Context-dependent roles of MDMX (MDM4) and MDM2 in breast cancer proliferation and circulating tumor cells
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Many human breast cancers overexpress the E3 ubiquitin ligase MDM2 and its homolog MDMX. Expression of MDM2 and MDMX occurs in estrogen receptor α-positive (ERα+) breast cancer and triple-negative breast cancer (TNBC). There are p53-independent influences of MDM2 and MDMX, and 80% of TNBC express mutant p53 (mtp53). MDM2 drives TNBC circulating tumor cells (CTCs) in mice, but the context-dependent influences of MDM2 and MDMX on different subtypes of breast cancers expressing mtp53 have not been determined.
To assess the context-dependent roles, we carried out MDM2 and MDMX knockdown in orthotopic tumors of TNBC MDA-MB-231 cells expressing mtp53 R280K and MDM2 knockdown in ERα+ T47D cells expressing mtp53 L194F. The corresponding cell proliferation was scored in vitro by growth curves and in vivo by orthotopic tumor volumes. Cell migration was assessed in vitro by wound-healing assays and cell intravasation in vivo by sorting GFP-positive CTCs by flow cytometry. The metastasis gene targets were determined by an RT-PCR array card screen and verified by qRT-PCR and Western blot analysis.
Knocking down MDMX or MDM2 in MDA-MB-231 cells reduced cell migration and CTC detection, but only MDMX knockdown reduced tumor volumes at early time points. This is the first report of MDMX overexpression in TNBC enhancing the CTC phenotype with correlated upregulation of CXCR4. Experiments were carried out to compare MDM2-knockdown outcomes in nonmetastatic ERα+ T47D cells. The knockdown of MDM2 in ERα+ T47D orthotopic tumors decreased primary tumor volumes, supporting our previous finding that estrogen-activated MDM2 increases cell proliferation.
This is the first report showing that the expression of MDM2 in ERα+ breast cancer and TNBC can result in different tumor-promoting outcomes. Both MDMX and MDM2 overexpression in TNBC MDA-MB-231 cells enhanced the CTC phenotype. These data indicate that both MDM2 and MDMX can promote TNBC metastasis and that it is important to consider the context-dependent roles of MDM2 family members in different subtypes of breast cancer.
KeywordsMDMX MDM2 CTC Metastasis TNBC
Circulating tumor cell
C-X-C chemokine receptor type 4
Estrogen receptor α
Mouse double minute 2
Mouse double minute x
Nonobese diabetic severe combined immunodeficiency gamma mouse, NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ
Prostaglandin-endoperoxide synthase 2
Red blood cell
Short hairpin RNA
Small interfering RNA
Triple-negative breast cancer
The Cancer Genome Atlas has determined the molecular portraits of breast cancer, which is the second leading cause of cancer-related deaths among women . It is well accepted that breast cancer is a heterogeneous disease. Five subtypes have been characterized on the basis of genes the cancers express . Luminal A and B subtypes are largely estrogen receptor α (ERα)-positive and/or progesterone receptor-positive; HER2-enriched subtypes are hormone receptor-negative and HER2-positive. The basal-like and claudin-low subtypes are largely triple-negative breast cancers (TNBCs), which have none of the above markers, are associated with poor survival, and are a heterogeneous group . Mutated pathways that are shared across breast cancer subtypes include mutant p53 (mtp53) and high expression of mouse double minute 2 (MDM2) . In fact, 80% of TNBCs express mtp53 . Increased MDM2 expression in breast cancer tissue is associated with poor prognosis . MDM2 is an E3 ubiquitin ligase that targets wild-type p53 for degradation but can also act as an oncogene through p53-independent pathways (reviewed in ). The involvement of MDM2 in promoting breast cancer through p53-independent pathways is becoming increasingly clear. A mouse model study showed that MDM2 promotes early-stage metastasis in TNBCs, providing the first in vivo evidence for a role of MDM2 in promoting circulating tumor cells (CTCs) . However, ERα+ breast cancer models often are not metastatic, and we and others have shown that estrogen signaling increases their cell proliferation in vitro through a p53-independent MDM2 pathway [6, 7].
The MDM2 homolog MDMX (also called MDM4) promotes breast cancer and can inhibit the transcriptional activity of p53 and promote p53 degradation by heterodimerizing with MDM2 [8, 9], but its p53-independent functions are understudied. MDMX interacts with MDM2 via the RING domain, which leads to more efficient auto-ubiquitination and degradation of both MDM2 and MDMX . Haupt and colleagues analyzed the METABRIC database set  and found that MDMX overexpression occurs at ~ 35% in ERα+ luminal A and B and ~ 20% in basal breast tumors . Co-occurrence of MDMX and MDM2 expression is 10% in the claudin-low subtype . Therefore, more studies are needed to understand the roles of MDM2 and MDMX in promoting breast cancer phenotypes in the context of different subtypes of breast cancer.
Studies with in vitro cell culture show that MDM2 can promote cellular invasiveness by degrading E-cadherin, upregulating SNAIL protein levels, and increasing MMP9 enzymatic activity regardless of p53 mutational status [13, 14, 15]. High levels of MDMX and low levels of MDM2 have been shown to correlate with acquisition of the mesenchymal phenotype associated with metastasis of breast cancers . MDMX knockdown has shown potential as a target for inhibiting the proliferation of breast cancers expressing wild-type p53 . Some breast cancer cells with gain-of-function mtp53 also show an MDMX proliferative role that is mediated in part by downregulation of p27 protein levels . To date, no study has been carried out to explore the role of MDMX in breast cancer metastasis. Solid tumor metastasis involves several steps, including tumor cell invasion and intravasation into the bloodstream, circulating and surviving cells in the blood, and extravasation of cells into secondary organs [19, 20].
We sought to stratify the biological functions of MDMX and MDM2 and their impacts on breast cancer development, comparing metastatic and nonmetastatic breast cancer subtypes. Using female nonobese diabetic severe combined immunodeficiency gamma (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) (NSG) immunodeficient inbred laboratory mice as the model, we assessed human breast tumor detection and development in response to MDMX or MDM2 knockdown. The tumor volume helps to assess cell viability and proliferation, whereas the number of CTCs quantitatively reflects the metastatic potential of cancer cells. We tested the role of MDM2 or MDMX knockdown in the metastatic TNBC MDA-MB-231 cells by assessing the tumor volumes and the number of endpoint CTCs. We found that MDM2 knockdown in MDA-MB-231 orthotopic tumors drastically increased MDMX protein levels and, in support of previously published data , also suppressed the number of CTCs. Importantly, we report, for the first time to our knowledge, that MDMX was indispensable in the metastasis cascade, because knocking down MDMX significantly blocked the presence of CTCs. Interestingly, although MDM2 or MDMX knockdown resulted in a trend toward smaller tumors, the decreases in size were only statistically significant at early time points and only with MDMX knockdown. Moreover, we identified that in primary MDA-MB-231 orthotopic tumors, there was increased expression of the human metastasis-promoting genes CXCR4 (C-X-C chemokine receptor type 4) and PTGS2 (prostaglandin-endoperoxide synthase 2) [21, 22]. However, the nonmetastatic ERα+ T47D (mtp53-expressing) orthotopic tumors showed no evidence of metastasis, but in vivo primary tumor growth was significantly decreased by the knockdown of MDM2. These findings highlight the importance of studying the MDMX and MDM2 signaling in the context of different breast cancer subtypes that express mtp53.
Materials and methods
2D cell culture
Human breast cancer cell lines T47D (mdm2 SNP309 G/G, mutant p53 L194F) and MDA-MB-231 (mdm2 SNP309 T/G, mutant p53 R280K) were purchased from the American Type Culture Collection (www.atcc.org; Manassas, VA, USA). Cells were maintained at 5% CO2 in DMEM (Life Technologies, Carlsbad, CA, USA) with 50 U/ml penicillin, 50 μg/ml streptomycin (Mediatech/Corning Life Sciences, Manassas, VA, USA), and supplemented with 10% FBS (Gemini Bio-Products, West Sacramento, CA, USA) in a 37 °C humidified incubator. T47D cells generated with inducible MDM2 knockdown were described previously . Constitutive MDM2 or MDMX knockdown cell lines were generated by retroviral infection with MLP.GFP vector (a generous gift from Scott Lowe) containing mir30 short hairpin RNA (shRNA)-expressing vector, mdm2 151656 shRNA, or mdmx 13023 shRNA. The mir30 shRNA inducible expressing vector has been used as a control for numerous previous high-impact studies [23, 24], and the only difference for the stable knockdown cell lines was a constitutively active promoter. Cell lines were generated and selected as previously described [7, 23]. All stable knockdown cell lines were used as selected pools.
3D Matrigel culture
Cells grown in regular culture conditions were trypsinized and counted. Cells (2000 per well) were seeded on top of 40 μl of solidified Matrigel (Cultrex; Trevigen, Gaithersburg, MD, USA) in DMEM containing 10% FBS and antibiotics. Medium was replenished every 3 days.
Cell proliferation assay
MDA-MB-231 cells (50,000/well) were seeded in a six-well plate in triplicate and were allowed to grow for 2, 4, 5, and 6 days. At each time point, cells were trypsinized, and the number of cells was determined by cell counting using a hemocytometer.
Cells (800,000/well) were plated in a six-well plate one night before the experiment. Scratches were created using a 200-μl pipette tip. Cells were then rinsed three times with fresh medium. Wound closure was observed within the scrape line and photographed by phase-contrast microscopy. Wound area was measured and quantified by using NIS-Elements software (Nikon Instruments, Melville, NY, USA). Thirty fields per condition were recorded, and three independent experiments were performed. Transient electroporation of small interfering RNA (siRNA) was carried out using an Invitrogen Neon transfection system (Life Technologies) with ON-TARGET siRNA smartpools obtained from Dharmacon (Lafayette, CO, USA): siGENOME™ Control Pool (catalogue no. D-001206-13-20), human mdm2 siRNA (catalogue no. L-003279-00), and human mdm4 siRNA (catalogue no. L-006536-02-0005).
RNA isolation, real-time qRT-PCR, and microarray analysis
RNA was extracted using QIAshredder columns and RNeasy Mini Kit (Qiagen, Hilden, Germany) following manufacturer’s protocol. Complementary DNA (cDNA) synthesis was carried out using the High-Capacity cDNA Archive Kit reagents (Applied Biosystems, Foster City, CA, USA). RT Master Mix and RNA were mixed and incubated at 25 °C for 10 min and then at 37 °C for 2 h. Amplification of gene transcripts was performed by qPCR with primer probes for mdm2(3–4) (Hs01069930_m1), mdmx (Hs00910358_s1), cxcr4 (Hs00607978_s1), ptgs2 (Hs00153133_m1), and gapdh (Ha02758991_g1) from Applied Biosystems. Primers were combined with 150 ng of cDNA and TaqMan Universal Master Mix (Applied Biosystems), and reactions were carried out using the standard program in the QuantStudio 7 sequence detection system (Applied Biosystems). cDNA (25 ng) from tumor samples was used in TaqMan™ Array Human Tumor Metastasis (Applied Biosystems) following the manufacturer’s protocol. The gene expression analysis was performed with ExpressionSuite software (Thermo Fisher Scientific, Waltham, MA, USA).
Cells were harvested at 1100 rpm for 5 min at 4 °C, then washed three times with ice-cold PBS. For extraction from tissues, samples were snap-frozen in liquid nitrogen and homogenized. Cells then were resuspended in radioimmunoprecipitation assay buffer (0.1% SDS, 1% IGEPAL Nonidet P-40, 0.5% deoxycholate, 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid, 0.5 mM ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid, 50 mM Tris-HCl, pH 8.0, 1 mM phenylmethylsulfonyl fluoride, 8.5 μg/ml aprotinin, and 2 μg/ml leupeptin). The cell suspension was incubated on ice for 30 min to lyse the cells, vortexing occasionally. Additional sonication of lysate three times for 30 s/30 s rest on ice at 98% amplitude was done after the incubation. Samples were centrifuged at 13,000 rpm for 30 min at 4 °C.
4× NuPAGE lithium dodecyl sulfate buffer (Life Technologies) and 20 mM dithiothreitol (DTT) were added to protein extracts, and samples were heated at 70 °C for 10 min. Iodoacetamide (100 mM; MilliporeSigma, Burlington, MA, USA) was then added to the samples when cooled down. For CXCR4 detection, extracts were incubated with the same buffer containing DTT and iodoacetamide at room temperature for 20 min. Ten percent SDS-PAGE or 4–12% gradient SDS-PAGE (Life Technologies) was used to separate samples, followed by electrotransfer onto nitrocellulose membrane or polyvinylidene fluoride membrane. The membrane was blocked with 5% nonfat milk (Bio-Rad Laboratories, Hercules, CA, USA) in either 1× PBS with 0.1% Tween 20 or 1× Tris-buffered saline TBS with 0.1% Tween 20 following incubation of primary antibody overnight at 4 °C. The next day, the membrane was washed with either 1× PBS with 0.1% Tween 20 or 1× TBS with 0.1% Tween 20 and then incubated with secondary antibody for 1 h at room temperature. Signal was detected by chemiluminescence with a Pierce Super Signal Kit (Thermo Fisher Scientific) and autoradiographed with HyBlot CL films (Thomas Scientific, Swedesboro, NJ, USA).
Cells were grown in 3D culture conditions described above. After 8 days of culturing, colonies were washed with 1× PBS and fixed with 4% paraformaldehyde (MilliporeSigma) for 15 min at room temperature. The plates were washed three times with 1× PBS, permeabilized with 0.5% Triton X-100 in PBS/1% FBS for 10 min and incubated with rhodamine-phalloidin (BK005; Cytoskeleton, Denver, CO, USA) for 1 h at room temperature. Alexa Fluor-conjugated secondary antibody (Life Technologies) was used, and cells were mounted with VECTASHIELD mounting medium (Vector Laboratories, Burlingame, CA, USA) containing 4′,6-diamidino-2-phenylindole (DAPI). Images were captured with a Nikon A1 confocal microscope at 200× magnification and analyzed by NIS-Elements AR Analysis software (Nikon Instruments).
Antibodies used were MDM2 (1:1:1 mix of mouse monoclonal 4B2, 2A9, 4B11 hybridoma supernatant), p53 (1:1:1 mix of mouse monoclonal 240,421,1801 hybridoma supernatant), and MDMX (Proteintech, Rosemont, IL, USA), actin-HRP (Santa Cruz Biotechnology, Dallas, TX, USA), E-cadherin (Cell Signaling Technology, Danvers, MA, USA), and CXCR4 (Abcam, Cambridge, MA, USA).
Orthotopic tumor implantation and measurement
For MDA-MB-231 study, 1 × 107 cells with constitutive MDM2 or MDMX knockdown were injected into the mammary fat pad of female NSG mice at 6 weeks of age. No additional drug was administered. Tumor growth was measured using calipers, and tumor volume was calculated as volume = π/6 (length × width × width). At ethical endpoint, mice were killed following institute guidelines. For T47D study, MDM2 knockdown was induced with 4 μg/ml doxycycline in cell culture conditions for 10 days before implantation. Tumor cells (1 × 107) were then injected into the mammary fat pad of female NSG mice at 6 weeks of age. Animals were provided with drinking water containing 2 mg/ml doxycycline (MilliporeSigma) dissolved in deionized water, 8 μg/ml 17β-estradiol (MilliporeSigma) dissolved in DMSO, and 2% sucrose (MilliporeSigma), replenished every other day.
Circulating tumor cell analysis
Cardiac punctures were performed at the endpoint of the experiment, and blood samples were stored temporarily in 1.5-ml microcentrifuge tubes coated with sodium heparin (Sagent Pharmaceuticals, Schaumburg, IL, USA) prior to CTC isolation procedure. Briefly, whole blood was subjected to centrifugation. After removal of plasma, the buffy coat layers were then collected and subjected to red blood cell (RBC) lysis to remove residual RBCs. Flow cytometric analysis was performed using a FACScan device (BD Biosciences, San Jose, CA, USA), and event counting was gated on the basis of size and GFP intensity from cultured cells as positive controls. The number of CTCs was obtained by dividing the number of positive events by individual blood volume. Statistical significance was calculated by two-sample permutation test, two-sided hypothesis after multiplicity adjustment (Hochberg procedure).
Tissue processing and histology
Animal tissues were harvested, fixed in 10% buffered formalin, and embedded in paraffin. Sections of primary tumors and lungs were cut at 5 μm and stained with H&E by the Laboratory of Comparative Pathology. The slides were analyzed by a board-certified veterinary pathologist (AP).
CTC data obtained from the MDA-MB-231 animal study were analyzed using R statistical software (version 3.4.2; R Foundation for Statistical Computing, Vienna, Austria). Datasets were tested for assumptions of normality using the Shapiro-Wilk test . If the normality was confirmed, a pairwise independent t test was carried out. Otherwise, for nonnormal data, we applied a permutation-based two-sample t test instead, which is appropriate for small samples from nonnormal distributions. Permutation tests were performed using DAAG (data analysis and graphics) version 1.22 in the R package. Hochberg correction [26, 27] was performed on the resulting p values for all multiple comparisons to control for the familywise error rate . All other graphs and statistical analysis were generated using Prism 7.01 software (GraphPad Software, La Jolla, CA, USA). In the box-and-whisker plots, each dot represents one mouse.
MDM2 and MDMX potentiate release of MDA-MB-231 circulating tumor cells
MDMX expression in MDA-MB-231 cells moderately influences tumor growth
We confirmed in vivo knockdown of MDMX and MDM2 from the tumors using qRT-PCR and Western blot analysis (Fig. 2c and d). Significant depletion of MDM2 in the tumor tissue was detected, and we also detected an increase in MDMX protein (Fig. 2c and d, lanes 4, 5, and 6). The shRNA-mediated decrease in mdmx mRNA was clear but had an insignificant p value resulting from two animals from the mir30 vector control group with random loss of mdmx expression (Fig. 2c). These random loss endpoint tumors corresponded to the largest and intermediate-sized masses (marked by arrows in Fig. 2b). The statistical conclusions were the same with or without the two random loss animals. Importantly, animals with clear MDMX protein depletion showed no associated change in MDM2 (Fig. 2d).
MDMX and MDM2 knockdown decrease cell migration in vitro for MDA-MB-231 cells
MDMX knockdown in TNBC tumors decreases transcription of CXCR4 and PTGS2
MDM2 facilitates ERα+ T47D xenograft primary tumor growth
Expression of MDM2, MDMX, and CXCR4 in the context of ERα+ versus TNBC tumors
MDMX and MDM2 are expressed in multiple subtypes of breast cancer . MDMX and MDM2 overexpression promote tumorigenic potential through blocking p53 and also through p53-independent influences [4, 36, 37, 38]. Mouse models addressing the p53-independent influence of MDM2 overexpression in mammary gland tumorigenesis show that p53−/− transgenic mice with MDM2 overexpression have an increased incidence of tumorigenesis . The role of MDMX overexpression in tumorigenesis appears to vary dependent on the system being studied. In one mouse model, the MDMX transgene increases mammary tumor development and enhances tumor development in a heterozygous mutant p53 or a p53-null background [40, 41]. However, in an alternative mouse model, overexpression of homozygous MDMX transgenes results in embryonic lethality, whereas the hemizygous animals are viable and do not have accelerated tumor formation . Mouse models vary and do not always recapitulate human disease. In the orthotopic model in the present study, both MDM2 and MDMX significantly enhanced the metastatic potential of the MDA-MB-231 cells, but they did not significantly increase the final tumor volume (Figs. 1 and 2).
In this report, we also uncovered CXCR4 and PTGS2 as two key target genes modulated by MDMX in primary tumors but not when the cells are grown in the culture dish. Interestingly, although MDM2 promotes the cancer cell release from the primary tumor into the circulating system, the observed downregulation of CXCR4 or PTGS2 expression by MDM2 knockdown was not statistically significant (Fig. 4b). One possible explanation for this is that when MDM2 was depleted, there was a consistent sharp increase in MDMX protein levels. This high MDMX in turn could upregulate CXCR4 and PTGS2. The increased MDMX upon MDM2 depletion may compensate for the depletion of MDM2. MDMX knockdown did not increase MDM2 levels, which could explain the significant result observed for decreased CTC release upon MDMX knockdown and the strongly observed inhibition of CXCR4 and PTGS2 transcription. Further experiments in the context of the microenvironment are required to explore this model.
Elevated CXCR4 expression has been documented in more than 23 different types of cancers with various origins and has been shown as a poor prognostic biomarker . CXCR4 overexpression in breast cancer has been shown to promote metastasis in an organ-specific manner, and new treatments targeting this pathway in TNBC have had some success . Inhibition of CXCR4 protein also leads to significantly less metastatic burden in mouse models [22, 46, 47]. It is known that in the tumor microenvironment, inflammation plays a significant role in activating CXCR4 signaling . In oral squamous cell carcinoma and glioblastoma, vascular endothelial growth factor has been shown to upregulate CXCR4 expression [49, 50]. Additionally, induction of CXCR4 and PTGS2 can be achieved through activation of NF-κB signaling . MDM2 modulates NF-κB signaling by directly inducing the transcription of p65 and increasing p100 transcripts, independently of p53 [52, 53]. However, there has been no investigation on deciphering the role of MDMX in relation to metastasis-promoting pathways. It is conceivable that inflammation and/or angiogenesis in the tumor microenvironment contributes to the activation of CXCR4 and PTGS2 in MDMX-overexpressing breast tumor cells. We documented that MDMX correlated with transcriptional activation of CXCR4 and PTGS2 in primary orthotopic tumors; however, the in vitro cell culture system expressed tenfold lower CXCR4 and PTGS2 transcripts that were unchanged by MDMX expression. This indicates that the tumor microenvironment provides stimulatory signals that activate the pathways. It is unclear what specific cue(s) in the tumor niche define(s) the activation in our model system and, more important, what role MDMX plays in facilitating and/or maintaining such induction.
In the TNBC cells in this preclinical mouse model, we observed a reduction in tumor volume only during early time point measurements when MDMX was knocked down. Thus, targeting MDM2 and MDMX in TNBCs may have more benefit for diagnosis through liquid biopsy and for targeting metastatic disease, rather than in treating patients’ primary tumors. Targeting MDM2 and MDMX may provide therapeutic value for patients with advanced stages of TNBC. In nonmetastatic ERα+ T47D breast cancer cells, we generated in vivo evidence that MDM2 promoted tumor growth in response to estrogen signaling without promoting tumor-invasive properties. Therefore, targeting MDM2 has promise for targeting primary ERα+ tumors. New studies suggest that an excellent strategy will be to combine treatments that block MDMX and MDM2 [12, 18]. Such combination trials will have potential positive benefits for all subtypes of breast cancer.
Importantly, our results showed that high levels of MDMX and MDM2 promoted a metastatic phenotype that correlated with increased CTCs and tumors expressing increased levels of CXCR4 and PTGS2. We found that MDMX had a strong influence on promoting CTCs, and upregulating CXCR4 and PTGS2. The fact that both CXCR4 and PTGS2 have been identified as key mediators of breast cancer metastasis to bone and lung [21, 22] provides a potential new combination targeting approach coupling MDM2, MDMX, CXCR4, and PTGS2 inhibition for a mechanism to block breast cancer metastasis.
Our findings provide novel insights into the roles of MDM2 and MDMX promoting CTCs of TNBC. We also documented that MDM2 promotes tumorigenesis of ERα+ breast cancers. Importantly, we discovered that MDMX correlates with the increased transcription of CXCR4 and PTGS2 in tumor tissue. Our observation that MDMX and MDM2 signaling pathways are different in TNBC and ERα+ breast cells has set the stage for suggesting the use of these biomarkers to more accurately define the nature of breast cancer subtypes.
Members of the Bargonetti laboratory are acknowledged for helpful discussion.
A grant to JB from the Breast Cancer Research Foundation supported this work. The mouse models and circulating tumor cell work were supported by a Pilot Project sub-award to JB for collaboration with OO from parent Grant Number MD007599 from the National Institute on Minority Health and Health Disparities (NIMHD) of the National Institutes of Health (NIH). The contents are solely the responsibility of the authors and do not necessarily represent the official views of the NIMHD or the NIH.
JB and CG wrote the manuscript. JB, CG, and OO conceptualized and designed the study and the experiments. CG, GX, and OO, carried out experiments. AP, CG, and GX carried out the pathological analysis. JG and CG carried out the statistical analysis. All authors read, critiqued, and approved the final manuscript.
Ethics approval and consent to participate
This study was given approval by the Institutional Animal Care and Use Committee of Weill Cornell Medical College.
Consent for publication
The authors declare that they have no competing interests.
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