Lipocalin 2 Reduces MET Levels by Inhibiting MEK/ERK Signaling to Inhibit Nasopharyngeal Carcinoma Cell Migration

Simple Summary Lipocalin-2 (LCN2) participates in multiple cellular processes, such as proliferation, survival, migration, invasion and inflammation. However, the molecular mechanisms involved in the LCN2-mediated cancer metastasis of human nasopharyngeal carcinoma remain poorly understood. Our study found that LCN2 negatively controlled cell invasion and metastasis by increasing the expression level of MET in NPC cells. Abstract Nasopharyngeal carcinoma (NPC) is the most common cancer that occurs in the nasopharynx, and it is difficult to detect early. The main cause of death of NPC patients is cancer metastasis. Lipocalin 2 (LCN2) has been shown to be involved in a variety of carcinogenesis processes. Here, we aimed to study the role of LCN2 in NPC cells and determine its underlying mechanism. We found that LCN2 was expressed differently in NPC cell lines, namely HONE-1, NPC-39, and NPC-BM. The down-regulation of LCN2 levels by siRNA targeting LCN2 (siLCN2) increased cell migration and invasion in HONE-1 cells, while the up-regulation of LCN2 levels by transfection with the LCN2 expression plasmid decreased cell migration and invasion in NPC-BM cells. Furthermore, LCN2 levels negatively regulated the phosphorylation of MEK/ERK pathways. The treatment of the specific MEK/ERK inhibitor, U0126, reduced cell migration in HONE-1 cells, whereas the treatment of tBHQ, an ERK activator, enhanced cell migration in NPC-BM cells. Based on the bioinformatics data, there was a moderately negative correlation between LCN2 and MET in metastatic NPC tissues (r = −0.5946, p = 0.0022). Indeed, the manipulation of LCN2 levels negatively regulated MET levels in these NPC cells. The treatment of U0126 reduced siLCN2-increased MET levels, while the treatment of tBHQ enhanced LCN2-enhanced MET levels. Interestingly, the down-regulation of MET levels by siMET further decreased siLCN2-enhanced MET levels and cell migration. Therefore, LCN2 inhibits NPC cell migration by reducing MET levels through MEK/ERK signaling.


Introduction
Nasopharyngeal carcinoma (NPC) is a malignant epithelial carcinoma of the head and neck region that occurs much more frequently in Southeast Asia [1]. Epidemiological data shows that NPC can occur in all age groups and is most commonly diagnosed in adults between the ages of 40 and 50 years old [2]. The incidence and mortality rate of NPC are two-to three-times higher in males compared with females. The main causes of death in NPC patients are local recurrence and distant metastasis. NPC is staged from stage 0 streptomycin and 10 U/mL penicillin (Invitrogen Life Technologies, Carlsbad, CA, USA) and cultured at 37 • C in a humidified atmosphere of a 5% CO2 incubator.

Small Interfering RNA (siRNA) Transfection
The human siRNAs targeting LCN2 (siLCN2) or MET (siMET) and negative control (NT) were purchased from Applied Biosystems Instruments (Foster City, CA, USA). The siLCN2 and siMET sequences targeted LCN2 (NM_005564) CCUCCGUCCUGUUUAG-GAAttUUCCUAAACAGGACGGAGGtg and MET (NM_000245) GCACUAGCAAAGUC-CGAGAttUCUCGGACUUUGCUAGUGCct, respectively. To silence the specific genes in HONE-1, siRNAs were transfected into cells using lipofectamine RNAiMAX reagents (Invitrogen Life Technologies) according to manufacturer instructions. Two days after transfection, these cells were used for the following experiments.

DNA Construction, Transient Transfection
Human full-length LCN2 and MET expression vectors were constructed as per our previous study [19]. Briefly, the open reading frame (ORF) of the human LCN2 genes from CaSki cells were amplified by polymerase chain reaction (PCR) using the forward primer 5 -GGATCCATGCCCCTAGGTCTCCTGT-3 followed by a BamHI site and the reverse primer 5 -CTCGAGCTCAGCCGTCGATACACT-3 followed by a stop codon and an XhoI site. The ORF of the human MET gene from pT3-EF1a-c-Met (addgene, Plasmid #31784) was amplified by PCR using the forward primer 5 -CCTGGTACCATGAAGGCCCCCGCTGTGCTT GCA-3 followed by a KpnI site and the reverse primer 5 -GAACTCGAGCTATGATGTCTC CCAGAAGGAGGCT-3 followed by an XhoI site. Both of the human LCN2 or MET cDNAs were cloned into the pcDNA expression vector. After cloning, the cDNA sequences were verified by DNA sequencing (Tri-I Biotech Inc., Taipei, Taiwan). The pcDNA-LCN2 expression vector, pcDNA-MET expression vector, or pcDNA control vector were transfected into NPC-BM cells using LipofectAMINE 2000 (Invitrogen) according to the manufacturer's protocols. Two days after transfection, these cells were used for the following experiments.

Cell Growth
Cell growth was assessed by a microculture tetrazolium colorimetric assay. At each respective time point, (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT) solutions were added into the treated cells for 2-4 h, and then the media were removed. The formazan crystals formed by the cells were dissolved using DMSO. The test wavelength at 570 nm and a reference wavelength at 630 nm absorbance was read by a spectrophotometer (Instruments, USA). The measured absorbance was the 630 nm background absorbance subtracted from the 570 nm measurement (OD570 nm-OD630 nm). The daily growth rates were calculated using the absorbance on day 1 as a baseline and measured for 4 days.

Cell Migration and Invasion Assays
Cell migration and invasion assays were performed using a Boyden chamber (pore size, 8 µm) (Neuro Probe, Cabin John, MD, USA) [20]. The Boyden chamber assay was used to study the migration (seeding cells with uncoated filter) and invasion (seeding cells with Matrigel-coated filter) abilities. After transfection, the cells were harvested and seeded in the upper chamber. The migrated and invaded cells were fixed by 100% methanol and stained with 10% Giemsa stain. The number of cells was counted using an Olympus CKX41 microscope (Olympus Corporation, Tokyo, Japan).

Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and Real-Time Quantitative PCR
Total RNAs were extracted using a Total RNA Mini Kit (Geneaid, New Taipei City, Taiwan) and reverse transcribed into complementary DNA (cDNA) using a high-capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). The cDNA synthesis and the PCR amplification assay (RT-PCR) were performed as described by previous studies [21]. Real-time quantitative PCR (qPCR) was performed using an ABI Cancers 2022, 14, 5707 4 of 15 7500 real-time PCR system (Applied Biosystems, Foster City, CA, USA) as per a previous study [19]. In brief, 20 µL of the reaction mixture was prepared with 10 µL 2× SYBR master mix, 1 µL primers, 2 µL cDNA and 7 µL nuclease-free water. The reaction mixture was initially denatured at 95 • C for 10 min followed by 40 cycles of denaturation at 95 • C for 15 s and annealing and extension at 60 • C for 30 s. For LCN2, the following forward (F) primers and reverse (R) primers were used: F: 5 -TGATCCCAGCCCCACCT-3 , R: 5 -CCAC TTCCCCTGGAATTGGT-3 . For MET, the following were used: F: 5 -ATACGGTCCTATGGCTGGTG-3 , R: 5 -TTGAGAGGTTCTTTCCACCAAGT-3 . The relative mRNA expression was analyzed through the Ct method and was normalized to GAPDH expression.

Western Blot Analysis
The total cell lysates were prepared as previously described in previous studies [22]. Briefly, an equal amount of proteins were fractionated on SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. The membranes were blocked using 5% skimmed milk in Tris buffer saline-0.1% Tween20 at room temperature for 1 h. After blocking, the membranes were incubated with various primary antibodies overnight at 4 • C. After washing, the membranes were incubated with the secondary antibody linked with horseradish peroxidase (Sigma-Aldrich, St. Louis, MO, USA) at room temperature for 1 h. After washing, the membranes were developed by an enhanced chemiluminescence kit (Sigma-Aldrich) and visualized by an imaging system. The intensity of the protein bands was measured and quantified was measured via an analysis system (AlphaImager 2000, Alpha Innotech Corporation, and San Leandro, CA, USA). The blots were normalized with their total protein levels. In the study, primary antibodies specific for p38, phosphorylated p38 and β-actin were obtained from BD Biosciences (San Jose, CA, USA). Primary antibodies specific for MET, MEK1/2, ERK1/2, JNK1/2, phosphorylated src, c-raf, MEK1/2, ERK1/2 and JNK1/2 were purchased from Cell Signaling Technology (Danvers, MA, USA). Primary antibodies specific for LCN2 were purchased from R&D Systems (Minneapolis, MN, USA).

Statistical Analysis
Statistically significant differences were calculated using one-way ANOVA followed by Tukey's test. When two groups were compared, the data were analyzed by using Student's t-test. Significance was set at p < 0.05. The presented values are the means standard deviation (SD) of at least three independent experiments.

NPC Cell Growth Is Not Affected by Differential LCN2 Levels
To study the role of LCN2 in NPC progression, we analyzed the dataset (GSE12452) from the Gene Expression Omnibus (GEO) database and found that the expression levels of LCN2 were significantly decreased in the tumor group (n = 31) compared with the normal group (n = 10) ( Figure 1A). In order to explore whether LCN2 was expressed in human NPC cell lines, the levels of LCN2 in three human NPC cell lines, HONE-1, NPC-39 and NPC-BM, were determined using RT-PCR, real-time qPCR and Western blot analysis. Among them, the HONE-1 cell line showed higher LCN2 mRNA and protein levels, while the NPC-BM cell line had lower LCN2 levels ( Figure 1B,C). To assess the effect of LCN2 levels on NPC cells, LCN2 expression was down-regulated by the transfection of siRNA targeting LCN2 (siLCN2) in the HONE-1 cells; conversely, LCN2 expression was up-regulated by the transfection of LCN2-expressing plasmids in the NPC-BM cells. As expected, both of the LCN2 mRNA and protein levels were efficiently reduced in the siLCN2-transfected HONE-1 cells compared to those in the HONE-1 cells transfected with control siRNA ( Figure 1D). The LCN2 mRNA and protein levels were significantly enhanced in the LCN2-overexpression NPC-BM cells compared to those in the control plasmid-transfected NPC-BM cells (pcDNA) ( Figure 1E). The whole Western blot can be found in Figure S1.
The effects of LCN2 levels on cell growth were first examined. As shown in Figure 1F,H, the reduced LCN2 levels in the HONE-1 cells did not interfere with the cell growth rates for up to 4 days, while the enhanced LCN2 levels in the NPC-BM cells also did not interfere with the cell growth rates ( Figure 1G,I). These data suggested that different LCN2 levels did not affect the cell growth of the NPC cells.

NPC Cell Migration and Invasion Are Negatively Regulated by LCN2 Levels
Next, we explored the effect of LCN2 levels on cell migration and invasion in the NPC cell lines. The knockdown of LCN2 levels significantly enhanced the migration and invasion of the HONE-1 cells (p < 0.05) (Figure 2A,B), while the overexpression of LCN2 levels significantly repressed the migration and invasion of the NPC-BM cells ( Figure 2C,D). To examine the direct effects of LCN2 protein levels on NPC migration, recombinant human LCN2 proteins (rhLCN2) were used in the NPC-BM cells. As shown in Figure 2E, the treatment of rhLCN2 efficiently suppressed the migration of the NPC-BM cells in a dose-dependent manner. These data demonstrated that the levels of LCN2 inversely correlated with the migration and invasion of the NPC cells. normal group (n = 10) ( Figure 1A). In order to explore whether LCN2 was expressed in human NPC cell lines, the levels of LCN2 in three human NPC cell lines, HONE-1, NPC-39 and NPC-BM, were determined using RT-PCR, real-time qPCR and Western blot analysis. Among them, the HONE-1 cell line showed higher LCN2 mRNA and protein levels, while the NPC-BM cell line had lower LCN2 levels ( Figure 1B,C). To assess the effect of LCN2 levels on NPC cells, LCN2 expression was down-regulated by the transfection of siRNA targeting LCN2 (siLCN2) in the HONE-1 cells; conversely, LCN2 expression was up-regulated by the transfection of LCN2-expressing plasmids in the NPC-BM cells. As expected, both of the LCN2 mRNA and protein levels were efficiently reduced in the siLCN2-transfected HONE-1 cells compared to those in the HONE-1 cells transfected with control siRNA ( Figure 1D). The LCN2 mRNA and protein levels were significantly enhanced in the LCN2-overexpression NPC-BM cells compared to those in the control plasmid-transfected NPC-BM cells (pcDNA) ( Figure 1E). The whole Western blot can be found in Figure S1. The effects of LCN2 levels on cell growth were first examined. As shown in Figure  1F,H, the reduced LCN2 levels in the HONE-1 cells did not interfere with the cell growth rates for up to 4 days, while the enhanced LCN2 levels in the NPC-BM cells also did not interfere with the cell growth rates ( Figure 1G,I). These data suggested that different LCN2 levels did not affect the cell growth of the NPC cells.

NPC Cell Migration and Invasion Are Negatively Regulated by LCN2 Levels
Next, we explored the effect of LCN2 levels on cell migration and invasion in the NPC cell lines. The knockdown of LCN2 levels significantly enhanced the migration and invasion of the HONE-1 cells (p < 0.05) (Figure 2A

LCN2 Reduces Cell Migration by Inhibiting the MEK/ERK Signaling
Previous studies have shown that mitogen-activated protein kinases (MAPKs), including ERK, p38 and Jun N-terminal kinase (JNK), play key roles in cell migration [23]. To explore the role of LCN2 in the molecular mechanisms of cell migration, we further analyzed these signaling molecules associated with cell migration. As shown in Figure 3A,B, the downregulation of LCN2 in the HONE-1 cells increased the phosphorylation levels of several signaling proteins, such as phospho-src (p-src), phospho-c-raf (p-c-raf), phospho-MEK (p-MEK), phospho-ERK (p-ERK) and phospho-p38 (p-p38) but not phospho-JNK (p-JNK), while the overexpression of LCN2 in the NPC-BM cells decreased the phosphorylation levels of these signaling proteins, including p-src, p-c-raf, p-MEK, p-ERK and p-p38. These data suggested that LCN2 may negatively regulate these motility-associated signal pathways, such the MEK/ERK signaling pathway, leading to a reduction in cell migration and invasion in the NPC cells.

LCN2 Reduces Cell Migration by Inhibiting the MEK/ERK Signaling
Previous studies have shown that mitogen-activated protein kinases (MAPK cluding ERK, p38 and Jun N-terminal kinase (JNK), play key roles in cell migration phospho-MEK (p-MEK), phospho-ERK (p-ERK) and phospho-p38 (p-p38) b phospho-JNK (p-JNK), while the overexpression of LCN2 in the NPC-BM ce creased the phosphorylation levels of these signaling proteins, including p-src, p-MEK, p-ERK and p-p38. These data suggested that LCN2 may negatively r these motility-associated signal pathways, such the MEK/ERK signaling pathway ing to a reduction in cell migration and invasion in the NPC cells. The NPC-B that were transfected with pcDNA or LCN2 plasmids and then treated with the ERK a tBHQ. After treatment, these cells were subjected to cell migration analysis. * p < 0.05 co with control. # p < 0.05 when compared with HONE-1 cells that were transfected with N U0126, or NPC-BM cells that were transfected with pcDNA plus tph. U0126 is a highly selective inhibitor of MEK [24] and is widely used as an in for the Ras/Raf/MEK/ERK pathway. To determine whether the LCN2-supp The migration-associated signal proteins, including their phosphorylation levels, were examined using Western blot analyses. (C) The HONE-1 cells that were transfected with siLCN2 or NT and then treated with the MEK/ERK inhibitor, U0126. After treatment, these cells were subjected to cell migration analysis. (D) The NPC-BM cells that were transfected with pcDNA or LCN2 plasmids and then treated with the ERK activator, tBHQ. After treatment, these cells were subjected to cell migration analysis. * p < 0.05 compared with control. # p < 0.05 when compared with HONE-1 cells that were transfected with NT plus U0126, or NPC-BM cells that were transfected with pcDNA plus tph.
Cancers 2022, 14, 5707 9 of 15 U0126 is a highly selective inhibitor of MEK [24] and is widely used as an inhibitor for the Ras/Raf/MEK/ERK pathway. To determine whether the LCN2-suppressed MEK/ERK signal pathway participates in the regulation of cell migration, HONE-1 cells with or without LCN2 knockdown were co-treated with U0126. The treatment of U0126 significantly reduced cell migration in the HONE-1 cells ( Figure 3C, left panel). Additionally, the treatment of U0126 also significantly decreased siLCN2-induced cell migration in the HONE-1 cells ( Figure 3C, right panel). Tert-butylhydroquinone (tBHQ) has been identified as an ERK activator [25]. The treatment of tBHQ enhanced cell migration in the NPC-BM cells with or without LCN2 overexpression ( Figure 3D). These data suggested that LCN2 downregulated cell migration by inhibiting the MEK/ERK signaling pathway.

LCN2 Negatively Regulated MET Levels by Inhibiting the MEK/ERK Signaling Pathway
Our recent study has shown that LCN2 suppresses MET expression to inhibit osteosarcoma cell metastasis [26]. We investigated the relationship between LCN2 and MET mRNA expression in samples from metastatic and non-metastatic NPC patients based on the microarray data (GSE103611) obtained from the GEO databases. We found a moderately negative correlation between LCN2 and MET mRNA in these NPC tissues (r = −0.3491, p = 0.015) ( Figure 4A). Further examination of the samples from metastatic NPC patients revealed a more significant negative correlation between LCN2 and MET mRNA (r = −0.5946, p = 0.0022) ( Figure 4B). Indeed, the MET mRNA and protein levels in the siLCN2-transfected HONE-1 cells were significantly enhanced ( Figure 4C-F), while the MET mRNA and protein levels in the LCN2-overexpression NPC-BM cells were reduced ( Figure 4C-F). The treatment of rhLCN2 proteins also suppressed the expression of MET proteins in the NPC-BM cells in a dose-dependent manner ( Figure 4G). Interestingly, the MET levels were repressed by the specific MEK/ERK inhibitor, U0126, in the HONE-1 cells transfected, or not, with siLCN2 ( Figure 4H), while the MET levels were slightly enhanced by the ERK activator, tBHQ, in the NPC-BM cells transfected, or not, with LCN2 ( Figure 4I). These data demonstrated that LCN2 negatively regulated MET levels through the MEK/ERK signaling pathway.

LCN2 Decreased Cell Migration by Reduced MET Expression
The level of MET in NPC tissues is directly and positively correlated with the clinical stage of NPC patients [27]. The downregulation of MET levels efficiently suppresses invasion and migration of NPC cells [28,29]. Thus, we further examined whether LCN2 negatively regulates cell migration by repressing MET levels. The MET protein was overexpressed in the NPC-BM cells ( Figure 5A). As expected, the overexpressed MET levels could enhance cell migration in the NPC-BM cells ( Figure 5B) similarly to previous studies. Conversely, the HONE-1 cells with reduced MET levels by a specific siRNA-targeting MET (siMET) exhibited a down-regulation of cell migration ( Figure 5C,D). It is worth noting that the reduced MET levels also decreased siLCN2-induced cell migration ( Figure 5C,D). The data suggested that LCN2 negatively regulated NPC cell migration by reducing MET levels.
of MET proteins in the NPC-BM cells in a dose-dependent manner ( Figure 4G). Interestingly, the MET levels were repressed by the specific MEK/ERK inhibitor, U0126, in the HONE-1 cells transfected, or not, with siLCN2 ( Figure 4H), while the MET levels were slightly enhanced by the ERK activator, tBHQ, in the NPC-BM cells transfected, or not, with LCN2 ( Figure 4I). These data demonstrated that LCN2 negatively regulated MET levels through the MEK/ERK signaling pathway.

Discussion
Local recurrence and distant metastasis are the main causes of death in patients with NPC. In this study, we demonstrated that LCN2 suppressed NPC cell migration by reducing MET levels through the MEK/ERK signaling pathway. In the NPC cells, LCN2 repressed the MET expression levels, leading to a reduction in the metastasis ability.
Here, we found that the manipulation of LCN2 expression levels did not affect cell growth in the HONE-1 and NPC-BM cell lines; however, Guo et al. showed that the overexpression of LCN2 inhibits cell proliferation in C666 and HNE-3 cell lines [10]. The possible reasons include: (1) The NPC-BM cell line is the first primary NPC cell line derived from a distant metastatic site, i.e., the bone marrow metastatic lesion [30]. It would help to study the real the progression of NPC. (2) Endogenous LCN2 expression of C666 and HNE-3 cells is higher than that of HONE-1 cells [10]. Ectopic overexpression of LCN2 in C666 and HNE-3 cells may induce other growth signals. (3) The C666-1 cell line is EBV-positive and shows a hypermutant phenotype. Therefore, the discrepancy in cell growth may be affected by the different cellular backgrounds. Regardless of the different cell growth results, our study and the study of Guo et al. [10] have shown that LCN2 negatively regulates the invasion and metastasis ability of NPC cells.
LCN2 has been show to participate in the initiation, progression and metastasis of various cancer types [31,32]. However, the role of LCN2 in the different processes of cancers is controversial [33,34]. In this study, we demonstrated that LCN2 negatively regulated the invasion and metastasis of NPC cells using the manipulation of LCN2 in a higher LCN2 expression of NPC-BM cells and lower LCN2 expression of HONE-1 cells. A recent study showed that a high expression of LCN2 was shown in NPC patients with a better prognosis using an immunohistochemical analysis of LCN2 tumor specimens [10]. Furthermore, LCN2 suppresses tumor metastasis in osteosarcoma and oral squamous cell carcinoma [35]. Using computational and immunohistochemistry examinations, LCN2 expression has been shown to be a suppressor of invasion and angiogenesis in six cancer types, such as bladder, colorectal, liver, lung, ovarian and pancreatic when compared to matched primary lesions [36]. On the contrary, LCN2 is positively associated with metastasis in breast cancer [37], anaplastic thyroid carcinoma cells [38] and prostate cancer [39]. LCN2 has also been shown to be unrelated to the metastasis of head and neck squamous cell carcinoma [40]. In addition, the overexpression of LCN2 is associated with radio-resistance and recurrence in NPC patients [9] Thus, the role of LCN2 in carcinogenesis and its underlying mechanism may depend on the various types of cancers.
We further identified that LCN2 negatively regulated the activation of several migrationassociated signal proteins, including src, c-raf, MEK, ERK and p38. Notably, we found that the LCN2 negatively regulated MET expression levels. An abnormal expression and activation of MET has been shown to promote the occurrence and progression of many cancer types [41]. Indeed, a high MET protein expression level correlates with poorer survival in late-stage NPC [27]. The expression of MET in NPC tissues from patients with lymph node metastasis was significantly higher than that in NPC tissues from NPC patients without lymph node metastasis [42]. It is currently known to affect the abnormal expression of MET and could promote the development and progression of NPC. Further research may explore how LCN2 affects MET expression during NPC metastasis, such as through transcriptional regulation or post-transcriptional modification.
Until now, many studies have demonstrated that several signal molecules may participate in the invasion and metastasis of NPC. For example, Capn4 promotes the invasion and metastasis of NPC [43]. Elevated serum c-Src levels are associated with a poor prognosis in NPC patients [44]. The expression of YBX3 is positively correlated with NPC metastasis [45]. An abnormal expression of COX-2 is associated with recurrence and a poor prognosis in NPC patients [46]. Additionally, the downregulation of TNFAIP3 is associated with distant metastasis and a worse patient prognosis [47]. In the present study, we found that a low LCN2 level increased the expression of MET, resulting in an increase in the invasion and metastasis ability of NPC cells. Future studies may explore the interaction of these molecules in clinical NPC samples and establish a novel prognostic panel for the early detection of metastatic NPC.
The current study still had some limitations. Here, we used a bioinformatic analysis of a nasopharyngeal carcinoma database to support the finding that LCN2 reduced MET levels to inhibit nasopharyngeal cancer cell migration. In the future, human nasopharyngeal carcinoma tissues or animal models could be used to further validate our findings.

Conclusions
In summary, we found that LCN2 negatively controlled cell invasion and metastasis by increasing the expression level of MET in NPC cells. To our knowledge, this is the first study to show that LCN2 inhibits MET expression, resulting in the prevention of NPC metastasis through a novel molecular mechanism. Our study indicated that LCN2 may be a novel target for monitoring and treating the metastatic ability of patients with NPC. The results from our study may help to develop new biomarker panels and provide therapeutic targets for metastatic NPC.

Data Availability Statement:
The datasets generated for this study are available on request to the corresponding authors.

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