ROR1C Regulates Differentiation of Myeloid-Derived Suppressor Cells

Abstract

Myeloid-derived suppressor cells (MDSCs) play a major role in cancer. MDSC expansion is closely associated with tumor progression, but molecular mechanisms of this expansion remain poorly understood. In this issue of Cancer Cell, Strauss and colleagues describe the roles of the nuclear receptor ROR1C in the regulation of MDSC differentiation and expansion.

The accumulation of non-lymphoid suppressive cells in cancer was first reported in the late 1970s, but these cells had attracted very little attention until 15 years ago, when their potential contribution to tumor progression became apparent. These cells have been named myeloid-derived suppressor cells (MDSCs) to reflect their origin and major functional feature—the ability to suppress T cell activation and function. In addition, the role of MDSCs in promoting tumor growth by supporting angiogenesis, tumor cell survival, metastases, and formation of pre-metastatic niches has been established (Condamine et al., 2015). Recent studies have provided ample evidence of the clinical relevance of MDSCs (Messmer et al., 2015). MDSCs are phenotypically distinct from terminally differentiated dendritic cells (DCs) and macrophages and represent a heterogeneous population of immature myeloid cells that include cells with granulocytic and monocytic morphology and phenotype. MDSCs are now divided into two major populations: polymorphonuclear-MDSCs (PMN-MDSCs) and monocytic-MDSCs (M-MDSCs) (Movahedi et al., 2008; Youn et al., 2008). In a majority of cancer types, PMN-MDSCs, which have a phenotype and morphology similar to those of neutrophils, represent 70%–80% of the total MDSC population. However, in contrast to neutrophils, PMN-MDSCs suppress T cell functions and have a distinct gene expression profile and a number of distinct functional characteristics. M-MDSCs share their phenotype and morphology with normal monocytes. In contrast to spleen monocytes in naive mice and blood monocytes in healthy individuals, M-MDSCs have a potent ability to suppress T cell functions, which is mediated by arginase-1, nitric oxide (NO), and different soluble factors (Gabrilovich et al., 2012). MDSCs arise from a common myeloid progenitor. Their development is supported by the same growth factors that are responsible for the normal myelopoiesis: granulocyte macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), and macrophage colony-stimulating factor (M-CSF) (Bayne et al., 2012; Dolcetti et al., 2010; Kowanetz et al., 2010). However, simple expansion of myeloid cells is not sufficient to generate bona fide MDSCs. MDSCs exist in the state of pathological activation, which is the result of persistent stimulation of the myeloid compartment with relatively low strength signals coming from tumors or sites of chronic inflammation. Myeloid cells generated under these conditions are unable to effectively differentiate into mature myeloid cells, are poorly phagocytic, and produce high levels of reactive oxygen species, myeloperoxidase, nitric oxide, and mostly anti-inflammatory cytokines. As a result, these cells acquire potent immune suppressive potential. The molecular mechanisms that govern such pathological expansion are subjects of intense investigations. Different factors were implicated in this process. They include signal transducer and activator of transcription 3 (STAT3) and 5, NF-κB, paired immunoglobulin-like receptor B, CCAAT/enhancer binding protein β (C/EBPβ), interferon regulatory factor 8 (IRF8), retinoblastoma protein (Rb), and other. In this issue of Cancer Cell Strauss et al. (2015) identified novel mechanisms that involved RORC1.

RORC1 and its splice variant RORC2 are master regulators of IL-17A gene transcription. Authors were interested in RORC1 because they found increased expression of IL-17A by PMN-MDSCs in tumor-bearing mice, although these cells failed to release IL-17. In contrast, M-MDSCs and macrophages lacked expression of IL-17A. The majority of blood and spleen PMN-MDSCs expressed RORC1. Tumor-bearing mice deficient for RORC1 showed a significant expansion of the granulocytic compartment, which was associated with a progressive contraction of the erythroid colonies and signs of dysmegakaryopoiesis. Data indicated that Rorc^−/− tumor-bearing mice effectively supported emergency hematopoiesis while displaying a defective induction of MDSCs.

To investigate in vivo relevance of RORC1-expressing myeloid cells, Strauss et al. (2015) transplanted RORC1-deficient bone marrow (BM) cells into lethally irradiated wild-type (WT) recipient mice. Tumor growth and metastasis were significantly reduced in these mice, and this was accompanied by a dramatic reduction of splenic MDSCs. These results implied that RORC1 promoted the expansion of splenic MDSCs. These conclusions were supported by the increase in the metastatic burden and the presence of splenic M-MDSCs and PMN-MDSCs in tumor-bearing mice treated with ROR1C agonist SR1078.

As compared to the recipients of WT BM, Rorc^−/− BM chimeras have a substantially higher number of hematopoietic stem and common myeloid progenitors but a decreased number of granulocyte/macrophage progenitors, suggesting a possible block in differentiation of early hematopoietic progenitors. In the presence of tumor-conditioned medium or in response to GM-CSF, BM progenitors from Rorc^−/− mice failed to differentiate to macrophages, displaying increased differentiation into granulocytes, while treatment with G-CSF resulted in reduced granulocytes production. These results suggested a key role of RORC1 in the myelopoietic activity of G- and GM-CSF. In vitro-generated Rorc−/− myeloid cells had a higher level of apoptosis than their WT counterparts, suggesting that RORC1 can be involved in regulation of the survival of these cells.

In an attempt to identify the mechanisms of the effect of ROR1C on myelopoiesis, Strauss et al. (2015) studied C/EBPβ, a major positive regulator of G-CSF- and GM-CSF-driven “emergency” myelopoiesis, and C/EBPα, a major regulator of “steady state” granulopoiesis. A modest but significant decrease of expression of PU.1, C/EBPβ, and C/EBPα was found in the spleen and BM of Rorc^−/− > WT tumor-bearing mice, which paralleled the decrease of C/EBPβ proteins in splenic PMN-MDSCs and M-MDSCs. The BM and spleen from Rorc^−/− > WT tumor-bearing mice had increased mRNA levels of the suppressor of cytokine signaling-3 (Socs3), which regulates STAT3 activity, and the transcriptional co-regulator B cell leukemia/lymphoma 3 (Bcl3), both potent inhibitors of G-CSF-driven granulopoiesis. Strauss et al. (2015) observed a decreased number of IRF8-expressing macrophages. RORC1-deficient macrophages displayed enhanced expression of M1 and decreased expression of M2 genes, suggesting that RORC1 may act as a negative regulator of M1 and a promoter of M2 polarization. A decreased number of tumor-associated macrophages (TAM) from Rorc^−/− > WT mice correlated with increased PMN-MDSC infiltration, suggesting that ROR1C pathways lead to terminal differentiation and M2-polarization of TAM and hamper neutrophil accumulation in tumors. To test this assumption, tumor-bearing WT mice were treated with CSFR1 antibody, which significantly depleted TAM. Macrophage depletion was paralleled by the inhibition of immature RORC1⁺ M-MDSCs and PMN-MDSCs and increased tumor infiltration of mature neutrophils. Treatment of mice with M-CSF antibody resulted in a similar increase of mature neutrophils. Administration of CSFR1 antibody led to reduced monocyte/macrophage precursors and increased granulocyte progenitors, supporting the role for reciprocal negative regulation of monocytes/macrophages and granulocytes in cancer inflammation. Treatment with G-CSF antibody significantly decreased RORC1 expression in PMN-MDSCs and partially in M-MDSCs and macrophages. This effect was significantly reduced in Rorc^−/− mice, supporting the hypothesis that G-CSF may work through RORC1.

Although the precise mechanism responsible for upregulation of ROR1C in cancer needs to be elucidated, this study provides exciting insight into potential regulation of pathological myelopoiesis by one of the members of the nuclear receptor superfamily and suggests that RORC1 could be a key driver of the differentiation of MDSCs and TAM (Figure 1).

Figure 1. RORC1 Involvement in Myeloid Lineage Differentiation.

Brown shows the general pathway of differentiation of neutrophils and mononuclear cells in healthy individuals. Hematopoietic stem cells (HSC) differentiate into common myeloid progenitors (CMP), then into granulocytes macrophage progenitors (GMP), which give rise to mature neutrophils via sequential steps of differentiation involving myeloblasts (MB), promyelocytes (PM), myelocytes (MC), metamyelocytes (MM), and band forms (BF). Differentiation of macrophages (MΦ) and DCs involve macrophage/dendritic cell progenitors (MDP), DC progenitors (CDP), pre-cDCs, as well as several types of monocytes. In cancer (blue), tumor-derived factors regulate various molecular pathways, including upregulation of RORC1, which affect all steps of granulocytic and monocytic cell differentiation. RORC1 promotes the accumulation of immature pathologically activated PMN-MDSC neutrophils for the expense of mature PMN. M-MDSCs differentiate to TAM in the tumor site. RORC1 promotes polarization toward the M2 phenotype of TAM.