以動物模型研究 Grb7 在乳癌惡性度中的角色

Abstract

乳癌長久以來佔據全世界女性的癌症死因首位。其中確診 HER2 陽性亞型或三陰性亞型的病患，因為現今治療措施效果有限以及其高復發、高轉移率等特徵，而面臨最差的預後結果。HER2 陽性乳癌的癌細胞因大量表達人類表皮生長因子受體2且具有ErbB2 基因異常擴增而得名。由先前研究得知，位於 ErbB2 基因擴增區段 (ErbB2amplicon)內的 Growth factor Receptor Bound protein7 (Grb7)，因為其在細胞功能性試驗與小鼠異種移植試驗中皆呈現與癌細胞惡性度正相關的現象，引起了我們的關注。Grb7是一種不具酵素活性的銜接蛋白，具有多重功能區域結構，可以與多種受體酪胺酸激酶(RTKs)或細胞因子受體結合，開啟或促進生長因子受體與細胞激素受體下游的訊息級聯。Grb7 涉入的訊息途徑包含細胞增殖、遷移、侵襲、抗凋亡與生存等。這些細胞生理功能訊號若在癌細胞內被增強，將會導致腫瘤生長、惡化、轉移、抗藥性與癌復發等可能。為了探討 Grb7 在乳癌惡化中的角色，我們首先利用線上分析程式 cBioPortal 整合來自The Cancer Genome Atlas (TCGA)資料庫中的四組乳癌患者基因原始資料，發現 Grb7 與ErbB2 共同異常突變的病患，有生存率較低、復發時間較早、惡化進程較短的統計結果。接著我們建立了 Grb7 基因剔除(knockout, KO)與具有自發乳腺腫瘤能力的小鼠品系，藉由觀察活體在不同 Grb7 基因背景下，所發展出的乳腺腫瘤生長與惡化進程的差異。我們發現 Grb7 KO 的乳癌小鼠相較於 Grb7 WT 小鼠，表現出腫瘤發展末期體積較大、顆數較少的趨勢，且 Grb7 表現量低的兩個組別診斷出腫瘤後的存活期較短。此結果有別於文獻內細胞試驗結果所推論出 Grb7 正向驅動癌症惡化的假設。令人意外的是，Grb7異質體(heterozygous)組的肺轉移發生率是其餘兩組的 2 至 3 倍。 在細胞功能性試驗中，我們初次探討 Grb7 表現迥異的兩種乳癌細胞株，是否具有不一樣的類球體(spheroid)成形能力與對 5-Fluorouracil (5FU)的抗藥性能力。同時我們也篩選出成功表達 overexpression-Grb7 質體之上皮細胞株，進行相同的試驗，確認非癌細胞在獲得 Grb7 的功能增益後，是否影響其類球體成形與藥物敏感度。我們發現 GRB7高表現的細胞相較於低表現細胞具有更高的 5FU 抗藥性，不論 GRB7 過表達為內生性或是透過誘導產生。在類球體試驗中，我們成功觀察到 3D 培養的細胞相較 2D 平面貼附培養的細胞，減少了表皮細胞標記 E-cadherin 的表現量，顯示此培養過程可以促使細胞進入表皮-間質轉換(EMT)狀態。我們希望未來繼續利用類球體試驗深入解構細胞EMT 狀態與抗藥性及癌幹細胞的關聯，以釐清 Grb7 參與細胞抗藥性能力時，是否亦涉入癌幹細胞幹性的訊息途徑，並解答 Grb7 對乳癌惡化程度的影響。

Breast cancer has long been the universal leading cause of death by cancer among women. Particularly, breast cancer patients diagnosed with HER2-positive subtypes or triple-negative subtypes usually face unfavorable prognoses due to the limited effect of current cancer therapy and higher recurrence and metastasis. HER2-positive breast cancer is defined as the tumor cells over-expressing the human epidermal growth factor receptor 2 (HER2) caused by abnormally increasing gene copies involving the ErbB2 (ErbB2 amplicon). According to the previous studies, Growth factor Receptor Bound Protein7 (Grb7) is among the genes within the ErbB2 amplicon and was correlated with cancer cell malignancy in several cell functional assays and xenograft animal studies. This has drawn our attention because the Grb7 protein, as an adapter protein without catalytic activity, interacts with diverse receptor tyrosine kinases (RTKs) or growth factor receptors to regulate cell survival, invasion, migration, anti-apoptosis, and proliferation signaling cascade. These pathways are of great importance in affecting tumor development progression, metastasis, and therapy failure in cancerous tissues. To study the implication of Grb7 in breast cancer malignancy, we first used the free online tool cBioPortal to make integrated patient tissue profile analysis from four datasets of The Cancer Genome Atlas (TCGA). We found that patients with mutations in Grb7 and ErbB2 genes had a shorter overall survival and progression-free survival rate, which indicate a bad prognosis and earlier recurrence. We also established a mouse model of Grb7 knockout (KO)/MMTV-PyMT and analyzed the development and progression of mammary tumors in mice with different Grb7 gene backgrounds. Our results reveal that the tumor fate of Grb7 mutant/MMTV-PyMT mice is not consistent with the previous studies referring Grb7 as a pro-tumor role developed from cell assays. Instead, MMTV/PyMT female mice with Grb7 depletion had larger tumor volumes and fewer tumor numbers at the late stage of the cancer course. Meanwhile, the overall survival rates of the Grb7 hetero and KO group were shorter than that of the Grb7 wild-type (WT) group. Far from our prediction, the incidence rate of lung metastasis in the Grb7 heterozygous group was twice to three times the rate in the other two groups. In cell functional assays, we showed that higher expression of Grb7 correlates with stronger resistance to 5FU, regardless of whether GRB7 is intrinsic or acquired. In the spheroid formation assay, we successfully observed the epithelial-mesenchymal transition (EMT) of cells through 3D culturing and demonstrated that spheroids are a good method to decode the EMT state of cancer cells. It has been mentioned that induction of EMT of cancer cells contributes to drug resistance and enrichment of cancer stem cell (CSC) markers. We hope to use spheroid formation assay in the future to decipher the relationships between EMT status, drug resistance, and CSC stemness, thus clarifying whether Grb7 is involved in these cellular pathways, and explaining the position of Grb7 in breast cancer malignancy.