Genomic instability in induced stem cells

The ability to reprogram adult cells into stem cells has raised hopes for novel therapies for many human diseases. Typical stem cell reprogramming protocols involve expression of a small number of genes in differentiated somatic cells with the c-Myc and Klf4 proto-oncogenes typically included in this mix. We have previously shown that expression of oncogenes leads to DNA replication stress and genomic instability, explaining the high frequency of p53 mutations in human cancers. Consequently, we wondered whether stem cell reprogramming also leads to genomic instability. To test this hypothesis, we examined stem cells induced by a variety of protocols. The first protocol, developed specifically for this study, reprogrammed primary mouse mammary cells into mammary stem cells by expressing c-Myc. Two other previously established protocols reprogrammed mouse embryo fibroblasts into induced pluripotent stem cells by expressing either three genes, Oct4, Sox2 and Klf4, or four genes, OSK plus c-Myc. Comparative genomic hybridization analysis of stem cells derived by these protocols revealed the presence of genomic deletions and amplifications, whose signature was suggestive of oncogene-induced DNA replication stress. The genomic aberrations were to a significant degree dependent on c-Myc expression and their presence could explain why p53 inactivation facilitates stem cell reprogramming.     

Differential remodeling of extracellular matrices by breast cancer initiating cells

Cancer initiating cells (CICs) have been the focus of recent anti‐cancer therapies, exhibiting strong invasion capability via potentially enhanced ability to remodel extracellular matrices (ECM). We have identified CICs in a human breast cancer cell line, MX‐1, and developed a xenograft model in SCID mice. We investigated the CICs' matrix‐remodeling effects using Second Harmonic Generation (SHG) microscopy to identify potential phenotypic signatures of the CIC‐rich tumors. The isolated CICs exhibit higher proliferation, drug efflux and drug resistant properties in vitro; were more tumorigenic than non‐CICs, resulting in more and larger tumors in the xenograft model. The CIC‐rich tumors have less collagen in the tumor interior than in the CIC‐poor tumors supporting the idea that the CICs can remodel the collagen more effectively. The collagen fibers were preferentially aligned perpendicular to the CIC‐rich tumor boundary while parallel to the CIC‐poor tumor boundary suggesting more invasive behavior of the CIC‐rich tumors. These findings would provide potential translational values in quantifying and monitoring CIC‐rich tumors in future anti‐cancer therapies.

CIC‐rich tumors remodel the collagen matrix more than CIC‐poor tumors.     

p53 in stem cells

p53 is well known as a "guardian of the genome" for differentiated cells,in which it induces cell cycle arrest and cell death after DNA damage and thus contributes to the maintenance of genomic stability.In addition to this tumor suppressor function for differentiated cells,p53 also plays an important role in stem cells.In this cell type,p53 not only ensures genomic integrity after genotoxic insults but also controls their proliferation and differentiation.Additionally,p53 provides an effective barrier for the generation of pluripotent stem celllike cells from terminally differentiated cells.In this review,we summarize our current knowledge about p53 activities in embryonic,adult and induced pluripotent stem cells.     

Genomic convergence and suppression of centrosome hyperamplification in primary p53-/- cells in prolonged culture

Chromosome instability, a major property of cancer cells, is believed to promote mutations that establish malignant phenotypes. Centrosome hyperamplification and the consequential increase in the frequency of aberrant mitoses are the major causes of chromosome instability in cancer cells that lack the functional p53 tumor suppressor protein. Here, we examined dynamic changes of chromosome and centrosome behaviors during long-term culturing of primary epithelial cells derived from p53-null mice. The heterogeneity in the number of chromosomes per cell in the early to mid passage cell population diminished in late passage cells, giving rise to distinct subpopulations of cells. Concomitantly, centrosome hyperamplification that was observed at a high frequency in early to mid passage cells was suppressed in late passage cells. These results provide an explanation for the frequent observations that some cancer cell lines and tissues that lack functional p53 show normal centrosome behaviors and altered, yet relatively stable, chromosomes. Moreover, our in vitro findings may provide a model for possible genomic convergence in cultured cells. This may be analogous to the genomic convergence model proposed for in vivo tumor progression in which chromosome instability initially imposed during tumorigenesis becomes suppressed when neoplastic cells have acquired chromosome compositions that promise an optimal growth in a given environment.     

The emerging functions of the p53-miRNA network in stem cell biology

The p53 pathway plays an essential role in tumor suppression, regulating multiple cellular processes coordinately to maintain genome integrity in both somatic cells and stem cells. Despite decades of research dedicated to p53 function in differentiated somatic cells, we are just starting to understand the complexity of the p53 pathway in the biology of pluripotent stem cells and tissue stem cells. Recent studies have demonstrated that p53 suppresses proliferation, promotes differentiation of embryonic stem (ES) cells and constitutes an important barrier to somatic reprogramming. In addition, emerging evidence reveals the role of the p53 network in the self-renewal, proliferation and genomic integrity of adult stem cells. Interestingly, non-coding RNAs, and microRNAs in particular, are integral components of the p53 network, regulating multiple p53-controlled biological processes to modulate the self-renewal and differentiation potential of a variety of stem cells. Thus, elucidation of the p53-miRNA axis in stem cell biology may generate profound insights into the mechanistic overlap between malignant transformation and stem cell biology.     

p53 regulates the proliferation, differentiation and spontaneous transformation of mesenchymal stem cells

Mesenchymal stem cells (MSC) have been extensively studied and gained wide popularity due to their therapeutic potential. Spontaneous transformation of MSC, from both human and murine origin, has been reported in many studies. MSC transformation depends on the culture conditions, the origin of the cells and the time on culture; however, the precise biological characteristics involved in this process have not been fully defined yet. In this study, we investigated the role of p53 in the biology and transformation of murine bone marrow (BM)-derived MSC. We demonstrate that the MSC derived from p53KO mice showed an augmented proliferation rate, a shorter doubling time and also morphologic and phenotypic changes, as compared to MSC derived from wild-type animals. Furthermore, the MSC devoid of p53 had an increased number of cells able to generate colonies. In addition, not only proliferation but also MSC differentiation is controlled by p53 since its absence modifies the speed of the process. Moreover, genomic instability, changes in the expression of c-myc and anchorage independent growth were also observed in p53KO MSC. In addition, the absence of p53 implicates the spontaneous transformation of MSC in long-term cultures. Our results reveal that p53 plays a central role in the biology of MSC.     

Long-term cultured mesenchymal stem cells frequently develop genomic mutations but do not undergo malignant transformation

Cultured human umbilical cord mesenchymal stem cells (hUC-MSCs) are being tested in several clinical trials and encouraging outcomes have been observed. To determine whether in vitro expansion influences the genomic stability of hUC-MSCs, we maintained nine hUC-MSC clones in long-term culture and comparatively analyzed them at early and late passages. All of the clones senesced in culture, exhibiting decreased telomerase activity and shortened telomeres. Two clones showed no DNA copy number variations (CNVs) at passage 30 (P30). Seven clones had ≥1 CNVs at P30 compared with P3, and one of these clones appeared trisomic chromosome 10 at the late passage. No tumor developed in immunodeficient mice injected with hUC-MSCs, regardless of whether the cells had CNVs at the late passage. mRNA-Seq analysis indicated that pathways of cell cycle control and DNA damage response were downregulated during in vitro culture in hUC-MSC clones that showed genomic instability, but the same pathways were upregulated in the clones with good genomic stability. These results demonstrated that hUC-MSCs can be cultured for many passages and attain a large number of cells, but most of the cultured hUC-MSCs develop genomic alterations. Although hUC-MSCs with genomic alterations do not undergo malignant transformation, periodic genomic monitoring and donor management focusing on genomic stability are recommended before these cells are used for clinical applications.     

Hyperproliferation and p53 status of lens epithelial cells derived from alphaB-crystallin knockout mice

alphaB-Crystallin, a major protein of lens fiber cells, is a stress-induced chaperone expressed at low levels in the lens epithelium and numerous other tissues, and its expression is enhanced in certain pathological conditions. However, the function of alphaB in these tissues is not known. Lenses of alphaB-/- mice develop degeneration of specific skeletal muscles but do not develop cataracts. Recent work in our laboratory indicates that primary cultures of alphaB-/- lens epithelial cells demonstrate genomic instability and undergo hyperproliferation at a frequency 4 orders of magnitude greater than that predicted by spontaneous immortalization of rodent cells. We now demonstrate that the hyperproliferative alphaB-/- lens epithelial cells undergo phenotypic changes that include the appearance of the p53 protein as shown by immunoblot analysis. Sequence analysis showed a lack of mutations in the p53 coding region of hyperproliferative alphaB-/- cells. However, the reentry of hyperproliferative alphaB-/- cells into S phase and mitosis after DNA damage by gamma-irradiation were consistent with impaired p53 checkpoint function in these cells. The results demonstrate that expression of functionally impaired p53 is one of the factors that promote immortalization of lens epithelial cells derived from alphaB-/- mice. Fluorescence in situ hybridization using probes prepared from centromere-specific mouse P1 clones of chromosomes 1 and 9 demonstrated that the hyperproliferative alphaB-/- cells were 30% diploid and 70% tetraploid, whereas wild type cells were 83% diploid. Further evidence of genomic instability was obtained when the hyperproliferative alphaB-/- cells were labeled with anti-beta-tubulin antibodies. Examination of the hyperproliferative alphaB-/- mitotic profiles revealed the presence of cells that failed to round up for mitosis, or arrested in cytokinesis, and binucleated cells in which nuclear division had occurred without cell division. These results suggest that the stress protein and molecular chaperone alphaB-crystallin protects cells from acquiring impaired p53 protein and genomic instability.     

Reactivating the ARF-p53 axis in AML cells by targeting ULF

The tumor suppressor ARF plays an essential role in the cellular response to oncogenic stress mainly through activation of p53. Nucleophosmin (NPM), a multifunctional protein, forms a stable protein complex with ARF in the nucleolus and protects ARF from the proteasome-mediated degradation. Notably, NPM is mutated in about one third of acute myeloid leukaemia (AML) patients and these mutations lead to aberrant cytoplasmic dislocation of nucleophosmin (NPM-c). Cytoplasmic NPM mutants lose their abilities to retain ARF in the nucleolus and fail to stabilize ARF. Thus, activation of the ARF-p53 axis is significantly compromised in these AML cells. We have recently identified the ubiquitin ligase of ARF (ULF) as a key factor that controls ARF turnover in human cells. Here, we found that the steady levels of both ARF and p53 are very low in human acute myeloid leukaemia OCI-AML3 cells expressing cytoplamsic dislocated nucleophosmin (NPM-c). As expected, ARF is very unstable and rapidly degraded by proteasome. Nevertheless, ULF knockdown stabilizes ARF and reactivates p53 responses in these AML cells. These results further demonstrate that ULF is a bona fide E3 ligase for ARF and also suggest that ULF is an important target for activating the ARF-p53 axis in human AML cells.Key words: ARF, ubiquitination, ULF, p53, NPM, B23, NPM-c     

Pluripotent stem cell-derived neural stem cells: From basic research to applications

Basic research on pluripotent stem cells is designed to enhance understanding of embryogenesis, whereas applied research is designed to develop novel therapies and prevent diseases. Attainment of these goals has been enhanced by the establishment of embryonic stem cell lines, the technological development of genomic reprogramming to generate induced-pluripotent stem cells, and improvements in vitro techniques to manipulate stem cells. This review summarizes the techniques required to generate neural cells from pluripotent stem cells. In particular, this review describes current research applications of a simple neural differentiation method, the neural stem sphere method, which we developed.     

Escape from premature senescence is not sufficient for oncogenic transformation by Ras

Resistance of primary cells to transformation by oncogenic Ras has been attributed to the induction of replicative growth arrest. This irreversible 'fail-safe mechanism' resembles senescence and requires induction by Ras of p19ARF and p53 (refs 3-5). Mutation of either p19ARF or p53 alleviates Ras-induced senescence and facilitates oncogenic transformation by Ras. Here we report that, whereas Rb and p107 are each dispensable for Ras-induced replicative arrest, simultaneous ablation of both genes disrupts Ras-induced senescence and results in unrestrained proliferation. This occurs despite activation by Ras of the p19ARF /p53 pathway, identifying pRb and p107 as essential mediators of Ras-induced antiproliferative p19ARF/p53 signalling. Unexpectedly, in contrast to p19ARF or p53 deficiency, loss of Rb/p107 function does not result in oncogenic transformation by Ras, as Ras-expressing Rb-/-/p107-/- fibroblasts fail to grow anchorage-independently in vitro and are not tumorigenic in vivo. These results demonstrate that in the absence of both Rb and p107 cells are resistant to p19ARF/p53-dependent protection against Ras-induced proliferation, and uncouple escape from Ras-induced premature senescence from oncogenic transformation.     

Cellular models of development of ovarian high‐grade serous carcinoma: A review of cell of origin and mechanisms of carcinogenesis

High‐grade serous carcinoma (HGSC) is the most common and malignant histological type of epithelial ovarian cancer, the origin of which remains controversial. Currently, the secretory epithelial cells of the fallopian tube are regarded as the main origin and the ovarian surface epithelial cells as a minor origin. In tubal epithelium, these cells acquire TP53 mutations and expand to a morphologically normal ‘p53 signature’ lesion, transform to serous tubal intraepithelial carcinoma and metastasize to the ovaries and peritoneum where they develop into HGSC. This shifting paradigm of the main cell of origin has revolutionarily changed the focus of HGSC research. Various cell lines have been derived from the two cellular origins by acquiring immortalization via overexpression of hTERT plus disruption of TP53 and the CDK4/RB pathway. Malignant transformation was achieved by adding canonical driver mutations (such as gain of CCNE1) revealed by The Cancer Genome Atlas or by noncanonical gain of YAP and miR181a. Alternatively, because of the extreme chromosomal instability, spontaneous transformation can be achieved by long passage of murine immortalized cells, whereas in humans, it requires ovulatory follicular fluid, containing regenerating growth factors to facilitate spontaneous transformation. These artificially and spontaneously transformed cell systems in both humans and mice have been widely used to discover carcinogens, oncogenic pathways and malignant behaviours in the development of HGSC. Here, we review the origin, aetiology and carcinogenic mechanism of HGSC and comprehensively summarize the cell models used to study this fatal cancer having multiple cells of origin and overt genomic instability.

The genetic alterations and known mechanism of transformation by ovulation and retrograde menstruation in the development of HGSC from the FTE.     

Reactivating the ARF-p53 axis in AML cells by targeting ULF

The tumor suppressor ARF plays an essential role in the cellular response to oncogenic stress mainly through activation of p53. Nucleophosmin (NPM), a multifunctional protein, forms a stable protein complex with ARF in the nucleolus and protects ARF from the proteasome-mediated degradation. Notably, NPM is mutated in about one third of acute myeloid leukaemia (AML) patients and these mutations lead to aberrant cytoplasmic dislocation of nucleophosmin (NPM-c). Cytoplasmic NPM mutants lose their abilities to retain ARF in the nucleolus and fail to stabilize ARF. Thus, activation of the ARF-p53 axis is significantly compromised in these AML cells. We have recently identified the ubiquitin ligase of ARF (ULF) as a key factor that controls ARF turnover in human cells. Here, we found that the steady levels of both ARF and p53 are very low in human acute myeloid leukaemia OCI-AML3 cells expressing cytoplamsic dislocated nucleophosmin (NPM-c). As expected, ARF is very unstable and rapidly degraded by proteasome. Nevertheless, ULF knockdown stabilizes ARF and reactivates p53 responses in these AML cells. These results further demonstrate that ULF is a bona fide E3 ligase for ARF and also suggest that ULF is an important target for activating the ARF-p53 axis in human AML cells.     

p53 and p21 form an inducible barrier that protects cells against cyclin E-cdk2 deregulation

BACKGROUND: Cyclin E, in conjunction with its catalytic partner cdk2, is rate limiting for entry into the S phase of the cell cycle. Cancer cells frequently contain mutations within the cyclin D-Retinoblastoma protein pathway that lead to inappropriate cyclin E-cdk2 activation. Although deregulated cyclin E-cdk2 activity is believed to directly contribute to the neoplastic progression of these cancers, the mechanism of cyclin E-induced neoplasia is unknown. RESULTS: We studied the consequences of deregulated cyclin E expression in primary cells and found that cyclin E initiated a p53-dependent response that prevented excess cdk2 activity by inducing expression of the p21Cip1 cdk inhibitor. The increased p53 activity was not associated with increased expression of the p14ARF tumor suppressor. Instead, cyclin E led to increased p53 serine15 phosphorylation that was sensitive to inhibitors of the ATM/ATR family. When either p53 or p21cip1 was rendered nonfunctional, then the excess cyclin E became catalytically active and caused defects in S phase progression, increased ploidy, and genetic instability. CONCLUSIONS: We conclude that p53 and p21 form an inducible barrier that protects cells against the deleterious consequences of cyclin E-cdk2 deregulation. A response that restrains cyclin E deregulation is likely to be a general protective mechanism against neoplastic transformation. Loss of this response may thus be required before deregulated cyclin E can become fully oncogenic in cancer cells. Furthermore, the combination of excess cyclin E and p53 loss may be particularly genotoxic, because cells cannot appropriately respond to the cell cycle anomalies caused by excess cyclin E-cdk2 activity.     

p14ARF inhibits the growth of p53 deficient cells in a cell-specific manner

While p14(ARF) suppression of tumorigenesis in a p53-dependent manner is well studied, the mechanism by which p14(ARF) inhibits tumorigenesis independently of p53 remains elusive. A variety of factors have been reported to play a role in this latter process. We report here that p14(ARF) displays different effects on the anchorage-dependent and -independent growth of p53-null/Mdm2 wild type cells. p14(ARF) blocks both the anchorage-dependent and-independent (soft agar) proliferation of 293T and p53(-/-) HCT116, but not p53-null H1299 lung carcinoma cells. While p14(ARF) had no effect on the anchorage-dependent proliferation of p53(-/-) MEFs and Ras12V-transformed p53(-/-) MEFs, it inhibited the growth of Ras12V-transformed p53(-/-) MEFs in soft agar. Furthermore, ectopic expression of p14(ARF) did not lead to degradation of the E2F1 protein and did not result in the reduction of E2F1 activity detected by two E2F1 responsible promoters, Apaf1 and p14(ARF) promoter, in 293T, p53(-/-) HCT116, and H1299 cells. This is consistent with our observations that p14(ARF) did not result in G1 arrest, but induced apoptosis via Bax up-regulation. Taken together, our data demonstrate that the response of p53-null cells to ARF is cell type dependent and involves factors other than Mdm2 and E2F1.