Characterization of Chromosome 7q 21-32 Amplification in Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is a highly malignant tumor that is associated with high morbidity and mortality. Liver carcinogenesis, often arising from chronic liver cirrhosis, is a process characterized by step-wise accumulation of genetic aberrations that confer cell growth advantages, malignant transformation, vascular invasion and metastatic potentials. Genome-wide analyses have illustrated some commonly altered genomic events in association with HCC metastasis. Specifically, common genomic gain on 7q arm is largely suggestive of progression to advanced stage HCC progression. Despite the fact that chromosomal 7q gain is of pivotal significance, little has been known about its underlying candidate genes in the process of hepatic tumorigenesis.;Our earlier gene mapping analysis on 7q21-22 region identified a few candidate proto-oncogenes, including PFTAIRE protein kinase 1 (PFTK1) that possesses a Cdc2-related serine/threonine kinase signature. Although PFTK1 protein has been shown to confer HCC cell migratory phenotypes, the prognostic value and mechanistic insight by which PFTK1 enhances HCC motile properties remain largely elusive. In this thesis, tissue microarray analysis showed that increased PFTK1 expression in human HCC is significantly associated with early age onset (≤40 years), differentiation to advanced tumor staging and histologic presence of microvascular invasion. Coupling 2D-PAGE mass spectrometry with immunoprecipitation validation, β-actin (ACTB) and transgelin2 (TAGLN2) were confirmed as downstream substrates of PFTK1 kinase. Nevertheless, the detection of serine-phosphorylated form of TAGLN2 would seem a downstream target of PFTK1 kinase. Since the tumor suppressor role of TAGLN2 has been widely ascribed in the control of cancer metastasis, I attempted to define if TAGLN2 is an immediate phosphorylated target of PFTK1 kinase. Knockdown experiments of TAGLN2 in PFTK1-abrogated cells showed a recovery on cellular motile and invasive properties, as well as actin stress fiber formation, proposing a potential mechanism that halts the actin cytoskeleton dynamics. Residues S83 and S163 on TAGLN2 were identified as target sites for PFTK1 protein in the control of the actin-binding property of TAGLN2 and thus HCC cell motility. The findings from this thesis highlighted a novel oncogene-tumor suppressor interplay where oncogenic PFTK1 confers HCC cell motile phenotypes through phosphorylations on tumor suppressor TAGLN2. The phosphorylation of TAGLN2 would in tum promote cell migratory properties through the reduced actin-binding property ofTAGLN2.;Given that TAGLN2 suppression in PFTK1-knockdown cells could not completely revert the effects of PFTK1, this might suggest that other interacting partners are involved in the PFTK1-modulated biological path. Previous studies on Cdc2-related kinase substrates showed caldesmon (CaD), an actin-stabilizing protein, in the actin cytoskeletal modeling and cell migratory enhancement. In line with previous reports, I found CaD is a plausible phosphorylation target of PFTK1 kinase in PFTK1-knockdown cells, there was loss of CaD phosphorylations and corresponding displacement from the F-actin fibers. Without the binding of CaD to actin, actin stress fibers were evidently dispersed in PFTK1-abrogated cells, implicating reduced migratory capabilities. In addition to TAGLN2, CaD was underscored as another intermediate participant in the PFTK1 biological cascade, suggesting the dynamics of actin organization by the kinase activity of PFTK1.;Recently, the concept of microRNA (miRNA) deregulations has been widely adopted as an essential regulatory event in cancer development, where their influence on multiple tumor suppressors and oncogenes has been well illustrated. Our group previously reported on the up-regulation of a miRNA cluster (miR-183/96/182) in HCC. This cluster located on 7q32 showed common overexpression in primary HCC tumors. In the present study, concomitant up-regulation of miR -183, -96 and -182 in primary HCC was shown to be significantly associated with poor prognosis of patients and clinical features of metastatic tumors including presence of microvascular invasion and advanced tumor differentiation. I also provided evidence that j3-catenin transactivity could activate miR -183/96/182 overexpression, which could confer a migratory advantage in malignant hepatocytes through convergent targeting on Forkhead BoxO1.;In summary, this thesis highlighted the metastatic implications of 7q21-32 amplification in the progression of HCC. Overexpression of the target gene(s) or miRNA(s), including PFTK1 and miR-183/96/182, was concordantly shown to confer HCC migratory phenotypes. Thus, detailed understanding of driver genes on the 7q21-32 region might provide key molecular informations underlying HCC metastasis, and hence aid development of more favourable therapeutic regimens for patients diagnosed with HCC.