探討人類粒線體NAD(P)+依賴型蘋果酸酶(ME2)在多發性骨髓瘤代謝重編程之研究

多發性骨髓瘤 (multiple myeloma, MM) 為血液性癌症的一種，主要是因為體內的漿細胞 (plasma cell) 出現病變所引起的惡性腫瘤，目前治療方式以藥物治療合併自體幹細胞移植為主，隨著新藥的發展，患者存活率已大幅延長，但當疾病再次復發時，死亡率也隨即增加，且初次治療時大部分的化療藥物都已經使用過，疾病已對藥物產生抗藥性，目前尚缺乏更好、更新的藥物來做治療，因此仍有必要開發新藥來克服這些困境。癌細胞代謝途徑改變是腫瘤增生的主要原因之一，與正常細胞相比，癌細胞因快速生長所需的能量亦增加，為了適應快速生長的環境，癌細胞重新編程他們的代謝途徑，從缺乏營養的環境中獲取必要的營養，並利用這些營養來保持生存的能力，和建立新的生物能量應對隨著代謝壓力的增加，以實現快速吸收和分解營養物質，從而有助於疾病轉化、維持和進展。而蘋果酸酶 (malic enzyme) 在過去研究被發現其功能為參與細胞能量調節將蘋果酸 (malate) 轉化為丙酮酸 (pyruvate)，產生NAD(P)H，因此粒線體蘋果酸酶 (m-NADP(H)-ME, ME2) 被認為對醣質新生、糖解、脂肪酸合成和能量生成息息相關，根據文獻也表明ME2與麩醯胺酸 (glutamine) 氧化還原、細胞分化、生長、增殖、侵襲、脂肪形成和活性氧物質 (reactive oxygen species, ROS) 生成等有關聯，因此進一步探討ME2在癌症中的角色將有助於抗癌新方法的研發。本研究發現3株多發性骨髓瘤細胞株 (H929、U266B1、RPMI8226) 在不一樣的營養狀況下，其生長狀況會受影響，H929、U266B1細胞株具有glutamine依賴性，而RPMI8226細胞株則是對glucose和glutamine皆有依賴性。當敲低 (knockdown) H929細胞株中ME2基因來降低其蛋白表現量後，使細胞生長速度變得緩慢，並在不同營養種狀態下，測量其丙酮酸、NADPH和ATP含量，Knockdown ME2表現量組 (shME2 group) 相對於對照組均有下降情形，而NAD+/NADH比值則是上升 (NADH 也是下降)，這顯示在敲低H929 的ME2表現量後出現了代謝重置 (metabolic reprogramming) 的情況，這一發現有助於未來針對癌細胞代謝路徑的療程開啟另一條道路，另外這也顯示ME2在細胞中能量代謝扮演重要角色，此外，我們的研究還發現了轉錄因子7類2 (transcription factor 7-like 2, TCF7L2) 與ME2有負向關係，而TCF7L2為Wnt/β-catenin 訊息傳遞路徑 (signaling pathway) 重要一員，所以此一發現也有助於探討ME2的調控機制。

Multiple myeloma (MM) is a kind of blood cancer. It is mainly a malignant tumor caused by the pathological changes of plasma cells in the body. At present, the treatment method is mainly drug therapy combined with autologous stem cell transplantation. With the development of new drugs, the survival rate of patients has been greatly prolonged. However, when the disease relapses again, the mortality also increases. Most of the chemotherapy drugs have been used during the initial treatment, and the disease has developed resistance to them, and there is still a lack of better and newer drugs to treat it. Therefore, it is still necessary to develop new drugs to overcome these difficulties. The change of metabolic pathways of cancer cells is one of the main causes of tumor proliferation. Compared with normal cells, cancer cells also need more energy for rapid growth. In order to adapt to the environment of rapid growth, cancer cells reprogram their metabolic pathways to obtain necessary nutrients from the nutrient deficient environment and use these nutrients to maintain their viability, and establish new bioenergy to cope with the increase of metabolic pressure, and to achieve rapid absorption and decomposition of nutrients, thereby contributing to disease transformation, maintenance and progression. Malic enzyme has previously been discovered to have a role in cell energy regulation, convert malate to pyruvate, and produce NAD(P)H. Therefore, m-NAD(P)-ME (ME2) is considered to be closely related to glycogenesis, glycolysis, fatty acid synthesis and energy production. Previous research reported that ME2 is also related to glutamine redox, cell differentiation, growth, proliferation, invasion, adipogenesis and ROS production. Therefore, further study on the role of ME2 in cancer will contribute to the development of new anti-cancer methods. This study found that the growth status of three multiple myeloma cell lines (H929, U266B1 and RPMI8226) was affected under different nutritional conditions. H929 and u266b1 cell lines were glutamine dependence, while RPMI8226 cell line was dependent on both glucose and glutamine. When ME2 expression in H929 cell line was knocked down, the growth rate of cells became slow. The contents of pyruvate, NADPH, and ATP were measured under different nutritional conditions. In the ME2 expression knockdown group were decreased, while NAD+/NADH were increased, indicating that H929 had metabolic reprogramming after ME2 expression knockdown. This discovery will help to provide another way for the treatment of cancer cells' metabolic pathway in the future. In addition, it also shows that ME2 plays an important role in cell energy metabolism. Moreover, we also found that TCF7L2 has a negative association with ME2, and TCF7L2 is member of Wnt/β-catenin signaling pathway, and this discovery is also helpful to understand the regulation mechanism of ME2.