Retinoid X receptor suppresses a metastasis-promoting transcriptional program in myeloid cells via a ligand-insensitive mechanism

Abstract

Retinoid X receptor (RXR) regulates several key functions in myeloid cells, including inflammatory responses, phagocytosis, chemokine secretion, and proangiogenic activity. Its importance, however, in tumor-associated myeloid cells is unknown. In this study, we demonstrate that deletion of RXR in myeloid cells enhances lung metastasis formation while not affecting primary tumor growth. We show that RXR deficiency leads to transcriptomic changes in the lung myeloid compartment characterized by increased expression of prometastatic genes, including important determinants of premetastatic niche formation. Accordingly, RXR-deficient myeloid cells are more efficient in promoting cancer cell migration and invasion. Our results suggest that the repressive activity of RXR on prometastatic genes is mediated primarily through direct DNA binding of the receptor along with nuclear receptor corepressor (NCoR) and silencing mediator of retinoic acid and thyroid hormone receptor (SMRT) corepressors and is largely unresponsive to ligand activation. In addition, we found that expression and transcriptional activity of RXRα is down-modulated in peripheral blood mononuclear cells of patients with lung cancer, particularly in advanced and metastatic disease. Overall, our results identify RXR as a regulator in the myeloid cell-assisted metastatic process and establish lipid-sensing nuclear receptors in the microenvironmental regulation of tumor progression.

Keywords: NCoR; metastasis; myeloid cell; premetastatic niche; retinoid X receptor.

Fig. 1.

RXRα deletion in myeloid cells enhances Lewis lung carcinoma metastasis. (A) Subcutaneous LLC tumor growth in mice with myeloid cell-specific deletion of RXRα (RXRα^fl/fl) and control (RXRα^+/+) mice (n = 5–7/group). (B) Number of macroscopic lung metastases in RXRα^fl/fl and RXRα^+/+ mice (*P < 0.05, Mann-Whitney test, n = 15/group, data combine two independent experiments). (C) Representative images of hematoxylin and eosin-stained midcoronal lung sections from RXRα^fl/fl and RXRα^+/+ mice. Arrowheads indicate metastatic nodules. (Scale bar, 1 mm.) Graphs on A and B show mean ± SEM.

Fig. 2.

RXRα deletion in myeloid cells results in enhanced lung colonization by B16-F10 melanoma cells. (A) Number of macroscopic lung metastases in RXRα^fl/fl and RXRα^+/+ mice (*P < 0.05, unpaired Student’s t test, n = 10–13/group, data combine two independent experiments). (B) Number of microscopic lung metastases on midcoronal lung sections in RXRα^fl/fl and RXRα^+/+ mice (*P < 0.05, unpaired Student’s t test, n = 8/group). (C) Representative images of hematoxylin and eosin-stained midcoronal lung sections from RXRα^fl/fl and RXRα^+/+ mice. (Scale bar, 6 mm.) (D) Size distribution of B16-F10 melanoma lung metastases with a diameter ≤1,000 μm in RXRα^fl/fl and RXRα^+/+ mice. Metastasis diameter data are pooled from eight lungs/group. (E) Representative images and quantification of Ki67 and cleaved caspase-3 stainings on midcoronal lung sections with B16-F10 melanoma metastases from RXRα^fl/fl and RXRα^+/+ mice (Scale bar, 100 μm.) (P > 0.05, unpaired Student’s t test, n = 5/group). Bar graphs on A, B, and E show mean ± SEM.

Fig. 3.

RXRα represses a prometastatic gene set in pulmonary myeloid cells. (A) Relative numbers of myeloid cells in the lungs of RXRα^+/+ and RXRα^fl/fl mice in the presence or absence of B16-F10 melanoma metastases (*P < 0.05, unpaired Student’s t test, n = 3/group). (B) Volcano plot showing transcriptomic differences in CD45⁺CD11b⁺ pulmonary myeloid cells from RXRα^fl/fl mice compared with RXRα^+/+. Red dots represent differentially expressed genes (adjusted P < 0.01, Wald test, n = 3/group). (C) Dot plot showing significantly enriched gene ontology biological processes within the up-regulated genes in RXRα^fl/fl pulmonary myeloid cells. The top five enriched GO terms by P value are highlighted. (D) Heat map showing expression changes of selected prometastatic genes in RXRα^+/+ and RXRα^fl/fl pulmonary myeloid cells. (E) Number of migrated LLC cells in the presence of RXRα^+/+ or RXRα^fl/fl iBMDMs in Transwell migration assay. The experimental setup is shown in SI Appendix, Fig. S2A (***P < 0.001, unpaired Student’s t test, n = 3/group). (F) Number of migrated LLC cells through Matrigel layer in the presence of RXRα^+/+ or RXRα^fl/fl iBMDMs in Transwell invasion assay. The experimental setup is shown in SI Appendix, Fig. S2B (*P < 0.05, unpaired Student’s t test, n = 3/group). Bar graphs on A, E, and F show mean ± SEM.

Fig. 4.

RXR-mediated repression of prometastatic genes is predominantly ligand-insensitive. (A) Heat map showing expression changes in response to synthetic RXR agonist LG268 in BMDMs among RXR-repressed genes identified in pulmonary myeloid cells (adjusted P < 0.01, Wald test, n = 2/time point). (B) Heat map showing expression changes in response to synthetic RXR agonist LG268 in BMDMs among selected RXR-repressed prometastatic genes identified in pulmonary myeloid cells. Genes with significant expression changes are shown in bold italics (adjusted P < 0.01, Wald test, n = 2/group). (C) Number of migrated LLC cells in the presence of control or LG268-pretreated iBMDMs in Transwell migration assay (P > 0.05, unpaired Student’s t test, n = 3/group).

Fig. 5.

RXR colocalizes with NCOR/SMRT corepressors to mediate repression of prometastatic genes. (A) NCoR and SMRT occupancy in BMDMs at the genomic sites of RXR-repressed genes identified in pulmonary myeloid cells with/without overlap with RXR (box plots show median and 5–95 percentiles). (B) Genomic loci of selected prometastatic genes showing overlapping RXR/NCoR/SMRT ChIP-seq peaks in BMDMs. (C) Expression of selected prometastatic genes in NCoR^+/+ and NCoR^fl/fl iBMDMs (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, unpaired Student’s t test, n = 7/group).

Fig. 6.

RXR signaling is down-modulated in PBMCs of patients with NSCLC. (A) Expression of RXR isotypes in PBMCs of patients with NSCLC and cancer-free control patients. (Graphs show mean. ***adjusted P < 0.001, Mann-Whitney test) (B) Top 10 upstream regulators by z-score predicted to be inhibited in PBMCs of patients with NSCLC compared with cancer-free control patients (P < 0.05). RXR heterodimeric partners are shown in bold italics.