**3. Results**

#### *3.1. Proteomics Profile of HO-1 Interactors in PCa Cells*

We have previously identified HO-1 molecular partners involved in cell–cell communication and cell adhesion through an integrative "omics" approach [28], establishing four molecular pathways (ANXA2/HMGA1/POU3F1; PLAT/PLAU; TMOD3/RAI14/VWF; NFRSF13/GSN). Further, we also described ANXA2, a protein associated with both bone physiology and in PCa bone progression [29,30], as an HO-1 interactor [20].

In light of the strong anti-tumoral role of HO-1 in PCa, we deepened our analyses in the search for other HO-1 interactors with clinical relevance for the disease. We carried out a proteomics analysis, in which PC3 cells, derived from a bone metastasis of PCa [31], were transfected with the HO-1 expression plasmid (pEBG-GST-HO-1) or with the empty vector as control (pEBG-GST) (Figure 1A). After 48 h, cells were treated with 200 μM H2O2 for 30 min, with the aim of generating an inflammatory and oxidative microenvironment similar to the one which characterizes PCa [7]. Subsequently, GST-HO-1 was immunoprecipitated together with its interactors. The eluates were digested for analysis by LC ESI-MS/MS (Figure 1A). We identified 41 HO-1 interactor proteins, including GSN (Gelsolin), FLNB (filamin B), 14-3-3 family proteins, TES (testin), TRIM28 (tripartite motif-containing 28), and SRSF3 (splicing factor rich in serine and arginine 3), with clinical relevance in PCa [32–37]. Supplementary Table S2 shows the differential proteins identified (GST-HO-1 vs. GST) together with the number of unique peptides, the name of the encoding gene, the score, and the percentage of coverage with respect to the complete sequence. Next, an interaction network was built using STRING [22] and Cytoscape [23], which is shown in Figure 1B. Significant protein hits (Supplementary Table S2) identified with more than 2 PSMs were selected for further analysis (Figure 1B, larger spheres). This filter resulted in the selection of 14 proteins. Among those, 11 proteins had been previously reported with nuclear localization, including tripartite motif-containing 28 (TRIM28), heterogeneous nuclear ribonucleoprotein A2/B1 (HNRNPA2B1), heat shock protein family B (small) member 1 (HSPB1), chromobox 1 (CBX1), chromobox 3 (CBX3), matrin 3 (MATR3), nucleophosmin 1 (NPM1), damage specific DNA binding protein 1 (DDB1), high mobility group AT-hook 1 (HMGA1), 14-3-3 zeta delta-like protein (14-3-3ζ/δ) and zinc finger CCCH-type containing, antiviral 1 (ZC3HAV1) [22] (Figure 1B, pink spheres). The gene ontology (GO) for the top biological processes (BP) categories of the genes encoding for the HO-1 interactor proteins with nuclear localization included response to stress and cellular response to DNA damage stimulus (Figure 1C).

#### *3.2. Gene Correlation between HMOX1 and the Genes Encoding for HO-1 Interactors with Nuclear Localization in PCa Cells*

To study the relevance of the HO-1 interactors in PCa, we assessed the clinical significance of these nuclear factors by using the GSE70770 dataset [25], which gathers transcriptomic and clinical data from PCa patients who had undergone radical prostatectomy and clinical follow-up of 9 years, including biochemical relapse. Next, we performed pairwise correlation analyses between *HMOX1* and the genes encoding for the 11 selected nuclear localized HO-1 interactors (Figure 2B). Results show a significant and positive intermediate/strong Spearman correlation between *HMOX1* and 6 of those genes (r = 0.4, *p* < 0.0001 for *HNRNPA2B1*; r = 0.3, *p* < 0.0001 for *HSPB1*; r = 0.4, *p* < 0.0001 for *NPM1*; r = 0.5, *p* < 0.0001 for *DDB1*; r = 0.6, *p* < 0.0001 for *HMGA1*; and r = 0.4, *p* < 0.0001 for *ZC3HAV1*). Interestingly, *HMOX1* only correlated negatively with *YWHAZ* (r = −0.4, *p* < 0.0001) (Figure 2A,B).

**Figure 1.** Construction of the HO-1 interactome in PCa cells. (**A**) Simplified schematic workflow of the construction of the HO-1 interactome in PC3 cells. GST-HO-1 immunoprecipitation assays were performed from PC3 cell extracts that had been previously treated with H2O2 (200 μM, 1 h). For the LC ESI-MS/MS analysis, peptides were desalted and concentrated on a C18 resin. Tandem mass spectra were extracted using the Xcalibur software. All MS/MS samples were analyzed by using Mascot. (**B**) Interactions network for the GST-HO-1 binding proteins. The network was built with Cytoscape 3.7.0. using the interactions data obtained with STRING, using a minimum combined interaction score of 0.400. The protein size increases proportionally to the PSMs obtained in the LC ESI-MS/MS analysis. Proteins in pink are proteins identified with PSM > 2 and previously reported in the cell nucleus. (**C**) Gene ontology (GO) (biological processes) categories significantly dysregulated for genes encoding for HO-1 interactor proteins with nuclear localization. The color gradient shows the adjusted *p*-value. FDR = false discovery rate.

#### *3.3. Clinical Relevance of HO-1 Interactors with Nuclear Localization in PCa*

In order to evaluate the clinical relevance of the HO-1 interactors that have been previously reported in the nucleus, we analyzed the biochemical relapse-free survival (RFS) of PCa patients associated with the expression of the genes encoding for those proteins. The analyzed genes were the ones which showed a significant (*p* < 0.05) and intermediate (0.30 < |r| < 0.66) or strong (|r| ≥ 0.66) mRNA correlation with *HMOX1* (*HNRNPA2B1, HSPB1, NPM1, DDB1, HMGA1, ZC3HAV1,* and *YWHAZ*). We also included *HMOX1* expression in this analysis. We plotted KM curves for each individual gene assessed (Figure 3). Patients were stratified into two groups based on the expression levels for each gene: high and low expression. The analysis showed that high expression of

*HNRNPA2B1*, *HSPB1*, *NPM1*, *DDB1*, *HMGA1*, *ZC3HAV1*, and *HMOX1* was associated with an increased RFS in PCa patients (HR = 0.467, Cox *p* = 0.003 for *HNRNPA2B1* (Figure 3A); HR = 0.364, Cox *p* = 0.001 for *HSPB1* (Figure 3B); HR = 0.377, Cox *p* < 0.0001 for *NPM1* (Figure 3C); HR = 0.52, Cox *p* = 0.01 for *DDB1* (Figure 3D); HR = 0.361, Cox *p* < 0.0001 for *HMGA1* (Figure 3E); HR = 0.266, Cox *p* < 0.0001 for *ZC3HAV1* (Figure 3F); and HR = 0.505, Cox *p* = 0.018 for *HMOX1* (Figure 3H)). However, high expression of *YWHAZ* was associated with a lower RFS (HR = 3.942, Cox *p* < 0.0001) (Figure 3G).

**Figure 2.** Gene correlation. (**A**) Pairwise Spearman correlation matrix analysis between *HMOX1* and all of the genes encoding for the HO-1 interactor proteins which were previously reported in the cell

nucleus (*TRIM28*, *HNRNPA2B1*, *HSPB1*, *CBX1, CBX3*, *MATR3*, *NPM1*, *DDB1*, *HMGA1*, *YWHAZ*, and *ZC3HAV1*). Rounded Spearman correlation values are included inside each color box. Color scale ranges from blue (r = −1) to white (r = 0) to red (r = 1). (**B**) Spearman correlation analysis for *HMOX1* and the genes encoding for the HO-1 interactor proteins, which were previously reported in the cell nucleus (*TRIM28*, *HNRNPA2B1*, *HSPB1*, *CBX1*, *CBX3*, *MATR3*, *NPM1*, *DDB1*, *HMGA1*, *YWHAZ*, and *ZC3HAV1*). Statistical significance was set at *p* < 0.05.

**Figure 3.** Relapse-free survival (RFS) of PCa patients using the Ross–Adams dataset, GSE70770, n = 206. (**A**–**H**) Kaplan–Meier curves for RFS of PCa patients segregated based on the gene expression levels for *HNRNPA2B1* (**A**), *HSPB1* (**B**), *NPM1* (**C**), *DDB1* (**D**), *HMGA1* (**E**), *ZC3HAV1* (**F**), *YWHAZ* (**G**), and *HMOX1* (**H**). RFS of patients with high (purple lines) vs. low (green lines) expression for each gene. HR = hazard ratios (95% confidence interval). All comparisons consider low expression patients as the reference group. Cox *p* = Cox proportional hazard model *p*-value. Statistical significance was set at Cox *p* < 0.05.

Next, we furthered our analysis by plotting the gene expression ratio between each of these seven genes and *HMOX1* in biochemical relapse (BCR) patients compared with non-BCR. Results show that *HNRNPA2B1/HMOX1*, *NPM1/HMOX1*, and *YWHAZ/HMOX1* were significantly higher in BCR compared with non-BCR patients (*p* = 0.028 for *HN-RNPA2B1/HMOX1* (Figure 4(Ai)); *p* = 0.018 for *NPM1/HMOX1* (Figure 4(Ci)); *p* < 0.0001 for *YWHAZ/HMOX1* (Figure 4(Gi))). Next, we plotted the RFS stratifying patients according to their gene expression ratio. PCa patients with higher *HSPB1/HMOX1*, *DDB1/HMOX1*, and *YWHAZ*/*HMOX1* showed a worse RFS compared with patients with lower ratios (HR = 2.291, Cox *p* = 0.006 for *HSPB1/HMOX1* (Figure 4(Bii)), HR = 1.888, Cox *p* = 0.014 for *DDB1/HMOX1* (Figure 4(Dii)), and HR = 3.764, Cox *p* < 0.0001 for *YWHAZ*/*HMOX1* (Figure 4(Gii))). Results evidenced that increased *HMOX1* expression in combination with high expressions of *HSPB1*, *DDB1*, and *YWHAZ*, improves RFS for PCa patients.

**Figure 4.** Expression ratios between *HNRNPA2B1*, *HSPB1*, *NPM1*, *DDB1*, *HMGA1*, *ZC3HAV1*, *YWHAZ*, and *HMOX1* and their association with biochemical relapse (BCR) and relapse-free survival

(RFS) using the Ross-Adams dataset, GSE70770, n = 206. (**i**) Violin plots depicting *HNRNPA2B1/HMOX1* (**A**), *HSPB1/HMOX1* (**B**), *NPM1/HMOX1* (**C**), *DDB1/HMOX1* (**D**), *HMGA1/HMOX1* (**E**), *ZC3HAV1/HMOX1* (**F**), and *YWHAZ/HMOX1* (**G**) expressions in BCR vs. non-BCR patients. (**ii**) Kaplan–Meier curves for RFS of PCa patients segregated based on the gene expression *HNRNPA2B1/HMOX1* (**A**), *HSPB1/HMOX1* (**B**), *NPM1/HMOX1* (**C**), *DDB1/HMOX1* (**D**), *HMGA1/HMOX1* (**E**), *ZC3HAV1/HMOX1* (**F**), and *YWHAZ/HMOX1* (**G**) ratios. RFS of patients with high (red lines) vs. low (blue lines) expression for each ratio. HR = hazard ratios [95% confidence interval]. All comparisons consider low expression patients as the reference group. Cox *p* = Cox proportional hazard model *p*-value. Statistical significance was set at Cox *p* < 0.05. \* *p* < 0.05; \*\*\* *p* < 0.001.

We then performed a Cox regression analysis and built a prognostic model for predicting the RFS, based on the expression (Expr) and coefficient (Coef) values of *HMOX1* and each of the three genes whose higher ratios with *HMOX1* showed a significant decrease in RFS compared with patients with lower ratios (*YWHAZ*, *DDB1*, and *HSPB1*) (Table 1). The risk score was calculated as follows: = *<sup>n</sup>* ∑ *i*=1 (*Coefi* × *Expri*). On the basis of the result for each PCa patient, the GSE70770 dataset was divided into two groups (high-risk group and low-risk group) according to the optimal cutoff value. Interestingly, we observed that patients with higher risk scores had a worse clinical outcome than patients with lower risk scores (HR = 4.807, Cox *p* < 0.0001) (Figure 5A).

**Table 1.** Detailed information of HO-1 interactors for the risk score model.


To validate *HSPB1*, *DDB1*, *YWHAZ*, and *HMOX1*'s clinical relevance in PCa, multivariable analyses were performed in the presence of clinic-pathological parameters previously associated with increased PCa relapse risk. These parameters included Gleason score (GS), PSA levels, patients' clinical and pathological stages, and *HMOX1* expression. *HSPB1* behaved independently from the patients' GS, PSA levels, clinical and pathological stages, and *HMOX1* expression (*p* = 0.03 for GS; *p* = 0.001 for PSA; *p* = 0.002 for the clinical stage; *p* = 0.005 for the pathological stage; *p* = 0.002 for *HMOX1* (Figure 5B)). *DDB1* behaved independently from the patients' GS, PSA levels, and clinical and pathological stages (*p* = 0.035 for GS; *p* = 0.018 for PSA; *p* = 0.014 for the clinical stage; and *p* = 0.001 for the pathological stage (Figure 5C)). When we further adjusted the model to include all variables simultaneously, the associations remained significant (*p* = 0.022) (Figure 5C). Further, *YW-HAZ* behaved independently from the patients' GS, PSA levels, clinical and pathological stages, and *HMOX1* expression (Figure 5D). When analyzing all variables simultaneously, the associations remained significant (*p* = 0.01) (Figure 5D). Figure 5E depicts *HMOX1* independence from all of the clinic-pathological parameters previously analyzed (*p* = 0.027 for GS; *p* = 0.019 for PSA; *p* = 0.019 for the clinical stage; *p* = 0.004 for the pathological stage; *p* = 0.023 for all the variables simultaneously (Figure 5E)). Altogether, the multivariable analyzes add support to the independence of variables to predict the patient outcome.

**Figure 5.** Risk score regression model and multivariable analyses for *HSPB1*, *DDB1*, *YWHAZ,* and *HMOX1* in PCa patients. (**A**) Kaplan–Meier curve for RFS in high-risk (purple lines) and low-risk

(green lines) groups, according to a risk score model based on the expression of *HSPB1*, *DDB1*, *YWHAZ*, and *HMOX1* in PCa patients. (**B**–**D**) Multivariable analyses presented by forest plots between each gene (*HSPB1* (**B**), *DDB1* (**C**), *YWHAZ* (**D**), and *HMOX1* (**E**)) and GS, PSA, clinical and pathological stage, *HMOX1* s expression, or all the variables together. Univariable analysis (light blue); multivariable analysis with GS (light green) = adjusted for the GS (6; 7 (3 + 4); 7 (4+3); 8-10); multivariable analysis with PSA (pink) = adjusted for the PSA serum levels at diagnosis (PSA (ng/mL): <4; 4-10; > 10); multivariable analysis with the clinical stage (dark green) = adjusted for the clinical stage; multivariable analysis with the pathological stage (red) = adjusted for the pathological stage; multivariable analysis with *HMOX1* (grey) = adjusted for the expression of *HMOX1*; multivariable analysis (purple) = adjusted for all the variables simultaneously. HR = hazard ratios (95% confidence interval). All comparisons consider low expression patients as the reference group. Cox *p* = Cox proportional hazard model *p*-value. Statistical significance was set at Cox *p* < 0.05.

#### *3.4. Validation of the Interaction between HO-1 and 14-3-3ζ/δ in PCa Cells*

Considering that: (1) *YWHAZ* has been reported to be an independent and strong predictor of aggressiveness in PCa [38]; (2) its expression showed a significant negative correlation with *HMOX1* expression; (3) PCa patients with high *YWHAZ/HMOX1* showed the highest HR; and (4) high *YWHAZ* was the only factor significantly associated with a higher risk of relapse; we validated the interaction between HO-1 and 14-3-3ζ/δ. PC3 cells were treated with H2O2 or vehicle, and protein eluates were subjected to co-immunoprecipitation. As seen in Supplementary Figure S1A, HO-1 and 14-3-3ζ/δ interact in PC3 cells.

In addition, by confocal microscopy, HO-1 and 14-3-3ζ/δ co-localization was evaluated in PC3 cells treated with H2O2 or vehicle. The images displayed in Supplementary Figure S1B showed that HO-1 and 14-3-3ζ/δ co-localize in the cell nucleus under the induction of HO-1 with H2O2 (Supplementary Figure S1B). When analyzing the Manders and Pearson co-localization coefficients, a significant increase is observed in PC3 cells treated with H2O2 compared with controls (Supplementary Figure S1C).

Altogether, we confirmed for the first time the interaction between HO-1 and 14-3-3ζ/δ, highlighting them as critical players in PCa, and potential targets for clinical intervention.

#### **4. Discussion**

HO-1 is a key player in the cellular defense system against pro-oxidative and proinflammatory insults [10]. Regarding its role in pathological conditions, this protein is commonly considered as a survival molecule that plays an important role in cancer [10]. However, there is controversy about its role in tumor development and progression, possibly because its expression profile is associated with the type of tissue in question and depends, in turn, on the context or the tumor microenvironment.

Previous reports from our laboratory documented for the first time that HO-1 is expressed in human primary prostate carcinomas and is localized in the cell nucleus [18]. In PCa cell lines, we found that the pharmacological and genetic induction of HO-1 inhibits proliferation, migration, and invasion in vitro; further, it slows down tumor growth, limits tumor-associated angiogenesis [12] and neovascularization [15], and boosts the antitumor response in vivo [15].

Given the pleiotropic anti-tumoral role of HO-1 in PCa, in this work, we set out to evaluate whether HO-1 interacted with proteins previously described with nuclear localization, enabling it to reprogram prostate tumor cells fate, favoring the acquisition of a less aggressive phenotype. After generating oxidative stress conditions, HO-1 coimmunoprecipitates were subjected to LC ESI-MS/MS, identifying 11 proteins reported with nuclear localization. Interestingly, GO analyses showcased response to stress and cellular response to DNA damage stimulus as the top significant biological processes categories, highlighting the non-canonical HO-1 potential nuclear function in PCa.

Our next aim was to evaluate the clinical relevance of such interactors in association with HO-1 in PCa patients that had undergone radical prostatectomy (GSE70770) [25]. Gene expression correlation analyses showed a significant and positive Spearman correlation between *HMOX1* and *HNRNPA2B1*, *HSPB1*, *NPM1*, *DDB1*, *HMGA1*, and *ZC3HAV1*. Of note, *HMOX1* and *YWHAZ* showed a significant negative correlation.

We next set out to study whether the ratios of *HNRNPA2B1*/*HMOX1*, *HSPB1*/*HMOX1*, *NPM1*/*HMOX1*, *DDB1*/*HMOX1*, *HMGA1*/*HMOX1*, *ZC3HAV1*/*HMOX1*, and *YWHAZ*/ *HMOX1* might affect patients RFS. Results confirmed that PCa patients with higher *HSPB1*/*HMOX1*, *DDB1*/*HMOX1*, and *YWHAZ*/*HMOX1* showed a worse RFS, highlighting the protective role of HO-1.

Further, we computed a risk score constituted by *HSPB1*, *DDB1*, *YWHAZ*, and *HMOX1,* evidencing a decrease in the RFS of patients with higher risk scores. Multivariable analyses supported the independence of variables to predict the patient outcome.

Interestingly, it has been reported that HSPB1 correlates with the overall survival of patients with several types of cancer. Particularly, HSPB1 induction is associated with highly aggressive disease and poor clinical outcomes in PCa. At early tumor stages, HSPB1 expression is inhibited, but it is re-expressed during PCa progression, leading to a more aggressive phenotype [39,40]. On the other hand, Zoubeidi et al. described a novel cooperative interaction between AR and HSPB1 that enhances AR stability and transcriptional activity, thereby increasing prostate cancer cell survival [41].

DDB1 is involved in DNA repairing and has been related to tumor suppression [42,43]. Regarding its role as a member of the CUL4A-DDB1 E3 ligase complex, it promotes ubiquitination-dependent AR degradation. Accordingly, DDB1 and AR protein levels negatively correlate in PCa cells [44]. 14-3-3ζ/δ is an adapter protein encoded by *YWHAZ*. Members of the 14-3-3 protein family are involved in the regulation of a wide spectrum of signaling pathways by binding to various proteins [45] and contributing to the regulation of crucial cellular processes such as protein trafficking, malignant transformation, and differentiation [46,47]. Particularly, the ζ/δ isoform constitutes a potential prognostic and therapeutic target since its high expression correlates with the progression of different cancers [38,48,49]. Previous studies from our group demonstrated *YWHAZ* relevance as a prognostic factor independent from the clinic-pathological parameters associated with the disease, such as age, GS, and TMPRSS2-ERG fusion [38]. Moreover, we observed that PCa patients with amplification, or increased mRNA or protein levels for *YWHAZ*, have significant alterations in key DNA repair genes [38].

In terms of PCa immunotherapy, different therapeutic mechanisms have been described for these 3 HO-1 interactors: HSPB1, DDB1, and 14-3-3ζ/δ. It has been reported a novel immune escape mechanism mediated by HSPB1, in which this protein expressed in the breast tumor microenvironment, promotes the differentiation of monocytes to macrophages with immune-tolerogenic phenotypes, which, in turn, trigger severe anergy in T-cells [50]. DDB1 has been proposed as a key factor for immunomodulatory drug sensitivity [51]. Moreover, DDB1 has been identified in high-throughput analyses as a potential target, showing sensitivity to Poly(ADP-ribose) polymerase (PARP) inhibition [52]. This therapeutic avenue might be combined together with different immunomodulatory drugs, enhancing their effect [53]. In the case of 14-3-3ζ/δ, Yu et al. [54] reported that immune-associated genes involved in interferon signaling, TLR-4 signaling, inflammasome network, antigen presentation/TCR recognition, and CD28 co-stimulation were found significantly downregulated in patients with urothelial carcinomas of the urinary bladder (UCUBs) presenting *YWHAZ* amplification/overexpression. However, there is no evidence of *YWHAZ* immunomodulation in PCa. Further studies are required in order to determine whether an anti-*YWHAZ* approach might be a useful strategy for improving the therapeutic efficacy of immunotherapy in PCa.

Remarkably, 14-3-3 proteins can affect various physical and functional aspects of their targets, such as: blocking nuclear localization or export signals affecting their subcellular localization, blocking their binding to other proteins, affecting their stability, and modulating their catalytic activity by modifying their conformation [47,55–58]. In most cases, the binding of 14-3-3 sequesters the target protein in a particular subcellular compartment, and the release of 14-3-3 allows the protein to relocate. Likewise, recently Chen et al. [58] revealed

through in vitro and in vivo studies that 14-3-3ζ/δ promotes invasion and metastasis of non-small-cell lung cancer by binding to the soluble form of β-catenin phosphorylated at Ser552, protecting it from ubiquitin-mediated degradation, suggesting a novel mechanism by which β-catenin accumulates in the cytoplasm and remains protected from degradation in tumoral cells. Hence, 14-3-3ζ/δ promotes the activation of the Wnt pathway and the transcriptional activity of β-catenin, which enters the nucleus and interacts with the TCF/LEF complexes, inducing epithelial-mesenchymal transition, proliferation, and cell migration. Furthermore, it is widely accepted that the canonical Wnt pathway modulates osteoblast function and participates in the induction of the osteoblastic phenotype in PCa bone metastasis [59]. These results show the importance of the 14-3-3ζ/δ/β-catenin axis in PCa progression.

In this work, we confirmed the HO-1/14-3-3ζ/δ interaction, performing a co-immunop recipitation assay. Further, through an immunofluorescence assay, we determined that HO-1 and 14-3-3ζ/δ co-localize in the nucleus of PCa cells under oxidative stress conditions. Remarkably, this is the first time that this interaction has been reported in this type of cell. Although Song et al. reported this interaction in hepatocellular carcinoma [55], they only observed co-localization of both proteins in the cell cytoplasm, demonstrating that the interaction inhibited the ubiquitination and subsequent degradation of HO-1, facilitating its stability.

#### **5. Conclusions**

In summary, the results obtained in this study describe for the first time the interaction between HO-1 and HSPB1, DDB1, and 14-3-3ζ/δ in PCa cells. Further work will be necessary to identify the fine molecular mechanisms tuning HO-1 towards the acquisition of a less aggressive phenotype and to delineate the role of its interactions in PCa.

**Supplementary Materials:** The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/antiox11020290/s1, Figure S1: Validation of the interaction between 14-3-3ζ/δ and HO-1 by co-immunoprecipitation and fluorescence microscopy; Table S1: Ross-Adams patients' characteristics at baseline (start of the follow-up survival analyses); Table S2: Differential proteins identified by mass spectrometry in GST-HO-1 vs. GST.

**Author Contributions:** Conceptualization, S.L.-V., P.S., J.C., E.V. and G.G.; methodology, S.L.-V., P.S., J.B., A.S., R.L., N.A., E.L., A.P., M.P.V., J.C., E.V. and G.G.; software, P.S., J.B. and M.P.V.; validation, S.L.-V., P.S., J.B., M.P.V., J.C., E.V. and G.G.; formal analysis, S.L.-V., P.S., J.B., M.P.V., J.C., E.V. and G.G.; investigation, S.L.-V., P.S., J.B., M.P.V., J.C., E.V. and G.G.; resources, N.N., M.P.V., J.C., E.V. and G.G.; data curation, S.L.-V., P.S., J.B., M.P.V., J.C., E.V. and G.G.; writing—original draft preparation, S.L.-V., P.S., J.C., E.V. and G.G.; writing—review and editing, S.L.-V., P.S., J.B., A.T., A.S., R.L., N.A., E.L., A.P., N.N., M.P.V., J.C., E.V. and G.G.; visualization, S.L.-V., P.S., J.C., E.V. and G.G.; supervision, J.C., E.V. and G.G.; project administration, J.C., E.V. and G.G.; funding acquisition, N.N., J.C., E.V. and G.G. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Agencia Nacional de Promocion de la Investigacion, el Desarrollo Tecnologico y la Innovación (ANPCyT), Argentina: PICT-2016-0056, PICT-RAICES-2018- 02639; PICT-2019-2019-03215; and Universidad de Buenos Aires, Argentina: 20020170100585BA.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data is contained in the manuscript and supplementary file.

**Conflicts of Interest:** The authors declare no conflict of interest.
