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      Integrative Analysis of Breast Cancer Cells Reveals an Epithelial-Mesenchymal Transition Role in Adaptation to Acidic Microenvironment

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          Abstract

          Early ducts of breast tumors are unequivocally acidic. High rates of glycolysis combined with poor perfusion lead to a congestion of acidic metabolites in the tumor microenvironment, and pre-malignant cells must adapt to this acidosis to thrive. Adaptation to acidosis selects cancer cells that can thrive in harsh conditions and are capable of outgrowing the normal or non-adapted neighbors. This selection is usually accompanied by phenotypic change. Epithelial mesenchymal transition (EMT) is one of the most important switches correlated to malignant tumor cell phenotype and has been shown to be induced by tumor acidosis. New evidence shows that the EMT switch is not a binary system and occurs on a spectrum of transition states. During confirmation of the EMT phenotype, our results demonstrated a partial EMT phenotype in our acid-adapted cell population. Using RNA sequencing and network analysis we found 10 dysregulated network motifs in acid-adapted breast cancer cells playing a role in EMT. Our further integrative analysis of RNA sequencing and SILAC proteomics resulted in recognition of S100B and S100A6 proteins at both the RNA and protein level. Higher expression of S100B and S100A6 was validated in vitro by Immunocytochemistry. We further validated our finding both in vitro and in patients' samples by IHC analysis of Tissue Microarray (TMA). Correlation analysis of S100A6 and LAMP2b as marker of acidosis in each patient from Moffitt TMA approved the acid related role of S100A6 in breast cancer patients. Also, DCIS patients with higher expression of S100A6 showed lower survival compared to lower expression. We propose essential roles of acid adaptation in cancer cells EMT process through S100 proteins such as S100A6 that can be used as therapeutic strategy targeting both acid-adapted and malignant phenotypes.

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          Most cited references38

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          Hypoxia-inducible expression of tumor-associated carbonic anhydrases.

          The transcriptional complex hypoxia-inducible factor-1 (HIF-1) has emerged as an important mediator of gene expression patterns in tumors, although the range of responding genes is still incompletely defined. Here we show that the tumor-associated carbonic anhydrases (CAs) are tightly regulated by this system. Both CA9 and CA12 were strongly induced by hypoxia in a range of tumor cell lines. In renal carcinoma cells that are defective for the von Hippel-Lindau (VHL) tumor suppressor, up-regulation of these CAs is associated with loss of regulation by hypoxia, consistent with the critical function of pVHL in the regulation of HIF-1. Further studies of CA9 defined a HIF-1-dependent hypoxia response element in the minimal promoter and demonstrated that tight regulation by the HIF/pVHL system was reflected in the pattern of CA IX expression within tumors. Generalized up-regulation of CA IX in VHL-associated renal cell carcinoma contrasted with focal perinecrotic expression in a variety of non-VHL-associated tumors. In comparison with vascular endothelial growth factor mRNA, expression of CA IX demonstrated a similar, although more tightly circumscribed, pattern of expression around regions of necrosis and showed substantial although incomplete overlap with activation of the hypoxia marker pimonidazole. These studies define a new class of HIF-1-responsive gene, the activation of which has implications for the understanding of hypoxic tumor metabolism and which may provide endogenous markers for tumor hypoxia.
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            A practical guide to the MaxQuant computational platform for SILAC-based quantitative proteomics.

            MaxQuant is a quantitative proteomics software package designed for analyzing large mass spectrometric data sets. It is specifically aimed at high-resolution mass spectrometry (MS) data. Currently, Thermo LTQ-Orbitrap and LTQ-FT-ICR instruments are supported and Mascot is used as a search engine. This protocol explains step by step how to use MaxQuant on stable isotope labeling by amino acids in cell culture (SILAC) data obtained with double or triple labeling. Complex experimental designs, such as time series and drug-response data, are supported. A standard desktop computer is sufficient to fulfill the computational requirements. The workflow has been stress tested with more than 1,000 liquid chromatography/mass spectrometry runs in a single project. In a typical SILAC proteome experiment, hundreds of thousands of peptides and thousands of proteins are automatically and reliably quantified. Additional information for identified proteins, such as Gene Ontology, domain composition and pathway membership, is provided in the output tables ready for further bioinformatics analysis. The software is freely available at the MaxQuant home page.
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              Neutralization of Tumor Acidity Improves Antitumor Responses to Immunotherapy.

              Cancer immunotherapies, such as immune checkpoint blockade or adoptive T-cell transfer, can lead to durable responses in the clinic, but response rates remain low due to undefined suppression mechanisms. Solid tumors are characterized by a highly acidic microenvironment that might blunt the effectiveness of antitumor immunity. In this study, we directly investigated the effects of tumor acidity on the efficacy of immunotherapy. An acidic pH environment blocked T-cell activation and limited glycolysis in vitro. IFNγ release blocked by acidic pH did not occur at the level of steady-state mRNA, implying that the effect of acidity was posttranslational. Acidification did not affect cytoplasmic pH, suggesting that signals transduced by external acidity were likely mediated by specific acid-sensing receptors, four of which are expressed by T cells. Notably, neutralizing tumor acidity with bicarbonate monotherapy impaired the growth of some cancer types in mice where it was associated with increased T-cell infiltration. Furthermore, combining bicarbonate therapy with anti-CTLA-4, anti-PD1, or adoptive T-cell transfer improved antitumor responses in multiple models, including cures in some subjects. Overall, our findings show how raising intratumoral pH through oral buffers therapy can improve responses to immunotherapy, with the potential for immediate clinical translation.
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                Author and article information

                Contributors
                Journal
                Front Oncol
                Front Oncol
                Front. Oncol.
                Frontiers in Oncology
                Frontiers Media S.A.
                2234-943X
                10 March 2020
                2020
                : 10
                : 304
                Affiliations
                [1] 1Department of Cell and Molecular Biology, Faculty of Science, Semnan University , Semnan, Iran
                [2] 2Department of Cancer Physiology, Moffitt Cancer Center and Research Institute , Tampa, FL, United States
                [3] 3Proteomics Core, Moffitt Cancer Center and Research Institute , Tampa, FL, United States
                [4] 4Molecular Biology Core, Moffitt Cancer Center and Research Institute , Tampa, FL, United States
                [5] 5Microscopy Core, Moffitt Cancer Center and Research Institute , Tampa, FL, United States
                [6] 6Department of Oncologic Sciences, Morsani College of Medicine, University of South Florida , Tampa, FL, United States
                Author notes

                Edited by: Mohit Kumar Jolly, Indian Institute of Science (IISc), India

                Reviewed by: Dongya Jia, Rice University, United States; Prashant Kumar, Institute of Bioinformatics (IOB), India

                *Correspondence: Mehdi Damaghi mehdi.damaghi@ 123456moffitt.org

                This article was submitted to Molecular and Cellular Oncology, a section of the journal Frontiers in Oncology

                †These authors have contributed equally to this work

                Article
                10.3389/fonc.2020.00304
                7076123
                239e4f48-8c7e-429d-9fea-f9bce9d52687
                Copyright © 2020 Sadeghi, Ordway, Rafiei, Borad, Fang, Koomen, Zhang, Yoder, Johnson and Damaghi.

                This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

                History
                : 03 November 2019
                : 20 February 2020
                Page count
                Figures: 5, Tables: 1, Equations: 1, References: 64, Pages: 14, Words: 9128
                Categories
                Oncology
                Original Research

                Oncology & Radiotherapy
                acid adaptation,emt,tumor microenvironment,breast cancer,s100 family proteins

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