Genetically Engineered Mouse Models to Study RTK Function in Melanoma

用于研究黑色素瘤 RTK 功能的基因工程小鼠模型

• 批准号: 8937664
• 负责人: GLENN T. MERLINO
• 金额: $ 118.73万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8937664
• 关键词: Address; Adult; Affect; Agar; Anabolism; Anchorage-Independent Growth; Animal Cancer Model; Architecture; Autophagocytosis; BRAF gene; Behavior; Burn injury; CDKN2A gene; Cancer Biology; Cancer Model; Cataloging; Catalogs; Cells; Childhood; Clinic; Collaborations; Communities; Complex; Cutaneous Melanoma; Cyclic AMP; DNA; DOPA decarboxylase; Data; Databases; Development; Differentiation Therapy; Disease; Dopamine; Dose; Drug Targeting; Drug resistance; Ectopic Expression; Embryo; Epidemiology; Excision; Exhibits; Fluorescence-Activated Cell Sorting; Gene Expression; Gene Expression Profiling; Genes; Genetic Engineering; Genetic Programming; Genetically Engineered Mouse; Genomics; Goals; Green Fluorescent Proteins; Growth; Guanine; Guanine Nucleotide Exchange Factors; Health; Health Hazards; Hepatocyte Growth Factor; Human; Immune; Immunocompetent; In Vitro; Incidence; Inflammatory; Influentials; Injection of therapeutic agent; Interferon Type II; Interferon-alpha; Invaded; Label; Laboratories; Lesion; Libraries; Ligands; Link; Malignant - descriptor; Malignant Neoplasms; Melanins; Melanocyte stimulating hormone; Melanocytic Neoplasm; Melanoma Cell; Metastatic Melanoma; MicroRNAs; Modeling; Molecular; Molecular Genetics; Molecular Profiling; Molecular Target; Morphology; Mus; Mutagenesis; Mutation; Myeloid Cells; Nature; Neonatal; Neoplasm Metastasis; Oncogenes; Oncogenic; Pathway interactions; Patients; Pattern; Pharmaceutical Preparations; Phenotype; Pigments; Primary Neoplasm; Process; Production; Program Development; Property; RNA Sequences; Radiation induced damage; Receptor Protein-Tyrosine Kinases; Recruitment Activity; Recurrent disease; Relative (related person); Relative Risks; Research; Resistance; Risk; Risk Factors; Role; Signal Pathway; Signal Transduction; Site; Skin; Skin tanning; Small Interfering RNA; Source; Staging; Stress; Sunburn; Sunscreening Agents; Tail; Testing; Tetracyclines; Therapeutic; Time; Tissues; Transgenic Mice; Transplantation; Tumor Suppressor Proteins; UV Radiation Exposure; UVB induced; Ultraviolet A radiation; Ultraviolet B Radiation; Ultraviolet Rays; Veins; Work; base; benign state; cancer genetics; clinically relevant; design; drug candidate; effective therapy; exome sequencing; falls; high throughput screening; human disease; in vivo; in vivo Model; inhibitor/antagonist; insight; interest; irradiation; knock-down; leukemia; macrophage; melanoblast; melanocyte; melanoma; member; mouse model; mutant; new therapeutic target; next generation sequencing; novel; overexpression; oxidative DNA damage; pre-clinical; preclinical study; prevent; relating to nervous system; success; therapeutic target; tumor; tumor initiation; tumorigenic

The Cancer Modeling Section seeks to elucidate the complex molecular/genetic program governing tumor genesis and progression through the development and analysis of genetically engineered mouse models of human cancer. Our efforts in this regard are focused primarily on cutaneous malignant melanoma. Exposure to ultraviolet radiation (UV) is a causal agent in the vast majority of melanoma. Previously, we tested this hypothesis in transgenic mice in which the receptor tyrosine kinase MET was deregulated by virtue of ectopic expression of its ligand, hepatocyte growth factor/scatter factor (HGF). We discovered that a single neonatal dose of burning UV in these mice was necessary and sufficient to induce tumors reminiscent of human melanoma with shortened latency (Noonan et al., Nature 413: 271-2, 2001). A critical role for the INK4A/ARF locus, which helps regulate the pRb and p53 pathways and is widely regarded as a key melanoma suppressor in human patients, was also confirmed in our animal model (Recio et al., Cancer Res. 62: 6724-30, 2002). There has been controversy surrounding the relative risks associated with UVB versus UVA radiation. We used albino HGF transgenic mouse to show that UVB, but not UVA, alone is able to induce the full melanoma phenotype in the absence of pigment (DeFabo et al., Cancer Res. 64: 6372-6, 2004). However, we more recently showed that UVA is highly melanomagenic in pigmented HGF/SF-transgenic mice (Noonan et al., Nat. Commun. 3: 884, 2012), demonstrating that melanin is associated with oxidative DNA damage and mutagenesis, and thus represents a double-edged sword with respect to melanoma risk. Our work suggests that indoor tanning, which is mostly UVA-based, could be a significant health risk. We have also employed in vitro and in vivo models based on genetically engineered melanocytes to identify novel regulators of differentiation, malignancy and metastasis. For example, we compared the oncogenic roles of the three major NRAS downstream effectors, RAF, PI3K and RAL guanine exchange factor (RalGEF) (Mishra et al., Oncogene 29:2249-2256, 2010). Although no single downstream pathway could recapitulate all the consequences of oncogenic NRAS, we discovered that constitutive RalGEF activation stimulated anchorage-independent growth, indicating that this often overlooked pathway should be evaluated as a possible therapeutic target. We have, in collaboration with Jim McMahon and Curt Henrich (Molecular Targets Development Program) also begun re-exploring the prospects of "differentiation therapy". INK4A/ARF-deficient melanocytes transformed by mutant NRAS were used in a high-throughput screen of the LOPAC drug library to identify agents that would induce re-differentiation of malignant melanoma cells, thus converting them to a more benign state. Eight candidate drugs were identified that induce a differentiated morphology. Interestingly, several of these candidates were related to dopaminergic signaling, including inhibitors of DOPA decarboxylase (DDC), which we found to be upregulated in melanomas. Dopamine not only controls the biosynthesis and secretion of melanotropins, but also helps regulate the activity of cAMP, which is also linked to melanin production. The differentiating activity of these compounds was confirmed in mouse and human melanoma cells. Notably, we found that several of these compounds effectively reduced the ability of melanoma cells to invade and grow in soft agar as well. The data suggest that re-differentiating drugs can also have anti-tumorigenic properties. Although an extensive accumulation of epidemiological evidence supports a fundamental role for UV in melanoma, the specific UV-affected molecular pathways and mechanisms remain largely unidentified. We have suggested that mechanisms other than UV-induced DNA mutagenesis may also be important in melanoma initiation. To determine the role(s) of UV in melanoma in vivo, we developed a mouse model (iDCT-GFP) that allowsmelanocytes, specifically and inducibly labeled with green fluorescent protein (GFP), to be highly purified from disaggregated mouse skin by FACS following UV irradiation in vivo. We identified a pattern of UVB induced gene expression changes in melanocytes isolated from mice that are consistent with inflammatory alterations and may spare melanocytes post-UV remodeling-associated destruction. We have identified an interferon (IFN)-gamma signaling signature arising in melanocytes after neonatal UV irradiation. The source was a type of macrophage recruited to the skin after UV exposure; IFN-gamma in turn activated melanocytes and the expression of genes that could facilitate immunoevasion. Transplanted neonatal macrophages were found to significantly enhance melanoma growth in vivo in an IFN-gamma-dependent fashion. This was surprising considering that IFN-alpha has been used to treat melanoma patients, albeit with limited success. We hypothesized that melanomas escape immune destruction by co-opting these pathways already hard-wired in melanocytes, and suggested that the IFN-gamma signaling pathway may represent a promising therapeutic target for melanoma patients (Zaidi et al., Nature 469:548-553, 2011). To address the UV mutation question we are also subjecting in vivo-exposed melanocytes to whole exome and RNA sequencing. We are isolating GFP-labeled melanocytes from all stages of melanoma development relevant to human disease in an attempt to catalog their precise genomic alterations. We anticipate that this in vivo model will provide novel insights into the nature of UV-induced damage, and the mechanisms by which UV provokes melanoma. We have also hypothesized that late stage melanoma cells can co-opt pathways hard-wired into normal developing melanoblasts to achieve a more aggressive and metastatic phenotype. Both the embryonic melanoblast and the metastatic melanoma cell must undergo a similar EMT and become invasive, highly migratory, and survive to colonize at a remote sites. We again employed our iDCT-GFP model to isolate embryonic melanoblasts from key stages of melanocyte development. RNA sequencing and microarray-based gene expression profiling have been performed from representative developmental stages. Genes have been identified whose expression is characteristically up-regulated (or down-regulated) in both melanoblasts and metastatic melanoma relative to adult melanocytes, which may represent new therapeutic targets against metastasis in melanoma. Novel candidates, which include genes that regulate ER stress, autophagy, and neural developoment have been evaluated for a role in metastasis using siRNA knockdown in metastatic human and mouse melanoma cells and tail vein injections. Several candidates have now been shown to regulate metastatic behavior, and to correlate with melanoma patient survival. These are being vigorously pursued. Finally, we integrated gene and microRNA expression data from our mouse models of highly and poorly malignant melanocytic tumors, and human melanoma databases, and discovered an important role for pathways centered on a new tumor suppressor, miR-32. Malignant tumors frequently exhibited poor expression of miR-32, the targets for which included NRAS, PI3K and notably, myeloid cell leukemia 1 (MCL1). MCL1 was highly expressed in melanomas, and when knocked down diminished oncogenic potential. MCL1 overexpression transformed immortalized primary mouse melanocytes, but only those expressing activating mutations in BRAF, CRAF or PI3K. The MCL1-specific antagonist sabutoclax was effective as a single agent, and acted synergistically in combination with vemurafenib in preclinical melanoma models. Our data showed that miR-32/MCL1 pathway members are novel candidate anti-melanoma drug targets, and suggested that their inhibition may enhance the efficacy of BRAFV600E inhibitors in the clinic.

癌症建模部分旨在通过开发和分析人类癌症的基因工程小鼠模型来阐明控制肿瘤起源和进展的复杂分子/遗传程序。我们在这方面的努力主要集中于皮肤恶性黑色素瘤。暴露于紫外线辐射（UV）是绝大多数黑色素瘤的因果剂。以前，我们在转基因小鼠中检验了这一假设，其中通过其配体的异位表达，肝细胞生长因子/散射因子（HGF）的异位表达对受体酪氨酸激酶进行了调节。我们发现，这些小鼠中的单一新生剂量燃烧的紫外线是必要的，足以诱导肿瘤，让人联想到潜伏期的肿瘤（Noonan等人，自然413：271-2，2001）。在我们的动物模型中也证实了INK4A/ARF基因座的关键作用，该INK4A/ARF基因座有助于调节PRB和p53途径，并被广泛认为是人类患者的关键黑色素瘤抑制剂（Recio等人，CancerRes。62：6724-30，2002）。与UVB与UVA辐射相关的相对风险存在争议。我们使用白化病HGF转基因小鼠来表明UVB（而不是UVA）仅在没有色素的情况下就能诱导全黑色素瘤表型（Defabo等，CancerRes。64：6372-6，2004）。但是，我们最近表明，UVA在色素色素的HGF/SF-转基因小鼠中具有高度黑色素的毒素（Noonan等，Nat。Commun。3：884，2012），表明黑色素与氧化性DNA损伤和诱变有关，因此代表了与糖浆瘤风险的双层剑相关的。我们的工作表明，主要基于UVA的室内晒黑可能是重大的健康风险。我们还基于基于基因工程的黑素细胞的体外和体内模型来识别分化，恶性和转移的新型调节剂。例如，我们比较了三个主要NRAS下游效应子RAF，PI3K和Ral鸟嘌呤交换因子（Ralgef）的致癌作用（Mishra等人，Oncogene 29：2249-2256，2010）。尽管没有单个下游途径可以概括致癌NRA的所有后果，但我们发现本构型ralgef激活刺激了锚固非依赖性的生长，这表明应将这种经常被忽略的途径评估为可能的治疗靶标。我们与吉姆·麦克马洪（Jim McMahon）和库特·亨利希（Curt Henrich）合作（分子目标开发计划）也开始重新探索“分化治疗”的前景。在Lopac药物文库的高通量屏幕中使用了INK4A/ARF缺陷型黑素细胞，用于鉴定会诱导恶性黑色素瘤细胞重新分化的药物，从而将其转化为更良性的状态。鉴定出八种诱导分化形态的候选药物。有趣的是，这些候选者中有几个与多巴胺能信号传导有关，包括DOPA脱羧酶（DDC）的抑制剂，我们发现它们在黑色素瘤中被上调。多巴胺不仅可以控制黑色素性蛋白的生物合成和分泌，而且还有助于调节cAMP的活性，这也与黑色素的产生有关。这些化合物的分化活性在小鼠和人黑色素瘤细胞中得到了证实。值得注意的是，我们发现其中几种有效地降低了黑色素瘤细胞在软琼脂中侵袭和生长的能力。数据表明，重新分化的药物也可以具有抗肿瘤特性。尽管流行病学证据的广泛积累支持了紫外线在黑色素瘤中的基本作用，但特定的受紫外线影响的分子途径和机制在很大程度上仍然不明显。我们已经提出，除紫外线诱导的DNA诱变以外的其他机制在黑色素瘤起始中也可能很重要。为了确定紫外线在体内黑色素瘤中的作用，我们开发了一种小鼠模型（IDCT-GFP），该模型允许使用绿色荧光蛋白（GFP）的特定和诱导标记的小鼠细胞在紫外小鼠皮肤上通过uv Inradiation Invivo中的面部高度纯化，以高度纯化绿色荧光蛋白（GFP）。我们确定了从小鼠分离的黑素细胞中UVB诱导的基因表达变化的模式，这些变化与炎症的改变一致，并可能避免使用后UV重塑相关的破坏后黑素细胞。我们已经确定了新生儿紫外线辐照后黑素细胞产生的干扰素（IFN） - 伽马信号传导信号。该来源是紫外线暴露后招募到皮肤的一种巨噬细胞。 IFN-gamma反过来激活了黑色素细胞和可以促进免疫驱虫的基因的表达。发现移植的新生儿巨噬细胞以IFN-Gamma依赖性方式显着增强体内黑素瘤的生长。考虑到IFN-Alpha已被用来治疗黑色素瘤患者，尽管成功有限，这令人惊讶。我们假设黑色素瘤通过选择已经在黑素细胞中的这些途径来逃脱免疫破坏，并建议IFN-GAMMA信号传导途径可能代表了黑色素瘤患者的有希望的治疗靶标（Zaidi等人，自然469：548-553，2011）。为了解决紫外线突变问题，我们还将体内暴露的黑素细胞进行整个外显型和RNA测序。我们正在将GFP标记的黑素细胞与与人类疾病有关的各个阶段分离出来，以试图分类其精确的基因组改变。我们预计，这种体内模型将为紫外线诱导的损伤的性质以及紫外线引起黑色素瘤的机制提供新的见解。我们还假设，晚期黑色素瘤细胞可以将途径与正常发育中的黑素细胞相连，以实现更具侵略性和转移性表型。胚胎黑色素细胞和转移性黑色素瘤细胞都必须经历类似的EMT，并变得侵入性，高度迁移并在偏远的位置殖民。我们再次采用了IDCT-GFP模型来分离胚胎发育的关键阶段的胚胎黑素细胞。从代表性的发育阶段进行了RNA测序和基于微阵列的基因表达分析。已经鉴定出基因在黑素细胞和转移性黑色素瘤相对于成年黑素细胞中的表达典型上上调（或下调），这可能代表了黑色素瘤中针对转移的新治疗靶标。新的候选者包括调节ER应激，自噬和神经发育的基因，已评估使用转移性人类和小鼠黑色素瘤细胞和尾静脉注射中的siRNA敲低在转移中的作用。现在已显示几名候选者调节转移性行为，并与黑色素瘤患者的存活相关。这些正在大力追捕。最后，我们从高度和恶性黑色素细胞肿瘤和人类黑色素瘤数据库的小鼠模型中整合了基因和microRNA表达数据，并发现了以新的抑制新肿瘤抑制器miR-32为中心的途径的重要作用。恶性肿瘤经常表现出miR-32的表达不佳，其中包括NRA，PI3K，尤其是髓样细胞白血病1（MCL1）。 MCL1在黑色素瘤中高度表达，当敲低的致癌潜力时。 MCL1的过表达转化了永生的原代小鼠黑素细胞，但仅在BRAF，CRAF或PI3K中表达激活突变的那些。 Mcl1特异性拮抗剂Sabutoclax作为单一药物有效，并在临床前黑色素瘤模型中与vemurafenib结合起作用。我们的数据表明，miR-32/mcl1途径成员是新型候选抗黑素瘤药物靶标，并建议它们的抑制作用可以增强BRAFV600E抑制剂在诊所中的疗效。