Alexandre Bruni‐Cardoso

Universidade de São Paulo

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Hernandes F. Carvalho

Universidade Estadual de Campinas (UNICAMP)

Antonio Carlos Manucci

Universidade de São Paulo

Ana Paula Zen Petisco Fiore

Universidade de São Paulo

Rebeka Tomasin

New York University

Taize Machado Augusto

Faculdade de Medicina de Jundiaí

Mina J. Bissell

Lawrence Berkeley National Laboratory

Conor C. Lynch

Moffitt Cancer Center

Danilo Marchete Damas-Souza

Universidade Estadual de Campinas (UNICAMP)

Deborah Schechtman

Universidade de São Paulo

Jamie L. Inman

Lawrence Berkeley National Laboratory

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Universidade de São Paulo

133

John Wiley & Sons (United States)

Hudson Institute

Universidade Estadual de Campinas (UNICAMP)

Universidade Cidade de São Paulo

Universidade Federal de São Paulo

Johns Hopkins University

Johns Hopkins Medicine

Universidade Estadual Paulista (Unesp)

Universidade Brasil

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Citrullination of actin-ligand and nuclear structural proteins, cytoskeleton reorganization and protein redistribution across cellular fractions are early events in ionomycin-induced NETosis

Redox Biology (2023)

Lorenna Rocha Reis Douglas Ricardo Souza Rebeka Tomasin Alexandre Bruni‐Cardoso Paolo Di Mascio

Neutrophil extracellular traps (NETs) are web-like structures of DNA coated with cytotoxic proteins and histones released by activated neutrophils through a process called NETosis. NETs release occurs through a sequence of highly organized events leading to chromatin expansion and rupture of nuclear and cellular membranes. In calcium ionophore-induced NETosis, the enzyme peptidylargine deiminase 4 (PAD4) mediates chromatin decondensation through histone citrullination, but the biochemical pathways involved in this process are not fully understood. Here we use live-imaging microscopy and proteomic studies of the neutrophil cellular fractions to investigate the early events in ionomycin-triggered NETosis. We found that before ionomycin-stimulated neutrophils release NETs, profound biochemical changes occur in and around their nucleus, such as, cytoskeleton reorganization, nuclear redistribution of actin-remodeling related proteins, and citrullination of actin-ligand and nuclear structural proteins. Ionomycin-stimulated neutrophils rapidly lose their characteristic polymorphic nucleus, and these changes are promptly communicated to the extracellular environment through the secretion of proteins related to immune response. Therefore, our findings revealed key biochemical mediators in the early process that subsequently culminates with nuclear and cell membranes rupture, and extracellular DNA release.

Citrullination

Ionomycin

Neutrophil Extracellular Traps

10.1016/j.redox.2023.102784

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Citations (9)

Data from Osteoclast-Derived Matrix Metalloproteinase-9 Directly Affects Angiogenesis in the Prostate Tumor–Bone Microenvironment

Alexandre Bruni‐Cardoso Lindsay C. Johnson Robert L. Vessella Todd E. Peterson Conor C. Lynch

<div>AbstractIn human prostate to bone metastases and in a novel rodent model that recapitulates prostate tumor–induced osteolytic and osteogenic responses, we found that osteoclasts are a major source of the proteinase, matrix metalloproteinase (MMP)-9. Because MMPs are important mediators of tumor-host communication, we tested the effect of host-derived MMP-9 on prostate tumor progression in the bone. To this end, immunocompromised mice that were wild-type or null for MMP-9 received transplants of osteolytic/osteogenic-inducing prostate adenocarcinoma tumor tissue to the calvaria. Surprisingly, we found that that host MMP-9 significantly contributed to prostate tumor growth without affecting prostate tumor–induced osteolytic or osteogenic change as determined by microcomputed tomography, microsingle-photon emission computed tomography, and histomorphometry. Subsequent studies aimed at delineating the mechanism of MMP-9 action on tumor growth focused on angiogenesis because MMP-9 and osteoclasts have been implicated in this process. We observed (a) significantly fewer and smaller blood vessels in the MMP-9 null group by CD-31 immunohistochemistry; (b) MMP-9 null osteoclasts had significantly lower levels of bioavailable vascular endothelial growth factor-A164; and (c) using an aorta sprouting assay, conditioned media derived from wild-type osteoclasts was significantly more angiogenic than conditioned media derived from MMP-9 null osteoclasts. In conclusion, these studies show that osteoclast-derived MMP-9 affects prostate tumor growth in the bone microenvironment by contributing to angiogenesis without altering prostate tumor–induced osteolytic or osteogenic changes. Mol Cancer Res; 8(4); 459–70. ©2010 AACR.</div>

10.1158/1541-7786.c.6542067.v1

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The role of SDF1 in prostate epithelial morphogenesis

Journal of Cellular Physiology (2018)

Guilherme Oliveira Barbosa Taize Machado Augusto Alexandre Bruni‐Cardoso Hernandes F. Carvalho

Abstract Androgens induce rat prostate induction from the urogenital sinus epithelium at embryonic day 17.5. Subsequent morphogenesis, including epithelial cord growth, branching, and canalization, results from concerted paracrine interactions with the stroma. A significant number of paracrine factors bind heparan sulfate (HS). We hypothesized that interfering with overall sulfation could disrupt the signaling mediated by HS‐binding factors and that the undersulfated environment would allow investigation of individual exogenous morphogens. First, we investigated whether acinar morphogenesis involved HS‐proteoglycan expression and found that syndecans 1 and 3 were upregulated in RWPE1 cells in the transition from two‐ to three‐dimensional (3D) Matrigel, capable of promoting spheroid formation. We then investigated whether sodium chlorate, a general sulfation inhibitor, interfered with spheroid formation by RWPE1 cells and acinar morphogenesis in ex vivo ventral prostate (VP) organ culture. As expected, treatment with sodium chlorate inhibited spheroid formation by RWPE1 cells in 3D culture. Chlorate also inhibited ex vivo VP epithelial branching and canalization, resulting in long branchless epithelial structures. We then investigated whether the HS‐binding factors, FGF10, TGFβ1, and SDF1, could reverse the effect of sodium chlorate. Although no effect was seen in the FGF10‐ and TGFβ1‐treated samples, SDF1 promoted epithelial canalization in the low sulfated environment, highlighting its specific role in lumen formation. Altogether, the results show that sodium chlorate perturbed prostate morphogenesis and allowed investigation of factors involved in branching and/or canalization, implicating SDF1 signaling in epithelial canalization.

Matrigel

10.1002/jcp.27447

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A practical and robust method to evaluate metabolic fluxes in pancreatic islets

bioRxiv (Cold Spring Harbor Laboratory) (2023)

Débora Santos Rocha Antonio Carlos Manucci Alexandre Bruni‐Cardoso Alicia J. Kowaltowski Eloisa Aparecida Vilas‐Boas

Abstract Aims/hypothesis Efficient mitochondrial oxidative phosphorylation is essential for pancreatic beta cell responses to nutrient levels. Consequently, the evaluation of mitochondrial oxygen consumption and ATP production is important to investigate essential aspects of pancreatic islet pathophysiology. Currently, most studies use cell lines instead of primary islets due to difficulties in measuring primary islet respiration, which requires specific equipment and consumables that are expensive, complicated to use, and poorly reproducible. The aim of this study is to establish a robust and practical method to assess primary islet metabolic fluxes using Extracellular Flux Technology and standard commercial consumables. Methods Pancreatic islets were isolated from 8 to 12-week-old mice and rats, and submitted to a dispersion protocol using trypsin. Dispersed islets were adhered overnight to pre-coated standard Seahorse microplates, and oxygen consumption rates were evaluated using a Seahorse Extracellular Flux Analyzer. We also validated the functionality of dispersed islets by analyzing glucose-stimulated insulin secretion (GSIS) and calcium (Ca 2+ ) influx in response to different modulators by fluorescence microscopy. Results We provide a detailed protocol with all steps necessary to optimize islet isolation and dispersion, in order to achieve a high yield of functional islets and perform metabolic flux analysis. With this method, which requires only a few islets per replicate, both rat and mouse islets present robust basal respiration and proper response to mitochondrial modulators (oligomycin, CCCP, antimycin and rotenone) and glucose addition. Both oligomycin and CCCP concentrations were titrated. Our method was also validated by other functional assays, which show these cells present conserved Ca 2+ influx and insulin secretion in response to glucose. Conclusions/interpretation We established a practical and robust method to assess ex vivo islet metabolic fluxes and oxidative phosphorylation. Our findings cover an important gap in primary islet physiology studies, providing a valuable tool we hope is useful to uncover basic beta cell metabolic mechanisms, as well as for translational investigations, such as pharmacological candidate discovery and islet transplantation protocols. Research in context What is already known about this subject? Pancreatic beta cells efficiently couple oxidative phosphorylation and ATP production with insulin secretion; mitochondrial ATP production is crucial for proper insulin secretion. Most studies of beta cell respiration use cell lines instead of primary islets, which are a much more robust model to evaluate beta cell function. The few works with primary islet respiration use specific equipment and consumables that are expensive, complicated, and poorly reproducible. What is the key question? Is it possible to develop a practical method to evaluate metabolic fluxes and ATP production in isolated islets, using the standard Seahorse Extracellular Flux Technology? What are the new findings? We optimized rodent islet isolation and functional analysis protocols using standard extracellular flux analysis equipment and consumables. Our method allows for increased islet yield and robust islet respiration measurements. How might this impact on clinical practice in the foreseeable future? Quantitative measurements of metabolic fluxes and oxidative phosphorylation are the cornerstone of new discoveries in beta cells, and can contribute toward the establishment of new cellular protocols, such as for cell transplantation, as well as the development of new pharmacological agents targeted to these cells.

Pancreatic Islets

Oligomycin

Respirometry

10.1101/2023.07.25.550538

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Distinct photo-oxidation-induced cell death pathways lead to selective killing of human breast cancer cells

Cell Death and Disease (2020)

Ancély Ferreira dos Santos Alex Inague Gabriel Santos Arini Letícia Ferreira Terra Rosangela A.M. Wailemann

Abstract Lack of effective treatments for aggressive breast cancer is still a major global health problem. We have previously reported that photodynamic therapy using methylene blue as photosensitizer (MB-PDT) massively kills metastatic human breast cancer, marginally affecting healthy cells. In this study, we aimed to unveil the molecular mechanisms behind MB-PDT effectiveness and specificity towards tumor cells. Through lipidomics and biochemical approaches, we demonstrated that MB-PDT efficiency and specificity rely on polyunsaturated fatty acid-enriched membranes and on the better capacity to deal with photo-oxidative damage displayed by non-tumorigenic cells. We found out that, in tumorigenic cells, lysosome membrane permeabilization is accompanied by ferroptosis and/or necroptosis. Our results also pointed at a cross-talk between lysosome-dependent cell death (LDCD) and necroptosis induction after photo-oxidation, and contributed to broaden the understanding of MB-PDT-induced mechanisms and specificity in breast cancer cells. Therefore, we demonstrated that efficient approaches could be designed on the basis of lipid composition and metabolic features for hard-to-treat cancers. The results further reinforce MB-PDT as a therapeutic strategy for highly aggressive human breast cancer cells.

10.1038/s41419-020-03275-2

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Citations (46)

Prognostic value of integrin αV expression and localization pattern in invasive breast carcinomas

Neoplasia (2022)

Otto Luiz Dutra Cerqueira Mayara Carolline Silva Botelho Ana Paula Zen Petisco Fiore Cynthia Aparecida Bueno de Toledo Osório Rebeka Tomasin

Invasion of surrounding stroma is an early event in breast cancer metastatic progression, and involves loss of cell polarity, loss of myoepithelial layer, epithelial-mesenchymal transition (EMT) and remodeling of the extracellular matrix (ECM). Integrins are transmembrane receptors responsible for cell-ECM binding, which triggers signals that regulate many aspects of cell behavior and fate. Changes in the expression, localization and pairing of integrins contribute for abnormal responses found in transformed epithelia. We analyzed 345 human breast cancer samples in tissue microarrays (TMA) from cases diagnosed with invasive breast carcinoma to assess the expression and localization pattern of integrin αV and correlation with clinical parameters. Patients with lower levels of integrin αV staining showed reduced cancer specific survival. A subset of cases presented a peripheral staining of integrin αV surrounding tumor cell clusters, possibly matching the remaining myoepithelial layer. Indeed, the majority of ductal carcinoma in situ (DCIS) components found in the TMA presented integrin αV at their periphery, whereas this pattern was mostly lost in invasive components, even in the same sample. The lack of peripheral integrin αV correlated with decreased cancer specific survival. In addition, we observed that the presence of integrin αV in the stroma was an indicative of poor survival and metastatic disease. Consistently, by interrogating publicly available datasets we found that, although patients with higher mRNA levels of integrin αV had increased risk of developing metastasis, high co-expression of integrin αV and a myoepithelial cell marker (MYH11) mRNA levels correlated with better clinical outcomes. Finally, a 3D cell culture model of non-malignant and malignant cells reproduced the integrin αV pattern seen in patient samples. Taken together, our data indicate that both the expression levels of integrin αV and its tissue localization in primary tumors have prognostic value, and thus, could be used to help predict patients at higher risk of developing metastasis.

Tissue microarray

10.1016/j.neo.2022.100803

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Citations (5)

Extracellular matrix stiffness regulates degradation of MST2 via SCF βTrCP

Biochimica et Biophysica Acta (BBA) - General Subjects (2022)

Ana Paula Zen Petisco Fiore Ana Maria Rodrigues Helder Veras Ribeiro Filho Antonio Carlos Manucci Pedro de Freitas Ribeiro

Hippo signaling pathway

Proteostasis

10.1016/j.bbagen.2022.130238

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Citations (6)

MMP‐2 regulates rat ventral prostate development in vitro

Developmental Dynamics (2010)

Alexandre Bruni‐Cardoso Rafaela Rosa‐Ribeiro Vinicius Pascoal André A. de Thomaz Carlos L. Cesar

Abstract We have hypothesized that epithelial growth, branching, and canalization in the rodent ventral prostate (VP) would require matrix remodeling, and hence matrix metalloproteinase (MMP) activity. Therefore, the aim of this study was to evaluate the impact of blocking MMP‐2, using whole organ culture. siRNA was employed to inhibit MMP‐2 expression, and this was compared to GM6001's (a broad‐spectrum MMP inhibitor) inhibition of general MMPs. These blocks impaired VP morphogenesis. MMP‐2 silencing reduced organ size, epithelial area, and the number of tips, as well as caused a dilation of the distal parts of the epithelium. Histology, 3‐D reconstruction, biochemistry, and second harmonic generation (SHG) revealed that MMP‐2 silencing affected VP architecture by interfering in epithelial cell proliferation, lumen formation, and cellular organization of both epithelium and stroma, besides intense accumulation of collagen fibers. These data suggest that MMP‐2 plays important roles in prostate growth, being directly involved with epithelial morphogenesis. Developmental Dynamics 239:737–746, 2010. © 2010 Wiley‐Liss, Inc.

Organ culture

Matrix metalloproteinase inhibitor

10.1002/dvdy.22222

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Citations (18)

Identification of a novel alternative splicing isoform of the Hippo kinase STK3/MST2 with impaired kinase and cell growth suppressing activities

Oncogene (2024)

Ana Maria Rodrigues Ana Paula Zen Petisco Fiore Gabriela D. A. Guardia Rebeka Tomasin André A. Teixeira

Hippo signaling pathway

10.1038/s41388-024-03104-2

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The SARS-CoV-2 Nsp3 macrodomain reverses PARP9/DTX3L-dependent ADP-ribosylation induced by interferon signalling

bioRxiv (Cold Spring Harbor Laboratory) (2021)

Lillian Cristina Russo Rebeka Tomasin Isaac de Araújo Matos Antonio Carlos Manucci Sven T. Sowa

Abstract SARS-CoV-2 non-structural protein 3 (Nsp3) contains a macrodomain that is essential for virus replication and is thus an attractive target for drug development. This macrodomain is thought to counteract the host interferon (IFN) response, an important antiviral signalling cascade, via the removal of ADP-ribose modifications catalysed by host poly(ADP-ribose) polymerases (PARPs). Here, we show that activation of the IFN response induces ADP-ribosylation of host proteins and that ectopic expression of the SARS-CoV-2 Nsp3 macrodomain reverses this modification in human cells. We further demonstrate that this can be used to screen for cell-active macrodomain inhibitors without the requirement for BSL-3 facilities. This IFN-induced ADP-ribosylation is dependent on the PARP9/DTX3L heterodimer, but surprisingly the expression of Nsp3 macrodomain or PARP9/DTX3L deletion do not impair STAT1 phosphorylation or the induction of IFN-responsive genes. Our results suggest that PARP9/DTX3L-dependent ADP-ribosylation is a downstream effector of the host IFN response and that the cellular function of the SARS-CoV-2 Nsp3 macrodomain is to hydrolyse this end product of IFN signalling, and not to suppress the IFN response itself.

Ectopic expression

ADP-ribosylation

10.1101/2021.04.06.438552

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Citations (9)