Purpose: Gastrointestinal stromal tumor (GIST) is a common type of soft-tissue sarcoma. Imatinib, an inhibitor of KIT, platelet-derived growth factor receptor alpha (PDGFRA), and a few other tyrosine kinases, is highly effective for GIST, but advanced GISTs frequently progress on imatinib and other approved tyrosine kinase inhibitors. We investigated phosphodiesterase 3 (PDE3) as a potential therapeutic target in GIST cell lines and xenograft models. Experimental Design: The GIST gene expression profile was interrogated in the MediSapiens IST Online transcriptome database comprising human tissue and cancer samples, and PDE3A and PDE3B expression was studied using IHC on tissue microarrays (TMA) consisting of 630 formalin-fixed human tissue samples. GIST cell lines were screened for sensitivity to 217 anticancer compounds, and the efficacy of PDE inhibitors on GIST was further studied in GIST cell lines and patientderived mouse xenograft models. Results: GISTs expressed PDE3A and PDE3B frequently compared with other human normal or cancerous tissues both in the in silico database and the TMAs. Anagrelide was identified as the most potent of the PDE3 modulators evaluated. It reduced cell viability, promoted cell death, and influenced cell signaling in GIST cell lines. Anagrelide inhibited tumor growth in GIST xenograft mouse models. Anagrelide was also effective in a GIST xenograft mouse model with KIT exon 9 mutation that may pose a therapeutic challenge, as these GISTs require a high daily dose of imatinib. Conclusions: PDE3A and PDE3B are frequently expressed in GIST. Anagrelide had anticancer efficacy in GIST xenograft models and warrants further testing in clinical trials.
BackgroundReceptor tyrosine kinases (RTK) are potential targets for the treatment of ischemic heart disease. The human RTK family consists of 55 members, most of which have not yet been characterized for expression or activity in the ischemic heart.MethodsRTK gene expression was analyzed from human heart samples representing healthy tissue, acute myocardial infarction or ischemic cardiomyopathy. As an experimental model, pig heart with ischemia-reperfusion injury, caused by cardiopulmonary bypass, was used, from which phosphorylation status of RTKs was assessed with a phospho-RTK array. Expression and function of one RTK, ROR1, was further validated in pig tissue samples, and in HL-1 cardiomyocytes and H9c2 cardiomyoblasts, exposed to hypoxia and reoxygenation. ROR1 protein level was analyzed by Western blotting. Cell viability after ROR1 siRNA knockdown or activation with Wnt-5a ligand was assessed by MTT assays.ResultsIn addition to previously characterized RTKs, a group of novel active and regulated RTKs was detected in the ischemic heart. ROR1 was the most significantly upregulated RTK in human ischemic cardiomyopathy. However, ROR1 phosphorylation was suppressed in the pig model of ischemia-reperfusion and ROR1 phosphorylation and expression were down-regulated in HL-1 cardiomyocytes subjected to short-term hypoxia in vitro. ROR1 expression in the pig heart was confirmed on protein and mRNA level. Functionally, ROR1 activity was associated with reduced viability of HL-1 cardiomyocytes in both normoxia and during hypoxia-reoxygenation.ConclusionsSeveral novel RTKs were found to be regulated in expression or activity in ischemic heart. ROR1 was one of the most significantly regulated RTKs. The in vitro findings suggest a role for ROR1 as a potential target for the treatment of ischemic heart injury.Electronic supplementary materialThe online version of this article (10.1186/s12872-018-0933-y) contains supplementary material, which is available to authorized users.
Gastrointestinal stromal tumour (GIST), the most common sarcoma of the gastrointestinal tract, can be treated effectively with tyrosine kinase inhibitors, such as imatinib. Cancer immune therapy has limited efficacy, and little is known about the immune suppressive factors in GISTs. Fibrinogen‐like protein 2 (FGL2) is expressed either as a membrane‐associated protein or as a secreted soluble protein that has immune suppressive functions. We found that GISTs expressed FGL2 mRNA highly compared to other types of cancer in a large human cancer transcriptome database. GIST expressed FGL2 frequently also when studied using immunohistochemistry in two large clinical series, where 333 (78%) out of the 425 GISTs were FGL2 positive. The interstitial cells of Cajal, from which GISTs may originate, expressed FGL2. FGL2 expression was associated with small GIST size, low mitotic counts and low tumour‐infiltrating lymphocyte (TIL) counts. Patients whose GIST expressed FGL2 had better recurrence‐free survival than patients whose GIST lacked expression. Imatinib upregulated FGL2 in GIST cell lines, and the patients with FGL2‐negative GIST appeared to benefit most from long duration of adjuvant imatinib. We conclude that GISTs express FGL2 frequently and that FGL2 expression is associated with low TIL counts and favourable survival outcomes.
Merkel cell carcinoma (MCC) is a rare cutaneous neuroendocrine carcinoma that is frequently divided into Merkel cell polyomavirus negative and positive tumors due their distinct genomic and transcriptomic profiles, and disease outcomes. Although some prognostic factors in MCC are known, tumorigenic pathways, which that explain outcome differences in MCC are not fully understood. We investigated transcriptomes of 110 tissue samples of a formalin-fixed, paraffin-embedded MCC series by RNA sequencing to identify genes showing a bimodal expression pattern and predicting outcome in cancer and that potentially could play a role in tumorigenesis. We discovered 19 genes among which IGHM, IGKC, NCAN, OTOF, and USH2A were associated also with overall survival (all p-values < 0.05). From these genes, NCAN (neurocan) expression was detected in all 144 MCC samples by immunohistochemistry. Increased NCAN expression was associated with presence of Merkel cell polyomavirus DNA (p = 0.001) and viral large T antigen expression in tumor tissue (p = 0.004) and with improved MCC-specific survival (p = 0.027) and overall survival (p = 0.034). We conclude that NCAN expression is common in MCC, and further studies are warranted to investigate its role in MCC tumorigenesis.
<p>Supplementary Figure S1. A box-whisker plot showing the relative KIT, ANO1, and PRKCQ (encodes for protein kinase C theta) mRNA expression in soft-tissue sarcoma samples in the MediSapiens database. Supplementary Figure S2. PDE3A (A) and KIT (B) expression in human intestinal smooth muscle tissue (magnification x400, scale bar 100 μm). Supplementary Figure S3 A, Effects of the combinations of PDE3 siRNAs and DMSO on the GIST882 and GIST48 cell lines. siPDE3B and siPDE3A/B reduced GIST882 cell viability, and none of the treatments influenced markedly GIST48 viability. SEM < 0.05 in all data points. B, Effects of PDE3 inhibitors cilostazol, milrinone, and amrinone given with DMSO on GIST882 and GIST48 cell line viability. SE < 0.05 in all data points. Supplementary Figure S4. A Sankey diagram of top SPIA pathways. A Sankey diagram showing differentially expressed miRNAs, their predicted target genes, and the biological pathways estimated to be significantly impacted by the expression change (SPIA pGFDR <0.1). The fold-change of pathways represent upregulation [2] or downregulation [-2] in the cell line 882. Supplementary Figure S5. Hematoxylin-eosin stainings to illustrate the histological responses to anagrelide, imatinib, and combination therapy in GIST xenograft models. Supplementary Figure S6. PDE3A and PDE3B are expressed in GIST xenograft models. PDE3A and PDE3B protein expression in Western blots from four GIST mouse xenograft models after treatment with imatinib, anagrelide, or both. Each column represents one mouse tumor (maximum two per mouse). The numbers of tumors available varied for the xenograft models, since some of the GIST2B, GIST882, and GIST3 model tumors shrank markedly or disappeared leaving no tissue material left for the Western blot. Low or lacking PDE3A or PDE3B expression in some treatment groups (notably anagrelide plus imatinib treatment for 36 GIST2B and GIST3) may be rather due to a high grade histological response than PDE3A or PDE3B downregulation. Supplementary Figure S7. Effects of the drug treatments on KIT signaling in GIST xenograft models. A Western plot showing the expression of KIT, phosphorylated KIT (Y703 and Y719), AKT, phosphorylated AKT, MAPK, phosphorylated MAPK, and tubulin (control). Supplementary Figure S8. Tumor PDE3A and PDE3B expression in four mouse GIST models. A, Tumor PDE3A and PDE3B mRNA levels measured with qPCR. The highest PDE3A levels and the lowest PDE3A to PDE3B ratio were found in the tumor from the GIST9 xenograft model that responded the poorest to anagrelide. The P values refer to the statistical difference between tumor PDE3A expression in the GIST9 model and the other GIST models, and the difference between tumor PDE3B expression in the GIST882 model and the other models. *P <0.5; **P < 0.01; ***P < 0.001. The bars represent the standard error of mean. The data represent mean {plus minus} s.e.m. B, Western blots showing expression of PDE3A and PDE3B in GISTs originating from the four xenograft models. Two untreated tumors were studied from each model. C, SLFN12 expression in GIST xenograft models. SLFN12 expression is increased in anagrelide treated tumors compared to the control group.</p>
<p>Supplementary Materials and Methods</p>
<p>Supplementary Table S1. The outlier mRNA expression profiles of the gene tissue index (GTI) outlier analysis. The list of 2415 outlier genes in GIST is ranked by the gene tissue index.</p>
<p>Supplementary Table S5. List of genes most affected by anagrelide treatment and protein expression data.</p>
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