Exosomes from Nischarin-Expressing Cells Reduce Breast Cancer Cell
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Published OnlineFirst January 11, 2019; DOI: 10.1158/0008-5472.CAN-18-0842 Cancer Molecular Cell Biology Research Exosomes from Nischarin-Expressing Cells Reduce Breast Cancer Cell Motility and Tumor Growth Mazvita Maziveyi1, Shengli Dong1, Somesh Baranwal2, Ali Mehrnezhad3, Rajamani Rathinam4, Thomas M. Huckaba5, Donald E. Mercante6, Kidong Park3, and Suresh K. Alahari1 Abstract Exosomes are small extracellular microvesicles that are secre- secreted by Nischarin-expressing tumors inhibited tumor ted by cells when intracellular multivesicular bodies fuse with growth. Expression of only one allele of Nischarin increased the plasma membrane. We have previously demonstrated that secretion of exosomes, and Rab14 activity modulated exosome Nischarin inhibits focal adhesion formation, cell migration, secretions and cell growth. Taken together, this study reveals a and invasion, leading to reduced activation of focal adhesion novel role for Nischarin in preventing cancer cell motility, kinase. In this study, we propose that the tumor suppressor which contributes to our understanding of exosome biology. Nischarin regulates the release of exosomes. When cocultured on exosomes from Nischarin-positive cells, breast cancer cells Significance: Regulation of Nischarin-mediated exosome exhibited reduced survival, migration, adhesion, and spread- secretion by Rab14 seems to play an important role in con- ing. The same cocultures formed xenograft tumors of signifi- trolling tumor growth and migration. cantly reduced volume following injection into mice. Exosomes See related commentary by McAndrews and Kalluri, p. 2099 Introduction rins to attach to extracellular matrix (ECM) proteins (6, 7). Each Nischarin, or imidazoline receptor antisera-selected (IRAS) integrin has designated ligand(s), and decreased expression of protein, is a protein involved in a number of biological processes. the ligand or receptor affects focal adhesion number. Integrins The Nisch gene is located on chromosome 3p21, which is fre- also bind to fibronectin-coated exosomes (8). Exosomes are quently lost in cancers (1). Most notably, Nischarin is an integrin smaller microvesicles (30–200 nm in diameter) secreted from a5b1 binding protein known to affect cell migration by antago- cells when multivesicular bodies (MVB) fuse with the plasma nizing the actions of cell signaling proteins that contribute to membrane (9–12). Although Nischarin's role has yet to be tumor cell migration and invasion (2). Furthermore, Nischarin linked to exosomes, previous studies have shown that the has also been shown to affect cytoskeletal reorganization, mainly Nischarin–Rab14 interaction promotes the maturation of þ by inhibiting Rac-induced lamellipodia formation (2). Consistent CD63 endosomes (13). Nischarin is an effector of the GTPase with this, Nischarin's inhibition of cell migration has been linked Ras-related protein Rab-14 (13). Although Rab14 is involved in to other proteins (3–5). vesicle sorting and trafficking (14), only one report has iden- During cell migration, cells adhere to its extracellular envi- tified Rab14 function in breast cancer exosomes (15). Nischarin ronment through focal adhesions. These complexes use integ- directly interacts with Rab14 to effect intracellular Salmonella survival (13). In the presence of Nischarin, there is triple 1 colocalization between the late endosome and exosome marker Department of Biochemistry and Microbial Sciences, Louisiana State University CD63, Rab14, and Nischarin (13). Health Sciences Center School of Medicine, New Orleans, Louisiana. 2Center of Biochemistry and Microbial Science, Central University of Punjab, Bathinda, While it is known that MVBs fuse with the plasma membrane Punjab, India. 3Division of Electrical and Computer Engineering, Louisiana State just before exosome release, the physiologic consequences of University, Baton Rouge, Louisiana. 4Wayne State University, Detroit, Michigan. this have yet to be determined in the breast cancer microen- 5Department of Biology, Xavier University of Louisiana, New Orleans, Louisiana. vironment. Furthermore, the proteins responsible for the MVB– 6 Louisiana State University Health Sciences Center School of Public Health, plasma membrane fusion are not well characterized. We Louisiana State University Health Sciences Center School of Medicine, New hypothesized that Nischarin may affect the migration of cancer Orleans, Louisiana. cells by controlling exosome release. Exosomes from 231 cells Note: Supplementary data for this article are available at Cancer Research promoted migration of 231 cells, while exosomes from 231 Online (http://cancerres.aacrjournals.org/). Nisch cells inhibited migration. These effects were due to the Corresponding Author: Suresh K. Alahari, LSUHSC School of Medicine, 1901 decreased number of exosomes released by 231 Nisch cells. In Perdido Street, New Orleans, LA 70112. Phone: 504-568-4734; Fax: 504-568- contrast, active Rab14 promotes exosome secretion and cell 2093; E-mail: [email protected] growth.Insummary,ourstudyhighlights the antitumoral doi: 10.1158/0008-5472.CAN-18-0842 function of Nischarin expression mediated by exosome- Ó2019 American Association for Cancer Research. dependent secretions in breast cancer. 2152 Cancer Res; 79(9) May 1, 2019 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2019 American Association for Cancer Research. Published OnlineFirst January 11, 2019; DOI: 10.1158/0008-5472.CAN-18-0842 Nischarin Regulates Exosome Production Materials and Methods Animal studies All mouse experiments were performed in accordance with the Cell culture protocols approved by the Institutional Animal Care and Use MDA-MB-231, MDA-MB-231 Nischarin, MDA-MB-231 GFP, Committee of the Louisiana State University Health Sciences MDA-MB-231 Rab14, MDA-MB-231 Rab14 S25N, MDA-MB- Center (New Orleans, LA). Four- to 10-week-old female Prkdc 231 Rab14 Q70L, MCF7, BT20, T47D, MDA-MB-468, and scid mice were used in the xenograft studies (4 mice per group). SUM185, and MCF7 cells were cultured in DMEM at 37 C, The exosomes used for all mouse coinjections were isolated 5% CO supplemented with 10% FBS and 1% penicillin/ 2 from 1.8 g of mouse mammary tumor. Therefore, the approxi- streptomycin (Gibco). MCF10A cells were cultured in þ þ mate number of Nisch / exosomes isolated were 1  108 while DMEM/F12 supplemented with 5% horse serum EGF, hydro- þ À the number of Nisch / exosomes was 1.9  108. After exosome cortizone, choleratoxin, insulin, and penicillin/streptomycin. isolation, animals were either injected with exosomes only, MDA-MB-231 Nisch, MDA-MB-231 GFP, MDA-MB-231 þ þ þ À þ þ 1  106 viable Nisch / or Nisch / cells alone, or Nisch / Rab14, MDA-MB-231 Rab14 S25N, and MDA-MB-231 Rab14 þ À þ þ or Nisch / cells that were cocultured with the Nisch / or Q70L were prepared as described previously (4). Briefly, þ À Nisch / exosomes for 4 days. Tumor growth was assessed every 231 Nisch cells were generated by amplifying human NISCH. three days with calipers and tumor volume was calculated as The 4545 base pair PCR product was then cloned into a p  length  width2/6. pCDH-CMV-MCS-EF1-copGFP vector. The viral particles were generated in HEK-293T cells along with the pCDH- GFP-Nisch plasmid. The supernatants containing the lentiviral Isolation of exosomes particles were collected 48 hours later, concentrated, then A total of 3  106 cells were seeded in T175 flasks and cultured reconstituted in serum-free media. The MDA-MB-231 GFP for 48 hours. The medium was centrifuged at 300  g for cells were generated similarly, except there was no cloning of 10 minutes, and then the supernatant was collected and centri- NISCH into the pCDH-CMV-MCS-EF1-copGFP vector. The fuged again at 2,000  g for 20 minutes. This was repeated again cells were sorted by FACS for GFP selection. Low passage for two more centrifugations both at 10,000  g for 30 minutes cells were used for all the experiments and the cell line purity and 1  100,000  g for 70 minutes. The supernatant was then was verified every two months using appropriate markers of discarded and the pellet was centrifuged with PBS for 70 the cell type. Transfected cells were selected using the antibi- minutes at 1  100,000  g. The pellet was finally resuspended otic puromcyin. in PBS for subsequent studies. To isolate exosomes from þ þ þ À þ þ Nischarin WT ( / ), Nischarin HET ( / ), and Nischarin Null whole tumors, we harvested breast tumors from Nisch / and À À þ À ( / ) animals were generated as described previously (16). Nisch / mice as described previously (17). Tumors were Briefly, exons 7 to 10 of Nischarin were deleted, and the disintegrated and incubated with collagenase for 3 hours. The resulting animals were intercrossed with animals expressing samples werecentrifuged for 5 minutes at 200  g.Thesuper- the mouse mammary tumor virus-polyma middle T transgene. natant was then used for the exosome isolation procedure by For mouse genotyping, mouse tail genomic DNA was extracted ultracentrifugation. and amplified by PCR and electrophoresed on 2% agarose gels. The Beckman Couter DelsaNanoC instrument was used to þ þ þ À Primary WT-PyMT (Nisch / ), HET-PyMT (Nisch / ), and measure diameter and molecular weight. The samples were dilut- À/À Null-PyMT (Nisch ) cells were isolated as described previ- ed in ddH2O, added to a cuvette, and inserted into the device. ously (17). Briefly, the mammary tumors were isolated and cut This instrument uses Photon Correlation Spectroscopy (PCS) to into small pieces with a razor blade and scissors. The tissues analyze the molecular weight of the sample as it relates to the were incubated with collagenase for 2 hours to allow for a and b ion particles in the solution. Basically, when laser light is enzymatic dissociation of the tissue. The resulting material was directed toward the particle, light will scatter in different direc- ultracentrifuged to remove debris and blood. The following tions. The intensities observed by the machine are a result of conditions were used for cell culture experiments of cells that the movement of ion particles due to Brownian motion.