纖維母細胞生長因子九（FGF9）是纖維母細胞生長因子的成員，對於睾丸發育中起重要作用。然而，高表達的纖維母細胞生長因子九與幾種人類癌症例如胃、肺、卵巢和前列腺腫瘤相關。當細胞受到外部壓力或異常調節時，可誘發腫瘤發生，從而引起過度的細胞增生，並伴有與轉移相關的上皮間質轉化 (Epithelial–mesenchymal transition)。先前的研究已經表示，局部黏合激酶 (Focal Adhesion Kinase) 的活性可以操縱腫瘤的表型，導致不受控制的增生和與轉移，且與腫瘤發生是高度相關的。先前我們實驗室已經證明FGF9可以在小鼠萊氏前驅細胞（TM3）中誘導體外細胞增生，細胞遷移和侵襲，而這可能與睾丸腫瘤的發展有關。因此，我們假設FGF9可以誘導萊氏前驅細胞的腫瘤發生。我們發現FGF9可以增強小鼠萊氏前驅細胞（TM3）細胞的增生和遷移以及間質標記物的表現增加。在TM3細胞中，FGF9還可以誘導FAK途徑中Y397位點磷酸化的FAK表達，以及絲裂原活化蛋白激酶 (Mitogen-Activated Protein Kinase) 途徑中ERK、 JNK、 p38的磷酸化，FGF9也可以誘導磷脂酰肌醇3-激酶 （PI3K）途徑中Akt的磷酸化。此外，使用FAK、MAPK和PI3K的特異性抑制劑分別抑制其相關途徑，抑制的結果進一步說明了由FGF9刺激影響的途徑中，FAK途徑位於上游，然後接著影響p-Akt、p-ERK和p-JNK途徑，然而磷酸化的ERK和JNK會影響下游p-p38途徑。此外，FAK抑制劑（PF573228） 、ERK抑制劑（U0126）和JNK抑制劑（SP600125）可以抑制TM3細胞的細胞增生，從而證明FAK、ERK和JNK途徑在FGF9刺激的TM3細胞增生中是至關重要的。在同種移植的小鼠模型中，對重症聯合免疫缺陷小鼠（NOD-SCID）皮下注射TM3細胞，從結果可以看到另外給予FGF9組別的腫瘤體積和重量明顯高於對照組，而使用ERK抑制劑的組別其小鼠的腫瘤生長顯著的被抑制。進一步使用免疫組織化學染色觀察小鼠的腫瘤切片，可以觀察到另外給予FGF9的組別，間質標記物的表現量增加。總結來說，FGF9 可以透過FAK、PI3K和MAPK 途徑的活化來誘導EMT的現象，從而使TM3前驅細胞的腫瘤發生。

Fibroblast growth factor 9 (FGF9), a member of the FGF family, plays an important role in testicular developments. However, high expression of FGF9 is associated with several human tumors, such as gastric, lung, ovarian, and prostate tumors. Tumorigenesis can be induced when cells are subjected to external stress or abnormal regulation, causing excessive cell proliferation with epithelial-mesenchymal transition (EMT) related to metastasis. Previous studies have also shown that focal adhesion kinase (FAK) activity can manipulate tumor phenotypes, leading to uncontrolled proliferation and metastasis highly associated with tumorigenesis. Previously, our lab has shown FGF9 could induce cell proliferation, migration, and invasion in vitro in TM3 mouse Leydig progenitor cells, which associated with testicular tumor development. Therefore, we hypothesized that FGF9 could induce testicular Leydig progenitor cell tumorigenesis. We found that FGF9 enhanced TM3 cell proliferation, migration and the increase in expression of mesenchymal markers. FGF9 could also induce the expressions of pY397-FAK, MAPK pathway (phosphor-ERK, phosphor-JNK and phosphor-p38), and PI3K pathway (phosphor-Akt) in TM3 cells in vitro. In addition, specific inhibitor for FAK, MAPK and PI3K could suppress its correlated pathway, respectively. These results illustrated a possible hierarchy among those pathways stimulated by FGF9 and showing FAK is in the upstream, which regulated p-Akt, p-ERK, and p-JNK pathways, and then p-ERK and p-JNK regulate downstream p-p38 pathway. Moreover, FAK inhibitor (PF573228), ERK inhibitor (U0126) and JNK inhibitor (SP600125) could inhibit TM3 cell proliferation, confirming that FAK, ERK, and JNK pathways were essential. In allograft in vivo study, results of subcutaneous injection of TM3 progenitor cells to the severe combined immunodeficiency (SCID) mice showed that tumor volumes and weights in FGF9-treated group were significantly higher than control groups. Moreover, ERK inhibitor could significantly inhibit tumor growth in SCID mice with TM3 cells in vivo. Immunohistochemistry results further showed that expressions of mesenchymal markers were increased in FGF9 group. In conclusion, FGF9 induces TM3 cell tumorigenesis with the induction of EMT via the activation of FAK, PI3K, and MAPK pathways.

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