Ran Brosh

When mutants gain new powers: news from the mutant p53 field

Ample data indicate that mutant p53 proteins not only lose their tumour suppressive functions, but also gain new abilities that promote tumorigenesis. Moreover, recent studies have modified our view of mutant p53 proteins, portraying them not as inert mutants, but rather as regulated proteins that influence the cancer cell transcriptome and phenotype. This influence is clinically manifested as association of TP53 mutations with poor prognosis and drug resistance in a growing array of malignancies. Here, we review recent studies on mutant p53 regulation, gain-of-function mechanisms, transcriptional effects and prognostic association, with a focus on the clinical implications of these findings.

Mutations in the p53 Tumor Suppressor Gene Important Milestones at the Various Steps of Tumorigenesis

Inactivation of the p53 tumor suppressor is a frequent event in tumorigenesis. In most cases, the p53 gene is mutated, giving rise to a stable mutant protein whose accumulation is regarded as a hallmark of cancer cells. Mutant p53 proteins not only lose their tumor suppressive activities but often gain additional oncogenic functions that endow cells with growth and survival advantages. Interestingly, mutations in the p53 gene were shown to occur at different phases of the multistep process of malignant transformation, thus contributing differentially to tumor initiation, promotion, aggressiveness, and metastasis. Here, the authors review the different studies on the involvement of p53 inactivation at various stages of tumorigenesis and highlight the specific contribution of p53 mutations at each phase of cancer progression.

p53‐repressed miRNAs are involved with E2F in a feed‐forward loop promoting proliferation

Normal cell growth is governed by a complicated biological system, featuring multiple levels of control, often deregulated in cancers. The role of microRNAs (miRNAs) in the control of gene expression is now increasingly appreciated, yet their involvement in controlling cell proliferation is still not well understood. Here we investigated the mammalian cell proliferation control network consisting of transcriptional regulators, E2F and p53, their targets and a family of 15 miRNAs. Indicative of their significance, expression of these miRNAs is downregulated in senescent cells and in breast cancers harboring wild‐type p53. These miRNAs are repressed by p53 in an E2F1‐mediated manner. Furthermore, we show that these miRNAs silence antiproliferative genes, which themselves are E2F1 targets. Thus, miRNAs and transcriptional regulators appear to cooperate in the framework of a multi‐gene transcriptional and post‐transcriptional feed‐forward loop. Finally, we show that, similarly to p53 inactivation, overexpression of representative miRNAs promotes proliferation and delays senescence, manifesting the detrimental phenotypic consequence of perturbations in this circuit. Taken together, these findings position miRNAs as novel key players in the mammalian cellular proliferation network.

Epigenetic polymorphism and the stochastic formation of differentially methylated regions in normal and cancerous tissues

DNA methylation has been comprehensively profiled in normal and cancer cells, but the dynamics that form, maintain and reprogram differentially methylated regions remain enigmatic. Here, we show that methylation patterns within populations of cells from individual somatic tissues are heterogeneous and polymorphic. Using in vitro evolution of immortalized fibroblasts for over 300 generations, we track the dynamics of polymorphic methylation at regions developing significant differential methylation on average. The data indicate that changes in population-averaged methylation occur through a stochastic process that generates a stream of local and uncorrelated methylation aberrations. Despite the stochastic nature of the process, nearly deterministic epigenetic remodeling emerges on average at loci that lose or gain resistance to methylation accumulation. Changes in the susceptibility to methylation accumulation are correlated with changes in histone modification and CTCF occupancy. Characterizing epigenomic polymorphism within cell populations is therefore critical to understanding methylation dynamics in normal and cancer cells.

p53 is balancing development, differentiation and de-differentiation to assure cancer prevention.

Many of the roles played by the tumor suppressor p53 in restraining cancer initiation and progression are well established. These include the ability of p53 to induce cell-cycle arrest, DNA repair, senescence and apoptosis. In addition, during the 30 years of p53 research, numerous studies have implicated p53 in the regulation of differentiation and developmental pathways. Here, we summarize the data on these relatively less-characterized functions of p53, including its involvement in embryogenesis and various differentiation programs, as well as its function in restraining de-differentiation of mature somatic cells. Besides the well-known functions of p53 as a cell-cycle regulator and a mediator of apoptosis, both coincide with differentiation processes, p53 was shown to exert its effects on various differentiation programs via direct regulation of specific key factors controlling these programs. The complex regulation by p53, which acts to suppress or to induce differentiation, is mainly the result of the specific cell type and fate. We argue that regulation of differentiation is pivotal for the tumor-suppressive activity of p53, which act to maintain the proper cellular state, preventing improper maturation or reprogramming. This conclusion is further supporting the notion that aberrant differentiation is associated with malignant transformation.

TMPRSS2/ERG Promotes Epithelial to Mesenchymal Transition through the ZEB1/ZEB2 Axis in a Prostate Cancer Model

Prostate cancer is the most common non-dermatologic malignancy in men in the Western world. Recently, a frequent chromosomal aberration fusing androgen regulated TMPRSS2 promoter and the ERG gene (TMPRSS2/ERG) was discovered in prostate cancer. Several studies demonstrated cooperation between TMPRSS2/ERG and other defective pathways in cancer progression. However, the unveiling of more specific pathways in which TMPRSS2/ERG takes part, requires further investigation. Using immortalized prostate epithelial cells we were able to show that TMPRSS2/ERG over-expressing cells undergo an Epithelial to Mesenchymal Transition (EMT), manifested by acquisition of mesenchymal morphology and markers as well as migration and invasion capabilities. These findings were corroborated in vivo, where the control cells gave rise to discrete nodules while the TMPRSS2/ERG-expressing cells formed malignant tumors, which expressed EMT markers. To further investigate the general transcription scheme induced by TMPRSS2/ERG, cells were subjected to a microarray analysis that revealed a distinct EMT expression program, including up-regulation of the EMT facilitators, ZEB1 and ZEB2, and down-regulation of the epithelial marker CDH1(E-Cadherin). A chromatin immunoprecipitation assay revealed direct binding of TMPRSS2/ERG to the promoter of ZEB1 but not ZEB2. However, TMPRSS2/ERG was able to bind the promoters of the ZEB2 modulators, IL1R2 and SPINT1. This set of experiments further illuminates the mechanism by which the TMPRSS2/ERG fusion affects prostate cancer progression and might assist in targeting TMPRSS2/ERG and its downstream targets in future drug design efforts.

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.

Prostate stromal cells produce CXCL-1, CXCL-2, CXCL-3 and IL-8 in response to epithelia-secreted IL-1

It is well accepted that tumor microenvironment is essential for tumor cells survival, cancer progression and metastasis. However, the mechanisms by which tumor cells interact with their surrounding at early stages of cancer development are largely unidentified. The aim of this study was to identify specific molecules involved in stromal-epithelial interactions that might contribute to early stages of prostate tumor formation. Here, we show that conditioned medium (CM) from immortalized non-transformed prostate epithelial cells stimulated immortalized prostate stromal cells to express cancer-related molecules. CM obtained from epithelial cells triggered stromal cells to express and secrete CXCL-1, CXCL-2, CXCL-3 and interleukin (IL)-8 chemokines. This effect was predominantly mediated by the cytokines of the IL-1 family secreted by the epithelial cells. Thus, prostate epithelial cells induced the secretion of proinflammatory and cancer-promoting chemokines by prostate stromal cells. Such interactions might contribute to prostatic inflammation and progression at early stages of prostate cancer formation.

Modulated expression of WFDC1 during carcinogenesis and cellular senescence

Fibroblasts located adjacent to the tumor [cancer-associated fibroblasts (CAFs)] that constitute a large proportion of the cancer-associated stroma facilitate the transformation process. In this study, we compared the biological behavior of CAFs that were isolated from a prostate tumor to their normal-associated fibroblast (NAF) counterparts. CAFs formed more colonies when seeded at low cell density, exhibited a higher proliferation rate and were less prone to contact inhibition. In contrast to the general notion that high levels of α-smooth muscle actin serve as a marker for CAFs, we found that prostate CAFs express it at a lower level compared with prostate NAFs. Microarray analysis revealed a set of 161 genes that were altered in CAFs compared with NAFs. We focused on whey acidic protein four-disulfide core domain 1 (WFDC1), a known secreted protease inhibitor, and found it to be downregulated in the CAFs. WFDC1 expression was also dramatically downregulated in highly prolific mesenchymal cells and in various cancers including fibrosarcomas and in tumors of the lung, bladder and brain. Overexpression of WFDC1 inhibited the growth rate of the fibrosarcoma HT1080 cell line. Furthermore, WFDC1 level was upregulated in senescent fibroblasts. Taken together, our data suggest an important role for WFDC1 in inhibiting proliferation of both tumors and senescent cells. Finally, we suggest that the downregulation of WFDC1 might serve as a biomarker for cellular transformation.

Wide-Scale Analysis of Human Functional Transcription Factor Binding Reveals a Strong Bias towards the Transcription Start Site

Background Transcription factors (TF) regulate expression by binding to specific DNA sequences. A binding event is functional when it affects gene expression. Functionality of a binding site is reflected in conservation of the binding sequence during evolution and in over represented binding in gene groups with coherent biological functions. Functionality is governed by several parameters such as the TF-DNA binding strength, distance of the binding site from the transcription start site (TSS), DNA packing, and more. Understanding how these parameters control functionality of different TFs in different biological contexts is a must for identifying functional TF binding sites and for understanding regulation of transcription. Methodology/Principal Findings We introduce a novel method to screen the promoters of a set of genes with shared biological function (obtained from the functional Gene Ontology (GO) classification) against a precompiled library of motifs, and find those motifs which are statistically over-represented in the gene set. More than 8000 human (and 23,000 mouse) genes, were assigned to one of 134 GO sets. Their promoters were searched (from 200 bp downstream to 1000 bp upstream the TSS) for 414 known DNA motifs. We optimized the sequence similarity score threshold, independently for every location window, taking into account nucleotide heterogeneity along the promoters of the target genes. The method, combined with binding sequence and location conservation between human and mouse, identifies with high probability functional binding sites for groups of functionally-related genes. We found many location-sensitive functional binding events and showed that they clustered close to the TSS. Our method and findings were tested experimentally. Conclusions/Significance We identified reliably functional TF binding sites. This is an essential step towards constructing regulatory networks. The promoter region proximal to the TSS is of central importance for regulation of transcription in human and mouse, just as it is in bacteria and yeast.