Experimental Study Of The Action Of Shh Signaling Pathway In Gastrointestinal Stromal Tumors

Gastrointestinal stromal tumors (GIST) are the most common gastrointestinal mesenchymal tumors. The abberant activation of c-kit or platelet-derived growth factor receptora(PDGFRA) receptor tyrosine kinase (RTK)s is the key factor leading to its generation. Surgery remains the major therapy currently, although the targeted medicine, Glivec, has been the first line of choice for patients with median to high risk or with recurring tumors, most of the patients develop drug resistance. At present, the search for common key signaling pathway downstream of c-kit/PDGFRA, or for targets on signaling pathways outside the c-kit/PDGFRA signaling pathway while crosstalking with the former, might be the direction for the development of targeted GIST therapy.The Sonic hedgehog (Shh) signaling transduction pathway plays a critical role in the tumorigenesis and development. Blocking the Shh signaling transduction pathway in tumor cells may provide a new targeted therapy option for treating human tumor. Currently there are only a small number of reports on the expression of Shh signaling pathway in GIST tissue, while to our knowledge, there is no literature on the relation between Shh and the clinical pathological factors of GIST and its prognosis, not to mention literatures on the exact action mechanism of Shh in GIST and its crosstalking with other signaling pathways. Furthermore, we were not able to locate any reports on the association between Shh and the proliferation and apoptosis of GIST cells.With the intention to explore the role of Shh signaling pathway in the tumorigenesis and development of GIST, and to verify that its possible action mechanism is to crosstalk with the PI3K and MAPK signaling pathway, this study sought to examine the expression of key proteins (Shh, Ptch, Smo and Gli-1) in the Shh signaling pathway in the biopsies of GIST using immunohistochemistry methods, and to analyze its relation with GIST pathological factors, reoccurrence and prognosis. The study used Western blot to assess the expression levels of Shh, PI3K, and MAPK pathways in GIST tissues, to analyze the correlation between the three signaling pathway expression; In vitro culture of the GIST-H1 cell line was the model chosen to activate these three signaling pathways mentioned above with corresponding activation factors; the three pathways were inhibited with targeted inhibitors separately or combined, the analysis of changes in the expression of key factors in the signaling pathway after inhibition was used to verify that these three signaling pathways cross talk to each other in GIST, and may play a role in the proliferation and apoptosis inhibition in GIST. This study is a preliminary attempt to identify the action mechanism of Shh signaling pathway in GIST and to explore the feasibility of targeted treatment by commissural inhibition of the Shh, PI3k and MAPK pathways.PartⅠThe expression of Shh signaling pathway factors in Gastrointestinal stromal tumor tissues and their associations with clinical pathological factorsObjective:To explore the expression of Shh signaling pathway factors in Gastrointestinal stromal tumor (GIST) tissues and their associations with clinical pathological factors.Methods:125 GIST patient cases that underwent surgery treatment at the first affiliated hospital of Anhui Medical University from January 2001 to December 2008 and with complete records were collected. The expression of Shh signaling pathway proteins Shh, Ptch, Smo and Gli-1 was assayed using immunohistochemistry methods, and the relation between these protein expression and the tumor site, diameter, mitotic figures, NIH risk classification, reoccurrence and prognosis of GIST was analyzed.Results:The expression rate of these four proteins, Shh, Ptch, Smo and Gli-1, in GIST tissues was 90.5%%,83.3%,85.7%, and 85.7% respectively; the expression level of Shh was positively associated that of Smo and Gli-1, the expression level of Ptch was positively associated with that of Smo, the expression level of Smo was positively associated with that of Gli-1, the expression level of Shh had no association with that of Ptch, the expression level of Ptch had no association with that of Gli-1; the expression level of Shh, Ptch, Smo or Gli-1 was not associated with patients’ gender or age, while it was associated with tumor size, mitotic figures, and risk classification. The positive expression of Smo and Gli-1 was also associated with the original tumor site and the histology category; the positive expression rate of Shh, Ptch, Smo and Gli-1 in GISTs with high and median risk according to NIH classification was substantially higher than those with extremely low and low risk, and the difference was significant. The 1-,3-, and 5-year survival rates of patients with positive expression of Shh, Ptch, Smo and Gli-1 were markedly lower than those with negative expression. Further multivariate analysis revealed that, the positive expression of Gli-1 was an independent risk factor of patients’ prognosis.Conclusion:The Shh signaling pathway was present and activated in the GIST tissues. The expression of critical proteins in the Shh signaling pathway was correlated, and they may participate in the origination and development of GIST through ligand-dependent activation. The expression of proteins in the Shh signaling pathway was associated with the reoccurrence and prognosis of GIST, particularly, the expression of Gli-1 protein may be used as an important indicator for predicting reoccurrence.Part ⅡThe expression of Gli-1, p-AMT and p-ERK in GIST tissues and its indicationsObjectives:To examine the expression levels of key factors in the Shh, PI3K/AKT and MAPK signaling pathways, Gli-1, p-AKT and p-ERK in GIST tissues, and to explore the correlation between the expression of these three signaling pathways.Methods:145 formalin-fixed and paraffin-embedded GIST tumor tissues from patients that underwent surgery treatment at the first affiliated hospital of Anhui Medical University from January 2013 to December 2013 and with complete records were collected. The expression levels of Gli-1, p-AKT and p-ERK in GIST tissues were assayed with western blot, and the correlation between these three protein expression levels were analyzed.Results:Gli-1, p-AKT and p-ERK were expressed in GIST of different risk classifications, and their expression levels were highly correlated and consistent.Conclusion:The key factors in the Shh, PI3K/AKT and MAPK signaling pathways, Gli-1, p-AKT and p-ERK were widely expressed in GIST, and these signaling pathways participated in the tumorigenesis and development of GIST. The Shh pathway may exert its effect on GIST through the mechanism of crosstalk regulation between the PI3K and MAPK pathways.Part ⅢStudies of the crosstalk regulation between Shh and the PI3K and MAPK signaling pathway and its impact on cell proliferation and apoptosis.Objective:To explore if there is crosstalk between the three signaling pathways in GIST, Shh, PI3K and MAPK, and its impact on cell proliferation and apoptosis.Methods:The GIST-H1 cell line was induced to activation by N-Shh and EGF, and specific inhibitors of each signaling pathway was used to block the corresponding pathway. The expression of key factors in each pathway, Gli-1, p-AKT and p-ERK was detected by western blot. The effect of Shh, PI3K/AKT and MAPK pathway inhibitors applied separately combined on the activity of each pathway in status of activation or inactivation was analyzed; The impact on GIST-H1 cell proliferation and apoptosis when these pathways were blocked by pathway inhibitors was studied.Results:In the GIST-H1 cell line, the expression levels of p-AKT, p-ERK and Glil were up-regulated by EGF and N-shh stimulation separately; the expression effect of p-AKT and p-ERK stimulated by EGF in the GIST-Hl cell line can be inhibited by wortmannin or PD98059, respectively, meanwhile this effect can be inhibited by cyclopamine too, and the combination of two inhibitors (cyclopamine + wortmannin, or cyclopamine+PD98059) can further inhibit the activity of these pathways; the expression effect of Gli-1 stimulated by N-Shh in the GIST-H1 cell line can be inhibited by cyclopamine, meanwhile this effect can be inhibited by wortmannin or PD98059 too, and the combination of two inhibitors (cyclopamine + wortmannin, or cyclopamine + PD98059) can further inhibit the activity of Gli-1 in the Shh pathway; under EGF treatment, the proliferation of GIST-Hl cells was notability faster, and this acceleration can be partially inhibited by PD98059 and cyclopamine-KAAD pretreatment; under N-Shh treatment, the proliferation of GIST-H1 cells was notability faster, and this acceleration can be partially inhibited by PD98059 and cyclopamine-KAAD pretreatment; the apoptosis of GIST-Hl cells can be induced by specific inhibitors of the Shh, PI3K and MAPK signaling pathways.Conclusions:The Shh, PI3K and MAPK signal transduction was present in GIST cells, and there was cross-links between each other. The conjunctive usage of inhibitors of these signaling pathways can synergistically inhibit cell proliferation or induce cell apoptosis, may be a new choice for targeted therapy in GIST.