探討肺癌中巨噬細胞影響的癌症進程及SPANXA抑制腫瘤轉移的機制

Abstract

在腫瘤微環境中的巨噬細胞被分類成M1及M2兩種型態。M1 巨噬細胞的表現在臨床上與肺癌病人有較佳的預後相關。因此，我們發現不同巨噬細胞對於肺癌細胞的影響。我們發現M2a/M2c巨噬細胞促進肺癌細胞A549侵襲能力及異種皮移植腫瘤生長。M1 巨噬細胞則是抑制血管新生。M1 巨噬細胞促進A549對於化療藥cisplatin的敏感度以及減少內皮細胞管柱生成活性，並藉由誘發細胞凋亡及衰老抑制細胞的生存能力。藉由微陣列表達基因晶片，我們鑑定出不同巨噬細胞對於A549的衝擊影響。巨噬細胞在A549中調控的基因與免疫反應，細胞骨架重組，凝結反應，細胞貼附及細胞凋亡路徑有關。被鑑定出的M1/M2基因特徵與肺癌病人較長的總體存活率有顯著相關性。另外，給予注射M1的培養基可以減小A549在老鼠皮下生長的腫瘤大小。M1培養基誘發細胞凋亡並且促進p53蛋白累積。更甚，M1藉由減少p53泛素化作用而延長p53蛋白的穩定度。接著，我們發現STAT1是一個主要的調控p53蛋白累積的基因。STAT1在M1培養基刺激下會大量表現並活化。STAT1減少p53泛素化作用而抑制STAT1則可以回復因為M1而增加的p53的穩定度。STAT1及p53的交互作用因M1而增加，但會因為外送STAT1-Y701F而減少。抑制STAT1的活化或是抑制IFNAR2, JAK1 及 TYK2基因表現都可以抑制M1誘發的細胞凋亡。總和來說，M1誘發的細胞凋亡及p53蛋白累積需要STAT1的訊息傳遞。M1誘發的細胞凋亡是與IFN-γ無關的路徑產生的。我們的發現指出M1巨噬細胞在癌症免疫中扮演很重要的角色，這角色是藉由引起在肺癌中STAT1的活化及p53蛋白的累積。 SPANXA(在X染色體上與細胞核相關精子蛋白家族成員A1/A2)是一個癌-睪丸抗原，表現在正常的睪丸中，但在各種腫瘤中也會不正常表現。它的角色在晶子發育跟癌症發展中都還未知。我們發現與高侵襲性的細胞CL1-5相比，SPANXA大量表現在低侵襲的CL1-0細胞中。因此，我們想調查SPANXA在肺腺癌中對於癌轉移的角色。SPANXA喜歡表現在腫瘤組織上並且與肺腺癌病人較長的存活率相關。SPANXA在試管內及生物體內能夠抑制肺癌細胞侵襲及轉移。藉由微陣列晶片表現基因及路徑分析，我們發現SPANXA 影響的基因集中在表皮-間質型態轉換 (EMT) 路徑。當SPANXA表示時細胞的塑性會朝向MET過程。Slug在肺癌中是一個很重要的轉錄因子，可誘發EMT同時抑制維持細胞黏附的蛋白E-cadherin. SPANXA可以抑制Slug表現導致E-cadherin上升。更甚，給予或抑制Slug表現可以回復因為SPANXA表現所抑制的細胞侵襲能力。c-JUN是一個正向調控EMT的基因。靜默SPANXA增加c-JUN mRNA的表現，抑制c-JUN則導致SNAI2降低。我們也發現SPANXA 減少Lamin A/C 而增加核內的E-cadherin。我們的結果清楚的定義在肺腺癌中SPANXA為一個EMT的抑制蛋白，是藉由抑制c-JUN-SNAI2。

Macrophages in a tumor microenvironment have been characterized as M1- and M2-polarized subtypes. M1 macrophage is associated with favorable outcome of lung cancer patients clinically. Herein, we discover the different macrophages’ influences on lung cancer cell. We found M2a/M2c subtypes promote A549 invasion and xenograft tumor growth. The M1 subtype suppressed angiogenesis. M1 enhanced the sensitivity of A549 to cisplatin and decreased the tube formation activity and cell viability of A549 cells by inducing apoptosis and senescence. By expression microarray, we identified the impacts from different macrophage subtypes on A549. The regulated genes were involved in the immune response, cytoskeletal remodeling, coagulation, cell adhesion, and apoptosis pathways in A549 cells. The identified M1/M2 gene signatures were significantly correlated with the extended overall survival of lung cancer patients. Administration of M1 conditioned media (CM) to A549-bearing mice significantly reduced the tumor size. Microarray analysis indicated that p53 was a critical regulator in M1-treated A549. M1 CM induced cell apoptosis and enhanced wt-p53 accumulation. Furthermore, M1 prolonged p53 protein stability by reducing its ubiquitination. Next, we revealed that STAT1 is a key mediator for p53 accumulation. STAT1 was up-regulated and activated upon M1 CM stimulation. STAT1 reduced p53 ubiquitination and knockdown STAT1 reversed the increase of p53 stability. The interaction of STAT1 and p53 was enhanced by M1 CM, but it was reduced by STAT1-Y701F. Inhibiting STAT1 activation or silencing the IFNAR2, JAK1 and TYK2 inhibited M1-induced apoptosis. Taken together, STAT1 signaling is required for M1 CM-induced apoptosis through p53 accumulation. The M1 CM-induced apoptosis was IFN-γ-independent. Our findings imply that M1 macrophages play an important role in the immune surveillance for cancer progression by inducing STAT1 activation and p53 accumulation in lung cancers. SPANXA (Sperm Protein Associated with the Nucleus on the X-chromosome family, A1/A2) is identified as a cancer-testis antigens expressed in normal testis but dysregulated in various tumors. It plays unknown roles in spermiogenesis and cancer progression. We discovered SPANXA highly expressed in the low-invasive CL1-0 cells compared with the isogenous high-invasive CL1-5 cells. Herein, we investigated the role of SPANXA in metastasis of lung adenocarcinoma. SPANXA was preferably expressed in tumor tissues and associated with the prolonged survival of lung cancer patients. SPANXA suppressed the invasion and metastasis of lung cancer cells in vitro and in vivo. By the expression microarray and pathway analysis, we found that the SPANXA-altered genes were enriched in the epithelial–mesenchymal transition (EMT) pathway. The cell plasticity undergoes MET while SPANXA expresses. Slug is an important transcription factor in lung cancer progression and also an EMT inducer which represses E-cadherin, a protein maintaining cell-cell adherin. SPANXA reduced Slug expression resulted in up-regulating E-cadherin. Furthermore, the restoration or inhibition of Slug could reverse the invasive suppression of SPANXA. c-JUN acts as the positive-regulator of EMT. Silencing SPANXA increased c-JUN mRNA expression and blockage of c-JUN led to SNAI2 down-regulation. We also discovered that the Lamin A/C decreased and E-cadherin increased in the nucleus. Our results clearly characterized SPANXA as an EMT inhibitor by suppressing c-JUN-SNAI2 axis in lung adenocarcinoma.