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挑战。越来越多的证据表明，炎症可能与癌症患者的不良临床预后有关。IL-1b是一种多效性促炎细胞因子，并诱导来自许多细胞的广泛基因组合。我们已经表明，转移性黑色素瘤细胞自发地分泌生物活性IL-1b在缺乏外源性刺激，因为组成型激活IL-1受体（IL-1 R）信号和多蛋白复合物，“炎性体”，表现出“自身炎症”的特征。我们假设来自黑色素瘤细胞的自身炎症有助于肿瘤微环境中的肿瘤生长和进展。我们建议解开的分子（目的1）和生物学（目的2）在人类黑色素瘤细胞的自体炎症机制。在目标3中，我们将研究自身炎症是否与黑色素瘤细胞亚群如癌症干细胞（CSC）相关。具体目标是：具体目标1。分析调节人黑色素瘤自身炎症的分子机制。我们将通过评估目标1.1中炎性小体的作用来描述自身炎症的分子机制。ASC、NLRP 1和NLRP 3将用短发夹RNA从人转移性黑色素瘤细胞系中沉默，并将评估其对分子信号传导和生物学功能的作用。然后，我们将在目标1.2中检查炎性小体非依赖性途径。具体目标2。探讨自身炎症反应在肿瘤微环境中的生物学作用。在目标2.1中，我们将通过将人黑色素瘤组织直接植入小鼠体内，利用直接患者肿瘤异种移植模型。这是人类癌症研究中重现复杂肿瘤微环境的最佳模型之一。此外，将在目标2.2中使用黑色素瘤的基因工程小鼠模型（GEMM），以更好地解决免疫活性同系小鼠中的肿瘤生物学。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以及肿瘤微环境中基质细胞的招募和激活的作用。具体目标3。评估自身炎症是否与黑色素瘤细胞亚群（如CSC）相关。我们报道了转移性黑色素瘤中IL-1b的异质性表达。CSC假说提出肿瘤细胞的亚群负责肿瘤组织的存活和维持生长。最近，我们确定了一个亚群的人黑色素瘤细胞，具有CSC的属性和表达高醛脱氢酶（ALDH）的活动。这些细胞表达独特的基因和IL-1信号网络，表明黑色素瘤自身炎症可能与CSC有关。我们将测量4种潜在CSC和非CSC的IL-1b产生和分泌（目标3.1），并通过在目标3.2中共培养这些群体来评估CSC对非CSC的自身炎症性质。显示自身炎症的亚群将在目标3.3中进一步进行表型和生物学分析。将使用人肿瘤和GEMM肿瘤。迄今为止，对肿瘤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在癌症生物学中起着重要作用，阐明癌症中自身炎症的生物学机制将有助于我们理解肿瘤的发生、进展和复发，并且对于成功治疗黑色素瘤至关重要。它还将导致发现癌症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.