探討第二型人類表皮生長因子受體陽性癌症之抗藥性機制

Abstract

第二型人類表皮生長因子接受體（human epidermal growth factor receptor 2; HER2）屬於表皮生長因子家族的一員。過度表現HER2的癌細胞常與細胞不正常增生、腫瘤快速生長、存活率降低以及對常規化療藥物有較差的療效及預後有關。目前臨床上常以標靶藥物賀癌平（trastuzumab，商品名Herceptin）治療HER2過度表現的乳腺癌及胃癌，但是隨著療程時間增長，有限的藥物治療效果使得患者治療後期開始出現藥物療效大幅降低的現象。 本研究共分成兩個部分，其一為探討對賀癌平具有抗藥性的HER2陽性癌細胞之抗藥機制，其二為研究奈米載體聯合治療平台用於HER2陽性卵巢癌之影響。 根據臺灣衛福部統計數據指出女性乳癌為十大癌症中死亡年增率之冠，癌細胞對藥物產生抗藥性或腫瘤發生復發為其中可能原因之一，迫使癌症難以受到控制造成罹患乳癌婦女預後不良及死亡率增加，因此，了解對於HER2標靶治療產生抗藥性的癌細胞之潛在機制並且預防腫瘤復發的治療策略至關重要。近年來，鐵依賴性細胞死亡（ferroptosis）被認為是一種新的程序性死亡形式，由細胞膜中積累的脂質活性氧物質引起，有研究指出抗藥性癌細胞在藥物的作用下能夠躲避此死亡模式。 在本研究的第一部分中，我們發現在賀癌平與AKT抑制劑MK2206的合併治療下，會下調賀癌平阻抗的HER2陽性乳腺癌BT-474 clone 5細胞中的磷酸化AKT和GPX4的蛋白表達量，並增強抗藥性細胞對賀癌平的敏感度。此外，我們也發現利用ferroptosis的誘導劑erastin或是將細胞轉換至缺乏胱胺酸的環境能增強賀癌平對抗藥性HER2陽性乳腺癌BT-474 clone 5細胞的抑癌效果且有良好的藥效協同性，推測可能的機制是由於合併治療抑制了SLC7A11/GPX4信號通路並誘導鐵依賴性細胞死亡。最後將BT-474 clone 5細胞中的GPX4基因靜默（gene silencing）也觀察到有效恢復賀癌平抑癌效果。綜上所述，本研究提供了以下觀點：（1）PI3K/AKT信號通路與HER2過表達的賀癌平阻抗的乳腺癌有關；（2）改變CBS介導的轉硫化途徑不會影響HER2陽性乳腺癌細胞對賀癌平的抗藥性機制；（3）賀癌平會誘導癌細胞走向鐵依賴性細胞死亡途徑；（4）賀癌平阻抗的HER2陽性乳腺癌細胞透過調節AKT和SLC7A11的信號傳遞路徑以增強GPX4表達量來減緩癌細胞走向鐵依賴性細胞死亡，侷限賀癌平對癌細胞的抑制作用。 卵巢癌為常見且致命的婦科癌症，儘管已經開發出許多新的治療方針，但患者的存活率仍然很低。根據統計指出卵巢癌病中HER2呈現陽性的占比約20%，雖然賀癌平已被FDA批准用於治療HER2陽性的乳腺癌和胃癌病患，但應用於卵巢癌病患中卻不見顯著療效。 綠茶萃取物中最豐富的多酚成分是表沒食子兒茶素沒食子酸酯（EGCG），多項研究顯示其功能在對抗心血管疾病、神經退行性疾病、糖尿病和癌症等方面表現皆具有良好的生物活性，因此近年來，人們關注開發EGCG參與的癌症靶向治療的抗癌策略。微胞奈米複合物（micellar nanocomplex）的設計核心是利用低聚合EGCG（Oligomerized EGCG, OEGCG）與抗癌蛋白藥物賀癌平自組裝形成，再將聚乙二醇修飾之EGCG（PEG-EGCG）複合形成外殼，全組合成新型奈米載體聯合療法並將之稱作nano-trastuzumab。利用此藥物靜脈注射至卵巢癌（SKOV3-Luc）小鼠異種移植模型中，給予EGCG作為載體並裝載賀癌平的nano-trastuzumab之組別顯示出比單賀癌平使用有更好的抑癌效果並有效抑制腫瘤生長，因此，在本研究的第二個部分中，發現奈米載體聯合療法nano-trastuzumab有望成為HER2陽性卵巢癌細胞的一個新興治療策略。

Human epidermal growth factor receptor 2 (HER2) belongs to the epidermal growth factor receptor family of proto-oncogenes. Its amplification is associated with rapid tumor growth, shortened survival, and poor response to conventional chemotherapeutic agents. The HER2-targeting antibody trastuzumab has shown effectiveness in treating HER2-positive breast and gastric cancers; however, its responses are limited. This study is divided into two parts. The first part is to investigate the molecular mechanisms of resistance in HER2-positive cancer treated with trastuzumab. The other is to investigate the anticancer effects of nanocarrier combination therapy platforms in ovarian cancer overexpressing HER2. Drug resistance makes it difficult for cancer therapies to achieve stable and complete responses. Emerging evidence suggests that amino acid reprogramming leads to cancer cells resistant to conventional chemotherapeutic drugs and plays an important role in the survival of residual drug-resistant cells. Targeting resistant cells thus provides a therapeutic opportunity to prevent tumor recurrence. Ferroptosis is a type of iron-dependent cell death caused by the accumulation of lipid reactive oxygen species in cell membranes. This new form of programmed cell death might play a role in drug resistance mechanisms. In the first part of this study, we reported that combining MK2206, an AKT inhibitor, with trastuzumab exerted the growth-inhibitory effects on trastuzumab-resistant HER2-positive breast cancer BT-474 clone 5 cells by dampening AKT and GPX4 signaling pathways. Moreover, We found that erastin, a ferroptosis inducer by inhibiting xCT (encoded by SLC7A11), or cystine deprivation enhanced the anticancer effects of trastuzumab on BT-474 clone 5 cells, which may be attributed to the upregulation of AKT/SLC7A11/GPX4 axis inhibited ferroptosis pathway and further conferred trastuzumab resistance in BT-474 clone 5 cells. Furthermore, silencing GPX4 expression enhanced the sensitivity to trastuzumab in trastuzumab-resistant HER2-positive breast cancer BT-474 clone 5 cells. In conclusion, the study provided insights into (1) PI3K/AKT pathway was involved in the resistance to trastuzumab in HER2-positive breast cancer; (2) CBS-mediated transsulfuration pathway may not be involved in HER2-positive breast cancer resistance to trastuzumab; (3) trastuzumab reduced GPX4 protein expression and induced ferroptosis; (4) mechanistic studies demonstrated that trastuzumab-resistant HER2-positive breast cancer cells prevent ferroptosis by regulating AKT and SLC7A11/GPX4 signaling pathway and enhancing GPX4 expression. The second part looked into how the nanocarrier combination therapy platform affected ovarian cancer that overexpressed HER2 at a rate similar to many malignant breast tumors. Ovarian cancer (OC) is a common and deadly gynecological cancer. Although new therapeutic agents for OC have been developed, patient survival remains low. Trastuzumab is approved for breast and gastric cancers with high HER2 expression, but it has not shown clinical success in OC. The most abundant polyphenolic constituent from green tea extract is epigallocatechin gallate (EGCG), which has demonstrated versatile bioactivities in combating cardiovascular diseases, neurodegenerative diseases, diabetes, and cancer. Because of its anticancer activity, there has been an increase in interest in developing effective strategies involving EGCG in cancer target therapy. The core of the micellar nanocomplex is formed by complexing oligomerized EGCG (OEGCG) with trastuzumab, followed by complexation of poly (ethylene glycol)-EGCG (PEG-EGCG) to form the shell, which is named nano-trastuzumab. The nano-trastuzumab showed better growth reduction than free trastuzumab when injected into the ovarian (SKOV3-Luc) xenograft mouse model. As a result, in the second part, we demonstrated that nano-trastuzumab could be a promising treatment for HER2-positive ovarian cancer.