探討PIM1激酶在肺腺癌中所扮演的角色

Abstract

肺腺癌(lung adenocarcinoma, LUAD)是最常見的肺癌類型，同時也是一種致命且惡性的呼吸疾病，涉及大量的腫瘤增生、轉移和不良的預後。瞭解能調節其腫瘤進展的相關機制對制訂有效的治療策略至關重要。絲氨酸/蘇氨酸激酶PIM1已知會頻繁表現在包括肺腺癌在內的各類癌症之中。近期的研究發現PIM1激酶的異常升高與腫瘤的發生有關。透過磷酸化下游許多的蛋白，PIM1能介導細胞內數個重要的功能，例如轉錄、代謝和細胞週期。然而，對於該蛋白在肺腺癌中所扮演的角色以及其相關的信息調控路徑至今仍不甚清楚。因此，為了釐清這個問題，我們首先從數據集GSE2514中下載了20筆人類肺腺癌組織基因表現量資料。接著我們將這些資料分為兩組(PIM1-high和PIM1-low)，並利用多種生物信息學工具進行分析。根據得到的結果，我們找到PIM1與腫瘤的血管新生、上皮間質轉化(epithelial–mesenchymal transition, EMT)以及發炎反應呈正相關。而我們也進一步以活體和細胞等方式來驗證上述的發現。從動物實驗中我們證明透過給予抑制劑降低PIM1可以有效減少小鼠腫瘤生長的情形。另一方面，細胞實驗顯示與生物信息學分析的結果相符。利用細胞活性測試我們發現抑制PIM1激酶可以誘導細胞凋亡；利用免疫細胞化學染色我們發現抑制PIM1激酶可以改變癌細胞間質型態的外形；利用傷口癒合試驗我們發現抑制PIM1激酶可以減少細胞遷徙的能力。此外，抑制PIM1還會影響到癌細胞中能調節免疫反應的重要分子──程序性細胞死亡配體1(programmed cell death-ligand 1, PD-L1)的表達。儘管PD-L1因其會表現在細胞膜表面上與PD-1相互作用進而達到免疫抑制的功能而聞名，但該蛋白近期被報導能夠易位到如細胞核等其他部位啟動MAPK及PI3K-AKT路徑進而調控腫瘤生成和上皮間質轉化。於是我們利用免疫組織化學染色來確認PIM1和PD-L1之間的臨床關聯。包含六十四個肺腺癌患者樣本的組織陣列玻片在經過染色後，被送由經專業訓練的病理醫師進行判讀。通過組織學影像數據顯示其結果與細胞實驗一致，即PIM1確實與PD-L1呈正相關。綜上所述，我們的研究證明PIM1對腫瘤發展至關重要。它可能與PD-L1作用進而介導如上皮間質轉化等會促進肺腺癌的事件。而我們也成功發現PIM1激酶所扮演的角色，為肺腺癌提供了具潛力的新治療靶點。

Lung adenocarcinoma (LUAD), the most prevalent type of lung cancer, is a fatal and malignant respiratory disease involving extensive tumor proliferation, metastasis, and poor prognosis. Understanding the mechanisms regulating the progression of LUAD is necessary for developing effective therapeutic strategies. PIM1, serine/threonine kinase-PIM family, has been found frequently expressed in different types of cancer including LUAD. Recent studies revealed that aberrant elevation of PIM1 kinase is associated with oncogenesis. By phosphorylating various downstream substrates, PIM1 mediates several important cellular processes, such as transcription, cell metabolism, and cell cycle. However, the role of PIM1 kinase in LUAD and its associated signaling pathways remain unclear. Therefore, in order to clarify this question, we downloaded twenty human lung adenocarcinoma gene expression profiles from the GSE2514 dataset first. Next, we sorted these profiles into two groups (PIM1-high vs. PIM1-low) and then utilized multiple bioinformatics tools to perform analyses. According to the results, we found that PIM1 positively correlated with angiogenesis, epithelial–mesenchymal transition (EMT), and inflammatory response. Here, we then determined the effects by in vivo as well as in vitro studies. Based on the animal experiment, we demonstrated that blocking PIM1 with a pharmacological inhibitor significantly alleviated tumor growth. On the other hand, cell experiments showed corresponded to the results of bioinformatic analyses. We explored that inhibition of PIM1 kinase could induce apoptosis by cell viability assay, change mesenchymal-form morphology by immunocytochemistry, and reduce cell migration ability by wound healing assay. Moreover, inhibition of PIM1 also affected immune response regulator programmed cell death-ligand 1 (PD-L1) expression in cancer cell. Despite the molecule was known for its immune inhibitory function which is expressed on the cell surface to interact with PD-1, it has been recently reported that PD-L1 could regulate tumorigenesis and EMT through translocating to other subcellular compartments like nucleus to initiate the MAPK (MEK-ERK) and PI3K-AKT signaling pathway. Accordingly, we adopted immunohistochemistry to figure out the clinical relevance between PIM1 and PD-L1. A tissue array containing 64 LUAD patient samples was stained and further assessed by a trained pathologist. Through histological images, the result was consistent with our in vitro data which PIM1 was positively correlated with PD-L1. Taken together, our research proved that PIM1 was crucial for tumor development. It may interact with PD-L1 to mediate events which promote LUAD such as EMT. In conclusion, we discovered a novel role of PIM1 kinase, and provided a potential therapeutic target for LUAD.