Ana M. Cabanillas

CCN6 (WISP3) decreases ZEB1-mediated EMT and invasion by attenuation of IGF-1 receptor signaling in breast cancer

During progression of breast cancer, CCN6 protein exerts tumor inhibitory functions. CCN6 is a secreted protein that modulates the insulin-like growth factor-1 (IGF-1) signaling pathway. Knockdown of CCN6 in benign mammary epithelial cells triggers an epithelial to mesenchymal transition (EMT), with upregulation of the transcription factor ZEB1/δEF1. How CCN6 regulates ZEB1 expression is unknown. We hypothesized that CCN6 might regulate ZEB1, EMT and breast cancer invasion by modulating IGF-1 signaling. Exogenously added human recombinant CCN6 protein was sufficient to downregulate ZEB1 mRNA and protein levels in CCN6-deficient (CCN6 KD) HME cells and MDA-MB-231 breast cancer cells. Recombinant CCN6 protein decreased invasion of CCN6 KD cells compared with controls. We discovered that knockdown of CCN6 induced IGF-1 secretion in HME cells cultivated in serum-free medium to higher concentrations than found in MDA-MB-231 cells. Treatment with recombinant CCN6 protein was sufficient to decrease IGF-1 protein and mRNA to control levels, rescuing the effect of CCN6 knockdown. Specific inhibition of IGF-1 receptors using the pharmacological inhibitor NVP-AE541 or short hairpin shRNAs revealed that ZEB1 upregulation due to knockdown of CCN6 requires activation of IGF-1 receptor signaling. Recombinant CCN6 blunted IGF-1-induced ZEB1 upregulation in MDA-MB-231 cells. Our data define a pathway in which CCN6 attenuates IGF-1 signaling to decrease ZEB1 expression and invasion in breast cancer. These results suggest that CCN6 could be a target to prevent or halt breast cancer invasion.

New insights in the DNA-[Cr(phen)2(dppz)]3+ binding and photocleavage properties by the complex with an intercalating ligand

Due to the key role of DNA in cell life and pathological processes, the design of specific chemical nucleases, DNA probes and alkylating agents is an important research area for the development of new therapeutic agents and tools in Biochemistry. Hence, the interaction of small molecules with DNA has attracted in particular a great deal of attention. The aim of this study was to investigate the ability of [Cr(phen)(2)(dppz)](3+) to associate with DNA and to characterize it as photocleavage reagent for Photodynamic Therapy (PDT). Chromium(III) complex [Cr(phen)(2)(dppz)](3+), (dppz = dipyridophenazine, phen = 1,10-phenanthroline), where dppz is a planar bidentate ligand with an extended pi system, has been found to bind strongly to double strand oligonucleotides (ds-oligo) and plasmid DNA with intrinsic DNA binding constants, K(b,) of (3.9+/-0.3)x10(5) M(-1) and (1.1+/-0.1)x10(5) M(-1), respectively. The binding properties to DNA were investigated by UV-visible (UV-Vis) absorption spectroscopy and electrophoretic studies. UV-Vis absorption data provide clearly that the chromium(III) complex interacts with DNA intercalatively. Competitive binding experiments show that the enhancement in the emission intensity of ethidium bromide (EthBr) in the presence of DNA was quenched by [Cr(phen)(2)(dppz)](3+), indicating that the Cr(III) complex displaces EthBr from its binding site in plasmid DNA. Moreover, [Cr(phen)(2)(dppz)](3+), non-covalently bound to DNA, promotes the photocleavage of plasmid DNA under 457 nm irradiation. We also found that the irradiated Cr(III)-plasmid DNA association is able to impair the transforming capacity of bacteria. These results provide evidence confirming the responsible and essential role of the excited state of [Cr(phen)(2)(dppz)](3+) for damaging the DNA structure. The combination of DNA, [Cr(phen)(2)(dppz)](3+) and light, is necessary to induce damage. In addition, assays of the photosensitization of transformed bacterial suspensions suggest that Escherichia coli may be photoinactivated by irradiation in the presence of [Cr(phen)(2)(dppz)](3+). In sum, our results allow us to postulate the [Cr(phen)(2)(dppz)](3+) complex as a very attractive candidate for DNA photocleavage with potential applications in Photodynamic Therapy (PDT).

Glypican-3 induces a mesenchymal to epithelial transition in human breast cancer cells

Breast cancer is the disease with the highest impact on global health, being metastasis the main cause of death. To metastasize, carcinoma cells must reactivate a latent program called epithelial-mesenchymal transition (EMT), through which epithelial cancer cells acquire mesenchymal-like traits. Glypican-3 (GPC3), a proteoglycan involved in the regulation of proliferation and survival, has been associated with cancer. In this study we observed that the expression of GPC3 is opposite to the invasive/metastatic ability of Hs578T, MDA-MB231, ZR-75-1 and MCF-7 human breast cancer cell lines. GPC3 silencing activated growth, cell death resistance, migration, and invasive/metastatic capacity of MCF-7 cancer cells, while GPC3 overexpression inhibited these properties in MDA-MB231 tumor cell line. Moreover, silencing of GPC3 deepened the MCF-7 breast cancer cells mesenchymal characteristics, decreasing the expression of the epithelial marker E-Cadherin. On the other side, GPC3 overexpression induced the mesenchymal-epithelial transition (MET) of MDA-MB231 breast cancer cells, which re-expressed E-Cadherin and reduced the expression of vimentin and N-Cadherin. While GPC3 inhibited the canonical Wnt/β-Catenin pathway in the breast cancer cells, this inhibition did not have effect on E-Cadherin expression. We demonstrated that the transcriptional repressor of E-Cadherin - ZEB1 - is upregulated in GPC3 silenced MCF-7 cells, while it is downregulated when GPC3 was overexpressed in MDA-MB231 cells. We presented experimental evidences showing that GPC3 induces the E-Cadherin re-expression in MDA-MB231 cells through the downregulation of ZEB1. Our data indicate that GPC3 is an important regulator of EMT in breast cancer, and a potential target for procedures against breast cancer metastasis.

Monoamine oxidase (MAO) activity and its forms in normal and pathological human thyroid tissues.

Monoamine oxidase (MAO) activity and its forms were measured in normal human thyroid glands (39 samples from necropsy and four from perinodular tissues) and in pathological thyroid glands (13 multinodular goitres, three carcinomas, seven chronic thyroiditis, five Graves’ disease and seven follicular, seven foetal and five embryonal adenomas). MAO activity in carcinomas was lower than in normal perinodular thyroid control tissue; in Graves’ disease, foetal and embryonal adenomas it was significantly higher than in the normal control. The use of clorgyline (a selective MAO A inhibitor) and deprenyl (selective MAO B inhibitor) allowed estimation of the relative proportion of MAO forms. A high proportion of MAO A (more than 90%) was observed, both in normal and in pathological thyroid tissues. Changes in total thyroid MAO activity, but not in its forms, were found in different pathologies of the thyroid gland.

Phosphorylation Regulates Functions of ZEB1 Transcription Factor

ZEB1 transcription factor is important in both development and disease, including many TGFβ‐induced responses, and the epithelial‐to‐mesenchymal transition (EMT) by which many tumors undergo metastasis. ZEB1 is differentially phosphorylated in different cell types; however the role of phosphorylation in ZEB1 activity is unknown. Luciferase reporter studies and electrophoresis mobility shift assays (EMSA) show that a decrease in phosphorylation of ZEB1 increases both DNA‐binding and transcriptional repression of ZEB1 target genes. Functional analysis of ZEB1 phosphorylation site mutants near the second zinc finger domain (termed ZD2) show that increased phosphorylation (due to either PMA plus ionomycin, or IGF‐1) can inhibit transcriptional repression by either a ZEB1‐ZD2 domain clone, or full‐length ZEB1. This approach identifies phosphosites that have a substantial effect regulating the transcriptional and DNA‐binding activity of ZEB1. Immunoprecipitation with anti‐ZEB1 antibodies followed by western analysis with a phospho‐Threonine‐Proline‐specific antibody indicates that the ERK consensus site at Thr‐867 is phosphorylated in ZEB1. In addition to disrupting in vitro DNA‐binding measured by EMSA, IGF‐1‐induced MEK/ERK phosphorylation is sufficient to disrupt nuclear localization of GFP‐ZEB1 fusion clones. These data suggest that phosphorylation of ZEB1 integrates TGFβ signaling with other signaling pathways such as IGF‐1. J. Cell. Physiol. 231: 2205–2217, 2016.

CLCA2 epigenetic regulation by CTBP1, HDACs, ZEB1, EP300 and miR-196b-5p impacts prostate cancer cell adhesion and EMT in metabolic syndrome disease: CLCA2 function in PCa EMT inhibition

Prostate cancer (PCa) is the most common cancer among men. Metabolic syndrome (MeS) is associated with increased PCa aggressiveness and recurrence. Previously, we proposed C‐terminal binding protein 1 (CTBP1), a transcriptional co‐repressor, as a molecular link between these two conditions. Notably, CTBP1 depletion decreased PCa growth in MeS mice. The aim of this study was to investigate the molecular mechanisms that explain the link between MeS and PCa mediated by CTBP1. We found that CTBP1 repressed chloride channel accessory 2 (CLCA2) expression in prostate xenografts developed in MeS animals. CTBP1 bound to CLCA2 promoter and repressed its transcription and promoter activity in PCa cell lines. Furthermore, we found that CTBP1 formed a repressor complex with ZEB1, EP300 and HDACs that modulates the CLCA2 promoter activity. CLCA2 promoted PCa cell adhesion inhibiting epithelial–mesenchymal transition (EMT) and activating CTNNB1 together with epithelial marker (CDH1) induction, and mesenchymal markers (SNAI2 and TWIST1) repression. Moreover, CLCA2 depletion in PCa cells injected subcutaneously in MeS mice increased the circulating tumor cells foci compared to control. A microRNA (miRNA) expression microarray from PCa xenografts developed in MeS mice, showed 21 miRNAs modulated by CTBP1 involved in angiogenesis, extracellular matrix organization, focal adhesion and adherents junctions, among others. We found that miR‐196b‐5p directly targets CLCA2 by cloning CLCA2 3′UTR and performing reporter assays. Altogether, we identified a new molecular mechanism to explain PCa and MeS link based on CLCA2 repression by CTBP1 and miR‐196b‐5p molecules that might act as key factors in the progression onset of this disease.

Abstract 1988: Role of N- ZEB1 in epithelial-mesenchymal transition (EMT)

Background: ZEB1 (Zn Finger E-box Binding Homeobox) transcription factor is important in both development and disease, including the TGFβ-induced epithelial-mesenchymal transition (EMT) by which many tumors undergo metastasis. ZEB1 contains 2 Zn finger domains (ZD1-ZD2) which bind DNA independently. ZEB1 expresses as two phosphorylated forms important for its biological role. We had characterized the role of phosphorylated C-term ZEB1. Aim: Our goal is to uncover the role of signaling pathway/s in the phosphorylation on N-term ZD1 (N-ZEB1) and characterize the functional role of N-ZEB1 in the process of EMT in mammary epithelial cells NMuMG. Results: A 728 aa fragment of N-ZEB1 was overexpressed in HEK293T cells and Immunoprecipitation with N-ZEB1 antibodies followed by western blot analysis with anti phospho kinases (AKT, MEK/ERK, PKC or PKA) substrate Antibodies. The results revealed that N-ZEB1 is solely phosphorylated by PI3K (AKT), MAPK and PKC pathways. IGF1-mediated phosphorylation can regulate nuclear localization of C-term ZEB1. Two N-ZEB1-GFP clones harboring first 490aa (eGFPZ1) and a 2nd clone of 130 aa, eGFPZ2 (included in the former) were transfected to CHO-K1 cells and treated with inhibitors and activators of kinase pathways for 15-60 minutes in serum free medium. Fluorescence microscopy techniques showed that IGF-1 induced relocation of a nuclear eGFPZ1 into the cytosol of CHO-K1 cells; on the contrary, eGFPZ2 was unresponsive to IGF-1. PMA/IONO (activator of PKC) treatment relocated both clones in the cytosol. Later on, NMuMG cells were used to investigate the role of N-ZEB1 in EMT. These epithelial cells, that can turn into mesenchymal type under TGFβ treatment, were stably transfected with eGFPZ2 or empty vector (control). The epithelial markers E-cadherin and actin assessed by Western blot and confocal microscopy were diminished or absent in eGFPZ2 cells. The activity of Matrix Metallo Proteases 2 was also increased in NMuMG-GFPZ2 cells. Also, proliferation, migration and invasion were significantly increased (P Conclusions: We have identified a small fragment in N-ZEB1 that is able to elicit the EMT program in a TGFβ independent manner. In addition, changes in ZEB1 cellular localization induced by either PKC or IGF-1 could regulate this transcription factor biological activity. Citation Format: M Candelaria Llorens, Ana M. Cabanillas. Role of N- ZEB1 in epithelial-mesenchymal transition (EMT). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1988. doi:10.1158/1538-7445.AM2015-1988

Abstract 37: CCN6 downregulation in invasive breast carcinomas: In situ characterization of changes in EMT proteins and metastatic disease

Background: The recognized heterogeneity of invasive breast carcinomas is a result of intrinsic characteristics of the tumor cells and their interaction with the microenvironment. CCN6, an extracellular matricellular protein involved in cell communication and epithelial-stromal interactions has a tumor suppressor role in breast cancer. CCN6 downregulation in benign breast cells induces an epithelial to mesenchymal transition marked by downregulation of E-cadherin and increased levels of the transcriptional regulators ZEB1 and Snai1. However, the relevance of these studies to human breast cancer needs further investigation. We hypothesized that CCN6 mRNA and protein levels in samples of breast cancer patients may be associated with metastasis and features of EMT. Methods: Forty clinical samples of invasive carcinoma and surrounding normal breast, frozen at the time of operation were collected. We extracted mRNA and protein from each sample and performed real time RT-PCR and Western blots for CCN6, respectively. All frozen samples were immunostained with antibodies against E-cadherin (BD Biosciences, 1:4000), ZEB1 (Santa Cruz Biotechnology, 1:400) and Snai1(Cell Signaling, 1:800). E-cadherin was analyzed as aberrant or normal. ZEB1 and Snai1 proteins were scored as positive or negative, either in the cancer or stromal cells. Results: Of the 40 frozen samples of breast cancer, 23 had high quality RNA and protein for further study. CCN6 mRNA and protein were both decreased in 18/23 (78%). Five of 23 (22%) samples exhibited normal or high expression. Low CCN6 was associated with the presence of distant metastasis. Four of 18 (22%) invasive carcinomas with low CCN6 had distant metastasis compared to none of the normal CCN6 expressing tumors (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 37. doi:1538-7445.AM2012-37

Abstract 3172: ZEB1 regulation in breast epithelial cells: Role of IGF-1 in the CCN6 modulation pathway

Background: During cancer progression, the transcription factor ZEB1 has been implicated in the regulation of key genes triggering the EMT (Epithelial to Mesenchymal Transition) program. We previously reported that the matricellular protein CCN6 is lost or reduced in highly aggressive breast carcinomas. Furthermore, we have identified CCN6 as a tumor suppressor in breast cancer whose down-regulation enhances IGF-1 signaling on breast epithelial cells. Recently, our lab observed that stable inhibition of CCN6 in benign mammary epithelial cells causes EMT and invasion with marked up-regulation of ZEB1. Since IGF-1 is shown to up-regulate ZEB1 expression in epithelial prostate cancer cells we hypothesize that CCN6 may regulate EMT in breast cancers by modulating IGF-1 signaling and thereby regulating ZEB1 expression. Methods: Stable CCN6-deficient HME (Human Mammary Epithelial cells)(HME-CCN6 KD) were incubated with 500 ng/mL rhCCN6 protein. Western blots (WB) for ZEB1 and vimentin, inmunofluorescence, invasion and proliferation assays were performed. ZEB1 mRNA levels were evaluated by RT-qPCR (Taqman). IGF-1 expression was analyzed by RT-qPCR and WB on MDA-MB-231 and HME-CCN6 KD (pre- and post-rhCCN6 treatment) while IGF-1R, phospho-IGF-1R, IRS-1 and phospho-IRS-1 expression were investigated by WB. Lentivirus-mediated (pLK.O1) short hairpin RNA targeting IGF1-R was performed in HME control and HME-CCN6 KD cells. WBs were done to assess expression of proteins of interest. mRNA content was evaluated by RT-qPCR. Results: CCN6 incubation of HME-CCN6 KD cells induced down-regulation of ZEB1 mRNA and protein levels as well as subcellular localization changes of the mesenchymal marker vimentin (“cap” pattern) evidenced as a decrease in vimentin expression by WB. hrCCN6 protein treatment also reduced proliferation and invasion abilities of HME-CCN6 KD cells compared to controls. In Addition, cell lines expressing low levels of CCN6 such us HME-CCN6 KD and MDA-MB-231 secreted IGF-1 in the conditioned media and showed activation of the IGF-1 signaling pathway. This phenomenon was reverted by CCN6 treatment, with reduction of both, IGF-1 protein and mRNA levels. IGF-1R down-regulation in HME-CCN6 KD cells decreased ZEB1 expression and induced the re-expression of cytokeratin-18 (epithelial marker). Conclusions: Our results suggest that CCN6 regulates ZEB1 expression and EMT in breast epithelial cells by modulating the IGF-1 signaling pathway. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3172.

Selective Inhibition by Monoamine Oxidase (MAO) Inhibitors of the Iodotyrosine Formation Induced by MAO Substrates in Bovine Thyroid Tissue

Monoamine oxidase (EC 1.4.3.4) (MAO) may probably be involved in the hydrogen peroxide generation required for the iodination process in the thyroid tissue (1,2). Biogenic amines have been demonstrated to be able to generate hydrogen peroxide and to increase iodine organification in thyroid tissue, and these effects were decreased by MAO inhibitors (3,4). On the other hand, it was observed that rat thyroid MAO activity was increased by TSH administration and reduced after hypophysectomy (2).