KIT Protein Expression Research Articles

Orchestrating Developmental Signaling to Balance Erythroblast Proliferation and Differentiation

The highly conserved exosome complex mediates the degradation and processing of multiple classes of RNAs. How this post-transcriptional RNA- and gene-regulatory machine impacts cell fate decisions and differentiation is poorly understood. Previously, we demonstrated that exosome complex subunits confer an erythroid maturation barricade, and the erythroid transcription factor GATA-1 dismantles the barricade by transcriptionally repressing the cognate genes. While dissecting requirements for the maturation barricade in mice, we discovered that the exosome complex is a vital determinant of a developmental signaling transition that dictates proliferation versus differentiation of erythroid precursor cells. Using shRNAs to downregulate exosome complex subunits, we developed conditions that disrupt exosome complex integrity and analyzed functional consequences. We discovered that erythroid precursor cells derived from E14.5 murine fetal liver exhibited significantly decreased survival when erythropoietin (Epo) was removed from the culture media. Live cells decreased from 66% in the control to 24% (p = 2 x 10-5) in the Exosc8-knockdown condition. The maturation-associated increase (1.5 fold, p = 0.003) in the Ter119+CD71+ (R3: early/late basophilic erythroblast) population upon Exosc8 knockdown did not occur without exogenous Epo. Thus, the survival and differentiation of erythroid precursors after downregulating Exosc8 is critically dependent on Epo. We tested whether Exosc8 downregulation impaired stem cell factor (SCF)-mediated proliferation/amplification of erythroid precursors. Using a phospho-flow cytometry assay, we quantitated the capacity of SCF or Epo to instigate cell signaling using the shared downstream substrate Akt. SCF induced maximal Akt phosphorylation in immature Ter119-CD71high erythroblasts (5.5 fold, p = 3 x 10-6). As erythroid maturation progressed to Ter119+CD71high, the SCF response was diminished. Exosc8 downregulation abrogated SCF-mediated induction of phospho-Akt. Epo induced maximal Akt phosphorylation in Ter119+CD71high erythroblasts, and Exosc8 downregulation accelerated acquisition of this signaling response. Whereas Epo did not affect Akt phosphorylation in control Ter119-CD71high erythroblasts, Epo increased phospho-Akt 4 fold (p = 0.003) upon Exosc8 downregulation. Thus, downregulating Exosc8 abrogated SCF signaling that supports erythroid precursor proliferation/amplification, while precociously inducing Epo-dependent pro-differentiation signaling. As downregulating Exosc8 abrogated SCF signaling, we tested whether the exosome complex or individual subunits confer Kit expression or promote signaling via a post-receptor mechanism. Downregulating Exosc8 or Exosc9 reduced Kit surface expression in the R2 (proerythroblast and early basophilic) cell population [14.6 (p = 3.6 x 10-4) and 6 fold (p= 1.3 x 10-6) decrease, respectively]. Exosc8 downregulation also reduced Kit mRNA and primary transcript levels at all erythroid maturation stages. By 24 h post-infection with shExosc8-expressing retrovirus, Kit protein decreased 10 fold (p = 0.046) in Ter119- erythroid precursor cells. We tested whether the Exosc8 requirement for Kit primary transcript, mRNA and protein expression involved alterations in the distribution of transcriptionally-competent serine 5-phosphorylated RNA polymerase II (Pol II) at Kit. Using quantitative ChIP analysis with control and Exosc8-knockdown Ter119- cells, Exosc8 downregulation reduced phospho-Ser5 Pol II occupancy within the coding region (+5 kb) and 3' UTR, but not at the promoter. Furthermore, Exosc9 occupied multiple regions of the Kit locus. These results support a model in which the exosome complex confers Kit expression and stem cell factor/Kit signaling via a transcriptional mechanism. Functioning as a gatekeeper of the SCF - Epo developmental signaling transition, the exosome complex controls the massive production of erythroid cells that ensures organismal survival in homeostatic and stress contexts. Studies are underway to establish the full repertoire of exosome complex targets in the developing erythroblast, how these targets relate to exosome complex targets in hematopoietic stem and progenitor cells and exosome complex-dependent strategies for translational applications. DisclosuresNo relevant conflicts of interest to declare.

Stem cell growth factor receptor in canine vs. feline osteosarcomas.

Osteosarcoma is considered the most common bone cancer in cats and dogs, with cats having a much better prognosis than dogs, since the great majority of dogs with osteosarcoma develop distant metastases. In search of a factor possibly contributing to this disparity, the stem cell growth factor receptor KIT was targeted, and the messenger (m)RNA and protein expression levels of KIT were compared in canine vs. feline osteosarcomas, as well as in normal bone. The mRNA expression of KIT was quantified by reverse transcription-quantitative polymerase chain reaction, and was observed to be significantly higher in canine (n=14) than in feline (n=5) osteosarcoma samples (P<0.001). KIT protein expression was evaluated by immunohistochemistry, which revealed that 21% of canine osteosarcoma samples did not exhibit KIT staining in their neoplastic cells, while in 14% of samples, a score of 1 (<10% positive tumour cells) was observed, and in 50% and 14% of samples, a score of 2 (10–50% positivity) and 3 (>50% positivity), respectively, was observed. By contrast, the cancer cells of all the feline bone tumour samples analysed were entirely negative for KIT. Notably, canine and feline osteocytes of healthy bone tissue lacked any KIT expression. These results could be the first evidence that KIT may be involved in the higher aggressiveness of canine osteosarcoma compared with feline osteosarcoma.

Abstract 191: LMTK3 is a novel regulator of oncogenic KIT in KIT-mutant cancers

Abstract Multiple cancers, such as gastrointestinal stromal tumors (GIST) and melanoma, have been shown to be caused by somatic activating mutations in the receptor tyrosine kinase KIT. The major cause of death in patients with advanced KIT-mutant cancers is due to KIT tyrosine kinase inhibitor-resistant metastatic disease. Resistance develops almost exclusively because of secondary mutations within KIT, highlighting the importance of KIT in the proliferation and survival of these tumors. Therefore, strategies that affect KIT protein expression and maturation may be therapeutic. In order to identify regulators of KIT that may represent novel targets, we performed an siRNA screen in multiple KIT-mutant cancer cell lines. From these screens we identified lemur tyrosine kinase 3 (LMTK3) as a novel KIT regulator and potential target in KIT-mutant cancers. LMTK3 has been implicated as an important protein in multiple cancers, including leukemia, breast, and colon cancer. However, the oncogenic function of LMTK3 is not entirely clear. Despite its name, lemur tyrosine kinase 3 is, in fact, a serine/ threonine kinase. Beyond its enzymatic kinase function, LMTK3 has also been shown to have important scaffolding functions. LMTK3 has not been previously known to interact with KIT, but does regulate the stability of other receptors. We have found that LMTK3 silencing reduced the viability of all KIT-mutant GIST and melanoma cells tested to date, including those with ATP binding pocket and activation loop resistance mutations. Importantly, LMTK3 silencing decreased the viability of KIT-mutant cells specifically, as silencing of LMTK3 in cells lacking KIT mutations did not affect their viability. Further, we found the decrease in cell viability was due to induction of apoptosis, as seen by cleavage of PARP within 96 hours of LMTK3 silencing. Because these cells depend so heavily on KIT and loss of KIT signaling results in cell death, we hypothesized that LMTK3 silencing may be affecting this pathway. Indeed, with LMTK3 silencing there was a significant decrease in phosphorylated KIT (Y719), which was accompanied by decreased phospho-AKT (S473). We also observed a robust decrease in total KIT protein levels. However, there was no decrease in KIT transcript levels, suggesting that LMTK3 plays a role in regulating KIT at the protein level. While other KIT protein regulators, such as HSP90, have been suggested as therapeutic targets for GIST, the ubiquitous functions of these targets makes these treatments sub-optimal, often causing toxicity. Silencing of LMTK3, on the other hand, specifically kills KIT-mutant cancer cells. Furthermore, as a kinase, inhibition of LMTK3 by a small molecule may be possible, and we are in the process of identifying LMTK3 inhibitors. LMTK3 is a positive regulator of oncogenic KIT in GIST and melanoma and represents a novel, tractable target in KIT-mutant cancers. Citation Format: Lillian R. Klug, Jeffery W. Tyner, Michael C. Heinrich. LMTK3 is a novel regulator of oncogenic KIT in KIT-mutant cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 191.

Expression and mutation of c-Kit in intracranial germ cell tumors: A single-centre retrospective study of 30 cases in China.

Although primary central nervous system (CNS) germ cell tumors (GCTs) are one of the most treatable types of malignant brain tumor, a subset of patients remain resistant to standard chemotherapy. Gain-of-function mutations of the c-Kit gene, and KIT protein expression, have been observed in a number of GCTs, including testicular seminoma, ovarian dysgerminoma and mediastinal seminoma in various ethnic groups. Although a small number of studies have reported the role of c-Kit in CNS GCTs, few have focused on Chinese patients exhibiting CNS GCTs. In the present study, the frequency and location of c-Kit mutations and KIT protein expression levels in CNS GCTs were investigated in 30 patients, between January 1994 and October 2014. Immunohistochemical assays suggested that KIT protein expression was present in 59.1% patients (66.7% in males and 42.9% in females); however, no statistically significant correlation was identified between KIT protein expression and patient clinicopathological features. By performing PCR amplification and direct sequencing, 4 mutational hot spots of the c-Kit gene (exons 9, 11, 13 and 17) were examined, and c-Kit gene mutation was identified in 1/17 (5.9%) CNS germinoma cases. This mutation was located in exon 11 at codon 557–558 WK (Tryptophan-Lysine). No c-Kit gene mutations were detected in non-germinomatous GCTs. Imatinib, a tyrosine kinase inhibitor, may be an effective treatment against standard chemotherapy-resistant CNS germinoma patients exhibiting c-Kit mutations.

Novel Mechanisms of Growth Inhibition By Histone Deacetylase Inhibitors in MPN

Histone deacetylase inhibitors (HDACi) are being evaluated in several clinical trials for the treatment of hematological malignancies and 2 of them have been approved for the treatment of T cell lymphoma. We have investigated the effectiveness of using 2 HDACi - Vorinostat (SAHA) and Panobionstat for treatment of systemic mastocytosis (SM) for which no treatments are available. SAHA and Panobinostat showed concentration dependent inhibition of proliferation in mastocytosis patient derived cell lines, HMC1.1 and HMC1.2 bearing either single mutation in the juxtamembrane domain KITG560V or double mutation also including the activation loop KITG560V,D816V, respectively. Interestingly HMC1.2 cells with 2 mutations in KITG560V, D816V were more sensitive to treatment with both SAHA (IC50 ~ 260 nM) and Panobinostat (IC50 ~11 nM) as compared to HMC1.1 cells (IC50 ~ 600 nM for SAHA and IC50 ~ 23 nM with Panobionstat) that have a single mutation in KIT. The inhibition of proliferation by HDACi treatment appeared to be associated with modulation of cell cycle and differentiation rather than induction of apoptosis. We further tested the effect of combining HDACi treatment with tyrosine kinase inhibitor, Dasatinib. Combination of Dasatinib (5 - 20 nM) with either SAHA or Panbionostat was more effective than any of the single drugs in HMC1.1 cells. However such a combination effect was not seen in HMC1.2 cells. Over 90% patients with aggressive systemic mastocytosis carry KITD816V mutation. This particular mutation of KIT is highly resistant to therapeutics and is associated with poor survival. Thus patients carrying the KITD816V mutation are likely to benefit from treatment with HDACi while juxtamembrane mutation bearing patients would benefit more from a combination of HDACi with kinase inhibitors such as Dasatinib. To further understand the mechanism behind HDACi inhibition of KITD816V mediated oncogenic signals, we expressed KITD814V (murine equivalent of human D816V) in myeloid cells and treated them with SAHA and Panobinostat. Cells expressing KITD814V showed cytokine independent proliferation and interestingly, cytokine independent proliferation was more sensitive to inhibition by both SAHA and Panbinostat as compared to the cytokine dependent proliferation. In contrast to HMC cells, inhibition of cytokine independent proliferation by HDACi in KITD814V oncogene expressing cells was associated with significant increase in apoptosis. The percent cells negative for Annexin V stain decreased from 74% in control to ~46% in 24h upon treatment with 500 nM SAHA or 10 nM Panbinostat (p< 0.001; n=3). Induction of apoptosis by HDACi in KITD814V oncogene expressing cells was significantly suppressed in presence of cytokine. Consistent with the in vitro data, treatment with SAHA prolonged the survival of mice transplanted with myeloid cells expressing KITD814V mutation as compared to the vehicle treated group (Median survival - 21d (veh), 34d (SAHA), p=0.0015, n=5). In addition to increased histone acetylation, treatment of cells with HDACi also leads to increase in acetylation of chaperone proteins such as HSP90 which can mediate proteasomal degradation of oncogenes and has been suggested as one of the mechanisms by which HDACi mediates growth inhibition. In contrast, we found no significant decrease in mutant KIT protein expression or AKT expression and phosphorylation upon treatment with either of the HDACi. Though expression of Pim1 a pro survival serine/threonine kinase was down-regulated by SAHA and Panobinostat. Treatment with HDACi was also associated with differential modulation of STAT3, STAT5 and ERK pathways. Treatment with both SAHA and Panobinostat down-regulated STAT5 phosphorylation and up-regulated ERK1/2 phosphorylation with more increase in ERK1/2 phosphorylation observed in cells with higher sensitivity to inhibition by HDACi. Though increased activation of the ERK pathway has been associated with leukemogenesis; there is some evidence to indicate that hyper activation of ERK pathway in absence of additional leukemogenic signals activates a pro senescence program. Differential expression of Pim1 and along with differential modulation of STAT and ERK pathways may contribute to differential sensitivity of the cells to HDACi and are likely to be of therapeutic value in aggressive systemic mastocytosis that has so far been associated with poor prognosis. DisclosuresNo relevant conflicts of interest to declare.

Abstract 2945: Nilotinib abrogates DNMT1-depenent DNA methylation: A novel mechanism for induction of AML leukemia regression

Abstract Nilotinib has been approved to treat newly diagnosed, imatinib-resistant or -intolerant leukemia, but the mechanisms underlying its antileukemia actions remain elusive. Here, we report that nilotinib blocks AML growth through its DNA hypomethylating activity. We showed that, in addition to inducing a reduction of KIT and FLT3 protein expression and phosphorylation, nilotinib downregulated DNMT1, 3a and 3b in Kasumi-1, MV4-11 and C1498 cells. Since DNMT1 is maintenance and de novo DNMT, we focused on DNMT1 and proved that nilotinib impaired DNMT1 expression via Sp1 dysfunction, since the luciferase activity driven by DNMT1 promoter containing Sp1 binding sites was decreased by 2.6 fold by nilotinib, while increased by 10.6 fold after Sp1 overexpression. Electrophoretic mobility shift assay disclosed that nilotinib abolished Sp1 binding on DNMT1 promoter. Dotblot using 5mC antibody revealed global DNA hypomethylation and qPCR showed p15INK4B upregulation in Kasumi-1 (3.4 fold) or MV4-11 (5.2 fold). Bisulfite sequencing found a decrease of DNA methylation on p15INK4B promoter from 100% to 89% in Kasumi-1 or 13% to 0% in MV4-11. The functional assays showed a decrease of colony formation in Kasumi-1 (60%) or MV4-11 (58%), and an increase of cell apoptosis in Kasumi-1 (6.9 fold) or MV4-11 (3.2 fold). We verified these findings in AML patient blasts (n = 3) by showing that nilotinib treatment led to the dephosphorylation of KIT, the downregulation of DNMT1, 3a, 3b and Sp1, the reduction of global DNA methylation and the re-expression of p15INK4B (4.4 fold) via promoter demethylation (from 14% to 6%), which was followed by suppressed colony formation (77%), promoted cell apoptosis (1.84 fold) and increased caspase-3 and caspase-8. To test if nilotinib induces AML regression via DNA hypomethylation in vivo, 0.1×106 C1498 cells were injected into C57BL/6 mice (n = 3 mice/group) via the tail vein. The leukemic mice were treated with 5, 10 or 15 mg/kg of nilotinib in PEG400 plus saline via intraperitoneal injection twice a week for a total of 6 doses. No toxicity was observed for the tested drug dosage and schedule, because of no evident change of mouse body weight. Compared to the control group, nilotinib-treated mice had a decrease in spleen size. H&amp;E staining showed that the control mice exhibited increased infiltration of AML cells into the spleen, lung and liver of recipients. The bone marrow (BM) histopathology identified a higher degree of mature post-mitotic cells containing metamyelocytes, bands and segmented neutrophils in nilotinib-treated mice than control. DNMT1 was downregulated (39%), p15INK4B was upregulated and global DNA methylation was reduced (60%) in BM cells from nilotinib-treated mice. Thus, our findings unveil DNA methylation machinery as an innovative target behind nilotinib's anticancer activities, and provide a new therapeutic avenue for nilotinib in overcoming aberrant epigenetics in AML. Note: This abstract was not presented at the meeting. Citation Format: Na Shen, Fei Yan, Jiuxia Pang, Naseema Gangat, Mark R. Litzow, Aref Al-Kali, Shujun Liu. Nilotinib abrogates DNMT1-depenent DNA methylation: A novel mechanism for induction of AML leukemia regression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2945. doi:10.1158/1538-7445.AM2015-2945

Molecular characterization of two candidate genes associated with coat color in Tibetan sheep (Ovis arise)

Coat color is a key economic trait in sheep. Some candidate genes associated with animal's coat color were found. Particularly, v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog (KIT) and microphthalmia-associated transcription factor (MITF) play a key role in the modulation of hair pigmentation in mammals. This study investigated those two candidate genes' mutations and expressions associated with wool color in Tibetan sheep. First, the gene polymorphisms of those two genes were analyzed, and then, relative mRNA expression levels of those two genes in skin tissue with different coat colors were compared. Thirdly, KIT and MITF protein expression levels were detected through Western blot and immune-histochemical. Allele C was predominant allele in the white coat color Tibetan sheep population of the MITF coding region g. 1548 C/T loci. The relative MITF mRNA expression in black coat skin tissue was significantly higher than white (P<0.01). However, no significant differences were detected in the KIT gene's mRNA expression of these two different coat color skin tissues (P>0.05), while the level of KIT protein expression in skin tissues of white and black coats was also roughly equivalent. Our study observed that, the level of MITF protein expression in black coat skin tissue was significantly higher than that in white coat skin tissue, and positive staining for MITF protein expression was detected mainly in the epidermis and the dermal papilla, bulb, and outer root sheath of hair follicles. We conclude that the black coat of Tibetan sheep is related to high MITF expression in the hair follicles, and MITF may be important for coat color formation of Tibetan sheep.

Feline intestinal mast cell tumours: clinicopathological characterisation and KIT mutation analysis.

Feline intestinal mast cell tumours (FIMCTs) are rare and reportedly characterised by poor differentiation, aggressive biological behaviour and lack of reliable therapeutic aids. KIT proto-oncogene-activating mutations have never been investigated in these tumours. This study describes the main clinicopathological and microscopic features observed in 17 FIMCTs. Tumour degree of differentiation, proliferative activity, Kit protein expression and KIT mutations were evaluated and correlated with survival to assess their prognostic relevance. Ten tumours were located in the small intestine, two in the ileocaecocolic junction, and five in the large intestine. Survival times ranged from 3-538 days. Fifteen tumours were evaluated histologically, and there were six well-differentiated, six moderately differentiated and three poorly differentiated FIMCTs. The last showed a medium-to-large deposition of collagen tissue (P <0.001), and significantly higher mitotic and Ki67 indexes compared with more differentiated tumours (P = 0.011). On survival analysis, tumour degree of differentiation (P <0.001) and a mitotic index >2 (P = 0.022) were significantly associated with decreased survival times. Twelve cases showed Kit protein immunoexpression. The Kit pattern was membranous in five cases (33.3%), focal paranuclear in five (33.3%) and diffuse cytoplasmic in two (13.3%). Cytoplasmic Kit patterns were associated with a lesser differentiation (P = 0.015). Mutation analysis was successfully performed on 12 primary tumours and four lymph node metastases; however, no encoding mutation was detected. Contrary to reports in the literature, FIMCTs seem to have an extremely variable biological behaviour. We propose a classification based on tumour degree of differentiation and proliferative activity. These findings need to be confirmed in larger series, and exploration of further genomic regions of KIT is warranted to clarify its role in the development and progression of these neoplasms.

Identifying therapeutic options in small cell cervical cancer by multiplatform evaluation of biomarker alterations

Small cell cervical cancer (SCCC) is an extremely rare and aggressive form of cervical cancer. Approximately 150 cases are diagnosed in the US each year, with few therapeutic options. We evaluated tumor samples in order to determine what percentage of patients may have targetable molecular aberrations. Methods: Seventy-five SCCC samples were profiled, 50 using a commercial multiple platform, including a combination of gene sequencing (Sanger, next generation sequencing (NGS)), protein expression (IHC) and gene amplification (CISH or FISH), and 25 at a cancer center using a 50 gene NGS platform (CMS50). The results were compared to ~800 HPV+ cervical cancers, neuroendocrine tumors (NET) from all sites, and small cell lung cancers. Results: TOP2A (85%), TOPO1 (55%), BCRP (91%), and MRP1 (100%) had high overexpression by IHC, while ERCC1 had low expression (11%). SCCC samples had higher protein expression of KIT (26%) than HPV+ cervical cancers (3%, p<0.05), but similar expression of KIT to small cell lung cancers (37%). HER2 amplification was identified in 4.5% of SCCC and 8% of HPV+ cervical cancers, while EGFR amplification was not seen in SCCC but was identified in 10% of HPV+ cervical cancers. Gene sequencing identified that SCCC samples had higher mutation rates for TP53 (23%) and KRAS (18%) compared to HPV+ cervical cancers (10% and 10%, respectively) but lower rates of PIK3CA (15% vs. 26%). Comparatively, small cell lung cancers had mutations in TP53 in 34% of cases and in KRAS in 5% of cases. NGS evaluation of 47 cases also identified 3 GNAS and RB1 mutations, 2 CTNNB1 and SMAD4 mutations, and single gene mutations in BRCA1, PTEN, MET, SMARCB1, APC, ATM, HNF1A, and FBXW7.

Clinical and Prognostic Significances of Cytokeratin 19 and KIT Expression in Surgically Resectable Pancreatic Neuroendocrine Tumors.

Background:Pancreatic neuroendocrine tumors (PanNETs) are malignant endocrine neoplasms that present diverse clinical behaviors. Therefore, identification of biomarkers of PanNETs is important for stratification of the prognosis of PanNET patients. Recently, cytokeratin 19 (CK19) and KIT expression were reported to have prognostic significance in PanNET patients.Methods:To identify their prognostic significance, CK19 and KIT protein expression were assessed in 182 surgically resected PanNETs and compared with clinicopathologic factors.Results:Of 182 PanNETs cases, CK19 and KIT expression was noted in 97 (53.3%) and 16 (8.8%) cases, respectively. PanNET patients with CK19 expression had larger tumors (p=.006), higher World Health Organization (WHO) grade (p=.002) and pT classification (p<.001), increased distant metastasis (p=.004), and lymphovascular (p=.012) and perineural (p=.019) invasion. Similarly, those with KIT expression had larger tumors (p=.030), higher WHO grade (p=.001), advanced pT classification (p<.001), distant metastasis (p=.001), and lymphovascular invasion (p=.014). The 5-year survival rate for PanNET patients with KIT expression was significantly lower (62%) than that of patients without KIT expression (77%, p=.011), as determined by univariate but not by multivariate analyses.Conclusions:CK19 and KIT expression correlate with higher metastatic potential and advanced disease stage, and KIT expression is associated with worse survival in PanNET patients.

Retinoic acid regulates Kit translation during spermatogonial differentiation in the mouse

In the testis, a subset of spermatogonia retains stem cell potential, while others differentiate to eventually become spermatozoa. This delicate balance must be maintained, as defects can result in testicular cancer or infertility. Currently, little is known about the gene products and signaling pathways directing these critical cell fate decisions. Retinoic acid (RA) is a requisite driver of spermatogonial differentiation and entry into meiosis, yet the mechanisms activated downstream are undefined. Here, we determined a requirement for RA in the expression of KIT, a receptor tyrosine kinase essential for spermatogonial differentiation. We found that RA signaling utilized the PI3K/AKT/mTOR signaling pathway to induce the efficient translation of mRNAs for Kit, which are present but not translated in undifferentiated spermatogonia. Our findings provide an important molecular link between a morphogen (RA) and the expression of KIT protein, which together direct the differentiation of spermatogonia throughout the male reproductive lifespan.

Mir-152 inhibits cell proliferation and colony formation of CD133(+) liver cancer stem cells by targeting KIT.

miR152 is involved in diverse biological functions and development of disease. This study investigates the role of mir-152 in cell proliferation and colony formation of liver cancer stem cells. We show that exogenous overexpression of mir-152 suppresses cell proliferation and colony formation in CD133(+) hep3B cells. We also show that KIT is a direct target of miR-152 and miR-152 downregulates protein expression of KIT by directly binding to 3' untranslated region of KIT. Downregulation of KIT by specific siRNAs inhibits proliferation and colony formation of CD133(+) hep3B cells, which is similar to inhibitory effects of miR-152. Moreover, exogenous expression of KIT compromises inhibitory effects of miR-152 on cell proliferation and colony formation. Our findings suggest that mir-152 inhibits cell proliferation and colony formation of CD133(+) hep3B cells by targeting KIT.

Comparison of molecular abnormalities in vulvar and vaginal melanomas

Malignant melanoma of the vulva and vagina is relatively uncommon and accounts for <5% of all melanomas in women. The aim of our study was to establish the biological properties and evaluate potential therapeutic targets in these tumors. We collected a series of 65 cases from three centers and re-evaluated the tumor tissue for predominant growth pattern (superficial spreading, nodular, and mucosal lentiginous) and tumor thickness. KIT (CD117) expression was detected immunohistochemically. In addition, tumors were screened for BRAF, NRAS, and KIT mutations by PCR and DNA sequencing as well as for KIT amplifications by fluorescence in situ hybridization. None of the cases contained BRAF mutations. NRAS mutations and KIT amplifications were detected in similar frequency (∼12%) in tumors of the vulva and vagina. In contrast, KIT mutations were present in 18% of primary melanomas of the vulva, but in none of the tumors arising in the vagina. Moderate or strong KIT protein expression was detected in 30 cases, including all tumors with KIT mutations and 6 of the 7 with KIT amplifications. In conclusion, BRAF mutations are virtually absent in melanomas originating from the vulva or vagina, whereas NRAS mutations and KIT amplifications occur in both locations. KIT mutations appear to be specific for melanomas of the vulva, suggesting that in spite of the anatomic proximity, the development of vulvar and vaginal melanomas involves different molecular alterations which may be targeted by novel treatment approaches.

Basaloid squamous cell carcinoma of esophagus expressing KIT: A case report with immunohistochemical analysis

Basaloid squamous cell carcinoma of esophagus (BSCC-E) is rare. This case report is the first demonstrating KIT protein expression of BSCC-E. A 74-year-old man presented with dysphagia. Endoscopy revealed a polypoid tumor (2×2×2.5cm) with a stalk in cervical esophagus. Biopsy showed squamous cell carcinoma with undifferentiated areas. An endoscopic submucosal dissection (ESD) was performed. Grossly, it was solid tumor with white cut surface. Histologically, the tumor was hypercellular carcinoma consisting of solid areas of island. The tumor cells were composed of basaloid malignant cells with hyperchromatic nuclei, scant cytoplasm, and basophilic cytoplasm. Many mitotic figures were recognized. Foci of comedonecrosis were scattered. Areas of squamous and glandular differentiations were scattered. Immunohistochemically, the tumor cells were positive for pancytokeratin (PCK) CAM5.2, PCK AE1/3, cytokeratin (CK) 7, KIT, CEA, CA19-9, EMA, Ki-67 (labeling index=80%). The tumor cells were negative for CK20, PDGFRA, NSE, vimentin, estrogen receptor, p53 protein, chromogranin, synaptophysin, CD56, and TTF-1.

Gastrointestinal Stromal Tumors: Diagnostic and Therapeutic Challenges

Background: Gastrointestinal stromal tumors (GIST) are the most common mesenchymal neoplasms of the digestive system. They originate from the interstitial cells of Cajal and are characterized by the over expression of KIT protein (Tyrosine Kinase), and they pose a diagnostic and therapeutic dilemma. Objective: A challenge in diagnosis and treatment of GIST Patients & Methods: This is a retrospective study of GIST cases that diagnosed and treated in our center during the past 5 years. These studies include clinical characteristics, imaging techniques, neoadjuvant therapy, surgical techniques, immunohistochemistry, and prognosis of such cases. Results: Sixteen patients were diagnosed as having GIST (12 males/4 females) with a mean age 62 years (31-83 years). Diagnosis was made preoperatively in 11 patients (69%) and intraoperatively with histopatholgical confirmation in five patients (31%). The site of the tumor was detected in the stomach in 6 cases (37.5%), one in duodenum (6.25%), five in small intestine (31.25%), one in mesentery (6.25%), two in colon (12.5%) and one rectal GIST (6.25%). The main presentation of the disease was anemia, GIT bleeding and abdominal mass. Fourteen patients considered resectable and they were operated upon (87.5%) and in two patients (12.5%) neadjuvant therapy was started with favorable response in one case and poor response in other one with advanced GIST. All patients received Imatinib as adjuvant therapy. Mean follow up period was 33 months (4-54 months). Conclusion: GIST is a complex and challenging disease that requires a multidisciplinary approach in specililized center for better prognosis of such disease.

MicroRNA-218 inhibits gastrointestinal stromal tumor cell and invasion by targeting KIT

The objectives of this study were to detect the expressions of microRNA-218 (miR-218) in human gastrointestinal stromal tumor (GIST) tissues and cells and explore its effects on the biological features of GIST-T1 cells and the expression of its target gene KIT, so as to provide new insights for GIST treatment. Using quantitative real-time polymerase chain reaction (qRT-PCR), we detected the expressions of miR-218 in the tissues and adjacent tissues of GIST and in the GIST cell lines including GIST882, GIST430, GIST48, and GIST-T1. Forty-eight hours after the miR-218 mimic was transfected into the GIST-T1 cells, the expression of miR-218 in the GIST-T1 cells was detected by qRT-PCR. The effect of miR-218 on the GIST-T1 cell viability was detected using MTT. The effect of miR-218 on the proliferation and apoptosis of GIST-T1 cell was analyzed using flow cytometry. Transwell invasion chamber was applied to detect the effect of miR-218 on the invasion of GIST-T1 cells. KIT was identified to be a target gene of miR-218 by the luciferase reporter enzyme system, and the effect of miR-218 on the expression of KIT protein in cells was determined using Western blotting. As shown by qRT-PCR, compared with that in the GIST adjacent tissue, the expressions of miR-218 in the tumor tissue and GIST cell lines were significantly decreased (P < 0.0001). Compared with the control group, the expression of miR-218 increased significantly in GIST-T1 cells transfected with miR-218 mimic for 48h (P < 0.01). MTT showed that the cell viability decreased significantly after the overexpression of miR-218 in the GIST-T1 cells (P < 0.01). Flow cytometry showed that the cell proliferation index significantly declined after the overexpression of miR-218 (P < 0.01); meanwhile, the apoptosis of cells also significantly increased (P < 0.01). Detection using the Transwell invasion chamber showed that the number of cells passing through the Transwell chamber significantly dropped after the enhanced expression of miR-218 (P < 0.01). Luciferase reporter gene assay showed that, compared with the control group, the relative luciferase activity significantly declined in the miR-218 mimic transfection group (P < 0.01). Compared with the control group, the expression of KIT protein in the GIST-T1 cells transfected with miR-218 mimic for 48h significantly decreased (P < 0.01). In conclusion, the expression of miR-218 decreases in human GIST tissue and cell lines. miR-218 can negatively regulate the expression of KIT protein and inhibit the proliferation and invasion of GIST cells. Treatment based on the enhanced expression of miR-218 may be a promising strategy for GIST.

114 Altered Expression of Tight Junction-Related Proteins and Increased Intestinal Permeability in a Rat Model of Post-Infectious Gut Dysfunction

Background & Aims: Reduced Kitl from the microenvironment and consequent loss of Kit expression contributes to ICC depletion e.g. in diabetic gastroparesis. It has been hypothesized that ICC that have lost Kit may be able to regain their phenotype. However, the fate of ICC has been difficult to track. Previously we developed conditionally immortalized cell lines (e.g., ICL2A) from Kithigh, hematopoietic marker-negative cells purified from gastric muscles of H-2Kb-tsA58 mice and used them to characterize the effects of chronic Kitl deprivation (DDW 2009, #270). These cells, while maintaining ICC-like morphology and ultrastructure, underwent progressive dedifferentiation resulting in a Kitlow/-Ano1low/-Cd44+Cd34phenotype and aberrant electrical activity consisting of small, highly irregular oscillations and rare, abnormally long slow waves. Resupplying membrane-bound Kitl did not increase ICCspecific gene expression. Here, we investigated whether epigenetic silencing could underlie these changes and whether pharmacological de-repression could restore ICC phenotype and function. Methods: ICL2A cells were treated with 2.5μM adenosine dialdehyde (Adox), an Ezh2 histone lysine methyltransferase inhibitor (HKMTI); 10μM BIX-01294, a G9a HKMTI; 1μM SAHA, a class I-II histone deacetylase inhibitor; or 5μM RG-108, a DNA methyltransferase inhibitor. Gene expression was studied by qRT-PCR and flow cytometry. Electrical activity was studied by whole-cell patch clamp in the current clamp mode. Occupancy of the Kit promoter (-1 to -1000 bp) by Ezh2 and Suz12, members of PcG repressive complex (PRC) 2, Ring1b, the histone ubiquityltransferase component of PRC1, as well as by the PRC2-established mark H3K27me3 (histone H3 trimethylated on K27) and the PRC1dependent mark H2AK119ub1 (monoubiquitylated histone H2A K119) was quantified by ChIP. Results: BIX-0129, SAHA and RG-108 applied for 2 weeks did not stimulate Kit protein expression. In contrast, Adox increased both Kit mRNA (P=0.001) and cell surface Kit and Cd44 expression. Ano1 mRNA also increased significantly (P,0.001), although its levels remained low. The role of PcG-mediated repression was verified by detecting occupancy of the 1000-bp Kit promoter by Ezh2, Suz12, Ring1b, H3K27me3 and H2AK119ub1. Adox reduced H3K27me3 and H2AK119ub1 occupancy by 47±6% and 33±3%, respectively. In Adox-treated ICL2A cells, current clamp recordings revealed steady, rhythmic, slow wavelike oscillations in 3 of 15 cells that were not seen in 64 control cells. Conclusions: Similarly to KitlowCd34+ ICC stem cells, Kit expression is repressed by PcG proteins in ICC chronically deprived of Kitl signaling. Inhibition of PcG activity may help restore ICC and their function in conditions that negatively impact the ICCmicroenvironment such as diabetic gastroparesis. Grant support: NIH DK58185.

Abstract 1035: Crenolanib, a highly selective platelet-derived growth factor receptor α/β (PDGFRA/B) tyrosine kinase inhibitor, destabilizes ETV1 protein in KIT-mutant gastrointestinal stromal tumor (GIST) cells.

Abstract Background &amp; Aims: Most GIST harbor oncogenic KIT or PDGFRA mutations, which are in most cases heterozygous. However, most GIST co-express both wild-type KIT and PDGFRA with the mutated KIT or PDGFRA. Signaling mediated by heterodimers of the wild-type receptors contribute to GIST growth even after blocking oncogenic signaling by KIT/PDGFRA inhibitors such as imatinib. Previously we found that crenolanib (CP-868596), a PDGFRA/B tyrosine kinase inhibitor with &amp;gt;100-fold selectivity over other tyrosine kinases including KIT and a potent inhibitor of oncogenic PDGFRA signaling in vitro, inhibited the proliferation of KIT+PDGFRA+PDGFRB− GIST-T1 cells with a potency comparable to that of imatinib (IC50: 5.3 nM; imatinib: IC50: 3.5 nM) but had very little effect in the KITlow/−PDGFRAlow/−PDGFRB+ GIST48B cells (maximum inhibition: &amp;lt;50%; IC50&amp;gt;75 nM) and in mouse stem cells for interstitial cells of Cajal (ICC-SC), a presumed cellular source for GIST, which share a phenotype with GIST48B cells (IC50 &amp;gt;600 nM; Hayashi et al., Gastroenterology 2012; 142:S30). Here we investigated whether the antiproliferative effect of crenolanib was dependent on KIT expression, and studied crenolanib's effect on KIT/PDGFRA signaling and on the stability of ETV1, a transcription factor required for GIST growth and survival. Methods: Protein expression and phosphorylation were studied by Western blotting. KITlow/−PDGFRAlow/+PDGFRB+ murine ICC-SC (2xSCS2F10) were retrovirally transduced to express wild-type murine KIT. Cell proliferation was studied by methyl-tetrazolium salt assay. Results: Expression of wild-type KIT in retrovirally transduced ICC-SC dramatically reduced crenolanib's IC50 from 1157 nM to 121 nM. In GIST-T1 cells, crenolanib significantly reduced PDGFRA and KIT protein expression, inhibited PDGFRA phosphorylation (IC50: 5 nM) but had no effect on KIT phosphorylation. Crenolanib applied at 8.3 nM concentration rapidly and profoundly inhibited phosphorylation of ERK1/2 MAP kinases and reduced ETV1 protein levels by &amp;gt;50% in 2 h. The MEK inhibitor PD98059 (50 μM) reproduced crenolanib's effect on ETV1 protein levels with an identical time-course. The inhibitory effects of both crenolanib and PD98059 on ETV1 protein expression could be reversed with the proteasome inhibitor MG132 (10 μM) and accentuated with the protein synthesis inhibitor cycloheximide (10 μg/mL). ETV1 was undetectable in the crenolanib-resistant, KITlow/−PDGFRAlow/−PDGFRB+ GIST48B cells. Conclusions: In KIT mutant, KIT+PDGFRA+PDGFRB− GIST cells, crenolanib induced ETV1 protein degradation by suppressing PDGFRA-dependent MAPK phosphorylation. Crenolanib's antiproliferative effect in these cells may depend on KIT-induced ETV1 expression and reflect the destabilization of ETV1. Grant support: NIH DK58185. Citation Format: Yujiro Hayashi, Michael R. Bardsley, Yoshitaka Toyomasu, Takahiro Taguchi, Brian P. Rubin, Monique Carter, Abhijit Ramachandran, Tamas Ordog. Crenolanib, a highly selective platelet-derived growth factor receptor α/β (PDGFRA/B) tyrosine kinase inhibitor, destabilizes ETV1 protein in KIT-mutant gastrointestinal stromal tumor (GIST) cells. [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 1035. doi:10.1158/1538-7445.AM2013-1035

Role of the Angotensin II Type 2 Receptor in thyroid hormone‐induced cardiac hypertrophy in a model of Ischemia/Reperfusion

We hypothesize that the cardioprotective effect of TH in ischemia/reperfusion (I/R) model may occur with the participation of AT2R. Mice were subdivided in four different groups: 1) Wild‐Type (treated with saline); 2) Hyperthyroid (treated with T3 0.7 μg/Kg BW/day, i.p., 14 days); 3) AT2R knockout (treated with saline) and 4) Knockout + T3 (treated with T3). Hearts were mounted on a Langendorff apparatus under a constant retrograde flow (2–2,5 ml/min) with oxygenated (95% O2, 5% CO2) Krebs–Henseleit buffer, 37ºC. Cardiac hypertrophy was determined based on heart weight (mg)/tibia's length (mm) ratio. Total T4 levels were determined using a commercial radioimmunoassay kit and cardiac protein expression was analyzed by Western Blotting. Data (mean±SEM) were analyzed by Student's t test, considering p&lt;0,05. T3 treatment was able to induced cardiac hypertrophy which was not changed by AT2R absence. Vasodilator effect of T3 treatment was observed in wild‐type mice while it was abolished in knockout mice. The p‐Akt (Thr 308) levels were significantly increased in T3 group compared to control group in wild‐type mice, but no difference was observed between knockout T3‐treated and AT2R knockout mice. These data point to a possible involvement of AT2R and Akt signaling pathway on cardioprotective effect induced by T3.

KIT gene exon 11 mutations in canine malignant melanoma

The proto-oncogene KIT encodes a receptor tyrosine kinase which has been shown to be upregulated in canine melanomas. KIT mutations lead to constitutive phosphorylation and activation of KIT in the absence of ligand binding. The presence of KIT mutations and KIT protein expression was examined in a cohort of 49 dogs with canine malignant melanoma. An exon 11 synonymous nucleotide 1743C→T mutation was identified in five cases in which one also harbored a L579P mutation. Tumors that harbored the KIT exon 11 mutation(s) correlated significantly with disease recurrence (P=0.05). All 36 melanomas available for immunohistochemical analysis showed either weak (16 cases, 44.4%) or strong (20 cases, 55.6%) expression of the KIT protein. The five KIT mutation carriers were all strongly positive for KIT by immunohistochemical staining. These findings suggest that a subset of canine malignant melanomas harbors a KIT exon 11 mutation.