miR‐199b‐5p directly targets PODXL and DDR1 and decreased levels of miR‐199b‐5p correlate with elevated expressions of PODXL and DDR1 in acute myeloid leukemia
Authors: Amanda J. Favreau, Erin L. Cross, Pradeep Sathyanarayana
Journal: American Journal of Hematology (2012)

Aberrant expression of Podocalyxin (PODXL), a CD34 ortholog, has been associated with acute myeloid leukemia (AML). In this study, via tissue microarray, the authors discovered elevated PODXL expression in M2, M4, and M1 FAB-subtype patients. Importantly, various investigations have linked aberrant miRNA expression with AML (Cammarata et al., Am J Hematol 85:331–339). A miRNA prediction algorithm identified PODXL as a conserved target for miR-199b, a significantly downregulated miRNA in AML. Further prediction of miR-199b-5p targets identified Discoidin domain receptor 1 (DDR1) as another highly conserved target. Initially, IHC analyses showed that DDR1 levels were also highly upregulated in AML and more significantly, were elevated in the same AML cases, where PODXL levels were increased. Experimental validation (via mimics) confirmed that both PODXL and DDR1 are targets of miR-199b-5p. Furthermore, 3′UTR-luciferase assays established that miR-199b-5p targets PODXL and DDR1. Most importantly, the authors found significant decrease in miR-199b-5p levels in most AML patients with elevated PODXL and DDR1 expressions. Importantly, overexpression of miR-199b-5p in K562 cells caused significant decrease in collagen IV induced migration. Taken together, our studies have identified concurrent increased expression of PODXL and DDR1 in AML and directly connect decreased miR-199b-5p to these novel targets and potential antigomir-mediated therapeutic implications in AML. AML represents a heterogeneous group of malignancies with huge variability in clinical course and response to therapy [2]. Discovery of new prognostic factors in AML is driving our understanding of disease biology and development of new therapeutic targets [2]. Podocalyxin (PODXL) is a highly glycosylated and sialylated transmembrane protein, and a CD34 ortholog normally expressed on hematopoietc stem cells, hemangioblasts, vascular endothelial cells, podocytes, and a subset of neural progenitors [3]. Recently, increased PODXL expression has been associated with a subset of aggressive cancers including acute myeloid and lymphoid leukemia, myeloid sarcomas, and certain breast, liver, pancreatic, and kidney tumors [3, 4]. PODXL's overexpression was detected earlier in AML, however, only 50% of M2 and M4 French American British subtypes exhibited aberrant PODXL expression [5]. Via the Tissue Bank at Maine Medical Center, the authors obtained n = 24 bone marrow-derived paraffin-embedded (PE) blocks of AML patients and n = 9 normal BM blocks (as controls). The clinical details regarding the AML cases are provided in Supporting Information Table S1. A custom-made tissue microarray (TMA) on a positively charged SuperFrost Plus glass slide was constructed using the services of US Biomax. Immunohistochemistry (IHC) analysis was performed on TMA using an anti-human PODXL antibody. The IHC stained slide was scanned and signal intensity was computed and measured using ImageScope, Aperio Scanning System. Data revealed that PODXL was highly expressed in most of the AML samples though the intensity of the expression varied among different patients (Fig. 1A and B). High PODXL expressions were detected in AML cases AML-2 (M1), AML-18 (M1), AML-4 (M2), AML-6 (M4) and AML-7, AML-10, -11, -12, -13 (AML – NOC WHO classification), respectively, when compared with BM samples from normal subjects (Fig. 1B). In addition, most of the remaining AML cases (AML – NOC WHO classification) also showed modest increases in PODXL expression when compared with normal/control samples (Fig. 1B). Interestingly, ectopic expression of PODXL in breast cancer cells (MCF) and prostate cancer (PC3) cell lines have lead to increase invasiveness, migration, elevated expressions of matrix metalloproteases 1 and 9 (MMP1 and MMP9) and increased activation of Erk1,2 and PI3K [6]. Significantly, these events were discovered to be mediated via PODXL's interaction with Ezrin, an actin binding protein known to promote neutrophil transmigration [6, 7]. Further, overexpression of PODXL in CHO cells has been shown to promote increased fibronectin binding, cell spreading, and migration [8]. Furthermore, PODXL has been implicated as a mediator of chemotactic signaling and cancer cell migration via interaction with CXCR4 [3]. In a related context, our previous studies discovered that PODXL was a potent erythropoietin responsive gene in erythroid progenitors and was found to facilitate/promote egression of immature reticulocytes from the bone marrow to the circulation possibly via its anti-adhesive function [9]. Therefore, it is tempting to speculate that increased PODXL expression in leukemic blasts may promote anti-adhesion and migration of blasts during onset and development of leukemogenesis. Podocalyxin is highly expressed in AML and is a target of miR-199b-5p. (A) Upon TMA generation, IHC analyses were performed using primary goat polyclonal anti-human PODXL antibody (R&D Systems, concentration 0.2 mg/mL) on n = 24 bone marrow derived PE blocks of AML patients and normal BM blocks (as controls). N = 13 AML samples showed high expression of PODXL (a–m) in comparison to normal samples (n–p). Images were recorded at 40× resolution using Aperio ImageScope system. (B) From the recorded images, (n = 33) total signal intensity was computed and scored via integrating three intensities from strong positive, positive, and weak positive respectively, which were measured using Aperio ImageScope system (left panel) and total mean (± SE) from n = 24 AML samples and n = 9 controls were also graphed (right panel). (C) TargetScan alignment of PODXL's 3′UTR with hsa-miR-199b-5p showing the highly conserved seed match. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] MicroRNAs (miRNAs) are a class of naturally occurring noncoding RNAs of 17–30 nucleotides, which are known to regulate gene expression by inducing translational inhibition and cleavage of their target mRNAs [10]. miRNAs have been shown to elicit several crucial roles during hematopoiesis that include lineage decisions, stem cell progenitor transitions, niche control, and other cellular functions [11-13]. Recent investigations have linked aberrant miRNA expression with acute myeloid leukemia [14-16]. A recent study evaluated miRNA expression in 122 newly diagnosed adult acute myeloid leukemia (AML) cases using a microarray platform [17]. This study identified a subset of miRNAs that were significantly downregulated in AML patients with respect to CD34+ cells obtained from 10 healthy donors. Interestingly, via a miRNA prediction algorithm (TargetScan), the authors identified PODXL as a highly conserved target of miR-199b, a significantly downregulated miRNA in AML (Fig. 1C). Experimental validation via transfecting hsa-miR-199b-5p mimics revealed that PODXL is indeed a bona fide target of miR-199b-5p (Fig. 2A). Further analyses of miR-199b-5p's predicted targets revealed that Discoidin domain receptor 1 (DDR1) is also a highly conserved target of miR-199b-5p (Fig. 2B). DDR1, is a class of collagen receptor, expressed on human leukocytes, including neutrophils, monocytes, lymphocytes, and podocytes [18]. This subfamily is distinct from other members of the large receptor tyrosine kinase group due to a homology domain to discoidin, a lectin first described during the cell aggregation process of the slime mold Dictyostelium discoideum [18]. DDR1′s autophosphorylation is achieved by all collagens tested (type I to type VI). Previous in situ hybridization studies and Northern blot analysis have shown that the expression of DDR1 is restricted to epithelial cells, particularly in the kidney, lung, gastrointestinal tract, and brain [18]. Importantly, similar to PODXL, DDR1 is also significantly overexpressed in several human tumors of breast, ovarian, esophageal, and brain origin [18]. Though considerable upregulation of DDR1 gene expression in cytokine-activated neutrophils has been reported [18], its elevated expression is not reported in any subset (or form) of leukemia including AML. Significantly, our IHC analyses in AML samples via a similar TMA approach revealed that DDR1 levels were upregulated in the same AML cases where PODXL levels were increased (Fig. 2C and D). Previously, overexpression of DDR1 in lung cancer cells resulted in a significant increase of cell motility and invasiveness, where DDR1–collagen interactions facilitated the invasiveness and importantly, was associated with poor prognosis of non-small cell lung cancer [19]. Further, in hepatocellular carcinoma cells and glioma cells (glioblastoma multiforme), overexpression of DDR1 promoted invasion and migration of tumor cells [20, 21]. Taken together, the presently observed increase of DDR1 expression on leukemic blasts suggests that upregulation of DDR1 may contribute toward migration and invasion of leukemic blasts to extramedullary sites. Interestingly, further comparative analyses of the IHC results revealed that PODXL and DDR1 were co-expressed at high levels in n = 9 AML patients (Fig. 3A). Interestingly, 55% of these cases were patients with persistent AML. Significantly, both PODXL and DDR1 have highly overlapping critical functional roles in maintaining slit diaphragms in the kidney. PODXL knockout mice fail to form foot processes and slit diaphragms and exhibit post-natal lethality with anuria, omphalocele, and perinatal lethality [3, 22]. Interestingly, DDR1-deficient podocytes also exhibits loss of cell–matrix communication that results in disturbed anchorage of foot processes and disruption of the slit diaphragm [23]. Further experimental investigations via transfection of miR-199b-5p mimics revealed that DDR1 was also a specific target of miR-199b-5p (Fig. 3B). To conclusively prove that miR-199b-5p targets PODXL and DDR1, the authors generated respective 3′UTR–luciferase constructs (both wild type and mutants that block miR-199b-5p binding) and co-transfected with miR-199b-5p mimics. The results clearly revealed that miR-199b-5p specifically targets PODXL and DDR1 via its predicted binding sites in respective 3′UTRs (Fig. 3C). Our results demonstrates for the first time that miR-199b-5p regulates two proteins with analogous biological functions and strongly implies that decreased miR-199b-5p expression in AML may lead to concurrent upregulation of PODXL and DDR1. miR-199b-5p silences PODXL expression and miR-199b's putative target DDR1 is also highly expressed in AML. (A) To experimentally test the PODXL silencing potential of miR-199b-5p, miRIDIAN Mimic hsa-miR-199b-5p and negative control #1 (Dharmacon) were transfected in HeLa cells using Dharmafect 1 and post 48 h transfection, RNA was isolated and PODXL expression examined using RT-qPCR. Data is representative of two independent experiments performed in triplicate (mean ± SEM). *P < 0.05, **P < 0.01. (B) TargetScan alignment of DDR1′s 3′UTR with hsa-miR-199b-5p showing the highly conserved match. (C) IHC analyses were performed on the generated TMA using primary goat polyclonal anti-human DDR1 antibody (R&D Systems, concentration 0.2 mg/mL) on the same set of n = 24 bone marrow derived PE blocks of AML patients (a–m) and normal BM blocks (n–p). Images were recorded as described in Figure 1. (D) From the recorded images, (n = 33) total signal intensity was computed and analyzed as described in Figure 1 (left panel) and total mean (± SE) from n = 24 AML samples and n = 9 controls were also graphed (right panel). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] miR-199b-5p specifically targets PODXL and DDR1 while decreased levels of miR-199b-5p correlates with increased PODXL and DDR1 expressions in AML and miR-199b-5p mediated silencing of PODXL and DDR1 affects cell migration. (A) Further comparative analyses of the IHC results revealed that PODXL and DDR1 were co-expressed at high levels in n = 9 AML patients. (B) To experimentally validate DDR1 as target of miR-199b-5p, miRIDIAN Mimic hsa-miR-199b-5p and negative control #1 (Dharmacon) were transfected in HeLa cells using Dharmafect 1 and post 48 h transfection, RNA was isolated and DDR1 expression examined using RT-qPCR. Data is representative of two independent experiments performed in triplicate (mean ± SEM). *P < 0.05, **P < 0.01. (C) To conclusively prove that miR-199b-5p targets PODXL and DDR1, luciferase-3′UTR constructs of the respective genes were generated both for wild type and mutants that lack miR-199b-5p binding sites. Luciferase-3′UTR constructs were co-transfected with miR-199b-5p and luciferase assays were performed 48 h post transfection. Data is representative of two independent experiments performed in triplicate (mean ± SEM). *P < 0.05, **P < 0.01. (D) miRNAs were extracted from bone marrow samples derived from PE sections of AML patients and control subjects and RT-qPCR assay for miR-199b-5p was performed. Values were normalized using RNU6 and data represent mean ± SE for triplicate values. *P < 0.05, **P < 0.01. (E) Silencing potential of miR-199b-5p on PODXL and DDR1 expressions at protein level was determined via western blotting. K562 cells stably expressing either miR-199b-5p or GFP empty vector control were lysed, electrophoresed, and blotted using anti-PODXL and anti-DDR1 antibodies and as internal controls, lysates were also blotted for beta-tubulin expression with anti-beta-tubulin antibody. (F) To examine the effect of PODXL and DDR1 silencing by miR-199b-5p on migration, cell migration assay was performed using CytoSelect Cell Migration Assay using K562 cells stably expressing either miR-199b-5p or GFP empty vector control. Data is representative of two independent experiments performed in triplicate (mean ± SEM). *P < 0.05, **P < 0.01. To definitely determine whether the increased PODXL and DDR1 expressions in the AML samples tested can be correlated to relative miR-199b-5p levels in the respective bone marrow samples, the authors extracted the miRNA from PE BM samples as described in methods and performed RT-qPCR. Importantly, the miR-199b-5p levels were significantly reduced in AML samples that demonstrated high PODXL and DDR1 levels (Fig. 3D). Notably, recent studies have revealed that 20% of individuals with AML harbor somatic mutations in DNMT3A (encoding DNA methyltransferase 3A) and genome-wide sequencing approaches have strengthened the notion that AML can be classified on the basis of patterns of promoter methylation [24]. Future investigations on methylation status of miR-199b-5p promoter might further unravel the link between aberrant epigenetic regulation and subsequent pathogenic consequences manifested via the dysregulated PODXL and DDR1 expressions. To further assess whether miR-199b-5p mediated targeting of PODXL and DDR1 affects protein expression, the authors generated K562 cell line that stably expresses GFP-miR-199b-5p. Analyses of PODXL and DDR1 protein expression via western blotting in this stable line revealed a significant decrease in PODXL and DDR1 expressions in presence of miR-199b-5p when compared with control K562 cells that expressed empty vector GFP control (Fig. 3E). To assess the functional consequence of decreased PODXL and DDR1 expressions due to miR-199b-5p, the authors next examined the migration potential of K562-miR-199b-5p as described in methods. Decreased expressions of PODXL and DDR1 resulted in significant decrease in the migration of K562 cells (Fig. 3F). Interestingly, in vitro studies have demonstrated the expression of DDR1 in human leukocytes and identified biologic role of DDR1 in adherence, migration, differentiation, and cytokine/chemokine production [18]. Notably, a recent investigation revealed that overexpression of miR-199b-5p in medulloblastoma (MB) cancer stem cells results in downregulation of CD133+ tumor initiating cells and causes depletion of side population compartment of MB tumor stem cell population via negatively regulating the Hes1-mediated Notch signaling pathway [25]. Therefore, it is possible that downregulation of miR-199b-5p in AML may also provide proliferative advantage for leukemic stem cells in addition to accelerating migration and invasion functions via upregulation of PODXL and DDR1. Taken together, the present findings identify PODXL and DDR1 as targets of miR-199b-5p and establish a strong inverse correlation for their dysregulated expressions in AML and in addition may advance antagomir-mediated therapeutic approaches in AML. A custom-made TMA for n = 24 PE bone marrow samples of AML patients and n = 9 control/normal samples was constructed using the services of US Biomax. The array was in a single core per case format. Tissue array sections were mounted on the positive charged SuperFrost Plus glass slide. The TMA sections were cut at 5 micron in thickness. Individual cores were 1.5 mm in diameter and spaced 0.25 mm apart. Anti-human PODXL or DDR1 antibodies suitable for PE-IHCs were obtained from R&D Systems. To optimize antibody concentration for IHC on PE tissue array slides, a pilot experiment was performed using serial dilutions of the primary antibody on testing slides to determine the optimal working condition. For IHC, standard protocol was followed. Briefly, antigen retrieval was performed with 1× antigen retrieval solution (Target Retrieval solution, S-1699, DAKO Cytomation). For detection, ImmPRESS™ Reagent [anti-Goat Ig (peroxidase)] (Vector Laboratories) and DAB as substrate chromogen (DAKO Cytomation) was used. Hematoxylin was used for counterstaining cell nuclei. IHC stained slides were scanned and the total positive cell numbers and intensity of anti-PODXL or anti-DDR1 staining was computed and measured by ImageScope from Aperio Scanning System. In addition, an independent pathologist also performed manual scoring of the intensity levels. miRNAs from the PE blocks of BM samples from AML patients and control subjects was extracted using miRNeasy FFPE Kit (Qiagen) following the manufacturer's instructions. As a precautionary measure, genomic DNA contamination from the PE materials was eliminated using a gDNA Eliminator spin column that rapidly removes genomic DNA. In addition, the authors also performed a DNase digestion step. The extracted miRNA was quantified by absorbance at 260 nm and ensured that total RNA concentration is greater than 0.5 mg/mL (at A260) and had an A260:A280 ratio between 1.9 and 2.1. miRNA samples that met all these criteria were selected for further analyses. The following primers (Quanta) were used for RT-qPCR analyses: HS-RNU6 (HS-RNU6 assay will detect both RNU6-1 and RNU6-2), RNU6-1(accession# NR_004394.1), sequence: GUGCUCGCUUCGGCAGCACAUAUACUAAAAUUGGAACGAUACAGAGAAGAUUAGCAUGGCCCCU GCGCAAGGAUGACACGCAAAUUCGUGA AGCGUUCCAUAUUUU; RNU6-2 (accession # NR_002752.1) RNU6-2 Sequence: CUGCGCAAGGAUGACACGCAAAUUCGU GAAGCGU UCCAUAUUUUU; HS-SNORD48 (accession # NR_002745.1) SNORD48 Sequence: AGUGAUGAUGACCCCAGGUAACUCUGAGUGUGUC GCUGAUGCCAUCACCGCAGCG CUCUGACC; hsa-miR-199b-5p (Cat#:HSMIR-0199B-5P), Mature sequence: CCCAGUGUUUAGACUAUCUGUUC (Tm PCR:75.0°C). To experimentally test the PODXL and DDR1 silencing potential of miR-199b-5p, miRIDIAN Mimic hsa-miR-199b-5p and negative control #1 (Dharmacon) were transfected in HeLa cells using Dharmafect 1 according to manufacturer's instructions. Post 48 h of transfection, RNA was isolated using RNeasy Mini Kit (Qiagen) and via RT-qPCR the transcript expression levels of PODXL and DDR1 were examined. Wild type and mutant 3′UTR luciferase reporter constructs in pMirTarget vector (Origene) were generated for PODXL (Origene) and DDR1 (Blue Heron). The four constructs were co-transfected with miRIDIAN Mimic hsa-miR-199b-5p in HeLa cells using Dharmafect Duo (Thermo Scientific) and 48 h post-transfection, luciferase assays were performed using Bright Glo reagent (Promega) according to manufacturer's instructions. K562 cells were transduced with miRidian shMIMIC lentiviral particles expressing hsa-miR-199b-5p or GFP empty vector control (Open Biosystems) and K562 cells stably expressing miR-199b-5p or GFP empty vector control were selected using puromycin (at 2 5μg/mL) 72 h post-transduction. For western blotting, cells were lysed in M-PER mammalian protein extraction lysis buffer (Thermo Scientific, Cat # 78501) containing Halt protease and phosphatase inhibitor cocktail (Thermo Scientific, Cat # 78442) and cleared lysates were assayed for protein content, denatured, electrophoresed, transferred to PVDF membranes and blotted. Primary antibodies (anti-human PODXL and anti-human DDR1) were from R&D Systems (Minneapolis, MN) and anti-beta-tubulin was from Cell Signaling (Boston, MA). Horseradish Peroxidase (HRP) conjugated secondary antibodies and electrochemiluminescence (ECL) reagents were used for Western blotting as described previously [26]. Migration assay was performed using CytoSelect Cell Migration Assay Kit (Cell Biolabs, San Diego, CA). K562 stable lines (hsa-miR-199b-5p and GFP empty vector control) were plated on Collagen IV coated plates (BD Biosciences) for 90 min in serum-free media and migration assay was performed as per manufacturer's protocol for suspension cells. Post 16 h of initial plating, the inner chamber was removed and three different 75 μL aliquots of the outer chamber media was examined on Moxi Z Cell Counter to determine the migrated cell counts. The significance of experimental results was determined by Student's t-test unless otherwise noted. This Research is supported in part through a Maine Cancer Foundation Grant and National Institute of Health National Center for Research Resources COBRE (P20RR018789, Project #7). Additional support was provided by Maine Medical Center Research Institute Core facilities in Genomics and Bioinformatics; and Flow Cytometry and Progenitor Cell Analysis (as supported by NIH P20RR018789, DM Wojchowski, PI); and Histopathology (as supported by NIH 1P30RR030927, R Friesel, PI and NIH P20RR018789, DM Wojchowski, PI). Additional Supporting Information may be found in the online version of this article. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article. Amanda J. Favreau* , Erin Cross*, Pradeep Sathyanarayana* , * The Graduate School of Biomedical Sciences, University of Maine, Orono, Maine, Center for Molecular Medicine, Maine Medical Center Research Institute, Scarborough, Maine, Department of Medicine, Tufts University School of Medicine, Boston, Massachusetts.