Insulin-Like Growth Factor 2 mRNA-Binding Protein 3 Modulates Aggressiveness of Ewing Sarcoma by Regulating the CD164-CXCR4 Axis

Ewing sarcoma (EWS) is the second most common bone and soft tissue-associated malignancy in children and young adults. It is driven by the fusion oncogene EWS/FLI1 and characterized by rapid growth and early metastasis. We have previously discovered that the mRNA binding protein IGF2BP3 constitutes an important biomarker for EWS as high expression of IGF2BP3 in primary tumors predicts poor prognosis of EWS patients. We additionally demonstrated that IGF2BP3 enhances anchorage-independent growth and migration of EWS cells suggesting that IGF2BP3 might work as molecular driver and predictor of EWS progression. The aim of this study was to further define the role of IGF2BP3 in EWS progression. We demonstrated that high IGF2BP3 mRNA expression levels correlated with EWS metastasis and disease progression in well-characterized EWS tumor specimens. EWS tumors with high IGF2BP3 levels were characterized by a specific gene signature enriched in chemokine-mediated signaling pathways. We also discovered that IGF2BP3 regulated the expression of CXCR4 through CD164. Significantly, CD164 and CXCR4 colocalized at the plasma membrane of EWS cells upon CXCL12 stimulation. We further demonstrated that IGF2BP3, CD164, and CXCR4 expression levels correlated in clinical samples and the IGF2BP3/CD164/CXCR4 signaling pathway promoted motility of EWS cells in response to CXCL12 and under hypoxia conditions. The data presented identified CD164 and CXCR4 as novel IGF2BP3 downstream functional effectors indicating that the IGF2BP3/CD164/CXCR4 oncogenic axis may work as critical modulator of EWS aggressiveness. In addition, IGF2BP3, CD164, and CXCR4 expression levels may constitute a novel biomarker panel predictive of EWS progression.

Ewing sarcoma (EWS) is the second most common bone and soft tissue-associated malignancy in children and young adults. It is driven by the fusion oncogene EWS/FLI1 and characterized by rapid growth and early metastasis. We have previously discovered that the mRNA binding protein IGF2BP3 constitutes an important biomarker for EWS as high expression of IGF2BP3 in primary tumors predicts poor prognosis of EWS patients. We additionally demonstrated that IGF2BP3 enhances anchorage-independent growth and migration of EWS cells suggesting that IGF2BP3 might work as molecular driver and predictor of EWS progression. The aim of this study was to further define the role of IGF2BP3 in EWS progression. We demonstrated that high IGF2BP3 mRNA expression levels correlated with EWS metastasis and disease progression in wellcharacterized EWS tumor specimens. EWS tumors with high IGF2BP3 levels were characterized by a specific gene signature enriched in chemokine-mediated signaling pathways. We also discovered that IGF2BP3 regulated the expression of CXCR4 through CD164. Significantly, CD164 and CXCR4 colocalized at the plasma membrane of EWS cells upon CXCL12 stimulation. We further demonstrated that IGF2BP3, CD164, and CXCR4 expression levels correlated in clinical samples and the IGF2BP3/CD164/CXCR4 signaling pathway promoted motility of EWS cells in response to CXCL12 and under hypoxia conditions. The data presented identified CD164 and CXCR4 as novel IGF2BP3 downstream functional effectors indicating that the IGF2BP3/CD164/CXCR4 oncogenic axis may work as critical modulator of EWS aggressiveness. In addition, IGF2BP3, CD164, and CXCR4 expression levels may constitute a novel biomarker panel predictive of EWS progression.
Keywords: IGF2BP3, metastases, CD164, CXCR4, Ewing sarcoma INTRODUCTION Ewing sarcoma (EWS) is a rare disease but it is still the second most common malignancy of bone and soft-tissues affecting pediatric age. It is characterized by a very aggressive behavior, high propensity for metastasis, specifically to bone and lung. Metastases occur in 30-40% of patients with localized disease, while 20-25% of patients present metastasis at diagnosis. The current standard treatment of EWS is a multimodal approach consisting of surgery and/or radiotherapy, and a multiagent chemotherapy, which confers a 5-years survival rate of 70% in patients with localized tumor. On the contrary, metastatic disease has a survival rate of 30%, independently of intensification of chemotherapeutic regimens (1). The identification of novel therapeutic strategies and reliable predictors of patient survival is therefore imperative to improve the outcome for metastatic patients.
While the genetic features of EWS are well-defined (2), as well as the contribution of the fusion gene EWS-FLI1 to oncogenesis (3), the molecular mechanisms underlying EWS metastases are still poorly understood (4,5).
EWS is characterized by one of the lowest mutation rates among all tumors (6)(7)(8) and this genomic stability is conserved in metastasis (9). On the contrary, epigenetic heterogeneity is prevalent in EWS, and even increased in the metastatic stage (10)(11)(12).
RBPs, along with microRNAs and long non-coding RNAs, represent major post-transcriptional regulators of gene expression, due to their ability to bind RNA sequences and finely tune nuclear export, translation/degradation rate, and intracellular localization of their multiple transcript targets (19).
We have recently identified insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3) as a major determinant of EWS aggressiveness (20,21). IGF2BP3 has a critical role in modulating multiple mRNAs, thereby regulating tumor initiation and progression (22). Accordingly, IGF2BP3 has recently emerged as putative prognostic biomarker for several tumors, including leukemia, carcinomas, and sarcomas (23).
In this study, we initially discovered that IGF2BP3 is significantly upregulated in metastatic lesions of EWS patients as compared to primary tumors, prompting us to investigate the molecular contribution of this RBP to the migration and dissemination of EWS cells. We then identified for the first time an oncogenic axis consisting of IGF2BP3/CD164 and CXCR4, which confers migratory advantage to EWS cells, particularly under stress-adaptive conditions.

Clinical Specimens
This study included EWS specimens from primary localized tumors and EWS metastatic lesions. EWS diagnosis and treatment were performed at the IRCCS Istituto Ortopedico Rizzoli (Bologna, ITALY). For diagnosis, histological, immunohistochemical, and molecular features were considered (24). For therapy, patients underwent local treatment (surgery and/or radiation therapy) and systemic induction chemotherapy. All the patients included in this study were enrolled in previously approved prospective studies (25,26). For those patients who underwent surgery, histologic response to chemotherapy was examined in accordance to Picci et al. (27). Clinical-pathological features of EWS patients, updated to 2018, are summarized in Table 1.

RNA-seq and Bioinformatics Analyses
RNA extraction, cDNA libraries, sequencing, reads alignment, and normalization were performed as previously described (21). Hierarchical supervised clustering was performed using GeneSpring 11.02 software on differentially expressed genes using Pearson's correlation. Enrichment analysis of differentially expressed genes was performed using MetaCore software (GeneGo, Thomson Reuters).

Motility Assay
Migration capability of EWS cells was established using Trans-well chambers (CoStar, Cambridge, MA, USA). 1 × 10 5 cells diluted in IMDM plus 1% FBS were seeded in the upper compartment, whereas IMDM plus 1% FBS and CXCL12 (100 ng/ml, ab9798, Abcam) were placed in the lower compartment of the chamber. After an overnight incubation, under normoxia or hypoxia, migrated cells were fixed in methanol. Cells were subsequently stained with Giemsa and counted.

Ribo-Immunoprecipitation (RIP) Assay
The RiboCluster Profiler RIP-Assay kit (MBL International, Woburn, MA, USA) was used to identify IGF2BP3/transcript interactions, according to the manufacturers' protocol. For immunoprecipitation, anti-IGF2BP3 antibody (MBL International) or normal IgG (MBL International), used as a negative control, were used. Obtained RNA was reverse transcribed and qRT-PCR on equivalent amounts of cDNA was performed.

Statistical Methods
Differences among means were tested using a one-way ANOVA, if more than two groups were present, or Student's t-test. Spearman's rank test was employed to establish correlation between continuous variables. Spearman's FIGURE 1 | Correlation between IGF2BP3 and metastatic disease in EWS patients. Scatter plot analysis of IGF2BP3 mRNA levels determined via (A,B) qRT-PCR or (C,D) microarray (GSE12102) in primary or metastatic EWS lesions. Differential expression between the groups was determined using (A,C) Student's t-test or (B,D) one-way ANOVA with respect to metastases. Mean ± SD of relative mRNA expression reported as log 2 is shown. Human mesenchymal stem cells were used as calibrator. Number of analyzed cases is reported below each plot. *p < 0.05; **p < 0.01. NS, not significant.

IGF2BP3 Is Associated With Metastasis Formation and With Chemokine Signaling
To initially explore a possible correlation between IGF2BP3 and EWS metastasis, we measured IGF2BP3 mRNA expression levels in 44 metastatic EWS lesions using as controls 48 primary untreated tumors from patients with localized disease at diagnosis ( Table 1). Metastatic specimens displayed significantly higher expression of IGF2BP3 mRNA as evaluated by qPCR ( Figure 1A).
Notably, when the subset of primary tumors was divided according to overall survival of patients (alive vs. dead from disease), we found that the significant difference of IGF2BP3 mRNA expression levels was only maintained when compared to tumors derived from patients with favorable overall outcome (alive; Figure 1B). This observation was confirmed in a different set of tumors previously analyzed by microarray analysis (29 primary tumors vs. 7 metastasis; Table 1) (9,21). We did not detect a significant difference in IGF2BP3 expression levels between primary and metastatic tumors ( Figure 1C). However, IGF2BP3 expression levels were upregulated in metastatic lesions as in primary tumors of patients dead from disease but they were significantly lower in primary tumors of patients who did not experience any recurrence or were alive at 10 years from diagnosis ( Figure 1D).
To further define whether IGF2BP3-regulated mechanisms might have clinical impact, we took advantage of another set of 14 tumors analyzed by RNAseq (21). We compared the genetic expression profile of three primary localized EWS cases with the highest expression of IGF2BP3 to three primary localized EWS cases with the lowest, if any, expression of IGF2BP3 and identified a signature of 814 differentially expressed genes (615 upregulated and 199 downregulated, P < 0.05; one-way ANOVA; Supplementary Table 1). This signature clearly separated the two groups with different IGF2BP3 expression levels when hierarchical supervised clustering was performed (Supplementary Figure 1). Enrichment analysis using GeneGo annotation revealed the specific involvement of immunological and chemokine-mediated signaling pathways ( Table 2).

IGF2BP3 Regulates the Expression of CXCR4 Through CD164
To confirm the functional association between IGF2BP3 and chemokine signaling pathways, we took advantage of IGF2BP3depleted experimental EWS cell models previously generated by shRNA approaches (21) (Supplementary Figure 2). We profiled control-transfected and IGF2BP3-depleted A673 EWS cells for genes encoding chemokine receptors and ligands using the RT 2 Profiler Cancer Inflammation and Immunity Crosstalk PCR Array. Notably, only CXCR4 was significantly downregulated in IGF2BP3-depleted A673 cells compared to controls (Figure 2A). Next, we confirmed by qRT-PCR and western blotting analyses downregulation of CXCR4 at mRNA and protein levels in IGF2BP3-silenced cells (Figure 2B), suggesting that CXCR4 might work as novel downstream effectors of IGF2BP3 action. Data from the literature indicate that IGF2BP3 modulates the expression of CD164 (33, 34), a type 1 integral transmembrane sialomucin involved in the regulation of adhesion and migration of tumor cells (35,36). Significantly, CD164 regulates CXCR4 function in different tumor types (36)(37)(38). Thus, we initially investigated a possible functional interaction between IGF2BP3 and CD164 mRNA by RIP assay. In both A673 and TC-71 EWS cells CD164 was significantly enriched in samples immunoprecipitated with anti-IGF2BP3 antibody as compared to IgG-immunoprecipitated control samples ( Figure 2C). In addition, stable depletion of IGF2BP3 in A673 and TC-71 cells (Supplementary Figure 2) was associated with a significant reduction of CD164 protein expression levels as demonstrated by immunoblot analysis (Figure 2D). Next, we analyzed by qRT-PCR IGF2BP3, CD164, and CXCR4 expression levels in clinical samples. We confirmed statistical association among the three molecules in both the 48 primary localized tumors and 44 metastatic lesions previously described (Figures 3A-F). Because Spearman coefficients (r) still indicated a weak to moderate correlation between IGF2BP3 and CD164 or CXCR4 while a strong correlation between CD164 and CXCR4 (32), we further investigated the IGF2BP3/CD164/CXCR4 association by IHC in an independent cohort of 50 primary tumors ( Table 1). The analyses confirmed a significant association at protein level between CD164 expression with both IGF2BP3 (p = 0.05, Chi-square test) and CXCR4 (p = 0.04, Chi-square test) (Table 3, Supplementary Figure 3).
Taken together these data support a role of IGF2BP3 in regulating the CD164/CXCR4 complex and demonstrate the evidence of an IGF3BP3-CD164-CXCR4 oncogenic axis critical for EWS progression.

The IGF2BP3/CD164/CXCR4 Axis Affects Migration of EWS Cells in Response to CXCL12 and Under Hypoxia Conditions
While the role of CXCR4 in regulating migration of EWS cells has been previously established (13,15), there are no data at the moment supporting the role of CD164 in modulating EWS cancer cells motility. Thus, we used siRNA approaches and transiently depleted CD164 in A673 and TC-71 cells. We obtained a robust CD164 depletion in both cell lines (Figure 4A), which determined a significant inhibition of EWS cell motility in condition of chemotactic stimulus toward a CXCL12 gradient (Figure 4B), supporting the notion that CD164 might act as an adjuvant factor of CXCR4 signaling in EWS cells.
We then investigated by confocal microscopy whether CD164 and CXCR4 might colocalize in A673 cell line. In CXCL12-unstimulated cells, a homogeneous distribution of CD164 and CXCR4 was observed in the cytoplasm and at the plasma membrane (Figures 5A,B). On the contrary, upon CXCL12 stimulation, CD164 and CXCR4 colocalized at the plasma membrane. To confirm that CD164 and CXCR4 indeed interacts at the plasma membrane, we repeated colocalization experiments supplementing CXCL12 with the general endocytosis inhibitor dynasore, a GTPase inhibitor that blocks dynamin activity, thus affecting both clathrin-dependent and -independent endocytic pathways (39). The combination of CXCL12 and dynasore enhanced colocalization of CD164 and CXCR4 (Figures 5A,B), confirming that this interaction likely occurs at the plasma membrane of A673 cells (Figure 5A, white arrows). Collectively these results suggest that CD164 and CXCR4 colocalize at the plasma membrane of A673 cells in CXCL12-dependent fashion.
Because CXCR4 is induced in EWS cells exposed to hypoxia (13), a common condition of human tumor microenvironment (40), we investigated the contribution of the IGF2BP3/CD164/CXCR4 axis on CXCL12-evoked biological responses of EWS cells under normoxic (21% O 2 ) or hypoxic conditions (1% O 2 ). In line with previous evidence (13), EWS cells exposed to hypoxia showed induced expression of CXCR4 and of the hypoxia inducible factor alpha (HIF-1α), used as control ( Figure 6A). Interestingly, IGF2BP3-silenced cells did not show CXCR4 expression, which was not increased under hypoxic conditions ( Figure 6A). From the functional standpoint, the inhibitory effect on cell migration associated with IGF2BP3 depletion was amplified under hypoxic conditions. In fact, A673 cells silenced for IGF2BP3 showed reduced migration in response to CXCL12 either in normoxic or under hypoxia conditions (Figure 6B). Of note, the reduction was more evident in hypoxia condition (p = 0.005, one-way ANOVA), indicating that the impact of reduced expression of the IGF2BP3/CD164/CXCR4 oncogenic pathway may be stronger in the tumor microenvironment compared to physiological conditions.

DISCUSSION
EWS is characterized by a very low somatic mutational load (6)(7)(8) and high levels of inter-and intratumor epigenetic heterogeneity (10)(11)(12). Analysis of DNA methylation has highlighted a large spectrum of alterations, which reflect disease heterogeneity in term of stem cell differentiation and clinical outcome, and preclude the possibility of identifying subset of patients with differential risk of progression (12). Treatment of EWS is still based on high dense chemotherapy, with relevant impact on quality of life of survivors, who may be overtreated, and on outcome of high-risk patients, who should be considered for alternative drug regimens.
We have recently demonstrated that the mRNA binding protein IGF2BP3 constitutes an important biomarker for EWS (20,21) as in fact high expression of IGF2BP3 in primary tumors is associated with poor prognosis of EWS patients (21). In addition, we demonstrated that IGF2BP3 increases anchorage-independent growth and migration of EWS cells  CXCR4 expression levels correlate in clinical samples. (F) The IGF2BP3/CD164/CXCR4 oncogenic axis promotes motility of EWS cells in response to CXCL12 and under hypoxia conditions. Previously published data from our laboratory indicates that IGF2BP3 may exert its oncogenic action in EWS in both IGFsdependent and -independent manner. IGF2BP3 loss promoted IGF1R downregulation and inhibited IGF1-evoked biological responses, thereby reducing cell growth and motility of EWS cells (20). IGF1R loss was associated with a compensatory mechanism driven by activation of the insulin receptor isoform A (IR-A) and its cognate ligand IGF2, which conferred enhanced sensitivity to dual IGF1R/IR inhibitors (20). On the other hand, IGF2BP3 expression is predictive of poor prognosis of EWS and regulate EWS aggressiveness independently of IGF1R action (21). The data presented here support the novel observation that in EWS cells IGF2BP3 might be a critical factor in regulating a specific cytokine pathway consisting of CD164 and CXCR4 signaling.
A role for CXCR4 in EWS has been previously demonstrated (13,15,41). Expression of CXCR4 is highly dynamic in EWS, and can be transiently induced by exposure to microenvironmental stress, like starvation, growth constraint and hypoxia (13). EWS cells characterized by high CXCR4 expression levels show increased invasion and migration capability, partially mediated by the intracellular activation of the Rho-GTPases, Rac1, and Cdc42 (13). Significantly, targeting the CXCL12/CXCR4 axis inhibited the aggressive phenotype, thereby indicating a potential contribution of CXCR4 signaling to EWS metastasis (13). In addition, in the model presented by Krook et al. stress induces the conversion of CXCR4-negative EWS cells to CXCR4positive cells, thereby supporting the role of the CXCL12/CXCR4 signaling pathway in tumor progression (15). This switch is mediated, at least in part, by epigenetic modifications of the CXCR4 promoter, which transitions from an inactive bivalent state to a univalent active state (15).
The adhesion receptor CD164 (endolyn), belonging to the sialomucin family, regulates the adhesion of CD34 + cells to bone marrow stroma, and the recruitment of those cells into cycle (37). CD164 associates with CXCR4 and cooperates with it in promoting CXCL12-mediated cell migration (37). CD164 depletion significantly attenuated the PI3K pathway but it did not alter MAPK activation, suggesting pathway  (B) Z-stacks were analyzed for colocalization by NIS Elements AR4.20.01 software (Nikon). Colocalization index is represented by histograms. Mean ± SE of an average of 30 independent fields is shown. ns, not significant; ***p < 0.01; ****p < 0.0001, one-way ANOVA.
specificity of CD164 action (37). A tumorigenic role of CD164 has been demonstrated in ovarian cancer where CD164 is upregulated in malignant ovarian cancer cell lines (38).
CD164 overexpression in human ovarian epithelial surface cells increased CXCL12/CXCR4 expression, enhanced cellular proliferation, and colony formation, and suppressed apoptosis EWS cells using a CXCL12 (100 ng/ml) gradient under normoxia (21% O2) or hypoxia (1% O2). Mean ± SE of three independent experiments is shown. **p < 0.01; ****p < 0.0001, one-way ANOVA. (38). Clinicopathological correlation analysis additionally indicated that CD164 upregulation was significantly associated with tumor grade and metastasis. In EWS, a putative role for CD164 in EWS transformation was suggested by Grunewald et al. who demonstrated that the thyroid receptor interacting protein 6 (TRIP6), belonging to the Zyxin family of proteins, is overexpressed in EWS and promotes cell growth, invasion, and migration through a transcriptional pro-invasive gene signature, which included CD164 (42). However, CD164 mechanisms of action in EWS cells were not further characterized and its impact on tumor progression has never been evaluated.
According to previous evidences (33,34), our data confirm a direct functional interaction between IGF2BP3 and CD164. In fact, IGF2BP3 and CD164 are part of a complex detected by RIP assays, suggesting that IGF2BP3 might regulate mRNA stability and therefore expression levels of CD164. In turn, CD164 functionally interacts with CXCR4, thus regulating CXCR4 activation and CXCL12-dependent motility of EWS cells. In ovarian cancer cells, CD164 was localized in the cytosol and nucleus suggesting that nuclear CD164 might regulate CXCR4 promoter activity (38). The definition of downstream mechanisms of action of this signaling axis in EWS cells deserves further studies. It is important to mention that, in addition to IGF2BP3, additional proteins may contribute to CD164/CXCR4 regulation at post-transcriptional or epigenetic level, as suggested by the moderate associations between these 3 molecules observed in EWS cases. For instance, CXCR4 is regulated by dynamic post-translational histone modifications (15) while CD164 is a direct target of miRNA124, whose role in EWS has been previously reported (43,44). Here, we put emphasis on the definition of an axis that may favor metastasis formation, the critical medical issue in the cure of EWS patients, and we provide evidence that support the possible use of drugs targeting IGF2BP3 and/or CXCR4 in high-risk patients with high expression of IGF2BP3/CD164/CXCR4 molecules. As recently reported, CXCL12 favors a pro-metastatic bone marrow niche in multiple myeloma, as well as in solid tumors with propensity to give bone metastases, including gastric, medullary thyroid, lung, prostate, and renal carcinomas (45). CXCR4blocking agents, such as the neutralizing antibody MDX1338 or Ulocuplumab, were reported to efficiently reduce migration and invasion of osteosarcoma, alveolar rhabdomyosarcoma and myeloma cells and suppress the CXCR4-driven Epithelial-tomesenchymal (EMT)-like phenotype (45)(46)(47), supporting the specific targeting of CXCR4 in therapy. More recently, the combination of MDX1338 and activated and expanded natural killer (NKAE) cell therapy was proposed as novel therapeutic approach to efficiently inhibit metastasis in mice (48). However, considering that CXCR4 may be up-regulated by epigenetic alterations or hypoxia-driven signaling which allow tumor cells to adapt and win the selection leading to tumor cell dissemination and metastasis in a new host environment, inhibition of IGF2BP3 may be more relevant. We have recently reported that inhibitors of Bromodomain and Extraterminal domain (BET) proteins can reduce expression of IGF2BP3 in EWS cells and synergize with vincristine (21). Further studies are necessary to develop more specific agents against this oncogenetic RBP.
In summary, the data presented in this work identified CD164 and CXCR4 as novel IGF2BP3 downstream functional effectors supporting the notion that the IGF2BP3/CD164/CXCR4 oncogenic axis may work as critical modulator of EWS aggressiveness. In addition, IGF2BP3, CD164, and CXCR4 expression levels may work as novel biomarkers predictive of EWS progression. Targeting of this axis may effectively prevent EWS disease dissemination.

DATA AVAILABILITY STATEMENT
The RNA-seq data discussed in this publication have been deposited in NCBI's Gene Expression Omnibus (49) and are accessible through GEO Series accession number GSE150722. Microarray data are accessible through GEO Series accession number GSE12102 (9).

ETHICS STATEMENT
The studies involving human participants were approved by Ethical Committee of the IRCCS Istituto Ortopedico Rizzoli, Bologna, Italy (0019012/2016, 0005175/2018, and 0006158/2020). Written informed consent to donate material to the IRCCS Istituto Ortopedico Rizzoli tissue biobank for research purposes was obtained.