Sumoylation in p27kip1 via RanBP2 promotes cancer cell growth in cholangiocarcinoma cell line QBC939.

BACKGROUND: Cholangiocarcinoma is one of the deadly disease with poor 5-year survival and poor response to conventional therapies. Previously, we found that p27kip1 nuclear-cytoplasmic translocation confers proliferation potential to cholangiocarcinoma cell line QBC939 and this process is mediated by crm-1. However, no other post-transcriptional regulation was found in this process including sumoylation in cholangiocarcinoma. RESULTS: In this study, we explored the role of sumoylation in the nuclear-cytoplasmic translocation of p27kip1 and its involvement of QBC939 cells' proliferation. First, we identified K73 as the sumoylation site in p27kip1. By utilizing plasmid flag-p27kip1, HA-RanBP2, GST-RanBP2 and His-p27kip1 and immunoprecipitation assay, we validated that p27kip1 can serve as the sumoylation target of RanBP2 in QBC939. Furthermore, we confirmed crm-1's role in promoting nuclear-cytoplasmic translocation of p27kip1 and found that RanBP2's function relies on crm-1. However, K73R mutated p27kip1 can't be identified by crm-1 or RanBP2 in p27kip1 translocation process, suggesting sumoylation of p27kip1 via K73 site is necessary in this process by RanBP2 and crm-1. Phenotypically, the overexpression of either RanBP2 or crm-1 can partially rescue the anti-proliferative effect brought by p27kip1 overexpression in both the MTS and EdU assay. For the first time, we identified and validated the K73 sumoylation site in p27kip1, which is critical to RanBP2 and crm-1 in p27kip1 nuclear-cytoplasmic translocation process. CONCLUSION: Taken together, targeted inhibition of sumoylation of p27kip1 may serve as a potentially potent therapeutic target in the eradication of cholangiocarcinoma development and relapses.

CRM-1 knockdown inhibits extrahepatic cholangiocarcinoma tumor growth by blocking the nuclear export of p27Kip1.

Cholangiocarcinoma is a deadly disease which responds poorly to surgery and conventional chemotherapy or radiotherapy. Early diagnosis is difficult due to the anatomical and biological characteristics of cholangiocarcinoma. Cyclin-dependent kinase inhibitor 1B (p27Kip1) is a cyclin­dependent kinase inhibitor and in the present study, we found that p27Kip1 expression was suppressed in the nucleus and increased in the cytoplasm in 53 samples of cholangiocarcinoma from patients with highly malignant tumors (poorly-differentiated and tumor-node-metastsis (TNM) stage III-IV) compared with that in samples from 10 patients with chronic cholangitis. The expression of phosphorylated (p-)p27Kip1 (Ser10), one of the phosphorylated forms of p27Kip1, was increased in the patient samples with increasing malignancy and clinical stage. Coincidentally, chromosome region maintenance 1 (CRM-1; also referred to as exportin 1 or Xpo1), a critical protein responsible for protein translocation from the nucleus to the cytoplasm, was also overexpressed in the tumor samples which were poorly differentiated and of a higher clinical stage. Through specific short hairpin RNA (shRNA)-mediated knockdown of CRM-1 in the cholangiocarcinoma cell line QBC939, we identified an elevation of cytoplasmic p27Kip1 and a decrease of nuclear p27Kip1. Furthermore, the viability and colony formation ability of QBC939 cells was largely reduced with G1 arrest. Consistent with the findings of the in vitro experiments, in a xenograft mouse model, the tumors formed in the CRM-1 knockdown group were markedly smaller and weighed less than those in the control group in vivo. Taken together, these findings demonstrated that the interplay between CRM-1 and p27Kip1 may provide potentially potent biomarkers and functional targets for the development of future cholangiocarcinoma treatments.

Immunohistochemically detected expression of Skp2, p27 kip1, and p-p27 (Thr187) in patients with cholangiocarcinoma.

The paper was aimed to detect the expression of Skp2, p27(kip1) (p27), and p-p27 (Thr187) in patients with cholangiocarcinoma (CCA). Western blot and immunohistochemistry were used to analyze the expression and subcellular localization of p27, Skp2, and p-p27 (Thr187) in tissue specimens of 53 patients with CCA and 10 with chronic proliferative cholangitis (CPC). Besides, the relationships of p27, Skp2, and p-p27 (Thr187) with clinicopathologic findings were examined, followed by analysis of the relationships of p27 expression with p-p27 (Thr187) and Skp2 in CCA tissue. The expression of p27 was lower in CCA than CPC tissue, and the expression of Skp2 and p-p27 (Thr187) was higher in CCA than CPC tissue (P < 0.05). The positive p27 and Skp2 proteins were mainly expressed in nucleus and cytoplasm of CCA, and p-p27 (Thr187) was only observed in nucleus. The expression of p27, Skp2, and p-p27 (Thr187) was closely associated with tumor grade and TNM stage (P < 0.05). A significantly negative correlation between the expression of p27 and Skp2 (r = -0.480, P < 0.05) and between the expression of p27 and p-p27 (Thr187) (r = -0.387, P < 0.05) was observed. Skp2-dependent proteasomal degradation of p27 plays role in the malignant transformation of CCA. Besides, the expression of p27, Skp2, and p-p27 (Thr187) may serve as markers for the progression of CCA.