Paola de Andrade Mello

Beth Israel Deaconess Medical Center

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Andréia Buffon

Universidade Federal do Rio Grande do Sul

Simon C. Robson

Beth Israel Deaconess Medical Center

Luiz Eduardo Baggio Savio

Universidade Federal do Rio de Janeiro

Robson Coutinho‐Silva

Universidade Federal do Rio de Janeiro

Haohai Zhang

Beth Israel Deaconess Medical Center

Yan Wu

Union Hospital

Guido Lenz

Universidade Federal do Rio Grande do Sul

Michaël Wink

Heidelberg University

Wenda Gao

Antagen Pharmaceuticals (United States)

Eva Csizmadia

Beth Israel Deaconess Medical Center

Cooperative Institutions

Universidade Federal do Rio Grande do Sul

Harvard University

Beth Israel Deaconess Medical Center

Universidade Federal de Ciências da Saúde de Porto Alegre

Universidade Federal do Rio de Janeiro

Universidade Federal do Rio Grande

Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro

Hadassah Medical Center

Universidade de São Paulo

Beth Israel Deaconess Hospital

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Cervical cancer stem‐like cells: systematic review and identification of reference genes for gene expression

Cell Biology International (2017)

Rafael Paschoal de Campos Iago Carvalho Schultz Paola de Andrade Mello Samuel Davies Manuela Sangalli Gasparin

Cervical cancer is the fourth most common cancer affecting women worldwide. Among many factors, the presence of cancer stem cells, a subpopulation of cells inside the tumor, has been associated with a worse prognosis. Considering the importance of gene expression studies to understand the biology of cervical cancer stem cells (CCSC), this work identifies stable reference genes for cervical cancer cell lines SiHa, HeLa, and ME180 as well as their respective cancer stem-like cells. A literature review was performed to identify validated reference genes currently used to normalize RT-qPCR data in cervical cancer cell lines. Then, cell lines were cultured in regular monolayer or in a condition that favors tumor sphere formation. RT-qPCR was performed using five reference genes: ACTB, B2M, GAPDH, HPRT1, and TBP. Stability was assessed to validate the selected genes as suitable reference genes. The evaluation validated B2M, GAPDH, HPRT1, and TBP in these experimental conditions. Among them, GAPDH and TBP presented the lowest variability according to the analysis by Normfinder, Bestkeeper, and ΔCq methods, being therefore the most adequate genes to normalize the combination of all samples. These results suggest that B2M, GAPDH, HPRT1, and TBP are suitable reference genes to normalize RT-qPCR data of established cervical cancer cell lines SiHa, HeLa, and ME180 as well as their derived cancer stem-like cells. Indeed, GAPDH and TBP seem to be the most convenient choices for studying gene expression in these cells in monolayers or spheres.

Reference Genes

HeLa

Glyceraldehyde 3-phosphate dehydrogenase

10.1002/cbin.10878

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Abstract B208: Reduction in P2X7 receptor expression is a marker of resistance to ATP treatment in human cervical carcinoma cell line.

Molecular Cancer Therapeutics (2013)

Paola de Andrade Mello Eduardo Cremonese Filippi–Chiela Jéssica Nascimento Franciele Kipper Aline Beckenkamp

Abstract Cervical cancer is the third most commonly diagnosed malignancy and the fourth leading cause of cancer death in females worldwide. The high mortality rate is largely due to the lack of effective therapies for eliminating disease in women with high-grade cervical cancer and the lack of response to chemotherapy of inoperable disease. Thus, to understand the mechanisms involved on cervical cancer development and find a molecular target to prevent it is strongly desirable. Until now, researchers show that HPV infection and suppression of cell death mechanisms like apoptosis are the most important factors involving of cervical carcinogenesis. Anyway, the mechanism that leads cells infected by HPV escape to apoptosis remains unknown. A role for purinergic signaling on control of cell growth and death, mainly through P2X7 receptor activation by extracellular ATP, has been described. We hypothesized that a disruption in this signaling could be involved with cervical cancer resistance to apoptosis and development. Previous work, using human carcinoma cell line SiHa, showed that ATP 5mM induces cell death through apoptosis by P2X7 activation. However, the mechanism involved in this cytotoxic effect remained unclear. To answers this question, we investigate the role of P2X7 on ATP induce cell death. We started analyzing P2X7 protein expression in cells resistant to ATP treatment. In this case, SiHa cells were treated with ATP 5mM for 24, 48, and 72h, and the adherent survival cells were removed and analyzed by Western Blot. After, we knockdown P2X7 receptor in SiHa cell line and evaluate this effect on cell viability after ATP treatment. In addition, we studied the mechanistic way involved in this cell death pathway using EGTA and caspase-3 inhibitor previous to ATP treatment. Unexpectedly, SiHa wild type (WT) cells that remained adherent after treatment with ATP showed less expression of P2X7, suggesting a defense way to apoptosis. Corroborating with this, SiHa cells knockdown (KD) for P2X7 showed increased cell viability when compared to SiHa WT and SiHa KD control, after exposure to ATP 5mM for 24, 48, and 72h. The mechanism of ATP induces apoptosis through P2X7 don't seems to be by increase intracellular calcium and caspase-3 activation, but in an independent caspase way. These findings suggest that P2X7 receptor could be involved with death and resistance cell mechanisms, indicating a possible new target on therapeutical research in cervical cancer and on tumor aggressiveness. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B208. Citation Format: Paola de Andrade Mello, Eduardo Cremonese Filippi-Chiela, Jessica Nascimento, Franciele Cristina Kipper, Aline Beckenkamp, Danielle Bertodo Santana, Alessandra Nejar Bruno, Guido Lenz, Andréia Buffon. Reduction in P2X7 receptor expression is a marker of resistance to ATP treatment in human cervical carcinoma cell line. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B208.

10.1158/1535-7163.targ-13-b208

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Correction: Hyperthermia and associated changes in membrane fluidity potentiate P2X7 activation to promote tumor cell death

Oncotarget (2022)

Paola de Andrade Mello Bian Shu Luiz Eduardo Baggio Savio Haohai Zhang Jingping Zhang

10.18632/oncotarget.27641

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Structural and functional characterization of engineered bifunctional fusion proteins of CD39 and CD73 ectonucleotidases

AJP Cell Physiology (2020)

Elizabeth H. Zhong Carola Ledderose Paola de Andrade Mello Keiichi Enjyoji Justin Mark Lunderberg

Extracellular diphosphate and triphosphate nucleotides are released from activated or injured cells to trigger vascular and immune P2 purinergic receptors, provoking inflammation and vascular thrombosis. These metabokines are scavenged by ectonucleoside triphosphate diphosphohydrolase-1 (E-NTPDase1 or CD39). Further degradation of the monophosphate nucleoside end products occurs by surface ecto-5'-nucleotidase (NMPase) or CD73. These ectoenzymatic processes work in tandem to promote adenosinergic responses, which are immunosuppressive and antithrombotic. These homeostatic ectoenzymatic mechanisms are lost in the setting of oxidative stress, which exacerbates inflammatory processes. We have engineered bifunctional enzymes made up from ectodomains (ECDs) of CD39 and CD73 within a single polypeptide. Human alkaline phosphatase-ectodomain (ALP-ECD) and human acid phosphatase-ectodomain (HAP-ECD) fusion proteins were also generated, characterized, and compared with these CD39-ECD, CD73-ECD, and bifunctional fusion proteins. Through the application of colorimetrical functional assays and high-performance liquid chromatography kinetic assays, we demonstrate that the bifunctional ectoenzymes express high levels of CD39-like NTPDase activity and CD73-like NMPase activity. Chimeric CD39-CD73-ECD proteins were superior in converting triphosphate and diphosphate nucleotides into nucleosides when compared with ALP-ECD and HAP-ECD. We also note a pH sensitivity difference between the bifunctional fusion proteins and parental fusions, as well as ectoenzymatic property distinctions. Intriguingly, these innovative reagents decreased platelet activation to exogenous agonists in vitro. We propose that these chimeric fusion proteins could serve as therapeutic agents in inflammatory diseases, acting to scavenge proinflammatory ATP and also generate anti-inflammatory adenosine.

Characterization

10.1152/ajpcell.00430.2020

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O papel dos nucleotídeos e nucleosídeos da adenina e do receptor P2x7 no controle da proliferação e morte celular e tumoral

Paola de Andrade Mello

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Exposição ocupacional a antineoplásicos citotóxicos : acompanhamento médico aos manipuladores

Ricardo Soares Gioda Edison Capp Helena von Eye Corleta Paola de Andrade Mello

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Glycoengineered anti-CD39 promotes anticancer responses by depleting suppressive cells and inhibiting angiogenesis in tumor models

Journal of Clinical Investigation (2022)

Haohai Zhang Lili Feng Paola de Andrade Mello Changchuin Mao Richard I. Near

Immunosuppressive cells accumulating in the tumor microenvironment constitute a formidable barrier that interferes with current immunotherapeutic approaches. A unifying feature of these tumor-associated immune and vascular endothelial cells appears to be the elevated expression of ectonucleotidase CD39, which in tandem with ecto-5′-nucleotidase CD73, catalyzes the conversion of extracellular ATP into adenosine. We glycoengineered an afucosylated anti-CD39 IgG2c and tested this reagent in mouse melanoma and colorectal tumor models. We identified major biological effects of this approach on cancer growth, associated with depletion of immunosuppressive cells, mediated through enhanced Fcγ receptor–directed (FcγR-directed), antibody-dependent cellular cytotoxicity (ADCC). Furthermore, regulatory/exhausted T cells lost CD39 expression, as a consequence of antibody-mediated trogocytosis. Most strikingly, tumor-associated macrophages and endothelial cells with high CD39 expression were effectively depleted following antibody treatment, thereby blocking angiogenesis. Tumor site–specific cellular modulation and lack of angiogenesis synergized with chemotherapy and anti–PD-L1 immunotherapy in experimental tumor models. We conclude that depleting suppressive cells and targeting tumor vasculature, through administration of afucosylated anti-CD39 antibody and the activation of ADCC, comprises an improved, purinergic system–modulating strategy for cancer therapy.

10.1172/jci157431

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Fc Receptor-Dependent Trogocytosis of CD39 Impacts Engraftment and Invasiveness of Acute Myeloid Leukemia Cells

Blood (2021)

Lili Feng Haohai Zhang Paola de Andrade Mello Dina Stroopinsky Wenda Gao

Bioluminescence imaging

10.1182/blood-2021-151021

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P2X7 receptor activation increases expression of caveolin-1 and formation of macrophage lipid rafts, thereby boosting CD39 activity

Journal of Cell Science (2020)

Luiz Eduardo Baggio Savio Paola de Andrade Mello Stephanie Alexia Cristina Silva Santos Júlia Costa de Sousa Suellen Darc Oliveira

ABSTRACT Macrophages are tissue-resident immune cells that are crucial for the initiation and maintenance of immune responses. Purinergic signaling modulates macrophage activity and impacts cellular plasticity. The ATP-activated purinergic receptor P2X7 (also known as P2RX7) has pro-inflammatory properties, which contribute to macrophage activation. P2X7 receptor signaling is, in turn, modulated by ectonucleotidases, such as CD39 (also known as ENTPD1), expressed in caveolae and lipid rafts. Here, we examined P2X7 receptor activity and determined impacts on ectonucleotidase localization and function in macrophages primed with lipopolysaccharide (LPS). First, we verified that ATP boosts CD39 activity and caveolin-1 protein expression in LPS-primed macrophages. Drugs that disrupt cholesterol-enriched domains – such as nystatin and methyl-β-cyclodextrin – decreased CD39 enzymatic activity in all circumstances. We noted that CD39 colocalized with lipid raft markers (flotillin-2 and caveolin-1) in macrophages that had been primed with LPS followed by treatment with ATP. P2X7 receptor inhibition blocked these ATP-mediated increases in caveolin-1 expression and inhibited the colocalization with CD39. Further, we found that STAT3 activation is significantly attenuated caveolin-1-deficient macrophages treated with LPS or LPS+BzATP. Taken together, our data suggest that P2X7 receptor triggers the initiation of lipid raft-dependent mechanisms that upregulates CD39 activity and could contribute to limit macrophage responses restoring homeostasis.

Lipid raft

Caveolin 1

Boosting

10.1242/jcs.237560

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Contribuição de diferentes rotas na biossíntese de gangliosídios nos dois fenótipos da linhagem celular GRX

Paola de Andrade Mello Aline Bohnenberger de Aguirres Cláudia Marlise Balbinotti Andrade Regina Maria Vieira da Costa Guaragna Radovan Borojević

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