植化物抑制Skp2過度表現乳癌細胞生長機制之研究

Abstract

乳癌是女性最常見的癌症，死亡率在女性癌症中為第二位。藉由觀察乳癌腫瘤是否表現雌激素接受器(ER-positive)或是HER2蛋白質有無過度表現，可做為專一性治療的指標。標靶治療(Targeted therapy) 藥物tamoxifen 及Transtuzumab (herceptin)可分別用於治療雌激素接受器陽性或是HER2蛋白質過度表現的乳癌。然而並無專一性的藥物可用於治療雌激素接受器陰性及HER2陰性的乳癌。 第二型S週期激酶相關蛋白（S phase kinase-associated protein 2，Skp2）是一種原致癌基因(proto-oncoprotein)，可藉由泛素（ubiquitin）參與來造成p27Kip1蛋白量下降。 在臨床研究中已證實，在雌激素接受器陰性及HER2陰性的乳癌中Skp2過度表現型佔很高的比例，約佔61－67％，而在雌激素接受器陽性的乳癌中Skp2也佔有相當高的比例，約佔15－23％。此外，Skp2過度表現會抑制抗雌激素(anti-estrogen)的作用，推測Skp2失去調控在抗雌激素治療無效的過程中可能扮演一個很重要的角色。因此，Skp2的過度表現可以當成預先診斷乳癌的生化指標，進一步可以發展出Skp2標靶治療藥物用於治療雌激素接受器陰性及HER2陰性和雌激素接受器陽性的乳癌病人。可惜的是目前並無專一性的標靶藥物可用於抑制Skp2的表現。 目前比較感興趣的是植化物（phytochemicals）用於乳癌的治療。流行病學研究結果已經顯示攝食蔬菜、水果及茶可以降低得癌症及罹患新血管疾病的風險，而蔬果當中主要成分植化物被認為是預防疾病最主要成分之一。這些研究結果顯示植化物可以當成癌症化學預防及化學治療的藥物。 在本文實驗中，我們評估在乳癌細胞中天然的植化物（例如：EGCG、 5gg、 quercetin、curcumin和lycopene）對skp2表現的影響。在本論文的第二章，我們評估在雌激素受體陽性反應的乳癌細胞MCF-7中EGCG對skp2的表現的影響。 我們發現在雌激素刺激的MCF-7細胞中不論是EGCG處理時間增加或是濃度增加都可以有效降低Skp2蛋白量，同時伴隨著p27Kip1的增加，推測EGCG可以抑制細胞生長主要是透過調控Skp2及p27Kip1蛋白量的表現。本論文是第一次證明EGCG可藉由調控Skp2蛋白量的表現進一步阻斷雌激素刺激的MCF-7細胞生長週期停止。有趣的是Skp2及p27Kip1 mRNA的表現量並不受到EGCG的影響。 此外，在MCF-7中過度表現Skp2可以避免p27Kip1蛋白量的增加，同時也可抑制EGCG造成細胞生長停止，由此結果推測Skp2是主要造成p27Kip1蛋白量增加的原因之一。進一步，我們發現EGCG併用抗癌藥物tamoxifen或是paclitaxel 具由協同加成的作用，可以更有效抑制抑Skp2的表現進一步抑制細胞生長。然而，EGCG併用抗癌藥物處理抑制Skp2的表現，但是並沒有同時促進p27Kip1蛋白量的增加，推測EGCG抑制Skp2的表現進而影響細胞生長可能透過很多不同的途徑。在臨床上抑制Skp2的表現可能是治療雌激素受體陽性反應乳癌很重要的一個分子標的。 在本論文的第三章，我們評估在ER/HER2-negative(MDA-MB-231)及 ER/HER2-positive breast (BT474)乳癌細胞中其它天然的植化物（例如： 5gg、 quercetin、 curcumin 和 lycopene）對skp2表現的影響。我們發現這四種植化物可以有效抑制MDA-MB-231細胞生長。其分子機制主要是藉由阻斷細胞生長週期進一步抑制細胞生長。5gg、 curcumin 和 lycopene 可以促使MDA-MB-231細胞停留在G1 時期，然而quercetin會促使細胞停留在G2時期。在BT474細胞中，5gg、 quercetin 和 lycopene可以促使細胞停留在G1時期及抑制細胞生長。相反的curcumin濃度在10μM以下並不會造成細胞生長週期停止及抑制細胞生長。進一步我們發現在MDA-MB-231細胞中quercetin和 curcumin可藉由抑制Skp2同時促進p27Kip1的增加而抑制細胞生長。然而，5gg 和 lycopene抑制Skp2的表現，但是並沒有同時促進p27Kip1蛋白量的增加。抑制Skp2的表現，並不一定會同時促進p27Kip1蛋白量的增加，我們推測抑制Skp2的表現進而影響細胞生長可能透過很多不同的途徑。 最近研究結果顯示植化物在疾病預防上扮演很重要的角色，然而其作用機制及其分子標的等等還不是很清楚。在本論文中我們提供一個新的機制探討，發現植化物可藉由調控Skp2的表現進一步影響乳癌細胞生長週期。由這些結果也說明了植化物在疾病預防上的作用機制及其分子標的，其望藉由本論文的研究結果，未來能夠提供化學預防臨床試驗一些資訊，使癌症化學預防有更多的選擇。

Breast cancer is the most common cancer in women and the second leading cause of cancer-related deaths in women. Estrogen receptor (ER) expression and HER2 amplification define specific subsets of breast tumors for which specific therapies exist. Targeted therapy with tamoxifen and trastuzumab (herceptin) is available for estrogen receptor–positive (ER-positive) and HER2–overexpressing breast tumors, respectively. However, no specific therapy has been identified in those tumors that are both ER and HER2 negative. The S-phase kinase-associated protein Skp2 is required for the ubiquitin mediated degradation of p27Kip1 and is a proto-oncoprotein. Clinical studies have demonstrated that Skp2 is highly expressed in 61–67% of the ER/HER2-negative tumors, and it is also expressed at higher levels in 15% - 23% of the ER-positive tumors. Additionally, overexpression of Skp2 abolished effects of antiestrogens, suggesting that deregulated Skp2 expression might play a role in the development of resistance to antiestrogens. Thus, Skp2 is desirable to identify prognostic biomarkers to aid in the development of targeted therapeutic strategies for ER/HER2-negative and ER-positive breast cancers. Unfortunately, specific drugs that target Skp2 are unavailable at present. There has been considerable interest in the use of phytochemicals for the treatment of breast cancer. Epidemiological studies have shown that the consumption of vegetable, fruits and tea is associated with a decreased risk of cancer and cardiovascular diseases, and phytochemicals are believed to play an important role in preventing these diseases. These data indicate that certain phytochemicals may be used as possible chemoprotective or chemotherapeutic agents. In the present study we examined the effect of natural phytochemicals, such as EGCG, 5gg, quercetin, curcumin, and lycopene, on Skp2 gene expression in breast cancer cells. In chapter II, we examined the effect of EGCG on Skp2 gene expression in ER-positive breast cancer cells. We found that EGCG, the main constituent of green tea, increased p27Kip1 and decreased Skp2 in a time- and dose-dependent manner, suggesting that p27Kip1 and Skp2 may be involved in the growth inhibition by EGCG in estrogen-stimulated MCF-7 cells. We demonstrated here for the first time that Skp2 is required for EGCG-induced cell cycle arrest in estrogen-stimulated MCF-7 cells. Interestingly, mRNA levels of p27 Kip1 and Skp2 did not significant change in estrogen-stimulated MCF-7 cells following EGCG treatments. Moreover, overexpression of Skp2 in MCF-7 cells prevented accumulation of p27 Kip1 and promoted resistance to the antiproliferative effects of EGCG. This suggests that the downregulation of the F-box protein Skp2 is the mechanism underlying p27 Kip1 accumulation. Furthermore, both tamoxifen and paclitaxel significantly and synergistically enhanced the growth inhibition of MCF-7 cells by EGCG through the downregulation of Skp2 protein. However, the downregulation of Skp2 was not always correlate with the upregulation of p27 Kip1, suggesting that EGCG dependent Skp2 downregulation can influence on cell growth in several ways. The therapeutic strategies designed to reduce Skp2 may therefore play an important clinical role in treatment of ER -positive breast cancer cells. In chapter III, we examined the effect of other natural phytochemicals, such as 5gg, quercetin, curcumin, and lycopene, on Skp2 gene expression in ER/HER2-negative(MDA-MB-231) and ER/HER2-positive breast (BT474) cancer cells. We found that all of the 4 phytochemicals induced cell growth inhibition in MDA-MB-231 cells. The mechanism of the initial growth inhibitory events is by blocking the the cell cycle progression. 5gg, curcumin and lycopene induced G1 phase arrest while quercetin caused G2M phase arrest. In BT474 cells, 5gg, quercetin and lycopene induced G1 phase arrest and cell growth inhibition. In contrast, curcumin at < 10μM did not induce G1 phase arrest and cell growth inhibition. Further, we found that quercetin and curcumin induces growth arrest by inhibition of Skp2, and induced p27 Kip1 expression in MDA-MB-231 cells. However, the decrease in Skp2 levels in cells treated with 5gg or lycopene did not translate to p27Kip1 upregulation. The downregulation of Skp2 was not always correlate with the upregulation of p27Kip1, suggesting that phytochemicals dependent Skp2 downregulation can influence on cell growth in several ways. Recent studies have demonstrated that despite the lack of changes in p27Kip1, Skp2 levels were independently associated with a significantly better disease-free survival. This supports the concept that Skp2 has other important oncogenic effects and that reduction in Skp2 levels is a rational therapeutic objective. Recent studies demonstrate that phytochemicals can protect humans against disease and how phytochemicals interfere with this mechanism is still unclear. In this dissertation, our findings provide additional insights into the mechanisms of action of phytochemicals on cell cycle arrest in breast cancer cells through direct down-regulation of Skp2 expression. The results presented here indicate that these phytochemicals are of potential value for the chemoprevention of breast cancer. However, our preclinical research might promising and ready for further study in clinical chemopreventive trials.