Nucleophosmin (Threonine234) is a novel mediator of tumor metastasis

Abstract

Metastasis is the major cause of death for cancer patients. Understanding the molecular mechanism of tumor metastasis is urgently awaited. Using hepatocellular carcinoma as our cancer model, the mechanism of tumor metastasis was studied by establishment of a pair of primary and its corresponding metastatic lung counterparts (PLC-PT and PLC-LM) by orthotopic injection of parental PLC into the liver of SCID mice. These two cell lines share the same genetic background but differ in invasive ability evidenced by invasion and wound healing assay. Since protein kinases and their phosphorylated substrates play key role in signalling process leading to cancer metastasis, we employed Celluspot™ Serine/Threonine peptide array to evaluate the phosphorylation profiling between these two matched HCC cell lines. Phosphorylation profiling revealed significant higher phosphorylated level of nucleophosmin (NPM) at Threonine 234 in PLC-LM than PLC-PT, which was phosphorylated by cyclin-dependent kinase (CDK1). Western blot analysis confirmed elevated phosphorylated level of NPM at Threonine 234 (Thr234) in PLC-LM using phospho-NPM (T234) antibody. Consistent NPM (Thr234) over-expression was also found in higher metastatic HCC cell line (MHCC-97H) when compared with its corresponding primary one (MHCC-97L), further suggesting the role of NPM (Thr234) in HCC metastasis. The role of NPM (Thr234) was further evaluated in 30 matched primary and metastatic HCC clinical samples by immunohistochemistry. Over-expression (24/30) of NPM (Thr234) was significantly correlated with HCC metastasis (p<0.001). Using a tissue microarray consisting of 31 cases of primary and their metastatic colorectal cancer, the role of NPM (Thr234) in metastasis was further evaluated in colorectal cancer. Tissue microarray revealed significant correlation between NPM (Thr234) over-expression (20/31) with tumor metastasis (p<0.001). Using immunohistochemical approach, prognostic significance of NPM (Thr234) was examined in 61 primary tissues derived from patients with prostate cancer. Interestingly, NPM (Thr234) over-expression not only correlated with tumor metastasis (p<0.001) but also with poor patients’ survival (5.6 months Vs 54.9 months for low and high NPM (Thr234) expression). Functional role of NPM (Thr234) in tumor invasion was examined by creation of non-phosphorylated mutant of NPM. Lentiviral-based transfection of NPM (Thr234) to Ala mutant in PLC-LM inhibited cell migration and invasion when compared with wild-type NPM and empty vector control. Wild-type NPM was found to physically interact with a novel metastatic gene, Rho-kinase II, thereby altering its transcriptional activity. The physical interaction between NPM (Thr234) and Rho-kinase II is being investigated. In conclusion, NPM (Thr234) is a novel inducer of metastasis and may be a novel phsophorylated substrate for prognosis and therapeutic intervention for cancer patients.