L-type amino acid transporter 1在肺癌幹細胞自我更新的角色探討

根據衛福部統計，肺癌為國人十大死因之首，近數十年來在臺灣地區的肺癌病人有顯著增加的趨勢。癌症幹細胞(cancer stem cells)，為癌細胞中有一群具有自我更新和分化能力的細胞，其在腫瘤生成和抗藥性扮演著重要的角色。L-type amino acid transporter 1 (LAT1)，主要轉運大分子支鏈胺基酸和芳香族中性胺基酸，其中包括必需胺基酸白胺酸(leucine)。近年來研究發現，LAT1在許多癌症中都有較高的表現量，包含食道癌、口腔癌、乳癌及肺腺癌，並且可藉由抑制LAT1表現量來抑制癌細胞的生長。本研究首先發現肺癌細胞株A549之Pemetrexed抗藥性細胞A400細胞中自我更新相關基因及LAT1的表現較A549細胞為高，因此我們推測LAT1可促進肺癌幹細胞的自我更新能力。除此之外，從Human Protein Atlas資料庫中顯示，高表現LAT1的肺癌病患其總生存期間較低表現者短。不論藉由小分子抑制劑JPH203或以慢病毒傳送專一性shRNA來抑制LAT1的活性或蛋白表現，都能有效抑制A400細胞中自我更新相關基因的表現和癌症球體(tumorsphere)的形成，並且造成細胞內mTOR和AKT磷酸化程度下降。為更進一步探討LAT1在肺癌幹細胞所扮演的角色，我們使用可穿透細胞之白胺酸類似物(L-Leucyl-L-Leucine methyl ester, Leu-Leu-OMe)處理A400細胞，則發現可以增加mTOR或AKT的磷酸化，並提高其癌症球體形成能力，而Leu-Leu-OMe處理更可降低JPH203對癌症球體形成的抑制效果，這些結果顯示LAT1在肺癌幹細胞的自我更新能力中扮演著重要角色。總結本研究結果，LAT1可能是藉由AKT/mTOR路徑來調控肺癌幹細胞的自我更新，且抑制LAT1的活性可能做為肺癌病患的潛在治療策略。

Lung cancer is the leading cause of death among cancers in Taiwan. Cancer stem cells (CSCs) are a subpopulation of cancer cells which participate in tumor initiation, drug resistance, and metastasis. L-type amino acid transporter 1 (LAT1/SLC7A5) is a membrane transporter, which delivers neutral amino acids into cells, such as leucine. Recent studies indicate that LAT1 is highly expressed in a variety of cancers, such as esophageal carcinoma, oral cancer, breast cancer, and lung adenocarcinoma and the inhibition of LAT1 reduces cell proliferation.In this study, we first discovered that the CSC activity of pemetrexed-resistant A549 cells (called A400) was higher than the parental A549 cells, including the increased number of tumorspheres and the upregulation of BMI1, Sox2, Oct4, and c-Myc. We also found that LAT1 expression in pemetrexed A400 cells was higher in comparison to the parental A549 cells. From Human Protein Atlas database, the expression level of LAT1 is positively associated with the shorter overall survival of lung cancer patients. Inhibition of LAT1 by a small molecule inhibitor, JPH203, or by lentiviral delivery of specific shRNAs led to a significant reduction in tumorsphere formation and downregulation of cancer stemness genes including BMI1, Sox2, Oct4, and c-Myc. We also found that the activation of AKT/mTOR was declined after LAT1 inhibition. The treatment of L-Leucyl-L-Leucine methyl ester (Leu-Leu-OMe), a cell-permeable leucine derivative, promoted mTOR/AKT phosphorylation and CSC activity in A400 cells. Leu-Leu-OMe also reduced the inhibitory effect of JPH203 to A400 cells in tumorsphere formation. In conclusion, our data reveal that LAT1 regulates self-renewal of lung CSCs through AKT/mTOR pathway and its inhibition may offer an effective therapeutic strategy in lung cancer patients.