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      Pyruvate carboxylase supports the pulmonary tropism of metastatic breast cancer

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          Abstract

          Background

          Overcoming systemic dormancy and initiating secondary tumor grow under unique microenvironmental conditions is a major rate-limiting step in metastatic progression. Disseminated tumor cells encounter major changes in nutrient supplies and oxidative stresses compared to the primary tumor and must demonstrate significant metabolic plasticity to adapt to specific metastatic sites. Recent studies suggest that differential utilization of pyruvate sits as a critical node in determining the organotropism of metastatic breast cancer. Pyruvate carboxylase (PC) is key enzyme that converts pyruvate into oxaloacetate for utilization in gluconeogenesis and replenishment of the TCA cycle.

          Methods

          Patient survival was analyzed with respect to gene copy number alterations and differential mRNA expression levels of PC. Expression of PC was analyzed in the MCF-10A, D2-HAN and the 4 T1 breast cancer progression series under in vitro and in vivo growth conditions. PC expression was depleted via shRNAs and the impact on in vitro cell growth, mammary fat pad tumor growth, and pulmonary and non-pulmonary metastasis was assessed by bioluminescent imaging. Changes in glycolytic capacity, oxygen consumption, and response to oxidative stress were quantified upon PC depletion.

          Results

          Genomic copy number increases in PC were observed in 16–30% of metastatic breast cancer patients. High expression of PC mRNA was associated with decreased patient survival in the MCTI and METABRIC patient datasets. Enhanced expression of PC was not recapitulated in breast cancer progression models when analyzed under glucose-rich in vitro culture conditions. In contrast, PC expression was dramatically enhanced upon glucose deprivation and in vivo in pulmonary metastases. Depletion of PC led to a dramatic decrease in 4 T1 pulmonary metastasis, but did not affect orthotopic primary tumor growth. Tail vein inoculations confirmed the role of PC in facilitating pulmonary, but not extrapulmonary tumor initiation. PC-depleted cells demonstrated a decrease in glycolytic capacity and oxygen consumption rates and an enhanced sensitivity to oxidative stress.

          Conclusions

          Our studies indicate that PC is specifically required for the growth of breast cancer that has disseminated to the lungs. Overall, these findings point to the potential of targeting PC for the treatment of pulmonary metastatic breast cancer.

          Electronic supplementary material

          The online version of this article (10.1186/s13058-018-1008-9) contains supplementary material, which is available to authorized users.

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          Most cited references17

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          Selective events in the metastatic process defined by analysis of the sequential dissemination of subpopulations of a mouse mammary tumor.

          To identify selective steps in metastasis, those that eliminate nonmetastatic tumor cells more efficiently than metastatic cells, we have evaluated the sequential dissemination of tumor cells from a mammary fatpad, using both metastatic (4T1 and 66cl4) and nonmetastatic (67NR, 168FARN, and 4TO7) subpopulations of a single mouse mammary tumor. Each of these variant subpopulations is resistant to one or more selective drugs so they could be quantitatively identified by colony formation in selective media. We found that the 2 metastatic cell lines metastasized by different routes and that the nonmetastatic tumor cell lines failed at different points in dissemination. Line 67NR did not leave the primary site; clonogenic tumor cells were not detected in the nodes, blood, or lungs during the experiment (7 weeks). Tumor line 168FARN disseminated from the primary tumor because clonogenic cells were cultured from the draining lymph nodes throughout the experiment. However, dissemination essentially stopped in the node as cells were rarely isolated from blood, lungs, or lives. Whether 168FARN cells failed to reach these tissues or were killed very rapidly after traversing the lymph node is unknown. Line 4TO7 cells disseminated via the blood and were consistently recovered from lungs by day 19 but failed to proliferate. This panel of 5 subpopulations thus identifies different points of selective failure in tumor cell dissemination and should be valuable in the assessment of antimetastatic therapies.
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            TScratch: a novel and simple software tool for automated analysis of monolayer wound healing assays.

            Cell migration plays a major role in development, physiology, and disease, and is frequently evaluated in vitro by the monolayer wound healing assay. The assay analysis, however, is a time-consuming task that is often performed manually. In order to accelerate this analysis, we have developed TScratch, a new, freely available image analysis technique and associated software tool that uses the fast discrete curvelet transform to automate the measurement of the area occupied by cells in the images. This tool helps to significantly reduce the time needed for analysis and enables objective and reproducible quantification of assays. The software also offers a graphical user interface which allows easy inspection of analysis results and, if desired, manual modification of analysis parameters. The automated analysis was validated by comparing its results with manual-analysis results for a range of different cell lines. The comparisons demonstrate a close agreement for the vast majority of images that were examined and indicate that the present computational tool can reproduce statistically significant results in experiments with well-known cell migration inhibitors and enhancers.
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              PDK1-Dependent Metabolic Reprogramming Dictates Metastatic Potential in Breast Cancer.

              Metabolic reprogramming is a hallmark of cellular transformation, yet little is known about metabolic changes that accompany tumor metastasis. Here we show that primary breast cancer cells display extensive metabolic heterogeneity and engage distinct metabolic programs depending on their site of metastasis. Liver-metastatic breast cancer cells exhibit a unique metabolic program compared to bone- or lung-metastatic cells, characterized by increased conversion of glucose-derived pyruvate into lactate and a concomitant reduction in mitochondrial metabolism. Liver-metastatic cells displayed increased HIF-1α activity and expression of the HIF-1α target Pyruvate dehydrogenase kinase-1 (PDK1). Silencing HIF-1α reversed the glycolytic phenotype of liver-metastatic cells, while PDK1 was specifically required for metabolic adaptation to nutrient limitation and hypoxia. Finally, we demonstrate that PDK1 is required for efficient liver metastasis, and its expression is elevated in liver metastases from breast cancer patients. Our data implicate PDK1 as a key regulator of metabolism and metastatic potential in breast cancer.
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                Author and article information

                Contributors
                ashinde@purdue.edu
                twilmans@purdue.edu
                chen2334@purdue.edu
                dteegard@purdue.edu
                765-494-0860 , mwendt@purdue.edu
                Journal
                Breast Cancer Res
                Breast Cancer Res
                Breast Cancer Research : BCR
                BioMed Central (London )
                1465-5411
                1465-542X
                13 July 2018
                13 July 2018
                2018
                : 20
                : 76
                Affiliations
                [1 ]ISNI 0000 0004 1937 2197, GRID grid.169077.e, Purdue University Center for Cancer Research, , Purdue University, ; West Lafayette, IN 47907 USA
                [2 ]ISNI 0000 0004 1937 2197, GRID grid.169077.e, Department of Medicinal Chemistry and Molecular Pharmacology, , Purdue University, ; West Lafayette, IN 47907 USA
                [3 ]Department of Nutrition Science, West Lafayette, IN 47907 USA
                Author information
                http://orcid.org/0000-0002-3665-7413
                Article
                1008
                10.1186/s13058-018-1008-9
                6045837
                30005601
                3310655c-5311-46ac-90c9-bbcea9aeb6d7
                © The Author(s). 2018

                Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

                History
                : 7 November 2017
                : 25 June 2018
                Funding
                Funded by: FundRef http://dx.doi.org/10.13039/100000054, National Cancer Institute;
                Award ID: R01CA207751
                Award ID: P30CA023168
                Funded by: FundRef http://dx.doi.org/10.13039/100000048, American Cancer Society;
                Award ID: RSG-CSM130259
                Funded by: FundRef http://dx.doi.org/10.13039/100000002, National Institutes of Health;
                Award ID: R25CA128770
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/100006975, Indiana Clinical and Translational Sciences Institute;
                Award ID: UL1TR001108
                Award Recipient :
                Categories
                Research Article
                Custom metadata
                © The Author(s) 2018

                Oncology & Radiotherapy
                breast cancer,metastasis,pyruvate carboxylase,metabolism,glycolysis,hypoxia,oxidative stress

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