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Exosomes from Nischarin-Expressing Cells Reduce Breast Cancer Cell Motility and Tumor Growth

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Exosomes from Nischarin-Expressing Cells Reduce Breast Cancer Cell Motility and Tumor Growth Author Manuscript Published OnlineFirst on January 11, 2019; DOI: 10.1158/0008-5472.CAN-18-0842 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Exosomes from Nischarin-Expressing Cells Reduce Breast Cancer Cell Motility and tumor growth Mazvita Maziveyi1,7, Shengli Dong1, Somesh Baranwal2, Ali Mehrnezhad3, Rajamani Rathinam4, Thomas M. Huckaba5, Donald E. Mercante6, Kidong Park3, Suresh K. Alahari1 1Department of Biochemistry and Molecular Biology, LSUHSC School of Medicine, New Orleans, LA, USA 2Center of Biochemistry and Microbial Science, Central University of Punjab, Bathinda-151001, India 3Department of Electrical Engineering and Computer Engineering, Louisiana State University, Baton Rouge, LA, USA 4Wayne State University, Detroit, MI, USA 5Department of Biology, Xavier University of Louisiana, New Orleans, LA, USA 6School of Public Health, LSUHSC School of Medicine, New Orleans, LA, USA 7Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, Texas *Corresponding author; Suresh K. Alahari, PhD; Fred G. Brazda Professor of Biochemistry, LSUHSC School of Medicine, New Orleans, LA 70112, USA; Tel: 504-568-4734 [email protected] Running Title: Nischarin regulates exosome production Conflicts of Interest No potential conflicts of interest were disclosed. Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on January 11, 2019; DOI: 10.1158/0008-5472.CAN-18-0842 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Abstract: Exosomes are small extracellular microvesicles that are secreted by cells when intracellular multivesicular bodies (MVB) fuse with the plasma membrane. We have previously demonstrated that Nischarin inhibits focal adhesion formation, cell migration, and invasion, leading to reduced activation focal adhesion kinase. In this study, we propose that the tumor suppressor Nischarin regulates the release of exosomes. When co-cultured on exosomes from Nischarin-positive cells, breast cancer cells exhibited reduced survival, migration, adhesion, and spreading. The same co-cultures formed xenograft tumors of significantly reduced volume following injection into mice. Exosomes secreted by Nischarin-expressing tumors inhibited tumor growth. Expression of only one allele of Nischarin increased secretion of exosomes, and Rab14 activity modulated exosome secretions and cell growth. Taken together, the present study reveals a novel role for Nischarin in preventing cancer cell motility, which contributes to our understanding of exosome biology. Significance: Regulation of Nischarin-mediated exosome secretion by Rab14 seems to play an important role in controlling tumor growth and migration. Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on January 11, 2019; DOI: 10.1158/0008-5472.CAN-18-0842 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Introduction: Nischarin, or imidazoline receptor antisera-selected protein (IRAS), is a protein involved in a number of biological processes. The Nisch gene is located on chromosome 3p21, which is frequently lost in cancers (1). Most notably, Nischarin is an Integrin α5β1 binding protein known to affect cell migration by antagonizing the actions of cell signaling proteins that contribute to tumor cell migration and invasion (2). Furthermore, Nischarin has also been shown to affect cytoskeletal reorganization, mainly by inhibiting Rac-induced lamellipodia formation (2). Consistent with this, Nischarin’s inhibition of cell migration has been linked to other proteins (3- 5). During cell migration, cells adhere to its extracellular environment through focal adhesions (FAs). These complexes use Integrins to attach to extracellular matrix (ECM) proteins (6,7). Each Integrin has designated ligand(s), and decreased expression of the ligand or receptor affects FA number. Integrins also bind to Fibronectin-coated exosomes (8). Exosomes are smaller microvesicles (30-200 nm in diameter) secreted from cells when multivesicular bodies (MVBs) fuse with the plasma membrane (9-12). Although Nischarin’s role has yet to be linked to exosomes, previous studies have shown that the Nischarin-Rab14 interaction promotes the maturation of CD63 positive endosomes (13). Nischarin is an effector of the GTPase Ras- related protein Rab-14 (13). Although Rab14 is involved in vesicle sorting and trafficking (14), only one report has identified Rab14 function in breast cancer exosomes (15). Nischarin directly interacts with Rab14 to effect intracellular Salmonella survival (13). In the presence of Nischarin, there is triple co-localization between the late endosome and exosome marker CD63, Rab14, and Nischarin (13). While it is known that MVBs fuse with the plasma membrane just before exosomes release, the physiological consequences of this have yet to be determined in the breast cancer microenvironment. Furthermore, the proteins responsible for the MVB-plasma membrane fusion are not well characterized. We hypothesized that Nischarin may affect the migration of cancer cells by controlling exosome release. Exosomes from 231 cells promoted migration of 231 cells, while exosomes from 231 Nisch cells inhibited migration. These effects were due to the decreased number of exosomes released by 231 Nisch cells. In contrast, active Rab14 promotes exosome secretion and cell growth. In summary, our study highlights the anti-tumoral function of Nischarin expression mediated by exosome-dependent secretions in breast cancer. Materials and Methods Cell Culture All cells (MDA-MB-231, MDA-MB-231 Nischarin, MCF10A, MCF7, BT20, T47D, MDA-MB-468, SUM185 and MCF7) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) at 37°C, 5% CO2 supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (Gibco, Waltham, MA). MDA-MB-231 Nisch, MDA-MB-231 GFP, MDA-MB-231 Rab14, MDA-MB-231 Rab14 S25N, and MDA-MB-231 Rab14 Q70L were prepared as previously described (4). Briefly, 231 Nisch cells were generated by amplifying human NISCH. The 4545 base pair PCR product was then cloned into a pCDH-CMV-MCS-EF1-copGFP vector. The viral particles were Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on January 11, 2019; DOI: 10.1158/0008-5472.CAN-18-0842 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. generated in HEK-293T cells along with the pCDH-GFP-Nisch plasmid. The supernatants containing the lentiviral particles were collected 48 hours later, concentrated, then reconstituted in serum-free media. The MDA-MB-231 GFP cells were generated similarly, except there was no cloning of NISCH into the pCDH-CMV-MCS-EF1-copGFP vector. The cells were sorted by FACS for GFP selection. Low passage cells were used for all the experiments, and the cell line purify was verified every two months using appropriate markers of the cell type. Transfected cells were selected using antibiotic puromcyin. Nischarin WT (+/+), Nischarin HET (+/-) and Nischarin Null (-/-) animals were generated as described before (16). Briefly, exons 7 to 10 of Nischarin were deleted, and the resulting animals were intercrossed with animals expressing the mouse mammary tumor virus-polyma middle T transgene. For mouse genotyping, mouse tail genomic DNA was extracted and amplified by PCR and electrophoresed on 2% agarose gels. Primary WT-PyMT (Nisch+/+), HET- PyMT (Nisch+/-) and Null-PyMT (Nisch -/-) cells were isolated as previously described (17). Briefly, the mammary tumors were isolated and cut into small pieces with a razor blade and scissors. The tissues were incubated with collagenase for two hours to allow for enzymatic dissociation of the tissue. The resulting material was ultra-centrifuged to remove debris and blood. The following conditions were used for cell culture experiments of cells that were seeded on ECM proteins, 10ug/ml of Fibronectin (BD Biosciences, San Diego, CA) was prepared in PBS. Bovine Collagen 1 ((BD Biosciences, San Diego, CA) was added to each well at 0.16ml/cm2. The ECM proteins were added to the wells and placed on a rocker for 2 hours at room temperature then washed two times with warm PBS. The cells were seeded onto the wells immediately after washing with PBS. Cell line authentication: MDA-MB-231 cells were obtained from ATCC and Nischarin expression in these cells was maintained by puromycin selection. Nischarin expression was monitored by immunoblotting using anti-Nischarin antibody. Cells were not used beyond passage five and mycoplasma was tested for all cell lines at least once every six months. The primary cells prepared from PyMT tumors were tested every time for Nischarin truncation genotype, PyMT expression by genomic PCR approach. The primary cells were never used beyond passage three. Animal Studies All mouse experiments were performed in accordance with the protocols approved by the Institutional Animal Care and Use Committee (IACUC) of the Louisiana State University Health Sciences Center, New Orleans. Four- to ten-week-old female Prkdc scid mice were used in the xenograft studies (4 mice per group). The exosomes used for all mouse co-injections were isolated from 1.8g of mouse mammary tumor.
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