Identification of PADI2 as a potential breast cancer biomarker and therapeutic target
© McElwee et al.; licensee BioMed Central Ltd. 2012
Received: 12 April 2012
Accepted: 27 October 2012
Published: 30 October 2012
We have recently reported that the expression of peptidylarginine deiminase 2 (PADI2) is regulated by EGF in mammary cancer cells and appears to play a role in the proliferation of normal mammary epithelium; however, the role of PADI2 in the pathogenesis of human breast cancer has yet to be investigated. Thus, the goals of this study were to examine whether PADI2 plays a role in mammary tumor progression, and whether the inhibition of PADI activity has anti-tumor effects.
RNA-seq data from a collection of 57 breast cancer cell lines was queried for PADI2 levels, and correlations with known subtype and HER2/ERBB2 status were evaluated. To examine PADI2 expression levels during breast cancer progression, the cell lines from the MCF10AT model were used. The efficacy of the PADI inhibitor, Cl-amidine, was tested in vitro using MCF10DCIS cells grown in 2D-monolayers and 3D-spheroids, and in vivo using MCF10DCIS tumor xenografts. Treated MCF10DCIS cells were examined by flow-cytometry to determine the extent of apoptosis and by RT2 Profiler PCR Cell Cycle Array to detect alterations in cell cycle associated genes.
We show by RNA-seq that PADI2 mRNA expression is highly correlated with HER2/ERBB2 (p = 2.2 × 106) in luminal breast cancer cell lines. Using the MCF10AT model of breast cancer progression, we then demonstrate that PADI2 expression increases during the transition of normal mammary epithelium to fully malignant breast carcinomas, with a strong peak of PADI2 expression and activity being observed in the MCF10DCIS cell line, which models human comedo-DCIS lesions. Next, we show that a PADI inhibitor, Cl-amidine, strongly suppresses the growth of MCF10DCIS monolayers and tumor spheroids in culture. We then carried out preclinical studies in nude (nu/nu) mice and found that Cl-amidine also suppressed the growth of xenografted MCF10DCIS tumors by more than 3-fold. Lastly, we performed cell cycle array analysis of Cl-amidine treated and control MCF10DCIS cells, and found that the PADI inhibitor strongly affects the expression of several cell cycle genes implicated in tumor progression, including p21, GADD45α, and Ki67.
Together, these results suggest that PADI2 may function as an important new biomarker for HER2/ERBB2+ tumors and that Cl-amidine represents a new candidate for breast cancer therapy.
KeywordsPeptidylarginine deiminase PAD2/PADI2 HER2/ERBB2 Breast cancer Luminal Cl-amidine Citrullination
PADIs are a family of posttranslational modification enzymes that convert positively charged arginine residues on substrate proteins to neutrally charged citrulline, and this activity is alternatively called citrullination or deimination. The PADI enzyme family is thought to have arisen by gene duplication and localizes within the genome to a highly organized cluster at 1p36.13 in humans. At the protein level, each of the five well-conserved PADI members shows a relatively distinct pattern of substrate specificity and tissue distribution [1, 2]. Increasingly, the dysregulation of PADI activity is associated with a range of diseases, including rheumatoid arthritis (RA), multiple sclerosis, ulcerative colitis, neural degeneration, COPD, and cancer [3–5]. While the presumptive function of PADI activity in most diseases is linked to inflammation, the role that PADIs play in cancer progression is not clear. We and others, however, have found that PADI4 appears to play a role in gene regulation in cancer cells via histone tail citrullination. For example, in MCF7 breast cancer cells estrogen stimulation enhances PADI4 binding and histone H4 citrullination at the canonical ER target gene, TFF1, leading to transcriptional repression . On the other hand, stimulation of MCF7 cells with EGF facilitates activation of c-fos via PADI4-mediated citrullination of the ELK1 oncogene . Additionally, others have shown that citrullination of the p53 tumor suppressor protein affects the expression of p53 target genes p21, OKL38, CIP1 and WAF1[8–10]. Interestingly, treatment of several PADI4-expressing cancer cell lines with the PADI inhibitor, Cl-amidine, elicited strong cytotoxic effects while having no observable effect on non-cancerous lines , suggesting that PADIs may represent targets for new cancer therapies.
Our current study suggests that PADI2 may also play a role in cancer progression, and this prediction is supported by several previous studies. For example, a mouse transcriptomics study investigating gene expression in MMTV-neu tumors found that PADI2 expression was upregulated ~2-fold in hyperplastic, and ~4-fold in primary neu-tumors, when compared to matched normal mammary epithelium . In humans, PADI2 is one of the most upregulated genes in luminal breast cancer cell lines compared to basal lines [13, 14]. Additionally, gene expression profiling of 213 primary breast tumors with known HER2/ERBB2 status identified PADI2 as one of 29 overexpressed genes in HER2/ERBB2+ tumors; thus, helping to define a HER2/ERBB2+ gene expression signature . Given these previous studies, our goal was to formally test the hypothesis that PADI2 plays a role in mammary tumor progression. For the study, we first documented PADI2 expression and activity during mammary tumor progression, and then investigated the effects of PADI inhibition in cell cultures, tumor spheroids, and preclinical in vivo models of breast cancer.
Cell culture and treatment with Cl-amidine
The MCF10AT cell line series (MCF10A, MCF10AT1kC1.2, MCF10DCIS.com, and MCF10CA1aC1.1) was obtained from Dr. Fred Miller (Barbara Ann Karmanos Cancer Institute, Detroit, MI, USA). This biological system has been extensively reviewed [16, 17] and culture conditions described [18–20]. The MCF7, BT-474, SK-BR-3, and MDA-MB-231 cell lines were from obtained from ATCC (Manassas, VA, USA) and cultured according to manufacturer’s directions. All cells were maintained in a humidified atmosphere of 5% CO2 at 37°C. For the experimental treatment of cell lines with Cl-amidine, cells were seeded in 6-well plates (2 × 104) and collected by trypsinization 5d post-treatment. Counts were performed using a Coulter counter (Beckman Coulter, Fullerton, CA, USA) and are represented as mean fold difference in cell number after treatment. Cl-amidine was synthesized as previously described .
MMTV mice and the generation of MCF10DCIS xenografts and multicellular tumor spheroids
Tissues from the MMTV-neu mouse were a generous gift from Dr. Robert S. Weiss, Cornell University, and the MMTV-Wnt-1 hyperplastic mammary glands and tumors were a gift of Dr. Louise R. Howe, Weill Cornell Medical College. MCF10DCIS xenograft tumors were generated by injecting 1 × 106 cells in 0.1 mL Matrigel (1:1) (BD Biosciences, San Jose, CA, USA) subcutaneously near the nipple of gland #3 in 6-week old female nude (nu/nu) mice (Taconic, Germantown, NY, USA). When the tumors reached ~200 mm3, intraperitoneal injections of Cl-amidine (50 mg/kg/day) or vehicle control (PBS) were initiated and carried out for 14 days. Tumor volume was calculated by the formula: (mm3) = (d2 × D)/2, where “d” and “D” are the shortest and longest diameters of the tumor, respectively. Tumor volume was measured weekly by digital caliper, and the differences between tumor volumes were evaluated by the non-parametric Mann–Whitney–Wilcoxon (MWW) test. Results are reported as mean ± SD. After 14 days, tumors were removed and either snap-frozen, placed in RNAlater (Qiagen Inc., Valencia, CA, USA), or added to 10% buffered formalin. Seven mice per group were used for each treatment. All mouse experiments were reviewed and approved by the Institutional Animal Care and Use Committees (IACUC) at Cornell University. Multicellular tumor spheroids were generated using the liquid overlay technique as previously described [22–24]. The spheroids were allowed to form over 48h and maintained up to 6–10 days for morphological analysis, then collected, rinsed with phosphate buffered saline, and fixed in 10% buffered formalin.
Assay of PADI activity
Cell lines were assayed for PADI activity as previously described [25, 26]. Briefly, citrulline levels were determined using BAEE (α-N-benzoyl-L-arginine ethyl ester) as a substrate. After incubating lysates for 1h at 50°C with BAEE substrate mixture, the reaction was stopped by the addition of perchloric acid. The perchloric acid-soluble fraction was subjected to a colorimetric reaction with citrulline used as a standard and absorbance measured at 464 nm.
Immunohistochemistry (IHC) and immunofluorescence (IF)
IHC and IF experiments were carried out using a standard protocol as previously described . Primary antibodies are as follows: anti-PADI2 1:100 (ProteinTech, Chicago, IL, USA), anti-ERBB2 (A0485) 1:100 (Dako, Carpentaria, CA, USA), anti-Cytokeratin 1:100 (Dako), and anti-p63 1:100 (Abcam, Cambridge, MA, USA). Sections prepared for IHC were incubated in DAB chromagen solution (Vector Laboratories, Burlingame, CA, USA) according to the manufacturer’s protocol, washed, and then counterstained with hematoxylin. The IF slides were incubated in streptavidin conjugated-488 (Invitrogen, Carlsbad, CA, USA), washed, and then mounted using Vectashield containing DAPI (Vector Laboratories). Negative controls for both IHC and IF experiments were either rabbit or mouse IgG antibody at the appropriate concentrations. Tumor sections were examined for general morphological differences after hematoxylin and eosin (H&E) staining. Basement membrane integrity was determined using periodic acid-Schiff (PAS) stained slides, and was scored by SM on a scale of 0–3: 0- continuous with no breaching, 1- a few small interruptions, 2- several interruptions with breaching by tumor cells, 3- extensive loss of basement membrane with invasion of tumor cells over the breached area; observations were performed under 10X magnification.
Immunoblotting was carried out as previously described . Primary antibodies were incubated overnight at 4°C using the following concentrations: anti-PADI2 1:1000 (ProteinTech) and anti-ErbB2 1:5000 (Dako). To confirm equal protein loading, membranes were stripped and re-probed with anti-β-actin 1:5000 (Abcam).
Quantitative real-time PCR (qRT-PCR)
RNA was purified using the Qiagen RNAeasy kit, including on-column DNAse treatment to remove genomic DNA. The resulting RNA was reverse transcribed using the ABI High Capacity RNA to cDNA kit according to the manufacturer’s protocol (Applied Biosystems, Foster City, CA, USA). TaqMan Gene Expression Assays (ABI) for human PADI2 (Hs00247108_m1) and GAPDH (4352934E) were used for qRT-PCR. Data were analyzed by the 2 -ΔΔ C(t) method . Data are shown as means ± SD from three independent experiments, and were separated using Student’s t-test. For the analysis of cell cycle gene expression, cDNA was synthesized (RT2 First Strand Kit, Qiagen) and samples analyzed for expression of 84 genes involved in cell cycle regulation by RT2 Profiler PCR Cell Cycle Array (PAHS-020A, Qiagen). For data analysis, the RT2 Profiler PCR Array software package was used and statistical analyses performed (n = 3). This package uses ΔΔ CT–based fold change calculations and the Student’s t-test to calculate two-tail, equal variance p-values.
Monolayers of MCF10DCIS and MCF10A cells were seeded into 25 cm2 flasks (2 × 106 cells) and treated with either Cl-amidine (200 μM or 400 μM), or 10μg/mL tunicamycin (apoptosis positive control). BT-474, SK-BR-3, and MDA-MB-231 cell lines were treated as previously described for MCF10DCIS and MCF10A; however, they were also treated with 100 μM Cl-amidine. Cells were harvested after 4d using Accutase (Innovative Cell Technologies, Inc, San Diego, CA, USA), fixed, then permeabilized, and blocked in FACS Buffer (0.1M Dulbecco’s phosphate buffered saline, 0.02% sodium azide, 1.0% bovine serum albumin, and 0.1% Triton X-100) containing 10% normal goat serum and stained (except the isotype controls) with rabbit anti-cleaved Caspase-3 antibody (Cell Signaling Technology, Inc, Danvers, MA, USA). Isotype controls were treated with normal rabbit IgG (Vector Laboratories) at 4 μg/mL. All samples were stained with secondary goat anti-rabbit IgG conjugated to Alexa-488 (Invitrogen) and DAPI (Invitrogen) according to the manufacturer’s instructions. Cells were analyzed on a FACS-Calibur (BD Biosciences) or a Gallios (Beckman Coulter) flow-cytometer and data analyzed for percent apoptotic cells (cleaved Caspase-3+) and cell cycle analysis with FlowJo software (TreeStar, Inc, Ashland, OR, USA). Data are shown as means ± SD from three independent experiments, and were separated using Student’s t-test.
RNA-seq analysis of breast cancer cell lines
Whole transcriptome shotgun sequencing (RNA-seq) was completed on breast cancer cell lines and expression analysis was performed with the ALEXA-seq software package as previously described . Briefly, this approach comprises (i) creation of a database of expression and alternative expression sequence ‘features’ (genes, transcripts, exons, junctions, boundaries, introns, and intergenic sequences) based on Ensembl gene models, (ii) mapping of short paired-end sequence reads to these features, (iii) identification of features that are expressed above background noise while taking into account locus-by-locus noise. RNA-seq data was available for 57 lines (17 basal, 5 basal-NM, 6 claudin-low, 29 luminal). An average of 70.6 million (76bp paired-end) reads passed quality control per sample. Of these, 53.8 million reads mapped to the transcriptome on average, resulting in an average coverage of 48.2 across all known genes. Log2 transformed estimates of gene-level expression were extracted for analysis with corresponding expression status values indicating whether the genes were detected above background level.
All experiments were independently repeated at least three times unless otherwise indicated. Values were expressed as the mean ± the SD. Means were separated using Student’s t-test or by Mann—Whitney-Wilcoxon (MWW) test, with a p-value less than 0.05 considered as significantly different. Subtype specific expression in the RNA-seq analysis was determined by Wilcoxon signed-rank test. Correlations were determined by Spearman rank correlation. Genes were considered significantly differentially expressed or correlated if they had a p-value less than 0.05.
PADI2 is overexpressed in transformed cells of the MCF10AT model of breast cancer progression
Levels of PADI2 correlate with the luminal breast cancer subtype and HER2/ERBB2 overexpression
Top 13 genes correlating with HER2/ERBB2 expression
Post-GPI attachment to proteins 3 (PERLD1)
CAMP responsive element binding protein 3-like 3
Chromosome 2 open reading frame 54
EF-hand calcium binding domain 4A
Rho GTPase activating protein 8
Growth factor receptor-bound protein 7
Transmembrane protein 210
E74-like factor 3 (ets domain transcription factor, epithelial-specific )
Tripartite motif containing 3
Protease, serine, 8
Peptidylarginine deiminase, type II
Inhibition of PADI activity reduces cellular proliferation in breast cancer cell lines
Cl-amidine alters the expression of cell cycle associated genes and induces apoptosis
Top 10 cell cycle genes up- and down-regulated in MCF10DCIS cells after Cl-amidine treatment
Cyclin-dependent kinase inhibitor 1A (p21, Cip1)
Growth arrest and DNA-damage-inducible, alpha
Ataxia telangiectasia mutated
DIRAS family, GTP-binding RAS-like 3
Hect domain and RLD 5
RAD17 homolog (S. pombe)
Ataxia telangiectasia and Rad3 related
Minichromosome maintenance complex component 5
Cyclin-dependent kinase 1
Cell division cycle 20 homolog (S. cerevisiae)
G-2 and S-phase expressed 1
Minichromosome maintenance complex component 2
Antigen identified by monoclonal antibody Ki-67
Baculoviral IAP repeat containing 5 (Survivin)
B-cell CLL/lymphoma 2
PADI2 is highly expressed in the luminal epithelium of xenograft tumors derived from MCF10DCIS cells
Next, we examined whether the observed correlation between PADI2 and HER2/ERBB2 expression also occurred in vivo. We found that both HER2/ERBB2 and PADI2 were expressed within the luminal epithelium of MCF10DCIS tumors (Figure 6a, panel III and IV). Interestingly, a previous report by Behbod et. al. found low levels of HER2/ERBB2 in MCF10DCIS tumors that were grown intraductally. The disparity between this data and our data may be due to differences in the microenvironment. We then quantified PADI2 mRNA in the MCF10DCIS xenografts by qRT-PCR, and found that PADI2 levels were significantly higher in the tumors when compared to monolayer cultures (Figure 6b). We also carried out immunofluorescence (IF) analysis of these tumors to examine PADI2 intratumoral localization, and found that PADI2 protein expression appears entirely limited to cytokeratin-positive luminal epithelial cells (Figure 6c, panel I and III, and Additional file 5, Figure S4), while no detectable PADI2 signal was observed in the p63 positive myoepithelial cells (Figure 6c, panel IV and VI, and Additional file 5, Figure S4).
Treatment of MCF10DCIS xenografts with Cl-amidine suppresses tumor growth
In this study, we show that PADI2 is specifically upregulated during mammary tumor progression and that the PADI inhibitor, Cl-amidine, is effective in inhibiting the growth of PADI2 overexpressing cell lines in both 2D and 3D cultures. In addition, we demonstrate here for the first time that Cl-amidine is successful in suppressing tumor growth in a xenograft mouse model of comedo-DCIS. Lastly, we document that PADI2 expression is highly correlated with HER2/ERBB2 overexpressing and luminal subtype breast cancers.
Given the previous correlations between PADI2 and the HER2/ERBB2 oncogene, the goal of this study was to carry out an initial test of the hypothesis that PADI2 plays a role in breast cancer progression. To accomplish this, we utilized the well-established MCF10AT model [16, 17] and found that PADI2 expression was highly upregulated in MCF10DCIS cells, a cell line that forms comedo-DCIS lesions that spontaneously progress to invasive tumors [30, 46]. Our finding that PADI2 expression is highest in comedo-DCIS lesions (defined by their necrotic centers) was perhaps not too surprising, given the close association of PADIs with inflammatory events. We are currently investigating the potential links between inflammatory signaling in these MCF10DCIS lesions and PADI2 activity.
Interestingly, PADI2 expression in the MCF10AT series coincided with HER2/ERBB2 upregulation which, again, was not entirely unexpected given previous reports correlating PADI2 expression with HER2/ERBB2. While we did find that HER2/ERBB2 and PADI2 protein expression correlated well across the MCF10AT cell lines, PADI2 protein levels are particularly high in the MCF10DCIS line, relative to HER2/ERBB2. We cannot currently explain this finding; however, it is possible that cell-line-specific factors are stabilizing the PADI2 transcript, thus allowing for increased protein expression [51, 52].
While our data show a potential relationship between PADI2 and HER2/ERBB2 in the MCF10AT model, we wanted to examine this correlation at higher resolution. To accomplish this we queried our RNA-seq dataset of 57 breast cancer cell lines with known subtype and HER2/ERBB2 status and found that: (a) PADI2 expression is highest in luminal cell lines and that (b) PADI2 expression is highly correlated with HER2/ERBB2 overexpression across the basal-NM, claudin-low, and luminal lines. The observation that PADI2 is upregulated in the luminal subtype confirms previous gene expression data where PADI2 was identified as one of the top upregulated genes in luminal breast cancer lines compared to basal lines [13, 14]. In order to test whether the observed correlation between PADI2 and HER2/ERBB2 would be retained at the protein level, we also tested a small sample of cell lines representing the four common breast cancer subtypes and found that PADI2 expression was only observed in the HER2/ERBB2+ BT-474 and SK-BR-3 lines. However, we did observe some discordance seen between PADI2 transcript and protein levels, but we predict this difference may be due to post-transcriptional regulatory mechanisms. This prediction is based, in part, upon the observation that PADI2 possesses a long 3’UTR  that contains several AU-rich elements [54, 55] that have been shown to bind the stabilizing regulatory factor HuR . HuR binding has been shown to enhance the stability of mRNAs involved in proliferation [57–59], while also playing a role in breast cancer, as cytoplasmic accumulation of HuR promotes tamoxifen resistance in BT-474 cells  and the stability of HER2/ERBB2 transcripts in SK-BR-3 cells . Interestingly, from these studies, the level of HuR was reported to be high in both BT-474 and SK-BR-3 cells, while it was relatively low in MCF7 cells. It is important to note that while we observed low levels of PADI2 protein expression in MCF7 (Additional file 1: Figure S1a), recent work from our lab has confirmed the expression of PADI2 in MCF7 cells [49, 50].
We also examined two mouse models of mammary tumorigenesis, the luminal MMTV-neu and the basal MMTV-Wnt-1, and found that, as predicted, PADI2 levels are highest in the HER2/ERBB2 overexpressing MMTV-neu mice compared to normal mammary tissue and to hyperplastic and primary MMTV-Wnt-1 tumors. Taken together, these findings indicate that PADI2 is predominantly expressed in luminal epithelial cells, and that there appears to be a strong relationship between PADI2 and HER2/ERBB2 expression in breast cancer. Subsequent studies are now underway to test whether PADI2 plays a functional role in HER2/ERBB2 driven breast cancers, potentially by functioning as an inflammatory mediator.
Previous studies have shown that the inhibition of PADI enzymatic activity by Cl-amidine is effective in decreasing the growth of several cancer cell lines (i.e. HL-60, HT-29, U2OS, and MCF7 cells), and that administering the drug in combination with doxorubicin or the HDAC inhibitor SAHA can have synergistic cytotoxic effects on cells [8, 9, 11, 45]. Cl-amidine is highly specific for all PADI enzymes, with dose-dependent cytotoxicity and little to no effect in non-cancerous cell lines (i.e. HL-60 granulocytes and NIH3T3 cells) . Our studies expand on these previous results by showing that Cl-amidine suppresses the growth of the transformed lines of the MCF10AT model, especially the MCF10DCIS cell line, in both 2D and 3D cultures. In addition, we show for the first time that Cl-amidine is successful in treating tumors in vivo using a mouse model of comedo-DCIS from xenografted MCF10DCIS cells. Given that the loss of basement membrane integrity is an important event during the progression of DCIS to invasive disease, it is significant that Cl-amidine treated xenografts maintain their basement membrane integrity and show reduced leukocytic infiltration across the basement membrane compared to the control group. These observations suggest that Cl-amidine treatment might enhance the ability of tumor ductular myoepithelial cells to deposit continuous and organized basement membranes.
While we chose the subcutaneous model of MCF10DCIS tumorigenesis, future studies on the effect of Cl-amidine could examine alternate methods of transplantation, such as the previously described intraductal method . In addition, different models of DCIS could be examined, such as xenografted SUM-225 cells, which show high HER2/ERBB2 and PADI2 levels (see Figure 3 for relative levels). Of note, we found that while Cl-amidine suppressed tumor growth, the drug was well tolerated by mice in this study. Similarly, our previous work found that doses of Cl-amidine up to 75 mg/kg/day in a mouse model of Colitis , and up to 100 mg/kg/day in a mouse model of RA , were well-tolerated without side effects. Further work into studying the pharmacokinetics and biodistribution of Cl-amidine, or perhaps the development of an isozyme specific inhibitor of PADI2, will be an important step in helping to find a potent drug for the treatment of DCIS patients.
The actual mechanisms by which Cl-amidine reduces cellular proliferation have yet to be fully elucidated, though evidence here suggests that PADI2 may play a role (direct or indirect) in regulating the expression of both cell cycle and tumor promoting genes. Previous reports have shown that Cl-amidine effectively upregulates a number of p53-regulated genes, including p21, PUMA, and GADD45[8, 45]. Our qRT-PCR cell cycle array results confirm that two of these genes, p21 and GADD45α, are upregulated after treatment of MCF10DCIS cells with Cl-amidine by 17.68- and 13.53-fold, respectively. Furthermore, we have identified additional genes downregulated by Cl-amidine, including MKI67, MCM5, and MCM2, each with known functions in cancer progression. We have also quantitatively analyzed for apoptosis levels (Caspase-3) after Cl-amidine treatment via flow-cytometry, and see a dose-dependent decrease in proliferation and increase in apoptosis. Moreover, we also show that the cells arrest in S-phase after Cl-amidine treatment, thus leading to S-phase coupled apoptosis, which is a known response to DNA damage . Taken together, the observed inhibitory effects of Cl-amidine on tumor growth may be due to the suppression of genes involved in oncogenesis and the activation of genes involved in apoptosis, though additional work is needed to define the mechanisms behind these potential relationships.
In summary, we provide here an important new line of evidence demonstrating that PADI2 may play a role in the oncogenic progression of cancer and, in particular, breast cancer. Using the MCF10AT model, we show that PADI2 is highly upregulated following transformation at both the mRNA and protein level, with highest levels in the cell line that recapitulates human comedo-DCIS. Furthermore, we show that, across a wide array of breast cancer cell lines, PADI2 is specifically overexpressed in the luminal subtype, while also being highly correlated with HER2/ERBB2 overexpression. This observation suggests that PADI2 may function as a biomarker for HER2/ERBB2+ lesions. Lastly, our preclinical mouse xenograft study suggests that the PADI inhibitor, Cl-amidine, could potentially be utilized as a therapeutic agent for the treatment of comedo-DCIS tumors.
Epidermal growth factor
Human epidermal growth factor receptor 2
v-erb-b2 avian erythroblastic leukemia viral oncogene homolog 2
Mouse mammary tumor virus
Ductal carcinoma in situ
Ribonucleic acid sequencing
Alternative expression analysis by sequencing
Chronic obstructive pulmonary disease
Quantitative real-time polymerase chain reaction
Hematoxylin and eosin
α-N-benzoyl-L-arginine ethyl ester.
This work was supported in part by funding through the DOD Era of Hope Award W871XWH-07-1-0372 to SAC, and through a National Institutes of Health Graduate Fellowship (Grant # T32HD057854) to JLM. In addition, work on the RNA-seq data was supported in part by the following funding sources: Director, Office of Science, Office of Biological & Environmental Research, of the U.S. Department of Energy under Contract # DE-AC02-05CH11231 and fellowship from the Canadian Institutes of Health Research to OLG. The RNA-seq work was also supported by the Department of the Army, award: W81XWH-07-1-0663 (The U.S. Army Medical Research Acquisition Activity, 820 Chandler Street, Fort Detrick, MD 21702–5014 is the awarding and administering acquisition office) and by the National Institutes of Health, National Cancer Institute grant, the U54 CA 112970 to JWG. The content of the information does not necessarily reflect the position or the policy of the Government, and no official endorsement should be inferred.
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