Papers by Leandro Venturutti
Molecular Endocrinology, 2015
Accumulated findings have demonstrated the presence of bidirectional interactions between progest... more Accumulated findings have demonstrated the presence of bidirectional interactions between progesterone receptor (PR) and the ErbB family of receptor tyrosine kinases signaling pathways in breast cancer. We previously revealed signal transducer and activator of transcription 3 (Stat3) as a nodal convergence point between said signaling pathways proving that Stat3 is activated by one of the ErbBs' ligands, heregulin (HRG)1 via ErbB2 and through the co-option of PR as a signaling molecule. Here, we found that HRG1 induced Stat3 recruitment to the promoters of the progestin-regulated cell cycle modulators Bcl-X L and p21 CIP1 and also stimulated Stat3 binding to the mouse mammary tumor virus promoter, which carries consensus progesterone response elements. Interestingly, HRG1-activated Stat3 displayed differential functions on PR activity depending on the promoter bound. Indeed, Stat3 was required for PR binding in bcl-X, p21 CIP1 , and c-myc promoters while exerting a PR coactivator function on the mouse mammary tumor virus promoter. Stat3 also proved to be necessary for HRG1-induced in vivo tumor growth. Our results endow Stat3 a novel function as a coregulator of HRG1-activated PR to promote breast cancer growth. These findings underscore the importance of understanding the complex interactions between PR and other regulatory factors, such as Stat3, that contribute to determine the contextdependent transcriptional actions of PR. (Molecular Endocrinology 29: 1468 -1485, 2015)
Oncogene, 2015
Membrane overexpression of the receptor tyrosine kinase ErbB-2 (MErbB-2) accounts for a clinicall... more Membrane overexpression of the receptor tyrosine kinase ErbB-2 (MErbB-2) accounts for a clinically aggressive breast cancer (BC) subtype (ErbB-2-positive) with increased incidence of metastases. We and others demonstrated that nuclear ErbB-2 (NErbB-2) also plays a key role in BC and is a poor prognostic factor in ErbB-2-positive tumors. The signal transducer and activator of transcription 3 (Stat3), another player in BC, has been recognized as a downstream mediator of MErbB-2 action in BC metastasis. Here, we revealed an unanticipated novel direction of the ErbB-2 and Stat3 interaction underlying BC metastasis. We found that Stat3 binds to its response elements (GAS) at the ErbB-2 promoter to upregulate ErbB-2 transcription in metastatic, ErbB-2-positive BC. We validated these results in several BC subtypes displaying metastatic and non-metastatic ability, highlighting Stat3 general role as upstream regulator of ErbB-2 expression in BC. Moreover, we showed that Stat3 co-opts NErbB-2 function by recruiting ErbB-2 as its coactivator at the GAS sites in the promoter of microRNA-21 (miR-21), a metastasis-promoting microRNA (miRNA). Using an ErbB-2 nuclear localization domain mutant and a constitutively activated ErbB-2 variant, we found that NErbB-2 role as a Stat3 coactivator and also its direct role as transcription factor upregulate miR-21 in BC. This reveals a novel function of NErbB-2 as a regulator of miRNAs expression. Increased levels of miR-21, in turn, downregulate the expression of the metastasis-suppressor protein programmed cell death 4 (PDCD4), a validated miR-21 target. Using an in vivo model of metastatic ErbB-2-postive BC, in which we silenced Stat3 and reconstituted ErbB-2 or miR-21 expression, we showed that both are downstream mediators of Stat3-driven metastasis. Supporting the clinical relevance of our results, we found an inverse correlation between ErbB-2/Stat3 nuclear co-expression and PDCD4 expression in ErbB-2-positive primary invasive BCs. Our findings identify Stat3 and NErbB-2 as novel therapeutic targets to inhibit ErbB-2-positive BC metastasis.Oncogene advance online publication, 27 July 2015; doi:10.1038/onc.2015.281.
Oncogene, 2014
Membrane overexpression of ErbB-2/HER2 receptor tyrosine kinase (membrane ErbB-2 (MErbB-2)) has a... more Membrane overexpression of ErbB-2/HER2 receptor tyrosine kinase (membrane ErbB-2 (MErbB-2)) has a critical role in breast cancer (BC). We and others have also shown the role of nuclear ErbB-2 (NErbB-2) in BC, whose presence we identified as a poor prognostic factor in MErbB-2-positive tumors. Current anti-ErbB-2 therapies, as with the antibody trastuzumab (Ttzm), target only MErbB-2. Here, we found that blockade of NErbB-2 action abrogates growth of BC cells, sensitive and resistant to Ttzm, in a scenario in which ErbB-2, ErbB-3 and Akt are phosphorylated, and ErbB-2/ErbB-3 dimers are formed. Also, inhibition of NErbB-2 presence suppresses growth of a preclinical BC model resistant to Ttzm. We showed that at the cyclin D1 promoter, ErbB-2 assembles a transcriptional complex with Stat3 (signal transducer and activator of transcription 3) and ErbB-3, another member of the ErbB family, which reveals the first nuclear function of ErbB-2/ErbB-3 dimer. We identified NErbB-2 as the major proliferation driver in Ttzm-resistant BC, and demonstrated that Ttzm inability to disrupt the Stat3/ErbB-2/ErbB-3 complex underlies its failure to inhibit growth. Furthermore, our results in the clinic revealed that nuclear interaction between ErbB-2 and Stat3 correlates with poor overall survival in primary breast tumors. Our findings challenge the paradigm of anti-ErbB-2 drug design and highlight NErbB-2 as a novel target to overcome Ttzm resistance.Oncogene advance online publication, 1 September 2014; doi:10.1038/onc.2014.272.
Breast Cancer Research, 2014
IntroductionThe transcription factor GATA3 is involved in mammary gland development and is crucia... more IntroductionThe transcription factor GATA3 is involved in mammary gland development and is crucial for the maintenance of the differentiated status of luminal epithelial cells. The role of GATA3 in breast cancer as a tumor suppressor has been established, although insights into the mechanism of GATA3 expression loss are still required.MethodsChromatin immunoprecipitation assays were conducted to study progestin modulation of recruitment of transcription factors to GATA3 promoter. We performed western blot and reverse RT-qPCR experiments to explore progestin regulation of GATA3 protein and mRNA expression respectively. Confocal microscopy and in vitro phosphorylation studies were conducted to examine progestin capacity to induce GATA3 serine phosphorylation in its 308 residue. GATA3 participation in progestin-induced breast cancer growth was addressed in in vitro proliferation and in vivo tumor growth experiments.ResultsIn this study, we demonstrate that progestin-activated progesterone receptor (PR) reduces GATA3 expression through regulation at the transcriptional and post-translational levels in breast cancer cells. In the former mechanism, the histone methyltransferase enhancer of zeste homolog 2 is co-recruited with activated PR to a putative progesterone response element in the GATA3 proximal promoter, increasing H3K27me3 levels and inducing chromatin compaction, resulting in decreased GATA3 mRNA levels. This transcriptional regulation is coupled with increased GATA3 protein turnover through progestin-induced GATA3 phosphorylation at serine 308 followed by 26S proteasome-mediated degradation. Both molecular mechanisms converge to accomplish decreased GATA3 expression levels in breast cancer cells upon PR activation. In addition, we demonstrated that decreased GATA3 levels are required for progestin-induced upregulation of cyclin A2, which mediates the G1 to S phase transition of the cell cycle and was reported to be associated with poor prognosis in breast cancer. Finally, we showed that downregulation of GATA3 is required for progestin stimulation of both in vitro cell proliferation and in vivo tumor growth.ConclusionsIn the present study, we reveal that progestin-induced PR activation leads to loss of GATA3 expression in breast cancer cells through transcriptional and post-translational regulation. Importantly, we demonstrate that GATA3 downregulation is required for progestin-induced upregulation of cyclin A2 and for progestin-induced in vitro and in vivo breast cancer cell growth.
FEBS Open Bio, 2013
The N-terminal stretch of human frataxin (hFXN) intermediate (residues 42-80) is not conserved th... more The N-terminal stretch of human frataxin (hFXN) intermediate (residues 42-80) is not conserved throughout evolution and, under defined experimental conditions, behaves as a random-coil. Overexpression of hFXN56-210 in Escherichia coli yields a multimer, whereas the mature form of hFXN (hFXN81-210) is monomeric. Thus, cumulative experimental evidence points to the N-terminal moiety as an essential element for the assembly of a high molecular weight oligomer. The secondary structure propensity of peptide 56-81, the moiety putatively responsible for promoting protein-protein interactions, was also studied. Depending on the environment (TFE or SDS), this peptide adopts α-helical or β-strand structure. In this context, we explored the conformation and stability of hFXN56-210. The biophysical characterization by fluorescence, CD and SEC-FPLC shows that subunits are well folded, sharing similar stability to hFXN90-210. However, controlled proteolysis indicates that the N-terminal stretch is labile in the context of the multimer, whereas the FXN domain (residues 81-210) remains strongly resistant. In addition, guanidine hydrochloride at low concentration disrupts intermolecular interactions, shifting the ensemble toward the monomeric form. The conformational plasticity of the N-terminal tail might impart on hFXN the ability to act as a recognition signal as well as an oligomerization trigger. Understanding the fine-tuning of these activities and their resulting balance will bear direct relevance for ultimately comprehending hFXN function.
European Journal of Cancer, 2012
proliferation assays determined the effective dose which inhibits growth by 50% (ED50) of cells w... more proliferation assays determined the effective dose which inhibits growth by 50% (ED50) of cells when treated with PF-00299804 alone and in combination with vincristine, cisplatin, lomustine, cyclophosphamide (CYC) and temozolomide. CompuSyn software was employed to analyse synergy between the drugs. Results: Variable expression of the ERBB receptors was observed in the different models, consistent with data from human MB. Cell proliferation assays revealed significant variability in sensitivity to PF-00299804 among the brain tumour cell lines (ED50 values ranged between 10−55 mM) with the nodular desmoplastic MB line being the most sensitive. Synergy was observed using the combination of PF-00299804 and CYC. Conclusions: Synergy between PF-00299804 and CYC in vitro suggests that combining these agents might be of therapeutic benefit. Current studies are focused on evaluating this approach pre-clinically in multiple mouse models to determine if PF-00299804 is a potential candidate for future clinical trials for a subset of patients with these tumours.
Breast Cancer Research, 2012
Introduction: Experimental and clinical evidence points to a critical role of progesterone and th... more Introduction: Experimental and clinical evidence points to a critical role of progesterone and the nuclear progesterone receptor (PR) in controlling mammary gland tumorigenesis. However, the molecular mechanisms of progesterone action in breast cancer still remain elusive. On the other hand, micro RNAs (miRNAs) are short ribonucleic acids which have also been found to play a pivotal role in cancer pathogenesis. The role of miRNA in progestin-induced breast cancer is poorly explored. In this study we explored progestin modulation of miRNA expression in mammary tumorigenesis. Methods: We performed a genome-wide study to explore progestin-mediated regulation of miRNA expression in breast cancer. miR-16 expression was studied by RT-qPCR in cancer cell lines with silenced PR, signal transducer and activator of transcription 3 (Stat3) or c-Myc, treated or not with progestins. Breast cancer cells were transfected with the precursor of miR-16 and proliferation assays, Western blots or in vivo experiments were performed. Target genes of miR-16 were searched through a bioinformatical approach, and the study was focused on cyclin E. Reporter gene assays were performed to confirm that cyclin E 3'UTR is a direct target of miR-16. Results: We found that nine miRNAs were upregulated and seven were downregulated by progestin in mammary tumor cells. miR-16, whose function as a tumor suppressor in leukemia has already been shown, was identified as one of the downregulated miRNAs in murine and human breast cancer cells. Progestin induced a decrease in miR-16 levels via the classical PR and through a hierarchical interplay between Stat3 and the oncogenic transcription factor c-Myc. A search for miR-16 targets showed that the CCNE1 gene, encoding the cell cycle regulator cyclin E, contains conserved putative miR-16 target sites in its mRNA 3' UTR region. We found that, similar to the molecular mechanism underlying progestin-modulated miR-16 expression, Stat3 and c-Myc participated in the induction of cyclin E expression by progestin. Moreover, overexpression of miR-16 abrogated the ability of progestin to induce cyclin E upregulation, revealing that cyclin E is a novel target of miR-16 in breast cancer. Overexpression of miR-16 also inhibited progestin-induced breast tumor growth in vitro and in vivo, demonstrating for the first time, a role for miR-16 as a tumor suppressor in mammary tumorigenesis. We also found that the ErbB ligand heregulin (HRG) downregulated the expression of miR-16, which then participates in the proliferative activity of HRG in breast tumor cells. Conclusions: In this study, we reveal the first progestin-regulated miRNA expression profile and identify a novel role for miR-16 as a tumor suppressor in progestin-and growth factor-induced growth in breast cancer.
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Papers by Leandro Venturutti