A Study On The Underlying Mechanism Of Imatinib-aquired Resistance In Gastrointestinal Stromal Tumors

Part one:The role of p55PIK in Imatinib-resistance in Gastrointestinal Stromal TumorsObjective To explore the relationship between p55PIK and Imatinib-resistant GISTs. And then observe whether p55PIK contributes to Imatinib-resistance in GIST.Methods (1) Selected patients with huge GISTs who took a oral Imatinib treatment after surgical resection and then accepted a second surgery because of relapse. By immunohistochemical staining we detected the expression of p55PIK in the tumor tissues before and after the resisitance.(2) Established a Imatinib-Resistant GIST cell line (GIST882-IR) by culturing the Imatinib-sensitive GIST cell line (GIST882) with gradually increased Imatinib in the medium daily. Then compared the expression of p55PIK between Imatinib-sensitive GIST cell line (GIST882) and Imatinib-Resistant GIST cell line (GIST882-IR) by Western Blot and Quantitative Realtime-PCR.(3) Proposed a model for imatinib in vivo resistance with imatinib treatment of nude mice bearing subcutaneous GIST882xenograft tumors resulted in significant reduction in tumor growth. GIST882parental tumors were termed as generation F0, after three weeks of Imatinib treatment, the parental tumors were cut into small pieces and placed subcutaneously in other nude mice to form xenograft tumors (F1). By generation F6, the tumors were characterized as resistant after growth comparison with control animals showed imatinib to be ineffective in inhibiting tumor growth. After the model was established, we compared the expression of p55PIK in these xenograft tumors from FO to F6by Western Blot and Quantitative Realtime-PCR.(4) To observe whether the over-expression of p55PIK in Imatinib-sensitive GIST cell line (GIST882) could promoted Imatinib-resistance, while whether the inhibtor of p55PIK could reverse Imatinib-resistance in Imatinib-resistant GIST cell line (GIST882-IR) by CCK8assay. Results (1) p55PIK expression was significantly up-regulated in Imatinib-resistance GIST clinical tissues, cell line and nude mice.(2) Over-expression of p55PIK could promoted Imatinib-resistance in Imatinib-Sensitive GIST cell line (GIST882), while knock down of p55PIK could reverse Imatinib-resistance in Imatinib-resistant GIST cell line (GIST882-IR).Conclusion p55PIK was significantly up-regulated in Imatinib-acquired resistance GISTs and contributes to Imatinib-resistance in GIST882. Part two:The mechanism of p55PIK promoted Imatinib-resistance in Gastrointestinal stromal tumoursObjective To investigate the molecular mechanism of how p55PIK, a regulatory subunit of phosphoinositide3-kinase, could activate NF-κB signaling pathway mediating Imatinib resistance in GISTs.Methods (1) Selected patients with huge GISTs who took a oral Imatinib treatment after surgical resection and then accepted a second surgery because of relapse. By immunohistochemical staining we detected the expression of KIT in the tumor tissues before and after the resisitance. Then we compared whether the expression of KIT and p55PIK had a consistency.(2) Detected the expression of p55PIK, KIT and NF-κB p65(Ser536) in imatinib-sensitive GIST cell line(GIST882) and imatinib-resistant GIST cell line(GIST882-IR) by western blot, and confirmed that p55PIK, KIT and NF-κB p65(Ser536) have a consistency of expression changes.(3) over-express p55PIK in imatinib sensitive GIST cell line(GIST882), then observe the expression of KIT and NF-κB p65(Ser536) by western blot; meanwhile, knock-down p55PIK in imatinib-resistant GIST cell line(GIST882-IR), also detected the expression of KIT and NF-κB p65(Ser536) by western blot.(4) in a condition of blocking NF-κB by its inhibitor, over-express p55PIK in imatinib-sensitive GIST cell line, then observe the expression of KIT by western blot.Results:(1) KIT was over-expressed in imatinib-resistant clinical samples, which was consistent with the change of p55PIK.(2) p55PIK, KIT and NF-κB p65(Ser536) were all over-expressed in imatinib-resistant GIST cell line (GIST882-IR).(3) over-expression of p55PIK in imatinib-sensitive GIST cell line (GIST882) promoted the expression of KIT and NF-κB p65(Ser536); and blocking of p55PIK in imatinib-resistant GIST cell line (GIST882-IR) reduced the expression of KIT and NF-κB p65(Ser536).(4) p55PIK can promote the transcription of KIT by activating NF-κB. Conclusion:p55PIK can promote the transcription of KIT by activating NF-κB, which contributes to imatinib resistance.