Studies on the Roles of 14-3-3 Proteins and Focal Adhesion Kinases in Cancer Metastasis

Introduction:
Hepatocellular carcinoma (HCC) is a serious health problem in Taiwan. Despite the progress in early diagnosis and aggressive treatment for HCCs, intrahepatic recurrence and extrahepatic metastasis still result in a high proportion of treatment failure. In our previous study, we found that a co-factor protein, 14-3-3, is related to increased migration of colon cancer cells, and synchronized over-expression of a cytoskeleton protein, focal adhesion kinase (FAK), with 14-3-3 was identified also. Because our preliminary data revealed that 14-3-3ε is over-expressed in some HCCs, we hypothesized that 14-3-3ε can increase HCC metastasis through a FAK-mediated mechanism. Furthermore, we also want to explore if any targeted therapy could be used against this signaling pathway.
Patients and Methods:
Two patient cohorts were established in our study, enrolling 55 and 114 HCC patients, respectively, receiving mainly surgical resection from January of 1999 to July of 2004 in a medical center in Taiwan. Eighteen (32.7%) and 34 patients (29.8%) experienced extrahepatic metastasis in the two cohorts, respectively. The protein expression levels of 14-3-3ε and FAK in pathological specimens were evaluated by a semi-quantitative immunohistochemical staining method, i.e. the Quick score (Q-score) method. Other laboratory methods in this thesis were all complied with the usual protocols and the manufacturer’s instructions.
Results:
In the two-chamber invasion assay, SK-Hep1 cells with over-expressed 14-3-3ε showed increased invasiveness over control. In the 2nd cohort, 14-3-3ε over-expression was found in 61.3% of primary HCC, which predicted worse 5-year disease-free survival rate (54.9±7.7% vs. 28.6±5.4%, p=0.004), worse 5-year overall survival rate (66.5±7.3% vs. 42.4±6.0%, p=0.007) and higher 5-year cumulative incidence of extrahepatic metastasis (48.4±6.8% vs. 14.0±5.9%, p<0.001). Furthermore, we demonstrated the correlated protein expression of 14-3-3ε and FAK in cell lines and patient samples, and 14-3-3ε over-expression in SK-Hep1 cells resulted in increased NF-κB binding activities on FAK promoters and then increased FAK gene transcription. Next we found 61.8% of primary HCC over-expressed FAK, which was associated with worse 5-year disease-free survival rate (51.5±8.7% vs. 90.2±6.6%, p=0.041), worse 5-year overall survival rate (51.5±8.7% vs. 90.2±6.6%, p=0.004) and higher 5-year cumulative incidence of extrahepatic metastasis (36.1±9.2% vs. 20.9±8.4%, p=0.045). Proteasome inhibitors, MG-132 or PS-341 (bortezomib), significantly delayed the closure of wound by H1299 cells on a culture dish. In further experiments, the expression of 14-3-3ε was reduced by MG-132 or PS-341 treatment, and the effect is due to decreased 14-3-3ε promoter activities and transcription. FAK expression, similarly, can be suppressed by MG-132 or PS-341, and the effects were derived from suppression of FAK promoter activities in a NF-κB-dependent manner.
Conclusions and Perspectives:
We conclude that 14-3-3ε over-expression results in over-expressed FAK and increase in invasiveness of HCC. Proteasome inhibitors suppress the transcription of both 14-3-3ε and FAK. This study raises the potentials of 14-3-3ε and FAK as biomarkers for predicting prognosis and metastasis of HCC. Bortezomib may also serve as an adjuvant therapy to prevent metastasis after curative treatment for HCC. Further clinical and laboratory studies are required to confirm the concepts.