p53, Aging, and Cancer

具体目的1：研究p53和miRNAs在复制应激和干细胞生物学中的分子节点作用假设：不同机制调节p53同工异构体的表达水平以协调复制性衰老我们报道了由内含子4的替代启动子转录产生的delta133p53的表达水平主要在蛋白质水平而不是转录水平上受到调节。与全长p53和p53beta受蛋白酶体降解不同，delta133p53的蛋白质周转是通过自噬蛋白降解特异性调节的。用巴菲霉素A1而不是MG-132治疗，恢复了复制性衰老的人成纤维细胞中的delta133p53蛋白。敲低自噬介导基因（即ATG5、ATG7和Bechlin-1）也恢复了delta133p53的表达减少。Delta133p53在C端赖氨酸残基泛素化，在自噬诱导下，与p62/SQSTM1（泛素化自噬底物的接头）和LC3-II（自噬体成分）共定位在细胞质中。这种同工异构体特异性的转换机制降解了delta133p53并消除了其对全长p53的作用，同时维持了全长p53的水平，协调了p53介导的复制性衰老。我们的数据显示，delta133p53与Hsp70伴侣、伴侣相关的E3泛素连接酶STUB1（但不包括与全长p53相互作用的MDM2）和伴侣调节因子BAG2相互作用，表明伴侣介导的自噬在delta133p53降解中起作用。由于在复制性衰老过程中，STUB1表达下调，BAG2表达上调，因此STUB1和/或BAG2可能调控了delta133p53的自噬降解。我们的目的是阐明delta133p53介导的细胞衰老和自噬之间的功能相互作用的分子基础和生物学意义，这两个主要过程涉及癌症和衰老。假设：p53亚型转换是复制性衰老的生理机制。为了研究p53亚型在体内的生理作用，我们使用循环CD8+ T淋巴细胞，这些细胞在体内经历一系列分化阶段，细胞表面抗原发生特异性变化（例如CD28的丢失和CD57的获得），从而走向复制性衰老。我们发现供体年龄依赖性CD8+ T淋巴细胞积累具有CD28 CD57+和衰老表型，包括增殖抑制，端粒缩短，HP1 γ和γ H2AX灶增加，衰老相关分泌表型（SASP）和衰老相关β -半乳糖苷酶活性。在这些体内积累的衰老或接近衰老的CD8+ T淋巴细胞中，内源性的delta133p53和p53beta分别下调和上调。在增殖能力差、delta133p53表达水平低的CD28阴性人群中，重建delta133p53和CD28的表达，恢复细胞增殖，延长复制寿命。相反，在高度增殖的CD28阳性群体中，敲低delta133p53或过表达p53beta会抑制细胞增殖并诱导衰老。Delta133p53和p53beta是CD8+ T淋巴细胞增殖和衰老的内源性调节因子，提示Delta133p53表达增强可能导致非增殖或最不增殖的CD8+ T淋巴细胞群的功能激活，这些细胞在老年人或人类免疫缺陷病毒感染患者中积累。重组后的delta133p53在mRNA和蛋白水平上上调CD28的表达。我们正在研究全长p53是否会在转录上抑制CD28，以及delta133p53对全长p53的抑制是否会导致CD28的转录上调。我们正在研究肿瘤微环境中肿瘤细胞与CD8+ T淋巴细胞之间可能的相互作用。我们从人肺癌组织中初步分离的肿瘤相关CD8+ T淋巴细胞表明，它们含有具有衰老表型（例如HP1-gamma灶和SASP）的CD28+ CD57+群体。肿瘤细胞对衰老表型和CD8+ T淋巴细胞中p53亚型表达的直接影响也将通过体外共培养这两种细胞类型来研究。假设：p53同工型是人类多能干细胞的生理调节因子。由于p53信号网络涉及干细胞生物学的各个方面，p53同工型可能在人类干细胞中具有生理作用。与先前报道的p53敲低相似，我们发现人成纤维细胞中delta133p53的过表达增加了iPSC重编程的效率。由于delta133p53优先抑制一组参与细胞衰老的p53诱导基因（如p21WAF1和miR-34a），而p53敲低会抑制所有p53诱导基因，包括参与DNA损伤反应的基因，因此我们假设delta133p53过表达是一种增强iPSC生成的方法，没有遗传畸变或恶性转化的风险。为了验证这一假设，我们通过常规核型、谱核型、mRNA和miRNA表达谱以及畸胎瘤形成分析来表征delta133p53诱导的iPSC。人类多能干细胞是内源性delta133p53蛋白表达最丰富的细胞类型。虽然全长p53的表达水平在hESC和iPSC系之间差异很大，但所有hESC和iPSC系的delta133p53蛋白表达水平始终高于人成纤维细胞，而delta133p53 mRNA表达水平仅略有增加。我们目前正在研究内源性delta133p53的上调是否源于抑制其自噬降解。为了阐明上调的delta133p53在hESC和iPSC的自我更新、多能性和分化潜能中的作用，我们将在这些细胞中进行内源性delta133p53 shRNA敲低。假设：p53同工异构体和突变体调节对称和非对称细胞分裂非对称细胞分裂使干细胞自我更新并产生致力于分化的细胞，从而促进正常组织稳态和癌症进展。我们之前证明了一小部分癌细胞具有不对称分裂其模板DNA链的能力，并且这种分离与细胞命运相关。为了进一步探讨癌细胞对称和非对称自我更新平衡的调控，我们研究了p53及其同型体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）也显著降低了癌细胞亚群不对称分裂其模板DNA链的频率。这种控制是短暂的，一旦p53被移除，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。利用监测干细胞和分化蛋白标记物表达的报告结构，我们目前正在研究p53、delta133及其各自突变调控的是自我更新、对称自我更新、对称分化还是不对称分裂的哪一种形式。具体来说，我们将使用SPC启动子的荧光报告子作为分化细胞的标记，使用OCT4启动子活性的荧光报告子作为干细胞的标记。我们之前已经证明，非模板DNA链与SPC共分离，而OCT-4是肺癌干细胞的标记物。

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.