Abstract 4920: FAP-α overexpression increases cancer cell migration
Authors: Mori Noriko , Jin Jiefu , Krishnamachary Balaji , Mironchik Yelena , Barnett James D. , Bhujwalla Zaver M. 
Published: September 2020

Abstract
Fibroblast activation protein-α (FAP-α) is attracting increasing interest because of its role in immune suppression and cancer metastasis. To understand the role of FAP-α in cancer cells we engineered triple negative human breast cancer MDA-MB-231 cells and fibrosarcoma HT1080 cells to stably overexpress FAP-α and compared cell migration of parental and FAP-α overexpressing cells using wound healing analysis. We observed a significant increase of migration with FAP-α overexpression in MDA-MB-231 cells. HT1080 cells displayed similar trends on migration with FAP-α overexpression.
We developed MDA-MB-231 and HT-1080 cells overexpressing FAP-α (231-FAP and HT-1080-FAP) by transducing these cells with lentivirus encoding the gene for human FAP (Accession No. NM_004460.3) that was subcloned into lentiviral vector pMA3211. Immunoblot analysis with FAP-α antibody confirmed the overexpression of FAP-α protein. To evaluate migration, wound healing analysis was performed. Cells were seeded and incubated in DMEM without FBS for 24h before wounds were made by scratching cells with a p200 pipet. Pictures were taken immediately (0h), and at 6h, 24h, and 48h after wound formation. The wound areas were measured using ImageJ and normalized to the area (100%) at 0h. We used a cell counting kit-8 (CCK-8) to determine if cell proliferation was altered by FAP-α overexpression.
231-FAP cells exhibited significantly increased migration from 6h (66%) to 48h (0.6%) after wound formation compared to parental cells (73% at 6h and 7.6% at 48h). HT1080-FAP cells showed increased migration only at 24h (8%) and 48h (1.9%) after wound formation compared to parental cells (19% at 24h and 5.3% at 48h) although this was not statistically significant. The CCK-8 assay showed that there were no differences in cell proliferation between MDA-MB-231 cells and 231-FAP cells with or without 10% FBS. Both cell lines showed a comparably small reduction of proliferation (approximately 30%) at 48h point in FBS free medium. In contrast, both HT1080 and HT1080-FAP cell proliferation was reduced significantly (approximately 75%) at 48h point in FBS free medium. HT1080-FAP cell proliferation was slightly higher than parental cells in medium with FBS (16%) and without FBS (19%) at 48h point than parental cells.
Our data are consistent with the known activities of FAP-α as an exopeptidase and endopeptidase/gelatinase/collagenase in tissue remodeling and repair, and in cell migration. We found that FAP-α significantly increased MDA-MB-231 breast cancer cell migration but had very little effect on cell proliferation. FAP-α overexpression in HT1080 fibrosarcoma cells also increased migration but not as much as in MDA-MB-231 cells. Ongoing work with xenografts derived from these cell lines will provide further insights into tumor microenvironment changes with FAP-α overexpression.
Citation Format: Noriko Mori, Jiefu Jin, Balaji Krishnamachary, Yelena Mironchik, James D. Barnett, Zaver M. Bhujwalla. FAP-α overexpression increases cancer cell migration [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4920.