Ectopic GR expression induced differentiation and reduced tumorigenesis
We first analyzed the effect of a synthetic ligand of GR, dexamethasone (DEX), and the RARα ligand all-trans retinoic acid (ATRA) on the viability of BE(2) cells stably expressing GR, named BE(2)-GR, and the empty vector carrying control BE(2)-EV, previously generated [18]. Following treatment with ATRA in BE(2)-GR cells, viability increased but no significant changes were found after treatment with either DEX or DEX + ATRA (Supplementary Fig. 1A).
Well-differentiated tumor cells are usually linked to a less aggressive phenotype [35]. We therefore asked whether DEX stimulated differentiation of BE(2)-GR cells. Retinoic acid (RA) is known for its differentiation mediating ability in NB [36] and was thus used as positive control. As expected, ATRA triggered neurite-like outgrowth, a morphological sign of neuronal differentiation. Activation of GR with DEX showed a similar phenotype and the DEX + ATRA combination slightly increased neurite formation compared to the single treatments in BE(2)-GR cells (Fig. 1A, Supplementary Fig. 1B and Supplementary Fig. 1C). In contrast, only ATRA provoked differentiation in BE(2)-EV cells. The levels of GR and RARα were analyzed by Western blot to determine expression changes upon treatment. We found that GR levels declined in BE(2)-GR cells incubated with DEX (Supplementary Fig. 1D), consistent with a well described negative feedback loop [18, 37]. The abundance of RARα was similar in both cell lines, and ATRA slightly increased the levels in BE(2)-GR cells while ATRA, DEX, or the combination led to a decrease in control cells (Supplementary Fig. 1D). Reduction of MYCN levels is a well-known effect during neuronal differentiation [38, 39]. Hence, MYCN protein was downregulated in BE(2)-EV cells treated with either ATRA or DEX + ATRA and mRNA levels were slightly reduced (Supplementary Fig. 1D, E). In BE(2)-GR cells, single treatment with ATRA reduced MYCN mRNA expression (Supplementary Fig. 1F). The neuronal differentiation marker tyrosine hydroxylase (TH) was elevated in BE(2)-GR cells upon DEX exposure, with a slight increase in the p75 neurotrophin receptor (p75NTR), while no changes were observed in secretogranin 2 (SCG2) or βIII-tubulin (Fig. 1B and Supplementary Fig. 1E). ATRA treatment alone or in combination with DEX resulted in an upregulation of differentiation markers, particularly in SCG2 in both cell lines. Expression analysis of mRNA levels of additional differentiation markers including Neurofilament L (NEFL) and Tropomyosin receptor Kinase A (TRKA), as well as the progenitor markers Nestin (NES) and Sex determining region Y-box 2 (SOX2), were performed by RT-qPCR to validate the induction of neuronal differentiation upon GR and RARα activation (Fig. 1C and Supplementary Fig. 1F). Treatment with DEX resulted in upregulated expression of NEFL and TRKA only in BE(2)-GR cells, while ATRA increased their levels in both cell lines. Both NES and SOX2 were significantly downregulated in BE(2)-GR cells in all conditions compared to control while in BE(2)-EV cells only SOX2 was significantly reduced when ATRA was added.
Moreover, we extended the differentiation studies by overexpressing GR in two additional cell lines, SH-SY5Y and SK-N-AS, both non-MYCN-amplified but expressing c-MYC. First, we assessed the protein levels of GR, RARα, and c-MYC after treatment with DEX, ATRA, or their combination (Supplementary Fig. 2A). In SH-SY5Y parental cells, GR protein levels were barely detected by Western blot analysis, while they were highly expressed in SH-SY5Y-GR cells, with all treatments. Furthermore, c-MYC levels decreased upon ATRA or DEX + ATRA in both cell lines while RARα was barely detectable in any condition in SH-SY5Y-GR cells (Supplementary Fig. 2A, B). We observed low GR expression in parental SK-N-AS. Exposure to DEX or DEX+ATRA reduced GR levels in both SK-N-AS parental and GR-overexpressing cells. Moreover, all treatments reduced RARα expression in both cell lines. c-MYC was downregulated in parental cells upon DEX + ATRA, and with DEX in SK-N-AS-GR cells (Supplementary Fig. 2A, B).
In SH-SY5Y parental and SH-SY5Y-GR cells, ATRA exposure resulted in neurite outgrowth, while DEX alone induced mild differentiation observed as a moderate neurite outgrowth in GR-overexpressing cells (Fig. 1D and Supplementary Fig. 2C). Moreover, SCG2 was increased in all ATRA conditions, while the other markers analyzed were barely detectable in parental cells (Fig. 1D, E and Supplementary Fig. 2D). In the SY5Y-GR cells, SCG2 was enhanced upon treatments, while TH increased only with DEX or DEX + ATRA, and p75NTR only slightly with all conditions (Fig. 1D, E and Supplementary Fig. 2D).
SK-N-AS cells are well known to differentiate into the glial lineage. Therefore, two glial differentiation markers, vimentin (VIM) and glial fibrillary acidic protein (GFAP), were analyzed. Treatment with DEX slightly increased vimentin abundance in parental SK-N-AS, and this effect was stronger in SK-N-AS-GR cells (Fig. 1F). All-trans retinoic acid (ATRA) and the combination with DEX induced vimentin expression in both cell lines, notably more pronounced in the GR-overexpressing cells, also evidenced by morphological changes (Fig. 1F), while GFAP protein levels were not changed (Fig. 1G and Supplementary Fig. 2E). As expected, the neural differentiation marker p75NTR was not expressed either in the parental or the GR-overexpressing cells, verifying the differentiation towards the glial lineage instead of neuronal of SK-N-AS cells.
To evaluate possible differences in tumor growth in vivo, we performed a xenograft experiment comparing BE(2)-EV and BE(2)-GR cells. Tumor volume was significantly decreased in mice injected with cells overexpressing GR compared to those generated by control cells (Fig. 2A, B, and Supplementary Fig. 3A). However, there was no significant difference in weight at experimental endpoint, since tumors from BE(2)-EV were filled with fluid with signs of necrosis, while BE(2)-GR derived tumors exhibited a solid texture (Fig. 2C). Analysis of tumor sections confirmed the presence of GR protein and low expression of Ki67, a proliferation marker associated with aggressiveness, in BE(2)-GR-derived tumors in contrast to in control tumors (Fig. 2D).
Together, our results showed that activation of GR-expressing cells resulted in increased expression of neuronal differentiation markers and neurite outgrowth, and in reduced tumor burden in vivo.
Triple activation of ERα, GR, and RARα induced differentiation
Combination treatments, i.e., therapeutic interventions with a cocktail of two or more drugs, is a cornerstone of cancer therapy. The amalgamation of anti-cancer medicines improves effectiveness compared to monotherapy since it targets key pathways in a characteristically additive or synergistic manner [40].
We found that treatment with 17-β-estradiol (E2) impaired the tumorigenic potential of NB cells overexpressing ERα [17] and that GR overexpression led to reduced tumorigenesis both in vitro and in vivo (Figs. 1 and 2, Supplementary Figs. 1-3) [18]. Although both effects were robust, they did not result in complete differentiation. To assess whether concomitant overexpression of GR and ERα would promote a stronger differentiation phenotype, we generated BE(2)-GR + EV and BE(2)-GR + ERα cells using lentiviral transduction (Supplementary Fig. 4A) and performed in vitro and in vivo assays. All-trans retinoic acid (ATRA) was used as positive control for neuronal differentiation and the impact of RARα activation on differentiation triggered by GR and ERα was analyzed.
GR and ERα expression were validated in BE(2)-GR + EV and BE(2)-GR + ERα cells by Western blot analysis (Supplementary Fig. 4A) and the IC50 value for each ligand was calculated. While DEX showed similar IC50 values in both cell lines, E2, as expected, barely affected viability of BE(2)-GR + EV cells (Supplementary Fig. 4B). The IC50 values for ATRA were similar in GR + ERα overexpressing and control cells. To evaluate possible cumulative effects upon activation of two or three ligands, we performed assays of single and combination treatments. All concentrations employed were below the IC50 values and ATRA was used in a significantly lower concentration (0.5 μM) than in many other studies (10 μM) [41, 42]. Single treatments using any of the concentrations analyzed did not affect cell viability (Supplementary Fig. 4C). Interestingly, 0.5 μM ATRA significantly increased viability of BE(2)-GR + ERα cells, while only slightly in BE(2)-GR + EV, similar as observed in BE(2)-GR cells (Supplementary Fig. 1A). While the triple combination of 10 nM E2 + 100 nM DEX + 0.5 μM ATRA did not affect cell viability in BE(2)-GR + EV cells, a reduction to 52% occurred when increasing the concentrations to 20 nM E2 + 250 nM DEX + 2 μM ATRA (Supplementary Fig. 4C). In BE(2)-GR + ERα cells, the incubation with E2 + DEX resulted in mild reduction of cell viability, which decreased to 63% upon triple combination using low concentrations of ligands, and it was strikingly reduced to 26% with higher concentrations (20 nM E2 + 250 nM DEX+ 2 μM ATRA) (Supplementary Fig. 4C). This demonstrated that ATRA decreased viability when combined with E2 + DEX in an additive and dose-dependent manner, even though ATRA alone at low concentration increased the percentage of viable cells.
Next, we examined the colony formation potential of BE(2)-GR + EV and BE(2)-GR + ERα cells and found that the latter formed significantly fewer colonies compared to control cells (Supplementary Fig. 4D). Treatment with ATRA or the triple combination led to formation of small, highly differentiated colonies that easily detached from the plate. Neither E2, DEX, nor E2 + DEX addition led to significant differences in colony number in control cells, while in BE(2)-GR + ERα cells numbers were reduced with either E2 or E2 + DEX.
Given the decrease in colonies between BE(2)-GR + ERα and control cells, we enquired whether activation of the three receptors together would trigger a stronger differentiation. To this end, we analyzed neurite outgrowth after single, double, or triple treatments. As expected, neurite-like protrusions appeared in BE(2)-GR + EV cells upon DEX and ATRA addition, whereas the morphology did not change with E2 alone (Supplementary Fig. 5A). The combined E2 + DEX cocktail also resulted in neurite outgrowth while the triple combination further enhanced neuronal differentiation. In contrast, all treatments promoted neurite outgrowth in BE(2)-GR + ERα cells, an effect that was most robust upon the triple condition (Supplementary Fig. 5A, B).
Neuronal differentiation was validated by immunofluorescence staining of the differentiation markers βIII-tubulin and SCG2. In control cells, βIII-tubulin increased upon exposure to DEX, ATRA, E2 + DEX, or E2 + DEX + ATRA, while SCG2 protein abundance was mainly induced with ATRA or the triple cocktail (Fig. 3A). Notably, in BE(2)-GR + ERα cells, single activation by any of the ligands resulted in elevation of both markers compared to control treatments. As observed, incubation with E2, ATRA, E2 + DEX, or E2 + DEX + ATRA, led to pronounced elongated neurite protrusions. Morphological changes also occurred in BE(2)-GR-EV cells albeit at a more modest level (Fig. 3A).
We confirmed that GR levels decreased following DEX treatment in both cell types (Supplementary Fig. 5C). In addition, BE(2)-GR + ERα cells showed higher RARα levels, which remained unchanged upon any treatment compared to control cells, where expression was decreased after incubation with E2, DEX, E2 + DEX, or the triple combination. None of the treatments affected MYCN protein expression in BE(2)-GR + EV cells, but RT-qPCR analysis revealed a significant downregulation of the MYCN gene upon DEX exposure (Supplementary Fig. 5C, D and Fig. 3C). Similarly, in BE(2)-GR + ERα cells, a considerable reduction in MYCN mRNA was observed after E2 or DEX exposure, although we did not detect any changes in protein levels. Note that the previous results included analysis on protein and mRNA abundance. A caveat of this approach is that mRNA and protein levels might not necessarily be correlated [43].
Moreover, we analyzed protein expression of the neuronal differentiation markers TH, SCG2, p75NTR, and βIII-tubulin by Western blot. Treatment with E2 + DEX slightly increased TH levels in control cells. In addition, ATRA alone or in combination with E2 + DEX upregulated all four markers. In BE(2)-GR + ERα cells, activation of ERα increased p75NTR, SCG2, and TH, and levels were robustly potentiated with triple treatment (Fig. 3B and Supplementary Fig. 5D).
Additionally, we confirmed neuronal differentiation by analyzing mRNA expression of other known neuronal differentiation and progenitor markers. Increased NEFL and TRKA were observed in all conditions with DEX or ATRA in BE(2)-GR + EV cells, whereas a downregulation in SOX2 and NES occurred upon ATRA and the double (E2 + DEX) and triple combinations. Likewise, all treatments increased the abundance of neuronal differentiation markers and diminished the levels of the progenitor markers in BE(2)-GR + ERα cells (Fig. 3C). The Discs Large MAGUK Scaffold Protein 2 (DLG2) gene was recently reported to regulate the differentiation phenotype induced by ATRA, and proposed as a tumor suppressor candidate in NB [44]. Our RT-qPCR analysis showed that all treatments, except E2 in BE(2)-GR + EV cells, elevated DLG2 levels in accordance with our differentiation results (Fig. 3C).
Furthermore, to validate the phenotype achieved by activation of the three NHRs, we expanded our analysis to two additional MYCN-amplified NB cell lines, IMR32 and KCN69n, both overexpressing GR + EV and GR + ERα. Equally to in BE(2) cells, neurite outgrowth was observed upon DEX, ATRA, double, and triple combination in GR + EV cells, as well as with all treatments in GR + ERα cells, in particular with E2 + DEX + ATRA in both IMR32-GR-ERα and KCN-GR + ERα (Supplementary Fig. 6A). These results were supported by phalloidin and SCG2 staining (Fig. 4A). Moreover, MYCN levels were significantly reduced with the double and triple combination in KCN-GR + ERα cells (Supplementary Fig. 6B, C). Notably, GR protein levels were downregulated upon all conditions containing DEX in GR + EV cells, and RARα expression diminished in all treatments. In contrast, all ligands increased ERα levels in KCN-GR + ERα cells (Supplementary Fig. 6B). Evaluation of the neuronal differentiation markers showed that TH was enhanced with DEX or ATRA in KCN-GR + EV and in all conditions in the double overexpressing cells. The levels of p75NTR were robustly increased after the triple combination in both cell lines while the change in SCG2 was only minor (Fig. 4B and Supplementary Fig. 6C).
IMR-32-GR+ERα cells showed the most robust differentiation phenotype of all cells analyzed, with a stronger increase in all neural differentiation markers after E2 + DEX or E2 + DEX + ATRA treatment (Fig. 4A, Supplementary Fig. 6A and 7B). In IMR32-GR + EV, SCG2 levels were elevated when treated with E2 + DEX + ATRA while TH was increased with both the double and triple combination. Furthermore, while no changes were observed in MYCN protein (Supplementary Fig. 7A, B), GR levels increased in both cell lines and ERα abundance was elevated in GR + ERα cells upon treatment. The endogenous protein levels of the receptors used in this study, GR, ERα, and RARα are shown in Supplementary Fig. 7C. Moreover, using RNAseq data from 39 common NB cell lines, we analyzed the expression of the three receptors and presented data as higher or lower than the average level in all samples. All cells used in our study were present except KCN69n. The levels of GR (NR3C1) were higher than the average in SH-SY5Y cells followed by SK-N-AS, while they were under the average in SK-N-BE(2) and IMR32, with the lowest levels in the latter. In comparison, we only detected GR protein in SK-N-AS cells by Western blot (Supplementary Fig. 2A and Supplementary Fig. 7D). All cells showed lower expression of ERα (ESR1) than average (Supplementary Fig. 7D). In accordance, we did not detect ERα levels in any of the cells (Supplementary Fig. 4A, 5C, 6B, 7A and 7C). For RARα (RARA), mRNA levels were high in SK-N-AS, similar to average in SH-SY5Y, and lower than average in SK-N-BE(2) and IMR32 (Supplementary Fig. 7D). However, we detected higher levels of RARα protein in the MYCN-amplified cells BE(2), IMR32, and KCN69n, compared to the non-MYCN-amplified cells (Supplementary Fig. 7C).
In summary, simultaneous activation of GR and ERα lead to reduced viability and colony forming ability, accompanied by a stronger induction of neurite outgrowth and neuronal differentiation markers. Expression data revealed that cells with MYCN-amplification have lower levels of NHRs receptors than non-MYCN amplified cells, supporting the notion that MYCN inhibits their expression.
NHR activation increased glycolytic capacity and induced lipid droplet accumulation
Cancer cells reprogram their metabolism to maintain proliferation [45, 46]. We have previously shown that the glycolytic and oxidative functions were impaired in MYCN-amplified NB cells overexpressing ERα [17]. To examine a putative cumulative effect of GR and ERα activation on the metabolic phenotype, we employed the Agilent Extracellular Flux Analyzer (Supplementary Fig. 8A, B) [47, 48]. We found that cells expressing GR were more glycolytic and, in addition, had a higher oxidative phosphorylation (OXPHOS) capacity than those co-expressing GR and ERα (Fig. 5A, B). Incubation with DEX increased the glycolytic parameters in control cells. As expected, both E2 and DEX single treatments raised glycolysis in BE(2)-GR + ERα cells, and it was further increased upon their combination (Fig. 5A). Similarly, treating with ATRA or the triple combination resulted in a stronger induction of glycolytic function, especially in control cells (Fig. 5B).
Conversely, analysis of oxygen consumption rate (OCR) revealed that mitochondrial respiration was significantly reduced in BE(2)-GR + ERα compared to control cells. Furthermore, activation of both receptors downregulated OXPHOS in BE(2)-GR + ERα. In contrast, neither E2, DEX, nor their combination affected mitochondrial activity in control cells (Fig. 5C). Notably, ATRA increased OXPHOS in both cell types whereas the mitochondrial parameters mildly decreased when combining E2 + DEX + ATRA, most likely due to a counter effect of E2 and DEX (Fig. 5B, D).
To investigate the metabolic potential in response to induced stress, we used oligomycin, inhibiting ATP synthase, and FCCP, an uncoupler of the electron transport chain. Upon stress induction, control cells shifted to a more energetic phenotype. BE(2)-GR + ERα cells moved towards an aerobic phenotype after ATRA treatment, with enhanced ECAR upon activation with ATRA or the triple cocktail (Fig. 5G and Supplementary Fig. 8C-F,). In BE(2)-GR + EV cells, ATRA alone or in combination with E2 + DEX increased glycolysis, whereas only a minor change was observed in OXPHOS (Fig. 5E, F). In contrast, in BE(2)-GR + ERα cells, E2 enhanced glycolysis and decreased respiration, an effect further potentiated when combining with DEX + ATRA for glycolysis while OXPHOS was unaffected compared to control. Notably, glycolysis was mildly enhanced with DEX but strikingly increased with ATRA but neither DEX nor ATRA influenced OXPHOS (Fig. 5G, H).
Lipids are stored in several cancer types as a consequence of metabolic reprogramming [49, 50]. We have previously demonstrated lipid droplet accumulation in ERα expressing NB cells upon E2-activation [17]. Hence, we explored lipid droplet formation upon simultaneous activation of GR, ERα, and RARα. Treatment with DEX, ATRA, their combination, or a mix of all three ligands resulted in lipid droplet accumulation in control cells. All treatments including E2 alone triggered lipid droplet deposition in BE(2)-GR + ERα cells while none of the vehicle conditions showed any sign of lipids (Fig. 5I).
Collectively, co-activation of GR, ERα, and RARα increased glycolysis and lipid droplet accumulation with minimal influence on mitochondrial respiration.
Concurrent overexpression of GR and ERα reduced angiogenesis and tumor burden
To evaluate the tumorigenic potential of combined expression of GR and ERα in vivo, cells were inoculated in nude mice, and tumor growth was followed until the control group reached the ethical endpoint volume. Overexpression of combined GR and ERα significantly reduced tumor burden and weight compared to tumors derived from GR expressing cells (Fig. 6A, B, and Supplementary Fig. 9A). We verified the presence of ERα and/or GR by analysis of tumor sections derived from control or BE(2)-GR + ERα cells. Ki67 staining showed that BE(2)-GR + ERα tumors had a less proliferative phenotype compared to controls (Fig. 6C). Staining of tumor sections showed higher levels of the differentiation markers, p75NTR, SCG2, and βIII-tubulin in tumors derived from BE(2)-GR + ERα cells compared to control tumors (Fig. 6D). Notably, tumors generated from BE(2)-GR + EV cells were more vascularized in appearance than those from BE(2)-GR + ERα cells. We confirmed extensive angiogenesis, with multiple and wide blood vessels, in BE(2)-GR + EV-derived tumors by staining for the endothelial marker endomucin in comparison with BE(2)-GR + ERα generated tumors (Fig. 6E and Supplementary Fig. 9B).
Together, these results demonstrated that co-activation of GR and ERα induced robust differentiation in combination with decreased angiogenesis and tumor burden in vivo, highlighting the potential of triggering both receptors as a putative therapeutic approach.
The levels of GR, ERα, and RARα correlated with high expression of differentiation markers and favorable prognosis in NB patients
Next, we interrogated the impact of the three receptors for survival of NB patients. Using the SEQC (n = 498) [27] and Kocak (n = 649) [28] NB cohorts, we analyzed the outcome of expression of GR, ERα, and RARα, either individually or in combination, for NB patient survival (Fig. 7). To this end, patients were divided in high or low GR, ERα, or RARα, according to their mRNA levels and separated in quartiles of expression: from quartile 1, patients with the highest levels, to quartile 4, patients with the lowest expression. We selected all patients in quartile 1 (HighGR, HighERα, or HighRARα) and in quartile 4 (LowGR, LowERα, or LowRARα), separating them from patients with intermediate expression levels (quartiles 2 and 3). Patients with higher GR, ERα, or RARα mRNA levels had a better event free and overall survival than patients with lower levels in the SEQC cohort (Supplementary Fig. 10A-C). However, only patients with HighGR mRNA expression correlated with a better overall survival in the Kocak dataset (Supplementary Fig. 10D-F). We therefore also analyzed the Oberthuer cohort (n = 251) [29], where patients with high levels of all the individual receptors presented with better prognosis (Supplementary Fig. 10G-I), in agreement with our previous data [16,17,18]. Additionally, since we already had demonstrated a negative correlation between MYCN and the three NHRs, we further studied the levels of GR, ERα, and RARα in the SEQC dataset separating the patients according to their MYCN status, either MYCN-amplified (MNA) or non-MYCN-amplified (NMNA). As expected, patients with MYCN-amplification showed lower levels of all three receptors than those lacking amplification (Supplementary Fig. 10 J).
Given these results, we investigated the clinical relevance of concurrent overexpression of GR and ERα. The SEQC and Kocak datasets were divided into patients with HighGR + ERα and LowGR + ERα mRNA levels. As expected, the high co-expression patient group was related to favorable prognosis with better event free and overall survival compared to the low expression group in both cohorts (Fig. 7A and Supplementary Fig. 11A).
As our in vitro and in vivo experiments showed strong induction of neuronal differentiation by GR and ERα co-expression, we compared levels of differentiation markers between the HighGR + ERα versus LowGR + ERα patient groups. Notably, expression of NGFR (p75NTR), TH, and SCG2, were significantly higher in HighGR + ERα patients in contrast to those with lower GR and ERα levels (Fig. 7B and Supplementary Fig. 11B). We next divided patients with HighGR + ERα and LowGR + ERα levels according to MYCN status using the SEQC dataset. Strikingly, no single patient with MYCN-amplification and HighGR + ERα was identified, further validating the role of MYCN in regulating NHRs expression. As expected, HighGR + ERα NMNA patients had a better survival than the LowGR + ERα NMNA patients (Fig. 7C).
Using Gene Set Enrichment Analysis (GSEA), we identified processes with significant differences between the HighGR + ERα versus LowGR + ERα patient groups (see Additional Files 1 and 2 containing the gene sets used for the analysis), including neuronal crest differentiation, regulation of actin cytoskeleton, early differentiation genes, and axon guidance. Moreover, we also found metabolism-related processes including fatty acid biosynthesis, and fatty acid β-oxidation although they did not reach the significant threshold (Supplementary Fig. 11C).
We anticipated that patients with high expression of the three receptors were associated with elevated levels of differentiation markers and a better prognosis than patients with high levels of two NHRs. Despite the relatively low number of patients in the HighGR + ERα + RARα (n = 24 in SEQC and n = nine in Kocak) versus LowGR + ERα + RARα (n = 36 in SEQC and n = eight in Kocak) mRNA expression groups, we observed very large differences in survival between these quartiles in both cohorts (Fig. 7D and Supplementary Fig. 11D), although for Kocak patients, differences were not statistically significant. Accordingly, neuronal differentiation markers showed higher expression in HighGR + ERα + RARα patients versus the LowGR + ERα + RARα patient group in both datasets (Fig. 7E and Supplementary Fig. 11E).
Collectively, in silico analyses demonstrated that combined high levels of GR, ERα, and RARα correlated with increased expression of neuronal markers and a more favorable outcome linked to a more differentiated state in tumors of NB patients.
Single-nuclei transcriptome analysis suggested subsequent GR, ERα, and RARα expression for signaling a transition from undifferentiated to adrenergic cells
To explore the physiological significance of GR, ERα, and RARα during development of the human sympathetic nervous system, we studied their expression in four embryonic and seven adult adrenal glands, using the Suntsova dataset [30]. Expression of some of the genes of interest were available, namely the genes encoding GR (NR3C1), p75NTR (NGFR), βIII-tubulin (TUBB3), and the Erb-B2 Receptor Tyrosine Kinase 3 (ERBB3). Of these, only GR showed differences, with higher expression in embryonic adrenal glands compared to adult (Supplementary Fig. 12A).
As a proxy to study neural differentiation in vitro and to overcome its limitations, we interrogated the role of GR, ERα, and RARα during chromaffin cell differentiation in single-cell-nuclei of embryonic and post-natal human adrenal glands [28, 29]. Expression was compared with reference genes including ERBB3 for progenitor cells, Dopamine Beta-Hydroxylase (DBH) as well as TH for nor-adrenergic population, and phenylethanolamine N-methyltransferase (PNMT) for adrenergic cells. We identified a significant expression of ERα (ESR1) in the progenitor population of chromaffin cells in post-natal adrenal gland (hC1, FDR = 0.008, one-tailed Welch’s t-test) (Fig. 8A). We found that GR (NR3C1) was significantly upregulated in chromaffin cells (hC4, FDR = 5.18 × 10-4, one-tailed Welch’s t-test) and their progenitor cluster (hC1, FDR = 2.82 × 10-3, one-tailed Welch’s t-test) in post-natal adrenal gland in accordance with the Suntsova dataset analysis (Supplementary Fig. 12A). In contrast, RARα was not significantly expressed in any post-natal human adrenal gland cell cluster (FDR > 0.01 for all clusters, one-tailed Welch’s t-test) (Fig. 8A).
We then examined the expression of GR, ERα, and RARα in the developing human adrenal gland using previously published data [32]. Following the corrected annotation [31, 51], a significant up-regulation of GR in chromaffin cells and of RARα in non-cycling chromaffin cells was reported (Fig. 8B).
Next, we generated a pseudotime reconstruction of differentiating chromaffin cells in post-natal human adrenal glands to identify the order of expression of the three NHR genes during neuronal development. Progenitor cells were separated from early to late cells, the former with expression of progenitor markers and a high differentiation potential, and the latter sourcing from chromaffin cells, characterized by nor- and adrenergic markers and a lower differentiation capacity. This trajectory showed that ESR1, NR3C1, and RARA were sequentially expressed during chromaffin development (Fig. 8C).
In conclusion, our data suggest that the three NHR genes, GR, ERα, and RARα, are sequentially expressed from progenitor to chromaffin cells during human adrenal gland differentiation, indicating important roles during maturation of the sympathetic nervous system.