p53, Aging, and Cancer

p53，衰老与癌症

• 批准号: 9343959
• 负责人: Curtis Harris
• 金额: $ 152.73万
• 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/9343959
• 关键词: Aging; Alzheimer' s Disease; Amyotrophic Lateral Sclerosis; Astrocytes; Autophagocytosis; Brain; CD8-Positive T-Lymphocytes; Cancer cell line; Cell Aging; Cell Line; Cell Proliferation; Cells; Cellular Stress; Coculture Techniques; Colonic Adenoma; DNA Damage; DNA Repair; DNA Repair Gene; Data; Degenerative Disorder; Development; Disease; Dominant-Negative Mutation; Down-Regulation; ES Cell Line; Embryo; Family member; Fibroblasts; Gene Expression Profile; Gene Targeting; Goals; Growth; Human; IL8 gene; In Vitro; Inflammatory; Insulin-Like Growth Factor I; Interleukin-6; Knock-in Mouse; Length; Life; Link; Longevity; Maintenance; Malignant Neoplasms; Malignant neoplasm of lung; Mediating; Medical; Messenger RNA; Metabolic; MicroRNAs; Molecular; Molecular Conformation; Molecular Profiling; Monitor; Mus; Nerve Degeneration; Neurodegenerative Disorders; Neurons; Normal Cell; Null Lymphocytes; Oncogene Activation; Oxidative Stress; Pathology; Pathway interactions; Patients; Phenotype; Physiological; Play; Pluripotent Stem Cells; Population; Premalignant; Premature aging syndrome; Production; Progeria; Protein Isoforms; Protein p53; RNA Splicing; Regulation; Repression; Research; Role; Serial Passage; Signal Transduction; Signaling Protein; Small Interfering RNA; Stress; Syndrome; TP53 gene; Therapeutic; Therapeutic Intervention; Toxic effect; Transfection; Undifferentiated; Variant; Werner Syndrome; base; brain tissue; cancer cell; carcinogenesis; cell type; cytokine; embryonic stem cell; functional restoration; gain of function; high throughput screening; human disease; in vivo; induced pluripotent stem cell; knock-down; mouse model; mutant; nano-string; nerve stem cell; neuron apoptosis; neuronal survival; neuroprotection; neurotoxic; neurotoxicity; overexpression; premature; protective effect; relating to nervous system; research study; response; self-renewal; senescence; small molecule; stem; stem cell biology; targeted treatment; telomere; tumor; tumorigenesis; ubiquitin-protein ligase

Specific Aim 1: Investigate p53 and microRNAs as molecular nodes in replicative stress and stem cell biology Hypothesis: p53 isoforms contribute to neurodegeneration through the regulation of cellular senescence. Neurodegenerative diseases, such as Alzheimer's disease (AD) and amyotrophic lateral sclerosis (ALS), are major medical challenges of the 21st century. Astrocytes are the most abundant cell type in the brain and play multiple key roles in providing structural, functional, and metabolic support to neurons. Astrocytes can exert both neuroprotective and neurotoxic roles. Their neurotoxic effect is mediated via senescence-associated secretory phenotype (SASP), including the production and secretion of pro-inflammatory cytokines IL-6 and IL-8, which increases with aging and neurodegenerative diseases. We have revealed that human astrocytes express p53 isoforms, delta133p53 and p53beta, and that these p53 isoforms regulate SASP in astrocytes and their protective and toxic effects on neurons. ALS and AD brain tissues, as well as senescent astrocytes in vitro after serial passaging, showed decreased delta133p53 and increased p53beta, which are the senescence-associated expression signature observed in other cell types including fibroblasts, CD8+ T-lymphocytes and pre-malignant colon adenoma. These changes were attributed to selective autophagy-mediated degradation of delta133p53 and SRSF3-mediated alternative mRNA splicing generating p53beta, which are again conserved across different cell types. Early-passage astrocytes with delta133p53 knockdown or p53beta overexpression (which reproduces the expression signature in neurodegenerative diseases and senescent astrocytes) were induced to show SASP and to exert neurotoxicity in co-culture with neurons. Importantly, restored expression of delta133p53 in near-senescent, neurotoxic astrocytes resulted in repressed SASP, elevated neurotrophic growth factors (NGF and IGF-1) and enhanced neuroprotection in co-culture (i.e., increased neuronal survival and decreased neuronal apoptosis). These findings, especially the delta133p53-induced reversion of neurotoxic astrocytes to neuroprotective ones, indicate that the p53 isoforms and their regulatory mechanisms are potential targets for therapeutic intervention in neurodegenerative diseases. We are initiating a high-throughput screening to identify small molecule compounds that modulate the expression or activity of delta133p53 and p53beta. Hypothesis: Delta133p53 rescues progeria cells from premature senescence and DNA damage. Werner syndrome (WS) and Hutchinson-Gilford progeria syndrome (HGPS) are human disorders characterized by aging-associated phenotypes that occur earlier in life than normal. Cells derived from WS or HGPS patients have decreased cell proliferation potential, show impaired DNA damage response, and are prematurely induced into cellular senescence. We have found that in vitro cultured WS and HGPS fibroblasts show the senescence-associated expression signature (i.e., decreased delta133p53 and increased p53beta) at an earlier passage than normal fibroblasts, prompting us to examine whether this p53 isoform expression signature contributes to premature senescent phenotypes in these progeria-derived cells. Our current data indicate that restored expression of delta133p53 in WS and HGPS fibroblasts rescues them from premature senescence and extends their replicative lifespan at least by 15 population doubling levels. These effects of delta133p53 are attributed to the downregulation of p21WAF1 and microRNA-34a through its dominant-negative inhibition of full-length p53. In addition, delta133p53 is suggested to function independently of full-length p53 to upregulate a set of DNA repair genes including Rad51. We have also found that delta133p53 exerts similar effects (i.e., inhibition of premature senescence and extension of replicative lifespan) in mouse fibroblasts derived from a mouse model of HGPS (Lmna G609G/G609G knock-in mice), which provides a basis towards generating a new mouse model to examine in vivo effects of delta133p53 on progeria pathologies. Senescence inhibition and lifespan extension in HGPS fibroblasts can be used as cellular phenotypes to identify small molecule compounds that upregulate or activate delta133p53. Hypothesis: p53 isoforms are physiological regulators of human pluripotent stem cells. We previously showed that delta133p53 increases the efficiency of reprogramming from human fibroblasts to iPS cells likely through repression of p53 target genes involved in cellular senescence (e.g., p21WAF1 and microRNA-34a). Our new data show that delta133p53, which is abundantly expressed in undifferentiated iPS, becomes downregulated during differentiation to neural stem cells and to mature neurons, further supporting a functional link between this p53 isoform and the acquisition and maintenance of an undifferentiated, self-renewing state in human pluripotent stem cells. To elucidate the role of abundant levels of endogenous delta133p53 in undifferentiated ES and iPS cells, we have successfully optimized the conditions for efficient transfection of siRNA specifically knocking down delta133p53. Our preliminary data showed that the siRNA-mediated knockdown of delta133p53 primed an ES cell line for neural differentiation and induced an iPS cell line into cellular senescence. Further experiments are ongoing to examine the molecular mechanisms by which delta133p53 controls differentiation and senescence in ES and iPS cells. Specific Aim 2: Define the Role of p53 Isoforms and Mutant Variants in Control of Cellular Division of Normal and Cancer Cells Hypothesis: p53 isoforms and mutants have gain-of-function activities. Cancer-associated mutants of full-length p53 can promote tumorigenesis in the absence of wild-type p53 (so-called "gain-of-function" mutants). Because of a possible mutant p53 conformation of delta133p53, we hypothesize that this p53 isoform and its mutant versions also have gain-of-function activities. We have generated p53-null cell lines (fibroblast and lung cancer cell lines) that inducibly express wild-type delta133p53 and mutants (V157F, R175H, R249S and R275H). We performed a microarray-based expression profiling of mRNA and a Nanostring-based expression profiling of microRNA upon induction of these wild-type and mutant delta133p53. The data are being analyzed to identify genes and signaling pathways regulated by their gain-of-function activities. Because gain-of-function mutants of full-length p53 can function by interacting with and inhibiting the p53 family members p63 and p73, we are examining whether wild-type and mutant delta133p53 also interact with p63 and p73.

具体目标1：在复制应激和干细胞生物学假说中研究p53和microRNA作为分子淋巴结：p53同工型通过调节细胞衰老而导致神经变性。神经退行性疾病，例如阿尔茨海默氏病（AD）和肌萎缩性侧面硬化症（ALS），是21世纪的主要医学挑战。星形胶质细胞是大脑中最丰富的细胞类型，在为神经元提供结构，功能和代谢支持方面起多个关键作用。星形胶质细胞可以发挥神经保护作用和神经毒性作用。它们的神经毒性作用是通过与衰老相关的分泌表型（SASP）介导的，包括促炎性细胞因子IL-6和IL-8的产生和分泌，随着衰老和神经退行性疾病的增加而增加。我们已经揭示了人类星形细胞表达p53同工型，delta133p53和p53beta，并且这些p53同工型调节星形胶质细胞中的SASP及其对神经元的保护性和毒性作用。串行传递后，ALS和AD脑组织以及在体外的衰老星形细胞显示出降低的Delta133p53和P53Beta的增加，这是在其他细胞类型中观察到的与衰老相关的表达特征，包括成纤维细胞，包括成纤维细胞CD8+ T-T-乳糖细胞和前膜型结肠癌。这些变化归因于Delta133p53和SRSF3介导的替代mRNA剪接生成P53BETA的选择性自噬介导的降解，它们再次在不同的细胞类型中保守。诱导了具有Delta133p53敲低的早期星形胶质细胞或P53BETA过表达（在神经退行性疾病中重现表达特征和衰老的星形胶质细胞中的表达特征）表现出SASP并与神经元共培养神经毒性。重要的是，在近期的神经毒性星形胶质细胞中，Delta133p53的恢复表达导致SASP抑制，神经营养生长因子升高（NGF和IGF-1）升高，并增强了共培养的神经保护作用（即神经元生存和降低的神经元和降低的神经元）。这些发现，尤其是Delta133p53诱导的神经毒性星形胶质细胞向神经保护细胞的逆转，表明p53同工型及其调节机制是神经退行性疾病中治疗干预的潜在靶标。我们正在启动高通量筛选，以鉴定调节Delta133p53和p53beta的表达或活性的小分子化合物。假设：Delta133p53从过早的衰老和DNA损伤中挽救了后代细胞。 Werner综合征（WS）和Hutchinson-Gilford Progeria综合征（HGP）是人类疾病，其特征是与正常人相比，生活在生活中发生的衰老相关表型。源自WS或HGPS患者的细胞具有降低细胞增殖潜力，显示出DNA损伤反应受损，并过早诱导细胞衰老。我们发现，在体外培养的WS和HGP成纤维细胞表明，与正常成纤维细胞相比，在早期通过时与衰老相关的表达特征（即减少的delta133p53和p53beta降低），并增加了p53beta），这促使我们检查了这种p53同生的表达签名对这些p53的表达签名是否有助于这些代表蛋白质的表达，以这些代表性的表现为这些代源。我们当前的数据表明，在WS和HGPS成纤维细胞中恢复了Delta133p53的表达，使它们从早期衰老中挽救了它们，并至少将其复制寿命延长至少15个人口加倍水平。 Delta133p53的这些作用归因于P21WAF1和MicroRNA-34A的下调，其主要阴性抑制全长p53。此外，建议使用Delta133p53独立于全长p53发挥作用，以上调一组包括RAD51在内的DNA修复基因。我们还发现，Delta133p53在小鼠成纤维细胞中产生相似的作用（即抑制过早衰老和复制寿命的扩展），该成纤维细胞源自HGP的小鼠模型（LMNA G609G/G609G/G609G敲击小鼠），从而产生了对新鼠标效果的基础，从而为VIV3的效果提供了Quiv的效果。病理。 HGP成纤维细胞中的衰老抑制和寿命扩展可以用作细胞表型，以鉴定上调或激活Delta133p53的小分子化合物。假设：p53同工型是人多能干细胞的生理调节剂。我们先前表明，Delta133p53提高了从人成纤维细胞到IPS细胞的重编程效率，这可能是通过抑制涉及细胞衰老的p53靶基因（例如p21WAF1和microRNA-34A）的p53靶基因。我们的新数据表明，在分化为神经干细胞和成熟神经元的过程中，在未分化的IP中大量表达的Delta133p53被下调，进一步支持了这种p53同工型与习得和维持人类pluripotent pluripotent pluripotent pluripotent pluripotent pluripotent pluripotent plulipotent nate的功能联系。为了阐明在未分化的ES和IPS细胞中，大量内源性Delta133p53水平的作用，我们成功地优化了有效转染siRNA的条件，专门击倒了Delta133p53。我们的初步数据表明，siRNA介导的Delta133p53的敲低启动了ES细胞系以进行神经分化，并将IPS细胞系诱导到细胞衰老中。正在进行进一步的实验，以检查Delta133p53控制ES和IPS细胞中的分化和衰老的分子机制。具体目标2：定义p53同工型和突变体在控制正常细胞和癌细胞细胞分裂中的作用：p53同工型和突变体具有功能良好的活性。全长p53的癌症相关突变体可以在没有野生型p53（所谓的“功能获得”突变体）的情况下促进肿瘤发生。由于可能具有delta133p53的突变体p53构象，我们假设该p53同工型及其突变体版本也具有功能奖励活性。我们已经产生了p53无效细胞系（成纤维细胞和肺癌细胞系），这些细胞系可表达野生型Delta133p53和突变体（V157F，R175H，R249S和R275H）。在诱导这些野生型和突变的delta133p53时，我们对mRNA进行了基于微阵列的表达分析和microRNA的基于纳米肌的表达分析。正在分析数据，以识别受功能收益活性调节的基因和信号通路。由于全长p53的功能收益突变体可以通过与p53家族成员p63和p73相互作用并抑制野生型和突变体delta133p53是否也与p63和p73相互作用。