Author Manuscript Published OnlineFirst on July 6, 2020; DOI: 10.1158/0008-5472.CAN-19-2269 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Title. Aquaporin-7 Regulates the Response to Cellular Stress in Breast Cancer Authors. Chen Dai1,2*, Verodia Charlestin1,2*, Man Wang1,2, Zachary T. Walker1,2, Maria Cristina Miranda-Vergara1,2, Beth A. Facchine1,2, Junmin Wu1,2, William J. Kaliney2, Norman J. Dovichi1,2, Jun Li2,3, and Laurie E. Littlepage1,2 Affiliations. 1Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556 2Harper Cancer Research Institute, South Bend, IN 46617 3Department of Applied and Computational Mathematics and Statistics, University of Notre Dame, Notre Dame, IN 46556 *Authors contributed equally Running title. Aquaporin-7 is a metabolic regulator in breast cancer Keywords. Aquaporin, breast cancer, tumor metabolism, correlation-based network analysis Corresponding Author. Laurie Littlepage, Ph.D., Harper Cancer Research Institute, University of Notre Dame, 1234 N Notre Dame Avenue, South Bend, IN 46617; Phone: (574) 631-4804 Fax: (574) 631-1165 Email: [email protected] Conflict of interest disclosure statement. The authors disclose no potential conflicts of interest. 1 Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on July 6, 2020; DOI: 10.1158/0008-5472.CAN-19-2269 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Abstract The complex yet interrelated connections between cancer metabolism, gene expression, and oncogenic driver genes have the potential to identify novel biomarkers and drug targets with prognostic and therapeutic value. Here we effectively integrated metabolomics and gene expression data from breast cancer mouse models through a novel unbiased correlation-based network analysis. This approach identified 35 metabolite and 34 gene hubs with the most network correlations. These hubs have prognostic value and are likely integral to tumor metabolism and breast cancer. The gene hub Aquaporin-7 (Aqp7), a water and glycerol channel, was identified as a novel regulator of breast cancer. AQP7 was prognostic of overall survival in breast cancer patients. In mouse breast cancer models, reduced expression of Aqp7 caused reduced primary tumor burden and lung metastasis. Metabolomics and complex lipid profiling of cells and tumors with reduced Aqp7 revealed significantly altered lipid metabolism, glutathione metabolism, and urea/arginine metabolism compared to controls. These data identify AQP7 as a critical regulator of metabolic and signaling responses to environmental cellular stresses in breast cancer, highlighting AQP7 as a potential cancer-specific therapeutic vulnerability. Statement of Significance Aquaporin-7 is identified as a critical regulator of nutrient availability and signaling that responds to cellular stresses, making it an attractive therapeutic target in breast cancer. 2 Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on July 6, 2020; DOI: 10.1158/0008-5472.CAN-19-2269 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Introduction Recent research established a new understanding of the Warburg effect and other metabolic pathways of cancer (1–3). Even with these advances in understanding cancer metabolism, therapeutic targeting of cancer metabolism has been difficult, mainly due to the complex, interrelated nature of metabolic pathways. Thus, understanding not only the factors that distinguish cancer metabolism from normal metabolism but also the crosstalk between metabolic pathways, gene expression, and the microenvironment is crucial for designing metabolism-based cancer therapies. Aquaporins facilitate the passive transport of water across plasma membranes and are candidate regulators of tumor metabolism. A subset of these membrane proteins, known as aquaglyceroporins, transport glycerol, water, and other small molecules (4). Aquaporin-7 (human AQP7/mouse Aqp7) is a transmembrane aquaglyceroporin and member of the aquaporin family (5–7). AQP7 also transports hydrogen peroxide, ammonia, urea, and arsenite (8–13). AQP7 has been particularly well studied in adipose tissue (6,7,14,15). During high energy demands and metabolic stress, lipolysis increases and converts triglycerides (TAG) into free fatty acids (FFA) and glycerol. AQP7 controls the efflux of glycerol under these conditions. The exported glycerol then is taken up by other cells and used as a backbone for energy needs during high energy demands. Aqp7 deficiency in animal models is associated with adipocyte hypertrophy, increased glycerol and TAG accumulation, insulin resistance, and increased 3 Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on July 6, 2020; DOI: 10.1158/0008-5472.CAN-19-2269 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. obesity in both mice and humans (6,7,14,15). Since obesity and the mammary adipose microenvironment promote aggressive breast cancers in both rodents and humans, Aqp7 may contribute to breast cancer pathogenesis (16,17). While other aquaporins regulate breast cancer invasion, Aqp7 has not been investigated in tumor pathogenesis (18–20). Materials and Methods Animals Mice were maintained under pathogen-free conditions in the University of Notre Dame Freimann Life Sciences animal facility. Animal experiments were conducted in accordance with the protocol guidelines approved by the Institution Animal Care and Use Committee (15-10-2724). Details on orthotopic, contralateral, and tail vein models are discussed in Supplementary Methods. Cell Culture All cells were maintained at 37℃ in humidified incubators with 5% CO2 at atmospheric oxygen levels. 4T1 cells were a gift from Dr. Siyuan Zhang, University of Notre Dame. EpH4 and NMuMG cells were a gift from Dr. Zena Werb, University of California, San Francisco. 4T1 cells were grown in RPMI1640 media (Sigma R6504) with 10% FBS. EpH4 cells were cultured in DMEM High Glucose media (Sigma D5648) with 5% FBS. NMuMG cells were cultured in DMEM High Glucose media with 10% FBS and 1 μg/mL insulin. All cells were 4 Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on July 6, 2020; DOI: 10.1158/0008-5472.CAN-19-2269 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. routinely tested for mycoplasma by PCR (Genlantis MY01050) and colorimetric detection kit (InvivoGen rep-pt1) according to manufacturer’s directions. Quality control of the cell lines was maintained by continual authentication of morphology and growth rate and were used less than 16 passages. Mouse cell lines were not authenticated genetically. Detailed information on various in vitro experiments - proliferation, scratch, 3-D organoid and contact inhibition assays, cell size, gene knockdown, dependence, ROS, NO detection, urea and NADP+ levels, glycerol quantification and stress tolerance assays can be found in the Supplementary Methods. RNA Extraction and Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR) For RNA extraction, cultured cells were scraped from culture dishes and washed with PBS before flash freezing with liquid nitrogen. Flash frozen tissue samples were ground with a pestle and mortar on liquid nitrogen. RNA was isolated with RNA Bee (Amsbio, CS-104B) following the manufacturer’s instructions. RNA analysis, cDNA synthesis and RT-qPCR were carried out as described in Supplementary Methods. Western Blot, Immunofluorescence and IHC Western blot, immunofluorescence and IHC conducted as described in the Supplementary Methods. 5 Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on July 6, 2020; DOI: 10.1158/0008-5472.CAN-19-2269 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Metabolism Experiments by Seahorse Assays Glycolysis (Seahorse 103020-100) and mitochondrial stress test (Seahorse 103015-100) were conducted following the manufacturer’s instructions using the XFe-96 analyzer from Seahorse Bioscience. Results from analysis were normalized to cell count. See Supplementary Methods for details. Metabolomics and Complex Lipid Profiling Cell and tumor sample preparation for metabolomics and complex lipid profiling according to previously described protocol (21). See Supplementary Methods for additional details. Oil Red O Staining Lipids and triglycerides were stained using Oil Red O (Sigma-Aldrich, O0625- 25G). See Supplementary Methods for details. MALDI Imaging Tumor tissues were embedded in gelatin and then sectioned into 10 µm -20 µm slices. Alternating slices were collected during cryosection for Matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-IMS) and H&E staining. MALDI-MSI spectra were carried out on an UltrafleXtreme (Bruker Daltonics, Billerica, MA). Depending on mounting reagent different MALDI methods were used. Data was processed using FlexImaging