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Nov 04, 2024

Atypical chemokine receptor 2 expression is directly regulated by hypoxia inducible factor-1 alpha in cancer cells under hypoxia | Scientific Reports

Scientific Reports volume 14, Article number: 26589 (2024) Cite this article Metrics details Lack of significant and durable clinical benefit from anti-cancer immunotherapies is partly due to the

Scientific Reports volume 14, Article number: 26589 (2024) Cite this article

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Lack of significant and durable clinical benefit from anti-cancer immunotherapies is partly due to the failure of cytotoxic immune cells to infiltrate the tumor microenvironment. Immune infiltration is predominantly dependent on the chemokine network, which is regulated in part by chemokine and atypical chemokine receptors. We investigated the impact of hypoxia in the regulation of Atypical Chemokine Receptor 2 (ACKR2), which subsequently regulates major pro-inflammatory chemokines reported to drive cytotoxic immune cells into the tumor microenvironment. Our in silico analysis showed that both murine and human ACKR2 promoters contain hypoxia response element (HRE) motifs. Murine and human colorectal, melanoma, and breast cancer cells overexpressed ACKR2 under hypoxic conditions in a HIF-1α dependent manner; as such overexpression was abrogated in melanoma cells expressing non-functional deleted HIF-1α. We also showed that decreased expression of ACKR2 in HIF-1α-deleted cells under hypoxia was associated with increased CCL5 levels. Chromatin immunoprecipitation data confirmed that ACKR2 is directly regulated by HIF-1α at its promoter in B16-F10 melanoma cells. This study provides new key elements on how hypoxia can impair immune infiltration in the tumor microenvironment.

Hypoxia is a well-known hallmark of solid tumors. Healthy tissues average about 5% oxygen, while hypoxic tumor tissues can exhibit a wide range of oxygen levels, which are often below 2%1. The response to hypoxic tumor microenvironment (TME) is mediated by the hypoxia-inducible factors (HIF), in particular HIF-1 alpha (HIF-1α), which promote an immunosuppressive TME and tumor immune escape2. In normoxia, HIF-1α is hydroxylated and bound to the von Hippel-Lindau (VHL) protein, which recruits an ubiquitine ligase that targets HIF-1α for degradation by the proteasome3. In hypoxia, HIF-1α is no longer hydroxylated and no longer degraded; instead, stabilized HIF-1α is translocated into the nucleus, where it can form a heterodimer with HIF-1β/Aryl hydrocarbon Receptor Nuclear Translocator4. The heterodimer HIF-1α/HIF-1β can bind on hypoxia response elements (HRE) defined by the binding site 5’-RCGTG-3’, where R can be A or G, and activate the transcription of target genes involved in cancer progression5.

Hypoxia also drives resistance to cancer therapies, such as chemotherapy6, radiotherapy7and immunotherapy8. Regarding the latter, it has been shown that hypoxic conditions create an immunosuppressive environment by multiple mechanisms; one of them is by decreasing the ratio of anti-tumor effector immune cells such as dendritic cells, CD4 + and CD8 + T cells, and Natural Killer (NK) cells9, relative to immunosuppressive cells such as myeloid-derived suppressor cells, tumor-associated macrophages with a pro-tumoral M2 phenotype and T-regulatory cells10. Trafficking of these critical immune cells is also mediated by chemokines and their corresponding chemokine receptors11. Studies have shown that hypoxia can impair immune cells trafficking and promote cancer progression by regulating chemokines and chemokines receptors. Indeed, in several cancer types, HIF-1 and HIF-2-mediated hypoxia can directly regulate the CC chemokines CCL2, CCL3, CCL5, CCL15, CCL16, CCL20 and CCL28, the CXC chemokines CXCL1, CXCL2, CXCL4, CXCL5, CXCL6, CXCL10, CXCL12, CXCL13, CXCL14 and CXCL16 ; as well as the chemokines receptors CCR1, CCR2, CCR5, CCR6, CCR7, CXCR1, CXCR2, CXCR4, CXCR5 and CXCR6, due to the presence of HRE on their promoters12,13. Hypoxia can also indirectly regulate the chemokines CCL7, CCL8, CCL11, CCL18, CXCL3, CXCL7, CXCL8 and CXCL11, and the chemokine receptors CXCR3 and CXCR7 through the regulation of other factors, such as zinc finger E-box-binding homeobox 1 (ZEB1), oncostatin M expression, activating protein-1 (AP-1) and NF-κB12,13.

An emerging class of chemokine receptors are Atypical Chemokine Receptors (ACKRs). Unlike the classical chemokine receptors, they present a modification on their second intracellular loop that prevents any G protein binding and classical GPCR signaling14. Instead, they regulate chemokine network through internalization and degradation or redirecting of their ligands15. The ACKRs family contains 4 members : Atypical Chemokine Receptors 1, 2, 3 and 4. More recently, additional atypical chemokine receptors have been considered : GPR182, CCRL2, GPR1, PITPNM3 and C5aR2, which posses similar features to ACKRs16. While the regulation of ACKRs and additional ACKRs is still under investigation, studies have shown that some of them could be regulated by hypoxia, such as ACKR3 (also called CXCR7)17,18and GPR18219. In our study, we focused on ACKR2, which can scavenge pro-inflammatory chemokines such as CCL2, CCL3, CCL4, CCL5, CCL7, CCL8, CCL11, CCL13, CCL14, CCL17, CCL22, CXCL2, CCL26 and CXCL10, thus regulating the infiltration of effector immune cells20,21. Studies have shown that ACKR2 expression on myeloid progenitor cells, mast cells, megakaryocytes, and dendritic cells is dependent on the transcription factor GATA122, and that ACKR2 is regulated by the miRNA miR-146b in psoriasis and thyroid cancer23,24and by the KRAS/BRAF/ERK pathway in Kaposi’s sarcoma25; however, there is no evidence of an hypoxia-mediated regulation of ACKR2. Based on evidence showing that hypoxia can regulate classical chemokine receptors as well as some ACKRs17,18,19, we hypothesized that hypoxia via HIF1-α could regulate ACKR2 expression.

In this study, we evaluated ACKR2 expression in hypoxia in various mouse and human cancer cell types: melanoma, triple-negative breast cancer and colorectal cancer. Our results revealed that ACKR2 expression was significantly increased in all cell types in hypoxia compared to normoxia, at both transcriptional and protein levels. In melanoma B16-F10 cells previously reported to express deleted HIF-1α26, ACKR2 expression was decreased and associated with increased CCL5 levels, providing further evidence that ACKR2 could be regulated by HIF-1α. Chromatin immunoprecipitation results confirmed that HIF-1α binds to at least two HRE motifs within ACKR2promoter in B16-F10 melanoma cells. In addition, we evaluated ACKR2 expression in B16-F10 tumors previously reported to express a transcriptionally inactive truncated form of HIF-1α26. In these tumors, ACKR2 expression was also significantly decreased and associated with an increase of CCL5 levels and immune infiltration.

B16-F10, CT26.WT, 4T1, A-375, MDA-MB-468 and Caco-2 cell lines were purchased from the American Type Culture Collection and cultured in Dulbecco’s Modified Eagle Medium or Roswell Park Memorial Institute 1640 Medium with 10% FBS and 1% Penicillin-Streptomycin, at 37 °C et 5% CO2. B16-F10 cells deleted for HIF-1α were generated by CRISPR-Cas9 as previously described26. Hypoxic conditions were performed in Whitley H35 Hypoxystation from Don Whitley Scientific at 0.1% O2, 5% CO2 and 80% relative humidity for 48 h.

Total RNA was extracted with a Nucleospin RNA Plus Kit (Macherey-Nagel) and reverse-transcribed with a Maxima First-Strand cDNA Synthesis Kit (Thermofisher Scientific). Synthetized cDNA was amplified by qPCR using SYBR Green PCR Master Mix (Eurogentec). Normalization was made with 18S (forward 5’-GAT-GGG-CGG-CGG-AAA-ATA-G-3’ and reverse 5’-GCG-TGG-ATT-CTG-CAT-AAT-GGT-3’) or Rpl13 (forward 5’-TGT-TGA-TGC-CTT-CAC-AGC-GT-3’ and reverse 5’-GGA-GGG-GCA-GGT-TCT-GGT-AT-3’). Other primers were purchased from Qiagen.

After collecting the supernatant, cells cultured in normoxia (21% O2) or hypoxia (0,1% O2) were washed with PBS and detached using 10 mM EDTA for 10 min at 37 °C, before collection with PBS and centrifugation at 400x g for 5 min, at 4 °C. Next, cells were incubated with LIVE/DEAD™ Fixable Near-IR Dead Cell Stain Kit (Invitrogen, L34976) for 20 min at 4 °C. After two washes in MACS buffer (Miltenyi, 130-091-221), cells were fixed and permeabilized using the Cyto-Fast™ Fix/Perm Buffer Set (Biolegend, 426803) according to the manufacturer’s instructions. After permeabilization, cells were incubated with anti-ACKR2 primary antibody (Invitrogen, CCRD6 Goat anti-Mouse PA121612 or CCRD6 Goat Polyclonal Antibody PA121614) and then the secondary antibody (Abcam, Donkey Anti-Goat IgG H&L, Alexa Fluor® 488, ab150129), both for 1 h at room temperature. Cells were washed twice in Cyto-Fast Perm Wash 1X in between antibodies and after staining. Samples were processed using the Beckman Coulter CytoFLEX Model A00-1-1102 and data were analyzed with the CytExpert software (Beckman Coulter) https://www.beckman.pt/flow-cytometry/research-flow-cytometers/cytoflex/software.

Cells were lysed using RIPA Lysis Buffer (Merck Millipore, 20–188) supplemented with 1x protease and phosphatase inhibitors (Thermo Scientific™, 78444). Collected samples were then vortexed and centrifuged at maximum power for 10 min at 4 °C. Supernatant was collected for protein assay with Bradford blue (Bio-Rad, 5000006) with the measure of optical density at 595 nm. The volume required to obtain 25 µg of protein was calculated and mixed with water and 4X Laemmli buffer, before boiling at 95 °C for 5 min. Samples were then placed on a 8% SDS-PAGE gel, alongside a molecular weight marker (Proteintech, PL00001). After migration, the gel was transferred onto a nitrocellulose membrane (Amersham™, GE10600004). Once transfer was complete, the membrane was blocked in 5% milk and then incubated in primary antibody for one hour at room temperature or overnight at 4 °C. After washing, the membrane was incubated again for one hour at room temperature with the secondary antibody. Antibodies used for protein detection by Western blot were anti-HIF-1α (Cell Signaling, HIF-1α (D2U3T) Rabbit mAb, 14179), anti-rabbit (Cell Signaling, Anti-rabbit IgG, HRP-linked Antibody, 7074) and anti-Actin (Sigma-Aldrich-Merck, Anti-β-Actin-Peroxidase antibody, Mouse monoclonal, reference: A3854). For revelation, chemiluminescent substrate was used (PerkinElmer, NEL113001EA). The membrane was revealed either by camera (ImageQuantLAS4000, GE Gealthcare Life Sciences) or using autoradiographic films (Cytiva, 28906843).

ELISAs were performed using DuoSet ELISA Ancillary Reagent Kit 2 (R&D Systems, DY008B) and Mouse CCL5/RANTES DuoSet ELISA (R&D Systems, DY478) following the protocol provided by the supplier.

ACKR2 and CCL5 staining were performed by HistoWiz Company (NY, USA) on tumors previously generated in26. Briefly, HIFΔ and CTRL B16-F10 cells were injected subcutaneously in seven-week-old C57BL/6 (Janvier Labs) mice. Mice were handled according to European Union guidelines and experimentation protocols were approved by the LIH ethical committee, Animal Welfare Society Luxembourg (agreements n. LECR-2018-12). The animal experiment described above has been conducted according the ARRIVE guidelines 2.0 (https://arriveguidelines.org). At the end of the experiment, the mice were euthanized by cervical dislocation after being anesthetized with isoflurane (2.5%). Quantification of ACKR2 and CCL5 staining was done using Aperio ImageScope software (https://www.leicabiosystems.com/digital-pathology/manage/aperio-imagescope/).

Chromatin immunoprecipitation was performed using the iDeal ChIP-qPCR Kit (Diagenode, C01010180) following the manufacturer’s instructions. Samples were sonicated with a M220 Focused-ultrasonicator (Covaris, 500295) and chromatin fragments size were analyzed on a 1.5% agarose gel. For immunoprecipitation, anti-IgG or anti-HIF-1α antibody (Novus Biologicals, NB100-479) were used. Primers for qPCR were designed using Primer-BLAST (NCBI). For the positive control, primers were designed specifically to amplify HRE motifs of Ca9 and Vefga. For the negative control, primers were designed specifically to amplify Ackr2 sequences without HRE. To identify the binding of HIF-1α on Ackr2 promoter, primers have been created specifically to amplify HRE motifs present in the promoter. At least two primer pairs were tested for each gene (primers sequences are provided in Supplementary Table 1). For each primer pair, INPUT was analyzed alongside the IgG negative control and immunoprecipitated samples. The cycle threshold values of the qPCR exponential phase were recorded for each sample and each INPUT, and the relative amount of immunoprecipitated DNA compared with INPUT DNA was calculated.

All statistical analysis were performed using unpaired two-tailed Student’s t-test or one-way ANOVA in GraphPad Prism version 10. All data were tested for normal distribution using a Shapiro-Wilk test on the GraphPad Prism software (https://www.graphpad.com/features) prior to testing via t-test.

Based on evidence showing that hypoxia regulates other atypical chemokine receptors and in silico data generated by JASPAR (https://jaspar.elixir.no/) and MCAST (https://meme-suite.org/meme/tools/mcast), which indicate that ACKR2 contains HRE motifs on its mouse and human promoters (Supplementary Fig. 1), we evaluated ACKR2 expression in various cancer cell types under normoxia and hypoxia.The studied cell lines included mouse melanoma B16-F10 (Fig. 1A), human melanoma A-375 (Fig. 1B), mouse colorectal CT26 (Fig. 1C), human colorectal Caco-2 (Fig. 1D), mouse triple-negative breast cancer 4T1 (Fig. 1E) and human triple-negative breast cancer MDA-MB-468 (Fig. 1F). For flow cytometry analysis, cells were permeabilized to determine intracellular staining, as no difference was observed at the cell surface (data not shown). This is consistent with the fact that ACKR2 is constitutively expressed at less than 5% on the cell surface, with the majority of its expression is found in internal endosomes, where the variable factor is the recycling rate27. The Vascular Endothelial Growth Factor A (Vegfa) and Carbonic anhydrase 9 (Ca9)are known downstream target genes of HIF-1α28,29, and their mRNA levels were determined by RT-qPCR in each experiments to verify hypoxic conditions. For each cell line, ACKR2 expression was increased at both transcriptional and protein levels in hypoxic conditions compared to normoxia, providing strong evidence that hypoxia upregulates ACKR2 expression (Fig. 1A-F).

ACKR2 protein and mRNA levels are upregulated in hypoxia in B16-F10, A-375, CT26, Caco-2, 4T1 and MDA-MB-468 cells. ACKR2 protein expression analysis by flow cytometry and RT-qPCR quantification of Ackr2 , Ca9 , and Vegfa mRNA levels in (A) B16-F10, (B) A-375, (C) CT26, (D) Caco-2, (E) 4T1 and (F) MDA-MB-468 cells cultured in normoxia (21% O 2 - N) or in hypoxia (0.1% O 2 - H) for 48 h. Ca9 and Vegfa gene expressions were analyzed to confirm the induction of hypoxic conditions. Results are reported as percentages of positive live cells or as fold change (FC) and represent the average of three independent experiments. Error bars indicate mean ± SEM. * = p < 0.05, ** = p < 0.01, *** = p < 0.001 and **** = p < 0.0001, determined by unpaired two-tailed Student’s t test.

To determine whether the upregulation of ACKR2 observed in hypoxia is mediated by HIF-1α transcription factor, we used B16-F10 melanoma cells deleted for HIF-1α (designated as B16-F10 HIFΔ) as previously described26. These clones were generated using CRISPR/Cas9 technology to cleave specifically the HIF-1α/ARNT dimerization domain. DNA sequencing confirmed a deletion of a 114 amino acid fragment, containing a substantial part of the HIF-1α heterodimerization domain (10 out 22 amino acids). We first validated our results by western-blot, confirming the accumulation of HIF-1α protein under hypoxic conditions, and with a truncated form of HIF-1α in B16-F10 HIFΔ cells (Fig. 2A). We next assessed ACKR2 protein expression by flow cytometry (Fig. 2B) and mRNA levels by RT-qPCR (Fig. 2C). Vegfa and Ca9 mRNA levels were verified by RT-qPCR for each experiments to confirm hypoxic conditions and to ensure that the truncated form of HIF-1α was no longer transcriptionnally active (Fig. 2C). Our data demonstrated a significant increase in ACKR2 protein in B16-F10 CTRL cells in hypoxia compared to normoxia, as observed previously with B16-F10 wt cells; however, for B16-F10 HIFΔ cells, there is still an increase of ACKR2 protein in hypoxia, but significantly less compared to hypoxic B16-F10 CTRL (Fig. 2B). This slight increase in hypoxic HIFΔ cells could be due to the regulation by other hypoxia-induced factor, e.g. HIF-2α. Notably, this increase was not observed at the mRNA level of Ackr2 under the same conditions (Fig. 2C), indicating a potential post-translational mechanism (e.g. phosphorylation, glycosylation, acetylation, or stimulation by ligand binding.). Regarding mRNA levels, Vegfa and Ca9 expressions were no longer induced in hypoxic B16-F10 HIFΔ cells compared to CTRL cells, confirming that the deletion of HIF-1α efficiently inhibits its transcription activity. The same pattern was observed for Ackr2 mRNA level, where the increased expression in hypoxic B16-F10 CTRL cells was absent in B16-F10 HIFΔ cells (Fig. 2C). In addition, increased ACKR2 expression was associated with a reduced levels of CCL5 under hypoxic conditions compared to normoxic conditions in B16-F10 CTRL cells, as confirmed by ELISA (Fig. 2D). In B16-F10 HIFΔ cells, there was a similar trend of CCL5 reduction under hypoxia compared to normoxia, although this change was not statistically significant (Fig. 2D). ACKR2 is known to scavenge the pro-inflammatory chemokine CCL530, but it is important to highlighted that in hypoxia, CCL5 is also regulated by HIF-2 and NF-κB12. CCL5 participates in the recruitment of immune cells such as macrophages, basophils, eosinophils and T cells, and is known to promote cancer cells proliferation and metastasis formation31. This result suggests the functional impact of ACKR2 downregulation in B16-F10 HIFΔ cells on CCL5, which could potentially lead to an increase of immune infiltration mediated by CCL5 or other chemokines scavenged by ACKR2.

It is essential to evaluate the functional impact of hypoxia-driven upregulation of ACKR2 on other chemokines known to be scavenged by this receptor. Notably, we have previously reported that, unlike CCL5, the release levels of CCL2 by HIFΔ-expressing cells remain unaffected26. This discrepancy may be explained by the higher affinity of CCL5 for ACKR2 compared to CCL2.

Inhibition of HIF-1α trancription activity impairs the hypoxia-induced ACKR2 expression and increases CCL5 levels in B16-F10 melanoma cells. (A) Western-blot analysis of HIF-1α protein expression in B16-F10 CTRL and HIFΔ cells cultured in normoxia (21% O2 - N) or hypoxia (0.1% O2 - H) for 48 h. The full-length blot is available in Supplementary Fig. 2. Actin was used as a loading control. (B) ACKR2 protein expression analysis by flow cytometry in permeabilized B16-F10 CTRL and HIFΔ cells cultured in normoxia (21% O2 - N) or hypoxia (0.1% O2 - H) for 48 h. Results are reported as the percentage of positive live cells and represent the average of three independent experiments. Error bars indicate mean ± SEM. * = p < 0.05, ** = p < 0.01, and *** = p < 0.001, determined by one-way ANOVA test. (C) RT-qPCR quantification of Ackr2 , Ca9 , and Vegfa mRNA levels in cells described in (B). Ca9 and Vegfa gene expressions were analyzed to confirm the induction of hypoxic conditions. Results are reported as fold change (FC) and represent the average of three independent experiments. Error bars indicate mean ± SEM. ns = not significant, * = p < 0.05, and ** = p < 0.01, determined by unpaired two-tailed Student’s t test. (D) ELISA quantification of CCL5 protein levels in the supernatant of cells described in (B) and (C). Results are reported as pg/ml and represent the average of three independent experiments. Error bars indicate mean ± SEM. * = p < 0.05 and *** = p < 0.001, determined by one-way ANOVA test.

Based on our results showing that the transcription factor HIF-1α is involved in the regulation of ACKR2, our goal was to determine whether the regulation is direct or mediated by other factors. To investigate this, we performed chromatin immunoprecipitation followed by qPCR (ChIP-qPCR) using an anti-HIF-1α antibody and primers designed to amplify HRE motifs within Ackr2 promoter (Supplementary Table 1). In B16-F10 CTRL, we observed a significant increase in the signal for two HRE of Ackr2 promoter, comparable to the signal for Vegfa HRE, which is a known downstream target gene of HIF-1α29, this signal was absent in B16-F10 HIFΔ cells and when using primers amplifying an Ackr2 sequence lacking HRE motifs (Ackr2-NegCTRL), confirming the specificity of the signal (Fig. 3A). We conclude that the transcription factor HIF-1α binds to both HRE motifs within the Ackr2 promoter (Fig. 3B). These results are consistent with our previous data showing an increased ACKR2 expression in hypoxic conditions, and a decreased ACKR2 expression in hypoxic cells deleted for HIF-1α.

HIF-1α binds to HRE motifs of Ackr2 promoter in B16-F10 cells. (A) Chromatin immunoprecipitation experiment performed using an anti-HIF-1α antibody on B16-F10 CTRL and HIFΔ cells cultured under hypoxia (0.1% O 2) for 48 h, followed by a qPCR using primers that amplify HRE motifs in the Vegfa and Ackr2 promoters. A sequence in Ackr2 lacking HRE motifs served as a negative control (Ackr2-NegCTRL), while Vegfa -HRE was used as a positive control. For each HRE motif in the Ackr2 promoter, two different primer pairs were used (Ackr2 HRE 1.1, 1.2, 2.1 and 2.2). For each gene, the signals obtained by qPCR were normalized to the IgG condition. (B) Combination of three independent chromatin immunoprecipitation experiments performed as described in (A). For each gene, qPCR signals were normalized to the IgG condition. Statistically significant differences between B16-F10 CTRL and HIFΔ cells are shown. Results are reported as fold enrichment with error bars representing the mean ± SEM of three independent experiments. ns = not significant, and * = p-value < 0.05, determined by an unpaired Student’s t-test.

To determine whether ACKR2 regulation by hypoxia is also be observed in B16-F10 tumors, we evaluated ACKR2 expression in HIF-1α-deleted B16-F10 tumors previously generated in26. Inhibition of HIF-1α transcription activity was confirmed by a significant decrease of Vegfa and Ca9 mRNA levels in B16-F10 HIFΔ tumors. In line with our previous results, Ackr2 mRNA levels were significantly decreased, while Ccl5 mRNA levels were significantly increased in B16-F10 HIFΔ tumors compared to CTRL tumors (Fig. 4A). We next assessed protein levels with an immunohistochemistry to assess ACKR2, and CCL5 proteins. A reduction in ACKR2 staining was observed in B16-F10 HIFΔ tumors relative to B16-F10 CTRL tumors, confirming that the expression of ACKR2 is not only decreased at the transcriptional level, but also at the protein level (Fig. 4B). Quantification of ACKR2 staining across three different tumors of each group confirmed that ACKR2 protein is significantly decreased in B16-F10 HIFΔ tumors compared to control tumors (Fig. 4C). In addition, a marked increase in CCL5 staining was seen in B16-F10 HIFΔ tumors compared to control (Fig. 4B and C), which is in line with the role of ACKR2 in scavenging CCL5 for degradation via the endosomal pathway30. This observation correlated with our previous data, where HIF-1α deletion in tumors resulted in a significant increase in the infiltration of live NK, CD4 + and CD8 + T cells in HIF-1α-deleted tumors compared to control tumors26. Overall, the increased immune infiltration is consistent with the role of ACKR2 in regulating the chemokine network, a key regulator of immune infiltration20.

ACKR2 expression is decreased in B16-F10 HIFΔ tumors and associated with increased CCL5 levels. (A) RT-qPCR analysis of Ackr2, Ccl5, Vegfa and Ca9 gene expression in B16-F10 CTRL and HIFΔ tumors. Results are reported as fold change (FC) with error bars representing the mean ± SEM of 3 tumors per group. * = p-value < 0.05 and **** = p-value < 0.0001, determined by unpaired Student’s t-test. (B) Immunohistochemistry images of ACKR2 and CCL5 staining (in red), in B16-F10 CTRL and HIFΔ tumors used in (A). Scale bars: 200 μm. (C) Quantification of ACKR2 and CCL5 staining by immunohistochemistry on B16-F10 CTRL and HIFΔ tumors described in (A) and (B). Results are reported as percentage of positively stained cells relative to total cell count. For quantification, three random areas of three tumors per group were analyzed and averaged. Error bars represent the mean ± SEM of 3 tumors per group. * = p-value < 0.05 determined by unpaired Student’s t-test.

Our study showed that hypoxia upregulates ACKR2 expression at both transcriptional and protein levels in melanoma, triple negative breast cancer and colorectal cancer murine and human cell lines. In B16-F10 cells with functional inhibition of HIF-1α, ACKR2 expression was decreased and associated with an increase in CCL5 protein levels, providing further evidence that ACKR2 may be regulated by HIF-1α and influences CCL5 levels. Our chromatin immunoprecipitation results confirmed that HIF-1α binds directly to at least two HRE motifs within the Ackr2 promoter in B16-F10 melanoma cells, in the same manner as with HRE within Vegfa promoter. This regulation was also observed in vivo, where ACKR2 expression was also significantly decreased in B16-F10 HIFΔ tumors compared to control tumors, and associated with increased CCL5 levels. Overall, our results identify HIF-1α as a novel direct regulator of ACKR2 in cancer, revealing a new mechanism by which HIF1-α-mediated hypoxia could impair immune infiltration within the TME and promote cancer progression.

In Kaposi’s syndrome, ACKR2 expression isdecreased by the K-ras/B-raf/ERK pathway, leading to an accumulation of tumor-associated macrophages TAMs25. A similar observation was made in thyroid cancer, where ACKR2 is targeted by microRNA miR-146a to support leukocyte trafficking in the TME23. In uveal melanoma and hepatocellular carcinoma, TCGA database analyses have associated ACKR2 expression with favorable progression-free survival and overall survival32. These findings suggest that in these cancers, regulation of ACKR2 plays a protective and anti-tumoral role.

In other studies, genetic targeting of Ackr2 lead to an increase in inflammatory chemokine receptors and was associated with a release of neutrophils with increased anti-metastatic activity in mice orthotopically transplanted with 4T1 mammary carcinoma, and intravenously injected with B16-F10 melanoma cells33. In another mouse model, only ACKR2, but not the other ACKRs, was significantly increased in the cancerous lesions of mice treated with the carcinogen 4-nitroquinoline-1-oxide. Furthermore, inhibition of ACKR2 in these mice induced an increase of CCL2, IL-6 and IL-17, suggesting that ACKR2 regulates inflammation in the TME34, which corroborates with the increased CCL5 levels we observe when HIF-1α is not transcriptionally active in our model. ACKR2 has also been involved in epithelial-mesenchymal transition (EMT) and the metastatic process viaCXCL1435. In in vitro and in vivo models of breast cancer, the CXCL14/ACKR2 pathway was able to stimulate EMT, tumor invasion and the metastatic process36. A similar role was observed in non-small cell lung cancer, in which CXCL14 and ACKR2 expressions are increased according to TCGA databases. In vitro and in vivo experiments showed that by inhibiting ACKR2, CXCL14-induced tumor cell migration was also inhibited. Furthermore, results established that ACKR2 had a role in the regulation of phospholipase Cβ3 by CXCL14, as well as the regulation of c-Src and consequently, NFқB, thus leading to the induction of cell migration and EMT37. Furthermore, ACKR2 and CCR2 have been identified as potential targets for modulating the metastatic process. In this study, ACKR2 limits CCR2 expression in NK cells, preventing their cytotoxic activity in B16-F10 melanoma tumors38. This finding could in part explain the increased cytotoxic activity in our HIFΔ tumors compared to control tumors26. Recently, a subpopulation of cervical cancer cells overexpressing ACKR2 in response to concurrent radiochemotherapy. It has been reported that the increased expression of ACKR2 by these tumor cells induced the production of transforming growth factor β to drive CD8 + T cell senescence, thereby compromising antitumor immunity39. Overall, while ACKR2 function and role may vary by tumor type and context, this highlights the importance of ACKR2 regulation in cancer and the translational potential of our results.

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

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This study was supported by the Luxembourg Institute of Health and grants from the Action LIONS Vaincre Le Cancer Luxembourg (2020) and FNRS Televie (7.4559.21 F).

Tumor Immunotherapy and Microenvironment (TIME) Group, Department of Cancer Research, Luxembourg Institute of Health (LIH), Luxembourg, L- 1210, Luxembourg

Alice Benoit, Audrey Lequeux, Phillip Harter, Guy Berchem & Bassam Janji

Department of Hemato-Oncology, Centre Hospitalier du Luxembourg, Luxembourg, L- 1210, Luxembourg

Guy Berchem

Department of Life Sciences and Medicine (DLSM), University of Luxembourg, Esch-sur-Alzette, Luxembourg

Guy Berchem

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A.B. designed and performed experiments, analyzed the data and wrote the manuscript. A.L. established cellular models. P.H. performed experiments. B.J. designed experiments and wrote the manuscript. All authors have read and agreed to the published version of the manuscript.

Correspondence to Bassam Janji.

The authors declare no competing interests.

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Supplementary Material 1: Fig. S1. MCAST and JASPAR analysis of ACKR2 mouse and human promoters. Fig. S2. Full-length blot corresponding to Figure 2A. Table S1. List of primers used for ChIP-qPCR results.

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Benoit, A., Lequeux, A., Harter, P. et al. Atypical chemokine receptor 2 expression is directly regulated by hypoxia inducible factor-1 alpha in cancer cells under hypoxia. Sci Rep 14, 26589 (2024). https://doi.org/10.1038/s41598-024-77628-8

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Received: 23 May 2024

Accepted: 23 October 2024

Published: 04 November 2024

DOI: https://doi.org/10.1038/s41598-024-77628-8

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