Retinoid X receptor suppresses a metastasis-promoting transcriptional program in myeloid cells via a ligand-insensitive mechanism Mate Kissa, Zsolt Czimmerera,b, Gergely Nagyb, Pawel Bieniasz-Krzywiecc,d, Manuel Ehlingc,d, Attila Papa, Szilard Poliskaa,e, Pal Botof, Petros Tzerpose, Attila Horvatha, Zsuzsanna Kolostyaka, Bence Danielg, Istvan Szatmarif, Massimiliano Mazzonec,d, and Laszlo Nagya,b,g,1 aNuclear Receptor Research Laboratory, Department of Biochemistry and Molecular Biology, University of Debrecen, 4032 Debrecen, Hungary; bMTA-DE “Lendület” Immunogenomics Research Group, University of Debrecen, 4032 Debrecen, Hungary; cLaboratory of Tumor Inflammation and Angiogenesis, Center for Cancer Biology, VIB, 3000 Leuven, Belgium; dLaboratory of Tumor Inflammation and Angiogenesis, Department of Oncology, KU Leuven, 3000 Leuven, Belgium; eUD-GenoMed Ltd., 4032 Debrecen, Hungary; fStem Cell Differentiation Laboratory, Department of Biochemistry and Molecular Biology, University of Debrecen, 4032 Debrecen, Hungary; and gGenomic Control of Metabolism Program, Sanford Burnham Prebys Medical Discovery Institute at Lake Nona, Orlando, FL 32827 Edited by Bert W. O’Malley, Baylor College of Medicine, Houston, TX, and approved August 18, 2017 (received for review January 18, 2017) Retinoid X receptor (RXR) regulates several key functions in myeloid and identified a network of RXR-bound enhancers that control cells, including inflammatory responses, phagocytosis, chemokine angiogenic genes including Vegfa, Hbegf, Litaf,andHipk2 (9). secretion, and proangiogenic activity. Its importance, however, in Activation of these enhancers by RXR induced a proangiogenic tumor-associated myeloid cells is unknown. In this study, we transcriptional program and phenotype (9). Interestingly, we demonstrate that deletion of RXR in myeloid cells enhances lung found that half of the 5,200 RXR-occupied genomic sites were metastasis formation while not affecting primary tumor growth. transcriptionally inactive in macrophages (9). This suggested that We show that RXR deficiency leads to transcriptomic changes in the the receptor may also possess extensive regulatory functions in lung myeloid compartment characterized by increased expression myeloid cells that are distinct from ligand recognition and acti- of prometastatic genes, including important determinants of pre- vation. These observations provided a rationale to investigate metastatic niche formation. Accordingly, RXR-deficient myeloid cells whether RXR influences tumor progression through regulating are more efficient in promoting cancer cell migration and invasion. the phenotype of recruited or tissue-resident myeloid cells. Our results suggest that the repressive activity of RXR on prometa- static genes is mediated primarily through direct DNA binding of Results the receptor along with nuclear receptor corepressor (NCoR) and RXR Deletion in Myeloid Cells Promotes Metastasis to the Lung. In silencing mediator of retinoic acid and thyroid hormone receptor myeloid cells, RXRα shows the highest expression and RXRβ is (SMRT) corepressors and is largely unresponsive to ligand activa- expressed at a lower level, while RXRγ is not expressed (5). tion. In addition, we found that expression and transcriptional ac- Specific deletion of RXRα in myeloid cells was achieved by tivity of RXRα is down-modulated in peripheral blood mononuclear crossing mice bearing lox-P-targeted RXRα (RXRαfl/fl) with cells of patients with lung cancer, particularly in advanced and mice carrying the lysozyme-M Cre (LysM-Cre) recombinase metastatic disease. Overall, our results identify RXR as a regula- transgene. To exclude the potential compensatory effects of tor in the myeloid cell-assisted metastatic process and establish RXRβ in the absence of RXRα resulting from the functional lipid-sensing nuclear receptors in the microenvironmental regu- lation of tumor progression. Significance INFLAMMATION retinoid X receptor | metastasis | premetastatic niche | myeloid cell | NCoR IMMUNOLOGY AND Metastasis formation from malignant tumors is the leading cause of cancer-related deaths. There is an increasing body of etinoid X receptor (RXR) is a member of the nuclear re- evidence indicating that immune cells in distant organs actively Rceptor superfamily of ligand-dependent transcription factors contribute to this process by establishing a tissue environment that bind various lipophilic hormones and lipid metabolites. that is hospitable for cancer cells. In this study, we show that Sensing the lipid milieu by RXR, in concert with other nuclear deletion of retinoid X receptor (RXR), a cellular sensor of vita- receptors, plays critical roles in physiology, including the regu- min A metabolites, specifically in the myeloid lineage of the lation of organogenesis, lipid metabolism, and skin homeostasis immune system, leads to an enhanced metastasis rate. We also (1). RXR is able to bind vitamin A derivatives 9-cis-retinoic acid demonstrate that RXR inhibits the expression of a number of and 9-cis-13,14-dihydroretinoic acid as well as fatty acids such as genes that encode proteins involved in the promotion of me- docosahexanoic acid and phytanic acid (1, 2). The receptor has tastasis formation. Surprisingly, our results suggest that this three isotypes, RXRα, RXRβ, and RXRγ, which appear to be activity of RXR is independent of the presence of its activators. functionally interchangeable in tissues in which more than one isotype is expressed (3). RXR has a unique integrative function Author contributions: M.K., Z.C., I.S., M.M., and L.N. designed research; M.K., Z.C., P.B.-K., M.E., A.P., S.P., P.B., P.T., Z.K., and B.D. performed research; M.K., Z.C., G.N., P.B.-K., M.E., in nuclear receptor signaling because of its ability to form S.P., A.H., and Z.K. analyzed data; and M.K. and L.N. wrote the paper. homodimers as well as heterodimers with several other members The authors declare no conflict of interest. of the superfamily, such as PPAR, LXR, RAR, and VDR. RXR This article is a PNAS Direct Submission. and its heterodimers have a profound effect on the function of myeloid cells by linking cellular metabolism and immune func- Freely available online through the PNAS open access option. Data deposition: The data reported in this paper have been deposited in the NCBI Se- tion (4, 5). Specific activation of RXR has been shown to quence Read Archive database, https://www.ncbi.nlm.nih.gov/sra (accession no. up-regulate chemokine expression, promote phagocytosis of SRP095164). apoptotic cells, and attenuate antiviral responses in myeloid cells 1To whom correspondence should be addressed. Email: [email protected]. – (6 8). To further delineate the function of the receptor, we re- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. cently mapped the genomic binding sites of RXR in macrophages 1073/pnas.1700785114/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1700785114 PNAS | October 3, 2017 | vol. 114 | no. 40 | 10725–10730 Downloaded by guest on October 1, 2021 redundancy of RXR isotypes (3, 10), we crossed LysM-Cre B16-F10 lung metastases fl/fl −/− A B B16-F10 lung metastases C RXRα with RXRβ mice. Given the low fertility and via- (macroscopic) (coronal section) αfl/fl β−/− 15 80 bility of the resulting LysM-Cre RXR RXR mice (11), * * we transplanted their bone marrow to syngeneic C57BL/6J mice. 60 This way, we were able to generate age-matched groups of mice 10 − − + + bearing LysM-Cre RXRαfl/fl RXRβ / and LysM-Cre RXRα / 40 − − RXRβ / bone marrow (hereafter referred to as RXRαfl/fl and 5 / coronal section 20 + + No. of metastases RXRα / , respectively). No. of surface metastases 0 0 RXR +/+ To investigate the effect of RXRα deletion in the myeloid in- RXR +/+ RXR fl/fl RXR +/+ RXR fl/fl filtrate on tumor progression, we s.c. implanted RXRαfl/fl and +/+ D RXRα mice with Lewis lung carcinoma (LLC), a murine model B16-F10 lung metastasis size distribution of non-small cell lung carcinoma showing abundant infiltration of 25 ( 1000 µm) RXR +/+ myeloid cells from early stages. Primary tumor growth remained 20 αfl/fl RXR unaltered (Fig. 1A), and the immune cell infiltration in RXR 15 mice was largely unaltered with a significantly lower abundance of + − − high 10 M1-like CD11b Ly6G Ly6C MHC-II macrophages (SI Ap- RXR fl/fl pendix,Fig.S1). However, the frequency of M2-like MHC-IIlow 5 0 macrophages and other immune cell populations was not affected % within total metastasis no. 0 by RXRα deficiency (SI Appendix,Fig.S1). Although primary 0-100100-200200-300300-400400-500500-600600-700700-800800-90900-1000 Diameter of metastasis (µm) tumor growth was not affected, we observed a higher frequency of animals with spontaneous lung metastases, as well as a higher E RXR +/+ RXR fl/fl 100 number of metastatic nodules in the RXRα knock-outs compared with controls (Fig. 1 B and C). Thus, we further investigated the 80 role of myeloid cell-expressed RXRα in lung colonization by using 60 ns an experimental metastasis model. Consistent with the results 40 Ki67 obtained in the LLC model, we found that B16-F10 melanoma % of Ki67+ cells in metastatic area 20 cells formed a higher number of macroscopic and microscopic fl/fl 0 metastases in the lungs of RXRα mice after i.v. injection (Fig. 2 RXR +/+ RXR fl/fl 8 A–C). RXRαfl/fl lungs contained a considerably higher frequency of micrometastases with diameters less than 100 μm, demon- 6 strating that extravasation followed by micrometastasis formation 4 in the lung is more efficient in the presence of RXRα-deficient 2 Cleaved casp-3 % of cleaved casp-3+ cells in metastatic area 0 RXR +/+ RXR fl/fl A LLC primary tumor growth B LLC lung metastases (macroscopic) Fig. 2. RXRα deletion in myeloid cells results in enhanced lung colonization 0.8 12 RXR +/+ * by B16-F10 melanoma cells. (A) Number of macroscopic lung metastases in fl/fl +/+ ) RXR fl/fl 6/15 11/15 α α < ’ = – 3 10 RXR and RXR mice (*P 0.05, unpaired Student s t test, n 10 13/ 0.6 group, data combine two independent experiments).