探討EGR1的磷酸化及其在乳癌細胞適應代謝壓力的功能

Abstract

癌細胞的失控性增殖往往超越血管新生的速度。此特性導致實體腫瘤中心經常處於營養及氧氣供應不足的狀態，從而引起腫瘤內代謝壓力的形成。為了生存於如此惡劣的微環境，癌細胞需啟動壓力適應反應，其中可藉由轉錄因子實現。實驗室先前研究發現，在多種乳癌細胞株中，營養剝奪會顯著誘導壓力反應轉錄因子Early Growth Response 1 (EGR1)的表現，而在葡萄糖缺乏條件下，EGR1的表現會受到ROS/p38訊號途徑的調控。當EGR1的表現抑制非壓力狀態下乳癌細胞的生長，EGR1的表現卻促進葡萄糖缺乏下乳癌細胞的存活，顯示EGR1在乳癌中會依細胞所面臨的壓力而扮演雙重角色，且能協助這些細胞適應代謝壓力。然而，EGR1調控代謝壓力適應的具體分子機制及其功能意涵仍有待深入闡明。 代謝壓力適應反應不僅能幫助癌細胞維持生存，更能協助其獲得遠端轉移的能力。在轉移過程中，癌細胞會脫離原發腫瘤部位，而失去其與細胞外基質的接觸，進一步引發失巢凋亡。因此，癌細胞是否能抵抗失巢凋亡會顯著影響癌症轉移。本研究亦探討EGR1在乳癌細胞抵抗失巢凋亡當中潛在的角色。 西方墨點法分析顯示，乳癌細胞葡萄糖缺乏下誘導的EGR1蛋白會多出現一條約100 kDa的條帶，並經過體外去磷酸酶處理後確認此為磷酸化修飾。這表明在代謝壓力下，EGR1除了被誘導表現外，還會進行磷酸化修飾。為找尋代謝壓力乏下EGR1磷酸化位點，我們建立了EGR1磷酸化位點突變並發現T391A突變使得100 kDa的條帶在葡萄糖充足以及缺乏的情況下都幾乎消失。這些結果表明EGR1在T391位點會被磷酸化，且在蛋白表現量上升時更加顯著，包括代謝壓力的條件下。 EGR1 T391的磷酸化雖然不會改變蛋白質在細胞內座落的位置，但顯著影響其轉錄活性。透過藥物抑制劑及基因改造等方法，我們發現EGR1代謝壓力下的磷酸化受ROS和AMPK的調控。未來我們將進一步探討AMPK對EGR1 T391磷酸化的調控機制，以及這修飾對於乳癌細胞適應代謝壓力的影響。這些發現將有助於理解乳癌細胞適應代謝壓力的反應機制，為乳癌治療提供新的策略方向。

Solid tumors frequently experience intratumoral metabolic stress due to rapid cell growth that exceeds the vascular supply. To maintain cell survival under this stress, cancer cells must elicit stress-adaptive responses, which can be achieved by activating transcription factors. Our previous studies identified that the stress-responsive transcription factor Early Growth Response 1 (EGR1) is significantly induced in various breast cancer cell lines under various nutrient depletion conditions. Under glucose deprivation, this induction was regulated via the ROS/p38 pathway. While EGR1 expression inhibited cell proliferation in non-stressed cells, it promoted cell viability in glucose-deprived breast cancer cells, denoting the capability of EGR1 in mediating metabolic stress adaptation. However, its precise regulatory mechanisms and detailed functional impact in this context remain unclear. In addition to supporting cell survival, metabolic stress adaptation in cancer cells may facilitate their metastatic escape. During metastasis, as cancer cells disseminate from the tumor mass, they become vulnerable to a programmed cell death triggered upon extracellular matrix detachment known as anoikis. Susceptibility toward anoikis highlights the importance of gaining anoikis resistance for cancer cells to metastasize. In this study, we delved into the possible contribution of EGR1 in the anoikis resistance of breast cancer cells. EGR1 was not only induced but also phosphorylated in breast cancer cells under glucose deprivation. This was evidenced by an additional EGR1 band migrating at 100 kDa in Western blot analysis, which ceased upon in vitro dephosphorylation. To locate the EGR1 phosphorylation site under metabolic stress, we employed site-directed mutagenesis. We identified that the additional EGR1 protein was nearly absent in the T391A mutant under both glucose-replenished and glucose-deprived conditions. This indicates that EGR1 is phosphorylated at T391, and this phosphorylation becomes evident as EGR1 expression increases under conditions such as metabolic stress. While EGR1 phosphorylation at T391 did not impact EGR1 nuclear localization, it was crucial for its transcriptional activity. Through pharmacological inhibition and genetic modification, we found that EGR1 phosphorylation under glucose deprivation was regulated by ROS and AMPK. However, further studies are required to elucidate the relation between AMPK and EGR1 T391 phosphorylation, as well as their detailed impact on breast cancer cells adapting to metabolic stress. Understanding how the cells respond to stress may provide a novel approach for breast cancer therapeutic intervention.