探討乳癌進程中透過表型可塑性適應代謝壓力之調控機制

Abstract

當腫瘤在生長過程中遭遇到壓力時，癌細胞會啟動壓力反應並透過調控轉錄路徑來調控細胞的行為，這樣的機制使得癌細胞能發展出表型可塑性(phenotypic plasticity)以因應壓力。在癌症進展過程中，癌細胞常因腫瘤微環境中營養供應不足而遭受代謝壓力。然而，目前對於癌細胞調控代謝壓力反應及代謝壓力如何影響表型可塑性的機制仍未完全釐清。本研究旨在探討乳癌細胞如何因應代謝壓力，並會聚焦於長鏈非編碼核糖核酸UBA6-AS1與轉錄因子EGR1在營養缺乏情況下所扮演的角色。 第一部分的研究主要是在探討UBA6-AS1在胺基酸缺乏下的功能。研究結果顯示，整合性壓力反應(integrated stress response)的核心路徑GCN2/ATF4會於麩醯胺酸(glutamine)和精胺酸(arginine)缺乏時上調UBA6-AS1的表現。而UBA6-AS1的上調會進一步促進PARP1的表現，進而增加細胞在代謝壓力下的存活能力。然而，UBA6-AS1的上調雖然促進了細胞的存活，卻也同時抑制了細胞的遷移能力，顯示其在代謝壓力下會調控細胞在生長與轉移之間的平衡(trade-off)。 第二部分的研究主要是在研究EGR1在葡萄糖缺乏下的表現與功能。研究結果顯示EGR1在代謝壓力之下的表現量會透過ROS/p38/Elk-1以及GCN2/ATF4兩條路徑被誘導。EGR1被上調後會抑制HERPUD1的表現，並且會部分抑制由IRE1所介導的未折疊蛋白反應(unfolded protein response)。這樣的機制能避免未折疊蛋白反應過度或延長活化，進而協助維持內質網恆定以及促進癌細胞於葡萄糖缺乏下的表型可塑性。不同的功能性試驗顯示EGR1除了能促進細胞在代謝壓力下的存活外，也會促進細胞對於失巢凋亡(anoikis resistance)的抗性，並同時抑制糖解作用與粒線體氧化磷酸化。由這些結果可以推論出在代謝壓力下EGR1能夠多面向地調控細胞的行為。 基於EGR1對代謝壓力的高度敏感性與快速反應性，本研究建立了一套以EGR1啟動子驅動Cre蛋白表現的報導系統。該系統可以在EGR1被誘發時將細胞由紅色螢光不可逆地轉換為綠色螢光，使曾經經歷代謝壓力的細胞能被追蹤其細胞命運與行為的變化，並作為一個可用來長期觀察細胞應對代謝壓力之過程及其轉移能力變化的工具。 總結來說，本研究發現乳癌細胞會分別透過UBA6-AS1以及EGR1調控代謝壓力反應並促進細胞於代謝壓力下的表型可塑性。這些結果顯示出癌細胞對不同營養缺乏所導致的代謝壓力調控具高度的特異性及複雜性，並為未來發展針對代謝壓力的乳癌治療策略提供新的標的與方向。

To adapt and survive from stress, cancer cells develop phenotypic plasticity through activating stress response which is mediated by diverse transcriptional programs to modulate phenotypic behaviors. During cancer progression, cancer cells are frequently exposed to metabolic stress due to insufficient nutrient supply in the tumor microenvironment. Nonetheless, the regulatory mechanism of metabolic stress response remains incompletely understood. This thesis would investigate how breast cancer cells respond to metabolic stress, which particularly focused on the role of long non-coding RNA UBA6-AS1 and transcription factor EGR1 under nutrient deprivation conditions. In the first part of the study, the role of UBA6-AS1 was investigated during amino acid deprivation. GCN2/ATF4 signaling axis, the core component of the integrated stress response, transcriptionally upregulated UBA6-AS1 upon glutamine and arginine starvation. The upregulation of UBA6-AS1 further enhanced the expression of PARP1, which promoted cell survival under stress conditions. Although UBA6-AS1 promoted cell survival under metabolic stress, it simultaneously suppressed the migratory ability of breast cancer cells, suggesting UBA6-AS1 mediated the trade-off between survival and metastasis in response to metabolic stress. In the second part, the role of transcription factor EGR1 under glucose deprivation was examined. EGR1 was induced by both ROS/p38/Elk-1 and GCN2/ATF4 signaling pathways upon glucose deprivation. After induction, EGR1 was found to suppress HERPUD1 expression and partially inhibited IRE1-mediated unfolded protein response (UPR) signaling, which prevented prolonged or excessive UPR activation, thereby maintaining ER homeostasis and promoting phenotypic plasticity under glucose starvation. EGR1 was demonstrated to regulate phenotypic plasticity through enhancing anoikis resistance and cell survival, whereas concurrently suppressing both glycolysis and oxidative phosphorylation, implying the multifaceted regulation of EGR1 on cell behavior upon metabolic stress. Based on the rapid responsiveness and sensitivity of EGR1 on metabolic stress, an EGR1 promoter-driven Cre reporter system was developed to visualize and trace the metabolic stress-experiencing cells. This reporter system irreversibly converted cells from red to green fluorescence upon EGR1 activation, and provided a lineage-tracing strategy to monitor how metabolic stress-exposed cells behaved or metastasized over time. Collectively, these findings revealed that breast cancer cells fine-tuned stress response signaling to regulate phenotypic plasticity under metabolic stress through UBA6-AS1 and EGR1. These mechanisms highlighted the complexity and specificity of cancer cell adaptation to metabolic stress induced by the deficiency of distinct nutrient, which provided the opportunity to develop future therapeutic strategies targeting metabolic vulnerabilities in breast cancer.