Stromal CAVIN1 Controls Prostate Cancer Microenvironment and Metastasis by Modulating Lipid Distribution and Inflammatory Signaling

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 2020 American Association for Cancer Research. CAV1 (24). Furthermore, CAVIN1 was shown to modulate the

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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 (NIH). were discarded and a new vial was thawed. RNA-sequencing Antibodies RNA was extracted with TRizol (Life Technologies) as per the CAVIN1 (catalog no. HPA049838) antibody was purchased from manufacturer’s instruction. Three biological samples were prepared Millipore Sigma. CAV1 (catalog no. ab2910), SV40 T antigen (catalog for each WPMY-1 clone. RNA sequencing (RNA-seq) library for no. ab16879), GAPDH (catalog no. ab8245), and alpha tubulin Illumina platform sequencing was prepared using Illumina TruSeq (catalog no. ab7291) antibodies were from Abcam. SREBP1 (catalog Stranded Total RNA Sample Kit following the manufacturer’s recom- no. NB600-582) antibody was from Novus Biologicals and FASN mended procedure. Briefly, 250 ng of total RNA was first depleted of (catalog no. 3180) antibody was from Cell Signaling Technology. rRNA using Ribo-Zero Gold (Epicentre) and fragmented. Fragmented RNA was converted to double-stranded cDNA with the second strand Gene knockdown marked. The resulting cDNA was polyA tailed and ligated to barcoded Lentiviral short hairpin RNA (shRNA) plasmids were purchased sequencing adaptors and amplified by PCR. The amplified libraries from Johns Hopkins High Throughput Biology Core Facility (shCA- were quality controlled and pooled. The pooled library was further VIN1_1 50-CCGCAACTTTAAAGTCATGAT-30 and shCAVIN1_2 quantitated using KAPA Library Quantification Kit (Kapa Biosystems) 50-GTGGAGGTTGAGGAGGTTATT-30). Following transfection in and sequenced on NextSeq 500 (Illumina) for 2 75 bp paired-end HEK293T cells for production of lentiviral particles, the viruses were reads. Sequencing data were analyzed using Tophat 2 and Cuffdiff 2.0, transduced into WPMY-1 cells, and selected for puromycin resistance respectively, for alignment to reference genome and differential to generate stable CAVIN1-knockdown clones. CAVIN1 targeting and expression detection. Pathway analyses were conducted using Gene scrambled control siRNA were purchased from Ambion (Assay ID: Set Enrichment Analysis (GSEA) as in ref. 31. The data are deposited to s49507 and s49508; Life Technologies). Cells were transfected with 10 Gene Expression Omnibus as GSE146229. nmol/L of siRNA before harvesting at 72 hours for subsequent experiments. qPCR RNA was extracted using TRizol as per the manufacturer’s instruc- Exogenous addition of lipids tion and converted into cDNA using Super Script II cDNA Synthesis Oleic acid (catalog no. O1383) and water-soluble cholesterol (cat- Kit (Life Technologies). qPCR was performed using iTaq Universal alog no. C4951) were purchased from Sigma-Aldrich and prepared SYBR Green Supermix purchased from Bio-Rad on a CFX6100 qPCR according to the manufacturer’s instructions. Instrument (Bio-Rad).

Conditioned medium Western blotting Cell culture medium was collected 48 hours after seeding and Protein lysate was collected from cells using RIPA buffer supplied centrifuged at 670 g for 5 minutes to remove cell debris before with Protease Inhibitor Cocktail and quantiﬁed using BCA Assay (Life being used for subsequent experiments. Technologies). Twenty micrograms of protein were electrophoresed

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on precast gel purchased from Life Technologies for 90 minutes. Statistical analyses Electrophoresed proteins were transferred onto Nitrocellulose Mem- Statistics were computed with GraphPad Prism 6 using Student brane (Bio-Rad) and blocked with 10% milk (Bio-Rad) for 1 hour at two-tailed t test, Mann–Whitney nonparametric t test, and one-way room temperature. Primary antibody was then added and incubated ANOVA with Tukey or Kruskal–Wallis post hoc test as indicated. N overnight. Following day, nitrocellulose membrane was washed thrice, represents biological replicates, unless otherwise stated. Data are 5 minutes each with TBST buffer before being incubated with horse- presented as mean SD. radish peroxidase (HRP)-conjugated secondary antibody (Agilent Dako) for 1 hour at room temperature. Membrane was then washed thrice, 5 minutes each, followed by addition of Lighting ECL Reagent Results (GE Amersham) and the membrane was visualized with Bio-Rad CAVIN1 KD impairs lipid uptake by prostate stromal cells GelDocþ System (Bio-Rad). Given that loss of CAVIN1 in prostate cancer stroma associates with aggressive prostate cancer and poor survival, we hypothesized that Multiplex cytokine array CAVIN1 could influence these events by modifying the lipid content in Multiplex cytokine array was performed at the Johns Hopkins the stroma. To study this, we knocked down CAVIN1 in an estab- Immune Monitoring Core Facility on a Bioplex 200 Platform (Bio- lished, well-studied WPMY-1 prostate stromal fibroblast line (32). For Rad) using Luminex Bead-based Immunoassays (Millipore) according this purpose, we used CAVIN1 targeting shRNAs to silence the to the manufacturer's protocols. The HAGE1MAG-20K panel was expression of CAVIN1 and generated two stable lines, shRNA- used to detect IL6 and IL18, HCMBMAG-22K panel for DKK1, CAVIN1_1 and shRNA-CAVIN1_2, and a nontargeting control, HCYP3MAG-63K panel for CSF-1 (MCSF), HCYP4MAG-64K for shCtrl (Fig. 1A). Unless otherwise noted, we chose to conduct IL32, and HMMP1MAG-55K for MMP3. IL6, IL18, and IL32 were subsequent experiments with the clone shRNA-CAVIN1_2 cells, below detection limit and are not reported. called hereafter shCAVIN1. To generate an alternative model, we used siRNAs to silence CAVIN1 in PrSc stromal fibroblasts (Fig. 1B). Site-directed mutagenesis Although PrSc cells are not fully characterized for all mesenchymal QuickChange II Site-directed Mutagenesis Kit (Agilent Technolo- markers, they provide another representation of prostate stromal gies) was used to generate CAVIN1 lentiviral plasmid resistant to fibroblasts. CAVIN1 silencing did not affect the growth of these cell shRNA degradation and sequence verified. The resulting plasmid was lines (Supplementary Fig. S1A and S1B). To test the robustness of our used to generate lentiviral particles in HEK293T cells and used to model, we reintroduced CAVIN1 expression in the shCAVIN1 stro- transduce stromal CAVIN1-knockdown (KD) cells. mal cells. Using site-directed mutagenesis, we generated a lentiviral CAVIN1 expression plasmid that is resistant to the shRNA used to KD Orthotopic model CAVIN1. After viral transduction, a mixed pool of cells was collected. The orthotopic mouse experiment was conducted under an We observed rescue of CAVIN1 expression in the shCAVIN1 cells approved protocol by the Johns Hopkins University Animal Care following transduction and selection of a stable clone, hereafter called and Use Committee. We generated PC3 cells stably expressing CAVIN-R (Fig. 1C). We then tested the presence of lipid droplets in firefly luciferase (PC3-Luc) and mixed them (1.25 105 cells, 1:1 these cells as determined by Oil Red O staining. To facilitate the ratio) with WPMY-1 shCtrl, shCAVIN1, or CAVIN1-R stromal detection of lipid droplets, we cultured the cells in 20% FCS. We found cells and implanted orthotopically to the right anterior prostate of that lipid droplets were dramatically reduced in the CAVIN1-silenced NSG (NOD-SCID) mice. Bioluminescence of the tumors was WPMY-1 and PrSc stromal cells compared with controls (Fig. 1D). determined using IVIS Spectrum In Vivo Imaging System (Perkin Lipid droplets were restored in the CAVIN1-R cells affirming that the Elmer) weekly or biweekly. At the end of the study, urogenital phenotype is CAVIN1 dependent (Fig. 1E). block, tumor, lungs, and liver were harvested. Invasion and metas- To further test the model, we fed the WPMY-1 shCAVIN cells with tasis were determined by counting visible metastases at necropsy. oleic acid, a fatty acid stored in lipid droplets. We observed that Frozen sections were prepared. Tissues were then fixed in 10% shCAVIN cells were not able to store the lipid, whereas the shCtrl cells formalin, and 4-mm sections were stained with hematoxylin and robustly did so (Fig. 1F). To assess whether the changes in intracellular eosin. The number of micrometastases were assessed in a blinded lipid droplets are due lipid uptake from the extracellular space, we used fashion. a cholesteryl ester uptake inhibitor, ML278, and found that this reduced lipid droplets in the WPMY-1 shCtrl cells (Supplementary IHC and quantitative analysis Fig. S1C). ShCAVIN1 cells remained devoid of lipid droplets. To ask CD163 (catalog no. ab182422) antibody was purchased from whether CAVIN1 KD leads to major perturbation in lipid synthetic Abcam and used to stain for the presence of M2 macrophages in pathways of the cells, we analyzed the levels of fatty acid synthase, tumor tissues. Briefly, paraffin-embedded slides were dewaxed, rehy- FASN, and the lipid metabolism controller, SREBP1, by Western drated, and antigen retrieval was performed with Antigen Unmasking blotting, but did not detect any obvious changes (Supplementary Buffer (Vector Laboratories). Slides were then treated Dual Endoge- Fig. S1D). These findings suggest that CAVIN1 depletion in stromal nous Enzyme Blocker (Agilent Dako). Primary antibody was then cells leads to defective lipid uptake. Given this, we postulated that added to the slides and incubated at room temperature for 1 hour. CAVIN1-depleted stromal cells could have augmented amounts of Slides were washed, stained with HRP-labeled secondary antibody lipids in their microenvironment. (catalog no. PV6119, Leica Microsystems) followed by detection using 3, 30-Diaminobenzidine (Sigma-Aldrich), counterstained with hema- Stromal CAVIN1-KD cells increase prostate cancer cell migration toxylin, dehydrated, and mounted with cover slip. Slides were then and invasion in coculture assays imaged using EVOS FL Auto microscope on three independent fields. To probe the impact of stromal cell lipid cycling on prostate cancer Signal intensity or presence of stained cells were quantified using cells, we used conditioned medium from WPMY-1 shCtrl and shCA- ImageJ software. VIN1 cells and applied the media to cultures of four prostate cancer cell

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Figure 1. CAVIN1-silenced prostate stromal cell lines lack lipid uptake. A, WPMY-1 prostate stromal cells were used to generate stable pools of CAVIN1-KD cells using lentiviral shRNAs targeting CAVIN1. B, PrSc primary prostate stromal cells were transfected with CAVIN1-targeting siRNAs. C, WPMY-1 shCAVIN1 cells were transfected with CAVIN1 expression vector with a mutation in the shRNA-targeting site to generate CAVIN1-R cells. A–C, Representative Western blots show expression of CAVIN1, CAV1, and GAPDH. D, Knocking down CAVIN1 abrogates the formation of lipid droplets in WPMY-1 and PrSc cells, as shown by Oil Red O staining. Data are from three independent experiments. Scale bar, 100 mm. E, Lipid uptake is restored in CAVIN1-R cells. Data are from four independent experiments. Scale bar, 100 mm. F, Oleic acid increases lipid droplet formation in WPMY-1 shCtrl cells, but has no effect on WPMY-1 CAVIN1 shRNA cells. Data are from three independent experiments. Scale bar, 100 mm. One-way ANOVA with Tukey post hoc test. Arrows denote lipid droplets. , P < 0.01; , P < 0.001.

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lines, PC3, DU145, MR49F, and CWR22RV1. We found that prostate ACTA2 (a-SMA), S100A4 (FSP1), and decorin (Supplementary cancer cell lines cultured in shCAVIN1 medium had significantly more Fig. S3D). Although other CAF markers such as SDF1, COL1A2, and lipid droplets compared with those cultured in shCtrl medium tenascin were downregulated and absolute markers for CAFs remain (Fig. 2A; Supplementary Fig. S2A–S2C). We also assessed for per- to be defined, these findings suggest that stromal cells with CAVIN1 turbations in prostate cancer cell growth by the conditioned medium KD had assumed CAF-like features. On the other hand, there was no but did not observe any changes in growth or expression of FASN or systematic activation of lipid synthetic pathways (Supplementary SREBP1 lipid synthesis proteins (Supplementary Fig. S2D–S2F). These Fig. S3E). experiments suggest that CAVIN1 KD in the stromal cells increases lipid uptake by the prostate cancer cells. Lipids drive aggressive and inflammatory phenotypes in To address how the stromal cells impact prostate cancer cell prostate cancer microenvironment phenotypes, we first cultured PC3 cells in shCtrl and shCAVIN1 Obesity and high fat diet are linked to prostate cancer progres- conditioned medium and performed a scratch wounding assay. We sion and mortality (33–35). To test whether extracellular lipids observed that PC3 cells applied with shCAVIN1 conditioned medi- promote prostate cancer cell migration or invasion, we added um had increased migration as measured by closure of the wound cholesterol or oleic acid to Transwell bottom wells and plated PC3 scratch (Fig. 2B). We then used a Transwell coculture model, where cells on the top inserts. We observed increased migration and we seeded WPMY-1 shCtrl or shCAVIN1 stromal cells in the invasion by the lipid addition in a dose-dependent manner bottom well and prostate cancer cells in the top inserts. Two (Fig. 4A and B). This simple experiment suggested that the lipid prostate cancer cell lines, PC3 and DU145, were tested. After content in the surrounding microenvironment influences prostate 14 hours of culture, the inserts with the prostate cancer cells were cancer cell malignant properties. We next explored potential inter- stained and analyzed for their ability to penetrate the insert vention strategies and used methyl-b-cyclodextrin (MBCD), a lipid membrane (migration) or invade the Matrigel (invasion). We scavenger, or cholesteryl ester uptake inhibitor, ML278. In a observed both increased migration and invasion of PC3 and DU145 Transwell assay, both agents reduced invasion of PC3 cells stim- cells cocultured with shCAVIN1 cells compared with shCtrl cells ulated by shCAVIN1 stromal cells, suggesting that targeting acces- (Fig. 2C). Using siRNA to silence CAVIN1 in PrSc stromal cells, we sibility of lipids by the prostate cancer cells restrains their metastatic repeated the Transwell invasion assay and observed increased susceptibility (Fig. 4C and D). To assess whether the excess of lipids invasion of PC3 cells cocultured in CAVIN1-silenced PrSc cells would be a driving factor that triggers inflammatory response, we compared with siRNA control cells (Supplementary Fig. S2G). treated WPMY-1 shCtrl cells with cholesterol and performed qPCR Furthermore, the invasion was decreased when PC3 cells were on the gene panel. We detected upregulation of gene expression cocultured with CAVIN1-R cells compared with coculture with of most transcripts observed in the shCAVIN1 cells (Fig. 4E). shCAVIN1 stromal cells (Fig. 2D), suggesting a successful rescue of Conversely, we used MBCD to scavenge cholesterol in shCAVIN1 the phenotype. These findings suggest that CAVIN1 downregula- cells and observed downregulation of several of the inflammatory tion in the stromal cells augments prostate cancer cell invasive genes (Fig. 4F). Taken together, this suggests that loss of CAVIN1 properties. in stromal cells triggers a lipid-induced inflammatory response in the tumor microenvironment. Loss of stromal CAVIN1 triggers inflammatory pathways We delineated the changes in transcriptome after knocking down Orthotopic coimplantation of stromal CAVIN1-knockdown cells CAVIN1 in WPMY-1 cells using RNA-seq. We detected significant and prostate cancer cells increases metastasis to distant organs alterations in over 6,000 transcripts as compared with the shCtrl cells. To assess how stromal CAVIN1 affects the metastatic ability of GSEA pathway analyses indicated significant enrichment of inflam- prostate cancer in vivo, we chose to use orthotopic coimplantation of matory and IFN response pathway transcripts (Fig. 3A and B). This the stromal and cancer cells to the mouse prostate. We used PC3 was highly interesting, as it suggested that CAVIN1 KD led, not only to prostate cancer cells for this purpose, given their robust metastatic a change in cellular lipid uptake, but also activation of an inflammatory capacity. We first generated a stable PC3 cell line that expresses firefly response including increased expression of several secreted cytokines luciferase (hereafter PC3-Luc cells) to facilitate bioluminescence mon- and proteases (Fig. 3C). We validated the RNA-seq results using qPCR itoring of the cells in vivo. PC3-Luc cells were then mixed 1:1 with and by biochemical analyses. We detected significant upregulation of either shCtrl or shCAVIN1 WPMY-1 stromal cells, and implanted gene transcripts such as CCL2, CSF1, DKK1, IL18, IL32, MX1, MMP3, orthotopically to the mouse anterior prostates. The growth of the and TLR3 in the shCAVIN1 fibroblasts (Fig. 3D). We used ELISA and tumors was determined using bioluminescence over the course of zymography to detect MMP3 and observed its robust upregulation 8 weeks (Fig. 5A and B) and by determining the urogenital block and (Supplementary Fig. S3A and S3B). To validate these findings in the tumor sizes at the end of the experiment. We observed that coinjection PrSc fibroblasts, we silenced CAVIN1 using siRNAs and observed of PC3-Luc cells with stromal fibroblasts with or without CAVIN1 did similar increases in gene transcripts (Supplementary Fig. S3C). Fur- not affect the growth rate or size of the tumors (Fig. 5B and C; thermore, as determined by qPCR, changes in the inflammatory gene Supplementary Fig. S4A). However, based on the macroscopic analysis signatures in shCAVIN1 cells were reversed in CAVIN1-R stromal during necropsy, we observed an increase in the number of lung cells (Fig. 3E). In addition, we performed multiplex cytokine assays to metastasis when PC3-Luc cells were coinjected with the shCAVIN1 determine the cytokines at protein level. Of those measurable, we stromal cells (Supplementary Fig. S4B and S4C). The lungs and livers found that MMP3, DKK1, and CSF-1 were upregulated in shCAVIN1 were harvested and subjected to histology (Fig. 5D). We observed a stromal cells. Conversely, the cytokine levels showed a trend of significant increase in micrometastases in both distant sites when the reduction in the CAVIN1-R stromal cells (Fig. 3F). We also explored orthotopic tumors were generated by coinjection with shCAVIN1 whether the change in inflammatory pathways was accompanied with stromal cells (Fig. 5E and F). Strikingly, assessment of the primary markers for cancer-associated fibroblasts (CAF). On the basis of RNA- tumors showed a significant increase in the lipid content in mice with seq, we observed robust increases in several CAF markers such as coimplantation of shCAVIN1 stromal cells as compared with shCtrl

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Figure 2. Stromal cells that lack CAVIN1 increase the migration and invasion of prostate cancer cells. A, Addition of stromal shCAVIN1 conditioned medium to PC3, DU145, MR49F, and CWR22Rv1 prostate cancer cells shows increased lipid droplet formation in prostate cancer cells as measured by quantitative analysis of Oil Red O staining. Data are from ﬁve independent experiments. B, Scratch assay of PC3 cells cultured in conditioned medium from WPMY-1 stromal cells. Data are from three independent experiments. C, Increased migration and invasion of PC3 and DU145 prostate cancer cells in Transwell cocultures with WPMY-1 shCAVIN1 cells. Data are from six independent experiments. D, Decreased PC3 cell invasion in coculture with WPMY-1 CAVIN1-R cells. Data are from four independent experiments. One-way ANOVA with Tukey post hoc test. Scale bars, 200 mm. , P < 0.05; , P < 0.01; , P < 0.001.

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Figure 3. CAVIN1 depletion in stromal cells activates inflammatory pathways. RNA-seq (A) and GSEA (Hallmark pathways, P < 0.05 and FDR < 0.1; B) comparing shCtrl or shCAVIN1 stromal cell transcriptomics revealed enrichment of inflammatory gene signatures. Representative enrichment plots. NES, normalized enrichment score. C, Selected inflammatory, cytokine, and protease transcripts. P < 0.01 and q < 0.02 for each transcript. D, Validation of RNA-seq data on the short-listed candidate genes by qPCR. Data are from five independent experiments. Student t test. E, Restoration of gene signatures in CAVIN1-R cells. Data are from four independent experiments. F, Multiplex cytokine assay for CSF1, DKK1, and MMP3. Data are from three independent experiments with three replicates each. One-way ANOVA with Tukey post hoc test. ns, not significant. , P < 0.05; , P < 0.01; , P < 0.001.

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Figure 4. Lipids drive prostate cancer cell invasion and stromal cell inflammation. Exogenous addition of cholesterol (A) or oleic acid (B) to PC3 cells increases their migration and invasion in a dose-dependent manner. Data are from four independent experiments. C, Cholesteryl ester uptake inhibitor, ML278 (10 nmol/L) reduces invasion of PC3 cells cocultured with shCAVIN1 stromal cells. Data are from three independent experiments. D, MBCD (20 mmol/L) reduces invasion of PC3 cells cocultured with shCAVIN1 stromal cells. Data are from three independent experiments. E, qPCR analysis for inflammation- and invasion-related genes following cholesterol addition (24 hours) in WPMY-1 shCtrl cells. Data are from three independent experiments. F, qPCR analysis for inflammation- and invasion-related transcripts following MBCD addition (24 hours) in WPMY-1 shCAVIN1 cells. Data are from three independent experiments. One-way ANOVA with Tukey post hoc test. Scale bars, 200 mm. , P < 0.05; , P < 0.01; , P < 0.001.

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Figure 5. Orthotopic coimplantation of shCAVIN1 stromal cells drives prostate cancer cell metastasis. PC3-Luc cells were injected into the prostate of male NSG mouse either alone or in combination with WPMY-1 shCtrl or shCAVIN1 cells (1:1 tumor:stromal cell ratio). Tumor growth was monitored using bioluminescence imaging. n ¼ 10 animals per group. A, Representative images. B, Tumor growth curves showing mean SEM. C, Plot showing tumor weights. Mean is shown, whiskers represent SEM. D, Histology of lung and liver sections and metastasis. M ¼ metastasis. Scale bar, 200 mm. E, Number of microscopic liver metastasis. F, Number of microscopic lung metastasis. One-way ANOVA with Kruskal–Wallis post hoc test. G, Oil Red O staining increases in primary tumors coinjected with shCAVIN1. n ¼ 5 animals analyzed (three fields per animal). One-way ANOVA with Kruskal–Wallis post hoc test. Scale bars, 100 mm. H, qPCR showed a trend of increased inflammatory gene expression in shCAVIN1 coinjected primary tumors. n ¼ 5 animals analyzed. Mann–Whitney nonparametric t test. ns, not significant. , P < 0.05; , P < 0.01; , P < 0.001.

cells (Fig. 5G). To analyze this further, we used qPCR to detect changes Reexpression of CAVIN1 in stromal cells reduces metastasis to in the inflammatory gene signatures. We observed up to 10-fold distant organs and abrogates macrophage infiltration to increases of CSF1 and MMP-3 in tumors coimplanted with shCAVIN1 primary tumors stromal cells albeit with an intracohort variation (Fig. 5H). These Prompted by these findings, we established another cohort of findings strongly suggest that stromal cells lacking CAVIN1 condition orthotopic PC3-Luc–bearing animals using coimplantation with stro- the tumor microenvironment and promote metastasis of the primary mal cells from shCtrl, shCAVIN1, and CAVIN1-R clones (Fig. 6A). tumor. Replication of the model was robust. We neither observed differences

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Figure 6. Reexpression of CAVIN1 rescues tumor metastasis and M2 macrophage infiltration to the primary tumor. PC3-Luc were orthotopically implanted into prostates in combination with shCtrl, shCAVIN1, or CAVIN1-R cells. A, Representative images. B, Urogenital block weight. C, Tumor weight. N ¼ 9 animals per group. One-way ANOVA with Kruskal–Wallis post hoc test. D, Number of macroscopic liver metastases. Mean SEM is shown. E, Number of macroscopic lung metastases. Mean SEM is shown. F, Number of microscopic liver metastases. Mean SEM is shown. G, Number of microscopic lung metastases. Mean SEM is shown. n ¼ 9 animals per group. One-way ANOVA with Kruskal–Wallis post hoc test. H, Tumor tissue lipid content is increased in shCAVIN1 coimplanted tumors and this phenotype is rescued with CAVIN1-R stromal cells. n ¼ 5 animals analyzed (three fields per animal). One-way ANOVA with Kruskal–Wallis post hoc test. I, M2 macrophage infiltration is increased in tumors with shCAVIN1 stromal cells and rescued with CAVIN1-R cells. n ¼ 5 animals analyzed (three fields per animal). One-way ANOVA with Kruskal– Wallis post hoc test. Scale bars, 100 mm. ns, not significant. , P < 0.05; , P < 0.01; , P < 0.001.

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Stromal CAVIN1 Represses Prostate Cancer Metastasis

between the cohorts in their rate of primary tumor growth during the increased intracellular lipids (43), further suggesting that lipids and study, nor tumor or urogenital block weights at the end of the study lipogenic programs contribute to prostate cancer progression and (Fig. 6B and C; Supplementary Fig. S5A). However, we observed metastasis. Genetic models have established that CAVIN1 is essential increased numbers of macrometastases in liver and lungs in the for caveolae formation and alterations in lipid content in adipo- shCAVIN1 cohort, and this phenotype was rescued in the cytes (8, 9, 18). CAVIN1 is highly expressed in normal prostate CAVIN1-R cohort (Fig. 6D and E). We analyzed the liver and lung stroma, and its loss confers poor clinical outcomes (24, 25). However, samples for histology and counted the number of micrometastases. As the function of CAVIN1 in the regulation of lipid metabolism has not previously, we observed an increase in micrometastases in the lungs been previously studied in the prostate stroma. Given our finding that and liver of shCAVIN1 cohort compared with shCtrl cohort. This loss of stromal CAVIN1 exposes prostate cancer microenvironment to phenotype was robustly rescued in the CAVIN1-R cohort (Fig. 6F an excess of lipids, and consequently, proinflammatory environment, and G; Supplementary Fig. S5B). our study identifies a molecular event that contributes to lipid-driven We analyzed the primary tumors for lipid content using Oil Red O. pathogenesis of prostate cancer. Again, we found a significant increase in the lipid content when Caveolar integrity is essential for cellular signal transduction and shCAVIN1 stromal cells had been used for coimplantation as com- cholesterol transport (3). Caveolae formation requires both CAVIN1 pared with shCtrl stromal cells. This phenotype was fully reverted and caveolins. Both are abundantly expressed in adipocytes, endothe- when CAVIN-R stromal cells were used (Fig. 6H). We then assessed lial, and stromal cells. They are typically not expressed by epithelial the primary tumors for M2 inflammatory macrophages using CD163 cells, but curiously, CAV1 expression varies broadly among cancer as a marker. We detected a significant increase in M2 macrophage types such that its high expression correlates with poor outcomes in infiltration in primary tumors from shCAVIN1 cohort as compared breast, prostate, and lung cancer and good outcomes in head and with shCtrl cohort and abrogation of macrophage infiltration in the neck and biliary cancer (3). In prostate cancer, CAV1 is bestowed CAVIN1-R cohort primary tumors (Fig. 6I). To assess whether the with protumorigenic properties that can be reset by ectopic human WPMY-1 stromal fibroblasts continued to reside in the expression of CAVIN1 (7, 25, 44, 45). Reintroduction of CAVIN1 primary tumors, we performed SV40 T-antigen immunostaining. We into prostate cancer cells reduced the aggressive phenotypes (migra- observed occasional staining of SV40 T-antigen–positive stromal cells tion, invasion, lymphangiogenesis, and angiogenesis) in vitro and in and around the primary tumors that varied from less than a few in vivo (7, 24, 26, 27). CAVIN1 is downregulated in prostate cancer hundred per tumor section to no detectable signal. Their presence in stroma (24, 25). Loss of CAV1 in tumor stroma also confers a poor the primary tumors was independent of CAVIN1 expression (Sup- outcome in prostate, breast, esophageal, gastric, and pancreatic can- plementary Fig. S5C). This suggested that it is unlikely that the cers (21, 25). Cav1-null mice have stromal abnormalities, increased persistent changes observed in the primary tumors were due to the epithelial hyperplasia, and support increased growth of ectopically continuous influence of the resident human stromal cells. These implanted breast cancer cells or tumors (21). Genetic ablation of Cav1 findings suggest that stromal cells cause permanent changes in the increases proliferation of primary and transformed fibroblasts via an primary tumors and their metastatic capacity. increase in MAPK signaling pathway (21). Knocking down CAV1 increases lipid uptake, the amount of cholesterol and testosterone in fibroblasts, and promotes cancer cell proliferation, primary tumor Discussion growth, and metastasis (21, 23). Here, we show that CAVIN1 KD Lipids fuel cancer cell metabolism, and serve as macromolecules for abrogates stromal lipid uptake and has no effect on proliferation of membranes and building blocks for hormone synthesis (36). Epide- either stromal cells or prostate cancer cells or primary tumor growth. miologic studies have linked obesity and high fat diet with cancer This suggests that even if functions of CAVIN1 and CAV1 are coupled incidence, and mechanistic studies have shown that cancer cells utilize through caveolae formation, their loss exerts different outcomes in the exogenous lipids for many of their pathologic pathways and functions. stromal cells, both of which may exacerbate the aggressiveness of the Our study provides a conceptual advance by showing that loss of primary tumor. CAVIN1, an essential factor for caveolae formation in the prostate Here, we found that loss of stromal CAVIN1 activated inflamma- stromal cells promotes an inflammatory microenvironment that fuels tory and CAF-like gene signatures and upregulated secretion of prostate cancer aggressive phenotypes. We demonstrated using in vitro MMP3, DKK1, and CSF1. Given that the addition of lipids caused coculture models that loss of stromal CAVIN1 expression negates the an inflammatory response, and that it was reverted by depleting ability of stromal cells to sequester lipids leading to upregulation of cholesterol, we suggest that these effects are lipid-enacted and reveal inflammatory signatures such as expression of cytokines, cytokine remodeling of the tumor microenvironment via CAVIN1. CAVIN1 receptors, matrix metalloproteinases, and markers for CAFs. We was recently reported to interact with suppressor of cytokine signaling showed that CAVIN1-deficient stromal cells promoted prostate cancer 3 (SOCS3), and is required for SOCS3 localization and function, cell migration and invasion, and in vivo, metastasis and M2 macro- suggesting at least one mechanism how CAVIN1 limits JAK–STAT phage infiltration. This study reaffirms the critical function of the inflammatory signaling (46). Cavin1 / mice have increased inflam- stromal component in tumorigenesis and reveals a metastasis- matory gene signatures including TNFa, IL6, F4/80, and CD11c in the suppressing role of CAVIN1. epididymal fat tissues, and these signatures are further upregulated in Prostate cancer is a lipid-rich tumor and periprostatic white the presence of high fat diet (17). The mechanisms that direct increases adipose tissue inflammation associates with prostate cancer in inflammatory pathways require further exploration. aggressiveness (37–39). Dietary fat promotes prostate cancer devel- We find here striking enrichment of M2 macrophages in the opment and progression through SREBP1-regulated lipogenic and primary prostate tumor when stromal cells lacking CAVIN1 had been MYC programs (34, 40, 41). Chemical inhibition of fatty acid synthase used for coinjection. Using SV40 T-antigen staining as a marker, we reprograms castration-resistant prostate cancer and reduces the detected little to no remaining human stromal fibroblasts in the expression androgen receptor and its treatment-resistant variant primary tumors, suggesting that the human stromal cells that were V7 (42). Circulating prostate tumor cells have high lipid uptake and initially grafted into the mouse prostate were priming the

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Low et al.

microenvironment and reprogrammed the prostate cancer cells. The know why and how stromal CAVIN1 is lost in prostate cancer stroma. M2 macrophage enrichment was accompanied by increased lipid This knowledge will be informative to pursue. Similarly, syngeneic deposition in the primary tumor cells, and both were reversed when genetic models that facilitate broad assessment of the immune and stromal cells reexpressing CAVIN1 were coinjected. CAVIN1 has been tumor microenvironment landscape will be needed. Further studies shown to regulate macrophage number and phenotype in lungs of that monitor CAVIN1 expression in tumor stroma and its prognostic Cavin1 / mice and demonstrate increased macrophage infiltration significance are warranted and may lead to early or novel intervention into the adipose tissues (47). Several studies suggest that presence of strategies to prevent metastatic disease. Although CAVIN1 was shown M2 macrophages in the prostate tumor leads to a worse prognosis in to regulate lipid metabolism and inflammatory cytokines, this study is men (48–50). Therefore, it is plausible that infiltration of M2 macro- the first to show these observations in the context of cancer. Collec- phages in the prostate tumors lacking stromal CAVIN1 may further tively, we established that CAVIN1 expression in the prostate stromal fuel the aggressive disease, and is consistent with the striking increase cells is critical in control of lipid content and inflammation in the in lung and liver metastases observed here. Strikingly, cancer cells have prostate microenvironment and suppresses metastatic disease. recently been shown to scavenge cholesterol from tumor-associated macrophages (TAM) leading to their polarization and reprogram- Disclosure of Potential Conflicts of Interest ming. Furthermore, inhibition of cholesterol efflux by deletion of the M. Laiho reports grants from Walsh Prostate Cancer Fund and DoD CDRMP fi ABC transporters led to reversion of macrophage TAM phenotype and PCRP during the conduct of the study; other from Blue eld Innovations outside the submitted work; has a patent to US 8,680,107; US 10,214,491; EU 2195316, Canada inhibition of tumor progression (51). Nevertheless, it is pertinent to 2,691,227; and Canada 2,912,456 issued, a patent to PCT/US2015/021699/15765295/ further validate these observations using additional metastatic in vivo 2,943,022 US, EU, and Canada pending, and a patent to PCT/US2017/052863, 16/ prostate cancer models, other than the androgen receptor–null PC3 335,737, 2017330390, and 17853955.7 pending. No potential conflicts of interest were cells, as they become available. Together with our findings, the disclosed by the other authors. emerging evidence points to intricate relationships between the cancer cells, TAMs, and stromal cells underlined by lipid metabolic needs, Authors’ Contributions inflammatory signals, and metastatic proneness where loss of CAVIN1 J.-Y. Low: Conceptualization, data curation, software, formal analysis, validation, in the stroma attracts inflammatory macrophages and promotes a investigation, visualization, methodology, writing-original draft, project administration, writing-review and editing. W.N. Brennen: Supervision, tumor-supportive microenvironment. investigation, methodology, project administration. A.K. Meeker: Resources, Different intervention strategies to alter the composition or amount formal analysis, validation, methodology. E. Ikonen: Conceptualization, funding of lipids in cancer have been explored, and most lipid-targeting acquisition, methodology, writing-review and editing. B.W. Simons: Investigation. compounds have failed to move beyond preclinical models (52). M. Laiho: Conceptualization, resources, supervision, funding acquisition, Because lipids were reported to drive prostate cancer progression, visualization, project administration, writing-review and editing. strategies aimed at reducing fatty acid uptake into prostate tumors may Acknowledgments provide a novel and viable therapeutic avenue for prostate cancer. We thank Drs. Ken Pienta and Helen Nicholson for critical review. We would also Statins have been shown to effectively reduce the growth and metas- like to thank Susan Dalrymple for her technical assistance in the orthotopic model. tasis of prostate cancer in preclinical models (34, 53, 54). In our study, This work was supported by Patrick C. Walsh Prostate Cancer Research Fund and we observed that scavenging lipids using MBCD and a lipid uptake Department of Defense CDMRP award W81XWH-17-1-0458 (to M. Laiho) and Jane inhibitor reduced invasion of PC3 cells. However, similar strategies and Aatos Erkko Foundation (to E. Ikonen). will need validation in preclinical models. It is also plausible that regulation of lipid and inflammatory pathways intersect and are both The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance culprits in driving aggressive prostate cancer. Nevertheless, this study, with 18 U.S.C. Section 1734 solely to indicate this fact. using a definitive model for CAVIN1 dependency emphasizes that loss of stromal CAVIN1 is a key event that contributes to a prometastatic Received April 23, 2020; revised May 1, 2020; accepted May 28, 2020; published first state. CAVIN1 can be epigenetically regulated (55), but we do not June 3, 2020.

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AACRJournals.org Mol Cancer Res; 2020 OF13

Stromal CAVIN1 Controls Prostate Cancer Microenvironment and Metastasis by Modulating Lipid Distribution and Inflammatory Signaling

Jin-Yih Low, W. Nathaniel Brennen, Alan K. Meeker, et al.

Mol Cancer Res Published OnlineFirst June 3, 2020.

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