血管內皮細胞在腫瘤微環境中促進透明腎細胞癌形成之研究;Study of the active role of vascular endothelial cells in the initiation and progression of clear-cell renal cell carcinoma

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Title:	血管內皮細胞在腫瘤微環境中促進透明腎細胞癌形成之研究;Study of the active role of vascular endothelial cells in the initiation and progression of clear-cell renal cell carcinoma
Authors:	阮陳孝輝;Hieu-Huy, Nguyen-Tran
Contributors:	系統生物與生物資訊研究所
Keywords:	腎細胞癌;von Hippel-Lindal;血管內皮細胞;抑癌蛋白M;腫瘤微環境;clear-cell renal cell carcinoma;von Hippel-Lindau;Endothelial cells;oncostatin M;Tumor microenvironment
Date:	2021-12-14
Issue Date:	2022-07-13 18:52:33 (UTC+8)
Publisher:	國立中央大學
Abstract:	腎細胞癌 (Renal cell carcinoma, RCC) 是十大癌症之一，占腎癌的 90% 以上。其中，透明腎細胞癌 (clear-cell RCC, ccRCC) 是最為常見的一種形態，占所有 RCC 病例的 75% 以上。ccRCC組織具有豐富的血管及免疫細胞。其發生率跟 von Hippel-Lindal (VHL)抑癌基因的突變有極高的相關性。 VHL蛋白是一個 E3 泛素連接酶，在氧氣充足時辨識缺氧誘導因子α(HIF-α)，將其破壞；突變後其抑制功能缺失，導致HIF-α在細胞中累積。然而，目前尚未清楚VHL基因的突變，是如何影響組織發炎和腫瘤發生。血管內皮細胞 (Endothelial cells,ECs) 是基質細胞的一種，也是每個器官不可少的一部分。傳統上，EC 被認為是一種將血液與組織分開的惰性隔離膜。然而，近來越來越多的證據表明，EC是一個非常活躍的器官，它積極參與多數疾病，尤其是發炎相關的疾病。然而，EC是如何參與發炎反應、從而誘發腫瘤的發生並不清楚。同時 VHL突變是否在這些病理生理的相互作用中所扮演的協調角色也尚未了解。在實驗室先前在腎小管中敲除Vhlh基因的小鼠模型 (VhlhKO)中發現被敲除Vhlh的腎小管除了有細胞增生和透明細胞的出現外，還引起嚴重的炎症和纖維化。該結果表明 VHL 的剔除可誘發慢性炎症，這可能是 ccRCC 發展的早期表現。我們意外地發現，在 Vhlh 基因敲除的組織中雖然EC沒有發生任何基因編輯但磷酸化c-Jun N末端激酶的表現量明顯增加。這發現給我們一個啟發，EC在炎症誘發腫瘤形成過程中可能會扮演重要的角色。此外，這些EC還與腫瘤血管具有相同的特徵，包括新血管生成及血管滲漏。在轉錄體研究中，從 VhlhKO組織來的 EC基因表現與對照組有很大的差異。這些有差異的基因主要是參與發炎反應以及間質轉化(mesenchymal transition)。重要的是，我們證明oncostatin M (OSM)是VHL突變的上皮細胞與血管內皮細胞之間的主要傳導物。OSM的表現在VHL缺陷的上皮細胞中增加，從而引發EC裡的OSMR增加，進而形成一個自我延續的積極信號循環。在體外實驗中，OSM 引發EC的活化和內皮間質細胞轉化（endothelial-mesenchymal transition, EndoMT），導致血管的通透性及腎臟癌細胞侵入穿透數量增加。被OSM激活過的ECs會誘導巨噬細胞變成M2/TAM形態。在HK-2正常腎臟細胞株中敲低 VHL 後會引發OSM表現。在上皮細胞和內皮細胞的共同培養系統中，被敲低 VHL的HK-2引發EC活化。在EC敲落OSMR 或在培養液中中和OSM可以衰減VHL缺陷的在動物實驗中，敲除 Osmr來阻擋 EC中的Osm 訊息傳導可以改善EC的表型包括降低血管增生、釋放E-Selectin、血管滲露以及腫瘤血管相關的形態。同時，可以改善VhlhKO小鼠的ccRCC相關表型包括降低發炎病灶，纖維化，細胞增生及巨噬細胞浸透。在藥理治療中，Janus激酶抑製劑Tofacitini可以顯著減輕VhlhKO組織中的EC活化指數和炎症表型，與基因改造的結果一致。最後，Vhlh突變微環境可以增強外源性腫瘤的轉移，包括小鼠黑色素瘤 (B16) 和人類 ccRCC (786-O) 細胞。因此，OSM 訊息傳導建構了VHL 缺陷腎小管細胞的發炎及致癌微環境，在 ccRCC的發生和轉移過程中扮演重要角色。我們的研究結果揭秘了EC在炎症相關癌症，並且指出OSM訊息傳導可用於預防、早發治療和ccRCC轉移治療的新標靶。 ;Renal cell carcinoma (RCC) is among the top ten cancers worldwide and accounts for >90% of cancers in kidney. Among them, clear-cell RCC (ccRCC) is the most common histological subtype accounting for more than 75% of all RCC cases. ccRCC is a highly vascularized and immunogenic tissue that is closely associated with inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene (found in ~80% of ccRCC patients). The VHL protein has been well-recognized as a key regulator of hypoxic response. However, it is still unclear how mutations in VHL, encoding the substrate-recognition subunit of an E3 ubiquitin ligase that targets the alpha subunit of hypoxia-inducible factor (HIF-α), can coordinate tissue inflammation and tumorigenesis. Endothelial cells (ECs) are a constituent of stromal cells, which are indispensable for every organ. ECs have traditionally been considered as a non-reactive barrier that separates blood from tissues. However, recently accumulating evidence has illustrated that ECs constitute a dynamic organ that actively participates in most if not all of the disease states, particularly in inflammatory response. Yet, how these ECs are involved in inflammatory regulation and tumorigenesis is less well documented, and whether VHL plays a role in coordinating these pathophysiological interactions is also unclear. Previously we generated a conditional mouse Vhlh gene knockout in kidney tubules (VhlhKO), which results in severe inflammation and fibrosis in addition to hyperplasia and appearance of transformed clear cells. These results imply that inactivation of VHL can induce chronic inflammation, which may be an early step in ccRCC development. Interestingly, we found that endothelial cells in Vhlh knockout kidney, although not harboring any genetic modifications, show an enrichment of phosphorylated c-Jun N-terminal kinase (p-Jnk)–an inflammatory marker–that suggested a role in inflammation induced tumorigenesis. In addition, these ECs in Vhlh knockout mice show abnormal phenotypes resembling tumor ECs, including neoangiogenesis and leakage. In the transcriptome study of these ECs, we found a profound change in gene expression program of ECs from VhlhKO tissue compared with those in wild-type mice (WT). These genes with altered expression are mainly involved in inflammatory response and mesenchymal transition. Importantly, we demonstrate that oncostatin M (OSM) is a major regulator that mediates the interaction between VHL mutant kidney tubule cells and the ECs. The expression of OSM is upregulated in VHL-deficient epithelial cells and can induce overexpression of OSMR in ECs, thus forming a self-perpetuating positive signaling loop. In vitro, OSM induces EC activation and endothelial-mesenchymal transition (EndoMT) that leads to increase of vascular permeability and the number of invaded kidney cancer cells. These OSM-activated ECs also induce macrophage polarization toward M2/TAM like phenotype. Knockdown of VHL in renal tubule cells (HK-2) results in upregulation of OSM. In coculture system, we found that HK-2 with VHL knockdown could induce activation of EC, which could be recused by attenuating OSM pathway in ECs by knockdown of OSMR receptor or neutralizing OSM in cocultured media. In vivo, attenuating OSM pathway in ECs by generating Osmr knockout in VhlhKO tissue (DBKO tissue) could recuse the abnormal phenotypes of ECs in VhlhKO tissue including reduced angiogenesis (CD31+ cells number), secreted E-Selectin (sSELE; marker of EC activation), vascular leakage and tumor-endothelial-cells related morphologies. In addition, DBKO also recuses the phenotypes of VhlhKO mice that are related to ccRCC, including inflammatory foci, fibrosis, proliferation index (Ki-67+ cells) and macrophage infiltration (F4/80+ cells). Consistent with the results in genetic modification, in the pharmacological treatment, tofacitinib (a Janus kinase inhibitor) could markedly alleviate the activated EC index and inflammatory phenotype of VhlhKO tissue. Finally, the Vhlh mutant microenvironment can enhance metastasis of exogenous tumors, both of the mouse melanoma (B16) and human ccRCC (786-O) cells. Thus, OSM signaling initiates reconstitution of inflammatory and tumorigenic microenvironment of VHL-deficient renal tubule cells, which play a critical role in ccRCC initiation and metastasis. Our results provide an insight regarding ECs in inflammation related cancer, and suggest OSM pathway as a novel target for prevention and treatment of early-onset and metastasis of ccRCC.
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