Stella Logotheti

The role of ATF‐2 in oncogenesis

Activating Transcription Factor‐2 is a sequence‐specific DNA‐binding protein that belongs to the bZIP family of proteins and plays diverse roles in the mammalian cells. In response to stress stimuli, it activates a variety of gene targets including cyclin A, cyclin D and c‐jun, which are involved in oncogenesis in various tissue types. ATF‐2 expression has been correlated with maintenance of a cancer cell phenotype. However, other studies demonstrate an antiproliferative or apoptotic role for ATF‐2. In this review, we summarize the signaling pathways that activate ATF‐2, as well as its downstream targets. We examine the role of ATF‐2 in carcinogenesis with respect to other bZIP proteins, using data from studies in human cancer cell lines, human tumours and mouse models, and we propose a potential model for its function in carcinogenesis, as well as a theoretical basis for its utility in anticancer drug design. BioEssays 30:314–327, 2008.

Environmental factors and genetic susceptibility promote urinary bladder cancer.

Cancer of the urinary bladder is the second most common malignancy of the genitourinary tract, currently accounting for up to 5% of all newly diagnosed tumours in the western world. Urinary bladder carcinogenesis seems to develop from the interaction of environmental exposure and genetic susceptibility. Smoking, specific industrial chemicals, dietary nitrates and arsenic represent the most important exogenous risk factors. Chromosomal abnormalities, silencing of certain genes by abnormal methylation of their promoter region, alterations in tumour suppressor genes and proto-oncogenes that induce uncontrolled cell proliferation and reduced apoptosis, are molecular mechanisms that have been reported in bladder carcinogenesis. In this article, we discuss the environmental risk factors of bladder cancer and we review the genetic and epigenetic alterations, including aberrant DNA methylation and deregulation of microRNAs expression. We also discuss the role of p53 and retinoblastoma suppressor genes in disease progression. Finally, we present recent reports on the use of molecular profiling to predict disease stage and grade and direct targeted therapy.

Global DNA hypomethylation-induced ΔNp73 transcriptional activation in non-small cell lung cancer

p73 possesses an extrinsic P1 promoter and an intrinsic P2 promoter controlling the expression of the pro-apoptotic TAp73 isoforms and the anti-apoptotic ΔΝp73 isoforms respectively. In this study, we investigated the DNA methylation status of both promoters as a means of epigenetic transcriptional control of their corresponding isoforms in 102 primary non-small cell lung carcinomas (NSCLCs). We demonstrated that while P1 hypermethylation-associated reduction of TAp73 mRNA levels is relatively infrequent, the P2 hypomethylation-associated over-expression of ΔΝp73 mRNA is a frequent event, particularly among squamous cell carcinomas. P2 hypomethylation strongly correlated with LINE-1 element hypomethylation, indicating that ΔΝp73 over-expression may be a passive consequence of global DNA hypomethylation.

Estrogen receptor α and β in uterine fibroids: a basis for altered estrogen responsiveness

OBJECTIVE To investigate the relative expression and the DNA-binding status of estrogen receptors alpha and beta in fibroids and normal myometrial tissue to explore the molecular basis of altered estrogen responsiveness of leiomyomas. DESIGN Biopsy samples from uterine fibroids and adjacent normal myometrial tissue at the follicular phase of the menstrual cycle. SETTING Aretaieio University Hospital and the National Hellenic Research Foundation, Athens, Greece. PATIENT(S) Thirty-five patients who underwent hysterectomy or myomectomy because of myoma symptoms. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Deoxyribonucleic acid-binding status of estrogen receptors alpha and beta. RESULT(S) The level of messenger RNA expression of estrogen receptor alpha and beta and the level of estrogen receptor as a whole are increased on average to a similar extent in leiomyomas compared with normal myometrium. Occasionally, however, estrogen receptor alpha is disproportionately increased in leiomyomas, and this appears to increase the amount of estrogen receptor alpha that binds to the estrogen-responsive element of estrogen target genes as homodimer rather than as heterodimer with estrogen receptor beta. CONCLUSION(S) The estrogen receptor alpha-to-estrogen receptor beta expression ratio rather than the individual expression levels determines the fraction of DNA-binding homodimers of estrogen receptor alpha and possibly the growth potential of myomas.

Functions, divergence and clinical value of TAp73 isoforms in cancer.

The p73 gene encodes the tumour suppressive full-length TAp73 and N-terminal-truncated DNp73 isoforms that act as dominant negative inhibitors of TAp73. The overall effect of p73 in oncogenesis is thought to depend on the TAp73 to DNp73 isoforms’ ratio. TAp73 isoforms include a number of C-terminal variants as a result of alternative splicing in 3′-end. TAp73 isoforms protect cells from oncogenic alterations in a multifaceted way since they are implicated in the suppression of all demonstrated hallmarks and enabling characteristics of cancer. Their best established role is in apoptosis, a process which seems to be differently affected by each TAp73 C-terminal variant. Based on previous findings and our thorough bioinformatics analysis, we highlight that TAp73 variants are functionally non-equivalent, since they present major differences in their transactivation efficiencies, protein interactions, response to DNA damage and apoptotic effects that are attributable to the primary structure of their C terminus. In this review, we summarise these differences and we unveil the link between crucial C-terminal motifs/residues and the oncosuppressive potential of TAp73 isoforms, emphasising on the importance of considering C terminus during the development of p73-based anticancer biologics.

Progression of mouse skin carcinogenesis is associated with the orchestrated deregulation of miR-200 family members, miR-205 and their common targets

MicroRNAs are small, non‐coding RNAs which regulate post‐transcriptionally hundreds of target mRNAs. Given that their expression is deregulated in several cancer types, they represent potential diagnostic, prognostic, and predictive biomarkers, as well as next‐generation therapeutic targets. Nevertheless, the involvement of miRNAs in non‐melanoma skin cancer, a cancer type with increasing prevalence, is not extensively studied, and their comprehensive characterization as regard to the initiation, promotion, and progression stages is missing. To this end, we exploited a well‐established multistage mouse skin carcinogenesis model in order to identify miRNAs consistently implicated in different stages of skin carcinogenesis. The cell lines comprising this model were subjected to miRNA expression profiling using microarrays, followed by bioinformatics analysis and validation with Q‐PCR, as well as treatment with miRNA modulators. We showed that among all deregulated miRNAs in our system, only a functionally coherent group consisting of the miR‐200 family members and miR‐205‐5p displays a pattern of progressive co‐downregulation from the early toward the most aggressive stages of carcinogenesis. Their overlapping, co‐regulated putative targets are potentially inter‐associated and, of these, the EMT‐related Rap1a is overexpressed toward aggressive stages. Ectopic expression of miR‐205‐5p in spindle cancer cells reduces Rap1a, mitigates cell invasiveness, decreases proliferation, and delays tumor onset. We conclude that deregulation of this miRNA group is primarily associated with aggressive phenotypes of skin cancer cells. Restoration of the miR‐205‐5p member of this group in spindle cells reduces the expression of critical, co‐regulated targets that favor cancer progression, thus reversing the EMT characteristics.

Progression of Mouse Skin Carcinogenesis Is Associated with Increased Erα Levels and Is Repressed by a Dominant Negative Form of Erα

Estrogen receptors (ER), namely ERα and ERβ, are hormone-activated transcription factors with an important role in carcinogenesis. In the present study, we aimed at elucidating the implication of ERα in skin cancer, using chemically-induced mouse skin tumours, as well as cell lines representing distinct stages of mouse skin oncogenesis. First, using immunohistochemical staining we showed that ERα is markedly increased in aggressive mouse skin tumours in vivo as compared to the papilloma tumours, whereas ERβ levels are low and become even lower in the aggressive spindle tumours of carcinogen-treated mice. Then, using the multistage mouse skin carcinogenesis model, we showed that ERα gradually increases during promotion and progression stages of mouse skin carcinogenesis, peaking at the most aggressive stage, whereas ERβ levels only slightly change throughout skin carcinogenesis. Stable transfection of the aggressive, spindle CarB cells with a dominant negative form of ERα (dnERα) resulted in reduced ERα levels and reduced binding to estrogen responsive elements (ERE)-containing sequences. We characterized two highly conserved EREs on the mouse ERα promoter through which dnERα decreased endogenous ERα levels. The dnERα-transfected CarB cells presented altered protein levels of cytoskeletal and cell adhesion molecules, slower growth rate and impaired anchorage-independent growth in vitro, whereas they gave smaller tumours with extended latency period of tumour onset in vivo. Our findings suggest an implication of ERα in the aggressiveness of spindle mouse skin cancer cells, possibly through regulation of genes affecting cell shape and adhesion, and they also provide hints for the effective targeting of spindle cancer cells by dnERα.

Unravelling a p73-regulated network: The role of a novel p73-dependent target, MIR3158, in cancer cell migration and invasiveness

The transcription factor p73 is homologous to the well-known tumor-suppressor p53. The p73-regulated networks are of significant clinical interest, because they may substitute for impaired p53-regulated networks which are commonly perturbed in cancer. Herein, we aimed to characterize a p73-regulated network that mediates cell migration and restores anti-oncogenic responses in p53-mutant cancer cells. In this study, we demonstrate that p73 regulates a network underlying cell migration, which consists of MIR34A/MIR3158/vimentin/β-catenin/lef1. The p73 isoforms transactivate the miRNA components (MIR34A/MIR3158) of this network, which in turn, downregulate their EMT-related mRNA co-targets (vimentin/β-catenin/lef1) to decrease cell-migration. Modulation of this network, by increasing the level of the novel p73-dependent target MIR3158, was found to induce anti-oncogenic/anti-invasive responses in p53-mutant cancer cells. Taken together, a p73-regulated, MIR3158-containing, network restores anti-invasive phenotypes in p53-mutant cancer cells; this property could be exploited towards the development of anticancer therapeutics.

ΔNp63α expression induces loss of cell adhesion in triple-negative breast cancer cells

Backgroundp63, a member of the p53 protein family, plays key roles in epithelial development and carcinogenesis. In breast cancer, p63 expression has been found predominantly in basal-A (epithelial-type) triple-negative breast carcinomas (TNBC). To investigate the functional role of p63 in basal-A TNBC, we created MDA-MB-468 cell lines with inducible expression of the two major N-terminal p63 isoforms, TAp63α and ∆Np63α.ResultsTAp63α did not have significant effect on gene expression profile and cell phenotype, whilst the main effect of ΔNp63α was reduction of cell adhesion. Gene expression profiling revealed genes involved in cell adhesion and migration whose expression relies on overexpression of ΔNp63α. Reduced cell adhesion also led to decreased cell proliferation in vitro and in vivo. Similar data were obtained in another basal-A cell line, BT-20, but not in BT-549 basal-B (mesenchymal-like) TNBC cells.ConclusionsIn basal-A TNBC cells, ∆Np63α has much stronger effects on gene expression than TAp63α. Although p63 is mentioned mostly in connection with breast cell differentiation and stem cell regulation, we showed that a major effect of p63 is regulation of cell adhesion, a process important in metastasis and invasion of tumour cells. That this effect is not seen in mesenchymal-type TNBC cells suggests lineage-dependent functions, mirroring the expression of ∆Np63α in primary human breast cancers.

N-bromotaurine surrogates for loss of antiproliferative response and enhances cisplatin efficacy in cancer cells with impaired glucocorticoid receptor

Glucocorticoids (GCs) are frequently used in anticancer combination regimens; however, their continuous use adds selective pressure on cancer cells to develop GC-resistance via impairment of the glucocorticoid receptor (GR), therefore creating a need for GC-alternatives. Based on the drug repurposing approach and the commonalities between inflammation and neoplasia, drugs that are either in late-stage clinical trials and/or already marketed for GC-refractory inflammatory diseases could be evaluated as GC-substitutes in the context of cancer. Advantageously, unlike new molecular entities currently being de novo developed to restore GC-responsiveness of cancer cells, such drugs have documented safety and efficacy profile, which overall simplifies their introduction in clinical cancer trials. In this study, we estimated the potential of a well-established, multistage, cell line-based, mouse skin carcinogenesis model to be exploited as an initial screening tool for unveiling covert GC-substitutes. First, we categorized the cell lines of this model to GC-sensitive and GC-resistant, in correlation with their corresponding GR status, localization, and functionality. We found that GC-resistance starts in papilloma stages, due to a dysfunctional GR, which is overexpressed, DNA binding-competent, but transactivation-incompetent in papilloma, squamous, and spindle stages of the model. Then, aided by this tool, we evaluated the ability of N-bromotaurine, a naturally occurring, small-molecule, nonsteroid anti-inflammatory drug which is under consideration for use interchangeably/in replacement to GCs in skin inflammations, to restore antiproliferative response of GC-resistant cancer cells. Unlike GCs, N-bromotaurine inhibited cell-cycle progression in GC-resistant cancer cells and efficiently synergized with cisplatin, thus indicating a potential to be exploited instead of GCs against cancer.