建立EGR1報導基因偵測系統以追蹤乳癌細胞在腫瘤內代謝壓力下的命運

Abstract

在實質固態瘤生長的過程中，癌細胞需消耗大量氧氣及養分以滿足其快速生長的特性。靠近腫瘤中心區域的癌細胞，由於血管新生不足，從血液獲得的氧氣及養分相對較少，經常面臨缺氧和營養不足的問題，導致腫瘤內代謝壓力的產生。在這樣不利生長的環境下，癌細胞會改變其代謝特徵以適應腫瘤內代謝壓力，維持細胞增殖與存活，甚至促使其轉移至其他組織。 早期生長反應蛋白1（Early Growth Response 1; EGR1）是一已知會受到多種刺激及壓力所誘導的轉錄因子，其活化將啟動下游基因的轉錄，影響癌細胞生長、凋亡、遷移與侵襲的調節。實驗室先前發現，EGR1在乳癌細胞中的表現量顯著低於正常乳腺細胞，而在營養缺乏時EGR1會被誘導上調，且此現象發生在多種不同乳癌細胞株中。我們認為EGR1可能參與調節乳癌細胞的壓力適應性，但目前仍不清楚代謝壓力所誘導的EGR1在乳癌細胞中扮演的角色，以及EGR1調節乳癌細胞壓力適應性的機制。 故本研究建立了在EGR1響應元件或EGR1啟動子控制下表現綠色螢光蛋白和螢光素酶的報導基因偵測系統，監測活細胞中EGR1表現量，並應用於探討對代謝壓力有不同反應性（EGR1上調程度不同）的細胞群體間的不同特徵。另一方面，我們期望未來能將此報導基因偵測系統應用於追蹤曾暴露於腫瘤內代謝壓力的乳癌細胞的命運，因此初步測試了三維細胞培養模型最適的培養及成像條件。 首先，我們建構數個不同的報導基因質體並透過轉導的方式送入細胞，建立帶有報導基因質體的穩定細胞株。接著，過表達EGR1或利用營養缺乏誘導EGR1上調來驗證此系統，確認報導基因的表現量與細胞內EGR1表現量的相關性。再來，我們利用流式細胞分選技術，將對代謝壓力感受性不同的MDA-MB-231乳癌細胞區分出來，探討不同群體間的表現型差異，並發現對代謝壓力有反應的細胞具有較差的增殖能力、細胞活性、群落形成能力、遷移能力及幹細胞性。 除二維細胞培養外，我們也利用三維細胞培養模擬實質固態瘤的腫瘤內代謝壓力。測試了不同的培養及染色條件，並嘗試以共軛焦顯微鏡觀察對代謝壓力有不同反應性的乳癌細胞。未來將進一步利用三維培養模型以及動物模型，探討代謝壓力下EGR1被誘導上調情形，對乳癌細胞命運的影響。這將有助於我們了解乳癌細胞產生代謝壓力抗性的潛在機制，對癌症治療的發展具有重大意義。

Cancer cells in the core region of solid tumors typically face hypoxia and nutrient deficiency because of their rapid growth and inadequate vascularization. These hostile conditions result in intratumoral metabolic stress and stimulate cancer cells to respond adaptively to maintain proliferation and survival. In our previous study, we discovered that Early Growth Response 1 (EGR1), a stress-responsive transcription factor playing multiple roles in cancer progression, can be induced in various breast cancer cell lines under several nutrient-deficient conditions. However, the functions of the stress-induced EGR1 and how it takes part in the stress-adaptive responses of breast cancer cells remain unclear. Hence, we developed a reporter system to monitor EGR1 levels in living cells and investigated how the difference in stress-responsiveness in breast cancer cells results in different phenotypes and affects their survival. First, we established a reporter system by constructing reporter plasmids with a destabilized green fluorescent protein (GFP) gene and a luciferase gene under the control of EGR1 response elements (ERE) or the EGR1 promoter (EP). Then, we introduced these reporter constructs to HEK293 cells and breast cancer cell line MDA-MB-231 to develop traceable cell models, where GFP and luciferase expression increased in response to EGR1 expression. To validate this EGR1 reporter system, we increased EGR1 expression in cells by overexpressing EGR1 or inducing EGR1 with nutrient deprivation and measured their GFP levels and luciferase activity. To investigate distinct phenotypes between stress-responsive and stress-non-responsive breast cancer cells, we sorted the cells by fluorescent-activated cell sorting (FACS) based on GFP levels upon nutrient deprivation. We found that the stress-responsive population of MDA-MB-231 cells featured declined cell proliferation, cell viability upon stress, colony formation ability, migratory ability, and cancer stemness. On the other hand, we utilized 3D spheroid models to mimic intratumoral metabolic stress, intending to fate-mapping metabolic stress-responsive breast cancer cells in spheroids with confocal microscopy. We modified the culture and staining conditions and generated spheroids using reporter cells to explore how EGR1 induction contributes to cell survival. Further investigation could provide greater insight into the underlying mechanisms of metabolic stress resistance in breast cancer cells, which could have tremendous implications for cancer treatment.