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.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIABC011496-01
Application #
8763568
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
1
Fiscal Year
2013
Total Cost
$802,217
Indirect Cost
Name
National Cancer Institute Division of Basic Sciences
Department
Type
DUNS #
City
State
Country
Zip Code
Nguyen, Giang Huong; Tang, Weiliang; Robles, Ana I et al. (2014) Regulation of gene expression by the BLM helicase correlates with the presence of G-quadruplex DNA motifs. Proc Natl Acad Sci U S A 111:9905-10