組蛋白去乙醯酶 2 與 HER2/neu 之互動在乳癌的角色與運用

The potential role and application of the interplay between HDAC2 and HER2/neu in breast cancer

林佳儀(Chia-Yi Lin)

指導教授 ： 侯明鋒

共同指導教授 ： 葉耀宗(Yao-Tsung Yeh)

高雄醫學大學/醫學院/醫學研究所/碩士(2011年)

https://doi.org/10.6832/KMU.2011.00128

摘要

截至目前為止，已被確認的組蛋白去乙醯酶家族共有18個。組蛋白去乙醯酶能夠修飾組蛋白及非組蛋白質中裸露出的離胺基酸基，進而促使蛋白質的不穩定及轉錄抑制作用。組蛋白去乙醯酶家族中，組蛋白去乙醯酶 2 (HDAC2) 會與其他蛋白質形成複合體，藉此來抑制轉錄作用、調控細胞的週期變化及細胞分化。文獻指出組蛋白去乙醯酶 2 會過度表現在許多的腫瘤，諸如前列腺癌、子宮頸癌、胃癌及口腔癌等。並且，組蛋白去乙醯酶 2 在口腔癌、前列腺癌、胃癌及卵巢癌的表現增加為一個較差的預後因子；異常的表現組蛋白去乙醯酶 2 也被指出與慢性發炎性疾病有關。有趣的是，許多研究指出組蛋白去乙醯酶抑制劑能夠協同或是提高許多治療人類癌症的藥物對抗腫瘤的能力。同時，組蛋白去乙醯酶抑制劑亦能夠增加雌激素受體陽性的乳癌細胞對 Tamoxifen 的敏感性。因此，釐清組蛋白去乙醯酶 2 在乳癌所扮演的角色或許可做為一個重要的預後指標或治療標的。
初步分析 104 位病患的乳癌組織檢體後，我們發現相較於鄰近的配對正常乳房組織，組蛋白去乙醯酶 2 在 69.8% 的癌組織中表現量增加。同時，癌組織中增加的組蛋白去乙醯酶 2 與 HER2 的表現狀態成正比 (p=0.018)。有趣的是，HER2 接受器陰性的病人在組蛋白去乙醯酶 2 的表現量下降時會有較差的存活率 (p=0.040)。cDNA 微陣列法分析 ER+/HER+ ZR75-1 在組蛋白去乙醯酶 2 特異性下降後所產生的變化，結果發現 HER2 下游因子 “TACSTD1/Ep-CAM” 的表現量也因此降低。免疫組織化學染色結果顯示組蛋白去乙醯酶 2 在細胞質中的表現與腫瘤分期 (stage) 呈現正相關 (p=0.009)；細胞核中的表現則與淋巴結的轉移成正相關性 (p=0.037)。總結以上的這些研究觀察，組蛋白去乙醯酶 2 和其與 HER2 間的交互作用可能在乳癌中扮演著重要的角色。
有趣的是，過度表現組蛋白去乙醯酶 2 能使正常乳腺細胞株 M10 增生並且增加其侵犯能力。然而，不論大量表現 HER2 或是抑制組蛋白去乙醯酶 2 的表現皆不影響彼此的總蛋白質表現量；免疫沉澱法發現，在建立穩定表現 HER2 的細胞株中，組蛋白去乙醯酶 2 蛋白質與 CK2α 的交互作用會增加；並且組蛋白去乙醯酶 2 在絲胺酸位置的磷酸化也會增加。這個結果顯示，過度表現 HER2 可能可以活化 CK2α，並進一步幫組蛋白去乙醯酶 2 在 serine 394 的位置加磷酸化。然而，出乎意料的是，增加組蛋白去乙醯酶 2 serine 394 磷酸化似乎會降低其與 p-STAT3 (tyrosine 705) 的交互作用，並且可能導致 STAT3 下游訊息的活化像是發炎因子 IL-6 的產生。
利用 Cytokine Ab array 進行初步的分析，發現有部份的發炎激素群在 Tamoxifen resistant 乳癌細胞 (TAMR-MCF-7) 中特異性的表現增加，並且，相對於原衍生乳癌細胞 (MCF-7)，TAMR-MCF-7 有較高的組蛋白去乙醯酶 2 與 HER2 的表現量。值得一提的是，HER2 下游訊息傳遞的活化可提升發炎激素的產量，並且，此現象與 Tamoxifen 抗性的產生可能有關。我們也發現 Tamoxifen resistant 乳癌細胞膜表面雌激素受體表現量明顯的增加，並且與 HER2 共位 (co-localization) 的數量亦相對增加。免疫螢光染色的實驗顯示在雌激素及膜不可滲透的 E2-BSA 刺激下，似乎可增加膜表面雌激素受體與 HER2 的交互作用，並進一步活化 HER2 下游訊息傳遞，像是發炎激素 IL-6 的產生。因此，我們進一步針對膜上雌激素受體設計一新型胜肽 (mER3)；在免疫螢光染色的結果下，確實可見到 mER3 可結合細胞膜表面雌激素受體，並且加入 E2-BSA後，可見到 TAMR-MCF-7 細胞膜表面 mER3 結合力的增加。
我們的研究結果顯示組蛋白去乙醯酶 2 的增加或許可做為治療乳癌的診斷標誌之一，然而更深入的作用機制仍須繼續的去研究探討，希望後續的研究能夠在乳癌的治療上提供臨床醫生治療個別乳癌病患一個新的治療方向。

關鍵字

組蛋白去乙醯酶2 ； HER2 ； 發炎激素 ； 細胞膜雌激素受體 ； 乳癌

並列摘要

Eighteen mammalian histone deacetylases (HDACs) have been identified so far. HDACs determine the acetylation status of histones as well as non-histone proteins. Among these HDACs, HDAC2 is ubiquitously expressed with nuclear localization and is one component of multiprotein complexes with transcriptional repression activity, participating in the regulation of cell cycle, differentiation and development, and is often significantly overexpressed in solid tumors such as prostate, endometrial, gastric or oral cancer, high levels of HDAC2 are independent markers of poor prognosis in patients suffering from oral, prostate, ovarian, endometrial or gastric cancer. Aberrant expression of HDAC2 has also been detected in chronically inflamed tissues. Mounting evidences have indicated that HDAC inhibitors (HDACIs) have synergistic or additive antitumor effects with a wide range of chemotherapy reagents. In addition, certain HDACIs sensitize cells to tamoxifen in ER-positive breast cancer cells. Beyond clarifying the role of HDAC2 in breast cancer may as a therapeutically important prognostic marker.
Our results showed that HDAC2 was increased in 69.8% of breast cancer tissues as compared with the matched non-cancer tissues from the same patient. Increased HDAC2 expression in the cancerous breast tissues was positively correlated with HER2/neu status (p=0.018). Intriguingly, HER2-negative patients with decreased HDAC2 had a poor survival rate (P=0.040). Meanwhile, the cDNA microarray data showed that TACSTD1/Ep-CAM, a downstream gene of HER2 signaling, was decreased upon knockdown of HDAC2 in ER+/HER2+ ZR-75-1 cells. Immunohistochemistry showed that cytoplasmic HDAC2 was positively correlated with tumor staging (p=0.009), and nuclear HDAC2 was positively correlated with LN metastasis (p=0.037). Based on these observations, it is presumably possible that HDAC2 and its interplay with HER2 signaling may play some roles in breast cancer.
Intriguingly, overexpression of HDAC2 increased the proliferation and invasion of the immortalized breast epithelium cells M10. Of note, overexpression of HER2 did not obviously alter the levels of HDAC2 protein. As well, knockdown of HDAC2 also did not change the levels of HER2 protein. Immunoprecipitation showed that HER2 overexpression increased the interaction between CK2 and HDAC2 and also the levels of serine phosphorylation of CK2 and HDAC2. It has been demonstrated that HER2 overexpression can activate CK2 and further activate HDAC2 via phosphorylation of ser394. However, unexpectedly, increased serine phosphorylation of HDAC2 seemed to decrease its interaction with p-tyr705-STAT3 and might lead to increased STAT3 signaling such as IL-6 production.
Immunofluorescence showed that membrane-impermeable E2-BSA was able to increase the interaction between membrane ER (mER) and HER2, and further transactivated HER2 signaling including IL-6 production. The Cytokine Ab array and cDNA microarray showed that abundant inflammatory cytokines were up-regulated in tamoxifen-resistant breast cancer cells TAM-R MCF-7, while the TAM-R MCF-7 had relatively high levels of HDAC2 and HER2 as compared with the parental cells. Of note, activation of HER2 signaling has been proven to increase production of inflammatory cytokines and associate with tamoxifen-resistance. We also developed a novel peptide (mER3) targeting mER. Immunofluoresce staining showed mER3 peptide could from dot-like merge images with mER, while E2-BSA increased the binding of mER3 to mER in TAM-R MCF-7.
Further investigations are required. Hopefully, the study will provide a better understanding for the carcinogenesis of breast cancer and provide useful information to the clinicians to decide the treatment for each individual breast cancer patient.

並列關鍵字

Histone deacetylase 2 ； HER2 ； inflammatory cytokines ； membrane ER ； breast cancer

參考文獻

參考文獻

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