Abstract
The PFTK1 gene encodes a cdc2-related serine/threonine protein kinase that has been shown to confer cell migratory properties in hepatocellular carcinoma (HCC). However, the prognostic value and biological mechanism by which PFTK1 promotes HCC motility remain largely unknown. Here, we showed from tissue microarray that common upregulations of PFTK1 in primary HCC tumors (n=133/180) correlated significantly with early age onset (40 years), advance tumor grading and presence of microvascular invasion (P0.05). To understand downstream phosphorylated substrate(s) of PFTK1, phospho-proteins in PFTK1 expressing and knockdown Hep3B cells were profiled by two-dimensional-polyacrylamide gel electrophoresis mass spectrometric analysis. Protein identification of differential spots revealed β-actin (ACTB) and transgelin2 (TAGLN2) as the two most profound phosphorylated changes affected by PFTK1. We verified the presence of TAGLN2 serine phosphorylation and ACTB tyrosine phosphorylation. Moreover, reduced TAGLN2 and ACTB phosphorylations in PFTK1-suppressed Hep3B corresponded to distinct actin depolymerizations and marked inhibition on cell invasion and motility. Given that TAGLN2 is a tumor suppressor whose function has been ascribed in cancer metastasis, we examined if TAGLN2 is an intermediate substrate in the biological path of PFTK1. We showed in PFTK1-suppressed cells that knockdown of TAGLN2 over-rode the inhibitory effect on cell invasion and motility, and a recovery on actin polymerization was evident. Interestingly, we also found that unphosphorylated TAGLN2 in PFTK1-suppressed cells elicited strong actin-binding ability, a mechanism that possibly halts the actin cytoskeleton dynamics. Site-directed mutagenesis of TAGLN2 suggested that PFTK1 regulates the actin-binding affinity of TAGLN2 through the S83 and S163 residues, which if mutated can significantly affect HCC cell motility. Taken together, our data propose a novel, oncogene-tumor suppressor interplay, where oncogenic PFTK1 confers HCC cell motility through inactivating the actin-binding motile suppressing function of TAGLN2 via phosphorylation.
Highlights
Hepatocellular carcinoma (HCC) is a highly aggressive tumor that is rapidly fatal
PFTK1 modulates HCC cell motility through TAGLN2 WKC Leung et al correlative analysis indicated that increased PFTK1 expressions were associated with early age onset of HCC (o40 years; P 1⁄4 0.021), advance tumor grading to poorly differentiated state (Po0.0001) and the histological presence of microvascular invasion (P 1⁄4 0.05) (Table 1)
We were able to establish a prognostic value for PFTK1 in human HCC, where common PFTK1 overexpressions correlated with advance tumor histology, the presence of micro-vascular invasion and the likely indication of an early age onset
Summary
Hepatocellular carcinoma (HCC) is a highly aggressive tumor that is rapidly fatal. It currently ranks the fifth most common cancer worldwide and the third leading cause of cancer-related deaths globally (Stewart and Kleihues, 2003). The development of HCC, often arising from chronic liver disease, is a multistep process characterized by progressive accumulation of genetic alterations that cause aberrant cell growth, malignant transformation, vascular invasion and metastasis. Genome-wide analysis has cataloged a number of causal genetic changes that underscore the molecular basis of HCC development and progression (Herath et al, 2006; Katoh et al, 2007). We reported previously on regional chr.7q21–q22 over-representations in close association with progression of HCC to advance metastatic tumors (Poon et al, 2006). The biological basis that underlies the action of PFTK1 in conferring cell migratory properties in HCC remains unknown
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