INHBE 在惡性橫紋樣瘤之表達與功能特性：結合臨床樣本與體外模式之研究

Abstract

惡性橫紋樣瘤（malignant rhabdoid tumor，MRT）是一種罕見且致死率極高的兒童癌症，其分化來源尚未確定，通常發生於嬰幼兒。雖然軟組織腫瘤是其主要表現形式，但臟器侵犯也很常見，尤其是腎臟、大腦和肝臟。此類腫瘤屬於高度侵襲性的惡性腫瘤，約有25%的患者於初次診斷時已發生遠端轉移，且需要強效的治療方案，包括多種化療、手術或放療以進行局部控制。雖然治療已使總生存率提高至75%，但治療過程中仍會產生顯著副作用，且轉移與復發的發生率依然較高，治癒率仍然偏低。 MRT的基因標誌是SMARCB1基因在22q11.2染色體區段的雙等位基因失活，這可能包括功能喪失突變、大範圍缺失，或無拷貝數變化的雜合性喪失。SMARCB1也稱為INI1，其所編碼的蛋白質已成為MRT病理診斷的重要依據。SMARCB1是SWI/SNF（又稱BAF）複合體中的核心成分，該複合體在染色質重塑中發揮關鍵作用，能夠鬆解抑制染色質結構，從而促進轉錄機制對DNA的作用。值得注意的是，SMARCB1缺失的狀態與這些肉瘤及癌症中所見的“橫紋樣特徵”有密切關聯。隨著對SMARCB1及其他SWI/SNF複合體成員在各類腫瘤中作用的了解日益加深，這些關鍵因子在對抗人類疾病中的重要性愈發凸顯。 我們先前的研究比較兩種MRT細胞株（其中一組強制表達SMARCB1/INI1，另一組為其原始細胞），RNA定序資料顯示，TGF-β/SMAD、AKT/mTOR以及VEGF訊息傳遞路徑可能在MRT的腫瘤發生中扮演重要角色。INHBE（TGF-β超級家族相關基因）在強制表達SMARCB1/INI1後，表現為下調幅度最大的基因，這與我們先前的ATAC-seq分析結果一致，顯示SMARCB1表達會抑制INHBE啟動子區域的染色質可及性。此外，TGF-β/SMAD訊息傳遞路徑在SMARCB1表達後顯著下調，暗示INHBE過度表達可能與MRT中該路徑的異常活化有關。然而，對於其具體機制的了解仍然不足。其他研究發現，肝癌細胞中INHBE表達下調，而其過度表達則能抑制細胞增殖，暗示其在細胞生長調控中的潛在作用。ATF4作為內質網壓力反應的關鍵轉錄因子，已被證明能特異性誘導INHBE的表達，這顯示INHBE是ATF4的一個重要靶基因。在我們的研究中，ATF4在SMARCB1強制表達後顯著下調，這提示SMARCB1可能藉由調控ATF4間接調控INHBE，而後者可能作為內質網壓力的新標誌物。對於TGF-β/SMAD訊號路徑在MRT中的具體角色，文獻尚不多，這一發現為進一步探索提供了契機。 本研究主要分為兩個部分。首先，我們利用G-402細胞株，通過Lentivirus載體分別含有INHBE的shRNA和強制表達SMARCB1/INI1的病毒，進行表達調控以建立實驗模型，並且以Western blot和qRT-PCR實驗確認該模型可用。接下來，我們在SMARCB1表達的MRT細胞、原始MRT細胞及shRNA介導INHBE表達降低的細胞分別使用tunicamycin誘導內質網壓力，並通過XBP1剪接及關鍵因子（如ATF4、ATF6、ATF3、DDIT3/CHOP、BIP）的即時RT-PCR定量分析，預測INHBE或SMARCB1表達是否與內質網壓力反應有關。最後，我們利用此模型，比較不同細胞對TGF-β/SMAD訊息傳遞路徑中重要標誌物的表現，並使用MTT測定法和傷口癒合實驗（wound healing assay）進行細胞差異的驗證。

Malignant rhabdoid tumor (MRT) is a rare, aggressive pediatric cancer of uncertain origin, primarily affecting infants and young children. Besides soft tissue, it frequently involves visceral organs such as the kidneys, brain, and liver. Despite multimodal therapy (chemotherapy, surgery, radiotherapy), high rates of metastasis and recurrence keep cure rates low and treatment toxicities high. The defining molecular hallmark of MRT is biallelic inactivation of the SMARCB1/INI1 gene, a core component of the SWI/SNF chromatin remodeling complex. Loss of SMARCB1 disrupts transcriptional regulation and is strongly associated with rhabdoid morphology. Increasing evidence implicates SWI/SNF complex dysregulation in a broad range of cancers. Our laboratory previously compared RNA sequencing profiles of MRT cell lines with and without SMARCB1/INI1 expression and identified multiple altered signaling pathways, including TGF-β/SMAD, AKT/mTOR, and VEGF. Notably, the TGF-β family member INHBE was markedly downregulated upon SMARCB1 restoration. ATAC-seq further revealed decreased chromatin accessibility at the INHBE promoter region, indicating that INI1 may directly regulate INHBE expression. Consistent with this, TGF-β/SMAD signaling was also suppressed in these cells, suggesting that the downregulation of INHBE may contribute to the aberrant activity of this pathway in MRT. We also investigated the relationship between INI1, INHBE, and the endoplasmic reticulum stress response. ATF4, a key transcription factor in ER stress, can induce INHBE expression; we observed that INI1 expression and INHBE knockdown produced distinct outcomes in ER stress responses, suggesting that INHBE may serve as a novel ER stress–responsive biomarker in MRT. To investigate these findings, we established a G-402 cell model with lentiviral SMARCB1/INI1 expression or INHBE knockdown. We validated gene modulation by Western blotting and qRT-PCR, then challenged cells with tunicamycin, an ER stress inducer. We measured XBP1 splicing and expression of ATF4, ATF6, ATF3, DDIT3/CHOP, and BIP to evaluate ER stress responses. Finally, we assessed TGF-β/SMAD signaling markers, cell viability (MTT assay), and migration (wound-healing assay) under different genetic conditions. Our data reveal distinct regulatory roles of SMARCB1 and INHBE in MRT, linking chromatin remodeling to TGF-β/SMAD signaling and ER stress responses. These findings provide a framework for further studies of SMARCB1-mediated pathways and may identify INHBE as a novel biomarker or therapeutic target in MRT.