Gankyrin Promotes The Initiation And Development Of Colorectal Cancer By Activating MTORC1

Objectives To explore the role of Gankyrin in the initiation and development of colorectal cancer(CRC).Methods1.Tissues from 268 patients were obtained in pathology department of Shanghai Ninth People’s Hospital Affiliated to Shanghai JiaoTong University School of Medicine from Jan 2004 to Jan 2008.30 normal colorectal tissues were acquired by endoscopy.Both 30 LIN adenoma tissues and 30 HIN adenoma tissues were acquired by endoscopy from patients who underwent endoscopic curative resection.178 malignant colorectal cancer and paired non-cancerous tissues were from patients who underwent curative colorectal resection.Expression of Gankyrin in tissues was evaluated using the IHC method.Kaplan-Meier survival analysis was used to examine the relationship between Gankyrin staining and patient survival.Western blot was used to observe the expression of Gankyrin in CRC cell lines and normal colorectal epithelial cells.2.We ectopically expressed Gankyrin in NIH3T3 cells.MTT assay and anchorage-independent growth assay were used to investigate the role of Gankyrin to cell growth and colony formation.Western blot was used to confirm the overexpression of Gankyrin and Gankyrin-related signaling molecules.We subcutaneously injected nude mice with NIH3T3 cells stably expressing Gankyrin or vector control to confirm the oncogenic transformation role of Gankyrin.Western blot and IHC were used to detect the expression of Gankyrin and P-S6K1 in the neoplasm.Three CRC cell lines(SW620,LOVO,RKO)were infected with increasing amounts(1ul,5ul,10ul)of plasmids introducing Gankyrin.Western blot was used to detect Gankyrin and mTORCl substrate P-S6K1 T389 and P-4E-BP1T37/46.MTT assay and anchorage-independent growth assay were used to investigate the role of Gankyrin on CRC cells growth and colony formation.We subcutaneously injected nude mice with CRC cells stably expressing Gankyrin or vector.Tumor volume was measured every 3days.Western blot and IHC staining were used to detect Gankyrin and P-S6K1 expression.Then we explore the effect of Rapamycin,a specific inhibitor of mTORC1,on Gankyrin induced mTORC1 avtivation in Gankyrin-NIH3T3 cells and Gankyrin-LOVO cells by using Western blot and anchorage-independent growth assay.3.Gankyrin siRNA was transfected in TSC1-/-and TSC2-/-MEF cells,Western blot was used to detect mTOR signaling.CRC cells stably expressing Gankyrin or vector and CRC cells stably expressing a shGANK or NC were used to detect TSC and mTOR signaling by Western blot.Gankyrin shRNA/vector were transfected in HEK293,Gankyrin siRNA/NC were transfected in CRC cells,Gankyrin plasmid/vector were transfected in CRC cells,After blocking new protein synthesis with cycloheximide(CHX),Western blot was used to detect TSC2.4.A panel of CRC cell lines stably expressing a Gankyrin shRNA(shGANK)or a negative control shRNA(NC)were constructed.MTT assay and anchorage-independent growth assay were used to investigate the role of Gankyrin to those CRC cell lines cell growth and colony formation.CRC cell lines(DLD-1,HCT116 and SW480)stably expressing a shGANK or NC were injected subcutaneously in nude mice.Tumor volume was measured every 3days.The effect of Gankyrin knockdown on mTORC1 signaling was detected by IHC staining.We developed three early passage PDX CRC tumors.After treatment with Gankyrin siRNA or NC,the volume of PDX CRC tumors were measured every 3days.The effect of Gankyrin knockdown on TSC2 and mTORCl signaling was determined by IHC staining of xenografrt tumor tissues.Results1.Increased expression of Gankyrin was observed in LIN,HIN and CRC compared to healthy colorectal tissue.Staining positive rate and Hscore were simalir in HIN and CRC tissues.Elevated expression of Gankyrin was also observed in CRC cell lines compared with normal colorecta epithelial cells.High Gankyrin staining was statistically significantly correlated with poor survival.2.Gankyrin promoted cell growth and anchorage-independent colony formation of NIH3T3 cells.After selection,stable clones were obtained and Western blot results confirmed mTORCl signaling pathway was significantly changed by Gankyrin overexpression.After injecting Gankyrin-NIH3T3 cells,nodular neoplasms could be observed while the vector control produced no detectable tumors.Histologically,tumors displayed significant Gankyrin overexpression and highly S6 phosphorylation.As determined by Western blot analysis,Phosphorylation of S6K1 increased with increasing Gankyrin level in all three CRC cell lines.Gankyrin overexpression promoted growth and colony formation of colorectal cancer cell lines in vitro and xenografrt tumor growth in nude mice.Gankyrin overexpressing xenografrts displayed significantly higher S6 phosphorylation and Ki67 staining,indicating activated mTOR signaling and cell proliferation.Rapamycim inhibits the formation of transformed foci by Gankyrin-NIH3T3 cells in soft-agar in a dose dependent manner and growth of CRC cells drnven by Gankyrin overexpression.In addition,Rapamycin abrogates the elevated mTORCl signaling(P-S6K1).3.Gankyrin regulates mTOR signaling through a TSC dependent pathway.Gankyrin overexpression dramatically decreased TSC2 protein levels while inducing mTORCl signaling.Knockdown of Gankyrin dramatically increased TSC2 protein levels while decreasing the activation of mTORCl.Knockdown of gankyrin significantly suppresses endogenous TSC2 degradation and increased the half-life of TSC2.Overexpression of gankyrin accelerated endogenous TSC2 degradation.4.In votro and in vivo,knockdown of Gankyrin significantly inhibited CRC cell growth,colony formation and tumor growth,with restored TSC2 expression and reduced mTORCl signaling.Conclusions Gankyrin promotes the initiation and development of colorectal carcinogenesis by activating mTORCl signaling.Knockdown of Gankyrin significantly attenuates mTORCl signaling and CRC growth.