分析Herstatin在乳癌細胞株中扮演的角色

Analyze the Role of Herstatin in Breast Cancer Cell Lines

程帆宜(Fan-Yi Cheng)

指導教授 ： 許金玉

國立臺灣大學/醫學院/生物化學暨分子生物學研究所/碩士(2004年)

https://doi.org/10.6342/NTU.2004.10119

摘要

乳癌 (breast cancer) 是歐美婦女常見且較惡化的癌症之一，根據統計，2002年單單在美國就有40,000名婦女死於乳癌，並且有200,000名新生乳癌患者的產生，高居美國癌症死亡的第二位。在台灣地區的婦女而言，雖然乳癌盛行率不及歐美國家高，但乳癌在女性十大死因的排行榜上亦高居第四位，而且罹患乳癌的年齡層有愈來愈年輕化的趨勢。
目前提出引起乳癌的危險因子有很多，就分子層次 (molecular level) 而言，一些致癌因子 (oncogene) 與抑癌因子 (tumor suppressor gene) 的變異常在乳癌檢體中出現，而被認為與乳癌的形成有關。在致癌基因方面，近年來以HER-2/neu基因愈來愈受重視。HER-2/neu屬於表皮生長因子受體家族 (EGF receptor family) 中的一員，EGF receptor family共有四個成員：EGFR (ErbB-1/HER-1)、HER-2 (human EGF receptor-2)/neu (又名ErbB-2)、HER-3 (ErbB-3)、HER-4 (ErbB-4)；彼此之間可形成homodimer或heterodimer並且在細胞增生 (proliferation) 和細胞分化 (differentiation)間扮演重要角色。HER-2/neu並不像EGFR一般以homodimer的形式執行受體的弁遄A而是經由不同的受質促發，HER-2/neu被當作coreceptor可與其他的EGF receptor family成員形成heterodimer，進而活化下游訊息傳遞路徑。當 ErbB receptor表現異常，特別是HER-2/neu時，則和一些人類癌症有關，根據文獻指出，HER-2/neu基因倍增/過量表現的情形在乳癌中約佔20 ~ 30%，且與癌症轉移有關；但也有不少的研究對此關聯性有所爭議，包括我們實驗室之前分析110乳癌檢體得到HER2/neu基因的過量表現與術後其他器官轉移復發 (systemic recurrence)之間沒有關聯的結論 (P=0.1491)。我們推想，可能有某種干擾因子干擾而導致上述不同結果的產生。
1999年，Herstatin (Hst) 被辨識出來，Hst為alternative HER-2/neu transcript所產生的一個secreted autoinhibitor，並且藉由和HER-2/neu蛋白的結合進而抑制HER-2/neu receptor的活化。根據上述研究，我們推測Hst是否為此干擾因子？實驗室再將之前所收集到的110個乳癌檢體分析HER2/neu及Hst的表現與術後其他器官轉移復發 (systemic recurrence) 三者之間的關係，發現當HER-2/neu表現過量但Hst表現微弱或幾乎不表現時，則和術後其他器官轉移復發 (systemic recurrence)之間有關聯 (P=0.0347)，符合我們的推論。因此我們對Hst 在乳癌中扮演的角色感到興趣並進一步利用一些癌化程度分析 (tumorigenicity assay) 的實驗觀察Hst 對乳癌細胞株癌化程度的影響。
我們得到在MCF-7與T47D細胞株中，大量表現Hst並不改變此兩株細胞株的生長速率、細胞飽和曲線、對血清的需求及細胞在沒有貼附時生長的情形。而Au565這株乳癌細胞株在加強Hst的表現之後，則發現Hst的大量表現對細胞的生長速率和細胞飽和程度似乎有些微影響，但效果並不顯著；但在細胞對血清的需求程度分析的實驗中，觀察到Au565雖然可以在含2% FBS之complete F12/DMEM培養液中緩慢生長，但Au565/Hst-46 subclone在相同環境中於第四天時減緩生長且下降，我們推測MCF-7和T47D此兩株乳癌細胞株的HER-2/neu receptor是幾乎不表現或表現很微弱的，故當處在Hst大量表現的情況下，並不會藉由Hst抑制HER-2/neu進而影響細胞的增生。Au565細胞株可表現過量的HER-2/neu receptor，故在含10% FBS之complete F12/DMEM培養液中即生長因子較多的環境下，Hst的大量表現並不會影響生長因子活化HER-2/neu receptor而促使細胞增生；而在含2% FBS之complete F12/DMEM培養液中即生長因子較少的環境下，Au565雖生長速度趨緩，卻仍可持續生長，但當加強Hst表現時，因為處在生長因子較少的環境中，故Hst的影響變的較顯著而導致Au565的生長於第四天時減緩且下降，進而導致細胞的癌化程度變低。
Hst的表現亦有可能是透過其它的影響來減緩乳癌細胞株的癌化程度，未來我們可以利用其他的癌化程度分析實驗，如觀察細胞轉移的能力或利用將細胞打入SCID mice或nude mice，觀察老鼠身上腫瘤生長的情形，分析Hst是否會對這類癌化程度的分析有減緩的趨勢。

關鍵字

乳癌細胞株

並列摘要

Breast is the most common fatal malignancy cancer of females in developing countries. Over 20,000 new cases of breast cancer are estimated to be diagnosed in 2002 alone with close to 41,000 women dying every year from the disease in the U.S.A. Although the incidence of breast cancer is lower in Taiwan, it is the fourth rank for overall female cancer-related deaths and the incidence of age in this disease tends to early onset.
The etiology of breast cancer involves a complex interplay of genetic, hormonal, and dietary factors that are superimposed on the physiological status of the host. At the molecular level, several oncogenes and tumor suppressor genes were shown to be involved in the development of breast cancer. In recent years, the expression of HER-2/neu (erbB-2) has gained increasing importance for the biology and prognosis of breast cancer. HER-2/neu is a member of the EGF receptor family. The members of the EGF receptor family including the EGF-receptor (HER-1, erbB-1), HER-2/neu (erbB-2), HER-3 (erbB-3) and HER-4 (erbB-4) are capable of forming homomeric as well as heteromeric interactions and play fundamental roles in proliferation and differentiation. HER-2/neu dominates as the preferred coreceptor that amplifies mitogenic signaling. Abnormalities in the expression levels of ErbB receptors, particularly HER-2/neu, are frequently encountered in a number of human cancers. The HER-2/neu oncogene is amplified in 20–30% of breast carcinomas, where it contributes to more aggressive disease, and several reports further indicate that HER-2/neu overexpression predicts significantly lower survival rates and shorter time to relapse. However, some studies disagree that HER-2/neu overexpression can be a poor prognostic factor in breast cancer, including the result of our previous study. We postulate that the conflicting consequence may be caused by some factors that interfere with it.
Herstatin (Hst), a secreted protein encoded by the alternative HER-2/neu transcript, is discovered in 1999. Hst, which behaves as a putative HER-2/neu receptor inhibitor, binds to the HER-2/neu receptor ectodomain and blocks receptor activation. According to this finding, we were interested in whether Hst would be the interfering factor which resulted in the distinct consequence. We analyzed the status of HER-2/neu and Hst of 110 breast tumor specimens from Taiwan. We observed that there was a correlation only between HER-2/neu without Hst overexpression and systemic recurrence. Our results suggested that Hst may play an important role in breast carcinogenesis by interfering the signaling pathway of HER-2/neu. Therefore, we investigate the impact of Hst activity on tumorigenicity of breast carcinoma cells lines.
The expression of Hst in MCF-7 cells (MCF-7/Hst-4, MCF-7/Hst-pool) or in T47D cells (T47D/Hst-6, T47D/Hst-12, T47D/Hst-14) does not alter the results of these tumorigenicity assays including cell growth rate, cell saturation density assay, serum requirement assay and cell anchorage-independent growth compared with parental cells. Although the tumorigenicity assays such as cell growth rate, cell saturation density assay and cell anchorage-independent growth of Hst-overexpressing Au565 cells (Au565/Hst-46) are indistinguishable from those of the parental cells, we find that there is difference between Au565/Hst-46 and Au565 parental cells in complete F12/DMEM medium with 2% FBS. We suppose that it is because MCF-7 cells and T47D cells express very low levels of HER-2/neu receptor, overexpression of Hst does not affect cell proliferation by inhibiting HER-2/neu. Au565 cells can overexpress HER-2/neu receptor by itself, and therefore overexpression of Hst does not impact HER-2/neu activation in complete F12/DMEM medium with 10%FBS because of the abundance of growth factors. However, in complete F12/DMEM medium with 2%FBS, because of the existence of a little growth factors, the impact of Hst on tumorigenicity assay becomes more apparent and causes that the cell growth of Hst-overexpressing Au565 cells slowed down and decreased in the fourth day as compared with Au565 parental cells. It results in the lower levels of tumorigenicity.
Hst expression may make other impact on decreasing the levels of tumorigenicity of breast carcinoma cells lines. In the future, we can use other experiments to detect the levels of tumorigenicity, such as the nude mouse tumorigenicity assay or cell metastasis assay and analyze whether these kinds of tumorigenicity assays have a tendency toward ease or not.

並列關鍵字

Herstatin ； breast cancer ； HER-2

參考文獻

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