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EGF receptor promotes prostate cancer bone metastasis by downregulating miR-1 and
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activating TWIST1
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Supplemental Information
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Supplemental Materials and Methods
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Reagents and Constructs
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EGF was from R&D Systems (MN). Matrigel was purchased from BD Biosciences (CA) for
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the invasion assay. miR precursors (empty vector (EV) and miR-1 precursor) and anti-miRs
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(control and anti-miR-1) were from GeneCopoeia (MD). siRNAs (control and siEGFR)
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were from Thermo Scientific Dharmacon ON‑TARGETplus siRNA with SMARTpool
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Reagents (Thermo Scientific, MA). The AT-rich minimal consensus sequence (ATRS) was
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located upstream of human primary hsa-mir-1-1 (pri-miR-1-1) and hsa-mir-1-2 (pri-miR-1-2)
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on chromosomes 20 and 18, respectively, at GRCh37 and is listed in Supplemental Table S1.
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The RFP reporter vectors were constructed using the Clone-it Enzyme Free Lentivectors Kit
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(System Biosciences, CA). The human TWIST1 full-length 3’UTR reporter was constructed
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using the psiHECKTM-2 vector (Promega, WI). The miRNA-binding site and pri-miR-1-1
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promoter-RFP mutations were made using a Site-Directed Mutagenesis System kit
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(Invitrogen, CA). All primers used for these constructs are listed in Supplemental Table S2.
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All constructs were verified by a DNA sequence analysis.
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Clinical Outcomes and Correlation Analyses using Human Datasets
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We used mRNA expression data from two public human prostate cancer datasets, the
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Memorial Sloan-Kettering Cancer Center (MSKCC) (1) and the Cancer Genome Atlas
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(TCGA). The study using the MSKCC dataset was conducted under MSKCC Institutional
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Review Board approval on 28 normal, 151 primary, and 19 metastatic samples. Additionally,
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miRNA expressions were determined on 111 tumors (98 primary and 13 metastasis) and 28
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matched normal samples. The study using the TCGA dataset was under National Cancer
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Institute, National Institutes of Health Review Board approval on 372 primary prostate
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cancer samples from patients treated by a radical prostatectomy. These expression data were
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log2-normalized. Gene set enrichment analysis (GSEA) software from the Broad (2) was
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used to assess the significance of EGFR signaling-responsive gene signatures (3,4) by the
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normalized enrichment score (NES) and false discovery rate (FDR). Up- and downregulated
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EGFR signaling signatures used in this study were gene profiles representing up- and
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downregulation by activated EGFR signaling. Concordant overexpression of all genes in a
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signature (compared to the mean expression of all genes) led to a high positive score and
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indicated the presence of the signature in the tumor.
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Real-time Reverse transcription (RT)-PCR
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Total RNA was isolated using the mirVana PARIS RNA isolation system (Ambion, TX).
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RT of a cDNA and miRNA PCR was performed as previously described (5). Clinical
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samples from patients with independent prostate tumors used in the qRT-PCR analyses were
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extracted from dissected tissues containing >70% tumor cell content. All reactions were run
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in triplicate using primers listed in Supplemental Table S3.
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Western Blot Analysis
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Cells were lysed with RIPA buffer containing complete protease inhibitors plus the
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phosphatase inhibitors (Roche, CA), 25 mM β‐ glycerophosphate, 10 mM sodium fluoride,
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and 1 mM sodium vanadate, as previously described (5). Lysis of the nuclear and
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cytoplasmic fractions was performed using the NE-PER Nuclear and Cytoplasmic
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Extraction Reagent kit (Thermo Scientific, IL). Primary antibodies were incubated overnight
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at 4 °C using dilutions listed in Supplemental Table S4.
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In Vitro Growth Assay
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Control/miR-1 precursors or the EGFR expression vector in RasB1 cells in response to EGF
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treatment (100 ng/ml) were expressed at a density of 2x103 cells/well. Each day, one plate
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was stained with a 0.5% crystal violet fixative solution for 15 minutes, rinsed in distilled
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water, and allowed to air-dry. At the end of the experiment, the crystal violet was dissolved
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by adding 100 µl of 50% ethanol containing 0.1 M sodium citrate to each well, and the
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absorbance was quantified at a wavelength of OD 550 nm on a plate reader.
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Chromatin Immunoprecipitation (ChIP)
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ChIP assays were performed using the EZ magna ChIP A kit (Millipore, CA) with a
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modified protocol. Cells were treated with or without EGF (100 ng/ml) for 24 hours.
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Cultured cells (107) were cross-linked with 1% formaldehyde at room temperature for 15
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minutes. Fixation was quenched with glycine, and cells were washed twice with cold PBS
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containing a complete protease inhibitor (Roche, CA). Cell pellets were resuspended in cell
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lysis buffer and incubated on ice for 15 minutes. Nuclei were collected by centrifugation at
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104 rpm and 4 °C for 10 minutes and resuspended in nuclear lysis buffer. Chromatin was
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sheared using a sonicator (Branson Sonifier 250, Germany) with a microtip in a 20-second
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burst followed by 1 min of cooling on ice for a total sonication time of 5 minutes/ample.
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This procedure resulted in DNA fragment sizes of 100~300 bp. Sheared chromatin was
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divided to perform immunoprecipitation with a rabbit IgG antibody (Santa Cruz
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Biotechnology, CA) or a primary antibody at 4 °C overnight. Immunoprecipitation, washing,
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elution, reverse cross-linking, and DNA purification steps were performed according to
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Millipore’s protocol. A qPCR was performed in triplicate with 2 µl of eluted chromatin.
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ChIP antibodies and PCR primers are listed in Supplemental Table S5.
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Immunohistochemical (IHC) staining
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IHC staining was performed using TWIST1 (Millipore, MA) and phosphorylated (p)-EGFR
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(Y1068) (Cell Signaling, MA) antibodies at respective 1:500 and 1:250 dilutions. In general,
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unstained sections were deparaffinized and rehydrated. Antigen retrieval was performed
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using the Target Antigen Retrieval Solution (DAKO, CA) and autoclaved for 10 minutes.
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Endogenous peroxidase was blocked using a 3% hydrogen peroxide solution. All sections
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were blocked with Cyto Q Background Buster Reagent (Innovex BioSciences, CA). Primary
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antibodies were incubated overnight at 4 °C in Antibody Diluent with Background Reducing
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Components (DAKO, CA). The secondary antibody, 1:250 HRP-labeled anti-mouse/rabbit
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(Vector Laboratories, CA), was incubated at room temperature for 30 minutes, and bound
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peroxidase was detected using the ABC Peroxidase Kit (Vector Laboratories, CA) and DAB
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(DAKO, CA). All IHC slides were counterstained with hematoxylin. For histomorphometric
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analysis of tissue sections, microscopic images were examined under 200x magnification
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using an Axioplan microscopy system (Zeiss, NY).
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Immunofluorescent (IF) Staining
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Adherent cells were fixed in 4% paraformaldehyde (PFA) in PBS for 10 minutes, followed
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by permeabilization with 0.5% Triton X-100 in PBS for 2 minutes. Non-specific sites were
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blocked by incubation in 2% BSA in PBS for 30 minutes. Cells were then incubated
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overnight at 4 °C with the specified antibodies in 2% BSA/PBS. IF staining used EGFR and
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p-EGFR (Y1068 and Y845) (Cell Signaling, MA) antibodies at 1:100 dilutions. Cells were
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washed with PBS containing 0.1% Tween-20, incubated with Alexa-488 and/or 568
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conjugated IgG in 2% BSA for 30 minutes at room temperature, and finally washed and
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mounted using the anti-fade reagent, Fluoro-gel II, with DAPI. Fluorescent signals and
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bright-field images were captured using an inverted and/or upright fluorescent Zeiss
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Axioplan microscope (Zeiss, NY).
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Supplemental Figure Legends
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Figure S1. EGFR signaling activation is associated with reduced miR-1 expression and
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prostate cancer progression. (A and B) GSEA showed enrichment of two prostate cancer
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datasets, MSKCC (A) and TCGA (B), with EGFR signaling downregulation-responsive
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genes in cancers expressing high levels of miR-1. NES, normalized enrichment score; FDR,
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false discovery rate. (C and D) Mean expression of miR-1 for signatures separated on the
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basis of up- and downregulated EGFR signaling-responsive genes in the MSKCC (C) and
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TCGA (D) datasets.
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Figure S2. Effects of miR-1 expression in response to EGFR signaling. (A) Endogenous
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levels of miR-1 in a panel of prostate cancer cell lines. * vs. RasB1. (B) Relative EGFR
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expression levels in various prostate cancer cell lines. * vs. LNCap. (C) Cellular migration
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and invasion of LNCap and 22Rv1 cells following EGF treatment for 6 hours and then
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fixation and staining with a 0.5% crystal violet fixative solution. (D) Representative
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histological images of bone metastases in mice from RasB1/miR-1 (miR-1/EV), and
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EGFR-rescued RasB1/miR-1 (miR-1/EGFR) cells. Bone metastases are indicated by arrows
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and red dotted lines.
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Figure S3. Nuclear EGFR translocation and direct binding to the pri-miR-1-1 promoter. (A)
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IF staining of RasB1 and PC3 cells with an antibody for EGFR following EGF and CI1033
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treatment. Scale bars represent 50 µm. (B) IHC staining of FFPE cell blocks of RasB1 and
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PC3 cells with an antibody for p-EGFR (Y1068) following EGF and CI1033 treatment. (C)
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The intensity of total EGFR was normalized to the internal control, histone 3 or β-actin,
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from Western blots of nuclear and cytoplasmic cell extracts of RasB1 cells following EGF
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treatment. (D) The intensity of p-ERK1/2 was normalized to total ERK1/2 from Western
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blots of nuclear and cytoplasmic cell extracts of RasB1 cells following EGF treatment. (E)
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ChIP analyses of predicted ATRSs in the pri-miR-1-1 promoter region of 22Rv1 cells
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following EGF treatment. Enrichment of each protein at each site is given as a percentage of
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the total input and then normalized to each IgG. Data are presented as the mean ± SEM, n=3.
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* vs. -EGF. ** p<0.01, *** p<0.001.
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Figure S4. EGFR signaling activation is correlated with induced TWIST1 and prostate
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cancer progression. (A) Mean TWIST1 levels in human normal (n=28), primary (n=98), and
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metastatic (n=13) prostate samples collected and analyzed at MSKCC. * vs. normal tissue; #
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vs. primary cancer tissues. * p<0.05, ** p<0.01, *** p<0.001. (B and C) GSEA analyses
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showing enrichment of the MSKCC (B) and TCGA (C) prostate cancer datasets with EGFR
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signaling downregulation-responsive genes in cancer patients expressing low levels of
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TWIST1. NES, normalized enrichment score; FDR, false discovery rate. (D and E)
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EGF-upregulated signature genes expressed as a summed z-score for samples separated on
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the basis of TWIST1 (D) and miR-1 (E) expressions above median (TWIST1 High and
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miR-1 Low) or below median (TWIST1 Low and miR-1 High) in the MSKCC dataset. (F)
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Immunoblotting of cell extracts from RasB1 and PC3 cells following EGF treatment. (G)
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Morphology of RasB1 and PC3 cells following EGF treatment.
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Figure S5. Induction of TWIST1 is correlated with lower miR-1 and with aggressive clinical
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outcomes. (A and B) TWIST1 (A) and miR-1 (B) expressions in patient samples at different
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clinical stages in the prostate cancer samples collected and analyzed at MSKCC. * vs. T2A.
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* p<0.05, ** p<0.01. (C and D) Kaplan-Meier curve showing the survival rate relative to
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TWIST1 (C) and miR-1 (D) expressions in prostate cancer samples collected and analyzed
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at MSKCC. Patient groups with high TWIST1 and low miR-1 levels (blue line) had a lower
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survival rate compared to groups with low TWIST1 and high miR-1 levels (black line).
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Hazard ratios (log rank) were 1.664 (TWIST1) and 1.907 (miR-1). X-axes show the time in
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months, and Y-axes show percentage survival (log rank (Mantel-Cox) test p=0.0026
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(TWIST1) and p=0.0017 (miR-1)). (E) H&E and IHC staining of bone metastases with an
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antibody specific for TWIST1 in mouse from tumor-bearing mice inoculated with parental
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RasB1
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(miR-1/EGFR) cells. B: bone; T: tumor; BM: bone marrow. Scale bars represent 50 µm.
(control),
RasB1/miR-1
(miR-1/EV),
and
EGFR-rescued
RasB1/miR-1
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Supplemental References
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1.
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Taylor BS, Schultz N, Hieronymus H, Gopalan A, Xiao Y, Carver BS, et al. Integrative
genomic profiling of human prostate cancer. Cancer Cell 2010;18(1):11-22.
2.
Subramanian A, Tamayo P, Mootha VK, Mukherjee S, Ebert BL, Gillette MA, et al.
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Gene set enrichment analysis: a knowledge-based approach for interpreting
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genome-wide expression profiles. Proc Natl Acad Sci U S A 2005;102(43):15545-50.
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3.
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genes by microarray. Arch Biochem Biophys 2002;399(2):212-24.
4.
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Ma Y, Croxton R, Moorer RL, Jr., Cress WD. Identification of novel E2F1-regulated
Amit I, Citri A, Shay T, Lu Y, Katz M, Zhang F, et al. A module of negative feedback
regulators defines growth factor signaling. Nat Genet 2007;39(4):503-12.
5.
Liu YN, Yin JJ, Abou-Kheir W, Hynes PG, Casey OM, Fang L, et al. MiR-1 and
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miR-200 inhibit EMT via Slug-dependent and tumorigenesis via Slug-independent
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mechanisms. Oncogene 2013;32(3):296-306.
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Supplemental Tables
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Table S1. Positions of AT-rich minimal consensus sequences (ATRSs)
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Site
Position
ATRS1
20: 61149019
ATRS2
20: 61150113
ATRS3
20: 61150287
ATRS4
20: 61151201
ATRS5
18: 19416238
ATRS6
18: 19417140
ATRS7
18: 19417749
ATRS8
18: 19418271
Table S2. Primer sequences of the 3'UTR and promoter reporter constructs
Primary hsa-mir-1-1 promoter ATRS-binding elements reporter construct
hsa-mir-1-1 ATRS P1
TGAAAACTTATCGGCAGTGG
hsa-mir-1-1 ATRS P2
TCCCAACAGAGGGAAGTCAC
hsa-mir-1-1 ATRS P3
GAGGCAGCAGAGACCGTGAAAACTTATCGGCAGTGG
hsa-mir-1-1 ATRS P4
CGAACAGAGAGAGACCGTCCCAACAGAGGGAAGTCAC
hsa-mir-1-1 ATRS1M F
GCGGCTGGGTGCTCTAGATCTATGCAGACG
hsa-mir-1-1 ATRS1M R
CCAGACGAACCTCCGCCGACCCACG
hsa-mir-1-1 ATRS2M F
AGTTTACTTAAACCCTCTAGAACAGGCTCAT
hsa-mir-1-1 ATRS2M R
CGGTGAGCGATTCAAATGAATTTGG
hsa-mir-1-1 ATRS4M F
GACAGGCGCTCGAGGAATTCTGGGGCTCACT
hsa-mir-1-1 ATRS4M R
GACCCCGGTCCCTGTCCGCGAGCTCCT
Primary hsa-mir-1-2 promoter ATRSs binding elements reporter construct
hsa-mir-1-2 ATRS P1
TGAAAAGTTAATACCACAACCACAA
hsa-mir-1-2 ATRS P2
CGCAGGAGTGCCTACTCAG
hsa-mir-1-2 ATRS P3
GAGGCAGCAGAGACCGTGAAAAGTTAATACCACAACCACAA
hsa-mir-1-2 ATRS P4
CGAACAGAGAGAGACCGCGCAGGAGTGCCTACTCAG
Human TWIST1 3'UTR reporter constructs primer sequence
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hTWIST1 3UTR F
atcgctcgagGGGCCGGAGACCTAGATGT
hTWIST1 3UTR R
attcgtttaaacTGAATGCATTTAGACACCGGA
hTWIST1 3’UTR M555 F
AAACTTAAAATACAAAAAACAAAAGGATATTTATTTATT
hTWIST1 3’UTR M555 R
TTTGTTTTTTTTTGAATTTTATGTTTTTTGT
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Table S3. Primer sequences for the qRT-PCR
Gene
5'-3'
TWIST1 F
CGGACAAGCTGAGCAAGAT
TWIST1 R
CTGGAGGACCTGGTAGAGGA
EGFR F
CATGTCGATGGACTTCCAGA
EGFR R
GGACAGCTTGGATCACACTTT
GAPDH F
CCAGTAGAGGCAGGGATGAT
GAPDH R
CTTTCATTGTCTTTTCCGCC
Table S4. Antibody information for Western blotting
Primary
antibody
Source
Dilution Secondary
antibody
Source
Dilution
p-EGFR
(Y1068)
Cell Signaling (#3777)
1/1000 anti-rabbit IgG
Jackson Lab
1/5000
EGFR
Cell Signaling (#4267)
1/1000 anti-rabbit IgG
Jackson Lab
1/5000
TWIST1
GeneTex (GTX127310)
1/1000 anti-rabbit IgG
Jackson Lab
1/5000
p-ERK1/2 Cell Signaling (#4376)
1/1000 anti-rabbit IgG
Jackson Lab
1/5000
ERK1/2
Cell Signaling (#9102)
1/1000 anti-mouse IgG
Jackson Lab
1/20000
Vimentin
Santa Cruz (sc-7557)
1/1000 anti-rabbit IgG
Jackson Lab
1/5000
Histone 3
Millipore (#07-690)
1/1000 anti-rabbit IgG
Jackson Lab
1/20000
GAPDH
Novus (NB300-221)
1/1000 anti-mouse IgG
Jackson Lab
1/5000
β-actin
GeneTex (GTX109639)
1/1000 anti-rabbit IgG
Jackson Lab
1/20000
Table S5. Antibody information and primer sequences of the ChIP assay
ChIP antibodies
Primary antibody
Source
Dilution
p-EGFR (Y1068)
Cell Signaling (#3777)
1/50
EGFR
Cell Signaling (#4267)
1/50
GAPDH
Novus (NB300-221)
1/50
Rabbit IgG
Santa Cruz (sc-2027)
1/50
Mouse IgG
Santa Cruz (sc-2343)
1/50
ChIP primers
Site
5'-3'
ATRS1 F
GGTATTCACCGCTGAAGAGC
ATRS1 R
AAAGGGCAGGAGAGTCACCC
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ATRS2 F
AGCCTTCTTTCCACCTCTCAC
ATRS2 R
AAAATGTGGAGAAAGAGCCAGA
ATRS3 F
CTGCTTCCCACTCAGAGACA
ATRS3 R
CCAGCCCCTGATCAATACCA
ATRS4 F
AGGGGGACAGGAAAGTGTTG
ATRS4 R
CTGTCTCACACACTCACACGA
ATRS5 F
GCAGCAGAGGGACTTCACTT
ATRS5 R
AGCACTGCCAAATAAAGCGG
ATRS6 F
TCCCCTCTTCTGAAGCATTTCA
ATRS6 R
TTCCCTCTCCTCCCCTCTTC
ATRS7 F
TGATTTTGCATCTCTAGTAAGTCACA
ATRS7 R
AGCAGTTTCAGCAATGCAGC
ATRS8 F
ACCCAGGTGCTCACAGACTA
ATRS8 R
TGCACTTTGATGCTTCTCTTTGG