The Role Of Gankyrin In Pancreatic Cancer Proliferation

【Background】Pancreatic cancer is one of the most common fatal malignancies in digestive system and is the fourth leading cause of death due to cancer in the United States. Despite considerable advances in neoadjuvant chemotherapy, surgical techniques and perioperative care, the prognosis for pancreatic cancer has not been improved significantly in the last 20 years. Although many genes and molecular pathways have been reported to be involved in pancreatic carcinogenesis, it is important to identify new oncogenes and therapeutic targets to improve the survival rate of patients with pancreatic cancer.The gankyrin gene is located on human chromosome Xq22.3. It encoded an oncoprotein with seven ankyrin repeats and is highly conserved in all mammals. It was previously thought that gankyrin was negatively or weakly expressed in normal organs of the digestive system but overexpressed in tumor tissues, such as those of hepatocellular carcinoma, esophageal squamous cell carcinoma and colorectal cancer. Gankyrin, a subunit of the 26S proteasome that interacts with the S6 ATPase of the 26S proteasome, was thought to functions as a dual-negative regulator of RB and p53. Gankyrin binds MDM2 and facilitates the ubiquitination of p53 to the proteasome for degradation. Gankyrin also binds RB to promote phosphorylation and degradation of RB. And gankyrin binds CDK4, enhanced RB phosphorylation and stimulation of E2F transcriptional activity, which causing G1/S cell cycle progression. Gankyrin is more commonly overexpressed in HCC than in normal hepatic tissues, and it could play an important role mainly at the early stages of human hepatocarcinogenesis. It also leads to cell cycle progression in normal hepatocytes and is important for liver regeneration of patients with fulminant hepatic failure. In human esophageal squamous cell carcinoma, its overexpression is associated with poor prognosis. Intriguingly, previous studies in our laboratory found a direct correlation between gankyrin expression and the malignant phenotype of colorectal cancer. These findings suggested that gankyrin might be an important protein that is involved in carcinogenesis. However, the role of gankyrin in pancreatic carcinogenesis remains unknown.【Aims】1. To evaluate the expression of gankyrin in pancreatic cancer tissues and its clinicalpathological significance.2. To investigate the effect of gankyrin on pancreatic cancer cell proliferation.3. To explore the possible mechanism by which gankyrin promotes pancreatic cancer cell proliferation.【Methods】1. Immunohistochemistry was performed to examine the expression of gankyrin in pancreatic cancer and the matched adjacent noncancerous tissues.2. Real-time PCR and western blot analysis were performed to examine the mRNA and protein expression of gankyrin in pancreatic cancer cell lines (SW1990, PC-2, BxPC-3 and PANC-1).3. Liposome 2000 was used to transfect full length human gankyrin vector and gankyrin-siRNA vector into PC-2 cells and SW1990 cells, respectively. G418 was used to select stable cell clones.4. MTT assay, soft agar assay and FCM were used to analyze the effect of gankyrin on pancreatic cancer cell proliferation in vitro.5. Subcutaneous tumor formative assay was used to investigate the effect of gankyrin on proliferative ability in vivo.6. Western blot analysis was carried out to detect cell cycle related molecules in transfected cells and control cells.【Results】1. The expression of gankyrin in pancreatic cancer tissues and pancreatic cancer cells.Gankyrin staining was found positive in 71.9%(46/64) cases of pancreatic cancer tissues, compared to 7.8%(5/64) cases of non-tumor tissues (P<0.01). The expression of gankyrin was higher in Poorly differentiated pancreatic cancer tissues, low degree TNM stage and large tumor size groups (P<0.05). The mRNA and protein expression of gankyrin was higher in SW1990 cell line and lower in PC-2 cell line.2. The effect of gankyrin on pancreatic cancer cell proliferative ability in vitro and in vivo.After the full length human gankyrin vector was successfully transfected into PC-2 cells, gankyrin enhanced PC-2 cell growth and tumorigenesis ability in vitro, and promoted more PC-2 cells entering into S phase from G1 phase. When gankyrin-siRNA vector was transfected into SW1990 cells, SW1990 cell growth, the ability of colony formation, G1/S cell cycle progression and tumorigenesis ability in vivo were decreased.3. Gankyrin regulated p53 and various cell cycle related proteins which control G1 to S transition.Suppression of gankyrin could down-regulate cyclin A, cyclin D1, cyclin E, CDK2, CDK4, PCNA and p-Rb, but up-regulate p27, Rb and p53. Overexpression of gankyrin led to opposite results.【Conclusions】1. The expression of gankyrin was significantly higher in pancreatic cancer tissues, compared with noncancerous tissues. The overexpression of gankyrin was associated with the malignant phenotype of pancreatic cancer.2. Gankyrin could enhance pancreatic cancer cell growth and proliferation in vitro and in vivo.3. Gankyrin enhance pancreatic cancer cell growth and proliferation partly by regulating cell cycle specific molecules.