-suppressive TGFβ signaling by inhibiting SMAD2 expression.

CircRNA-3302 promotes endothelial-to-mesenchymal transition via sponging miR-135b-5p to enhance KIT expression in Kawasaki disease

Endothelial-to-mesenchymal transition (EndMT) is implicated in myofibroblast-like cell-mediated damage to coronary artery wall of Kawasaki disease (KD) patients, which subsequently increases the risk of coronary artery aneurysm. Many circular RNAs (circRNAs) have been reported to be associated with cardiovascular diseases. However, the roles and underlying molecular mechanism of circRNAs in KD-associated EndMT remains indefinite. In this research, we screened out circRNA-3302 from human umbilical vein endothelial cells (HUVECs) treated by sera from healthy controls (HCs) or KD patients via circRNA sequencing (circRNA-seq). In addition, circRNA-3302 upregulation was verified in endothelial cells stimulated by KD serum and pathological KD mice modeled with Candida albicans cell wall extracts (CAWS). Moreover, in vitro experiments demonstrated that overexpression of circRNA-3302 could markedly induce EndMT, and silencing of circRNA-3302 significantly alleviated KD serum-mediated EndMT. To further explore the molecular mechanisms of circRNA-3302 inducing EndMT, RNA sequencing (RNA-seq), a dual-luciferase reporter system, nuclear and extra-nuclear RNA isolation, RT-qPCR and Western blot analyses and so on, were utilized. Our data demonstrated that circRNA-3302 contributed to the KD-associated EndMT via sponging miR-135b-5p to enhance KIT expression. Collectively, our results imply that circRNA-3302 plays an important role in KD-associated EndMT, providing new insights into minimizing the risks of developing coronary artery aneurysms.