Stromal CAVIN1 Controls Prostate Cancer Microenvironment and Metastasis by Modulating Lipid Distribution and Inflammatory Signaling
Total Page：16
File Type：pdf, Size：1020Kb
Published OnlineFirst June 3, 2020; DOI: 10.1158/1541-7786.MCR-20-0364 MOLECULAR CANCER RESEARCH | TUMOR MICROENVIRONMENT AND IMMUNOBIOLOGY Stromal CAVIN1 Controls Prostate Cancer Microenvironment and Metastasis by Modulating Lipid Distribution and Inflammatory Signaling Jin-Yih Low1, W. Nathaniel Brennen2, Alan K. Meeker2,3,4, Elina Ikonen5,6, Brian W. Simons7, and Marikki Laiho1,2 ABSTRACT ◥ Lipid uptake occurs through caveolae, plasma membrane invagi- accumulation and increases inflammation. Stromal cells lacking nations formed by caveolins (CAV) and caveolae-associated protein 1 CAVIN1 enhance prostate cancer cell migration and invasion. (CAVIN1). Genetic alterations of CAV1N1 and CAV1 modify lipid Remarkably, they increase lipid uptake and M2 inflammatory mac- metabolism and underpin lipodystrophy syndromes. Lipids contrib- rophage infiltration in the primary tumors and metastasis to distant ute to tumorigenesis by providing fuel to cancer metabolism and sites. Our data support the concept that stromal cells contribute to supporting growth and signaling. Tumor stroma promotes tumor prostate cancer aggressiveness by modulating lipid content and proliferation, invasion, and metastasis, but how stromal lipids influ- inflammation in the tumor microenvironment. ence these processes remain to be defined. Here, we show that stromal CAVIN1 regulates lipid abundance in the prostate cancer microen- Implications: This study showed that stromal CAVIN1 suppresses vironment and suppresses metastasis. We show that depletion of prostate cancer metastasis by modulating tumor microenvironment, CAVIN1 in prostate stromal cells markedly reduces their lipid droplet lipid content, and inflammatory response. Introduction Mice and humans with targeted disruption or mutations of CAVIN1 are affected by numerous abnormalities including lipodystrophy, Caveolae, ultrastructural microdomains at the plasma membrane, muscular dystrophy, cardiovascular disease, and diabetes (8, 13–16). are involved in protein and lipid trafficking and function as scaffolds Cavin1-knockout mice have decreased insulin-dependent glucose for signaling proteins (1–3). Caveolae also provide a physical buffering uptake, reduced lipid storage and impaired lipid tolerance, adipose capacity for cells under mechanical stress (4). Caveolae are formed by tissue fibrosis, and increased macrophage infiltration (12, 13). Fur- the assembly of caveolins (CAV) and caveolae-associated protein 1 thermore, loss of Cavin1 impairs insulin-mediated focal adhesion (CAVIN1; also known as polymerase-1 and transcript release factor, formation and remodeling required for a mechanical stress response, PTRF), and are rich in lipids, in particular cholesterol (5–10). Intrigu- concomitant with activation of ERK and p38 stress signaling (17). In ingly, CAVIN1 was first identified as RNA polymerase I transcription adipocytes, CAVIN1 also adjusts ribosomal activity to the nutrient termination factor (11), and subsequently, as a critical component of state suggesting that CAVIN1 functions both in the regulation of caveolae (7, 8). CAVIN1 is involved in adipocyte lipid storage and lipid metabolism and RNA polymerase I transcription (18). Also, hence contributes to energy metabolism. Adipocytes deficient of either CAVIN1 may behave as an adipokine and partially contribute to the CAVIN1 or caveolins are defective in lipid uptake and storage, have well-known detrimental effects of visceral fat accumulation (19). reduced cholesterol transport, and contribute to increased levels of Under starvation or exercise, fatty acids from lipid droplet triacylgly- circulating triglycerides and free fatty acids (2, 8, 9, 12). Although cerol stores are released as a source of energy (20). Given that caveolin-1 (CAV1) is also involved in lipid trafficking (9), less is known certain lipids are cytotoxic, the uptake of the lipids by caveolae how CAVIN1 and CAV1 regulate lipid droplet formation. also has cytoprotective functions. CAVIN1 deficiency may, hence, drastically reprogram both cellular energy metabolism and the 1Department of Radiation Oncology and Molecular Radiation Sciences, Johns microenvironment (21). Hopkins University School of Medicine, Baltimore, Maryland. 2Department of Both CAVIN1 and CAV1 are largely absent in the normal prostate Oncology, Johns Hopkins University School of Medicine, Baltimore, Maryland. epithelium but present in the stroma (3, 22). During prostate cancer 3Department of Pathology, Johns Hopkins University School of Medicine, progression, the expression of CAV1 in the tumor cells increases, but Baltimore, Maryland. 4Department of Urology, Johns Hopkins University School the expression of both CAV1 and CAVIN1 is lost in the tumor 5 of Medicine, Baltimore, Maryland. Faculty of Medicine, Anatomy and Stem Cells stroma (23–25). The decrease in stromal CAV1 and CAVIN1 corre- and Metabolism Research Program, University of Helsinki, Helsinki, Finland. 6Minerva Foundation Institute for Medical Research, Helsinki, Finland. 7Center lates with reduced relapse-free survival, higher Gleason score, and poor for Comparative Medicine, Baylor College of Medicine, Houston, Texas. outcome (23). Ectopic expression of CAVIN1 in prostate cancer cells reduces their aggressive phenotypes (proliferation, anchorage- Note: Supplementary data for this article are available at Molecular Cancer Research Online (http://mcr.aacrjournals.org/). independent growth, migration, and invasion), lymphangiogenesis, and angiogenesis in vitro and in vivo (24, 26, 27). In contrast to Corresponding Author: Marikki Laiho, Johns Hopkins University School of CAVIN1, expression of CAV1 in prostate cancer cells increases their Medicine, 1550 Orleans St, Baltimore, MD 21287. Phone: 410-502-9748; Fax: 410-502-2821; E-mail: [email protected] anchorage-independent growth, invasive and angiogenic potential, and castration resistance (3, 22, 28). The anchorage-independent Mol Cancer Res 2020;XX:XX–XX growth is reversed by coexpression of CAVIN1, suggesting that their doi: 10.1158/1541-7786.MCR-20-0364 functional association can mitigate the oncogenic activity of Ó2020 American Association for Cancer Research. CAV1 (24). Furthermore, CAVIN1 was shown to modulate the AACRJournals.org | OF1 Downloaded from mcr.aacrjournals.org on September 27, 2021. © 2020 American Association for Cancer Research. Published OnlineFirst June 3, 2020; DOI: 10.1158/1541-7786.MCR-20-0364 Low et al. dynamics of cholesterol and actin cytoskeleton and impair prostasome Oil Red O staining secretion in prostate cancer (29). Oil Red O was purchased from Sigma-Aldrich and was used as per The function of stromal CAVIN1 in prostate cancer has not been the manufacturer's protocol. Cells were cultured in culture media studied before. Given the abundant changes of CAVIN1 in prostate supplemented with 20% FBS. Following, culture medium was aspi- cancers, we implemented in vitro and in vivo orthotopic tumor models rated and cells were washed with PBS, followed by fixing with 10% to systematically analyze how stromal CAVIN1 affects tumor growth, formalin for 45 minutes. Formalin was then removed and cells were phenotype, and lipid regulation. We show in coculture models that washed with deionized water followed by incubation with 60% iso- prostate stromal cells lacking CAVIN1 increase prostate cancer cell propanol for 5 minutes. Isopropanol was then removed and cells were lipid content, cause an inflammatory tumor microenvironment, and stained with Oil Red O solution for 5 minutes before rinsing under promote invasion and metastasis. We propose that stromal fibroblasts running tap water. Cells were then visualized with EVOS FL Auto contribute to prostate cancer aggressive phenotypes through control of Microscope (Life Technologies) and images were analyzed with ImageJ lipid and cytokine balance. (NIH). Oil Red O staining was quantified from five fields per sample and normalized for total cellular area. Materials and Methods Scratch assay Cell lines PC3 cells were seeded into a 6-well plate and incubated overnight. Normal prostate stromal line WPMY-1, HEK293T cells, and pros- Culture medium was removed and cells were scratched with a 1 mL tate cancer cell lines PC3, DU145, and CW22Rv1 were purchased from pipette tip and conditioned medium from stromal cells was added. ATCC. Normal primary prostate stromal line, PrSc was purchased Images were taken at 8, 16, and 24 hours with EVOS Microscope from Lonza. MR49F cell line was a kind gift from Dr. Martin Gleave (Thermo Fisher Scientific) and images were analyzed with ImageJ (Vancouver Coastal Health Institute, Vancouver, British Columbia, (NIH). Canada; ref. 30). All cells were maintained in the culture media as per the manufacturer's instruction, cultured at 37C in a humidified Transwell migration and invasion assay atmosphere containing 5% CO2, and were authenticated by short Stromal cells were seeded into a 24-well plate. Following day, tandem repeat analyses at the Johns Hopkins Genetic Resources Core Transwell inserts were inserted into the 24-well plate and prostate Facility (Baltimore, MD). Cell lines were tested for Mycoplasma with cancer cells were seeded into the inserts. After 14 hours, the inserts Venor GeM Mycoplasma Detection Kit (Sigma-Aldrich) for negativ- were washed, fixed with 3.5% PFA, and stained with 0.5% crystal violet. ity. Upon thawing from liquid nitrogen, cell lines were passaged once Images were taken with EVOS FL Auto microscope and images were before being used for experiments. Before reaching 20 passages, cells analyzed with ImageJ