Macrophage HIF‐2α regulates tumor‐suppressive Spint1 in the tumor microenvironment

In solid tumors, tumor‐associated macrophages (TAMs) commonly accumulate within hypoxic areas. Adaptations to such environments evoke transcriptional changes by the hypoxia‐inducible factors (HIFs). While HIF‐1α is ubiquitously expressed, HIF‐2α appears tissue‐specific with consequences of HIF‐2α expression in TAMs only being poorly characterized. An E0771 allograft breast tumor model revealed faster tumor growth in myeloid HIF‐2α knockout (HIF‐2αLysM−/−) compared with wildtype (wt) mice. In an RNA‐sequencing approach of FACS sorted wt and HIF‐2α LysM−/− TAMs, serine protease inhibitor, Kunitz type‐1 ( Spint1) emerged as a promising candidate for HIF‐2α‐dependent regulation. We validated reduced Spint1 messenger RNA expression and concomitant Spint1 protein secretion under hypoxia in HIF‐2α‐deficient bone marrow–derived macrophages (BMDMs) compared with wt BMDMs. In line with the physiological function of Spint1 as an inhibitor of hepatocyte growth factor (HGF) activation, supernatants of hypoxic HIF‐2α knockout BMDMs, not containing Spint1, were able to release proliferative properties of inactive pro‐HGF on breast tumor cells. In contrast, hypoxic wt BMDM supernatants containing abundant Spint1 amounts failed to do so. We propose that Spint1 contributes to the tumor‐suppressive function of HIF‐2α in TAMs in breast tumor development.

of TAMs are often associated with poor prognosis and chemoresistance in breast cancer. 8 Their protumor functions comprise the secretion of growth factors, stimulation of angiogenesis, and immunosuppression, while the secretion of proteolytic enzymes promotes tumor cell invasion and migration, and thus, supports metastasis. Several of these TAM-characteristic features are facilitated by hypoxia-inducible factors (HIFs). 9 HIFs are heterodimeric transcription factors, sensing and coordinating cellular responses to hypoxia. 10,11 Their α-subunits (1α, 2α, or 3α) bind a common β-subunit (aryl hydrocarbon receptor nuclear translocator, [ARNT]) to induce a set of hypoxia-responsive genes. Both, α and β subunits belong to the PAS family of basic helixloop-helix Per-ARNT-Sim proteins. While the β-subunit is constitutively expressed, the α-subunits are rapidly degraded under normoxic conditions. Specifically, prolyl hydroxylases (PHD) target the αsubunits for ubiquitin-dependent proteasomal degradation by hydroxylation of two conserved proline residues. As this process critically depends on oxygen as a cofactor, PHD enzymes are inactive under hypoxia. Upon stabilization, α-subunits dimerize with the β-subunit, followed by the translocation of the dimer to the nucleus and recognition of hypoxia-responsive elements to induce transcription of specific genes. 12,13 HIF-1α was identified first and found to be ubiquitously expressed in almost all cells. Classical HIF-1 target genes affect energy metabolism and glycolysis. The expression of HIF-2α, also known as endothelial PAS protein 1 (EPAS1), appears tissue-restricted, being highly abundant in endothelial and renal cells.
HIF-2α target genes characterized so far mainly affect angiogenesis and erythropoiesis. 14 Of note, high expression of HIF-2α was also observed in macrophages suggesting a role in myeloid cell function. 15,16 As both α-subunits generate overlapping, but also distinct transcription profiles, their differential activities contribute to shape the versatile macrophage phenotypes specifically. 9,17,18 The importance of myeloid HIF-1α for inflammatory and tumorpromoting properties of TAMs was pointed out by numerous studies.
As myeloid HIF-1α is needed for adenosine triphosphate production, the energy-demanding processes of invasion and motility are severely impaired in TAMs lacking HIF-1α. 19,20 Attenuating proliferation and activity of tumor-infiltrating T cells appeared also to be HIF-1α-dependent, in part, by controlling the expression of L-arginine consuming enzymes such as inducible nitric oxide synthase (iNOS) and arginase 1 (Arg-1). 21,22 During inflammation HIF-1α is required for a full-blown bactericidal activity of macrophages, regulating the release of granule proteases and antimicrobial peptides. 23,24 In contrast, information on myeloid HIF-2α is limited, especially concerning its role in TAMs. Casazza et al 25 suggested HIF-2α to activate nuclear factor-κB (NF-κB) and to repress neuropilin 1 (Nrp1) to trap TAMs in hypoxic areas inside the tumor. Imtiyaz et al 26 analyzed macrophages during acute and tumor-associated inflammation and identified HIF-2α-dependent proinflammatory cytokine production. They further showed that the loss of myeloid HIF-2α reduced infiltration of TAMs into both murine hepatocellular and colitis-associated colon carcinoma models, attenuating tumor cell proliferation as well as tumor progression. Work from Roda and colleagues explored expression of HIF-2α and secretion of a soluble form of the vascular endothelial growth factor (VEGF) receptor (sVEGFR-1) in TAMs. sVEGFR-1 binds and neutralizes VEGF, which decreased vascularization of melanoma-bearing mice and reduced tumor growth. 27 It is also hypothesized that HIF-2α blocks the mitochondrial reactive oxygen species-Marco signaling axis to suppress the phagocytic activity of resting macrophages. 28 Although the wealth of existing data suggests that both HIF isoforms are important for macrophage function, we still lack a complete picture to understand the overlapping vs distinct roles of each isoform for myeloid biology. To expand our understanding, especially of the role of HIF-2α in macrophages during tumor progression, we compared wildtype (wt) and myeloid HIF-2α knockout mice (HIF-2α LysM−/− ) in an allograft breast cancer model. Herein, we identified the serine protease inhibitor Spint1 (hepatocyte growth factor activator inhibitor type-1 [HAI-1]), as a novel HIF-2α target in macrophages. Furthermore, we observed that HIF-2αdependent expression of Spint1 in TAMs limits tumor growth by suppressing the activation of hepatocyte growth factor (HGF).

| Flow cytometry
For the analysis of macrophage polarization the following antibodies were used: anti-CD80-PE and anti-CD206-FITC.

| RNA sequencing
Tumors from allograft mice were isolated and dissociated as described  resulting candidates were screened for HIF-1α-and HIF-2α-binding sites using a previously published chromatin immunoprecipitation (ChIP)-seq data sets. 17 Binding sites residing within 250 kb of the respective genes were taken as indicator for a potential transcription regulatory-binding site.

| Statistical analysis
Each experiment was performed at least three times and results are expressed as mean ± SEM. Statistical analyses were performed using the Student t test, the Wilcoxon signed-rank test or a two-way analysis of variance (ANOVA).

| HIF-2α-dependent gene expression changes in TAMs
As immune responses did not contribute to enhanced tumor growth in myeloid HIF-2α knockout mice, we determined HIF-2α-dependent differences in gene expression of TAMs. To allow for cell typespecific gene expression profiling, we isolated TAMs from tumors with a size of 1.2 cm by FACS using the gating strategy depicted in Spint1 expression increased in tumors growing in wt mice, comparing early vs late tumor growth. In contrast, Spint1 levels remained unaltered in tumors of myeloid HIF-2α knockout mice ( Figure 5C).
Interestingly, while Spint1 protein also increased in tumor lysates of wt mice during tumor progression, it was markedly reduced at both TP in tumors of HIF-2α LysM−/− as compared with wt mice ( Figure 5D).
Our experiments therefore establish Spint1 as a novel hypoxiaresponsive HIF-2α target in macrophages.

| HIF-2α-dependent expression of Spint1 is tumor suppressive
Spint1 is characterized as an inhibitor of the serine protease HGF activator (HGFA), which is critically required for activation of HGF, which enhances tumor cell proliferation. 36  BMDMs, while Spint1-containing supernatants of wt BMDMs did not enhance proliferation, even when pro-HGF was added ( Figure 6C and 6D). To gain further evidence for the importance of the HGFactivating function of HIF-2α BMDM supernatants, we next aimed to overcome the proliferative properties of pro-HGF supplemented HIF-2α BMDM supernatants by adding either recombinant Spint1 or an inhibitor for the HGF receptor c-Met (PF-04217903). Indeed, both Spint1 and PF-04217903 reduced CCND1 and Ki67 expression to control levels ( Figure 6E and 6F). In summary, our data suggest that

| DISCUSSION
In the present study, we provide evidence for a tumor-suppressive function of HIF-2α in TAMs. We observed that HIF-2α is strongly properties of HIF-2α. 43 Whereas our data also support a tumorsuppressive role of myeloid HIF-2α, it neither appeared to depend on differences in tumor vascularization nor on changes in immune cell infiltration, as previously suggested for hepatocellular and colitisassociated colon carcinoma mouse models. 26 Furthermore, while myeloid HIF-2α was previously shown to be essential for an alternative macrophage phenotype in thioglycollate-elicited peritoneal macrophages, 44 macrophage polarization was not altered in HIF-2α knockout macrophages (BMDMs and TAMs) in our study. In contrast, our data suggest that the newly identified HIF-2α target Spint1 contributes to reduced tumor cell proliferation. Spint1 is well characterized to inhibit HGF-dependent cell proliferation and consequently attenuates tumor growth. 35,45 Specifically, Spint1 inhibits HGFA, which is required in the extracellular space to activate pro-HGF. Activated HGF exerts its effects on target cells by binding to and activating its receptor c-Met. 46 Activation of c-Met, in turn, enhances proliferation, cell survival, organ morphogenesis, neovascularization, tissue repair, and regeneration. 47  Interestingly, while we found many genes to be differentially expressed between wt and HIF-2α knockout TAMs, only roughly a third showed reduced expression in HIF-2α-deficient macrophages, indicative for a potential direct transcriptional regulation. Moreover, only a small fraction of these emerged as HIF-2-exclusive candidates.
Thus, it will be interesting to see, if the differentially expressed genes lacking a previously identified HIF-binding site might share characteristics that allow for parallel HIF-2α-dependent regulation.
In summary, our study supports a novel tumor-suppressive function of HIF-2α in TAMs in breast tumors. In detail, HIF-2αdependent regulation of the HGF-activation inhibitor Spint1 limited tumor cell proliferation, thereby reducing tumor growth.
Our findings not only provide new insights into the role of HIF-2α in tumor development but also add another facet to the impact of