Grb7於HER2陽性乳癌化療抗藥性中之功能性探討

Abstract

乳癌是全球女性最常被診斷的惡性腫瘤，亦為台灣女性癌症中發生率最高的癌別。約有15–20%的乳癌呈現人類表皮生長因子受體2（human epidermal growth factor receptor 2, HER2）過度表現，通常與較具侵襲性的腫瘤特徵相關。生長因子受體連接蛋白7（growth factor receptor-bound protein 7, Grb7）常與HER2共同過度表現，並作為銜接蛋白參與多條訊息傳遞路徑。Grb7亦已被鑑定為壓力顆粒（stress granules, SGs）相關蛋白，可能參與SG的組裝與解散。然而，Grb7是否參與化療壓力下的SG相關反應及癌細胞適應能力仍未明確。因此，本研究旨在探討Grb7與5-氟尿嘧啶（5-fluorouracil, 5-FU）誘導之SG-like結構形成、細胞適應反應及乳癌細胞長期存活能力之間的關係。公開資料庫分析顯示，GRB7表現與HER2型乳癌具有相關性。在HER2陽性的SKBR3細胞中，5-FU處理可引起Grb7蛋白表現隨濃度與時間而呈現動態變化，並促進G3BP1陽性細胞質顆粒形成。相較於shLuc對照組，Grb7 knockdown（Grb7 KD）細胞在5-FU處理後呈現較不明顯的G3BP1陽性SG-like結構，顯示Grb7可能參與這些結構的形成或維持。功能性分析顯示，Grb7 KD造成較低的基礎MTT訊號及細胞生長動態改變，提示Grb7可能與SKBR3細胞的基礎代謝或增殖狀態有關。因此，MTT-based細胞存活率結果不應單獨解讀為5-FU敏感性改變。進一步以colony formation assay評估長期存活與增殖能力，結果顯示Grb7 KD在未處理及5-FU處理條件下皆明顯降低SKBR3細胞的colony形成能力，支持Grb7在維持長期clonogenic capacity中的角色。短暫前處理Grb7 SH2 domain胜肽抑制劑G7-18NATE亦可降低basal colony formation；然而，G7-18NATE前處理後再給予5-FU雖呈現平均colony數下降的趨勢，但未達統計顯著，故目前尚不能證明G7-18NATE可顯著提升5-FU敏感性。在另一個獨立建立的EO771/5-FU抗藥性模型中，EO771/5-FU-R細胞於再次接受5-FU處理後仍維持較高的相對存活率，並伴隨較高的Grb7表現及較低的總G3BP1表現。此外，本研究建立結合DeepPurpose與DiffDock-L的探索性藥物篩選流程，並篩選出Tacrolimus與Olcegepant作為可能作用於Grb7 SH2相關區域的候選化合物。綜合以上結果，Grb7可能參與5-FU誘導之G3BP1陽性SG-like反應、基礎細胞適應狀態及長期colony formation能力。雖然目前結果尚未建立Grb7介導SG調控與化療抗藥性之間的直接因果關係，但本研究顯示Grb7可能參與乳癌細胞對化療壓力的反應與恢復，並為後續探討Grb7相關訊息傳遞作為潛在治療標靶提供研究基礎。

Breast cancer is the most commonly diagnosed malignancy among women worldwide and has the highest incidence among female cancers in Taiwan. Approximately 15–20% of breast cancers exhibit human epidermal growth factor receptor 2 (HER2) overexpression, which is generally associated with aggressive tumor behavior. Growth factor receptor-bound protein 7 (Grb7) is frequently co-overexpressed with HER2 and functions as an adaptor protein involved in multiple signaling pathways. Grb7 has also been identified as a stress granule (SG)-associated protein; however, whether Grb7 contributes to SG-associated responses under chemotherapeutic stress remains unclear. This study investigated the relationship between Grb7, 5-fluorouracil (5-FU)-induced SG-like structure formation, cellular adaptation, and long-term survival in breast cancer cells. Public database analyses showed that GRB7 expression was associated with the HER2 breast cancer subtype. In HER2-positive SKBR3 cells, 5-FU treatment induced dynamic concentration- and time-dependent changes in Grb7 protein expression and promoted the formation of G3BP1-positive cytoplasmic puncta. Grb7 knockdown reduced the formation of G3BP1-positive SG-like structures following 5-FU exposure, suggesting that Grb7 may contribute to their formation or maintenance. Grb7 knockdown also resulted in lower basal MTT signals and altered growth behavior, indicating that MTT-based viability should be interpreted cautiously due to potential differences in basal metabolic activity. Colony formation assays further showed that Grb7 depletion reduced clonogenic capacity under both basal and 5-FU-treated conditions. Transient treatment with the Grb7 SH2-domain peptide inhibitor G7-18NATE also reduced basal colony formation; however, sequential G7-18NATE pretreatment followed by 5-FU exposure did not significantly enhance the suppressive effect of 5-FU. In an independently established EO771/5-FU-resistant model, resistant cells retained higher viability following renewed 5-FU exposure and exhibited increased Grb7 expression together with reduced total G3BP1 expression. In addition, an exploratory computational workflow integrating DeepPurpose and DiffDock-L prioritized tacrolimus and olcegepant as candidate compounds predicted to bind near the G7-18NATE-associated region of the Grb7 SH2 domain. Collectively, these findings suggest that Grb7 is associated with 5-FU-induced G3BP1-positive SG-like responses, clonogenic fitness, and cellular adaptation to chemotherapeutic stress. Although the present results do not establish a direct causal relationship between Grb7-mediated SG regulation and chemoresistance, they provide a foundation for further mechanistic investigation of Grb7 and for evaluating Grb7-associated signaling as a potential therapeutic target in chemotherapy-stressed breast cancer cells.