14-3-3蛋白與 Focal Adhesion Kinases 在癌症轉移中的角色及其相關研究

Abstract

緒論： 肝癌是台灣地區的嚴重健康問題。雖然近年來在肝癌的早期診斷及積極治療方面有長足的進步，肝內復發以及肝外轉移依然還是許多病患治療失敗的原因。在我們先前的研究中發現，一種可以做為蛋白質co-factor的蛋白14-3-3，和大腸癌細胞的移動有密切的關係，而且其和細胞內一種和cytoskeleton關係密切的蛋白FAK (focal adhesion kinase)，的表現有相當高的一致性。由於在初步實驗中我們也發現14-3-3ε蛋白在部分肝癌細胞中有過度表現的情形，我們因此在本研究中假設14-3-3ε可以透過增加FAK蛋白的表現來增加肝癌的轉移，並嘗試加以證實；另外我們也將嘗試找尋可以抑制14-3-3ε-FAK途徑的標靶治療藥物，以期可以在臨床上應用本研究的成果。 病患及研究方法 在本研究中我們建立了兩個肝癌世代，收集了自西元1999年1月至西元2004年7月在台灣某醫學中心接受主要為手術切除治療的肝癌病患，兩個世代各有55人及114人，其中各有18人 (32.7%) 及34人 (29.8%) 後續發生肝外轉移。在本研究中，對病患檢體中14-3-3ε及FAK蛋白的表現量是以免疫組織染色 (immunohistochemical staining)檢驗，並以半定量的Quick score (Q-score) method方式判讀。其它的實驗方法則依照一般慣用的protocol及廠商提供的說明書加以進行。 結果 首先我們確認了在肝癌細胞株SK-Hep1中，14-3-3ε的過度表現會使其在two-chamber invasion assay中表現的細胞侵襲性明顯增加。而在病患世代研究中，14-3-3ε可以在61.3%病患的原發肝癌中發現其表現量增加，同時這些病患會有明顯較差的5年無疾病存活率 (54.9±7.7% vs. 28.6±5.4%，p=0.004)，明顯較差的5年整體存活率 (66.5±7.3% vs. 42.4±6.0%，p=0.007)，以及較高的5年累積肝外轉移發生率 (48.4±6.8% vs. 14.0±5.9%，p<0.001)。接著我們確認了在肝癌細胞株及病患檢體中14-3-3ε和FAK蛋白的表現有極高的一致性，同時在SK-Hep1細胞中14-3-3ε的過度表現會增加NF-κB 和FAK promoter的結合，因而增加FAK基因的轉錄及蛋白表現。另一方面，FAK蛋白也可以在61.8%的肝癌病患中過度表現，同時FAK的過度表現也和肝癌病患較差的5年無疾病存活率 (51.5±8.7% vs. 90.2±6.6%，p=0.041)，明顯較差的5年整體存活率 (51.5±8.7% vs. 90.2±6.6%，p=0.004) 以及較高的5年累積肝外轉移發生率 (36.1±9.2% vs. 20.9±8.4%，p=0.045) 明顯相關。在進一步的研究中，蛋白體抑制劑(proteasome inhibitors)如MG-132及PS-341 (bortezomib) 等，可以明顯在wound healing assay中抑制癌細胞的移動。而MG-132或PS-341均可以降低14-3-3ε和FAK promoter的活性，並進而抑制基因轉錄及蛋白表現。我們同時也證實MG-132或PS-341對FAK promoter的抑制是透過降低NF-κB和其promoter的結合而達成的。 結論及展望 在本研究中，我們確認14-3-3ε的過度表現可透過增加FAK的表現來促進肝癌的轉移，而且這個途徑具有重要的臨床意義；蛋白體抑制劑則具有抑制這個途徑的效果。根據這些結果，14-3-3ε及FAK蛋白的表現量可以做為肝癌病患的預後及發生肝癌轉移的重要預測因子，而蛋白體抑制劑也有可能可以做為肝癌病患接受根治性治療後預防再發或轉移的輔助治療 (adjuvant therapy) 之用；但這些概念的實際應用仍需待進一步的臨床試驗加以證實。

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.