Use of Mouse Models of Human Rhabdomyosarcoma to Study Progression to Metastasis

使用人类横纹肌肉瘤小鼠模型研究转移进展

• 批准号: 8552867
• 负责人: GLENN T. MERLINO
• 金额: $ 8.24万
• 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/8552867
• 关键词: Accounting; Acetylation; Adolescent; Adoptive Immunotherapy; Alveolar Rhabdomyosarcoma; Animal Model; Behavior; C57BL/6 Mouse; CDK4 gene; Cancer Biology; Cancer Model; Cancer Patient; Cancer Vaccines; Cell Differentiation process; Cell Line; Cell Proliferation; Cells; Cessation of life; Child; Childhood; Childhood Soft Tissue Sarcoma; Classification; Clinical; Collaborations; Complex; Cutaneous Melanoma; Data; Dendritic Cells; Development; Disease; Dose; Embryonal Rhabdomyosarcoma; Epigenetic Process; Event; FGFR4 gene; Gene Expression; Gene Family; Genes; Genetic; Genetic Programming; Genetically Engineered Mouse; Genomic Instability; Goals; Hematopoietic Neoplasms; Hepatocyte Growth Factor; Histology; Histone Deacetylase Inhibitor; Histones; Human; IL2RA gene; Immune; Immunocompetent; Infant; Injection of therapeutic agent; Laboratories; Lesion; Lung; M cell; MDM2 gene; Malignant Childhood Neoplasm; Malignant Neoplasms; Mediating; Methylation; Modeling; Molecular; Molecular Genetics; Molecular Profiling; Mus; Muscle Development; Muscle Fibers; Mutate; Mutation; Nature; Neoplasm Metastasis; Oncogenes; PAX3 gene; PAX7 gene; Pathogenesis; Pathway interactions; Patients; Pediatric Neoplasm; Pharmaceutical Preparations; Physiologic pulse; Primary Neoplasm; Process; Publishing; Receptor Protein-Tyrosine Kinases; Regulatory T-Lymphocyte; Research; Resources; Rhabdomyosarcoma; Role; Skeletal Muscle; Staging; T cell response; T-Lymphocyte; Tail; Therapeutic; Therapeutic Intervention; Tissue Sample; Tissues; Transgenes; Transgenic Organisms; Transplantation; Tumor Burden; Tumor-Derived; Vaccination; Work; base; cancer genetics; chromatin modification; combat; forkhead protein; homeodomain; immunogenic; immunogenicity; in vivo; insight; member; mouse model; mutant; novel; preclinical efficacy; prevent; progenitor; sarcoma; t(2; 13)(q35; q14); therapeutic target; transcription factor; tumor; tumor growth; tumor initiation; young adult

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 on two tumor types, cutaneous malignant melanoma and the pediatric malignancy rhabdomyosarcoma. Rhabdomyosarcoma, accounting for up to 10% of all pediatric neoplasms and for more than 50% of pediatric soft tissue sarcomas, is believed to arise from imbalances in skeletal muscle cell proliferation and differentiation (Merlino and Khanna, Genes & Dev. 21: 1275-9, 2007). There are two major subtypes of rhabdomyosarcoma. Embryonal rhabdomyosarcoma, the most common subtype, typically occurs in infants and young children. There is no distinct molecular event that characterizes embryonal rhabdomyosarcoma, but they tend to demonstrate severe genomic instability. Alveolar rhabdomyosarcomas are highly aggressive tumors of adolescents and young adults, with classification based on translocations involving the genes encoding the forkhead transcription factor, and either PAX3 or PAX7. Detailed molecular pathways associated with rhabdomyosarcoma had been poorly characterized, due in part to the lack of a rhabdomyosarcoma-prone animal model. We have developed the first relevant model for embryonal rhabdomyosarcoma, showing that virtually all mice harboring a hepatocyte growth factor/scatter factor transgene (which deregulates its tyrosine kinase receptor MET) and deficient in Ink4a/Arf rapidly succumbed to highly invasive rhabdomyosarcoma (Sharp et al., Nature Med. 8: 1276-80, 2002). Highly comparable molecular lesions have also been described for human rhabdomyosarcoma. These data provide genetic evidence that MET and INK4a/ARF pathways represent critical and synergistic targets in rhabdomyosarcoma pathogenesis, and suggest a rational therapeutic combination to combat this pediatric sarcoma. Our more recent in vivo analyses of the pathways downstream of INK4a/ARF revealed that it was the ARF-MDM2-p53 pathway, and not the INK4a-CDK4-pRb pathway that was the key regulator of this disease (Ha et al., Proc. Natl. Acad. Sci. 104: 10968-73, 2007).A panel of highly and poorly metastatic cell lines was subsequently established from the many rhabdomyosarcoma tumors arising in our novel mouse model, and used in concert with microarray-based expression profiling to identify a set of genes associated with enhanced metastatic behavior. Multiple functional in vivo studies confirmed that the cytoskeletal linker EZRIN and the homeodomain-containing transcription factor SIX1 both have essential roles in determining the metastatic fate of rhabdomyosarcoma cells (Yu et al., Nature Med. 10: 175-81, 2004). SIX1 was especially intriguing, as it is known to be required for skeletal muscle development. Notably, EZRIN and SIX1 expression levels were also both enhanced in human rhabdomyosarcoma tissue samples, significantly correlating with clinical stage. Remarkably, subsequent molecular analyses showed that the EZRIN gene was in fact a direct transcriptional target of SIX1, and indispensable for SIX1-mediated rhabdomyosarcoma metastasis (Yu et al., Cancer Res. 66: 1982-9, 2006). Recently, we have found that SIX1 regulates EZRIN expression, at least in part, through epigenetic modification of the chromatin around the EZRIN gene locus, including regulating the states of methylation and acetylation within the histone tails. Studies are currently underway to more fully elaborate mechanisms by which the EZRIN gene is regulated, and also to determine the efficacy of preclinical drug studies that use histone deacetylase inhibitors or demethylating agents in concert with EZRIN knockdown to treat rhabdomyosarcoma. EZRIN appears to represent a very promising therapeutic target for patients with advanced stage rhabdomyosarcoma (Yu et al., PLoS One 5(9):e12710).Our RMS cell lines were recently employed in the identification and characterization of activating FGFR4 mutations in human RMS tumors, indicating that FGFR4 can function as an oncogene in RMS (Taylor et al., J. Clin. Invest. 119:3395-407, 2009). FGFR4 knockdown in a human RMS cell line reduced tumor growth and experimental lung metastases when the cells were transplanted into mice. FGFR4 mutants enhanced tumor proliferation and metastatic potential when expressed in a murine RMS cell line. These findings support the potential therapeutic targeting of FGFR4 in RMS.In collaboration with Dr. Crystal Mackall, a new RMS cell line (M3-9-M) was derived from an embryonal RMS occurring in a C57BL/6 mouse transgenic for HGF/SF and heterozygous for mutated p53. Primary tumors and metastases derived from M3-9-M were studied for similarities to human embryonal RMS, and for immunogenicity and immune responsiveness. Primary and metastatic tumors were found to develop after orthotopic injection of M3-9-M into immunocompetent C57BL/6 mice, which mirror human embryonal RMS with regard to histology, gene expression, and metastatic behavior. Whole cell vaccination using irradiated M3-9-M cells or M3-9-M-pulsed dendritic cells (DC)-induced tumor-specific T-cell responses that prevent tumor growth following low-dose tumor injection, and slow tumor growth following higher doses. Administration of anti-CD25 moAbs to deplete CD4(+) CD25(+) FOXP3(+) regulatory T cells prior to tumor vaccination enhanced the potency of the RMS tumor vaccine. Adoptive immunotherapy with M3-9-M primed T cells plus DC-based vaccination resulted in complete eradication of day 10 M3-9-M derived tumors. We conclude that M3-9-M derived murine RMS line is immunogenic and immunoresponsive, regulatory T cells contribute to immune evasion by murine RMS, and adoptive immunotherapy with DC vaccination can eradicate low tumor burdens. This work was recently published (Meadors et al., Pediatr. Blood Cancer 57:921-9, 2011).

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集中在两种肿瘤类型上，即皮肤恶性黑色素瘤和儿科恶性纹状性肉瘤。横纹肌肉瘤最多占所有儿科肿瘤的10％，以及50％以上的小儿软组织肉瘤，据信是由于骨骼肌细胞增殖和分化的失衡而引起的（Merlino和Merlino和Khanna，Genes，Genes＆Dev。21：1275-9-9，2007年，2007年）。横纹肌肉瘤有两个主要的亚型。 胚胎横纹肌肉瘤是最常见的亚型，通常发生在婴儿和幼儿中。没有明显的分子事件来表征胚胎横纹肌肉瘤，但它们倾向于表现出严重的基因组不稳定性。肺泡横纹肌肉瘤是青少年和年轻人的高度侵略性肿瘤，基于涉及编码叉子转录因子的基因的易位，以及PAX3或PAX7。与横纹肌肉瘤相关的详细分子途径的特征很差，部分原因是缺乏横纹肌肉瘤模型。我们已经开发了胚胎横纹肌肉瘤的第一个相关模型，表明几乎所有具有肝细胞生长因子/散射因子转基因（消除其酪氨酸激酶受体满足的小鼠），并且在Ink4a/Arf中不足，迅速屈服于高度的rhabyosaryosarcoma（sharp and sapl and sapl and and and and and and and and and and and and and and and and and and and and and and and and and and and and and and and and and and and and and and and and。也已经描述了高度可比的分子病变。这些数据提供了MET和INK4A/ARF途径代表横纹肌肉瘤发病机理中的关键和协同靶标的遗传证据，并提出了一种合理的治疗组合来对抗这种儿科肉瘤。我们最近对Ink4a/arf途径的体内分析表明，这是ARF-MDM2-P53途径，而不是Ink4a-CDK4-PRB途径是该疾病的关键调节剂（Ha等人，Proc。Natl。Natl。Natl。Natl。Acad。Sci。104：104：104：10968-73，MAPERESES the MAPERESTY MATERESTY MATERSITY MATERSITY MATERSITY MATERSESTY MATERYSTYSTAITS MATERSY and andy cally and andy Panly andly Panely andy aa aa aa aa aa aa aa aa aa aa aa aa aa aa。在我们的新型小鼠模型中产生的许多横纹肌肉瘤肿瘤，并与基于微阵列的表达分析一起使用，以识别一组与增强的转移行为相关的基因。多个功能性的体内研究证实，细胞骨架接头Ezrin和含同源域的转录因子Six1均在确定横纹肌肉瘤细胞的转移性命运方面具有重要作用（Yu等人，自然医学，10：175-81，2004）。 Six1特别有趣，因为已知骨骼肌发育是必需的。值得注意的是，在人横纹肌肉瘤组织样品中，Ezrin和Six1表达水平也均得到增强，与临床阶段显着相关。值得注意的是，随后的分子分析表明，ezrin基因实际上是第六1个直接的转录靶标，对于六1介导的横纹肌肉瘤转移是必不可少的（Yu等人，CancerRes。66：1982-9，2006）。最近，我们发现SIX1至少部分通过对Ezrin基因基因座的染色质的表观遗传修饰，包括调节组蛋白尾部内的甲基化和乙酰化状态，从而调节Ezrin的表达。目前正在进行研究Ezrin基因受调节的更全面的机制，并确定使用组蛋白脱乙酰基酶抑制剂或与Ezrin敲低治疗rhabdomyosarcoma一起使用组蛋白脱乙酰基酶抑制剂的临床前药物研究的功效。对于晚期横纹肌肉瘤患者（Yu等人，PLOS One 5（9）：E12710），Ezrin似乎是一个非常有前途的治疗靶标。最近，使用RMS细胞系在人类RMS肿瘤中激活FGFR4突变的识别和表征中，表明FGFR4的fgfr4 clin n. clin an act ancoge and and and and and and an。 119：3395-407，2009）。当细胞移植到小鼠中时，人类RMS细胞系中的FGFR4敲低可减少肿瘤的生长和实验性肺转移。当在鼠RMS细胞系中表达时，FGFR4突变体增强了肿瘤的增殖和转移潜力。这些发现支持RMS中FGFR4的潜在治疗靶向，在与Crystal Mackall博士合作的情况下，新的RMS细胞系（M3-9-M）源自HGF/SF的C57BL/6小鼠转基因中的胚胎RMS，用于HGF/SF，并用于突变的p53。研究了来自M3-9-M的原发性肿瘤和转移酶与人类胚胎RMS的相似性以及免疫原性和免疫反应性。在原位注射M3-9-M中，原发性和转移性肿瘤在免疫能力的C57BL/6小鼠后发育，这些小鼠在组织学，基因表达和转移性行为方面反映了人类胚胎RMS。全细胞疫苗接种使用辐照的M3-9-M细胞或M3-9-M脉冲的树突状细胞（DC）诱导的肿瘤特异性T细胞反应，可防止低剂量肿瘤注射后肿瘤的生长，并且较高剂量后肿瘤的生长缓慢。在肿瘤疫苗接种之前，给予抗CD25摩押术以耗尽CD4（+）CD25（+）FOXP3（+）FOXP3（+）调节性T细胞，从而增强了RMS肿瘤疫苗的效力。使用M3-9-m引发T细胞加上基于DC的疫苗接种的收养免疫疗法可完全消除第10 m3-9-m衍生的肿瘤。我们得出的结论是，M3-9-m衍生的鼠RMS系具有免疫原性和免疫响应性，调节性T细胞有助于鼠RMS免疫逃避，而通过直流疫苗接种的收养免疫疗法可以消除低肿瘤负担。这项工作最近发表了（Meadors等，Pediatr。HloodCancer 57：921-9，2011）。