Igor Shats

Activated p53 suppresses the histone methyltransferase EZH2 gene

Replicative senescence is an irreversible cell cycle arrest that limits the proliferation of damaged cells and may be an important tumor suppression mechanism in vivo. This process is regulated at critical steps by the tumor suppressor p53. To identify genes that may regulate the senescence process, we performed cDNA microarray analysis of gene expression in senescent, young proliferating, and hTERT-immortalized primary human fibroblasts. The histone methyltransferase (HMTase), EZH2, was specifically downregulated in senescent cells. Activated p53 suppressed EZH2 gene expression through repression of the EZH2 gene promoter. This activity of p53 requires intact p53 transactivation and DNA binding domains. Furthermore, the repression of EZH2 promoter by p53 is dependent on p53 transcriptional target p21Waf1 inactivating RB/E2F pathways. In addition, the knockdown of EZH2 expression retards cell proliferation and induces G2/M arrest. We suggest that the p53-dependent suppression of EZH2 expression is a novel pathway that contributes to p53-mediated G2/M arrest. EZH2 associated complex possesses HMTase activity and is involved in epigenetic regulation. Activated p53 suppresses EZH2 expression, suggesting a further role for p53 in epigenetic regulation and in the maintenance of genetic stability. Suppression of EZH2 expression in tumors by p53 may lead to novel approaches to control cancer progression.

Regulation of AIF expression by p53

The tumor suppressor p53 plays a pivotal role in suppressing tumorigenesis by inducing genomic stability, cell cycle arrest or apoptosis. AIF is a mitochondrial protein, which, upon translocation to the nucleus, can participate in apoptosis, primarily in a caspase-independent contexts. We now report that AIF gene expression is subject to positive transcriptional regulation by p53. Interestingly, unlike most known p53 target genes, the AIF gene is regulated by basal levels of p53, and activation of p53 by genotoxic stress does not result in a substantial further increase in AIF expression. The AIF gene harbors a p53 responsive element, which is bound by p53 within cells. p53 drives efficient induction of large-scale DNA fragmentation, a hallmark of AIF activity. Importantly, caspase-independent death is compromised in cells lacking functional p53, in line with the known role of AIF in this process. Thus, in addition to its documented effects on caspase-dependent apoptosis, p53 may also sensitize cells to caspase-independent death through positive regulation of AIF expression. Moreover, in the absence of overt apoptotic signals, the constitutive induction of AIF by p53 may underpin a cytoprotective maintenance role, based on the role of AIF in ensuring proper mitochondrial function.

p53 plays a role in mesenchymal differentiation programs, in a cell fate dependent manner.

Background The tumor suppressor p53 is an important regulator that controls various cellular networks, including cell differentiation. Interestingly, some studies suggest that p53 facilitates cell differentiation, whereas others claim that it suppresses differentiation. Therefore, it is critical to evaluate whether this inconsistency represents an authentic differential p53 activity manifested in the various differentiation programs. Methodology/Principal Findings To clarify this important issue, we conducted a comparative study of several mesenchymal differentiation programs. The effects of p53 knockdown or enhanced activity were analyzed in mouse and human mesenchymal cells, representing various stages of several differentiation programs. We found that p53 down-regulated the expression of master differentiation-inducing transcription factors, thereby inhibiting osteogenic, adipogenic and smooth muscle differentiation of multiple mesenchymal cell types. In contrast, p53 is essential for skeletal muscle differentiation and osteogenic re-programming of skeletal muscle committed cells. Conclusions These comparative studies suggest that, depending on the specific cell type and the specific differentiation program, p53 may exert a positive or a negative effect, and thus can be referred as a “guardian of differentiation” at large.

hTERT-Immortalized Prostate Epithelial and Stromal-Derived Cells: an Authentic In vitro Model for Differentiation and Carcinogenesis

Prostate cancer is the most commonly diagnosed type of cancer in men, and there is no available cure for patients with advanced disease. In vitro model systems are urgently required to permit the study of human prostate cell differentiation and malignant transformation. Unfortunately, human prostate cells are particularly difficult to convert into continuously growing cultures. We report here the successful immortalization without viral oncogenes of prostate epithelial cells and, for the first time, prostate stromal cells. These cells exhibit a significant pattern of authentic prostate-specific features. In particular, the epithelial cell culture is able to differentiate into glandular buds that closely resemble the structures formed by primary prostate epithelial cells. The stromal cells have typical characteristics of prostate smooth muscle cells. These immortalized cultures may serve as a unique experimental platform to permit several research directions, including the study of cell-cell interactions in an authentic prostate microenvironment, prostate cell differentiation, and most significantly, the complex multistep process leading to prostate cell transformation.

The promoters of human cell cycle genes integrate signals from two tumor suppressive pathways during cellular transformation.

Deciphering regulatory events that drive malignant transformation represents a major challenge for systems biology. Here, we analyzed genome‐wide transcription profiling of an in vitro cancerous transformation process. We focused on a cluster of genes whose expression levels increased as a function of p53 and p16INK4A tumor suppressors inactivation. This cluster predominantly consists of cell cycle genes and constitutes a signature of a diversity of cancers. By linking expression profiles of the genes in the cluster with the dynamic behavior of p53 and p16INK4A, we identified a promoter architecture that integrates signals from the two tumor suppressive channels and that maps their activity onto distinct levels of expression of the cell cycle genes, which, in turn, correspond to different cellular proliferation rates. Taking components of the mitotic spindle as an example, we experimentally verified our predictions that p53‐mediated transcriptional repression of several of these novel targets is dependent on the activities of p21, NFY, and E2F. Our study demonstrates how a well‐controlled transformation process allows linking between gene expression, promoter architecture, and activity of upstream signaling molecules.

Transcriptional Programs following Genetic Alterations in p53, INK4A, and H-Ras Genes along Defined Stages of Malignant Transformation

The difficulty to dissect a complex phenotype of established malignant cells to several critical transcriptional programs greatly impedes our understanding of the malignant transformation. The genetic elements required to transform some primary human cells to a tumorigenic state were described in several recent studies. We took the advantage of the global genomic profiling approach and tried to go one step further in the dissection of the transformation network. We sought to identify the genetic signatures and key target genes, which underlie the genetic alterations in p53, Ras, INK4A locus, and telomerase, introduced in a stepwise manner into primary human fibroblasts. Here, we show that these are the minimally required genetic alterations for sarcomagenesis in vivo. A genome-wide expression profiling identified distinct genetic signatures corresponding to the genetic alterations listed above. Most importantly, unique transformation hallmarks, such as differentiation block, aberrant mitotic progression, increased angiogenesis, and invasiveness, were identified and coupled with genetic signatures assigned for the genetic alterations in the p53, INK4A locus, and H-Ras, respectively. Furthermore, a transcriptional program that defines the cellular response to p53 inactivation was an excellent predictor of metastasis development and bad prognosis in breast cancer patients. Deciphering these transformation fingerprints, which are affected by the most common oncogenic mutations, provides considerable insight into regulatory circuits controlling malignant transformation and will hopefully open new avenues for rational therapeutic decisions.

Hypoxia-dependent regulation of PHD1: cloning and characterization of the human PHD1/EGLN2 gene promoter.

The recent identification of hypoxia‐inducible‐factor (HIF) prolyl hydroxylases (PHD1, 2, and 3), which modify HIF‐1α in an oxygen‐dependent manner, provided an important link between oxygen availability and hypoxia‐induced gene expression. However, little is known about the regulation of the PHDs. To investigate the transcriptional regulation of PHD1, we cloned the PHD1 gene promoter. Here, we report that the expression of PHD1 is reduced under hypoxic conditions. Furthermore, we identified binding sites for aryl hydrocarbon nuclear translocator (ARNT/HIF‐1β) within the PHD1 promoter, and showed that ARNT is associated in vivo with the PHD1 promoter following hypoxia, which implies a role for ARNT in the hypoxia‐dependent regulation of PHD1. Taken together, our findings suggest a hypoxia‐induced regulatory loop of PHD1 expression, mediated by ARNT.

p53 is a regulator of macrophage differentiation

AbstractWhile it is well accepted that p53 plays a role in apoptosis, less is known as to its involvement in cell differentiation. Here we show that wild-type p53 facilitates IL-6-dependent macrophage differentiation. Treatment of M1/2 cells expressing the temperature-sensitive p53 143 (Val to Ala) mutant, at the wild-type conformation, facilitated the appearance of mature macrophages that exhibited phagocytic activity. Enhancement of differentiation by the p53 143 (Val to Ala) in the wild-type conformation was coupled with the inhibition of apoptosis induction by this protein. In agreement with previous studies, we found that p53 levels were reduced during p53-dependent macrophage differentiation. This occurred when p53 levels before IL-6 stimuli were high. Interestingly, the p53 143 (Val to Ala) protein, at the mutant conformation, enhanced macrophage differentiation, as did the wild-type conformation, whereas the p53 273 (Arg to His) core mutant exerted an inhibitory effect on this pathway. The transcription-deficient p53 molecules, p53 (22–23) and p53 22,23,143, could not induce p53-dependent differentiation. Moreover, the p53 (22–23) protein inhibited the p53-independent differentiation pathway. Interestingly, the p53 (22–23) protein not only blocked IL-6-mediated differentiation, but also induced significant apoptotic cell death, upon IL-6 stimulation. Taken together, our data show that wild-type p53 enhances macrophage differentiation, while various p53 mutant types exert different effects on this differentiation pathway.

Myocardin in Tumor Suppression and Myofibroblast Differentiation

The malignant transformation process is associated with defects in cell cycle regulation and disruption of the normal differentiation programs in both neoplastic and adjacent stroma cells. However, the relationships between the cell cycle, differentiation and cancer are very complex and tissue specific. Recently we have demonstrated a previously unrecognized role in human carcinogenesis for the important regulator of cardiac and smooth muscle differentiation, myocardin. Myocardin expression is frequently repressed during human malignant transformation contributing to a differentiation defect in the premalignant mesenchymal cells. TGFβ treatment, serum deprivation and intact contact inhibition response all contribute to myocardin induction and differentiation. Positive regulation of myocardin mRNA levels and activity by the p16/Rb pathway provides a molecular link between cell cycle and differentiation defects during cancer development. In addition, we show that myocardin represses its own expression in human fibroblasts. This negative autoregulatory loop might be potentially important for restraining myocardin activity and allowing reversibility of fibroblast-myofibroblast phenotypic conversion.Here we discuss the emerging role of myocardin in tumor suppression as well as novel aspects of its regulation in normal and malignant conditions.

Upregulation of survivin during immortalization of nontransformed human fibroblasts transduced with telomerase reverse transcriptase

These investigations demonstrate that expression of the inhibitor of apoptosis family member, survivin, is dramatically increased during immortalization of nontransformed human fibroblasts that were transduced with telomerase reverse transcriptase (hTERT). Expression of survivin in immortalized fibroblasts peaked during G2/M phase of the cell cycle. However, the upregulation of survivin was dissociated from the rate of proliferation and proportion of G2/M cells. Depletion of survivin from immortal fibroblasts increased sensitivity to stress-induced apoptosis and resulted in an accumulation of cells with 4N DNA content. Conversely, overexpression of survivin in mortal fibroblasts conferred resistance to apoptosis. In contrast, very low levels of survivin in proliferating parental fibroblasts had no bearing on sensitivity to apoptosis. The upregulation of survivin did not appear to be a direct consequence of hTERT transduction. However, repression of hTERT resulted in the rapid downregulation of survivin in telomerase-immortalized fibroblasts and tumor cell lines, but not in cells immortalized via an Alternative Lengthening of Telomeres mechanism. These results have important therapeutic implications, as telomerase and survivin are both broadly expressed in human cancers. Selection during the immortalization process for cells expressing high levels of survivin may account for the abundance of survivin in diverse tumor types.