Alexander Polyzos

A new tumor suppressor role for the Notch pathway in bladder cancer

The Notch signaling pathway controls cell fates through interactions between neighboring cells by positively or negatively affecting the processes of proliferation, differentiation and apoptosis in a context-dependent manner. This pathway has been implicated in human cancer as both an oncogene and a tumor suppressor. Here we report new inactivating mutations in Notch pathway components in over 40% of human bladder cancers examined. Bladder cancer is the fourth most commonly diagnosed malignancy in the male population of the United States. Thus far, driver mutations in fibroblast growth factor receptor 3 (FGFR3) and, less commonly, in RAS proteins have been identified. We show that Notch activation in bladder cancer cells suppresses proliferation both in vitro and in vivo by directly upregulating dual-specificity phosphatases (DUSPs), thus reducing the phosphorylation of ERK1 and ERK2 (ERK1/2). In mouse models, genetic inactivation of Notch signaling leads to Erk1/2 phosphorylation, resulting in tumorigenesis in the urinary tract. Collectively our findings show that loss of Notch activity is a driving event in urothelial cancer.

Chronic p53-independent p21 expression causes genomic instability by deregulating replication licensing

The cyclin-dependent kinase inhibitor p21WAF1/CIP1 (p21) is a cell-cycle checkpoint effector and inducer of senescence, regulated by p53. Yet, evidence suggests that p21 could also be oncogenic, through a mechanism that has so far remained obscure. We report that a subset of atypical cancerous cells strongly expressing p21 showed proliferation features. This occurred predominantly in p53-mutant human cancers, suggesting p53-independent upregulation of p21 selectively in more aggressive tumour cells. Multifaceted phenotypic and genomic analyses of p21-inducible, p53-null, cancerous and near-normal cellular models showed that after an initial senescence-like phase, a subpopulation of p21-expressing proliferating cells emerged, featuring increased genomic instability, aggressiveness and chemoresistance. Mechanistically, sustained p21 accumulation inhibited mainly the CRL4–CDT2 ubiquitin ligase, leading to deregulated origin licensing and replication stress. Collectively, our data reveal the tumour-promoting ability of p21 through deregulation of DNA replication licensing machinery—an unorthodox role to be considered in cancer treatment, since p21 responds to various stimuli including some chemotherapy drugs.

Genomic Analysis Reveals a Novel Nuclear Factor-κB (NF-κB)-binding Site in Alu-repetitive Elements

Background: The transcription factor NF-κB is a critical regulator of immune response pathways. Results: 11% of total NF-κB binding events in humans occur at the Alu-κB element present in Alu repeats. Conclusion: NF-κB has a primate-specific function and a role in human evolution. Significance: Repetitive elements expand the repertoire of binding sites to engage new genes into regulatory networks. The transcription factor NF-κB is a critical regulator of immune responses. To determine how NF-κB builds transcriptional control networks, we need to obtain a topographic map of the factor bound to the genome and correlate it with global gene expression. We used a ChIP cloning technique and identified novel NF-κB target genes in response to virus infection. We discovered that most of the NF-κB-bound genomic sites deviate from the consensus and are located away from conventional promoter regions. Remarkably, we identified a novel abundant NF-κB-binding site residing in specialized Alu-repetitive elements having the potential for long range transcription regulation, thus suggesting that in addition to its known role, NF-κB has a primate-specific function and a role in human evolution. By combining these data with global gene expression profiling of virus-infected cells, we found that most of the sites bound by NF-κB in the human genome do not correlate with changes in gene expression of the nearby genes and they do not appear to function in the context of synthetic promoters. These results demonstrate that repetitive elements interspersed in the human genome function as common target sites for transcription factors and may play an important role in expanding the repertoire of binding sites to engage new genes into regulatory networks.

Therapeutic Effects of an Anti-Myc Drug on Mouse Pancreatic Cancer

BACKGROUND Pancreatic ductal adenocarcinoma (PDA) is frequently driven by oncogenic KRAS(KRAS*) mutations. We developed a mouse model of KRAS*-induced PDA and, based on genetic results demonstrating that KRAS* tumorigenicity depends on Myc activity, we evaluated the therapeutic potential of an orally administered anti-Myc drug. METHODS We tested the efficacy of Mycro3, a small-molecule inhibitor of Myc-Max dimerization, in the treatment of mouse PDA (n = 9) and also of xenografts of human pancreatic cancer cell lines (NOD/SCID mice, n = 3-12). Tumor responses to the drug were evaluated by PET/CT imaging, and histological, immunohistochemical, molecular and microarray analyses. The Students t test was used for differences between groups. All statistical tests were two-sided. RESULTS Transgenic overexpression of KRAS* in the pancreas resulted in pancreatic intraepithelial neoplasia in two-week old mice, which developed invasive PDA a week later and became moribund at one month. However, this aggressive form of pancreatic tumorigenesis was effectively prevented by genetic ablation of Myc specifically in the pancreas. We then treated moribund, PDA-bearing mice daily with the Mycro3 Myc-inhibitor. The mice survived until killed at two months. PET/CT image analysis (n = 5) demonstrated marked shrinkage of PDA, while immunohistochemical analyses showed an increase in cancer cell apoptosis and reduction in cell proliferation (treated/untreated proliferation index ratio: 0.29, P < .001, n = 3, each group). Tumor growth was also drastically attenuated in Mycro3-treated NOD/SCID mice (n = 12) carrying orthotopic or heterotopic xenografts of human pancreatic cancer cells (eg, mean tumor weight ± SD of treated heterotopic xenografts vs vehicle-treated controls: 15.2±5.8 mg vs 230.2±43.9 mg, P < .001). CONCLUSION These results provide strong justification for eventual clinical evaluation of anti-Myc drugs as potential chemotherapeutic agents for the treatment of PDA.

Composite macroH2A/NRF-1 Nucleosomes Suppress Noise and Generate Robustness in Gene Expression.

The histone variant macroH2A (mH2A) has been implicated in transcriptional repression, but the molecular mechanisms that contribute to global mH2A-dependent genome regulation remain elusive. Using chromatin immunoprecipitation sequencing (ChIP-seq) coupled with transcriptional profiling in mH2A knockdown cells, we demonstrate that singular mH2A nucleosomes occupy transcription start sites of subsets of both expressed and repressed genes, with opposing regulatory consequences. Specifically, mH2A nucleosomes mask repressor binding sites in expressed genes but activator binding sites in repressed genes, thus generating distinct chromatin landscapes that limit genetic or extracellular inductive signals. We show that composite nucleosomes containing mH2A and NRF-1 are stably positioned on gene regulatory regions and can buffer transcriptional noise associated with antiviral responses. In contrast, mH2A nucleosomes without NRF-1 bind promoters weakly and mark genes with noisier gene expression patterns. Thus, the strategic position and stabilization of mH2A nucleosomes in human promoters defines robust gene expression patterns.

Profiling of Discrete Gynecological Cancers Reveals Novel Transcriptional Modules and Common Features Shared by Other Cancer Types and Embryonic Stem Cells

Studies on individual types of gynecological cancers (GCs), utilizing novel expression technologies, have revealed specific pathogenetic patterns and gene markers for cervical (CC), endometrial (EC) and vulvar cancer (VC). Although the clinical phenotypes of the three types of gynecological cancers are discrete, the fact they originate from a common embryological origin, has led to the hypothesis that they might share common features reflecting regression to early embryogenesis. To address this question, we performed a comprehensive comparative analysis of their profiles. Our data identified both common features (pathways and networks) and novel distinct modules controlling the same deregulated biological processes in all three types. Specifically, four novel transcriptional modules were discovered regulating cell cycle and apoptosis. Integration and comparison of our data with other databases, led to the identification of common features among cancer types, embryonic stem (ES) cells and the newly discovered cell population of squamocolumnar (SC) junction of the cervix, considered to host the early cancer events. Conclusively, these data lead us to propose the presence of common features among gynecological cancers, other types of cancers, ES cells and the pre-malignant SC junction cells, where the novel E2F/NFY and MAX/CEBP modules play an important role for the pathogenesis of gynecological carcinomas.

Mutational signatures reveal the role of RAD52 in p53-independent p21-driven genomic instability

BackgroundGenomic instability promotes evolution and heterogeneity of tumors. Unraveling its mechanistic basis is essential for the design of appropriate therapeutic strategies. In a previous study, we reported an unexpected oncogenic property of p21WAF1/Cip1, showing that its chronic expression in a p53-deficient environment causes genomic instability by deregulation of the replication licensing machinery.ResultsWe now demonstrate that p21WAF1/Cip1 can further fuel genomic instability by suppressing the repair capacity of low- and high-fidelity pathways that deal with nucleotide abnormalities. Consequently, fewer single nucleotide substitutions (SNSs) occur, while formation of highly deleterious DNA double-strand breaks (DSBs) is enhanced, crafting a characteristic mutational signature landscape. Guided by the mutational signatures formed, we find that the DSBs are repaired by Rad52-dependent break-induced replication (BIR) and single-strand annealing (SSA) repair pathways. Conversely, the error-free synthesis-dependent strand annealing (SDSA) repair route is deficient. Surprisingly, Rad52 is activated transcriptionally in an E2F1-dependent manner, rather than post-translationally as is common for DNA repair factor activation.ConclusionsOur results signify the importance of mutational signatures as guides to disclose the repair history leading to genomic instability. We unveil how chronic p21WAF1/Cip1 expression rewires the repair process and identifies Rad52 as a source of genomic instability and a candidate therapeutic target.

A prototypical non-malignant epithelial model to study genome dynamics and concurrently monitor micro-RNAs and proteins in situ during oncogene-induced senescence

BackgroundSenescence is a fundamental biological process implicated in various pathologies, including cancer. Regarding carcinogenesis, senescence signifies, at least in its initial phases, an anti-tumor response that needs to be circumvented for cancer to progress. Micro-RNAs, a subclass of regulatory, non-coding RNAs, participate in senescence regulation. At the subcellular level micro-RNAs, similar to proteins, have been shown to traffic between organelles influencing cellular behavior. The differential function of micro-RNAs relative to their subcellular localization and their role in senescence biology raises concurrent in situ analysis of coding and non-coding gene products in senescent cells as a necessity. However, technical challenges have rendered in situ co-detection unfeasible until now.MethodsIn the present report we describe a methodology that bypasses these technical limitations achieving for the first time simultaneous detection of both a micro-RNA and a protein in the biological context of cellular senescence, utilizing the new commercially available SenTraGorTM compound. The method was applied in a prototypical human non-malignant epithelial model of oncogene-induced senescence that we generated for the purposes of the study. For the characterization of this novel system, we applied a wide range of cellular and molecular techniques, as well as high-throughput analysis of the transcriptome and micro-RNAs.ResultsThis experimental setting has three advantages that are presented and discussed: i) it covers a “gap” in the molecular carcinogenesis field, as almost all corresponding in vitro models are fibroblast-based, even though the majority of neoplasms have epithelial origin, ii) it recapitulates the precancerous and cancerous phases of epithelial tumorigenesis within a short time frame under the light of natural selection and iii) it uses as an oncogenic signal, the replication licensing factor CDC6, implicated in both DNA replication and transcription when over-expressed, a characteristic that can be exploited to monitor RNA dynamics.ConclusionsConsequently, we demonstrate that our model is optimal for studying the molecular basis of epithelial carcinogenesis shedding light on the tumor-initiating events. The latter may reveal novel molecular targets with clinical benefit. Besides, since this method can be incorporated in a wide range of low, medium or high-throughput image-based approaches, we expect it to be broadly applicable.

Mobile-phone radiation-induced perturbation of gene-expression profiling, redox equilibrium and sporadic-apoptosis control in the ovary of Drosophila melanogaster

ABSTRACT The daily use by people of wireless communication devices has increased exponentially in the last decade, begetting concerns regarding its potential health hazards. Drosophila melanogaster four days-old adult female flies were exposed for 30 min to radiation emitted by a commercial mobile phone at a SAR of 0.15 W/kg and a SAE of 270 J/kg. ROS levels and apoptotic follicles were assayed in parallel with a genome-wide microarrays analysis. ROS cellular contents were found to increase by 1.6-fold (x), immediately after the end of exposure, in follicles of pre-choriogenic stages (germarium - stage 10), while sporadically generated apoptotic follicles (germarium 2b and stages 7–9) presented with an averaged 2x upregulation in their sub-population mass, 4 h after flys irradiation with mobile device. Microarray analysis revealed 168 genes being differentially expressed, 2 h post-exposure, in response to radiofrequency (RF) electromagnetic field-radiation exposure (≥1.25x, P < 0.05) and associated with multiple and critical biological processes, such as basic metabolism and cellular subroutines related to stress response and apoptotic death. Exposure of adult flies to mobile-phone radiation for 30 min has an immediate impact on ROS production in animals ovary, which seems to cause a global, systemic and non-targeted transcriptional reprogramming of gene expression, 2 h post-exposure, being finally followed by induction of apoptosis 4 h after the end of exposure. Conclusively, this unique type of pulsed radiation, mainly being derived from daily used mobile phones, seems capable of mobilizing critical cytopathic mechanisms, and altering fundamental genetic programs and networks in D. melanogaster.

The histone variant macroH2A blocks cellular reprogramming by inhibiting mesenchymal-to-epithelial transition

ABSTRACT Transcription factor-induced reprogramming of somatic cells to pluripotency is mediated via profound alterations in the epigenetic landscape. The histone variant macroH2A1 (mH2A1) is a barrier to the cellular reprogramming process. We demonstrate here that mH2A1 blocks reprogramming and contributes to the preservation of cell identity by trapping cells at the very early stages of the process, namely, at the mesenchymal-to-epithelial transition (MET). We provide a comprehensive analysis of the genomic sites occupied by the mH2A1 nucleosomes in human fibroblasts and embryonic stem (ES) cells and how they affect the reprogramming of fibroblasts to pluripotency. We have integrated chromatin immunoprecipitation sequencing (ChIP-seq) data with transcriptome sequencing (RNA-seq) data using cells containing reduced levels of mH2A1 and have inferred mH2A1-centered gene-regulatory networks that support the fibroblast and ES cell fates. We found that the exact positions of mH2A1 nucleosomes in regulatory regions of specific network genes with key regulatory roles guarantee the functional robustness of the regulatory networks. Using the reconstructed networks, we can predict and validate several components and their interactions in the establishment of stable cell types by limiting progression to alternative cell fates.