Brachyury, a Driver of the Epithelial–Mesenchymal Transition, Is Overexpressed in Human Lung Tumors: an Opportunity for Novel Interventions Against Lung Cancer

Published OnlineFirst May 18, 2012; DOI: 10.1158/1078-0432.CCR-11-3211

Clinical Cancer Cancer Therapy: Preclinical Research

Brachyury, a Driver of the Epithelial–Mesenchymal Transition, Is Overexpressed in Human Lung Tumors: An Opportunity for Novel Interventions against Lung Cancer

Mario Roselli2, Romaine I. Fernando1, Fiorella Guadagni3, Antonella Spila3, Jhessica Alessandroni3, Raffaele Palmirotta3, Leopoldo Costarelli4, Mary Litzinger1, Duane Hamilton1, Bruce Huang1, Joanne Tucker1, Kwong-Yok Tsang1, Jeffrey Schlom1, and Claudia Palena1

Abstract Purpose: The epithelial–mesenchymal transition (EMT) is emerging as a critical factor for the progression and metastasis of carcinomas, as well as drug resistance. The T-box transcription factor Brachyury has been recently characterized as a driver of EMT in human carcinoma cells. The purpose of this study was to characterize Brachyury as a potential target for lung cancer therapy. Experimental Design: The expression of Brachyury was evaluated by PCR and by immunohistochemistry in human lung tumors and adult normal tissues. Brachyury gene copy number and promoter methylation status were analyzed in tumor tissues with various levels of Brachyury expression. Lung carcinoma cells’ susceptibility to T-cell lysis and EGF receptor (EGFR) kinase inhibition were also evaluated relative to the levels of Brachyury. Results: Our results showed Brachyury protein expression in 41% of primary lung carcinomas, including 48% of adenocarcinomas and 25% of squamous cell carcinomas. With the exception of normal testis and some thyroid tissues, the majority of normal tissues evaluated in this study were negative for the expression of Brachyury protein. Brachyury-speciﬁc T cells could lyse Brachyury-positive tumors and the level of Brachyury corresponded to resistance of tumor cells to EGFR kinase inhibition. Conclusion: We hypothesize that the elimination of Brachyury-positive tumor cells may be able to prevent and/or diminish tumor dissemination and the establishment of metastases. The ability of Brachyury-speciﬁc T-cell lines to lyse Brachyury-positive tumor cells, in vitro, supports the development of Brachyury-based immunotherapeutic approaches for the treatment of lung cancer. Clin Cancer Res; 18(14); 3868–79. 2012 AACR.

Introduction patients involve surgery, chemotherapy, radiotherapy, Lung cancer is the leading cause of cancer-related death and more recently developed targeted therapies against, worldwide (1). Depending on the type and stage of for example, the EGF receptor (EGFR; refs. 2, 3). Despite disease, current therapeutic options for lung cancer advances in therapeutic interventions, the overall 5-year survival rate for all lung cancer stages combined remains at only 16%; the rate of tumor recurrence is high even in the group of patients with early-stage lung cancer (stage Authors' Afﬁliations: 1Laboratory of Tumor Immunology and Biology, Center for Cancer Research, NCI, NIH, Bethesda, Maryland; 2Department I),inwhichtherecurrencerateisapproximately50% of Internal Medicine, Medical Oncology Unit, Tor Vergata University; within 5 years from the time of diagnosis (4). Compli- 3 Interinstitutional Multidisciplinary Biobank (BioBIM), Department of Lab- cating the outcome of chemotherapy or targeted therapies oratory Medicine and Advanced Biotechnologies, IRCCS San Raffaele Pisana; and 4Department of Pathology, San Giovanni Addolorata Hospital, for the management of lung cancer, drug resistance Rome, Italy mechanisms are often exhibited by the tumor cells, either Note: Supplementary data for this article are available at Clinical Cancer as an intrinsic property or one that is acquired along with Research Online (http://clincancerres.aacrjournals.org/). treatment (5). Ideally, a novel therapeutic intervention

M. Roselli and R.I. Fernando contributed equally to this work. against lung cancer should address tumor recurrence while simultaneously minimizing tumor resistance for Corresponding Author: Jeffrey Schlom, Laboratory of Tumor Immunology and Biology, Center for Cancer Research, National Cancer Institute, NIH; improved response to therapy. 10 Center Drive, Room 8B09, MSC 1750, Bethesda, MD 20892. Phone: The epithelial–mesenchymal transition (EMT) has 301-496-4343; Fax: 301-496-2756; E-mail: [email protected] recently emerged as a process of relevance during carcinoma doi: 10.1158/1078-0432.CCR-11-3211 progression and metastasis (6–8). During tumor EMT, 2012 American Association for Cancer Research. epithelial tumor cells lose the expression of proteins

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Brachyury, a Driver of EMT, Is a Novel Lung Cancer Target

tissues were evaluated. It is also reported here, for the first Translational Relevance time, that overexpression of Brachyury in human lung The epithelial–mesenchymal transition (EMT) has carcinoma lines positively correlates with resistance to recently emerged as a process of relevance during carci- EGFR kinase inhibition. Moreover, we show that Brachy- noma progression and metastasis and drug resistance. In ury-positive lung cancer cells can be effectively lysed by this study, we show, for the first time, the expression of Brachyury-specific CTLs, further supporting the develop- the Brachyury protein, a driver of human carcinoma ment of Brachyury-based cancer vaccine approaches for EMT, in 41% of primary lung carcinomas, and its ability the treatment of human lung cancer. to confer resistance to EGF receptor kinase inhibition. We also show that Brachyury-specific T cells expanded from the blood of cancer patients can lyse Brachyury- Materials and Methods positive lung carcinoma cells in vitro, in an MHC-restrict- Patient information and tissue collection ed manner. These results support the use of Brachyury as Thirty-nine patients with histologically diagnosed prima- a novel target for therapeutic interventions against lung ry lung cancer were enrolled in the Interinstitutional Mul- cancer. It could be hypothesized that, if used at early tidisciplinary BioBank (BioBIM) of the Department of stages of the disease, a Brachyury-based cancer vaccine Laboratory Medicine and Advanced Biotechnologies, approach could prevent or diminish the establishment IRCCS San Raffaele Pisana, Rome, Italy, in collaboration of metastatic disease in lung cancer patients and poten- with the Surgical and Pathology Department of San Gio- tially alleviate resistance to therapy. vanni Addolorata Hospital and Medical Oncology Unit of the "Tor Vergata" Clinical Center, Rome, Italy. Lung tumor tissue samples were collected at the time of surgery (Tables 1, 2). Twenty-four histologically normal lung tissues adja- involved in cell-to-cell adhesion, such as E-cadherin, and cent to tumors were also obtained from lung cancer gain expression of proteins typically associated with mes- patients. No patient received neoadjuvant chemotherapy enchymal cells, including Fibronectin, N-cadherin, and or radiation therapy previous to surgery and tissue collec- Vimentin (9, 10). The phenotypic switch also results in tion. In addition, 34 samples corresponding to 11 types of enhanced tumor cell motility and invasiveness and, as a normal tissues obtained from noncancer subjects have been consequence, tumor cells undergoing EMT are thought to be evaluated in this study. Informed consent was obtained able to detach from the primary tumor and to initiate the from each participating subject; the study was carried out cascade of events leading to the establishment of metastases under the appropriate institutional ethics approvals and in (11). Several recent studies have also shown yet another accordance with the principles embodied in the Declaration interesting aspect of tumor EMT, which involves the acqui- of Helsinki. sition of cancer stem-like features by the tumor cells (12), including tumor resistance to a range of therapeutic inter- Immunohistochemistry ventions (13–17). Sections of paraffin-embedded, formalin-fixed tissues We recently characterized the T-box transcription factor were tested for Brachyury (Brachyury homolog, T) antigen Brachyury as a driver of EMT in human carcinoma cells expression using the avidin–biotin complex method as (18–21). Brachyury was shown to induce the expression previously described (22). Briefly, tissue sections were of molecules associated with the mesenchymal pheno- deparaffinized in xylene, rehydrated in a series of graded type, tumor cell motility, and invasiveness in vitro,aswell ethanol, and treated with 0.3% H2O2 in methanol to block as metastatic propensity in xenograft models of lung endogenous peroxidase activity. Microwave-citrate buffer cancer (19). We also showed that multiple human tumor antigen retrieval method was carried out to unmask the tissues and carcinoma cell lines have elevated levels of antigen. The sections were blocked in 10% horse serum Brachyury mRNA, in contrast to most human normal (Invitrogen) for 1 hour at room temperature and then tissues in which Brachyury mRNA is rarely detected (18, 19). The expression of Brachyury mRNA was also shown in Table 1. Brachyury protein expression analyzed primary lung tumor tissues, predominantly in tumors of higher stages (stages II–IV) than among those of stage I or by IHC in human primary lung carcinoma tissues histologically normal lung. In this study, we sought to characterize Brachyury as a potential target for lung can- Brachyury cer therapy by analyzing its protein expression levels in Lung tumor tissues positive (%) primary lung tumors and various human normal tissues. Adenocarcinoma 10/21 (48) By using a Brachyury-specific, murine monoclonal anti- Squamous carcinoma 3/12 (25) body (mAb), we show, for the first time, Brachyury Undifferentiated carcinoma 2/4 (50) protein expression in human lung tumors, including Bronchioloalveolar carcinoma 1/1 (100) adenocarcinomas and squamous cell carcinomas. In Small cell lung carcinoma 0/1 (0) addition, genetic and epigenetic processes that may con- Total 16/39 (41) tribute to the expression of Brachyury in human tumor

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Table 2. Lung tumor tissues positive for Brachyury protein expression analyzed by IHC

Tumor cells

Nuclear Cytoplasmic Adjacent tissue

# Histology Grade Stage % positive intensity % positive intensity % positive intensity 1 Adenocarcinoma G2 pT2N0 85 þþþ 85 þ neg neg 2 Adenocarcinoma G3 pT2N2 80 þþþ 80 þ 30 þþþ 3 Adenocarcinoma G2 pT1aN0 80 þþ 80 þ 10 þ 4 Adenocarcinoma G2 pT1aN0 60 þþ 60 þ neg neg 5 Adenocarcinoma G3 pT2N1 60 þ 60 þ 50 þ 6 Adenocarcinoma G2 pT2N0 40 þþ 20 þ neg neg 7 Adenocarcinoma G3 pT2N0 30 þ 30 þ neg neg 8 Adenocarcinoma G2 pT1bN0 30 þ neg neg neg neg 9 Adenocarcinoma G2 pT2N0 10 þþ 10 þþ 30 þþþ 10 Adenocarcinoma G2 pT2N1 10 þ 10 þ neg neg 11 Squamous cell carcinoma G2 pT3N0 80 þþ neg neg neg neg 12 Squamous cell carcinoma G1 pT3N0 50 þþ neg neg 40 þ 13 Squamous cell carcinoma — rec 10 þ 10 þ neg neg 14 Undifferentiated carcinoma G3 pT2N1 80 þþ 60 þ 10 þ 15 Undifferentiated carcinoma G3 pT2N1 25 þ 15 þ 50 þ 16 Bronchioloalveolar carcinoma G1 pT1bN0 90 þþ 90 þ neg neg

NOTE: rec ¼ recurrence; neg ¼ negative.

incubated overnight at 4C with a mouse anti-Brachyury Tumor cell lines mAb (ab57480; Abcam) at a 1:100 dilution. In addition, a The human lung cancer cell lines used in this study were positive control antibody (mouse anti-Cytokeratin mAb; obtained from the American Type Culture Collection and BD) and an isotype-matched mouse mAb (MOPC 21; propagated in RPMI-1640 medium with 2 mmol/L gluta- Sigma-Aldrich) were used to verify accurate staining meth- mine, 1 solution of penicillin/streptomycin (Mediatech, od. Antibodies specific for E-cadherin and Vimentin were Inc.) and 10% FBS (Invitrogen). H460 cells were stably purchased from BD Biosciences. Immunostaining was car- transfected with a control short hairpin RNA (shRNA)- or a ried out using the Vectastaining ABC kit (Vector Laborato- Brachyury-specific shRNA-encoding vector; A549 cells were ries) following the manufacturer’s instructions; color was stably transfected with a control pCMV or a vector encoding developed with 3,30-diaminobenzidine (DAB) peroxidase for the full-length human Brachyury protein (pBrachyury), substrate (Vector Laboratories). Sections were counter- as previously described (19). stained with hematoxylin, dehydrated in ethanol, cleared in xylene, and mounted under a coverslip using Permount Brachyury expression analysis by Western blot (Fisher Scientific). For detection of Brachyury in human tumor tissues, total protein extracts from frozen tissues were prepared in lysis Scoring method buffer: 10 mmol/L HEPES pH ¼ 8.0, 350 mmol/L NaCl, 0.1 Two pathologists independently evaluated the tumor and mmol/L EDTA pH ¼ 8.0, Complete Protease Inhibitor normal tissue samples in a blinded, randomized way. For Cocktail (Roche Applied Science). For separation of cyto- each slide, 3 to 5 random fields were evaluated; for each plasmic and nuclear protein fractions, tissues were lysed in a field, the percentage of DAB-positive tumor cells was cal- hypotonic buffer (10 mmol/L HEPES pH ¼ 8.0, 50 mmol/L culated as: [(number of DAB-positive tumor cells/total NaCl, 1 mmol/L EDTA pH ¼ 8.0, 0.2% Triton X-100, 500 number of tumor cells) 100], and the relative staining mmol/L Sucrose, Complete Protease Inhibitor Cocktail) intensity was scored as weak (þ) for pale brown intensity, followed by 30 seconds of centrifugation at 16,000 g at moderate (þþ) for intermediate brown intensity, and 4C. Soluble, cytosolic proteins were collected; nuclear strong (þþþ) for intense, dark brown immunoprecipitate. proteins were extracted in lysis buffer from the pellet frac- For normal tissues, the percentage of reactivity was indi- tions. Protein extracts (total, cytoplasmic, and nuclear) vidually evaluated for each cell type and calculated as: were purified by precipitation with chloroform/methanol, [(number of DAB-positive cells/total number of cells of followed by resuspension in PBS/Tween 0.05% buffer. the same type) 100]. Staining was recorded as negative For immunoblotting, 30 mg of tissue protein extracts if less than 5% of the tumor or parenchymal cells from were separated by SDS-PAGE, blotted onto nitrocellulose normal tissues stained positive for Brachyury expression. membranes, and subsequently incubated with primary

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Brachyury, a Driver of EMT, Is a Novel Lung Cancer Target

anti-Brachyury (Abcam) and anti-Actin (C-2, Santa Cruz Survival for treated wells was calculated as a percentage of Biotechnology Inc.) mAbs. A secondary horseradish perox- the values representing wells of untreated cells. idase–conjugated anti-mouse Ab was subsequently used; chemiluminescent signal was detected with an ECL kit Cytotoxic assay (EuroClone). Brachyury-specific CTLs were generated from the blood of a normal donor (designated as CTL line I) or 2 different Real-time PCR prostate cancer patients postvaccination with a PSA-TRI- Commercially available normal and tumor lung tissue COM-based vaccine (designated as CTL lines II and III), as cDNA panels were analyzed (TissueScan Lung Cancer previously described (18). No Brachyury-specific T cells qPCR Arrays I and II; Origene Technologies, Inc.) by could be detected in these patients before vaccination. using the Gene Expression Master Mix and the following Tumor cells (lung H226, H441, and H1703 and pancreatic TaqMan Gene Expression Assays (Applied Biosystems): ASPC1) were labeled with 50 mCi of 111Indium-labeled Brachyury (Hs00610080), Twist 1 (Hs00361186), Snail oxyquinoline and subsequently incubated with Brachyury- (Hs00195591), PAP (Hs00173475_m1), CEACAM5 specific CTLs at the indicated effector-to-target ratios. For (Hs00944023_m1), and GAPDH (4326317E). PCR was cold target inhibition assays, unlabeled K562 cells trans- carried out according to the manufacturer’s recommenda- fected with A2.1 were added to the labeled targets (10) tions on the 7300 Real-Time PCR System (Applied Bio- either unpulsed or pulsed with 20 mg/mL of the Brachyury 111 systems). Mean Ct values for target genes were normalized peptide. Following a 16-hour incubation, In released to mean Ct values for the endogenous control GAPDH was measured by gamma counting and the percent specific [DCt ¼ Ct (GAPDH) Ct (target gene)]. The ratio of lysis was calculated as previously described (18). mRNA expression of target gene versus GAPDH was D defined as 2 Ct. Results Invasion assay Detection of Brachyury protein in tumor cells In vitro invasion assays were conducted as previously The specificity of a murine mAb directed against the described (19). Cells on the bottom side of the filters were human Brachyury protein was first shown by its ability to counted in 5 random 100 microscope objective fields. specifically recognize the 48.2-kDa recombinant His6- Brachyury protein purified from baculovirus-directed Brachyury gene copy number analysis overexpression in insect cells (Fig. 1A). To determine the Real-time PCR reactions were carried out with 5 ng of ability of the mAb to recognize the Brachyury protein in genomic DNA using a TaqMan Copy Number Assay whole-cell protein lysates obtained from human lung (Hs01038891_cn) with the reference assay for RNase P carcinoma cells, a tumor cell pair was used consisting of (Applied Biosystems). Copy numbers were calculated using H460 cells that were stably transfected with a control the CopyCaller software (Applied Biosystems). shRNA- or a Brachyury-specific shRNA-encoding vector. Evaluation of the Brachyury protein by immunoblotting Brachyury promoter methylation with the anti-Brachyury mAb showed a single protein The Brachyury promoter (–2,000 to þ500) was analyzed band of expected molecular weight in the H460 control. for the presence of CpG islands and suitable restriction sites. shRNA cells (Fig. 1B) with reduced intensity (70% Methylation status of an identified CpG island was evalu- reduction) in H460 cells stably inhibited for Brachyury ated by using the Promoter Methylation PCR kit (Affyme- expression. These results confirmed the specificity of the trix). Briefly, genomic DNA (3 mg) was digested with the StuI mAb and its ability to selectively detect Brachyury protein restriction enzyme, followed by purification of methylated in human tumor cells. DNA fragments by binding to the methyl CpG-binding protein 2 (MeCP2, also designated as MBP). Eluted DNA Brachyury is overexpressed in human primary lung was subjected to PCR amplification using the following tumors primers: forward 50-TCAGGAGGGTCCGGCGTCAG-30 and Brachyury protein expression was detected in 16 of 39 reverse 50-GCACACCTCGCCCATTCGCT-30. PCR products (41%) of primary lung tumor tissues analyzed, including 10 (expected size: 581 bp) were resolved on 1.5% agarose gels; of 21 adenocarcinomas (48%) and 3 of 12 (25%) squamous the presence of a band on the gel indicates methylation of carcinomas (Table 1). For each of the 16 lung tumor tissue the Brachyury promoter in the genomic DNA sample. samples that were positive for Brachyury expression in this study, Table 2 indicates the tumor grade, tumor–node– Susceptibility to EGFR inhibition metastasis stage, and the positivity observed in tumor cells Indicated tumor cells were seeded in 96-well plates, as well as in the histologically normal lung tissue adjacent to allowed to attach overnight, and treated with various doses the tumor, in terms of percentage of Brachyury-positive cells (1–50 mmol/L) of AG1478 (Sigma-Aldrich) for 2 days. as well as the intensity of the staining (see Materials and Viable cells in culture were assayed by the MTT assay as Methods for a description of the scoring method used). previously described (19). Assays were carried out in trip- Moreover, staining of tumor cells in the nuclear versus licate; data shown are representative averages, with SE. cytoplasmic compartment is indicated. As shown, the

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A MWBrachy HSA C 62 i ii 49 Brachyury 38 t t B H460 Control Brachyury MW shRNA shRNA s 64 t s 50 Brachyury 36 iii iv 50 β-Actin t 36

0.2

β -actin 66% 0.1 Reduction t

Brachyury/ 0.0 Control Brachyury v shRNA shRNA H460 D t

Patient 7 Patient 17 s 50 50

37 37 n Tumor Adjacent Tumor Adjacent tissue tissue Nuclear lysates Nuclear lysates

Figure 1. Detection of Brachyury protein in lung tumor cells and tissues. Western blot analysis using anti-Brachyury mAb for detection of (A) recombinant His6-Brachyury fusion protein derived from insect cells versus purified human serum albumin (HSA; Sigma-Aldrich) or (B) Brachyury protein in H460 lung carcinoma cells stably transfected with a control- or a Brachyury-specific shRNA-encoding vector. b-Actin was used as protein loading control; shown in the graph is the ratio Brachyury/b-actin. C, representative tissue sections stained with anti-Brachyury mAb corresponding to a grade 3 adenocarcinoma, patient 2 (i); a grade 1 squamous carcinoma, patient 12 (ii); and a grade 1 bronchioloalveolar carcinoma, mucinous type, patient 16 (iii). Tumor cells positive for Brachyury expression are indicated with (t); positive cells in the stroma adjacent to the tumor are indicated with (s). iv, staining of a grade 2 lung adenocarcinoma (patient 10) showing Brachyury-positive tumor cells invading a blood vessel. Arrows indicate endothelial cells. v, staining of a grade 2 adenocarcinoma (patient 9) showing Brachyury positivity in a fraction of tumor cells (t), and cells in the stroma directly adjacent to the tumor (s). Indicated with (n) are tumor cells negative for Brachyury expression (magnification 20: panels i, ii; 40: panels, iii–v). D, Western blot analysis of Brachyury expression in nuclear protein lysates prepared from primary lung tumor tissue (tumor) and corresponding normal lung adjacent to the tumor from patients 7 and 17.

percentage of lung tumor cells that stained positive with the ed cytosolic localization, a phenomenon that will be further anti-Brachyury mAb ranged from 10% to 90%; in all cases, investigated in the context of Brachyury. staining was localized in the nuclear compartment although cytoplasmic staining was also observed in 13 of 16 positive Brachyury-positive cells are detected in lung tissue tissues. Although the intensity of the nuclear staining ran- adjacent to the tumor ged from weak (þ) to intense (þþþ), the cytosolic staining In all 16 cases evaluated, no stromal cells stained positive was scored as weak (þ) in all but one case. Similar staining for Brachyury expression in the lung tissue distal to the pattern has been reported for the Twist protein in the cytosol Brachyury-positive tumor mass. However, microscopic and nuclei of gastric carcinoma cells (23). Representative evaluation of the 16 cases of lung tumors that were positive images of lung tumor sections positive for Brachyury pro- for Brachyury expression showed the presence of Brachyury- tein expression, corresponding to an adenocarcinoma positive cells in the stroma adjacent to the tumor in 7 of the (patient 2, Fig. 1C–i), a squamous carcinoma (patient 16 cases (Table 2 and Fig. 1C–v). Detection of Brachyury- 12, Fig. 1C–ii), and a bronchioloalveolar carcinoma, positive cells in the tissue adjacent to the tumor seemed to mucinous type (patient 16, Fig. 1C–iii) showed the intense be independent of the level of Brachyury positivity observed staining of Brachyury protein in the nucleus of tumor cells, at the tumor site. For example, Brachyury-positive cells have and the weak, more diffuse pattern of cytoplasmic staining been observed in the stroma adjacent to tumor from in the same cells. Intense Brachyury staining was also patients 2 and 3, in which 80% of the tumor cells scored observed in the cytosol, but not the nuclei, of tumor cells strongly positive for Brachyury, as well as in patient 9 in invading into blood vessels (patient 10, Fig. 1C–iv). No data which only 10% of the cells in the tumor were Brachyury are available at this time with regard to the mechanism that positive (Table 2). Figure 1C–v shows a representative promotes cytosolic compartmentalization of Brachyury or image of an area of transition between tumor and stroma the biologic signiﬁcance, if any, of this observation. A for patient 9, showing a focal area of strong positivity for previous report (24) has shown that the activity of the EMT Brachyury expression in the tumor mass and in the stroma driver Snail is downregulated by phosphorylation-mediat- adjacent to the tumor. The anti-Brachyury mAb was also

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Brachyury, a Driver of EMT, Is a Novel Lung Cancer Target

tested for its ability to detect Brachyury protein in lysates enchymal Vimentin by immunohistochemistry (IHC) in derived from human lung tumor tissues. Although no serial tissue sections. Two representative cases are shown reactivity was observed with cytosolic protein fractions in Fig. 2A. In one example (adenocarcinoma patient 2, top (data not shown), a single band at the expected molecular panels), the epithelial tumor compartment (indicated with weight was observed in nuclear fractions derived from the white arrowheads) showed intense expression of Brachyury primary tumor of 2 lung cancer patients evaluated (patients and simultaneous expression of epithelial E-cadherin and 7 and 17, Fig. 1D). In addition, histologically normal lung mesenchymal Vimentin. Another example of an adenocar- tissue adjacent to the tumor obtained from patient 17 cinoma (patient 6, bottom panels) showed expression of showed intense Brachyury positivity in Western blot (Fig. Brachyury in the epithelial tumor compartment (white 1D). The potential explanations as to why tissue directly arrowheads), with weak expression of E-cadherin and no adjacent to lung cancer cells is positive in some cases expression of Vimentin in the tumor cells. In both cases, whereas distal tissue and lung tissue from noncancer single disseminated cells within the adjacent stroma (indi- patients are always negative will be discussed below. There cated with black arrowheads) stained positive for Brachyury were also 4 cases (patients 5, 7, 9, and 10) in which strong but negative for E-cadherin. These results thus indicated that but focal staining of a minority of chondrocytes in distal the pattern of expression of Brachyury and the EMT markers, bronchial cartilage was detected with the anti-Brachyury E-cadherin and Vimentin, is not necessarily consistent mAb (data not shown). among various lung tumor specimens and points out at a potentially complex connection between these markers in Expression of EMT markers in primary lung tumor lung cancer. tissues To evaluate whether the expression of Brachyury corre- Brachyury upregulation in tumor tissues and cell lines lates with expression of EMT-related proteins in vivo, 4 cases To investigate whether a genomic rearrangement could of lung cancer with various levels of Brachyury were eval- account for the enhanced expression of Brachyury in lung uated for the expression of epithelial E-cadherin and mes- tumors, a qPCR-based method was used to evaluate

A Brachyury E-cadherin Vimentin (patient 2) Adenocarcinoma (patient 6) Adenocarcinoma

B Tumor specimen 212019181714131275 pos neg SM

650 bp 500 bp

Figure 2. Expression of EMT markers and mechanism of Brachyury overexpression. A, representative tissue sections stained for Brachyury, E-cadherin, and Vimentin. In one example (adenocarcinoma patient 2, top), the epithelial tumor compartment (indicated with white arrowheads) showed intense expression of Brachyury and simultaneous expression of epithelial E-cadherin and mesenchymal Vimentin. Another example of an adenocarcinoma (patient 6, bottom) showed expression of Brachyury in the epithelial tumor compartment (white arrowheads), with weak expression of E-cadherin and no expression of Vimentin in the tumor cells. In both cases, single disseminated cells within the adjacent stroma (indicated with black arrowheads) stained positive for Brachyury but negative for E-cadherin (magniﬁcation 20). B, results of qPCR analysis for Brachyury copy number in indicated tumor specimens. Pos ¼ positive control reaction with genomic DNA; neg ¼ negative control reaction without DNA. SM ¼ size marker.

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Brachyury gene copy number variations in Brachyury-pos- obtained from lung cancer patients (1 of 8 positive, itive versus Brachyury-negative lung tumor tissues. Five of 12.5%), the expression of Snail or Twist mRNA was equally 5 Brachyury-negative and 4 of 5 Brachyury-positive elevated in lung normal or tumor tissues and, in the case of tumors revealed a normal diploid status for the Brachyury Snail, the average expression among normal lung tissues gene; anomalous Brachyury gene copy number (3 copies) was approximately 10-fold higher than that of lung tumors. was only detected in one Brachyury-positive case (Supple- Similar results were obtained at the protein level; all 3 mentary Table S1). These results would rule out gene molecules were detected in protein lysates from normal amplification as a mechanism responsible for Brachyury testis, used as a positive control, whereas the expression of upregulation in lung tumors. Snail and Twist, but not Brachyury, was also detected in The methylation status of a specific CpG island on the lysates from human normal lung (Fig. 3C). Brachyury promoter was also investigated. As shown We have also compared the expression of Brachyury in Fig. 2B and Supplementary Table S1, the Brachyury mRNA in normal testis and thyroid tissues to that of 2 promoter was methylated in 2 of 5 Brachyury-positive tumor-associated antigens that have been extensively eval- tumors and 1 of 5 Brachyury-negative tumors, which uated in clinical studies without any evidence of autoim- showed a PCR amplification product at the expected size munity. As shown in Fig. 3D, mRNA expression levels of (Fig.2B).Theseresultssuggested that methylation of this prostatic acid phosphatase (PAP) and carcinoembryonic particular region of the Brachyury promoter is unlikely to antigen (CEA) are higher to those of Brachyury in normal be involved in the regulation of Brachyury expression in thyroid. lung cancer. Brachyury mRNA expression in tumor cell lines Brachyury expression in human normal tissues The expression of Brachyury mRNA was comparatively The expression of Brachyury protein was also evaluated evaluated by real-time PCR in multiple human primary lung in a set of human normal tissues obtained from adult tumors and various lung carcinoma cell lines. As shown noncancer subjects. Expression of Brachyury was negative in Fig. 4A and Supplementary Table S3, 42 of 80 lung among most normal tissues analyzed, including lung, tumors (52.5%) and 7 of 10 lung cancer cell lines (70%) heart, brain, liver, kidney, spleen, skeletal muscle, adrenal evaluated were positive for Brachyury mRNA. Interestingly, gland and skin, with the major exception of testis that was when comparing the levels of expression in the lung tumor positive in 3 of 3 cases and normal thyroid that seemed samples and the various lung carcinoma lines used for positive in 4 of 6 cases evaluated (Supplementary Table experimental preclinical studies, a comparable range of S2). In testis, cells in the germinal epithelium of the Brachyury mRNA expression can be observed that spans a seminiferous tubules stained weakly positive for the Bra- 4-log range, suggesting that available human tumor cell chyury protein (Fig. 3A–i). The staining was prevalently line models reflect the heterogeneity seen in patients and nuclear although weak cytoplasmic staining was also may be useful in developing strategies attempting to target observed in the same cells. The expression of Brachyury the EMT process. protein in normal testis was in agreement with our previous studies showing Brachyury mRNA in cDNA sam- Brachyury overexpression confers resistance to EGFR ples prepared from human normal testis (18). Here we kinase inhibitors also report, for the first time, the expression of Brachyury EGFR kinase inhibitors are commonly used for the man- protein in some biopsies of human normal thyroid tis- agement of lung cancer. We next investigated whether sues. As shown in Fig. 3A–ii, follicular cells stained expression of Brachyury in lung carcinoma lines could have strongly positive for Brachyury expression in the nucleus a significant impact on their ability to withstand EGFR and, to a lesser extent, the cytoplasmic compartment. In inhibition mediated by Tyrphostin AG1478, a cell-perme- contrast, as depicted in representative images in Fig. 3A, able, reversible, ATP-competitive inhibitor of the EGFR all other human normal tissues evaluated were negative tyrosine kinase. Two human lung carcinoma line pairs with for Brachyury expression in all cell types, including nor- high versus low levels of Brachyury expression were gener- mal lung (Fig. 3A–iii, iv), normal spleen (Fig. 3A–v), and ated by stable transfection of (i) A549 cells with a control normal skeletal muscle (Fig. 3A–vi). pCMV or a pBrachyury vector or (ii) H460 cells with a Examples of other previously characterized transcription control shRNA versus a Brachyury shRNA. As shown in Fig. factors able to drive EMT in various tumor models include 4B, A549 pBrachyury cells had a survival advantage com- the zinc finger protein Snail (25, 26) and the helix-loop- pared with control A549 pCMV cells in response to treat- helix (HLH) transcription factor Twist (27, 28). Using a ment with various doses of the EGFR inhibitor, AG1478. commercial panel of cDNAs obtained from 40 lung tumor Similarly, H460 cells inhibited for the expression of Bra- tissues and 8 histologically normal lung tissues obtained chyury were found to be more susceptible to treatment with from lung cancer patients, we comparatively evaluated the various doses of inhibitor, as compared with control cells. expression of mRNA encoding for Brachyury, Snail, and To further characterize the role of Brachyury on the resis- Twist. As shown in Fig. 3B, whereas the expression of tance of lung carcinoma cells to EGFR inhibition, Brachy- Brachyury mRNA was selectively enhanced in lung tumor ury-low A549 cells were exposed in culture to a nonlethal tissues (21 of 40 positive, 52.5%) versus normal lung concentration (1 mmol/L) of AG1478. Long-term exposure

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Brachyury, a Driver of EMT, Is a Novel Lung Cancer Target

A i ii

iii iv

Figure 3. Expression of Brachyury in human normal tissues. A, representative pictures of normal (i) positive testis, (ii) positive thyroid, (iii) negative lung alveoli, (iv) negative lung bronchioles, (v) negative spleen, vvi and (vi) negative skeletal muscle (magniﬁcation 40). B, real-time PCR analysis of Brachyury, Snail, and Twist mRNA expression in commercial panels of cDNA (, normal tissue adjacent to tumor). C, Western blot analysis of Brachyury, Snail, and Twist protein expression in commercially available B whole-tissue protein lysates 100 100 100 obtained from normal testis and Brachyury Snail Twist 10–1 10–1 10–1 normal lung. The following 10–2 10–2 10–2 antibodies were used: Snail clone H- 10–3 10–3 10–3 130, Twist clone H-81, GAPDH clone 10–4 10–4 10–4 0411 (Santa Cruz Biotechnology). D, 10–5 10–5 10–5 Brachyury real-time PCR analysis of , Gene expression –6 –6 –6

relative to GAPDH 10 10 10 PAP, and CEA mRNA expression in 0 0 0 cDNA from normal testis and thyroid tissues. Normal* Stages I-IV Normal* Stages I-IV Normal* Stages I-IV 1/8 21/40 8/8 39/40 8/8 40/40

C Human normal tissues D 0.005 Brachyury PAP CEA Testis Lung 0.004 Brachyury 0.003

Snail 0.002

Twist 0.001 Gene expression relative to GAPDH GAPDH 0 Testis Thyroid Testis Thyroid Testis Thyroid

of A549 to AG1478 markedly induced (>3-log) the expres- Brachyury-specific T-cell lines have been used to lyse, in sion of Brachyury mRNA above the level found in dimethyl vitro, H226, H441, and H1703 lung carcinoma cells. As sulfoxide (DMSO)-treated cells (Fig. 4C). In addition, A549 shown in Fig. 5A, normal donor–derived Brachyury-specific tumor cells that survived exposure to AG1478 had signif- T cells were able to lyse H441 tumor cells that express icantly enhanced invasive capacity in vitro, compared with Brachyury and are HLA-A2 positive, but not H226 cells DMSO-treated or untreated cells (Fig. 4D). which express elevated levels of Brachyury but lack the appropriate MHC class I expression. Using a different Bra- Brachyury as target for immunotherapeutic chyury-specific T-cell line derived from a prostate cancer interventions against lung cancer patient, a cold target inhibition assay with H441 tumor cells þ We have previously shown that human CD8 Brachyury- using peptide-pulsed K562-A2.1 cells as competitors specific T cells could be expanded in vitro from peripheral showed the epitope specificity of the lysis (Fig. 5B). Using þ blood mononuclear cells of cancer patients and normal tetramer-isolated, CD8 Brachyury-specific T cells derived donors by using a 9-mer peptide of Brachyury that specif- from a different prostate cancer patient, a high level of ically binds to the HLA-A2 molecule (18). Here, various specific lysis was observed with H441 and H1703 lung

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Roselli et al.

A B 0 10 140 A549 pcMV (Brachyury LOW) 140 H460 control shRNA (Brachyury HIGH) 42/80 A549 pBrachyury (Brachyury HIGH) H460 Brachyury shRNA (Brachyury LOW) (52.5%) 10-1 120 120

10-2 100 100

10-3 80 80

-4 10 % Survival 60 60

10-5 40 40 Brachyury vs. GAPDH

10-6 20 20

0 0 0 01105550 0 5

H226 H460 SW900 H520 A549 H441 H1703 H157 H596 Calu-1 EGFR kinase inhibitor (AG1478) EGFR kinase inhibitor (AG1478) Lung Lung tumors carcinoma lines

C Brachyury D Invasion 1.5 x 100 20 *** ***

100 6 x 10-4 10

Ratio vs. GAPDH -4 3 x 10 * Number of cells (x100 field) Number of cells (x100 0 0 DMSO AG1478 Parental DMSO AG1478 A549 A549

Figure 4. Brachyury and susceptibility to EGFR inhibition. A, real-time PCR analysis of Brachyury mRNA in human primary lung tumors and lung carcinoma lines. B, A549 and H460 lung carcinoma cell pairs were treated with various doses (mmol/L) of the EGFR kinase inhibitor AG1478 for 2 days and assayed for survival by the MTT assay. C, real-time PCR analysis of Brachyury expression in A549 cells grown for 2 weeks in the presence of control DMSO- or 1 mmol/L AG1478-containing medium. D, extracellular matrix invasion assay of A549 cells untreated (parental) or treated as indicated for 2 weeks in culture. , P < 0.05; , P < 0.005.

carcinoma cells (both Brachyury positive/HLA-A2 positive), Snail and Twist, 2 previously described drivers of EMT, as compared with the control HLA-A2–negative ASPC1 cells which were expressed at equally high levels in normal and (Fig. 5C). lung cancer tissues. It is also shown here, for the first time, a negative association between Brachyury expression in lung cancer cell lines and their susceptibility to EGFR inhibition. Discussion In addition, data are presented on the analysis of copy In this study, we have shown for the first time the number and promoter methylation status of the Brachyury expression of Brachyury protein, a transcriptional regulator gene, in multiple lung tumor specimens. of human carcinoma EMT, in 41% of primary lung carci- The presence of Brachyury-positive tumor cells in human nomas, including adenocarcinomas and squamous cell lung cancer tissues could be indicative of tumor cells under- carcinomas, which together account for approximately going EMT in vivo, which are expected to have enhanced 80% of all non–small cell lung cancer diagnoses. Brachyury migratory and invasive potential and greater drug resis- positivity varied from 10% to 90% of the tumor cells and, in tance. It is important to emphasize, however, that the a fraction of cases, staining was also seen in scattered cells process of EMT is one of plasticity in which epithelial tumor localized in the stroma adjacent (but not distant) to the cells can be rendered mesenchymal-like by a number of tumor. These studies were carried out by IHC and confirmed tumor environmental factors, such as interleukin (IL)-8 by Western blot analysis using a mAb against Brachyury. We (21) and TGF-b (9, 29), leading to greater migration and have also contrasted here for the first time the highly tumor- invasion. Thus, one cannot predict when analyzing a pri- associated pattern of Brachyury expression, compared with mary tumor by IHC or PCR, which reflects the state of

3876 Clin Cancer Res; 18(14) July 15, 2012 Clinical Cancer Research

Brachyury, a Driver of EMT, Is a Novel Lung Cancer Target

C A B 25 80 30 20 60

20 15 H441 (HLA-A2 pos) 40 10 H1703 (HLA-A2 pos) 10

Specific lysis (%) 20

5 Specific lysis (%) Specific lysis (%) ASPC1 (HLA-A2 neg) 0 0 0 Cold competitor: None K562 K562 A2.1 25.0:1 12.5:1 6.3:1 A2.1 + Bra pep Effector: target ratio

Figure 5. Brachyury as a target for antitumor interventions. A, CTL-mediated lysis of H226 and H441 lung carcinoma cells with a normal donor–derived Brachyury-specific T-cell line I (25:1 effector-to-target ratio). B, lysis of H441 tumor cells with a Brachyury-specific T-cell line II derived from a prostate cancer patient in the presence of cold, competitor K562 A2.1 cells unpulsed or pulsed with the specific Brachyury peptide (Bra pep). C, lysis of H441 and þ H1703 lung carcinoma and control ASPC1 cells by tetramer-isolated, CD8 Brachyury-specific T-cell line III derived from a different prostate cancer patient. individual tumor cells at only one point in time, whether a domas, in which duplication of the Brachyury gene is subpopulation of cells had previously undergone the EMT commonly observed (33), most lung tumors analyzed process and had already metastasized. Our results indicated showed no deviations from the normal diploid status. no simple association between the expression of Brachyury Moreover, epigenetic control mediated by methylation of and the EMT markers E-cadherin and Vimentin in lung the Brachyury promoter was also ruled out, as no correla- tumors. It should be pointed out that EMT is a dynamic tion was observed between Brachyury levels and methyla- process and that IHC depicts only one point in time of this tion of its promoter. These results indicate that the expres- process. The clinical relevance of Brachyury expression in sion of Brachyury in lung cancer might result from a diverse lung cancer is unknown at this time and further studies need mutational or epigenetic mechanism, or via signaling ini- to define whether its expression has any impact on lung tiated in the extracellular compartment by components of cancer progression or survival. In support of the role of the tumor microenvironment. Brachyury in human carcinoma progression, for example, It is important to point out that a previous study using a the expression of Brachyury has been recently identified as a polyclonal Ab against Brachyury has reported that a variety poor prognostic factor in early-stage (T1-2N0M0, Dukes A) of human carcinomas were negative for Brachyury protein colon cancer (30). expression, including lung carcinomas (34). In our study, The identification of tumor cells undergoing EMT in vivo we have used a mAb that was extensively evaluated by IHC has been reported, for example, in colon cancer in which and Western blot, showing its ability to (i) specifically react tumor cells with features indicative of EMT have been with purified Brachyury protein, (ii) react with a band at the observed as dissociated tumor cells at the invasive front expected molecular weight in lysates from lung carcinoma (31, 32). In this study, we have shown that Brachyury cell lines, (iii) react with a band at the expected molecular positive, single disseminated cells can be detected in the weight in lysates from lung tumor tissues, and (iv) be able to lung stroma adjacent to the tumor in a fraction of cases stain lung tumors by IHC. Differences in epitope specificity analyzed, although no cellular components reacted with the as well as in the avidity of the used antibodies could anti-Brachyury mAb in the normal stroma distal to the potentially explain the difference in results between these tumor. Although further studies are needed to characterize studies. In addition, the presence of Brachyury protein in the origin of these Brachyury-positive cells in the transition lung tumor cell lines was shown here by using Brachyury- tumor stroma, one possibility is that they correspond to specific T-cell lines that were able to lyse, in an MHC- invasive cells that detached from the tumor mass and restricted manner, human lung carcinoma lines in vitro. invaded the surrounding tissue. Another possibility is that The present article also reinforces the tumor-associated soluble mediators secreted by the tumor such as IL-8 (21) character of Brachyury. Expression of Brachyury protein was are able to induce normal cellular components of the undetectable in most human normal tissues analyzed here, adjacent stroma to upregulate the expression of Brachyury. with the exception of testis that was previously reported to From a clinical point of view, identification of the be positive at the mRNA (18) and protein levels (34) and, mechanisms that control the expression of Brachyury in for the first time, in normal thyroid tissue (4 of 6, 67%). As lung cancer could lead to improved therapeutic options for the testis is known to be immune privileged, it is anticipated patients with Brachyury-positive tumors. Unlike with chor- that the expression of Brachyury protein in this organ will

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Roselli et al.

not pose a problem in the context of a Brachyury-based an extended period of time. In view of these data, it is immunotherapeutic approach. With regard to the expres- possible that Brachyury-mediated EMT could be a major sion of Brachyury in normal thyroid tissue, we have shown factor contributing to the development of resistance to here that the tumor-associated antigens PAP and CEA, EGFR kinase inhibitors. which have been investigated extensively in clinical studies One strategy to eliminate mesenchymal-like, Brachy- with no evidence of thyroid dysfunction, are expressed at ury-positive tumor cells is via immune targeting with higher levels in thyroid tissue compared with the expression Brachyury-based cancer vaccines. The Brachyury protein of Brachyury mRNA. is shown here to fulfill a fundamental requisite as a target In addition to its role as a driver of EMT, we have recently for vaccine strategies against the tumor, owing to its characterized another important function of Brachyury in highly tumor-associated pattern of expression. Moreover, human carcinoma cells. Brachyury expression positively we have shown that Brachyury-specific T cells can be correlates with the acquisition of tumor stemness features, expanded from the blood of cancer patients (18) and as including the ability to self-renew and to withstand treat- we have shown here, those T cells can lyse, in vitro, ment with various chemotherapy agents as well as radiation Brachyury-positive lung carcinoma cells in an MHC- (Huang and colleagues; manuscript in preparation). In this restricted manner. A phase I clinical study with a Brachy- study, we have shown for the first time the effect of Brachy- ury vaccine has now been initiated in patients with ury expression on resistance to EGFR inhibition. EGFR is carcinomas. The results presented here support the inclu- overexpressed in approximately 60% of lung cancers and sion of lung cancer patients in this phase I clinical study activation of this receptor has been shown to be associated as well as in future phase II clinical studies using Brachy- with poor clinical outcome (35). Pharmacologic inhibition ury-based vaccines. of EGFR is being explored as a therapeutic option for lung fl cancer patients, with 2 EGFR kinase small molecule inhi- Disclosure of Potential Con icts of Interest No potential conflicts of interest were disclosed. bitors, erlotinib and gefitinib, currently approved for use in the clinic (35). Unfortunately, despite an initial response, Acknowledgments most treated patients develop resistance to EGFR kinase The authors thank Margie Duberstein for technical assistance and Debra inhibition (36). As shown here for the first time, over- Weingarten for editorial assistance. expression of Brachyury in lung carcinoma cells also associ- ates with resistance to cell death mediated by EGFR kinase Grant Support The work was supported by Intramural Research Program of the Center for inhibition. Both lung cancer lines used in our study, A549 Cancer Research, National Cancer Institute, NIH and partially supported by and H460, express wild-type EGFR and harbor a K-Ras Italian Ministry of Instruction, University and Research (MIUR): MERIT, mutation; however, their sensitivity to EGFR inhibition was grant code B11J10000180008. The costs of publication of this article were defrayed in part by the different. Whereas H460 cells with high Brachyury levels payment of page charges. This article must therefore be hereby marked were resistant to EGFR inhibition with AG1478, Brachyury advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate low A549 cells were more responsive to the same treatment. this fact. Another interesting observation was the acquisition of an Received December 13, 2011; revised April 9, 2012; accepted May 14, EMT-like phenotype by A549 cells exposed to AG1478 for 2012; published OnlineFirst May 18, 2012.

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Brachyury, a Driver of the Epithelial−Mesenchymal Transition, Is Overexpressed in Human Lung Tumors: An Opportunity for Novel Interventions against Lung Cancer

Mario Roselli, Romaine I. Fernando, Fiorella Guadagni, et al.

Clin Cancer Res 2012;18:3868-3879. Published OnlineFirst May 18, 2012.

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