探討Hippo訊息傳遞路徑於貓注射部位肉瘤之致腫瘤機制以及verteporfin對該腫瘤之療效

Abstract

貓注射部位肉瘤（Feline injection-site sarcoma，FISS）是一種經常在醫療注射行為之後發生的嚴重不良反應。此種腫瘤具有高度局部侵犯性，並且即使在手術切除後局部復發率仍然極高，雖然放射治療能夠有效降低復發機率，但目前台灣放射治療尚未普及，故仍無法有效控制此種疾病。普遍認為FISS主要是由慢性炎症反應所引起，但造成腫瘤生成的確切分子調控機制仍不完全明確。另一方面，Hippo訊息傳遞路徑在人類醫學中已被證實是調控細胞存亡與腫瘤生長有關的關鍵因子而被頻繁探討；然而，此訊息傳遞路徑在獸醫腫瘤學（尤其是貓的間質性腫瘤）中仍鮮少被討論。Verteporfin（VP，中文商品名維視達）是一種臨床上用於治療人類黃斑部病變的光敏感藥物，其作用原理是透過特定波長的光源刺激後造成活性氧（Reaction oxygen species，ROS）的累積而對細胞造成光化學性損傷。近期研究顯示，VP 在沒有光活化的情況下可以抑制 Hippo訊息傳遞路徑的不活化，也因為Hippo訊息傳遞路徑的失調與多種腫瘤的臨床預後不良密切相關，所以VP作為抗腫瘤治療的潛在標靶藥物而備受討論。 為了探討 Hippo訊息傳遞路徑在FISS腫瘤生成中所扮演的角色，本篇研究重點討論了此路徑中兩個重要的調控蛋白：YAP（Yes-associated protein）以及TAZ （Transcriptional co-activator with PDZ-binding motif）。我們分析了三株 FISS初代細胞株中 YAP 與 TAZ 的基因序列、mRNA和蛋白表現量與正常貓組織的差異。此外，我們透過一系列體外功能性實驗，包括量測細胞活性、遷移能力、群落形成能力、細胞凋亡與壞死及細胞週期分析，在「非光活化」與「光活化」兩種條件下來評估VP同時作為抑制 Hippo訊息傳遞路徑失調以及光動力療法的抗腫瘤療效。最後，本研究試圖優化原有的雞絨毛尿囊膜（chicken chorioallantoic membrane）實驗流程，期望建立FISS初代細胞株之雞胚蛋模型，並藉此評估VP治療是否能有效縮小腫瘤體積。 研究成果顯示，與健康貓組織相比FISS 細胞株中存在YAP與TAZ 蛋白過度表現的現象。同時藉由免疫細胞化學與免疫組織化學染色實驗證實這些蛋白存在於細胞核內，顯示Hippo訊息傳遞路徑的下游轉錄在FISS確實有被啟動。此外，本研究經由基因定序分析，發現YAP1（YAP）與WWTR1（TAZ）基因中有多個尚未被發表過的核苷酸及胺基酸突變點，但其mRNA表現量未出現對應的上升，推測Hippo訊息傳遞路徑在FISS的失調並不僅源於YAP和TAZ基因擴增，而可能是更複雜的轉錄後調控機制。在藥物研究方面，我們發現非光活化的VP顯著抑制了FISS 細胞株的活性、遷移能力與群落形成，誘發腫瘤細胞凋亡與壞死，同時也會導致G1週期停滯（G1 phase arrest）；而光活化VP則可在遠低於非光活化VP的藥物劑量下，在所有FISS細胞株中展現出相當的效果，顯示出其作為光動力療法下的抗腫瘤潛力。最後我們成功優化CAM實驗流程，將FISS腫瘤生成的成功率提升至51.2%，同時也利用該模型驗證VP可縮小腫瘤體積。綜上所述，本研究揭示了Hippo訊息傳遞路徑在FISS腫瘤生成機制中扮演著調控的作用，並且為Hippo 訊息傳遞路徑的標靶治療以及光動力療法作為未來治療FISS疾病上提供了一些重要資訊，以其未來能應用於臨床治療上為動物醫學做出貢獻。

Feline injection-site sarcoma (FISS) is a serious adverse effect following injection event, causing the growth of highly invasive mesenchymal neoplasm characterized by a high local recurrence rate. Although radiotherapy has been proved to effectively reduce the tumor recurrence, it has not been widely available in Taiwan, thus leading to the difficulties of FISS managements in clinics. While its association with chronic inflammation is well-documented, the precise regulatory mechanism effecting FISS tumorigenesis remain undefined. On the other hand, the Hippo signaling pathway has been recognized as a key regulator of cellular homeostasis and tumor progression in humans; however, its role in veterinary oncology remains largely unexplored. Verteporfin (VP) is a human medicine that is originally used for treating age-related macular degeneration. It causes accumulation of reactive oxygen species (ROS) after light activation, resulting in photochemical damage of the mitochondria. Recently, VP has been proved to affect the Hippo pathway in the absence of light activation. In human medicine, the dysregulation of Hippo pathway has been reported to correlate with poor clinical outcomes and poor prognosis in varies tumors. Thus, VP has emerged as a potential therapeutic target of antitumor treatments. To determine the effect of Hippo pathway in the tumorigenesis of FISS, we investigated the involvement of two Hippo pathway core effectors, Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ), by characterizing the mutational status, mRNA expression, and protein expression of YAP and TAZ in multiple FISS primary cell lines. Furthermore, the anti-tumor efficacy of VP, a known Hippo pathway inhibitor and a photosensitive drug, under both non-photoactivated and photoactivated conditions through a series of in vitro functional assays was evaluated, including cell viability, cell migration, clonogenic ability, apoptosis and necrosis rate, and cell cycle analysis. An optimized chorioallantoic membrane (CAM) assay was also used for the evaluation of tumor size reduction efficacy after non-photoactivated VP treatment. Our results demonstrate a generalized overexpression of YAP and TAZ proteins in FISS cell lines compared to healthy feline tissues. Immunocytochemical and immunohistochemical analyses confirmed nuclear localization of these effectors, indicating transcriptional activation of the Hippo pathway. Despite genomic sequencing identified several novel nucleotide mutations and amino acid substitutions within YAP1(YAP) and WWTR1(TAZ), no alternation of their mRNA expression levels suggested complex post-translational regulation rather than simple gene amplification. Pharmacological intervention with Hippo regulation effects of non-photoactivated VP significantly inhibited cell viability, cell migration, and colony formation, and induced apoptosis, necrosis, and G1 phase arrest of FISS cells. For the photodynamic effects of photoactivated VP, it also exhibited comparable anti-tumor effects in all FISS cell lines with a way lower dosage compared to non-photoactivated VP. Finally, by modifying the CAM assay protocol in primary FISS cell lines, we achieved an overall success rate of 51.2% for generating evaluable tumor on chicken CAM, and also successfully validated the efficacy of non-photoactivated VP in reducing tumor volume. In summary, these findings highlighted the regulatory role of Hippo pathway in the tumorigenesis of FISS, and also provided important insights for the future development of therapeutic strategies for FISS via Hippo pathway inactivation and photodynamic treatments.