The MCF7, T47D, MDA-MB-231, 4T1 and 293T cell lines were purchased from American Type Culture Collection and were cultured under conditions specified by the manufacturer. The SUM149, SUM159, SUM229 and SUM1315 cell lines were from S. Ethier and cultured as described (SUM149, SUM159 and SUM229 cell lines, http://www.asterand.com/Asterand/human_tissues/149PT.htm
; SUM1315 cell line, http://www.asterand.com/Asterand/human_tissues/1315M02.htm
). Mouse and human LIF ligands were purchased from Gibco and Millipore, respectively. For LIF stimulation, cells were starved in serum-free medium and then treated with mouse (for 4T1 cells, 10 ng/ml for 30 min) or human (for MDA-MB-231 cells, 50 ng/ml for 1 h) LIF, according to the manufacturer's instructions.
Plasmids and shRNA
The human mir-9-3
genomic sequence was PCR amplified from normal genomic DNA and cloned into the MDH1-PGK-GFP 2.0 retroviral vector as previously described14
. A LIFR
3′ UTR fragment (801 bp) was cloned into the pMIR-REPORT luciferase construct10
using the following cloning primers: forward, 5′-TGCACACTAGTCAGTGTCACCGTGTCACTTCA-3′; reverse, 5′-CTAGTAAGCTTGTCTCTAGTCTTAGAAGTGTA-3′. The shRNA and ORF clones were from Open Biosystems through MD Anderson's ShRNA and ORFeome Core, and the clone numbers are as follows: human LIFR shRNA, V3LHS-347493 (designated as `A8') and V3LHS-347496 (designated as `F3'); human YAP shRNA, V2LHS_65508 and V3LHS-306101; mouse gp130 shRNA, V2LMM-219118 and V3LMM-503552; human LIFR ORF, PLOH-100016429; mouse LIFR fully sequenced complementary DNA (cDNA), 4159053; mouse YAP ORF, MMM1013-7510984; and mouse CTGF ORF, MMM1013-64071. The shRNA sequences can be found at https://www.openbiosystems.com/
using the clone numbers. The LIFR 3′ UTR mutant and the YAP nonphosphorylatable mutant were generated using a QuikChange Site-Directed Mutagenesis Kit (Stratagene). The vectors used in this study are listed in Supplementary Table 4
Four individual siRNAs that target human LIFR were purchased from Sigma and Dharmacon. The siRNA sequences are as follows: (i) GUUGCAAUCAAGAUUCGUA (Sigma, SASI_Hs02_00330115); (ii) CGAUUAACAGUGUCACCGU (Sigma, SASI_Hs02_00330118); (iii) CCA CACCGCUCAAAUGUUA (Dharmacon, J-008017-06); and (iv) GAAC AAAACGUUUCCUUAA (Dharmacon, J-008017-08). Cells were transfected with 150 nM of the indicated oligonucleotide using the Oligofectamine reagent (Invitrogen). Forty-eight hours after transfection, cells were plated for migration and invasion assays, and the remaining cells were harvested for western blot analysis.
RNA isolation and real-time RT-PCR
Total RNA, with efficient recovery of small RNAs, was isolated using the mirVana miRNA Isolation Kit (Ambion) and was then reverse transcribed with an iScript cDNA Synthesis Kit (Bio-Rad). The resulting cDNA was used for qPCR using the TaqMan Gene Expression Assays (Applied Biosystems), and data were normalized to an endogenous control, β-actin. Quantification of the mature form of the miRNAs was performed using the TaqMan MicroRNA Assay Kit (Applied Biosystems) according to the manufacturer's instructions, and U6 small nuclear RNA was used as an internal control. Real-time PCR and data collection were performed on a CFX96 instrument (Bio-Rad).
Tumor metastasis PCR array analysis
The Tumor Metastasis RT2
Profiler PCR Array, consisting of 84 genes known to be involved in metastasis, was used to profile LIFR-expressing 4T1 cells according to the manufacturer's instructions (http://www.sabiosciences.com/rt_pcr_product/HTML/PAMM-028Z.html
). Briefly, total RNA was extracted and reverse transcribed into cDNA using an RT2
First Strand Kit (Qiagen). The cDNA was combined with an RT2
SYBR Green qPCR Master Mix (Qiagen), and then equal aliquots of this mixture (25 μl) were added to each well of the same PCR Array plate that contained the predispensed gene-specific primer sets. Real-time PCR and data collection were performed on a CFX96 instrument (Bio-Rad).
Lentiviral and retroviral transduction
The production of lentivirus and amphotropic retrovirus and the infection of target cells were performed as described previously41
miRNA target analysis
Genes that contained the miR-9-binding site(s) in their 3′ UTR were obtained using the TargetScan program18
; version 5.1). The RNAhybrid program42
was used to predict duplex formation between human LIFR
3′ UTR and miR-9.
Cell growth and viability assays
To determine growth curves, we plated equal numbers of cells in 6-cm dishes. Beginning the next day, cells were trypsinized and counted every day. To determine cell viability, we trypsinized cells and diluted them with 0.4% trypan blue staining solution. Cell counts and the percentages of viable cells were obtained from a TC10 Automated Cell Counter (Bio-Rad).
Migration and invasion assays
Transwell migration and Matrigel invasion assays were performed as described previously10
Luciferase reporter assay
Dual luciferase reporter assays were performed as described previously10
Western blot analyses were performed with precast gradient gels (Bio-Rad) using standard methods. Briefly, cells were lysed in the radio-immunoprecipitation assay (RIPA) buffer containing protease inhibitors (Roche) and phosphatase inhibitors (Sigma). Proteins were separated by SDS-PAGE and blotted onto a nitrocellulose membrane (Bio-Rad). Membranes were probed with the specific primary antibodies and then with peroxidase-conjugated secondary antibodies. The bands were visualized by chemiluminescence (Denville Scientific). The following antibodies were used: antibodies to LIFR (1:1,000, Santa Cruz Biotechnology, sc-659), gp130 (1:500, Upstate, 09-261), E-cadherin (1:1,000, BD Transduction Laboratories, 610182), pSTAT3 (phosphorylated at Tyr705; 1:1,000, Cell Signaling Technology, 9131), STAT3 (1:1,000, Cell Signaling Technology, 9132), pYAP (Ser127; 1:1,000, Cell Signaling Technology, 4911), YAP (1:500, Cell Signaling Technology, 4912), pLATS1 (Ser909; 1:500, Cell Signaling Technology, 9157), LATS1 (1:1,000, Cell Signaling Technology, 3477), pMST1 (Thr183)/MST2 (Thr180) (1:500, Cell Signaling Technology, 3681), MST1 (1:500, Cell Signaling Technology, 3682), histone H3 (1:1,000, Millipore, 06-755), HSP90 (1:3,000, BD Transduction Laboratories, 610419), CTGF (1:500, Abcam, ab6992; and 1:500, Santa Cruz Biotechnology, sc-34772), Scribble (1:500, Santa Cruz Biotechnology, sc-11048), Na/K ATPase (1:500, Santa Cruz Biotechnology, sc-21712), vimentin (1:2,000, NeoMarkers, MS-129-P), β-actin (1:5,000, Sigma, A5441), cyclophilin B (1:2,000, Thermo, PA1-027A) and GAPDH (1:3,000, Thermo, MA5-15738). The ImageJ program (http://rsbweb.nih.gov/ij/download.html
) was used for densitometric analyses of western blots, and the quantification results were normalized to the loading control.
Fractionation of nuclear and cytoplasmic proteins was done using the NE-PER Nuclear and Cytoplasmic Extraction Kit (Thermo) according to the manufacturer's protocol. After fractionation, 30 mg of protein was used for western blot analysis of YAP in the cytoplasm and nucleus. HSP90 and histone H3 were used as markers of cytoplasm and the nucleus, respectively. Plasma membrane proteins were isolated using the Plasma Membrane Protein Extraction Kit (Abcam) according to the manufacturer's protocol. Twenty-five micrograms of protein was used for the western blot analysis of plasma-membrane–localized Scribble. HSP90 and Na/K ATPase were used as markers of cytoplasm and the plasma membrane, respectively.
Cells were cultured in chamber slides overnight and fixed with 3.7% formaldehyde in PBS for 20 min at 4 °C and then permeabilized with 0.5% Triton X-100 in PBS for 30 min. Cells were then blocked for nonspecific binding with 5% milk in PBS and Tween-20 (PBST) overnight and incubated with YAP-specific antibody (1:300, Cell Signaling Technology, 4912) at 37 °C for 1 h and then incubated with Alexa Fluor 488 goat anti-rabbit IgG (1:500, Invitrogen, A11008) or Alexa Fluor 594 goat anti-rabbit IgG (1:500, Invitrogen, A11012) at 37 °C for 1 h. Cover slips were mounted on slides using antifade mounting medium with DAPI. Immunofluorescence images were acquired on a Zeiss Axio Observer Z1 microscope.
RNA sequencing analysis
Ten micrograms of total RNA from each sample was used to construct RNA-Seq libraries using the Solexa kit (Illumina) according to the manufacturer's instructions. Images acquired from the Solexa sequencer were processed through the bundled Solexa image extraction pipeline version 1.6. RNA-Seq reads were aligned to the human reference sequence NCBI Build 36.1 (hg18) using ELAND (as implemented in CASAVA version 1.6). Briefly, the first 25 bases of a read were used as a seed. Each matched seed was then extended to 36 bases and scored to break any ties between multimatches. For mRNA expression counts, unique reads in the genome that landed within any exons of NCBI gene models (v37.1) were counted. The counts were normalized to the mRNA length and then further normalized to one million total reads to obtain the reads per kilobase of exon per million mapped reads (RPKM) values. The RPKM value was considered the final expression of a given sample. A list of genes (`list A') was determined by the following criteria: (i) the count of a gene was more than 1.5 times higher in mock-infected cells (SUM159_MDH1) than in miR-9–expressing SUM159 cells (SUM159_miR-9); and (ii) the count was no less than five in the SUM159_MDH1 sample. A second list of genes (`list B') that contain the miR-9 binding site(s) was obtained using the TargetScan program (http://www.targetscan.org/
; version 5.1). Genes that contain miR-9–binding site(s) and are downregulated by miR-9 (`list C') were then determined by comparing list A to list B.
All animal experiments were performed in accordance with a protocol approved by the Institutional Animal Care and Use Committee of MD Anderson Cancer Center. Six- to eight-week-old female NOD-SCID (for orthotopic injection of human cells), nude (for intravenous injection of human cells) or BALB/c (for all injections of mouse cells) mice were used for tumor cell implantation. For orthotopic injection, mice were anesthetized, and the skin was incised; tumor cells (0.5 × 106) in 25 μl growth medium (mixed with Matrigel at a 1:1 ratio) were injected into the inguinal mammary fat pad using a 100-μl Hamilton microliter syringe, and the incision was then closed using wound clips. For intravenous injection, mice were placed in a restrainer, and tumor cells (4T1 cells, 0.5 × 106 cells in 100 μl PBS; SUM159 cells, 2 × 106 cells in 200 μl PBS) were injected through the tail vein using a 1-ml syringe. Mice were euthanized when they met the institutional euthanasia criteria for tumor size and overall health condition. The mammary tumors were removed and weighed; the freshly dissected primary tumors, lungs, livers, spleens, kidneys and macroscopic metastases were examined and photographed using a Zeiss SteREO Discovery V20 stereomicroscope equipped with bright-field and fluorescence imaging. Tissue samples were fixed in 10% buffered formalin overnight and then washed with PBS, transferred to 70% ethanol and then embedded in paraffin, sectioned and stained with H&E. The immunohistochemistry detection using the GFP-specific (1:1,000, Invitrogen, A6455) and vimentin-specific (human specific, 1:2,000, Dako, M0725) antibodies was performed on paraffin sections in the Histology Core Lab at Memorial Sloan-Kettering Cancer Center. Stained sections were photographed using a Zeiss Axio Observer Z1 microscope.
All TMAs were purchased from the NCI Cancer Diagnosis Program. These TMAs have associated pathological and clinical outcome data from the Cooperative Breast Cancer Tissue Resource (CBCTR). The Progression TMAs consist of three different case sets, including 134 analyzable cases of invasive breast carcinoma, 17 analyzable cases of DCIS and 34 analyzable cases of normal breast tissue. The Prognostic TMAs consist of five nonoverlapping stage I case sets (590 specimens), four stage II case sets (398 specimens) and two stage III case sets (181 specimens); these nonmetastatic stage I–III breast tumor specimens have a long-term clinical follow-up record (mean follow-up time of 122 months; longest follow-up time of 284 months). Samples were deparaffinized and rehydrated. Antigen retrieval was done using 0.01 M sodium-citrate buffer (pH 6.0) in a microwave oven. To block endogenous peroxidase activity, the sections were treated with 1% hydrogen peroxide in methanol for 30 min. After 1 h of preincubation in 10% normal serum to prevent nonspecific staining, the samples were incubated with LIFR-specific antibody (1:1,500, Santa Cruz Biotechnology, sc-659) at 4 °C overnight. The sections were then treated with a biotinylated secondary antibody (Vector Laboratories, PK-6101, 1:200) and then incubated with avidin-biotin peroxidase complex solution (1:100) for 1 h at room temperature. Color was developed with the 3-amino-9-ethylcarbazole (AEC) solution. Counterstaining was carried out using Mayer's haematoxylin. All immunostained slides were scanned on the Automated Cellular Image System III (ACIS III, Dako, Denmark) for quantification by digital image analysis. A total score of protein expression was calculated from both the percentage of immunopositive cells and the immunostaining intensity. High and low protein expression were defined using the mean score of all samples as a cutoff point. Negative expression indicated no detectable immunoreactivity. Spearman rank correlation was used for statistical analyses of the correlation between LIFR and the clinical parameters. Kaplan-Meier survival analysis and the log-rank test were used for statistical analyses of the correlation between LIFR and clinical survival outcomes.
Oncomine's gene search function (https://www.oncomine.org/resource/login.html
) was used to assess and visualize the differential expression of a selected gene across all available datasets (updated March 2011). We searched for LIFR
in human cancer using the following threshold values: P
value of 0.05, fold change of 2 and gene rank in the top 10% among all differentially expressed genes. Oncomine then listed all differential expression analyses in which LIFR
was included. For each listed analysis, the statistical results were provided and linked to graphical representations of the original microarray dataset.
TCGA data analysis
We obtained mRNA and miRNA expression data of clinical breast cancer from the TCGA data portal (http://cancergenome.nih.gov/
). mRNA expression was measured using the Agilent 244K Custom Gene Expression G4502A-07-3 platform, and miRNA expression was measured using the Illumina (Genome Analyzer or HiSeq 2000) miRNA sequencing platform. We used the level three data provided by TCGA: log2 scale normalized data for mRNA expression, and `reads per million miRNA reads' for miRNA expression. There are 536 tumor samples with available mRNA expression data (updated July 2011), 788 tumor samples with available miRNA expression data (updated October 2011) and 512 samples with both mRNA and miRNA expression data. Among the three precursors of miR-9 (mir-9-1, mir-9-2 and mir-9-3), the expression data of mir-9-3 in the vast majority of the samples were zero and were therefore excluded from further analyses. Spearman rank correlation was used to quantify the correlation of any miRNA-mRNA or mRNA-mRNA pair.
Unless otherwise noted, each sample was assayed in triplicate. Each in vitro experiment was repeated three to five times or more, and each in vivo experiment was repeated two or three times. Unless otherwise noted, data are presented as means ± s.e.m., and Student's t test (unpaired, two-tailed) was used to compare two groups of independent samples. Correlations of LIFR with clinical parameters and correlation of miRNA-mRNA and mRNA-mRNA pairs were analyzed using Spearman rank correlation tests. The log-rank test was used to compare Kaplan-Meier survival curves.