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Impact of ETV7 on chemoresistance and cancer stem-like cell plasticity in breast cancer
Get PDFETV7 is a poorly characterized transcriptional repressor that belongs to the large family of ETS transcription factors, whose members have been associated with several cancer-related processes. ETV7 is a well-recognized Interferon-stimulated gene (ISG), and it was shown that its expression can be synergistically induced by the combined treatment with the chemotherapeutic drug Doxorubicin and the inflammatory cytokine TNFa in different cancer cell lines, including the breast cancer-derived MCF7 cells. Recently, it has been shown that ETV7 expression is significantly increased in breast cancer tissues, compared to the normal breast; however, the roles and the impact of ETV7 expression in breast cancer have still to be elucidated. This project aimed at understanding the effects caused by increased ETV7 expression on breast cancer (BC) progression and resistance to conventional anti-cancer drugs.
We first observed that ETV7 expression can be induced by different stimuli, particularly by the treatment with several chemotherapeutic drugs able to induce DNA damage. We also demonstrated that the expression of ETV7 could affect the sensitivity of BC cell lines to standard anti-cancer therapies, such as Doxorubicin, 5-Fluorouracil and radiotherapy, and this evidence was correlated with an increase in ABC transporters and anti-apoptotic proteins expression. By investigating the possible mechanism responsible for ETV7-dependent Doxorubicin resistance we identified a novel target gene of ETV7, DNAJC15, which is a co-chaperone protein whose repression was previously associated with drug resistance.
Given the ability of cancer stem cells (CSCs) to be more chemoresistant, we analyzed the effects of ETV7 expression on the sub-population of breast CSCs. We found that ETV7 expression could exert a strong effect on breast cancer cells stemness, confirmed by both an increase in CD44+/CD24low population and mammosphere formation efficiency. In order to investigate the mechanisms responsible for these effects, we performed an RNA-seq analysis, which revealed significant repression of a signature of Interferon-stimulated genes, suggesting a possible negative feedback mechanism in the regulation of the response to Interferon. Finally, prolonged treatment of breast cancer cells with IFNb was able to rescue the effects on CSCs content.
Taken collectively, our data revealed that ETV7 can affect the sensitivity of breast cancer cells to some chemotherapeutic drugs and we propose ETV7 as an important contributor to the tumor-initiating capabilities of BC cells
p53 Functional Interactions: the Study of a New Crosstalk with Estradiol Pathway in Transcriptional Responses to Chemotherapeutics
No full textBACKGROUND: Objective of this thesis has been the analysis of the sequence specific transcription factor p53, a critical tumor suppressor protein, specifically, the crosstalk (or functional interactions) with other transcription factors, namely, the estrogen receptors, and the modeling in reconstituted assays of the interaction of p53 with positive and negative cofactors (e.g. MDM4 and 53BP1) and the impact of small molecules, including chemotherapeutic drugs, on such interactions. Previous reports have revealed a complex, often negative, crosstalk between p53 and estrogen receptors (ERs) related in part to the physical interaction between the two proteins. An example of transcriptional cooperation mediated by cognate, non-canonical cis-elements was instead discovered for the angiogenesis related VEGFR1, FLT1 promoter.
MAIN TASK: Transcriptional cooperation between p53 and ERs was sought out on a global scale using the human breast adenocarcinoma MCF7 cells as a model and transcriptome analyses. Cells were subjected to single or combinatorial treatments with the chemotherapeutic agent doxorubicin (able to induce p53 protein stabilization) and the ER ligand 17β-estradiol (E2). 201 differentially expressed genes, that showed limited responsiveness to either doxorubicin treatment or ER ligand alone, but were up-regulated in a greater than additive manner following combined treatment were identified. Among sixteen genes chosen for validation using quantitative real-time PCR (qPCR), seven (INPP5D, TLR5, KRT15, EPHA2, GDNF, NOTCH1, SOX9) were confirmed to be novel direct targets of p53, based on responses in stable MCF7 clone cells silenced for p53, or cooperative targets of p53 and ER. Based on exposure to 5-fuorouracil (another genotoxic drug) and nutlin-3a (a non-genotoxic p53-specific activator), the combined response identified genes that were consistently regulated, although with different kinetics (e.g. INPP5D, CDH26, KRT15), while others (e.g. TLR5, SOX9) were treatment selective.
Promoter pattern searches and chromatin IP experiments for the INPP5D, TLR5, KRT15 genes were also performed to interrogate a direct, cis-mediated p53 and ERs regulation. While these analyses confirmed the identification of novel direct p53 targets, the important contribution of ER in their transcriptional modulation and the role of non-canonical response elements, the correlation between occupancy levels and gene expression varied.
SECONDARY TASK: Using a newly developed miniaturized yeast-based assay, functional interactions between p53 and its regulators MDM4 and 53BP1 was investigated. MDM4 was confirmed as a p53 negative regulator and the impact of nutlin-3a or RITA (apoptosis inducer through p53 binding) on the p53-MDM4 interaction was explored. Instead, no stimulatory effect of the p53 co-activator 53BP1 was detected.
CONCLUSIONS: Collectively, the results indicate that combinatorial activation of p53 and ER can induce novel gene expression programs which have implications for cell-cell communications, adhesion, cell differentiation, development and inflammatory responses as well as cancer treatments. The yeast-based assay represents a versatile tool to study p53 interactions with cofactors
ETV7 can trigger breast cancer chemoresistance
Get PDFChemotherapy still represents the most common and sometimes the only possible therapeutic option for advanced breast cancer. Its efficacy is profoundly threatened by intrinsic or acquired chemoresistance, which, in some cases, can be unexpectedly promoted by the chemotherapeutic drugs used. ETV7, a poorly characterized ETS factor with no established roles in breast cancer so far, is reported here to be activated at the transcriptional level by chemotherapy and be able to promote breast cancer cells chemoresistance. This project proposes a novel drug resistance circuitry in breast cancer cells, specifically to Doxorubicin, governed by the ETV7 repressive action on DNAJC15, a gene whose low expression was previously associated with drug resistance in breast and ovarian cancer. Moreover, the impact of ETV7 in causing drug resistance is proved here to extend also to another type of drug, 5-Fluorouracil (5-FU), a chemotherapeutic agent commonly used in combination with Doxorubicin for breast cancer treatment. In this case, additional novel ETV7 targets (DPYD and DPEP1) were established and can represent important mediators for ETV7-mediated resistance to 5-FU. Additional relevant data, relative to biological implications for ETV7 in breast cancer progression, is provided here by the ETV7 ChIP-seq analysis, which reveals the first reported in vivo mapping of ETV7 occupancy. Through ChIP-seq novel ETV7 direct targets have been identified, some of them yet unexplored in breast cancer; notably, their expression revealed to be capable of predicting breast cancer patients outcome. Furthermore, a possible control exerted by ETV7 on the TGF-β pathway, discovered by enrichment analysis on ChIP-seq ETV7 targets, suggests a different and opposing role for ETV7 activation in either advanced stage cancers or normal and early stage breast cancers.
Taken collectively, the results from this project propose an important key role for ETV7 in triggering breast cancer multi-drug resistance phenotype in response to chemotherapy, by controlling the expression of specific targets
Abstract 3402: ΔN-p63α and TA-p63α exhibit intrinsic differences in transactivation specificities that depend on distinct features of DNA target sites
No full textTP63 is a member of the p53 gene family that encodes for up to ten different TA- and ΔN- isoforms through differential promoter usage and alternative C-terminal splicing. The TA isoforms (TA-p63α, β, γ, δ and ε) contain the N-terminal transactivation domain (TA1), whereas the ΔN isoforms (ΔN-p63α, β, γ, δ and ε) are transcribed from an internal promoter (P2) and lack the TA1 domain. A second C-terminal transactivation domain (TA2) present in all p63α and β isoforms has been reported. Besides being a master regulator of gene expression for squamous epithelial proliferation, differentiation and maintenance (germline TP63 mutations are causative for a subset of human ectodermal dysplasia syndromes -EDs-), TA- and ΔN-p63 isoforms play an important role in tumorigenesis. More recently, p63 was shown to modulate apoptosis in the female and male germ line in response to DNA damage. All isoforms share a large, immunoglobulin-like folded DNA binding domain that is responsible for binding to sequence-specific response elements (REs), whose overall consensus sequence is similar to the p53 RE. Since the ΔN-p63 isoforms lack the N-terminal transactivation domain, it was originally proposed that these proteins might act primarily as oncogenes through dominant-negative mechanisms. However, different studies indicate that ΔN-p63 protein itself can be transcriptionally active. For example, ΔN-p63α may directly contribute to tumorigenesis by up-regulating the chaperone protein Hsp70, which displays proliferative and anti-apoptotic functions or by repressing pro-apoptotic genes. Transcriptional activation of specific genes by ΔN-p63α, namely VDR and Id-3, has been also associated with an anti-tumorigenic role, i.e. a decrease in cell invasion. Using a defined functional assay in yeast where p63 isoform and RE sequence are the only variables (more than 80 different REs were tested), as well as mammalian-transcription assays (gene reporter assays, qPCR measurements, western blotting), we demonstrated that human TA- and ΔN-p63α proteins exhibited differences in transactivation specificity. In fact 21 REs were identified that exhibited higher or selective responsiveness to ΔN-p63α. These differences were not observed with the related p73 and p53 proteins isoforms and were dependent on specific features of the RE sequences. Based on gene annotations, we propose that cis-element sequence features might have been selected along with evolutionarily conserved, intrinsic differences in cooperative DNA binding of p63 proteins, to establish tighter control of the apoptotic processes
P63 modulates the expression of the WDFY2 gene which is implicated in cancer regulation and limb development
No full textTP63 is a member of the TP53 gene family, sharing a common gene structure that produces two groups of mRNAs’ encoding proteins with different N-terminal regions (∆N and TA isoforms); both transcripts are also subjected to alternative splicing mechanisms at C-terminus, generating a variety of isoforms. p63 is a master regulator of epidermal development and homoeostasis as well as an important player in tumorigenesis and cancer progression with both oncogenic and tumour suppressive roles. A number of studies have aimed at the identification of p63 target genes, allowing the dissection of the molecular pathways orchestrated by the different isoforms. In the present study we investigated in more detail the p63 responsiveness of the WDFY2 (WD repeat and FYVE domain containing 2) gene, encoding for an endosomal protein identified as a binding partner of the PI-3K/AKT signalling pathway. We showed that overexpression of different p63 isoforms was able to induce WDFY2 expression in TP53-null cells. The p63-dependent transcriptional activation was associated with specific response elements (REs) that have been identified by a bioinformatics tool and validated by yeast- and mammal-based assays. Interestingly, to confirm that WDFY2 belongs to the p63 network of cancer regulation, we analysed the impact of WDFY2 alterations, by showing its frequent deletion in different types of tumours and suggesting its expression level as a prognostic biomarker. Lastly, we identified a chromosomal translocation involving the WDFY2 locus in a patient affected by a rare congenital limb anomaly, indicating WDFY2 as a possible susceptibility gene placed downstream p63 in the network of limb development
ETV7 regulates breast cancer stem-like cell features by repressing IFN-response genes
Get PDFAbstract Cancer stem cells (CSCs) represent a population of cells within the tumor able to drive tumorigenesis and known to be highly resistant to conventional chemotherapy and radiotherapy. In this work, we show a new role for ETV7, a transcriptional repressor member of the ETS family, in promoting breast cancer stem-like cells plasticity and resistance to chemo- and radiotherapy in breast cancer (BC) cells. We observed that MCF7 and T47D BC-derived cells stably over-expressing ETV7 showed reduced sensitivity to the chemotherapeutic drug 5-fluorouracil and to radiotherapy, accompanied by an adaptive proliferative behavior observed in different culture conditions. We further noticed that alteration of ETV7 expression could significantly affect the population of breast CSCs, measured by CD44+/CD24low cell population and mammosphere formation efficiency. By transcriptome profiling, we identified a signature of Interferon-responsive genes significantly repressed in cells over-expressing ETV7, which could be responsible for the increase in the breast CSCs population, as this could be partially reverted by the treatment with IFN-β. Lastly, we show that the expression of the IFN-responsive genes repressed by ETV7 could have prognostic value in breast cancer, as low expression of these genes was associated with a worse prognosis. Therefore, we propose a novel role for ETV7 in breast cancer stem cells’ plasticity and associated resistance to conventional chemotherapy and radiotherapy, which involves the repression of a group of IFN-responsive genes, potentially reversible upon IFN-β treatment. We, therefore, suggest that an in-depth investigation of this mechanism could lead to novel breast CSCs targeted therapies and to the improvement of combinatorial regimens, possibly involving the therapeutic use of IFN-β, with the aim of avoiding resistance development and relapse in breast cancer
ETV7 reduces inflammatory responses in breast cancer cells by repressing the TNFR1/NF-κB axis
Get PDF: The transcription factor ETV7 is an oncoprotein that is up-regulated in all breast cancer (BC) types. We have recently demonstrated that ETV7 promoted breast cancer progression by increasing cancer cell proliferation and stemness and was also involved in the development of chemo- and radio-resistance. However, the roles of ETV7 in breast cancer inflammation have yet to be studied. Gene ontology analysis previously performed on BC cells stably over-expressing ETV7 demonstrated that ETV7 was involved in the suppression of innate immune and inflammatory responses. To better decipher the involvement of ETV7 in these signaling pathways, in this study, we identified TNFRSF1A, encoding for the main receptor of TNF-α, TNFR1, as one of the genes down-regulated by ETV7. We demonstrated that ETV7 directly binds to the intron I of this gene, and we showed that the ETV7-mediated down-regulation of TNFRSF1A reduced the activation of NF-κB signaling. Furthermore, in this study, we unveiled a potential crosstalk between ETV7 and STAT3, another master regulator of inflammation. While it is known that STAT3 directly up-regulates the expression of TNFRSF1A, here we demonstrated that ETV7 reduces the ability of STAT3 to bind to the TNFRSF1A gene via a competitive mechanism, recruiting repressive chromatin remodelers, which results in the repression of its transcription. The inverse correlation between ETV7 and TNFRSF1A was confirmed also in different cohorts of BC patients. These results suggest that ETV7 can reduce the inflammatory responses in breast cancer through the down-regulation of TNFRSF1A
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
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