Tumor Biol. DOI 10.1007/s13277-016-4857-9

ORIGINAL ARTICLE

Expression of the anti-inflammatory suppressor of cytokine signaling 3 (SOCS3) in human clear cell renal cell carcinoma Anja Urbschat 1 & Svenja Stumpf 1 & Jörg Hänze 1 & Patrick Paulus 2 & Thorsten J. Maier 3 & Christine Weipert 4 & Rainer Hofmann 1 & Axel Hegele 1

Received: 7 October 2015 / Accepted: 13 January 2016 # International Society of Oncology and BioMarkers (ISOBM) 2016

Abstract The oncogenic transcription factor signal transducer and activator of transcription 3 (STAT3) is a cytokineactivated transcription factor controlling inflammation, cell proliferation, survival, and differentiation in normal tissue as well as in tumor growth. One of its most important negative regulators is the suppressor of cytokine signaling 3 (SOCS3). Here, we analyzed SOCS3 and other tumor-associated local immune regulators in human clear cell renal cell carcinoma (ccRCC). Analyses were performed in tumor and adjacent tumor-free healthy renal tissue from 35 patients with ccRCC. For functional analysis, ccRCC Caki-1 cell lines were stimulated with IL-6 and IFNγ in cell culture assays. We observed significantly lower SOCS3 messenger RNA (mRNA) levels in tumor tissue compared to healthy tissue. SOCS3 mRNA strongly correlated within tumor and healthy tissue. Interestingly vice versa, SOCS3 protein levels were significantly higher in tumor tissue than in healthy tissue. IL-22 and IL-22R1 mRNA displayed no differences in tumor and healthy tissue. Stimulation of Caki-1 cells with IFNγ resulted in markedly increased SOCS3 mRNA levels. We conclude that SOCS3 along with STAT3 participates in regulatory

* Anja Urbschat [email protected]

1

Department of Urology and Pediatric Urology, University Hospital of the Philipps-University Marburg, Baldinger Strasse, Marburg, Germany

2

Department of Anesthesiology and Operative Intensive Care Medicine, Kepler University Hospital Linz, Linz, Austria

3

Department of Biomedicine, Aarhus University, Aarhus, Denmark

4

Clinic of Urology and Andrology, Landeskrankenhaus Hall in Tirol, Hall, Austria

mechanisms in ccRCC, which certainly features only one of multiple factors involved but nevertheless merits further attention. Keywords Clear cell renal cell carcinoma . SOCS3 . STAT3 . JAK2

Introduction The renal cell carcinoma (RCC) occurs with an estimated 338, 000 new cases yearly worldwide [1]. Hereof, the clear cell renal cell carcinoma (ccRCC) is the most frequently diagnosed histological entity with 80 % of cases [2]. Although noticeable efforts have been made to decode molecular pathogenesis of ccRR, the detailed molecular mechanisms underlying the oncogenesis and progressions remain largely to be elucidated. Great attention in this context is paid to the capacity of immunity to control and shape cancer [3]. Tumor microenvironments having stimulatory effects in malignancy include tumor cells, malignant transforming cells next to immune cells and macrophages. Different cellular and molecular pathways that coordinate tumor-promoting and tumorantagonizing effects of inflammation are known to act in tight communication between immune and malignant cells [4]. Recent studies have indicated a crucial role of STAT3 in oncogenesis, progression, and therapy of renal tumors [5–7]. STAT3 is a transcriptional factor that mediates cytokine and growth factor signaling [8] thereby influencing cell survival, proliferation, and angiogenesis [7]. One of the main negative regulators of cytokine-mediated induction of the JAK-STAT pathway is the suppressor of cytokine signaling 3 (SOCS3) belonging to the suppressor of cytokine signaling (SOCS) proteins, a family of eight cytokine-inducible negative regulators of cytokine signaling [9]. The receptors to which

Tumor Biol.

SOCS3 binds activate primarily STAT3; therefore, SOCS3 can be considered as an inhibitor that is relatively selective to STAT3 [10]. SOCS3 was found to suppress inflammatory reactions by inhibiting STAT3 [11, 12]. In experimental immune complex glomerulonephritis, SOCS3 was found to modulate cell signaling. In this study, SOCS3 was induced in infiltrating cells as well as in renal resident cells in the tubulointerstitium [13]. Therefore, particularly SOCS3 may play an important role not only in renal disease states but also in malignancies by antagonizing STAT3. So far, SOCS3 was associated with suppressed tumor growth in lung cancer [14], gastric cancer [15], and hepatitis-induced hepatocarcinogenesis [16]. Recently, the presence of SOCS3 in RCC was detected by immunohistochemistry, and its expression was associated with accelerated progression, the presence of metastases, and reduced survival [17]. To better understand the role of SOCS3 in tumor-associated local immune response and tumor proliferation, we investigated the expression of SOCS3, JAK2, STAT3, and related cytokines in clear cell renal cell carcinoma.

Materials and methods Participants The study protocol and consent documents were approved by the local Ethic Committee of the Goethe-University Hospital Frankfurt am Main (file number 04/09 UGO 03/10) and Philipps-University Hospital Marburg (file number 122/14). Patients gave their informed consent prior to surgery. Tumor and corresponding adjacent healthy tissues were obtained from 35 patients with histopathological diagnosis of ccRCC: 12 women (34 %) and 23 men (66 %) aged 42–84 years (mean age 64). Clinical characteristics of these patients are listed in Table 1. Patient exclusion criteria comprised immunodeficiency, autoimmune diseases, or second malignancy in order to eliminate factors interfering with the measurements. All patients underwent imaging (CT and/or MRI) for preoperative staging and surgery before receiving any other therapy. Specimens of the tumor tissue as well as adjacent healthy renal tissues were collected from each patient immediately after radical nephrectomy. Samples were stored at −80 °C until further processing. The pathological examination was performed according to the UICC TNM classification of malignant tumors [18]. Gene expression analysis by real-time quantitative reverse transcription-PCR Total RNA was isolated using Tri-Reagent (Sigma-Aldrich), and integrity was assessed on a denaturing agarose gel stained with ethidium bromide. SOCS3 possesses only 1 exon;

Table 1 Characteristics of the study population

Parameter Number of patients

35

Mean age

64

Median age Age x-x

63 42–84

Female

34 %

Male

66 %

pT1/2 pT3/4

57 % 43 %

G1/2 G3/4

83 % 17 %

cM0

77 %

cM+

23 %

therefore, DNA digestion was performed. Intact RNA was transcribed using random hexameric primers and reverse transcriptase (Applied Biosystems). The following TaqMan sondes were used: RPL-PO: Hs99999902_m1, SOCS3: Hs00269575_s1, CAIX: Hs00154208_m1, IL-6: Hs00985639_m1, IL-22: Hs00220924_m1, and IL-22R1: Hs00222035_m1 (LifeTechnologies). Taq Man Fast Advanced Master Mix (Applied Biosystems) was used as Assay Mix. Real-time PCR was performed on AbiPrism 7500 Fast Sequence Detector (Applied Biosystems). Calculation of threshold cycles (Ct values) and data analyses were performed by the Sequence Detector software. Relative changes in messenger RNA (mRNA) expression were calculated by normalizing the values to their corresponding RPLPO expression using the 2−ddCt method. Cell culture Human ccRCC cell line Caki-1 (LGC Promochem, Wesel, Germany) was cultured in DMEM supplemented with 100 U/ml streptomycin and 10 % FCS (GIBCO-BRL) at 37 °C in 5 % CO2. For experiments with conditioned media, cell lines were seeded on 6-well polystyrene plates at equal density (Greiner) and incubated in the aforementioned cell culture medium. All incubations were performed at 37 °C and 5 % CO2. Protein isolation and western blot analysis For detection of intracellular proteins, a lysis buffer supplemented with protease inhibitor cocktail (Roche Diagnostics), DTT, Na3V04, PMSF, and NaF was added to human tissue samples and Caki-1 cells. The extracts were clarified by centrifugation, and tissue extracts were stored at −80 °C. Total protein concentrations of tissue homogenates were determined by the method according to Bradford. For western blotting,

Tumor Biol.

50 μg of total protein was used per lane. SDS-PAGE and antibody conditions for SOCS3 and STAT3 detection: 10 % SDS-PAGE, SOCS3 antibody (#55155 AnaSpec (dilution 1:500) and sc-9023 Santa Cruz (dilution 1:500)), β-tubulin (#sc-555-29, Santa Cruz (dilution 1:5000)), phospho-STAT3 (Tyr705, #9145, Cell Signaling (dilution 1:1000)), and total STAT3 (#9139 Cell Signaling (dilution 1:1000) and Y705 rabbit monoclonal Cell Signaling (dilution 1:1000)). For detection of total STAT3 (tSTAT3), blots were stripped and reprobed after detection of phosphorylated STAT3 (pSTAT3). Detection was achieved by horseradish peroxidase-labeled secondary antibodies (Bio-Rad, Munich, Germany) and a chemoluminescence detection kit (GE Healthcare, Munich, Germany) according to the manufacturer’s instructions. SDS-PAGE and antibodies condition for JAK detection: 7.5 % SDS-PAGE, JAK antibody (#3230 Cell Signaling (dilution 1:500)), and β-actin (#sc-47778 Santa Cruz (dilution 1:1000). Detection was achieved by horseradish peroxidaselabeled secondary antibodies (#1514021 R&D) and a chemiluminescent HRP detection reagent (#1511223 Millipore) according to the manufacturer’s instructions. Relative protein expression of SOCS3 (23 kDa) was calculated by normalizing the values to their corresponding β-tubulin (55 kDa) expression. Relative protein expression of JAK (125 kDa) was calculated by normalizing the values to their corresponding βactin (43 kDa) expression.

Immunhistochemical staining Immunohistochemistry was performed for SOCS3 from frozen sections (7 μm). For specific staining, slides were incubated with a primary SOCS3 antibody overnight at 4 °C (AnaSpec, #55155 (dilution 1:200) and sc-9023 Santa Cruz (dilution 1:200)). For signal development, a biotinylated secondary antibody goat anti-rabbit (sc-2014, Santa Cruz) was incubated for 1 h at room temperature prior to incubation with streptavidin-HRP complex (AXXORA) and subsequent DAB incubation. Counterstaining was carried out with hematoxylin.

Statistical analysis Statistical analysis and artwork were performed with GraphPad Prism® 5.02 software (GraphPad Software, Inc.). The distribution of variables was tested for normality. The Wilcoxon matched pairs test (two-tailed) was used for paired analysis of tumor and adjacent healthy tissue. The Spearman’s rank correlation coefficient analysis was used to determine the significance of correlations. Descriptive results were expressed as medians ± quartiles. In all analyses, a p value of less than 0.05 was considered significant.

Results SOCS3 mRNA and protein are significantly and inversely regulated in human renal cell carcinoma tissue compared with adjacent healthy renal tissue Messenger RNA expression of SOCS3 normalized by its RPLPO content was detectable in both tumor and adjacent healthy tissue, but a significantly lower expression was observed in tumor tissue compared to adjacent healthy tissue (n = 35, p < 0.01) (Fig. 1a). Interestingly, individual SOCS3 mRNA in tumor tissue correlated strongly with its expression in healthy tissue (Spearman r = 0.7571; p < 0.001) (Fig. 1a). Female subjects generally tended to display higher SOCS3 mRNA level in tumor as well as in adjacent healthy renal tissue compared to male subjects (ns) (Fig. 1a). In contrast to the gene expression of SOCS3, protein levels were significantly higher in tumor tissue than in healthy tissue (n = 22; p < 0.01) (Fig. 1b). Immunohistochemical staining demonstrated SOCS3 expression in healthy as well as in tumor tissue (Fig. 1c). No distinct variations in JAK2 and STAT3 expression As SOCS3 is a negative regulator of oncogenic STAT3 signaling, phosphorylated and total STAT3 protein levels were analyzed next using western blotting. In analyzed tissue samples of nine patients, varying levels of pSTAT3 protein relative to tSTAT3 were observed without accentuation of either tumor or healthy tissue (ns) (figure not shown). As SOCS3 is a negative regulator of the JAK/STAT pathway, we next investigated JAK2 protein levels using western blotting. In analyzed paired tissue samples of 16 patients, a tendency to higher JAK2 protein expression in tumor tissue was obvious but lacking significance (ns) (Fig. 1b). Validation of tumor tissue via measurement of carboanhydrase IX mRNA Carboanhydrase IX (CAIX) is a HIF-1α regulated transmembrane protein upregulated in more than 90 % of ccRCC [19]. To confirm this in our tissue samples, CAIX mRNA expression was measured. CAIX standardized to RPL-PO was significantly upregulated in tumor tissue in comparison with healthy renal tissue as expected (p < 0.001) (Fig. 2a). IL-6 but neither IL-22 nor IL-22R1 was alternated in ccRCC tissue IL-6 is known to be elevated in renal cell carcinoma [20]. Moreover, transcription of the SOCS3 gene is increased rapidly in response to IL-6, in vitro and in vivo [21]. Therefore, we examined the gene expression of IL-6 mRNA in human

Tumor Biol.

Fig. 1 a SOCS3 mRNA expression normalized to its RPL-PO content in tumor and adjacent healthy renal tissue (n = 35) and their correlation (n = 35) (Spearman’s rank correlation). SOCS3 mRNA in female and male subjects. b Protein expression of SOCS3 (23 kDa) and β-tubulin (55 kDa) (n = 22) as well as JAK2 (125 kDa) and β-actin (43 kDa) (n = 16) with representative western blots. c Exemplary immunohistochemical staining in 400fold magnification of SOCS3 in tumor (sinistral) and adjacent healthy renal tissue (dexter) with negative

control on the respective left side. SOCS3 mRNA and SOCS3 protein expression are presented in box blots to depict the distributions of the relative expression values. Horizontal lines represent the medians, boxes represent the interquartile range (25-75 %). Whiskers above and below the box indicate the 90th and 10th percentiles. Outliers are depicted as dots. Statistical calculation was performed using the Wilcoxon matched pairs test for paired analysis of tumor and adjacent healthy tissue. Pvalues refer to the Wilcoxon matched pairs test. **p < 0.01

clear cell renal cell carcinoma in comparison to healthy renal tissue. IL-6 mRNA was significantly more abundant in tumor tissue compared to healthy tissue (p < 0.05) (Fig. 2a). Apart from IL-6, IL-22 represents a potent mediator of cellular inflammatory responses and a strong activator of STAT3 which also induces SOCS3 expression [22]. However, IL-22 and IL22-receptor 1 (IL-22R1) mRNA were equally low expressed without any significant differences in tumor versus healthy tissue (ns) (Fig. 2b). A lack of any significant correlation was seen with SOCS3 mRNA expression and IL-22, IL22R1, or IL-6 mRNA expression neither in tumor tissue nor in healthy tissue (data not shown).

and healthy tissue samples from patients without distant metastases (M0) were compared to tissues from patients with distant metastases (M+). In tumors with pathological tumor stage, pT3/4 SOCS3 mRNA expression was significantly impaired in tumor tissue in comparison to adjacent healthy tissue (p < 0.01; n = 15). In pT1/2, a similar trend was obvious, albeit not significant (ns; n = 20) (Fig. 3). Considering the tumor grading, SOCS3 mRNA was significantly reduced in G1/2 tumor tissue in comparison to healthy tissue (p < 0.01; n = 29). In G3/4 tumors, the same trend was evident (ns, n = 6) (Fig. 3). Regarding the existence of distant metastases, significantly higher SOCS3 mRNA levels were observed in healthy tissue of patients without distant metastases (p < 0.01; n = 27) but not in patients with distant metastases (ns) (Fig. 3).

Differences in SOCS3 mRNA expression between tumor and healthy tissue persist throughout pathological features Pathological tumor stages 1 and 2 were summarized into pT1/ 2, and tumor stages 3 and 4 were summarized into pT3/4. Equally, tumor grades 1 and 2 were summarized into G1/2, and tumor grades 3 and 4 were summarized into G3/4. Tumor

Induction of SOCS3 in clear cell renal cell carcinoma cell line Caki-1 We assessed next whether the SOCS3 signaling pathway is functional and is capable of exerting any significant biological

Tumor Biol.

Fig. 2 a CAIX and IL-6 mRNA expression normalized on its RPL-PO content in tumorous and adjacent healthy renal tissue (n = 31). b IL-22 and IL-22R1 mRNA expression normalized on its RPL-PO content in tumorous and adjacent healthy renal tissue (n = 31). All data are presented in box blots to depict the distributions of the relative expression values. Horizontal lines represent the medians, boxes

represent the interquartile range (25-75 %). Whiskers above and below the box indicate the 90th and 10th percentiles. Outliers are depicted as dots. Statistical calculation was performed using the Wilcoxon matched pairs test for paired analysis of tumor and adjacent healthy tissue. Pvalues refer to the Wilcoxon matched pairs test. *p < 0.05; ***P < 0.001

effects in a cultured ccRCC line. Therefore, expression of SOCS3 mRNA in Caki-1 clear cell renal cell carcinoma cells treated with IL-6 and IFNγ was investigated using RT-PCR. As pro-inflammatory IL-6 was already found to be elevated in tumor tissues (Fig. 2a), increasing concentrations of IL-6 and IFNγ were added to the medium for 30 min. Analysis revealed a rapid induction of SOCS3 mRNA in IFNγ-stimulated cells detectable already after 30 min of incubation (Fig. 4). As expected, no induction of SOCS3 mRNA upon IL-6 stimulation was detected since Caki-1 cells lack the co-receptor gp130 and are therefore not susceptible to IL-6 (Fig. 4) [23].

with these findings, SOCS3 mRNA expression levels were lower in 80 samples of gastric tumor tissues than in their matched normal mucosa [15]. Reduced expression of SOCS3 in human hepatocellular carcinoma (HCC) compared to non-tumorous tissue was also seen in a study with 20 hepatitis C virus infected patients [16]. Our results are in concordance with these findings in ccRCC as mRNA expression of SOCS3 displayed a significant lower expression in tumor tissue compared to non-tumorous tissue. Interestingly, individual SOCS3 mRNA in tumor tissue correlated strongly with the respective expression in healthy tissue with a tendency to higher levels in females compared to male subjects. As contamination of histological normal tissue with tumor cells can be excluded, patients may generate individually differing inflammatory responses to renal malignancies which may than involve tumor as well as surrounding renal healthy tissue. Female subjects generally tended to display higher SOCS3 mRNA level in tumor as well as in adjacent healthy renal tissue compared to male subjects. In this regard, it was proven in a study that male and female kidneys respond differently to injury [25]. Therefore, the here observed gender-specific changes in gene expression are in agreement with the literature. However, contrary to the findings with mRNA expression SOCS3 protein expression was significantly higher in tumor tissue than in healthy tissue but not detectable in all

Discussion STAT proteins and STAT3 in a leading role are transcriptional regulators of diverse tumor-promoting factors, including genes that regulate cell proliferation and survival, as well as angiogenesis and metastasis [24]. Hereof, SOCS3 is an inhibitor preferentially suppressing STAT3 [10]. Thus, the role of SOCS3 has been investigated in human primary non-smallcell lung cancer (NSCLC) tissue samples [14]. Eight matched pairs of surgically resected early-stage NSCLC were analyzed. Either lacking or significantly reduced SOCS3 mRNA compared to the respective normal samples was found. In line

Tumor Biol.

Fig. 4 SOCS3 expression in clear cell renal cell carcinoma cell line Caki1 upon stimulation with IL-6 and IFNγ in increasing concentrations of 1, 10 and 100 ng/ml medium. Cell culture experiments were performed 3 times

Fig. 3 SOCS3 mRNA expression in tumorous and healthy tissue throughout pathological features. Pathological tumor stage 1 and 2 were summarized into pT1/2, tumor stages 3 and 4 were summarized into pT3/ 4. Equally tumor grade 1 and 2 were summarized into G1/2, tumor grades 3 and 4 were summarized into G3/4. Tissue samples of patients without distant metastases (M0) were compared to those of patients with distant metastases (M+). All data are presented in box blots to depict the distributions of the relative expression values. Horizontal lines represent the medians, boxes represent the interquartile range (25-75 %). Whiskers above and below the box indicate the 90th and 10th percentiles. Outliers are depicted as dots. Statistical calculation was performed using the Wilcoxon matched pairs test for paired analysis of tumor and adjacent healthy tissue. P-values refer to the Wilcoxon matched pairs test. **p < 0.01

tissue samples (detected in 91 %). This would be in line with the hypothesis that SOCS proteins are rapidly induced by activated STATs upon phosphorylation and act to block cytokine signaling [14]. Additionally, investigations on proximal renal tubule cells (HK-2) indicate that SOCS3 inhibits phosphorylation of pSTAT3 in these cells [26]. This is insofar relevant as the ccRCC origins from the proximal tubule cells [27]. Taking into consideration that pSTAT3 protein expression is described to be elevated and relevant in renal tumors [5–7, 28] consequently higher SOCS3 protein levels seem to be conclusive. SOCS3 has been shown to inhibit STAT3 phosphorylation by binding to and inhibiting JAK2 kinases or competing with STAT3 for phosphotyrosine binding sites on cytokine receptors. In order to obtain a more complete picture of the tumor growth regulation by the system of SOCS3/JAK2/STAT3, we investigated phosphorylated (pSTAT) and unphosphorylated STAT3 (tSTAT3). Yet, in our tissue samples, we observed varying levels of phosphorylated STAT3 protein relative to total (tSTAT3) without accentuation of either tumor or healthy tissue, which might be due to the small number of samples though. The feedback inhibition of cytokine receptor inhibition signaling is in part mediated by the JAK2 family kinases [29]. Concerning JAK2, in our analyzed paired tissue samples of 16 patients, a trend to higher JAK2 protein expression in tumor tissue was obvious but lacking significance. Recently, the presence of SOCS3 in different histological types of RCC tissue (94 % ccRCC) was examined by immunohistochemistry showing that SOCS3 was co-expressed with pSTAT3 [17] confirming our present findings on posttranscriptional expression of SOCS3 and pSTAT3. The observed trend to higher JAK2 protein expression in tumor tissues might be a result of this enhanced SOCS3 and STAT3 co-expression. SOCS3 mRNA expression has also been investigated in vitro in IL22 activated lung epithelial cells after 30 and 60 min, and 2, 4,

Tumor Biol.

8, and 24 h of incubation [22]. This time course analysis revealed rapid induction of SOCS3 mRNA detectable within 1 h of incubation and upregulation translated well into cellular SOCS3 protein expression. SOCS3 mRNA expression decreased in later time points. In contrast, increased SOCS3 protein expression was also detectable after 24 h [22]. Thus, a feedback loop through longer lasting suppressor of cytokine signaling protein may lead to reduced gene expression of SOCS3 as observed in the present study. Although a trend to lower SOCS3 mRNA expression in tumor tissues persists throughout pathological features in the present study, no significant association with clinical features could be detected. SOCS3 mRNA expression did not differ significantly between pT1/2 and pT3/4 or G1/2 and G3/4 tumor tissues nor in tumor tissues of patients without or with distant metastasis. This may be due to the smaller patient number within the subgroups. The pro-tumorigenic function of IL-6 is well established. As transcription of the SOCS3 gene is increased rapidly in response to IL-6 in vitro and in vivo [21] and also STAT3 was reported to mediate IL-6-induced proliferation of renal cancer cells [7], we examined IL-6 in this context. According to the literature, we could observe higher mRNA expressions of IL-6 in tumor tissue in comparison to healthy renal tissue; however, no association with SOCS3 expression was detected. Apart from IL-6, IL-22 represents a strong activator of STAT3 which also induces SOCS3 expression [22]. In particular, the distribution pattern of the IL-22 receptors suggests that important target cells might not be immune cells but rather non-leukocytic cells such as kidney cells [30, 31], and possibly renal cell carcinoma cells. Therefore, we aimed at corroborating this hypothesis for our tumor tissue samples. However, both ccRCC tissue and adjacent healthy tissue exhibited similar broad IL-22 and IL-22R1 expression levels without accentuation on either tumor or healthy tissue. Though, we could observe a considerable interpersonal variability with bimodal distribution in resulting in a nonsignificant difference between those tissues. One might hypothesize that activation of both IL-22 as well as IL-6 might be more elevated in the peripheral zone of tumor tissue participating in the angiogenetic process encompassed by the microenvironment, rather than in the inner zone where our tumor samples derive from. Additionally, our in vitro cell culture assays on ccRCC cell lines revealed increased mRNA expression of SOCS3 upon IFNγ stimulation; therefore, SOCS3 signaling within ccRCC might be influenced by interferon therapy. In conclusion, the present study supports the hypothesis that human renal cells and ccRCC express SOCS3 thereby impacting signaling pathways with potential relevance for tumor growth. Further studies are needed to precise and accurate the role of these mechanisms in oncogenesis and their possible use as therapeutic target in human ccRCC.

Acknowledgments technical support.

We especially thank Ecatarina Oplesch for her

Compliance with ethical standards Conflicts of interest None Ethical approval All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Informed consent Informed consent was obtained from all individual participants included in the study. Funding This study was financially supported by a junior researchers grant to A.U. (Fokus Programm, Förderlinie A from the GoetheUniversity Frankfurt) and by the German Research Foundation (DFG projects MA-5825/1-1) to T.J.M. The founders of the study had no role in design, interpretation of the data, nor in the decision to publish.

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