Author contributions: M.M.K., P.S., S.K.S., P.O.Z., and A.L.M.: conception and design, collection and/or assembly of data, data analysis and interpretation, manuscript writing, and final approval of manuscript; S.L.: conception and design, data analysis and interpretation, and manuscript writing; J.G. and D.S.: provision of study material or patients, collection and/or assembly of data, and data analysis and interpretation; H.O.E.: financial support and administrative support; H.C.: provision of study material or patients; G.N.F. and F.F.L.: conception and design, provision of study material or patients, collection and/or assembly of data, and data analysis and interpretation; S.M.: conception and design, financial support, administrative support, data analysis and interpretation, manuscript writing, and final approval of manuscript. M.M.K. and P.S. contributed equally to this article. S.K.S. and P.O.Z. contributed equally to this article.
Disclosure of potential conflicts of interest is found at the end of this article.
First published online in STEM CELLSEXPRESS December 29, 2011.
Glioblastoma multiforme (GBM) tumors are the most common malignant primary brain tumors in adults. Although many GBM tumors are believed to be caused by self-renewing, glioblastoma-derived stem-like cells (GSCs), the mechanisms that regulate self-renewal and other oncogenic properties of GSCs are only now being unraveled. Here we showed that GSCs derived from GBM patient specimens express varying levels of the transcriptional repressor repressor element 1 silencing transcription factor (REST), suggesting heterogeneity across different GSC lines. Loss- and gain-of-function experiments indicated that REST maintains self-renewal of GSCs. High REST-expressing GSCs (HR-GSCs) produced tumors histopathologically distinct from those generated by low REST-expressing GSCs (LR-GSCs) in orthotopic mouse brain tumor models. Knockdown of REST in HR-GSCs resulted in increased survival in GSC-transplanted mice and produced tumors with higher apoptotic and lower invasive properties. Conversely, forced expression of exogenous REST in LR-GSCs produced decreased survival in mice and produced tumors with lower apoptotic and higher invasive properties, similar to HR-GSCs. Thus, based on our results, we propose that a novel function of REST is to maintain self-renewal and other oncogenic properties of GSCs and that REST can play a major role in mediating tumorigenicity in GBM. STEM CELLS2012;30:405–414
Glioblastoma multiforme (GBM) tumors are the most common malignant primary brain tumors in adults [1–3] However, despite decades of basic science and clinical research, the outcome for GBM patients remains dismal, with average median survival of only 12–16 months [4–15]. The lack of progress in GBM research can be attributed to the highly heterogeneous, invasive, necrotic, and chemotherapy- and radiation-resistant nature of GBM tumor cells . Thus, even after aggressive multimodal therapy, the invading tumor cells often survive and cause tumor relapse. The generation of invaluable animal models has provided new insights into GBM biology and alternate therapeutic approaches [17–19]. In addition, the recent advent of the “cancer stem cell” hypothesis has brought a new perspective to our understanding of GBM biology and therapy [16, 20–23]. According to this hypothesis, tumors contain stem-like cells, which have the capacity for long-term self-renewal. This hypothesis further proposes that stem-like cells are the main cancer-initiating cells due to their long-term self-renewal capacity. However, the mechanisms that lead to self-renewal and tumorigenicity in glioblastoma-derived stem-like cells (GSCs) are still unclear.
The transcriptional repressor REST was originally discovered as a repressor of many neuronal differentiation genes in non-neural cells and results derived from Rest mutant mice showed that it was indeed a repressor of neurogenesis [24–26]. However, more recent genome-wide analysis revealed that REST can bind to the chromatin of more than a thousand genes and can potentially affect many biological processes of different tissue types . Our previous studies indicated that REST maintains self-renewal of embryonic stem cells (ESCs)  and that this role of REST was dependent on the specific cell line as well as the cellular environment (unpublished data). More recently, REST was found to maintain stemness of normal adult neural stem cells . Our previous studies indicated that REST is involved in medulloblastoma tumorigenesis, in which it functions by blocking differentiation of neuronal stem/progenitor cells [30–32]. Thus, we reasoned that REST might have a critical role in maintaining self-renewal and other oncogenic properties of GSCs. Our studies indicate that REST indeed regulates such functions in GSCs. Our studies also suggest that targeting REST can potentially represent a novel therapeutic strategy in GBM.
MATERIALS AND METHODS
Cells and Cultures
GSCs used in this study were obtained by acute cell dissociation of human GBM surgical specimens from patients treated at The University of Texas MD Anderson Cancer Center, Houston, TX, as described previously [33–38]. The specific high REST-expressing GSCs (HR-GSC1, HR-GSC2) and/or low REST-expressing GSC (LR-GSC) lines have been described previously [33–38]. These cell lines were maintained under proliferation conditions in Dulbecco's modified Eagle's medium/F12 supplemented with B27 1:50 (Invitrogen, Carlsbad, CA, http://www.invitrogen.com), 20 ng/ml epidermal growth factor (Sigma), and 20 ng/ml basic fibroblast growth factor as neural stem cell-permissive medium, according to procedures described elsewhere [39, 40]. These cell lines form neurospheres and demonstrate the ability to express multiple lineage markers and reliably form tumors that recapitulate many of the pathological features of the original tumor after orthotopic xenografting [36, 37, 41].
Immunofluorescence and Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling (TUNEL) Assay
Cells were fixed with 4% paraformaldehyde in phosphate buffered saline (PBS) for 15 minutes. After washing with PBS, cells were blocked with 4% goat serum and 0.3% Triton X for 1 hour at room temperature. Primary antibodies were added overnight at 4°C, rabbit anti-REST (Sigma, 1:100), mouse anti-REST (1:100), Sox-2 (Stemgent, 1:50), rabbit anti-YKL40 (Quidel, 1:500). After washing with PBS, cells were incubated with Alexa fluor secondary antibodies (Invitrogen) for 1 hour at room temperature. Cells were mounted with mounting media containing 4,6 diamino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA, http://www.vectorlabs.com) and photographed using a Nikon TE2000 controlled with Metamorph software (Molecular Devices Corp., Union City, CA, http://www.moleculardevices.com).
Paraffin sections were hydrated through alcohol grades after clearing with CitriSolv (Fisher Scientific International, Hampton, NH, http://www.fisherscientific.com). Antigen retrieval was performed with citrate buffer at 90°C for 20 minutes. Sections were blocked with 4% goat serum followed by human specific primary antibody (Nestin 1:200 [Millipore, Billerica, MA, http://www.millipore.com]) overnight at 4°C. After washing twice with PBS, sections were incubated with secondary antibodies followed by mounting with DAPI. In vivo TUNEL assay was conducted as per manufacturer's recommendations and positive cells were counted from 10 different fields (Roche cat no 11684817910).
Neurosphere and Self-Renewal Assay
For GSCs, neurosphere assay was done as previously described [33–38]. In brief, cells were dissociated into single cell suspensions, 10 cells per well for self-renewal assay for multiple generations or 100 cells per well for single generation were seeded in 96-well plates, and the number of neurospheres was counted after 15 days. For serial neurosphere assay, cells were collected after the first passage and dissociated into single cells and plated at the same density as the first passage.
In Vitro Invasion Assay
Control and Rest altered lines were counted, and 5 × 103 cells were seeded into the upper compartment of either the invasion or control chambers; 10% fetal bovine serum (FBS) containing media was added to the lower chamber as chemo-attractant. Cells were incubated for 24 hours. The nonadherent cells were removed by washing with PBS, and the noninvading cells were removed by cotton swabs. The control and invasion membranes were then fixed with 100% methanol and stained with crystal violet. Cells were counted under the microscope and a ratio of invading cells on the Matrigel matrix to invasive cells on the control membranes of the control versus the Rest altered lines was compared.
Cell Transfections and Transductions
Stable knockdown of REST in GSCs was done using Mission-lentiviruses expressing short hairpin RNA (shRNA) (Sigma), either nontargeting shRNA (shNT) as control or targeting Rest (shRest). Cells were incubated the day prior to transduction with 8 μg/ml hexadimethrine bromide. Cells were then incubated with the virus particles at one multiplicity of infection in normal proliferation media for 48 hours. Cells were puromycin geneticin (G418, Invitrogen) (150 μg/ml) selected. Five individual clones for each line were selected, cultured, and REST knockdown efficiency was determined by Western blot analysis. The clone with maximum knockdown for each line (shRest construct no 1 for HR-GSC1: 42% knockdown; shRest construct no 3 for HR-GSC2: 70% knockdown) was selected for further experimentation.
GSCs were transfected with plasmids expressing a control vector or Rest using the Amaxa nucleofector kit. In brief, 4 × 106 cells were suspended in 90 μl of nucleofector solution with REST or control plasmids and then transfected using an Amaxa transfection device. Cells were puromycin (1.5 μg/ml) selected, and REST overexpression was confirmed by Western blot analysis.
RNA Preparation and Real-Time Polymerase Chain Reaction (qRT-PCR)
Total RNA was prepared using Trizol (Invitrogen) according to the manufacturer's instructions. Approximately 500 ng total RNA was used as a template for cDNA synthesis using the Verso cDNA kit (Thermo-Scientific, Waltham, MA). Real-time PCR was done using SYBR green master-mix (Applied Biosystems, Foster City, CA, http://www.appliedbiosystems.com) as per the manufacturer's instructions. All real-time PCR experiments were done on an ABI7900HT sequence detection system (Applied Biosystems).
Genome-Wide Expression Analysis of GSCs with Loss- and Gain-of-Function Manipulations
Total RNA was used and Affymetrix HG-133 Plus 2.0 whole genome microarrays were performed (LC sciences, Houston) for two HR-GSCs and one LR-GSC plus the corresponding control lines. Genes with differential fold expression in HR- versus LR-GSCs were determined using the Agilent GeneSpring GX 11.5 software. Robust multiarray average was used for normalization and genes greater than 1.5-fold differentially expressed were included to determine gene function and disease ontologies using ingenuity pathway analysis (http://www.ingenutiy.com).
Western Blot Procedures
Whole cell extracts were prepared from cell lines, and 30 μg protein was loaded on gradient sodium dodecyl sulfate polyacrylamide gel electrophoresis [(SDS)-PAGE] gel (Pierce, Rockford, IL, http://www.piercenet.com). Then proteins were electrophoretically transferred to nitrocellulose membranes. The membranes were probed with the following antibodies: Rabbit anti-REST (1:1000, Upstate), mouse anti-Sox2 (1:350, R&D Systems Inc., Minneapolis, MN, http://www.rndsystems.com), and rabbit anti-Nestin (Millipore, 1:10,000), mouse anti Bmi-1 (Millipore, 1:500), and goat anti-YKL40 (Santacruz, 1:200). Mouse anti-actin (1:10,000, Sigma) was used as the internal control. The membranes were incubated with the primary antibodies overnight before they were incubated with corresponding fluorescent-labeled secondary antibodies (1:15,000, Licor) for 1 hour at room temperature. The protein–antibody complexes were visualized by scanning the membrane using the Odyssey detection system (LI-COR Biosciences).
Proliferation assays for HR-GSC1 and HR-GSC2 cell lines were done using colorimetric bromodeoxyuridine (BrdU) cell proliferation assay kit (Roche Diagnostics, Basel, Switzerland, http://www.roche-applied-science.com, 11647229-01).The assay was performed according to manufacturer's instructions. In short, HR-GSC1 and HR-GSC2 cells were seeded at 3,000 cells per well in a 96-well plate, allowed them to grow for overnight, and incubated with BrdU labeling agent for 24 hours. Cells were fixed for 30 minutes and incubated with anti-BrdU for 2 hours, and the substrate was added and on color development, colorimetric measurements were performed at 492 nm.
Intracranial Tumorigenicity Assay
Intracranial transplantation of GSCs into nude male mice was done as described previously . A total of 56 animals (seven animals per treatment condition) were used. To standardize GSC transplantation into nude mice brain, a guide-screw system was implanted 2.5 mm lateral and 1 mm anterior to the bregma accepting a 26-gauge Hamilton syringe. GSCs cultured were collected and dissociated into single cells. Approximately, 50,000 cells (or 5,000 cells for Supporting Information Fig. 2) in 5 μl of media were simultaneously injected using an automated syringe pump (Harvard Apparatus). Animals were carefully monitored for survival.
Values are reported as mean ± SEM. Microsoft Excel 2010 and SPSS (IBM) software was used to conduct t test or analysis of variance for each of the experiments. Microarray data were analyzed with Agilent GeneSpring GX11.5 and the Genepattern suite (Broad Institute).
Patient-Derived Glioblastoma Stem Cells Show Varying Levels of the REST Protein
To understand the role of REST in GBM, we quantified REST protein expression in 10 patient-derived GSC lines. These lines showed varying levels of REST protein with several fold differences between the lines (Fig. 1A). High REST expression (HR-GSCs) coincided with high levels of Sox2, a known neural stem cell self-renewal regulator [42, 43], whereas low-REST expression (LR-GSCs) with low levels of Sox2, suggesting that REST plays a role in self-renewal of GSCs. Interestingly, only one of the GSC lines expressed YKL-40, a mesenchymal marker . To further understand the regulation of REST expression in GSCs, we measured the Rest transcript levels using real time RT-PCR. Comparison of the relative levels of REST protein and Rest transcript levels (Fig. 1B, left panel) and a scatter plot (Fig. 1B, right panel) are shown. The data indicated that the transcript levels did not correlate with the REST protein levels (correlation coefficient = .04, Fig. 1B). To investigate the lack of correlation between protein and RNA levels, we treated GSCs with the proteasomal inhibitor (MG132) for 24 hours. Our results indicated significant REST protein accumulation in most of the 11 different GSC lines after MG132 treatment compared with the control dimethyl sulfoxide (DMSO) treatment (Fig. 1C). These results strongly suggested that REST expression is controlled mainly at the protein degradation level in GSCs, similar to what was observed in other cell types [26, 44, 45].
REST Protein Levels Correlate with Expression of Stemness Markers and Neurosphere Formation Capacity of GSCs
We selected two GSC lines with high levels of REST, HR-GSC1 and HR-GSC2 (GSC11 and GSC23, respectively), and one line with low levels of REST, LR-GSCs (GSC20), for further characterization by Western blotting (Fig. 2A) and double-immunofluorescence (Fig. 2B) analysis. As shown, both HR-GSC lines, compared with LR-GSCs, showed higher expression of the neural stemness markers Nestin, Sox2, and Bmi1 and absence of YKL-40. These findings further confirmed the association of REST with stemness markers of GSCs.
To determine whether high levels of REST in GSCs corresponded to increased functional stem-like properties, we measured their self-renewal efficiency using neurosphere assays. Our results showed greater neurosphere formation ability of both HR-GSC1 and HR-GSC2 compared with LR-GSCs (Fig. 2C). Thus, our data indicated that high REST expression is associated with high stemness of GSCs both at marker and functional levels.
REST Maintains Self-Renewal of GSCs
To determine the functional role of REST in GSCs, we generated stable lines expressing either shNT control or shRest in HR-GSC1 and HR-GSC2. As shown in Figure 3A, 3B, Western blot analysis confirmed REST protein knockdown in the shRest clones in both HR-GSC lines. The knockdown of REST corresponded to increased expression of neuronal β-tubulin, a direct target gene of REST [46, 47], indicating that shRest caused a functional REST knockdown. The loss of REST also produced concomitant loss of the stemness regulator Sox2. To determine whether loss of REST resulted in functional loss of stem-like properties of GSCs, we performed self-renewal assays in both HR-GSC lines. Our results showed that loss of REST resulted in decreased self-renewal in both the high REST lines over multiple passages (Supporting Information Fig. 1A for HR-GSC1 and Fig. 1B for HR-GSC2), indicating that REST maintains self-renewal in both HR-GSC lines.
To further establish the role of REST in GSCs, we performed complementary experiments in which we transfected LR-GSCs with expression vectors encoding either nontargeting control or Rest, confirmed the increased REST protein expression by Western blot, and determined the cells' self-renewal capacity (Fig. 3C). As shown, higher REST expression produced lower expression of its target gene neuronal β-tubulin and caused a significant increase in self-renewal. As expected from the loss-of-function experiments, additional REST caused an increase in the expression of Sox2 levels (Supporting Information Fig. 2). Thus, taken together, these results showed that REST maintains self-renewal of GSCs.
HR-GSCs and LR-GSCs Produce Distinct Brain Tumors in Mice
To determine the tumor-producing properties of the HR- and LR-GSCs, we transplanted HR-GSC1 and LR-GSC cells (5 × 104 cells) into the brains of nude mice and performed mouse survival assays (Fig. 4A) and histopathological examination of the resultant tumors (Fig. 4B). HR-GSC1 cells produced shorter survival in mice compared with LR-GSCs. This corresponded to their respective self-renewal capacity (Fig. 2C). While both HR- and the LR-GSC lines produced tumors, HR-GSC1 tumors morphologically showed a marked difference in growth pattern when compared with the LR-GSC tumors. The HR-GSC1 tumors were more diffusely infiltrative than the LR-GSC tumors, which grew as relatively circumscribed expansive masses.
REST Regulates Survival in Orthotopic Mouse Models
We performed loss-of-function experiments, in which we transplanted 5 × 104 cells of the stable lines of HR-GSC1 (Fig. 4C) and HR-GSC2 (Fig. 4D) expressing either shRest or shNT into the brains of nude mice and assayed for their survival. Under these conditions, shRest treatment, when compared with shNT treatment, increased the survival of tumor-bearing mice. Although the effect in mouse survival was statistically significant, it was not robust and perhaps points to the notion that survival depended on multiple factors. To further confirm that these experiments were performed under conditions, in which the survival correlated with the number of cells injected, we compared the survival of mice harboring HR-GSC1 cells expressing either shRest or shNT and either 5 × 104 (Fig. 4C) or 5 × 103 (Supporting Information Fig. 3) per mouse. All mice bearing shNT control cells died within 64 days for 5 × 104 cells compared to within 112 days for 5 × 103 cells, indicating that survival corresponded inversely to the number of tumor cells transplanted. Thus, these results suggest that by regulating self-renewal in GSCs, REST expression also correlated with survival of GSC tumor-bearing mice.
To further establish the role of REST on survival, we performed gain-of-function experiments, in which we transplanted LR-GSCs expressing exogenous REST into the brains of nude mice and then assayed for survival (Fig. 4E). As we previously found, these cells express higher levels of the REST protein, lower levels of its target gene, neuronal beta-tubulin, and concomitant higher self renewal ability. We now find that the addition of exogenous REST, compared with that of nontargeting control, to LR-GSCs caused decreased survival in tumor-bearing mice. Thus, REST controls survival in mice bearing GSC-mediated brain tumors.
REST Suppresses Apoptosis in GSC-Mediated Tumors
To determine whether the tumorigenic effects of REST in mouse brain tumors were partly regulated by promoting GSC survival, we analyzed our orthotopic mouse tumor sections for apoptosis using TUNEL assays. The data showed increased cell death upon REST knockdown in both HR-GSC lines (Fig. 5A, 5B) and, conversely, decreased cell death upon expression of exogenous REST in the LR-GSC line (Fig. 5C). Quantification of the TUNEL assay is shown in Figure 5D. The in vivo TUNEL staining was concordant with the BrdU incorporation in cultured cells and showed a decrease in cell proliferation for the two HR-GSC lines upon REST knockdown (Supporting Information Fig. 4). These results indicated that REST suppresses apoptosis in GSC tumors.
Genome-Wide mRNA Expression Analysis Reveals Further Potential Oncogenic Properties of REST
Because REST regulates numerous genes, it is likely that REST controls tumorigenicity of GSCs at multiple regulatory points. To determine what pathways could be affected by REST alteration in GSCs, in addition to self-renewal and cell viability, we compared genome-wide mRNA expression profiling of the six GSC lines that we characterized above: HR-GSC1+shNT, HR-GSC1+shRest, HR-GSC2+shNT, HR-GSC2+shRest, LR-GSC + green fluorescent protein (GFP), and LR-GSC+Rest. We identified 301 genes that were more than 1.5-fold differentially expressed across the HR- versus the LR-GSC lines. We then identified core biological pathways and cellular functions associated with REST expression using the ingenuity pathway analysis (Table 1). The pathways involving cellular movement, cellular assembly and organization, cellular development, cell-to-cell signaling and interaction, and cellular growth and proliferation were most significantly associated with REST.
Table 1. Potential pathways regulated by REST in glioblastoma-derived stem-like cells (GSCs)
REST Regulates Invasion of GSC-Mediated Orthotopic Tumors
Since the genome-wide results suggested REST to play a major role in cellular movement, we further investigated whether REST regulated invasion of GSCs using in vitro invasion assays. Our data showed a statistically significant decrease of invasion in the two HR-GSC lines upon REST knockdown with HR-GSC1 showing comparatively higher degree of infiltration than HR-GSC2 (Supporting Information Fig. 5).
To further establish the role of REST in GSC-mediated invasion, we re-examined the tumor sections generated during orthotopic mouse experiments described in Figure 4 by staining the tumor sections with a human nestin antibody to distinguish the human tumor-derived cells from the surrounding mouse cells (Fig. 6A–6C) . This approach helps in visualizing the injected human cells in the midst of mouse brain cells present in the mouse xenograft tumors but cannot determine the cellular status of the injected cells, such as their stemness or differentiation properties. Our results indicated that the shNT-treated HR-GSC1 and HR-GSC2 lines showed very high degree of migration with cell invasion to the pial surface from the core of the tumor. In contrast, the corresponding shRest-treated cells showed a decreased migratory phenotype with a circumscribed tumor growth, where cells rarely reached the pial surface. In the complementary experiment, exogenous REST expression in LR-GSCs resulted in a diffuse tumor growth and significant increase of cellular invasion compared with the control cells, in which GFP was expressed. Remarkably, the expression of additional REST in LR-GSCs converted the tumors such that they were similar to HR-GSCs. Taken together, these results suggest a major in vitro and in vivo role of REST in regulating tumor phenotype and invasion in GSCs.
Here we show that REST promotes oncogenic properties of GSCs with respect to self-renewal, cellular viability, and invasion and affects survival in mice bearing GSC-xenograft tumors. Our work does not reflect the GBM tumor, which will presumably contain many other types of cells than just the GSCs. Previously, we found that REST, which is also overexpressed in a major subtype of human medulloblastoma tumors that are mostly in the neuronal pathway, caused tumorigenesis by blocking the differentiation of cerebellar stem/progenitor cells [30–32]. Thus, these results suggest that the conservation of stemness through REST contributes to both neuronal and glial tumors and that deregulation of normal REST expression is a major factor in producing these tumors. Our results also indicate that REST expression in GSCs is mainly regulated at the protein degradation level suggesting that patient outcome should be determined based on REST expression at the protein and not transcript levels. Thus, the public datasets, which are based on the transcript levels, cannot be used to determine the prognostic values of the HR- versus LR-GBM patient population.
Our results indicate that expression of exogenous REST in LR-GSCs produce more invasive tumors in the mouse brain. The mechanism by which REST regulates invasive properties is unclear. Neural stem cells carrying exogenous genes were found to specifically target mouse intracranial glioma tumors either when transplanted at a site distant from the tumor or injected into the tail veins, providing a promising means of delivery of therapeutic molecules to the tumor . The targeting activity of the NSCs is presumably regulated by cell–cell and cell–microenvironment interactions. Whether the high-level expression of REST in HR-GSCs would affect the tumor-targeting process of NSCs is unknown. We are currently exploring ways to answer these questions.
Studies in ESCs have revealed that self-renewal in these cells is regulated by an interconnected regulatory circuit consisting of many factors, including Oct4, Sox2, and Nanog, rather than by a single molecule . Similarly, REST was found to be a part of the interconnected network regulating ESC self-renewal and pluripotency [50–52]. This suggests that self-renewal in GSCs, as well as in normal neural stem cells, might be controlled by a circuit, in which there is constant crosstalk between the regulators of self-renewal and the microenvironment (niche), producing a dynamic equilibrium; when individual regulators of the circuit are influenced by the niche, the equilibrium is re-set. To understand GBM tumorigenesis and to design a more appropriate targeted therapy for GBM than what is currently available, it will be important to identify and characterize the other players in this self-renewal circuit.
Our studies show that GSCs exhibit varying levels of the REST protein. Further, loss- and gain-of-function experiments establish that REST maintains self-renewal of GSCs in vitro and it causes lowered apoptosis and increased invasion in GSC-mediated brain tumors in orthotopic mouse models and shorter survival in these mice. Thus, REST is a critical regulator of GSC-mediated tumorigenesis.
This work was partly supported by grants from the NCI (CA97124 and CA81255 to S.M.). P.S. is the recipient of Dodie P. Hawn Fellowship in Genetics. H.C. is currently affiliated with Department of Neurosurgery and Hunstman Cancer Institute, University of Utah, Salt Lake City, UT.
DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
The authors indicate no potential conflicts of interest.