Autoinflammation in Human Melanoma

描述(由申请人提供)： 黑色素瘤仍然是临床肿瘤学的一个挑战。越来越多的证据表明，炎症可能与癌症患者不良的临床预后有关。IL-1b是一种多效性促炎细胞因子，可从多种细胞诱导广泛的基因组合。我们已经证明，转移性黑色素瘤细胞在没有外源性刺激的情况下会自发地分泌生物活性的IL-1b，这是因为IL-1受体(IL-1R)信号的结构性激活和一个多蛋白复合体--炎症体，呈现出一种“自体炎症”的特征。我们假设黑色素瘤细胞的自体炎症在肿瘤微环境中促进了肿瘤的生长和进展。我们建议揭开人类黑色素瘤细胞自身炎症的分子(目标1)和生物学(目标2)机制。在目标3中，我们将调查自身炎症是否与黑色素瘤细胞亚群相关，例如癌症干细胞(CSCs)。具体目的有：1.分析人黑色素瘤自身炎症的分子调控机制。我们将通过评估炎症小体在AIM 1.1中的作用来描述自体炎症的分子机制。ASC、NLRP1和NLRP3将从具有短发夹状RNA的人转移性黑色素瘤细胞系中沉默，并将评估其在分子信号转导和生物学功能中的作用。然后，我们将在AIM 1.2中研究非炎症体依赖途径。具体目的2.探讨自身炎症在肿瘤微环境中的生物学作用。在Aim 2.1中，我们将利用直接患者肿瘤异种移植模型，将人黑色素瘤组织直接移植到小鼠体内。这是人类癌症研究中概括复杂肿瘤微环境的最佳模型之一。此外，AIM 2.2将使用黑色素瘤的基因工程小鼠模型(GEMM)，以更好地解决免疫能力同基因小鼠的肿瘤生物学问题。我们将研究黑色素瘤来源的IL-1b和自身炎症对黑色素瘤细胞存活和肿瘤微环境中基质细胞的招募和激活的作用。具体目的3.评估自身炎症是否与黑色素瘤细胞亚群(如CSCs)相关。我们报道了IL-1b在转移性黑色素瘤中的异质性表达。CSC假说提出，肿瘤细胞亚群负责肿瘤组织的存活和生长。最近，我们鉴定了一种具有CSC特性并表达高醛脱氢酶(ALDH)活性的人黑色素瘤细胞亚群。这些细胞表达独特的基因和IL-1信号网络，提示黑色素瘤自身炎症可能与CSCs有关。我们将测量4个潜在的CSCs和非CSCs(目标3.1)的IL-1b的产生和分泌，并通过在Aim 3.2中共同培养这些群体来评估CSCs在非CSCs上的自炎性质。在Aim 3.3中，将对表现为自身炎症的亚群进行进一步的表型和生物学分析。人类肿瘤和GEMM肿瘤都将被使用。到目前为止，对肿瘤细胞等非免疫细胞的炎性小体和自身炎症的机制和作用的研究还很少。由于IL-1b在肿瘤生物学中发挥着重要作用，阐明肿瘤自身炎症的生物学机制将有助于我们理解肿瘤的发生、发展和复发，并对成功治疗黑色素瘤至关重要。它还将导致发现抗癌药物开发的新分子靶点。

DESCRIPTION (provided by applicant): Melanoma remains a challenge in clinical oncology. Evidence increasingly suggests that inflammation can be associated with unfavorable clinical prognosis in cancer patients. IL-1b is a pleiotropic pro-inflammatory cytokine and induces a broad portfolio of genes from many cells. We have shown that metastatic melanoma cells spontaneously secrete biologically active IL-1b in the absence of exogenous stimuli because of constitutive activation of IL-1 receptor (IL-1R) signaling and a multi-protein complex, "inflammasome", exhibiting a feature of "auto-inflammation". We hypothesize that auto-inflammation from melanoma cells contributes to tumor growth and progression in tumor microenvironment. We propose to unravel the molecular (in Aim 1) and biological (in Aim 2) mechanisms of auto-inflammation in human melanoma cells. In Aim 3, we will investigate if auto-inflammation is associated with a subpopulation of melanoma cells such as cancer stem cells (CSCs). The specific aims are: Specific Aim 1. To analyze molecular mechanisms regulating auto-inflammation in human melanoma. We will delineate molecular mechanisms of auto-inflammation by assessing the role of inflammasomes in Aim 1.1. ASC, NLRP1 and NLRP3 will be silenced from human metastatic melanoma cell lines with short hairpin RNA and its role on molecular signaling and biological function will be assessed. We will then examine inflammasome-independent pathway in Aim 1.2. Specific Aim 2. To examine the biological role of auto-inflammation in tumor microenvironment. We will utilize a direct patient tumor xenograft model by implanting human melanoma tissues directly into mice in Aim 2.1. This is one of the best models in human cancer research that recapitulate complex tumor microenvironment. In addition, a genetically engineered mouse model (GEMM) of melanoma will be used in Aim 2.2 to better address tumor biology in immunocompetent syngeneic mice. We will examine the role of melanoma-derived IL-1b and auto-inflammation on survival of melanoma cells and recruiting and activating stromal cells in tumor microenvironment. Specific Aim 3. To assess if auto-inflammation is associated with a subpopulation of melanoma cells such as CSCs. We reported heterogeneous IL-1b expression in metastatic melanoma tumors. The CSC hypothesis proposes that a subpopulation of tumor cells is responsible for survival and maintaining of the growth of neoplastic tissue. Recently, we identified a subpopulation of human melanoma cells that possess CSC properties and express high aldehyde dehydrogenase (ALDH) activity. These cells express unique genes and IL-1 signaling network, suggesting that melanoma auto-inflammation may be associated with CSCs. We will measure IL-1b production and secretion from 4 potential CSCs and non-CSCs (Aim 3.1) and assess autoinflammatory nature of CSCs on non-CSCs by co-culturing these populations in Aim 3.2. The subpopulation that shows auto-inflammation will be further analyzed phenotypically and biologically in Aim 3.3. Both human tumors and GEMM tumors will be used. Until now, little has been studied on the mechanisms and roles of inflammasome and auto-inflammation from non-immunological cells such as tumor cells. Since IL-1b plays an important role in cancer biology, elucidating biological mechanisms of auto-inflammation in cancer will help our understanding of tumor initiation, progression and recurrence and is critical for a successful treatment against melanoma. It will also lead to the discovery of novel molecular targets for cancer drug development.