探討 WNT/β-catenin 分子機制於貓注射部位肉瘤之致腫瘤機制

Abstract

貓注射部位肉瘤（Feline injection-site sarcoma, FISS） 是一種高度惡性且具有 強烈組織侵犯性的軟組織腫瘤， 即使在接受大範圍手術切除後， 復發率仍相當高。 儘管已有研究指出， 大範圍手術切除合併放射線治療可延長無病生存期， 但目前 在獸醫領域的放射線治療資源有限，故 FISS 的治療在臨床上仍相當有挑戰性。 此外，雖然慢性發炎已被廣泛被認為是 FISS 的誘發因子，但其確切致病機轉尚 未完全釐清， 目前已有多項研究指出，多種發炎相關的訊息傳遞路徑， 例如 COX- 2、 NF-κB 及 STAT3， 可能與 FISS 的發生和進展有密切相關。 另一方面， WNT/β- catenin 訊息傳遞路徑在維持細胞恆定中扮演重要角色，並日益被認為與人類及 獸醫腫瘤之形成有關，其中 β-catenin 基因（CTNNB1）的突變已知可促使多種 人類癌症的腫瘤轉化。為探討此訊息傳遞路徑是否參與 FISS 的腫瘤生成，我們 檢測 β-catenin 在 mRNA 與蛋白質層級的表現 ，並進行 CTNNB1 全長定序。 結果 顯示 ，在福馬林固定石蠟包埋組織與 FISS 初代細胞中皆可觀察到 β-catenin 在細 胞質內的累積，而且所有 FISS 細胞株的 β-catenin 蛋白質表現量皆顯著高於正常 貓皮膚組織，然而即時定量聚合酶連鎖反應（RT-qPCR） 的結果顯示，FISS 細胞 中 β-catenin mRNA 表現量低於正常組織，推測可能與轉譯後調控有關。進一步 分析 CTNNB1 序列可發現數個點突變，部分突變會導致胺基酸置換，包含一處 位於 β-TrCP 結合區之人類癌症突變熱點， 已知可增強 β-catenin 的穩定性與促癌 訊號活性。為了進一步評估 β-catenin 抑制劑 MSAB 對於 FISS 的治療效果 ，我們 以 FISS 初代細胞進行多種功能測試， 結果發現 MSAB 能顯著抑制細胞增殖、 聚 落形成與細胞移行。 綜上所述 ，本研究結果支持 FISS 發展過程中 WNT/β-catenin 訊息傳遞路徑可能會發生異常， 凸顯此訊息傳遞路徑具備作為藥物研發靶點的潛 力。此外，我們亦嘗試建立雞胚尿囊絨毛膜 （chorioallantoic membrane, CAM） 模 型， 作為替代性動物模型來評估藥物療效 ，此模型相較於傳統鼠類模型在實驗動 物減量與實驗操作方面具有實用優勢， 可應用於新型 FISS 治療藥物的快速篩選。

Feline injection-site sarcoma (FISS) is a highly malignant and locally invasive soft tissue sarcoma characterized by a high recurrence rate even after radical surgical resection. Although combination of wide-margin excision and radiation therapy has been reported to prolong disease-free intervals, the limited availability of radiotherapy in veterinary medicine continues to impede optimal clinical management. Moreover, the precise pathogenesis of FISS remains unclear, although chronic inflammation is widely considered as a key factor. Multiple studies have indicated that various inflammatory signaling pathways, such as COX-2, NF-κB, and STAT3, may contribute to the initiation and progression of FISS. Meanwhile, the WNT/β-catenin signaling pathway, which plays a fundamental role in maintaining cellular homeostasis, has also been increasingly associated with tumorigenesis in both human and veterinary tumors, and mutations in the β-catenin gene (CTNNB1) are known to promote neoplastic transformation in several human cancers. To investigate the potential involvement of this pathway in FISS, we evaluated β-catenin expression at both the mRNA and protein levels and analyzed the full-length sequence of CTNNB1 gene. Our findings demonstrated increased amounts of β-catenin accumulated in the cytoplasm of formalin-fixed paraffin-embedded FISS tissues and primary FISS cells. Additionally, western blot analysis revealed that β-catenin protein levels of all FISS cell lines were markedly elevated when compared to those of normal feline skin tissue. However, the mRNA expression levels of β-catenin in FISS cells were lower than in normal tissue by using quantitative real-time PCR, suggestive of possible post-transcriptional regulation. Several point mutations, some of which lead to amino acid substitution, in CTNNB1 were identified. One mutation located in the β-TrCP binding domain was the reported hotspot in human cancers and known for enhancing β-catenin stability and oncogenic signaling. To investigate the potential therapeutic potential of MSAB, a β-catenin inhibitor, on FISS, several functional assays were conducted. Our results revealed that MSAB could significantly inhibit cell proliferation, clonogenesis, and cell migration. , These findings support that WNT/β-catenin signaling is dysregulated during FISS development and might be a potential target for drug intervention. On the other hand, we sought to establish a chick embryo chorioallantoic membrane (CAM) model as an alternative in vivo platform to evaluate drug efficacy. This model offers several practical and ethical advantages over traditional rodent models, and may facilitate the screening of novel therapeutic candidates against FISS.