探討發炎與缺氧微環境之交互作用與其對腫瘤發生之影響

Studies on the potential crosstalk between inflammatory and hypoxic microenvironment linking to cancer development

葉彥秀(Yen-Hsiu Yeh)

指導教授 ： 李財坤

國立臺灣大學/醫學院/微生物學研究所/博士(2014年)

https://doi.org/10.6342/NTU.2014.02434

摘要

2013年國人十大死因之首，惡性腫瘤（癌症），連續三十一年蟬聯冠軍。癌症已成為致死率最高的疾病，因此，全世界許多科學研究者致力於探討癌症發生與進程的可能分子機制。其中，慢性發炎（chronic inflammation）更是近年來，被許多分子及病理學證實，可藉由改變宿主的免疫機轉而影響癌化的進行。慢性發炎，有如缺氧微環境，除被認為是一關鍵癌化的微環境因素外，更是人體內的沉默殺手，可導致心臟病、腦中風等慢性疾病的生成。本論文探討不同的微環境的交互作用與對腫瘤進程的的影響分為兩部分：（一）干擾素刺激基因15（interferon-stimulated gene15，ISG15）和泛素（ubiquitin）結合路徑在缺氧和發炎所營造的發炎缺氧微環境（inflammatory hypoxia）扮演重要的角色。為了探究兩者癌症環境間的交互作用，我們研究ISG15對影響缺氧誘導因子（hypoxia- inducible factor-1α，HIF-1α）的活性影響。利用Interferon和模擬缺氧藥物desferoxamine的刺激，誘發ISG15和HIF-1α的產生，發現干擾素刺激基因15（ISG15）可藉由蛋白質後修飾作用來影響HIF-1α的功能性。探討其中的機制，我們發現HIF-1α不僅和ISG15有物理性的交互作用，也是同時亦是干擾素引發的蛋白質後修飾作用(ISGylation) 的受質蛋白 (Substrate)，在多處區域有修飾點。過度表現ISG15瓦解HIF-1α和HIF-1β的偶合作用（dimerization）。緊接著，HIF-1α引發的基因表現和癌症生長也會被ISG15和ISGylation所弱化。綜合以上結果，一個全新的缺氧負回饋機制被我們提出，干擾素可透過對HIF-1α進行ISGylation達成對缺氧的微環境調節。（二）延續第一部分的發現，我們探討慢性發炎和癌症發生的相關性。多方的證據顯示，癌細胞可藉由駕馭訊息傳遞路徑在艱困微環境下適應生存。HIF-1α在匯集發炎和缺氧通往癌症的調控網域上扮演一個重要角色，因此HIF-1α在許多癌症都有過度表現的現象。利用抗病毒的interferon模擬慢性發炎環境，觀察到干擾素向上調控細胞中HIF-1α的表現。並探究背後機制及其生理意義。在769-P腎臟癌細胞中，我們發現HIF-1α路徑可被發炎干擾素所調控，而誘發的HIF-1α累積是透過PI3K/AKT/mTOR/GSK3β路徑。為了確認干擾素誘發HIF-1α產生的可能機制，一系列藥理學方法被應用來揭開其中所蘊含的調控機制。結果顯示，干擾素也藉由促進HIF-1α的轉譯作用增加其表現量。更重要的是，干擾素藉由誘發的HIF-1α來促進上皮細胞中胚轉化（epithelial-mesenchymal transition，EMT）和細胞侵襲性。同樣的，利用RNA interference干擾方式壓制干擾素所誘發之HIF-1α。綜合以上結果，干擾素在有些細胞可以促進缺氧誘導因子表現，並且透PI3K/AKT/mTOR/GSK3β路徑影響上皮細胞中胚轉化（epithelial-mesenchymal transition，EMT）的發生。此為第一次報導干擾素可經由誘發缺氧誘導因子累積，進而影響癌症發生，可提供臨床上，癌症治療策略使用的考慮因素。

關鍵字

發炎 ； 缺氧 ； 干擾素 ； 干擾素刺激基因15 ； 缺氧誘導因子

並列摘要

Because cancer has become the highest mortality among diseases, researchers around the world are committed to uncover the potential tumorigenic mechanisms and related intervention of concurring cancer. In recent years, inflammation has regarded as the enhancing characteristics of cancer hallmarks and been proven to contribute to tumor initiation and progression. Inflammation, like hypoxia microenvironment, is also a critical factor for a variety of diseases such as heart disease, stroke and diabetes. Here, we have investigated interactions between microenvironments and relation with cancer development. Thus, my study has divided into two major parts. [I] We found that ISG15 (interferon-stimulated gene 15) modulates hypoxia-inducible factor-1α (HIF-1α) functions. ISG15 conjugation (ISGylation) and ubiquitylation systems play critical roles in hypoxic inflammation. Interferon and hypoxia-mimetic desferoxamine were used to induce ISG15 and HIF-1α expression respectively and to study effects of ISG15 on the HIF-1α activity. We observed free-form HIF-1α is regulated by interferon, and expression of ISG15 is lower in the hypoxic state. Further mechanistic investigation reveals HIF-1α not only physically interacts with ISG15 but also is a substrate for ISGylation at multiple sites. Overexpression of ISG15 disrupted HIF-1α/HIF-1β dimerization and subsequently HIF-1α-induced gene expression and tumor growth in xenograft mouse models were attenuated by ISG15 and ISGylation expression. Based on the above results, we concluded and proposed a novel negative feedback mechanism of hypoxia where ISG15 regulates HIF-1α via ISGylation. [II] Accumulating evidence demonstrate that tumor cells highjack signaling pathways to exploit the machinery for tumor microenvironments and to adapt and help survival under stress environments. Among these regulatory networks, HIF-1α plays a crucial role linking hypoxia and inflammation to cancer, thus elevated expression of HIFs are often observed in tumors. Here, we have found that the HIF-1α pathway is modulated by the inflammatory cytokine IFN-α in 769-P renal cancer cell and our mechanistic study has determined the involvement of PI3K/AKT/mTOR/GSK3β pathways in the IFN-induced HIF-1α accumulation. HIF-1α expression was elevated in a time- and concentration-dependent manner by IFN-α and requires transcription. Moreover, cells were exposed to a serial of pharmacological inhibitors to investigate their effects on the IFN-mediated HIF-1α accumulation and revealing that the de novo synthesis is also involved in the IFN-induced HIF-1α. Importantly, IFN-α also triggers epithelial-to-mesenchymal transition (EMT) via up-regulation of HIF-1α as evident by the fact that IFN-α-induced EMT is attenuated by silencing of HIF-1α expression. To our knowledge, the above results demonstrate for the 1st report that IFN-α is a contributing factor for cancer development through modulating of HIF-1α expression and thus providing considerable strategy in clinical for tumor-therapy with IFN-α.

並列關鍵字

Inflammation ； hypoxia ； interferon ； ISG15 ； HIF-1α

參考文獻

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