po 290 etv7 regulates breast cancer stem cells content and chemoresistance

Introduction Cancer stem cells (CSCs) are considered the population of cells within the tumour able to drive tumorigenesis and known to be highly resistant to conventional chemotherapy. ETV7 is a poorly studied transcription factor member of ETS large family, known to be an interferon-stimulated gene. It has been recently found over-expressed in breast cancer (BC), with higher expression levels in the more aggressive BC subtypes. In this work, we investigated the effects of ETV7 increased expression on breast CSCs population and resistance to chemotherapy in BC cells. Material and methods We generated MCF7 and T47D BC-derived cells stably over-expressing ETV7 and obtained ETV7 KO in MDA-MB-231 BC cells using CRISPR/Cas9 technology. We analysed breast CSCs content via CD44/CD24 staining and FACS analysis, as well as mammospheres formation assay. We measured expression of ABC transporters and anti-apoptotic proteins via RT-qPCR and western blot. We finally assessed sensitivity to Doxorubicin and 5-Fluorouracil (5-FU) via MTT assay and AnnexinV/PI staining at FACS. Results and discussions We observed that the expression of ETV7 could be induced by various stimuli, particularly by chemotherapeutic drugs able to induce DNA damage. We then analysed the impact of ETV7 expression on the sensitivity to Doxorubicin and 5-FU and we could observe a significantly decreased sensitivity to these drugs upon ETV7 over-expression. We could also appreciate an increase in ABC transporters and BCL2 anti-apoptotic protein expression following ETV7 over-expression. We further observed that alteration of ETV7 expression could significantly affect the population of breast cancer stem cells (CD44+/CD24low cells) in different BC cell lines. Conclusion We propose a novel role for ETV7 in breast cancer stem cells plasticity and associated resistance to conventional chemotherapy. We finally suggest that an in-depth investigation of this mechanism could lead to novel breast CSCs targeted therapies and to the improvement of combinatorial regimens with the aim of avoiding resistance and relapse in breast cancer

06N-P63\u3b1 and TA-P63\u3b1 exhibit intrinsic differences in transactivation specificities that depend on distinct features of DNA target sites.

TP63 is a member of the TP53 gene family that encodes for up to ten different TA and 06N isoforms through alternative promoter usage and alternative splicing. Besides being a master regulator of gene expression for squamous epithelial proliferation, differentiation and maintenance, P63, through differential expression of its isoforms, plays important roles in tumorigenesis. All P63 isoforms share an immunoglobulin-like folded DNA binding domain responsible for binding to sequence-specific response elements (REs), whose overall consensus sequence is similar to that of the canonical p53 RE. Using a defined assay in yeast, where P63 isoforms and RE sequences are the only variables, and gene expression assays in human cell lines, we demonstrated that human TA- and 06N-P63\u3b1 proteins exhibited differences in transactivation specificity not observed with the corresponding P73 or P53 protein isoforms. These differences 1) were dependent on specific features of the RE sequence, 2) could be related to intrinsic differences in their oligomeric state and cooperative DNA binding, and 3) appeared to be conserved in evolution. Since genotoxic stress can change relative ratio of TA- and 06N-P63\u3b1 protein levels, the different transactivation specificity of each P63 isoform could potentially influence cellular responses to specific stresses

Regulatory Crosstalk of Doxorubicin, Estradiol and TNFα Combined Treatment in Breast Cancer-derived Cell Lines

We present a new model of ESR1 network regulation based on analysis of Doxorubicin, Estradiol, and TNF alpha combination treatment in MCF-7. We used Doxorubicin as a therapeutic agent, TNF alpha as marker and mediator of an inflammatory microenvironment and 17 beta-Estradiol (E2) as an agonist of Estrogen Receptors, known predisposing factor for hormone-driven breast cancer, whose pharmacological inhibition reduces the risk of breast cancer recurrence. Based on the results of transcriptomics analysis, we found 71 differentially expressed genes that are specific for the combination treatment with Doxorubicin +Estradiol +TNF alpha in comparison with single or double treatments. The responsiveness to the triple treatment was examined for seven genes by qPCR, of which six were validated, and then extended to four additional cell lines differing for p53 and/or ER status. The results of differential regulation enrichment analysis highlight the role of the ESR1 network that included 36 of 71 specific differentially expressed genes. We propose that the combined activation of p53 and NF-kappa B transcription factors significantly influences ligand-dependent, ER-driven transcriptional responses, also of the ESR1 gene itself. These results provide a model of coordinated interaction of TFs to explain the Doxorubicin, E2 and TNF alpha induced repression mechanisms

PO-111 Capicua suppresses hepatocellular carcinoma progression by controlling etv4-mmp1 axis

Introduction T-ALL is a malignancy characterised by aberrant Notch signalling, sustained by activating mutations in Notch1 as well as over-expression of Notch3, a Notch paralog physiologically subjected to lysosome-dependent degradation in human cancer cells. Given some limitations of existing drugs blocking Notch signalling, it is important to get new insights into the biology of Notch3 to further stimulate the development of Notch-targeted therapies in cancer. Material and methods T-ALL cell lines and cells obtained from patient-derived xenografts (PDX) were treated in vitro with HDAC inhibitors and other drugs or HDAC6-specific shRNA and effects on Notch expression and activity were investigated by standard methods. Confocal microscopy was used for intracellular localization studies. PDX models were also utilised to investigate effects of HDAC6 silencing on leukaemia growth. Results and discussions We initially found that treatment with the pan-HDAC inhibitor Trichostatin A (TSA) strongly decreases Notch3 full-length protein levels in T-ALL cell lines and primary human T-ALL cells xenografted in mice without substantially reducing NOTCH3 mRNA levels. Moreover, TSA markedly reduced the levels of Notch target genes, including pTa, CR2 and DTX-1, and induced apoptosis of T-ALL cells. We further observed that Notch3 was post-translationally regulated following TSA treatment, with reduced Notch3 surface levels and increased accumulation of Notch3 protein in the lysosomal compartment. Surface Notch3 levels were rescued by inhibition of Dynein with Ciliobrevin D. Pharmacologic studies with HDAC1, 6 and 8-specific inhibitors disclosed that these effects were largely due to inhibition of HDAC6 in T-ALL cells. HDAC6 silencing by specific shRNA was followed by reduced Notch3 expression and increased apoptosis of T-ALL cells. Finally, HDAC6 silencing impaired leukaemia outgrowth in mice, associated with reduction of Notch3 full-length protein in vivo. Conclusion These results connect HDAC6 activity to regulation of total and surface Notch3 levels by a mechanism involving endosomal sorting and suggest HDAC6 as potential novel therapeutic target to lower Notch signalling in T-ALL and other Notch3-addicted tumours

Dissecting molecular mechanisms of resistance to NOTCH1-targeted therapy in T-cell acute lymphoblastic leukemia xenografts

Despite substantial progress in treatment of T-cell acute lymphoblastic leukemia (T-ALL), mortality remains relatively high, mainly due to primary or acquired resistance to chemotherapy. Further improvements in survival demand better understanding of T-ALL biology and development of new therapeutic strategies. The Notch pathway has been involved in the pathogenesis of this disease and various therapeutic strategies are currently under development, including selective targeting of NOTCH receptors by inhibitory antibodies. We previously demonstrated that the NOTCH1-specific neutralizing antibody OMP52M51 prolongs survival in TALL patient-derived xenografts bearing NOTCH1/FBW7 mutations. However, acquired resistance to OMP52M51 eventually developed and we used patient-derived xenografts models to investigate this phenomenon. Multi-level molecular characterization of T-ALL cells resistant to NOTCH1 blockade and serial transplantation experiments uncovered heterogeneous types of resistance, not previously reported with other Notch inhibitors. In one model, resistance appeared after 156 days of treatment, it was stable and associated with loss of Notch inhibition, reduced mutational load and acquired NOTCH1 mutations potentially affecting the stability of the heterodimerization domain. Conversely, in another model resistance developed after only 43 days of treatment despite persistent down-regulation of Notch signaling and it was accompanied by modulation of lipid metabolism and reduced surface expression of NOTCH1. Our findings shed light on heterogeneous mechanisms adopted by the tumor to evade NOTCH1 blockade and support clinical implementation of antibody-based target therapy for Notch-addicted tumors