p53, Aging, and Cancer

p53，衰老与癌症

• 批准号: 8763568
• 负责人: Curtis Harris
• 金额: $ 80.22万
• 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/8763568
• 关键词: Aging; Alternative Splicing; Autophagocytosis; Autophagosome; Biological; Biological Assay; C-terminal; CD28 gene; CD8-Positive T-Lymphocytes; Cell Aging; Cell Line; Cell Proliferation; Cell division; Cells; Cellular Stress Response; Coculture Techniques; Commit; Cytoplasm; DNA; DNA Damage; Data; Dominant-Negative Mutation; Elderly; Equilibrium; Fibroblasts; Frequencies; Generations; Genes; Genetic Risk; Genetic Transcription; HIV; Homeostasis; Human; In Vitro; Introns; Karyotype determination procedure; Lead; Length; Longevity; Lysine; MDM2 gene; MG132; Malignant - descriptor; Malignant Neoplasms; Malignant neoplasm of lung; Mediating; Messenger RNA; Methods; MicroRNAs; Molecular; Molecular Chaperones; Molecular Profiling; Monitor; Multiprotein Complexes; Mutation; Normal Cell; Normal tissue morphology; Pathway interactions; Patients; Phenotype; Physiological; Play; Pluripotent Stem Cells; Population; Process; Protein Isoforms; Proteins; Regulation; Reporter; Reporting; Repression; Research; Role; Series; Signal Transduction; Somatic Mutation; Spectral Karyotyping; Staging; Stem cells; Stress; Surface Antigens; Telomere Shortening; Teratoma; Testing; Tissues; Trans-Activators; Tumor Suppression; Tumor Suppressor Proteins; Up-Regulation; Variant; Virus Diseases; age related; bafilomycin A1; base; beta-Galactosidase; cancer cell; cancer stem cell; carcinogenesis; cell type; human embryonic stem cell; human stem cells; in vivo; induced pluripotent stem cell; lymphocyte proliferation; mutant; neoplastic cell; overexpression; pluripotency; promoter; protein degradation; reconstitution; response; segregation; self-renewal; senescence; small hairpin RNA; stem; stem cell biology; tumor; tumor microenvironment; tumor progression; ubiquitin-protein ligase

Specific Aim 1: Investigate p53 and miRNAs as molecular nodes in replicative stress and stem cell biology Hypothesis: Differential mechanisms regulate the expression levels of p53 isoforms to coordinate replicative senescence We reported that the expression level of delta133p53, which is produced via transcription from an alternative promoter in intron 4, is mainly regulated at the protein level rather than at the transcriptional level. In contrast to full length p53 and p53beta that are subject to proteasomal degradation, the protein turnover of delta133p53 was specifically regulated through autophagic protein degradation. Treatment with bafilomycin A1, but not MG-132 , restored the delta133p53 protein in replicatively senescent human fibroblasts. Knockdown of autophagy-mediating genes (i.e., ATG5, ATG7 and Bechlin-1) also restored diminished expression of delta133p53. Delta133p53 was ubiquitinated at C terminal lysine residues and, upon autophagy induction, became colocalized in the cytoplasm with p62/SQSTM1 (adaptor for ubiquitinated autophagy substrates) and LC3-II (autophagosome component). This isoform-specific turnover mechanism degrades delta133p53 and removes its effect against full-length p53, while maintaining full length p53 levels, orchestrating p53 mediated replicative senescence. Our data show that delta133p53 interacts with Hsp70 chaperones, a chaperone-associated E3 ubiquitin ligase STUB1 (but not MDM2, which full-length p53 interacts with) and a chaperone regulator BAG2, suggesting that chaperone-mediated autophagy plays a role in delta133p53 degradation. Because STUB1 was downregulated and BAG2 was upregulated at replicative senescence, STUB1 and/or BAG2 may regulate the autophagic degradation of delta133p53. We aim to clarify the molecular basis and biological significance of a delta133p53-mediated functional interaction between cellular senescence and autophagy, two major processes involved in cancer and aging. Hypothesis: p53 isoform switching is a physiological mechanisms of replicative senescence To investigate in vivo physiological roles of the p53 isoforms, we use circulating CD8+ T lymphocytes, which undergo a series of differentiation stages toward replicative senescence in vivo with specific changes in cell surface antigens (e.g., loss of CD28 and gain of CD57). We found a donor age dependent accumulation of CD8+ T lymphocytes with CD28 CD57+ and senescent phenotypes, including inhibited proliferation, shortened telomeres, increased HP1 gamma and gamma H2AX foci, senescence associated secretory phenotypes (SASP) and senescence-associated beta galactosidase activity. In these in vivo accumulated senescent or near senescent CD8+ T lymphocytes, endogenous delta133p53 and p53beta were downregulated and upregulated, respectively. In poorly proliferative CD28 negative populations with low expression levels of delta133p53, reconstituted expression of delta133p53, as well as that of CD28, restored cell proliferation and extended replicative lifespan. Conversely, knockdown of delta133p53 or overexpression of p53beta in highly proliferative CD28 positive populations inhibited cell proliferation and induced senescence. Delta133p53 and p53beta are endogenous regulators of CD8+ T lymphocyte proliferation and senescence, and suggest that enhanced expression of delta133p53 may lead to functional activation of non- or least proliferative populations of CD8+ T lymphocytes, which accumulate in the elderly or patients with human immunodeficiency virus infection. The reconstituted expression of delta133p53 upregulated CD28 expression at both mRNA and protein levels. We are examining whether full length p53 transcriptionally represses CD28 and whether the inhibition of full length p53 by delta133p53 leads to transcriptional upregulation of CD28. We are investigating possible interactions between tumor cells and CD8+ T lymphocytes in tumor microenvironment. Our initial isolation of tumor associated CD8+ T lymphocytes from human lung cancer tissues suggests that they contain CD28+ CD57+ populations with senescent phenotypes (e.g., HP1-gamma foci and SASP). A direct effect of tumor cells on senescent phenotypes and p53 isoform expression in CD8+ T lymphocytes will also be examined by co culturing these two cell types in vitro. Hypothesis: p53 isoforms are physiological regulators of human pluripotent stem cells Since the p53 signaling network has been implicated in various aspects of stem cell biology, p53 isoforms may have physiological roles in human stem cells. Similar to p53 knockdown as reported, we found that overexpression of delta133p53 in human fibroblasts increased the efficiency of reprogramming to iPSC. Because delta133p53 preferentially represses a set of p53 inducible genes involved in cellular senescence (e.g., p21WAF1 and miR-34a), in contrast to p53 knockdown that represses all p53 inducible genes including ones involved in DNA damage response, we hypothesize that delta133p53 overexpression is a method of enhanced iPSC generation without a risk of genetic aberrations or malignant transformation. To test this hypothesis, we are characterizing delta133p53 induced iPSC by conventional karyotyping, spectral karyotyping, mRNA and miRNA expression profiling, and teratoma formation assay. Human pluripotent stem cells are the cell type that expresses the most abundant levels of endogenous delta133p53 protein. While the expression levels of full length p53 widely varied among hESC and iPSC lines all hESC and iPSC lines consistently expressed much higher levels of delta133p53 protein than human fibroblasts, with only a slight increase in delta133p53 mRNA. We are currently examining whether the upregulation of endogenous delta133p53 results from repression of its autophagic degradation. To clarify the role of upregulated delta133p53 in self-renewal, pluripotency and differentiation potential of hESC and iPSC, shRNA knockdown of endogenous delta133p53 will be performed in these 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 regulate symmetric and asymmetric cell division Asymmetric cell division enables stem cells to self-renew and to generate cells committed to differentiation, thus contributing to normal tissue homeostasis and cancer progression. We previously demonstrated that a sub-fraction of cancer cells had the ability to asymmetrically divide their template DNA strands and that this segregation correlated with cell fate. To further explore the regulation of the symmetric and asymmetric self-renewal balance in cancer cells we examined the role of p53, its isoforms and somatic mutations in lung cancer. Full length p53 decreased the frequency of asymmetric division, while mutants (V157F, R175H, R249S and R275H) also significantly decreased the frequency at which a sub-population of cancer cells asymmetrically divide their template DNA strands. This control was transient, and the frequency of asymmetric division increased again once p53 was removed. Regulation of asymmetric division was not modulated by the p53 isoforms p53beta and delta133. Using reporter constructs that monitor the expression of stem and differentiation protein markers, we are currently examining which form of self renewal, symmetric self renewal, symmetric differentiation or asymmetric division, p53, delta133 and their respective mutations regulate. Specifically, we will use a fluorescent reporter for the SPC promoter as a marker of differentiated cells, and a fluorescent reporter for OCT4 promoter activity as a marker for stem cells. We have shown previously that non-template DNA strands co-segregate with SPC, while OCT-4 is a marker of lung cancer stem cells.

Specific Aim 1: Investigate p53 and miRNAs as molecular nodes in replicative stress and stem cell biology Hypothesis: Differential mechanisms regulate the expression levels of p53 isoforms to coordinate replicative senescence We reported that the expression level of delta133p53, which is produced via transcription from an alternative promoter in intron 4, is mainly regulated at the protein level rather than at the transcriptional level.与受蛋白酶体降解的全长p53和p53beta相反，Delta133p53的蛋白质更新是通过自噬蛋白质降解的。用Bafilomycin A1而非MG-132治疗，在复制衰老的人成纤维细胞中恢复了Delta133p53蛋白。自噬中断基因的敲低（即ATG5，ATG7和Bechlin-1）也恢复了Delta133p53的表达降低。 Delta133p53在C末端赖氨酸残基上被泛素化，并在自噬诱导后与p62/SQSTM1（泛素化自噬底物的衔接子）和LC3-II（自phagagosome组）在细胞质中共定位。这种同工型特异性的周转机制降低了Delta133p53，并消除了对全长p53的影响，同时保持了全长P53水平，策划了p53介导的复制性衰老。 Our data show that delta133p53 interacts with Hsp70 chaperones, a chaperone-associated E3 ubiquitin ligase STUB1 (but not MDM2, which full-length p53 interacts with) and a chaperone regulator BAG2, suggesting that chaperone-mediated autophagy plays a role in delta133p53 degradation.由于Stub1被下调，并且在复制衰老时BAG2被上调，因此Stub1和/或Bag2可能会调节Delta133p53的自噬降解。我们旨在阐明Delta133p53介导的细胞衰老与自噬之间的功能相互作用的分子基础和生物学意义，这是癌症和衰老涉及的两个主要过程。假设：p53同工型切换是一种复制衰老的生理机制，用于研究p53同工型的体内生理作用，我们使用循环的CD8+ T淋巴细胞，在CD.G27和CD5的特定体内损失CD28和CD28的特定变化中，经过一系列差异化阶段，CD28和CD28的特定变化。我们发现，CD8+ T淋巴细胞具有CD28 CD57+和衰老表型的供体年龄依赖性积累，包括抑制的增殖，缩短端粒，增加HP1 Gamma和Gamma H2AX焦点，衰老，衰老，相关的分类表型（SASP）（SASP）和SENESESSESSESS BETA-BETA-BETA-GALASSOSSOSESAPSE sAPSICSIDASE。在这些体内累积的衰老或接近衰老的CD8+ T淋巴细胞中，内源性Delta133p53和p53beta分别下调并上调。在较低的Delta133p53表达水平低的CD28阴性群体中，Delta133p53的重构表达以及CD28的表达恢复了细胞增殖和扩展的复制寿命。相反，在高度增殖的CD28阳性人群中敲低Delta133p53或p53beta的过表达抑制了细胞增殖并诱发衰老。 delta133p53和p53beta是CD8+ T淋巴细胞增殖和衰老的内源调节剂，并表明增强的Delta133p53的表达可能会导致非cd8+ t淋巴细胞的非或最少增殖性群体的非肿瘤群体的功能激活，而这些CD8+ T淋巴细胞的患者在失败的患者中积累了cd8+ T淋巴细胞的患者。 Delta133p53的重构表达在mRNA和蛋白质水平上都上调CD28表达。我们正在研究全长p53是否在转录中抑制CD28以及delta133p53对全长p53的抑制是否导致CD28的转录上调。我们正在研究肿瘤细胞与肿瘤微环境中CD8+ T淋巴细胞之间可能的相互作用。我们对肿瘤相关的CD8+ T淋巴细胞的最初分离表明，它们包含具有衰老表型的CD28+ CD57+种群（例如，HP1-Gamma foci和SASP）。肿瘤细胞对CD8+ T淋巴细胞中衰老表型和p53同工型表达的直接作用也将通过在体外培养这两种细胞类型来检查。假设：p53同工型是人类多能干细胞的生理调节剂，因为p53信号网络已与干细胞生物学的各个方面有关，p53同工型可能在人类干细胞中具有生理作用。与报道的p53敲低相似，我们发现在人成纤维细胞中的Delta133p53过表达提高了对IPSC的重新编程的效率。 Because delta133p53 preferentially represses a set of p53 inducible genes involved in cellular senescence (e.g., p21WAF1 and miR-34a), in contrast to p53 knockdown that represses all p53 inducible genes including ones involved in DNA damage response, we hypothesize that delta133p53 overexpression is a method of enhanced iPSC generation without a risk of genetic畸变或恶性转化。为了检验这一假设，我们正在表征Delta133p53通过常规核分型，光谱核分型，mRNA和miRNA表达谱分析以及畸胎瘤形成分析引起的IPSC。人多能干细胞是表达最丰富水平的内源性Delta133p53蛋白的细胞类型。尽管hESC和IPSC线的全长p53的表达水平差异很大，而hESC和IPSC线的表达水平始终如一地表达的delta133p53蛋白水平比人成纤维细胞高得多，而delta133p53 mRNA只有略有增加。我们目前正在研究内源性Delta133p53的上调是否是由于其自噬降解的抑制而导致的。为了阐明上调delta133p53在hESC和IPSC的自我更新，多能和分化潜力中的作用，这些细胞将执行内源性delta133p53的shRNA敲低。具体目标2：定义p53同工型和突变体变异在控制正常和癌细胞的细胞分裂中的作用：p53同工型和突变体调节对称和非对称细胞分裂细胞不对称细胞不对称细胞使干细胞可自我更新，并导致分化为分化，从而对正常的组织组织和癌症进行造成促进。我们先前证明，癌细胞的亚逆转能力具有不对称的模板DNA链的能力，并且这种分离与细胞命运相关。为了进一步探索癌细胞中对称和不对称自我更新平衡的调节，我们检查了p53，其同工型和体细胞突变在肺癌中的作用。全长p53降低了不对称分裂的频率，而突变体（V157F，R175H，R249S和R275H）也显着降低了癌细胞不对称地分裂其模板DNA链的频率。该对照是短暂的，一旦去除p53，不对称分裂的频率再次增加。不对称分裂的调节不受p53同工型p53beta和delta133的调节。使用记者构造的构造来监测茎和分化蛋白标记的表达，我们目前正在研究哪种形式的自我更新，对称自我更新，对称分化或不对称分裂，p53，delta133及其各自的突变。具体而言，我们将使用荧光记者为SPC启动子作为分化细胞的标记，而荧光报告基OCT4启动子活性作为干细胞的标记。我们先前已经表明，非板板DNA链与SPC共隔离，而OCT-4是肺癌干细胞的标志。