Human Homologue of Cement Gland Protein, a Novel Metastasis Inducer Associated with Breast Carcinomas

Research Article

Dong Liu,1 Philip S. Rudland,1,2 D. Ross Sibson,3 Angela Platt-Higgins,2 and Roger Barraclough2

1Cancer Tissue Bank Research Centre and 2School of Biological Sciences, University of Liverpool, Liverpool, United Kingdom and 3Clatterbridge Cancer Research Trust, J.K. Douglas Laboratories, Clatterbridge Hospital, Wirral, United Kingdom

Abstract PCR-selected suppression subtractive hybridization (11) has A suppression subtractive cDNA library representing mRNAs been used to identify cDNAs representing mRNAs differentially a a expressed at a higher level in the malignant human breast expressed (12–14) between an estrogen receptor (ER )–negative cancer cell line, MCF-7, relative to a benign breast tumor- benign human mammary epithelial cell line, Huma 123 (15), and a derived cell line, Huma 123, contained a cDNA, M36, which the ER -positive malignant human mammary epithelial cell line, was expressed in estrogen receptor A (ERA)–positive breast MCF-7 (16). The resulting subtracted libraries contained well- carcinoma cell lines but not in cell lines from normal/benign/ characterized, differentially expressed cDNAs that have been ERA-negative malignant breast lesions. M36 cDNA had an associated previously with tumor progression (12). In this article, identical coding sequence to anterior gradient 2 (AGR2), the we report one novel cloned cDNA, M36, which matches identically the coding sequence of human anterior gradient 2 (AGR2) cDNA, human homologue of the cement gland–specific gene (Xeno- pus laevis). Screening of breast tumor specimens using reverse the human homologue (previously hAG-2) of the Xenopus laevis transcription-PCR and immunocytochemistry with affinity- cement gland–specific gene, XAG-2 (17). The XAG-2 gene product purified anti-AGR2 antibodies showed that the presence of has developmental significance in Xenopus embryos (18). Here, we AGR2 mRNA and protein were both statistically significantly show that the human homologue of this developmentally associated with ERA-positive carcinomas (P = 0.007, Fisher’s associated protein is differentially expressed between benign and exact test) and with malignancy (P V 0.025). When an malignant human breast carcinoma specimens. Furthermore, its expression vector for AGR2 cDNA was introduced into benign cDNA, when introduced into a benign, nonmetastatic, rat nonmetastatic rat mammary tumor cells, and three separate mammary cell line, confers a metastatic phenotype on benign clones and two pools of cells were transferred to the nonmetastatic cells. mammary glands of syngeneic hosts, there were no consistent differences in the mean latent periods of tumor formation. Materials and Methods However, metastases occurred in the lungs of animals Cell lines and cell culture. The normal human mammary epithelial cell receiving the AGR2 transfectants in 77% to 92% of animals line, Huma 7, was subcloned from primary cultures of reduction with primary tumors (P = 0.0001) compared with no mammoplasty specimens of normal breast tissue immortalized with metastases in the control groups. The AGR2 transfectants SV40 (19). The benign human mammary epithelial cell line, Huma 123, exhibited enhanced rates of adhesion to a plastic substratum and the derivative benign human mammary myoepithelial-like cell line, and extracellular AGR2 enhanced the rate of attachment of Huma 109 (15), were derived from HMT-3522, itself obtained from a AGR2-negative but not AGR2-positive cells. These experi- primary cell culture of human benign breast disease displaying prominent ments are the first to link mechanistically the developmental epithelial hyperplasia (20). These cell lines, and the malignant human gene product, AGR2, with metastasis in vivo. (Cancer Res 2005; mammary epithelial cell lines derived from pleural effusions of breast 65(9): 3796-805) cancer patients, MCF-7, T47D, ZR-75, and MDA-MB-231 (21), were cultured as described previously (12, 15, 19). The benign rat mammary epithelial cell line, Rama 37, was cultured as described previously (22). Introduction The transfected derivative cell lines were grown in medium containing Altered levels of gene products are thought to be associated 1 mg/mL geneticin. All cells were passaged on reaching 70% confluency. with the changed behavior of cancer cells (1). Modern array Culture in medium depleted of steroid hormones was carried out as techniques are now being used to relate patterns of gene described previously (23, 24). Subtractive hybridization and Northern hybridization screening. A expression to both pathologic appearance and malignant behavior suppression subtractive (11) library consisting of PCR products represent- of breast cancer (1, 2) and other cancers (3–5). However, it is also ing mRNAs expressed at a higher level in the malignant breast epithelial important to identify the key changes in gene expression that cell line, MCF-7, relative to a benign human breast-derived cell line, Huma cause the malignant properties of cancer cells. Such an approach 123, was constructed using a PCR-Select cDNA Subtraction kit (Clontech, has led to the discovery of the metastasis-inducing proteins Palo Alto, CA) as described previously (14). Reverse Northern screening of S100A4 (6, 7) and osteopontin (8) that have been shown to have the subtracted cDNA library was carried out as described previously (12). such a dramatic effect on patient survival in a group of breast Total cellular RNA was prepared using the guanidinium-isothiocyanate- cancer patients (9, 10). cesium chloride method (25–27). Poly(A)-containing RNA was isolated from total RNA using the Fast Track mRNA isolation kit (Invitrogen, Groningen, the Netherlands). Northern hybridization procedures were done as described previously (12). cDNA probes were radioactively labeled to 1 109 dpm/Ag DNA by random-primed DNA synthesis (28) using a Requests for reprints: Roger Barraclough, Biosciences Building, University of labeling kit (Roche Molecular Biochemicals, Mannheim, Germany). The Liverpool, Crown Street, Liverpool L69 7ZB, United Kingdom. Phone: 44-151-795-4469; Fax: 44-151-795-4406; E-mail: [email protected]. constitutive probe, 36B4, a cDNA to human acidic ribosomal phospho- I2005 American Association for Cancer Research. protein PO mRNA (29), was used to normalize RNA loading on the gel.

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Coupled transcription and translation assay in vitro. Transcription PCR was done as follows: 94jC for 3 minutes followed by 25 cycles at and translation assays in vitro were carried out using a TNT T7/T3-coupled 94jC for 30 seconds, 60jC for 30 seconds, and 72jC for 1.5 minutes. PCR reticulocyte lysate system (Promega, Madison, WI) to produce a protein products were visualized with ethidium bromide following agarose gel product labeled with [35S]methionine in vitro. DNA template (2 Ag) was electrophoresis (31). transcribed and translated in 50 AL containing 40 units RNAsin RNase Transfection of plasmid DNA into rat cells. Exponentially growing inhibitor, 25 AL TNT rabbit reticulocyte lysate, 20 Amol/L amino acid benign rat mammary epithelial Rama 37 cells were harvested, seeded at a mixture without methionine, 20 units (T3 or T7) RNA polymerase, 20 ACi density of 0.5 to 0.7 106 cells per 9 cm diameter culture dish in routine [35S]methionine (>1,000 Ci/mmol at 10 mCi/mL), and 2 AL TNT reaction medium, and incubated for 24 hours at 37jC. pcDNA-M36 construct (5 Ag) buffer. The mixture was incubated at 30jC for 90 minutes. The resulting 35S- containing full-length M36 cDNA, which had been verified by DNA labeled proteins and nonradioactive standards were fractionated on urea- sequencing, was transfected into Rama 37 cells using calcium phosphate containing SDS, 15% (w/v) polyacrylamide gels (SDS-PAGE) with 6% (w/v) as described previously (32). Empty pcDNA vector (5 Ag) was transfected polyacrylamide stacking gels (30). The gels were stained with Coomassie into Rama 37 cells as a negative control. Colonies were visible after f7 days blue, destained with 40% (v/v) methanol, 7% (v/v) acetic acid, dried under following selection in 1 mg/mL geneticin. Three single colonies and two vacuum, and autoradiographed with Kodak X-Omat film (Eastman Kodak, pools of cells were picked, expanded, and subsequently transferred several Rochester, NY) at 70jC for 3 to 10 days. times before being frozen for storage. Production and purification of recombinant protein anterior Tumorigenesis and metastasis. Cultured cells were harvested by gradient 2 and its antiserum. The full-length M36 cDNA or one with trypsinisation and centrifugation and washed twice before being resus- the M36 signal sequence deleted was cloned into the expression vector, pended in cold PBS (4jC) at a concentration of 107 viable cells/mL. Female pET-16b (Novagen, Madison, WI), downstream of the His tag, to yield a Ludwig Furth-Wistar rats (5-6 weeks old), maintained on expanded rat recombinant cDNA construct designated pET-M36, which was first and mouse diet no.1 (B.P. Ltd., Essex, United Kingdom) and tap water verified by automated DNA sequencing and then transformed into ad libitum, were injected s.c. with 2 106 viable cells at the site of the left or Escherichia coli BL21DE3 cells. Induction of recombinant protein was right inguinal mammary fat pad. All rats were observed at 3- to 4-day carried out by adding isopropyl-L-thio-h-D-galactopyranoside (1 mmol/L) intervals up to 5 months. Tumor-bearing rats were necropsied when their to the culture medium (A 600 = 0.5) for 2 hours. Purification of primary tumors reached 10% of body mass or earlier if the tumors ulcerated recombinant AGR2 protein to a single band on SDS-PAGE gel was or caused serious morbidity. The lungs, liver, axillary lymph nodes, spleen, carried out using His-Bind resin (Novagen). The amino acid sequence of kidney, and heart were examined for gross metastases. Samples of the the purified recombinant AGR2 protein was confirmed by an automated primary tumors and lungs with abnormal appearance were fixed in sequencer. The production of rabbit anti-AGR2 serum was conducted by Methacarn [methanol/trichloroethane/acetic acid 6:3:1 (v/v)] and processed Eurogentec (Seraing, Belgium). The anti-AGR2 antibodies were affinity for histology (7). Animal experiments were conducted according to the purified by their binding to antigen immobilized on a polyvinylidene United Kingdom Coordinating Committee for Cancer Research guidelines difluoride (PVDF) membrane. Briefly, recombinant AGR2 (1 mg) was and the New York Academy of Sciences Committee on Animal Research subjected to SDS-PAGE and electrophoretically transferred to a PVDF under Home Office Project Licenses PPL 40/1515 and 40/2395 to Prof. Philip membrane and the part of the membrane containing the immobilized S. Rudland. antigen was incubated with serum from a rabbit immunized with Human breast specimens. Human breast specimens, normal specimens recombinant AGR2. Bound antibody was eluted with a 100 mmol/L from reduction mammoplasties, benign fibroadenomas, and invasive ductal glycine buffer (pH 2.5) followed by neutralization with 1 mol/L Tris buffer carcinoma of no special type were obtained from the Cancer Tissue Bank (pH 8.0). Research Centre (Liverpool, United Kingdom) with full and informed Western blot analysis. Cells were grown to 70% to 80% confluence, patient consent and with ethical approval. The carcinomas were subdivided washed twice with ice-cold PBS buffer, and lysed in lysis buffer [50 mmol/L into two groups based on immunocytochemical staining for ERa, a cutoff at Tris-HCl (pH 7.5), 150 mmol/L NaCl, 1% (v/v) NP40, 0.5% (w/v) sodium 5% of the carcinoma cells stained by antibodies to ERa divided the negative deoxycholate, 1 mmol/L EDTA, 0.1% (w/v) SDS], and a protease inhibitor from the positive group. cocktail tablet (Roche Molecular Biochemicals) was added. The cleared Histology and immunocytochemistry. The histology of 4 Am tissue lysates were collected by centrifugation at 12,000 g for 20 minutes at 4jC. sections was determined after staining with H&E. Immunocytochemical The protein concentration in the lysate was measured by Bio-Rad protein staining for vimentin, skeletal muscle actin, myoglobin, and ERa was assay (Bio-Rad Laboratories, Hemel Hempstead, Herts, United Kingdom). carried out as described previously (33, 34). Immunocytochemical staining Lysates containing equal amounts of total proteins were resolved by SDS- for AGR2 was done with affinity-purified AGR2 antibodies with or without PAGE. The proteins were electrotransferred onto PVDF membranes using a prior incubation with 0.1 mg/mL recombinant AGR2 protein either for Bio-Rad semidry transfer apparatus. The membranes were incubated with sections of human specimens [1:500 dilution in PBS buffer containing 2% the affinity-purified, in-house rabbit polyclonal anti-AGR2 antibody. After (w/v) bovine serum albumin incubated at room temperature for 2 hours] or washing and incubating with anti-rabbit horseradish peroxidase–conjugated for sections of rat specimens [1:200 dilution in PBS buffer containing 0.5% IgG, the membranes were washed and detected by the Supersignal West Pico (w/v) bovine serum albumin incubated at room temperature overnight]. Chemiluminescent Substrate (Pierce Biotechnology, Inc., Perbio Science, The bound antibodies were detected using biotinylated donkey anti-rabbit Cramlington, Northumberland, United Kingdom) according to the manu- serum followed by ABC complex/horseradish peroxidase kit (DAKO Ltd., facturer’s instructions. The membranes were reprobed with mouse Cambridgeshire, United Kingdom). The sections were visualized as a brown monoclonal anti-actin antibody (Sigma, Poole, Dorset, United Kingdom) to stain by incubating with 3,3V-diaminobenzidine (Sigma, Dorset, United ensure equal protein loading. Kingdom) and 0.075% (v/v) H2O2, counterstained with Mayer’s hemalum, Reverse transcription-PCR. Total RNA (2 Ag) was reverse transcribed in and mounted in dibutyl polystyrene xylene (Merck, Dorset, United 10 AL with 200 units SuperScript RNase H reverse transcriptase (Invitrogen Kingdom). All staining results were examined by three independent Ltd., Paisley, United Kingdom). Subsequently, the first-strand cDNA reaction observers and scored as plus/minus using 5% of carcinoma cells staining mixture (1 AL) was amplified by PCR with Taq DNA polymerase (Invitrogen). as a cutoff. Photography was carried out as described previously (33). For M36 cDNA, the forward primer (5V position at nucleotide 87, Genbank Cell adhesion assays. The cells were grown to 70% to 80% confluence, accession no. NM_006408.2) was 5V-GCTCCTTGTGGCCCTCTCCTACAC-3V washed twice with PBS, trypsinized, and counted using a Coulter counter and the reverse primer (5V position at nucleotide 440, Genbank accession (Beckman-Coulter UK Ltd., High Wycombe, Buckinghamshire, United no. NM_006408.2) was 5V-ATCCTGGGGACATACTGGCCATCAG-3V.For Kingdom). The cells were resuspended at 2 105 cells/mL and counted the human glyceraldehyde-3-phosphate dehydrogenase cDNA, the forward again to check the concentration before adding 1 mL cells to each well of primer 5V-ACCACAGTCCATGCCATCAC-3V and the reverse primer 5V-TCCA- a 24-well plate. After incubation for 30 minutes at 37jC, the cells were CCACCCTGTTGCTGTA-3V were used to provide a normalization control. washed thrice with PBS buffer to remove any cells in suspension, and www.aacrjournals.org 3797 Cancer Res 2005; 65: (9). May 1, 2005

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2005 American Association for Cancer Research. Cancer Research cells adhering to the wells were trypsinized and counted. The number of on urea-containing SDS-PAGE (data not shown), a size that adherent cells was calculated as the percentage of the total number of corresponds to the 20 kDa derived from the amino acid sequence. cells that had adhered after 30 minutes. In some experiments, the cell Quantitative reverse Northern hybridizations using as probes A culture plates were coated with either 2 or 20 g of recombinant AGR2 double-stranded mixed cDNAs showed that the level of AGR2 lacking the signal sequence before carrying out cell attachment assays as mRNA was >15-fold higher in the RNA from MCF-7 cells than in described above. Three independent experiments were carried out using triplicate wells and error bars represent the SDs of the means of the three that from the Huma 123 cells (data not shown). Northern separate experiments. hybridization experiments showed that the M36 probe hybridized Statistical analysis. Statistical analyses were done by the two-tailed to a major band of RNA with a molecular size of 0.9 kb (mean of Fisher’s exact test or Mann-Whitney U-test using Arcus Pro-Stat Dos version three independent experiments) corresponding to the AGR2 3.28 software (Medical Computing, Aughton, United Kingdom). mRNA and to an additional faint band at 1.6 kb in all the positive lanes (Fig. 1). The 1.6-kb band corresponds in size to the recently updated 1.7-kb variant mRNA of AGR2 containing a Results longer untranslated 3V-end (Genbank accession no. NM-006408). Overexpressed M36 cDNA in the malignant human breast The AGR2 mRNA was present in all three ERa-positive breast cancer cell lines corresponds to anterior gradient 2 mRNA. A cancer cell lines tested, MCF-7, T47D, and ZR-75, but undetect- suppression subtractive library was constructed containing cDNAs able in the ERa-negative MDA-MB-231 breast cancer cell line and expressed at a higher level in the malignant mammary cell line, in the SV40-immortalized normal human breast cell line, Huma 7, MCF-7, than in the cell line, Huma 123, derived from a benign the benign human breast tumor cell line, Huma 123, and its mammary lesion (Materials and Methods). Four cloned cDNAs myoepithelial-like convert, Huma 109 (Fig. 1). The same (M36, M40, M202, and M234) of 174 cloned cDNAs sequenced each distribution was found for the AGR2 protein using Western blot- exhibited 100% identity to the coding sequence of human cDNA ting with the AGR2 antibody (see Materials and Methods; Fig. 1), AGR2 (Genbank accession no. NM_006408). AGR2 is the human the signal being abolished by prior incubation of the antibody homologue of the X. laevis AGR2 (XAG-2) gene, which is expressed with 0.1 mg/mL recombinant AGR2 (data not shown). These by the cement gland of the developing Xenopus embryo (18). The results suggest that the expression of AGR2 mRNA and protein nucleotide sequences for M36 or M202 cDNA contained an open correlates with the presence of ERa at least in these cell lines. reading frame of 175 amino acids that was identical in amino acid AGR2 mRNA was present at a 7.3 F 0.2-fold (mean F SD of sequence to that of AGR2 protein (Genbank accession no. three independent experiments) higher level in MCF-7 cells grown NP_006399). In a T7/T3 RNA polymerase–coupled transcription/ in the presence of estrogen than in cells grown in estrogen- translation system, clones, M36 and M202, but not empty vector, depleted conditions, whereas the level of a previously described, yielded a single 35S-labeled primary translation product of 20 kDa estrogen-responsive mRNA, that of pS2 (35), was increased only

Figure 1. Northern and Western blots of AGR2 mRNA and protein in human mammary cell lines derived from benign and malignant breast tumors. A, total RNAs isolated from the SV40-immortalized normal human mammary cell line, Huma 7, the benign human mammary derived cell line, Huma 123, a benign myoepithelial-like convertant cell line, Huma 109, derived from Huma 123, the ER-negative, malignant, human mammary epithelial cell line, MDA-MB-231, and the ER-positive, human mammary cell lines MCF-7, T47D, and ZR-75, were subjected to Northern hybridization (Materials and Methods) using a 32P-labeled probe to AGR2 mRNA (A, top) or to the mRNA for ribosomal phosphoprotein cDNA, 36B4 (A, bottom) for normalization purposes. B, protein (10 Ag) from Huma 7, Huma 123, MCF-7, T47D, ZR-75, and MDA-MB-231 or recombinant, His-tagged AGR2 (1 Ag) was subjected to SDS-PAGE and blotted onto PVDF membranes as described in Materials and Methods. The membranes were incubated with antibodies to actin (B, top) or AGR2 (B, bottom) and visualized by chemiluminescence. nAGR2, natural AGR2; rAGR2, recombinant AGR2. The recombinant AGR2 is larger than the natural AGR2 due to the presence of a histidine tag and a protease factor X cleavage site.

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3.4 F 0.1-fold. In these quantitative results, mRNA levels were nor- specimens (Fig. 3F). Overall, AGR2 protein immunocytochemical malized with respect to 36B4 mRNA, a mRNA that is not dependent positivity (defined as >5% of epithelial cells staining) was found in on the presence of estrogen and its receptor for its production (29). only 2 of 5 (40%) normal specimens and 7 of 15 (47%) benign breast Identification of anterior gradient 2 mRNA and protein in tumor specimens, but 33 of 44 (75%) breast carcinoma specimens human breast tumor specimens. The occurrence of AGR2 mRNA were positive for AGR2 protein. This proportion was significantly in human benign breast lesions and malignant breast carcinomas different from normal and benign specimens (P = 0.025, Fisher’s was examined by reverse transcription-PCR (RT-PCR; Fig. 2). Using exact test). Twenty-six of 29 (90%) ERa-positive specimens were 25 cycles of PCR, only 3 of 9 (33%) normal and 13 of 25 (52%) benign positively stained, whereas only 7 of 15 (47%) ERa-negative samples were positive for AGR2 mRNA (positivity defined as a single carcinomas were positive for AGR2 protein, significantly different PCR band of 354 bp), whereas 44 of 56 (79%) breast carcinoma from the ERa-positive carcinomas (P = 0.0033, Fisher’s exact test). samples were positive for AGR2 mRNA (Table 1). This proportion These experiments showed quantitative results for AGR2 protein was significantly different from the normal and benign specimens that were similar to those obtained for mRNA by RT-PCR. (P = 0.0029, Fisher’s exact test). Moreover, 31 of 34 (91%) ERa- Identification of anterior gradient 2 as a novel metastasis positive carcinoma specimens yielded a strong PCR product, inducer. The full-length M36 (AGR2) cDNA was inserted into the whereas only 13 of 22 (59%) ERa-negative carcinomas were positive multiple cloning site of the mammalian expression vector pcDNA3, for AGR2 mRNA, values that were also significantly different downstream of the cytomegalovirus promoter, to yield a recom- (P = 0.007, Fisher’s exact test). These results show that AGR2 mRNA binant cDNA construct designated pcDNA-M36. The pcDNA-M36 is dependent on the presence of ERa in the majority of breast expression construct, or the same amount of empty pcDNA vector carcinoma specimens as well as in the breast carcinoma cell lines. as a negative control (pcDNA), was transfected into the benign rat The affinity-purified AGR2 antibodies (Materials and Methods) mammary epithelial cell line, Rama 37 (Materials and Methods). were used to stain immunocytochemically histologic sections of Following selection in geneticin, single colonies from the Rama 37 human breast specimens. The epithelial cells of human normal pcDNA-M36 transfectants were picked and expanded along with breast tissue and benign lesions either were stained modestly for two independent pools from the same transfectants. Using PCR or AGR2 protein or were unstained (Fig. 3A and B), but the epithelial Southern hybridization, RT-PCR, or Northern hybridization, AGR2 cells of ERa-positive breast carcinomas were stained strongly for sequences were shown to be present in the DNA and RNA from the AGR2 protein (Fig. 3C). The positive staining for AGR2 protein was transfectant pools and clones but absent from the Rama 37 cells completely abolished by prior incubation of the antibodies with and the Rama 37 cells transfected with empty vector summarized recombinant AGR2 protein (Fig. 3D). The immunocytochemical in Table 2. staining for AGR2 protein showed a granular appearance, The incidences of primary tumors and metastases produced by reminiscent of secretory granules (Fig. 3E). There was little or no AGR2 transfection are shown in Table 2 along with statistical staining for AGR2 in >50% of ERa-negative breast carcinoma analyses. A single s.c. injection of cells transfected with the empty

Figure 2. Identification of AGR2 mRNA from human breast tumor specimens using RT-PCR. RNA from human breast carcinoma specimens (lanes 1-28) was amplified by RT-PCR using primers specific for AGR2 cDNA (A) or human glyceraldehyde-3-phosphate dehydrogenase cDNA (B) to yield PCR products of 354 and 452 bp, respectively. The ERa status of specimens was recorded (+ or -) as described in Materials and Methods. The resulting RT-PCR products were subjected to agarose gel electrophoresis and stained with ethidium bromide as described in Materials and Methods. Lanes M, DNA molecular weight markers. www.aacrjournals.org 3799 Cancer Res 2005; 65: (9). May 1, 2005

Table 1. Identification of AGR2 mRNA and protein in breast specimens using RT-PCR and immunocytochemistry

Clinical specimens RT-PCR screening of specimens Immunocytochemical screening of specimens

No. positive* No. negative* Total No. positivec No. negativec Total

Normal 3 6 9 2 3 5 Benign 13 12 25 7 8 15 Total 16 18 34 9 11 20

ERa-positive carcinomas 31 3 34 26 3 29 b ERa-negative carcinomas 13 9227k 815 Total 44x 12 56 33{ 11 44

*Positive RT-PCR is defined as a single band of molecular weight 354 bp for AGR2 on the gel; negative RT-PCR is defined as no clear band on the gel, all have a band of 452 bp for control GPDH on the gel. cPositive immunocytochemistry is defined as >5% of epithelial cells staining; negative immunocytochemistry is defined as <5% of epithelial cells staining. bP = 0.007, statistically significantly different from ERa positive (Fisher’s exact test). x P = 0.0029, statistically significantly different from normal and benign specimens (Fisher’s exact test). k P = 0.0033, statistically significantly different from ERa positive (Fisher’s exact test). { P = 0.0025, statistically significantly different from benign specimens (Fisher’s exact test). vector yielded primary tumors in the mammary glands with a tumor-bearing rats exhibited lung metastases, and this was mean latent period of 49 F 11 days and with a tumor incidence confirmed by subsequent histologic examination of selected of 50% (Table 2). This incidence was not significantly different tissues, including lung and lymph nodes. However, the mean from that obtained with Rama 37 cells (Table 2). None of the latent period for palpable primary tumors of the pcDNA

Figure 3. Immunocytochemical staining of human breast specimens for AGR2. Histologic sections showing a normal human breast containing ducts and lobules (A), a benign fibroadenoma (B), an ERa-positive (C-E), and an ERa-negative (F) invasive ductal carcinomas stained immunocytochemically by affinity-purified antibodies to recombinant AGR2 protein (A-C, E, and F)orby antibodies preincubated with 0.1 mg/mL recombinant AGR2 protein (D) as described in Materials and Methods. For A-D and F, bar (shown on A), 50 Am; for E, bar,20Am.

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Table 2. Incidence of tumors and metastases produced by M36 cDNA transfected cells

Cell lines Presence Primary tumors Metastases or absence of AGR2 mRNA Mean F P Mean F P Mean F P Incidence P Incidence of P SD appearance (Mann- SD growth (Mann- SD weight (Mann- of primary (Fisher’s metastases (Fisher’s (days after Whitney period Whitney (g)b Whitney tumors exact (%)k exact test)k injection U-test)* (d)c U-test)c U-test)b (%)x test)x of cells)*

Rama 37 Negative N/A — N/A — N/A — 22/46 (48) — 0/22 (0) — 1st test Rama 37 Negative 31 F 7—15F 5 — 5.5 F 2.1 — 12/18 (67) — 0/12 (0) — 2nd test pcDNA Negative 49 F 11 0.0002 15 F 2 0.07 N/A — 10/20 (50) 1, 0.5 0/10 (0) — vector Pool 1 Positive 38 F 18 0.46 37 F 14 <0.0001 5.4 F 2.6 0.99 13/23 (57) 0.6, 0.5 12/13 (92) <0.0001 Pool 2 Positive 54 F 15 <0.0001 30 F 18 0.009 4.8 F 1.7 0.32 14/26 (54) 0.8, 0.2 12/14 (86) <0.0001 Clone 1 Positive 81 F 6 <0.0001 18 F 5 0.49 N/A — 30/60 (50) 0.9, 0.3 23/30 (77) <0.0001 Clone 2 Positive 37 F 14 0.39 16 F 5 0.94 6.8 F 2.4 0.25 9/14 (64) 0.4, 1 7/9 (78) 0.0003 Clone 3 Positive 46 F 22 0.014 42 F 20 0.0003 5.9 F 2.7 0.88 11/37 (30) 0.1, 0.02 9/11 (82) <0.0001

NOTE: Fifteen percent of injected rats were omitted from the analysis due to the presence of ascites or ulceration. N/A, not applicable. *Mean time of appearance of primary tumors (P values < 0.05, significantly different from Rama 37, second test). cMean growth period from primary tumor appearance to culling (P values <0.05, significantly different from Rama 37, second test). bMean weight of primary tumor (all AGR2 transfectants not significantly different from Rama 37, second test). x Incidence of primary tumors: % (no. rats with tumors/no. rats injected), all transfectants not significantly different from Rama 37, first test, and all but one transfectant not significantly different from Rama 37, second test. k Incidence of metastases: % (no. rats with metastases/no. rats with tumors), all AGR2 transfectants significantly different from Rama 37, first test, Rama 37, second test, and pcDNA vector transfectant.

transfectants was statistically different from Rama 37 (Table 2). between metastatic potential and the length of time the tumors Rats injected with the two independent pools or three took to grow following detection, nor any significant relationship independent clones of pcDNA-M36-transfected, AGR2-expressing when latent period plus tumor growth period was plotted against cells yielded tumors with a range of mean latent periods to metastatic potential (least squares regression analysis of a fit of the palpability (Table 2). All of these were longer, some significantly points to a straight line yielded probabilities in the range 0.12-0.55), longer than Rama 37 cells (Table 2), as were the growth periods ruling out the possibility that metastasis arose due to a longer between tumor appearance and culling (Table 2). There was no period of tumor growth. The results show that transfection of the relationship between latent period and growth period for the nonmetastatic cells with an expression vector containing AGR2 individual clones and pools. The weights of tumors at culling for induces metastasis in two pools and three separate clones of cells. any of the pools and clones tested were not significantly different Histology and immunocytochemistry of tumors produced by from Rama 37 cells (Table 2). transfected cells. Some of the primary tumors from rats injected The incidences of primary tumors for the AGR2-transfected with the AGR2-expressing Rama 37 cells transfected with pcDNA- clones and pools in the mammary glands ranged from 30% to 64% M36 were composed of cuboidal cells, many forming cords that of injected rats (Table 2), none were statistically different from were surrounded by neoplastic spindle cells, whereas others pcDNA vector-transfected cells (Table 2), Rama 37 cells first test consisted predominantly of neoplastic spindle cells. Many tumors (Table 2), and all but one not significantly different from Rama 37 showed central necrotic cores. In many primary tumors arising second test (Table 2). However, 92% and 86% of rats injected with from cells transfected with the pcDNA-M36 construct, extensive pool 1 and pool 2 cells, respectively, and 77% to 82% of rats numbers of blood vessels were seen. Some tumor cells had injected with the three clones of AGR2-transfected cells developed breached the surrounding connective tissue capsules and had either gross metastases in the lungs, which were visible at invaded the adjacent host skeletal muscle (Fig. 4A). In general, the necropsy, or micrometastases evident on subsequent histologic histology of the metastases was the same as that of the primary examination (Table 2). These values for the incidences of tumor. Both cannonball metastases and tumor cells penetrating metastases were significantly different from the control group of the surrounding lung tissue were evident (Fig. 4B). The primary pcDNA empty vector-transfected Rama 37 cells and two separate tumors and metastases were also extensively stained by antibodies groups of animals injected with Rama 37 cells (all P V 0.0003, to vimentin (Fig. 4B), and in that case, tumor cells in endothelial Fisher’s exact test), in which no lung macrometastasis or micro- cell–lined spaces, possibly lymphatics (Fig. 4C) and in blood vessels metastasis was found. There was no significant correlation (data not shown), were also observed. The primary tumor cells and www.aacrjournals.org 3801 Cancer Res 2005; 65: (9). May 1, 2005

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2005 American Association for Cancer Research. Cancer Research lung metastases also exhibited staining for milk fat globule the primary tumor and its metastases, sometimes forming large membrane antigen (data not shown) and by pan-keratin antibodies multinucleate cells. These skeletal muscle–like elements were (Fig. 4D) and by peanut lectin (data not shown). Differentiation of immunocytochemically stained by antisera to skeletal muscle actin tumor cells to skeletal muscle–like elements was common in both (data not shown) and to myoglobin (Fig. 4E). Skeletal muscle

Figure 4. Histology and immunocytochemical staining of primary rat tumors and metastases produced by M36 cDNA transfectants. Histologic sections of tissues from animals injected with Rama 37 cells transfected with the pcDNA3-M36 construct were stained with H&E or immunocytochemically stained. Carcinoma cells (T) of a primary tumor in the mammary gland locally invading muscle tissue (M), visualized by H&E staining (A). A cannonball metastasis (M) in the lung stained by antibodies to vimentin (L; B). Metastatic tumor cells in the lungs (large arrow) possibly in a lymphatic space adjacent to a blood vessel (small arrow) stained by antibodies to vimentin (C). Tumor cells in a lung metastasis stained by antibodies to pan-keratin (D). Skeletal muscle elements (arrow) within a lung metastasis stained by antibodies to myoglobin (E). Histologic sections of primary tumors from animals injected with Rama 37 cells transfected with pcDNA3 empty vector (F) or primary tumors (G and I) and metastases in the lungs (H) produced from animals injected with Rama 37 cells transfected with vector containing M36 cDNA (G-I) were stained with affinity-purified antibodies to recombinant AGR2 protein (F-H) or with antibodies preincubated with 0.1 mg/mL recombinant AGR2 protein (I) as described in Materials and Methods. Bar,50Am(A), 200 Am(B), and 20 Am(C); (D-I)as(A).

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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2005 American Association for Cancer Research. AGR, a Novel Metastasis Inducer in Breast Cancer elements were not found in any of the primary tumors arising from cells examined relative to parental Rama 37 cells (P range, 0.009 to cells transfected with the control pcDNA vector. <0.0001, Student’s t test) and by all pools and two of three clones Although there was no immunocytochemical staining for AGR2 relative to Rama 37 cells transfected with empty vector (P range, protein in the histologic sections of primary tumors arising from 0.0074-0.001, Student’s t test). Although there was a broad animals injected with Rama 37 cells transfected with empty pcDNA correlation between the metastatic and the adhesive potential of plasmid vector (Fig. 4F), both carcinoma cells of primary tumors the different transfectants, the similar levels of AGR2 in the pcDNA- (Fig. 4G) and metastases in the lungs (Fig. 4H) produced from M36-transfected cells precluded determining any significant animals injected with the AGR2-expressing Rama 37 cells trans- relationship between AGR2 expression and adhesive/metastatic fected with pcDNA-M36 were stained positively by the affinity- potential of the individual pools/clones of cells. To further identify purified antibodies directed against recombinant AGR2 protein. the mechanism by which AGR2 affects adhesion, coating of the The immunocytochemical staining by the antibodies to AGR2 was tissue culture dishes with either 2 or 20 Ag of recombinant AGR2 blocked completely by their prior incubation with recombinant protein lacking a signal peptide increased the rate of attachment of AGR2 protein (Fig. 4I). the AGR2-negative Rama 37 cells or Rama 37 cells transfected with Enhanced adhesion is associated with the anterior gradient empty vector but had little effect on the rate of attachment of the 2–transfected cells. The AGR2 transfectants did not exhibit any AGR2-transfected cell lines or pools (Fig. 5). significantly different growth rates in vitro compared with vector- transfected or parental Rama 37 cells (P = 1.0, Student’s t test), nor Discussion any altered invasive potential as measured by their behavior in an A subtracted library of cloned cDNAs expressed at a higher level invasion assay using Matrigel-coated Transwell chambers (P = 1.0, in the malignant human mammary cell line, MCF-7, relative to a Student’s t test). In contrast, a statistically significant increase in the benign cell line, Huma 123, derived from benign breast disease, rate of cellular adhesion to a plastic substratum (Fig. 5) was shown yielded among others, a cDNA, M36, identical to the coding by all the AGR2 cDNA-transfected pools and transfected clones of sequence of human AGR2, the anterior gradient 2 homologue (36), produced by the cement gland of the developing X. laevis embryo (18). Furthermore, the size of the primary translation products of M36 and the AGR2 protein were identical and the proposed product additionally exhibited 91% identity to the first 175 amino acids of the mouse gob-4 gene, which is expressed in intestinal mucin-secreting goblet cells (37). Transfection of M36 cDNA in an expression vector into a benign rat mammary cell line, Rama 37, induced a metastatic phenotype in vivo when the cells were injected into the mammary fat pads of syngeneic rats, whereas similar transfection of empty vector failed to induce a metastatic phenotype. The nonmetastatic Rama 37 cells have been shown previously to be converted to a metastatic phenotype by genes encoding proteins S100A4 (7), osteopontin (8), and cutaneous fatty acid-binding protein (38) but not by oncogenes, such as Ha-ras or DNA virus-transforming genes (39). Elevated levels of immunore- active S100A4 (9) or osteopontin (10) in the breast cancers of patients have been shown to correlate with markedly reduced patient survival, whereas cutaneous fatty acid-binding protein occurs preferentially in malignant as opposed to benign prostatic lesions (38). Use of the Rama 37 cell line has thus identified previously cloned genes whose products are biologically relevant to the metastatic process in human cancer, and our present results suggest strongly that AGR2 may also be involved in metastasis. Human AGR2 was reported previously to be expressed in the ERa- positive breast cancer cell line, MCF-7, and not in the ERa-negative cell line, MDA-MB-231 (17), but this is the first report of its direct involvement with metastasis and/or malignancy. The apparent

Figure 5. Adhesive and metastatic properties of AGR2-transfected and inconsistency of the involvement of a strongly ER-dependent gene/ untransfected rat mammary cell lines. A, Rama 37 or Rama 37 cells transfected gene product being associated with the process of metastasis of with empty pcDNA3 vector (Vector control) or pcDNA3-M36 (Pool 1, Pool 2, breast tumor cells is supported by the observation that in a group Clone 1, Clone 2, and Clone 3) were tested in vitro for their rate of adhesion to a plastic substratum (white columns) and for their metastatic ability (black of 225 tamoxifen-treated patients with ER-positive breast cancers, columns) as described in Materials and Methods. White columns, mean of three those with AGR2 in their breast cancer cells exhibited a statistically separate experiments; bars, SD. No black columns are shown for Rama 37 cells significantly poorer survival than those without AGR2 in their and Rama 37 transfected with vector alone because they did not produce metastases. B, Rama 37, Rama 37 cells transfected with empty vector, or cancer cells. In contrast, the similarly treated 126 patients with ER- Rama 37 cells transfected with expression vector containing AGR2 cDNA (pools negative breast cancers showed no such relationship.4 and clones) were allowed to attach to the substratum in 24-well culture dishes and the percentage of cells adhering in 30 minutes was recorded. The experiments were carried out in untreated tissue culture wells (white columns) and with wells coated with 2 Ag(gray columns)or20Ag(black columns) recombinant AGR. 4 Innes et al., in preparation. www.aacrjournals.org 3803 Cancer Res 2005; 65: (9). May 1, 2005

The AGR2-induced tumors in the experimental rats differed To identify a possible mechanism for AGR2-induced metastasis, somewhat from those induced by S100A4. Whereas S100A4 the effect of added recombinant AGR2 protein on Rama 37 cells produced primarily cannon ball metastases in the lungs/lymph was tested. AGR2 protein lacking the signal sequence up to a nodes (7), AGR2 induced both cannon ball metastases and concentration of at least 24 Amol/L6 and pcDNA-M36 transfection micrometastases in the lungs, similar to those observed failed to affect the growth rate of Rama 37 cells in culture. These previously for osteopontin (8). The presence of AGR2-induced results support the lack of a consistent effect of AGR2 on the micrometastases in blood, and possibly in lymphatic vessels, latent period of tumor formation in vivo (Table 2). Furthermore, suggests that AGR2 may induce metastasis to the lungs by both pcDNA-M36-transfected cell pools and clones did not show any blood-borne and lymphatic routes. enhanced invasive ability through Matrigel compared with The Xenopus AGR2 protein is a product of the mucin-producing parental and empty vector-transfected Rama 37 cells. However, cement gland, which is the first ectodermal organ to appear in the the pcDNA-M36 transfectants exhibited an increased adhesive developing Xenopus embryo (18). AGR2 is up-regulated in potential to a plastic substratum. The fact that AGR2 contains an experimentally dorsalized embryos and down-regulated in exper- active secretory signal is consistent with the punctate staining imentally ventralized embryos (18). Although the precise role of pattern for AGR2 observed in human breast cancers at higher AGR2 is presently unknown, its injection into early cleavage stage magnification (Fig. 3E) and suggests that AGR2 functions embryos results in enhanced cement gland development (18), and extracellularly. An extracellular mechanism of AGR2 is shown ectopically produced AGR2 not only can signal dorsoanterior by the observation that extracellularly added AGR2 enhances the ectodermal fate but also can induce neural markers in embryo rate of attachment of two AGR2-negative cell lines to that cells, suggesting that extracellular AGR2 is able to alter the observed with five independent AGR2-producing cell clones and differentiation potential of its target cells (18). It is not yet clear pools but had no effect on the AGR2-producing cell clones and whether AGR2 has similar activities in human development or pools. Although the mechanistic link between AGR2-induced whether such activities are related to its metastasis-inducing increased adhesion to plastic and metastasis is not yet known, properties. However, in the transfection experiments, some of the another well-characterized metastasis-inducing (8, 10) secreted cells containing the AGR2 (M36) construct exhibited muscle, and protein, osteopontin (41), has also been shown to increase the not neuronal, patterns of differentiation. The reason for this adhesion of cells (42), including Rama 37 cells, to plastic substrata pattern of differentiation being evident in the Rama 37 cells (43). Taken together, these results strongly suggest that AGR2 transfected with AGR2 is not known; however, this pattern of might also cause metastasis by enhancing this adhesive property differentiation has been described before in derivative cells of of the Rama 37 cells. distinct morphologic appearance, representing possible pluripo- In summary, this article describes the first demonstration of tent intermediates in differentiation of the Rama 37 epithelial cells metastasis-inducing properties of the developmentally important to a myoepithelial-like phenotype (40). Thus, the capability to form protein, AGR2. The presence of detectable AGR2 mRNA and muscle elements might be an intrinsic property of the recipient protein above a threshold in breast carcinoma cells significantly Rama 37 cell derivatives that is enhanced by the AGR2 (M36) correlates with carcinoma in preference to benign/normal tissue, gene/gene product. The mechanism of such enhancement is not and ERa-positive in preference to ERa-negative carcinomas, known. suggesting that the metastasis-inducing properties of AGR2 may Previously, the XAG-2 (AGR2) protein has been shown to be contribute, in some way, toward the malignant progression of some secreted when expressed in Xenopus oocytes (18). In the present ERa-positive breast cancers. Identification of the receptor for experiments, a granular cytoplasmic appearance of immunocyto- AGR2 will provide the means to identify the signaling pathways chemical staining for AGR2 observed in some human carcinoma that link the enhancement of cell attachment to the process of specimens suggests that AGR2 might be secreted by some metastasis. carcinoma cells. Pilot experiments using an AGR2 COOH-terminal GFP fusion cDNA5 have shown that the fusion protein is secreted Acknowledgments at least in HeLa cells, strongly suggesting that the AGR2 secretory Received 10/25/2004; revised 3/1/2005; accepted 3/3/2005. signal is active. Grant support: Clatterbridge Cancer Research Trust studentship and UK Committee of Vice Chancellors and Principals ORS scholarship award (D. Liu), Cancer and Polio Research Fund, and North West Cancer Research Fund grant CR532. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 5 D. Liu, P.S. Rudland, R. Barraclough, unpublished data. We thank Joe Carroll, Barry Cotterill and Karen Collard for excellent technical 6 D. Liu, P.S. Rudland, R. Barraclough, unpublished results. assistance.

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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2005 American Association for Cancer Research. Human Homologue of Cement Gland Protein, a Novel Metastasis Inducer Associated with Breast Carcinomas

Dong Liu, Philip S. Rudland, D. Ross Sibson, et al.

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