Sonic hedgehog pathway在造釉細胞瘤的致病機轉所扮演的角色

Abstract

按照本團隊之前使用77 例造釉細胞瘤的Sanger 定序研究出來的台灣造釉細胞瘤（ameloblastoma）突變景觀，發現有78% (60/77)BRAF(V600E)的突變，但沒有測到BRAF以外的其他分子(如MAPK路徑的FGFR2(C382R)、KRAS(G12R) 或 SHH 路徑的SMO(L412F 或 W535L) )基因突變熱點的突變。為了釐清Sonic hedgehog（SHH）途徑在其發生機轉中的角色，針對 30 例造釉細胞瘤進行即時反轉錄聚合酶連鎖反應的以測量 SHH pathway下游分子 Gli1 的mRNA表現量，並用4個發炎性囊腫和1個鈣化齒源性囊腫組成的對照組做比較，之後又使用 9 例造釉細胞瘤進行單細胞 RNA 定序，以了解Gli1的高度表達會在造釉細胞瘤細胞可能產生什麼影響。 由Gli1即時反轉錄聚合酶連鎖反應結果發現，雖然在台灣未能偵測到 SHH 路徑的SMO基因突變，但與由根尖囊腫及鈣化牙原性囊腫所組成的陰性對照組相比，造釉細胞瘤中 SHH 路徑終端轉錄效應分子 Gli1 的 mRNA 表現量顯著上升。值得一提的是棘皮樣型造釉細胞瘤（acanthomatous ameloblastoma）中的 Gli1 mRNA 表現量又明顯高於對照組及其他組織學亞型的造釉細胞瘤，顯示 Gli1 mRNA 的表現量與造釉細胞瘤中的角蛋白形成呈正相關。雙重免疫染色結果顯示，Gli1 與 CK17 在造釉細胞瘤腫瘤巢中呈現相鄰（juxtaposition）而非共定位（co-localization），此結果顯示 Gli1 蛋白可能透過旁分泌（paracrine）機制調控成釉細胞瘤中的角蛋白生成。 此外，Gli1 mRNA 表現與腫瘤復發、傳統型（conventional type(solid/multicystic type)）腫瘤及較高的病患年齡具有顯著相關性。在單細胞 RNA 定序的分析中，Gli1 的 mRNA 表現量於非典型（atypical type）造釉細胞瘤(展現出細胞非典型性與增加的有絲分裂活性，但未達明顯惡性的標準)中最高，其次為傳統型（conventional type），最少見於單囊型（unicystic type）。另外，Gli1 mRNA 的高度表現在單細胞層級亦被發現與某些調控增生、遷移、侵襲及幹性(stemness)的基因以及pathway有關。 本研究顯示 Gli1 overexpression 與較具侵襲性之病理特徵及 Gli1-high tumor epithelial cell transcriptomic profile 相關，支持 SHH-related signaling 可能參與造釉細胞瘤進展。然而，其是否為 canonical SHH pathway activation 或 MAPK pathway-driven secondary event，仍需進一步功能性實驗證實。

Based on our previous study of mutational landscape of 77 ameloblastoma cases in Taiwan with Sanger sequencing, we identified a BRAF(V600E) mutation in 78% (60/77) of the tumors, while no hotspot mutations were detected in other genes, including FGFR2 (C382R) and KRAS (G12R) in MAPK pathway, or SMO (L412F or W535L) in Sonic hedgehog (SHH) pathway. To elucidate the role of the SHH pathway in tumorigenesis, we conducted real-time quantitative RT-PCR on 30 ameloblastoma cases to measure the expression of Gli1, the terminal transcription factor of the SHH signaling axis, and compared the results with a control group consisting of four radicular cysts and one calcifying odontogenic cyst. Subsequently, single-cell RNA sequencing was conducted on nine ameloblastoma cases to further investigate the potential effects of elevated Gli1 expression on ameloblastoma cells. Although no SHH pathway mutation can be detected, mRNA overexpression of the transcriptional effector at the terminal end of this pathway- Gli1 in ameloblastoma was noted when compared with the negative control group consisting of radicular cysts and a calcified odontogenic cyst. Notably, obvious up-regulation of Gli1 mRNA were observed in acanthomatous variants of ameloblastomas than in controls and other histological types, indicating a positive correlation between Gli1 expression and keratin formation. Double immunostaining revealed the spatial proximity of Gli1 and CK17, showing they were adjacent rather than co-localized within the tumor epithelial nests. Such findings imply a paracrine signaling axis where Gli1-positive cells influence the keratinization of neighboring tumor cells. . Furthermore, elevated Gli1 expression profiles showed statistically significant associations with recurrent disease status, solid/multicystic (conventional) histopathological type, and advanced patient age. In addition, the Gli1 mRNA expression of single cell level was highest in the atypical type (Presence of cellular atypia and increased mitotic activity, but falls short of frank malignancy), followed by the conventional type, and lowest in the unicystic type of ameloblastoma. Gli1 mRNA overexpression in ameloblastoma is also discovered to be associated with some proliferation, migration and invasion and stemness-related genes and pathways in single cell sequencing assay. This study shows that Gli1 overexpression correlates with aggressive pathological features and a Gli1-high tumor epithelial transcriptomic profile, supporting a role for SHH signaling in ameloblastoma progression. However, further functional assays are needed to clarify whether this reflects canonical SHH activation or a MAPK-driven secondary event.