research article · effect of conditional knockout of the type ii tgf-bbb receptor gene in mammary...

Effect of Conditional Knockout of the Type II TGF-bbb Receptor Gene

in Mammary Epithelia on Mammary Gland Development

and Polyomavirus Middle T Antigen Induced Tumor

Formation and Metastasis

Elizabeth Forrester,1Anna Chytil,

1,3Brian Bierie,

1Mary Aakre,

1,3Agnieszka E. Gorska,

1,3

Ali-Reza Sharif-Afshar,1William J. Muller,

4and Harold L. Moses

1,2,3

Departments of 1Cancer Biology and 2Medicine; 3Vanderbilt-Ingram Cancer Center, Nashville, Tennessee; and 4Departments of Medicineand Biochemistry, McGill University, Montreal, Quebec, Canada

Abstract

Transforming growth factor–B (TGF-B) isoforms are growthfactors that function physiologically to regulate development,cellular proliferation, and immune responses. The role of TGF-Bsignaling in mammary tumorigenesis is complex, as TGF-B hasbeen reported to function as both a tumor suppressor andtumor promoter. To elucidate the role of TGF-B signaling inmammary gland development, tumorigenesis, and metastasis,the gene encoding type II TGF-B receptor, Tgfbr2 , wasconditionally deleted in themammary epithelium (Tgfbr2MGKO).Loss of Tgfbr2 in the mammary epithelium results in lobular-alveolar hyperplasia in the developing mammary gland andincreased apoptosis. Tgfbr2MGKO mice were mated to themouse mammary tumor virus-polyomavirus middle T antigen(PyVmT) transgenic mouse model of metastatic breast cancer.Loss of Tgfbr2 in the context of PyVmT expression results in ashortened median tumor latency and an increased formationof pulmonary metastases. Thus, our studies support a tumor-suppressive role for epithelial TGF-B signaling in mammarygland tumorigenesis and show that pulmonary metastases canoccur and are even enhanced in the absence of TGF-B signalingin the carcinoma cells. (Cancer Res 2005; 65(6): 2296-302)

Introduction

Transforming growth factor–h (TGF-h) acts as a potent growthinhibitor of most epithelial cell types (1). It arrests the cell cycle inthe G1 phase thereby tightly regulating cell proliferation andpotentially exerting a tumor-suppressive role (reviewed in ref. 2). Atumor suppressor role for TGF-h signaling is supported bytransgenic mouse studies demonstrating that overexpression ofTGF-h1 [mouse mammary tumor virus (MMTV)-TGF-h1] in themammary epithelium suppresses mammary tumor formation (3).Additionally, overexpression of a dominant-negative mutant form ofthe type II TGF-h receptor in mammary epithelium results inlobular-alveolar hyperplasia and formation of mammary tumorsafter a long latency (4–6). Although inactivating mutations inTGFBR2 or other TGF-h signaling pathway genes, such as Smads ,are quite rare in human breast cancers, loss or down-regulated

expression of ThRII in human breast cancers has been shownthrough immunohistochemistry, and this reduced expressioncorrelates with a higher tumor grade (7, 8). Furthermore, decreasedexpression of the type II receptor (ThRII) may be an early event inhuman breast tumorigenesis because women with breast biopsiescontaining epithelial hyperplasia lacking atypia and a reduced levelof ThRII immunostaining cells had a 3.41-fold increased risk fordeveloping invasive breast cancer compared with women withsimilar lesions having high levels of ThRII immunostaining (9).Paradoxically, it has been documented that many tumor cells,

including breast carcinoma cells, become resistant to TGF-h-mediated growth inhibition, and the tumor cells often increasethe production of one or more of the TGF-h isoforms. In responseto increased production of TGF-h, tumor cells, which are nowresistant to TGF-h’s growth inhibitory effects, become moreinvasive and metastatic (reviewed in refs. 10, 11). In addition,several studies using inhibitors of TGF-h have shown that systemicinhibition of TGF-h signaling suppresses metastasis of mammarycarcinomas (12, 13).To further elucidate the complex role of TGF-h signaling in

mammary carcinogenesis, we conditionally inactivated ThRII usingCre/Lox technology (14). We report here that conditional loss ofThRII in the mammary gland epithelium results in lobular-alveolarhyperplasia, similar to the phenotype seen in MMTV-DNIIRanimals (5). In addition, MMTV-PyVmT transgene expression inthe context of Tgfbr2 knockout results in a shortened latency oftumor formation and an increase in the size of metastatic lunglesions. These studies support the role of TGF-h as a tumorsuppressor in mammary gland development and tumorigenesisand show that TGF-h signaling in the mammary epitheliumfunctions to inhibit mammary tumor formation. The data furthershow that pulmonary metastases are enhanced with loss of TGF-hsignaling in carcinoma cells.

Materials and Methods

Generation and Characterization of Tgfbr2flox/flox, Tgfbr2MGKO,MMTV-PyVmT/Tgfbr2flox/flox, and MMTV-PyVmT/Tgfbr2MGKO Mice.Generation of the Tgfbr2flox/flox mice (C57/B6) has been described

previously (14). These mice were mated with MMTV-Cre mice (FVB) to

generate Tgfbr2mammary gland knockout (Tgfbr2MGKO) mice (FVB/C57/B6; refs.

15, 16). The Tgfbr2flox/flox mice were bred 10 generations into the FVBgenetic background. Female Tgfbr2flox/flox (FVB) and Tgfbr2MGKO mice

(FVB) were mated with MMTV-PyVmT male mice (FVB) to generate

MMTV-PyVmT/Tgfbr2flox/flox (FVB) and MMTV-PyVmT/Tgfbr2MGKO mice(FVB). All mice were housed in the Animal Care Facility at Vanderbilt

University following the Association for the Assessment and Accreditation

of Laboratory Animal Care guidelines.

Note: Supplementary data for this article are available at Cancer Research Online(http://cancerres.aacrjournals.org).

Requests for reprints: Harold L. Moses, Department of Cancer Biology,Ingram Cancer Center, Vanderbilt University School of Medicine, 698 PrestonResearch Building, 2220 Pierce Avenue, Nashville, TN 37232-6838. Phone: 615-936-1782; Fax: 615-936-1790; E-mail: [email protected].

I2005 American Association for Cancer Research.

Cancer Res 2005; 65: (6). March 15, 2005 2296 www.aacrjournals.org

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Identification of Transgenic Mice. Genotypes of Tgfbr2flox/flox,Tgfbr2MGKO, MMTV-PyVmT/Tgfbr2flox/flox, and MMTV-PyVmT/Tgfbr2MGKO

animals were determined by using oligonucleotide primers as described

previously (14, 17).

Southern Blot Analysis. Genomic DNA was extracted from mammarytumors and lung metastases and probed as described previously (14).

Isolation and Culture of PyVmT Cells.Mammary tumors were removed

and digested at 37jC for 4 hours in serum-free DMEM: F12 + 100 units/mL

pen-strep, 250 Ag/mL ampho B, 10 mg/mL gentamicin, 2 mg/mL collagenase,and 100 units/mL hyaluronidase. Cells were washed five times with PBS

containing 5% adult bovine serum. Cells were plated in flasks coated with

50 Ag/mL of collagen in 0.02 N acetic acid. Cells were maintained in DMEM/

F12 medium containing 2% adult bovine serum.Cell Proliferation Assays. For sequential cell-counting experiments,

cells were plated at 50,000 cells per well in 6-well plates in complete growth

medium and incubated for 24 hours. Cells from three replicate wells were

then counted (day 0) using a Coulter Counter (Beckman Coulter, Inc.,

Fullerton, CA). The remaining wells were treated and counted in triplicate

at 24-hour intervals for 7 days.

Reverse Transcription-PCR Analysis for Tumor-Derived Cell Lines.

Primers used for PCR were TBRII-R (exon 6) 5V-AATTTCCG-

GCCGCCCTCGGTCT-3V, TBRII-F (exon 4) 5V-CCCGGGGCATCGCT-

CATCT-3V, TBRII-F (exon 2) 5V-TAACAGTGATGTCATGGCC-3V, TBRII-R

(exon 3) 5V-GGAAGTACTGTGTGAACCC-3V; TBRIII-F 5V-AACTTCTCCTT-

GACAGCAGA-3V, TBRIII-R 5V-CACTCTCTTTTCCAAAGCC-3V; GAPDH-F 5V-

CTGGCATGGCCTTCCGTG-3V, 5V-GAAATGAGCTTGACAAAG-3V.Histologic Analysis and Immunohistochemistry. Mammary glands

and tumors were harvested and immediately fixed in 4% paraformaldehyde/

PBS at 4jC overnight. Hematoxylin-stained whole mounts of #4 inguinal

glands were sectioned and stained as described previously (18). Apoptosiswas visualized with the DeadEnd Colorimetric TUNEL System (Promega

Co., Madison, WI). For proliferating cell nuclear antigen staining, sections

were incubated with rabbit anti-proliferating cell nuclear antigen (SantaCruz Biotechnology, Santa Cruz, CA). h-Galactosidase activity assays were

modified from a previous report (19).

Western Analyses. Whole cell extracts were prepared as previously

described (20). For whole tumor lysates, ice-cold lysis buffer (describedabove) was added and tissue was homogenized on ice. Tissue lysates were

clarified by centrifugation for 15 minutes at 12,000 rpm, 4jC. Proteinconcentrations were determined as described above. Immunoblotting, for

the analysis of Smad2/Smad3 (Santa Cruz Biotechnology), Smad2phosphorylated on Ser465 (Cell Signaling, Beverly, MA), AKT (Cell Signaling),

AKT phosphorylated on Ser473 (Cell Signaling), and h-tubulin was done as

previously described (20). The h-tubulin monoclonal antibody developed by

Michael Klymkowsky was obtained from the Developmental StudiesHybridoma Bank developed under the auspices of the National Institute

of Child Health and Human Resources and maintained by the Department

of Biological Sciences, University of Iowa, Iowa City, IA 52242.

Results

Mice with Conditional Knockout of Tgfbr2 Using MMTV-Cre(Tgfbr2MGKO) Lack Expression of Tgfbr2 in the MammaryEpithelium. MMTV-Cre mice were bred to Tgfbr2flox/flox/Rosa26reporter mice to generate Tgfbr2MGKO/Rosa26r mice to verifyrecombination of the mammary epithelium (21). In whole mountpreparations and sections of 6-week-old virgin mammary glands,LacZ staining was observed only in the ducts and terminal endbuds of Tgfbr2MGKO/Rosa26r mice, whereas no staining wasdetected in Tgfbr2flox/flox/Rosa26r mammary glands (Fig. 1A).Furthermore, recombination in the ductal epithelial cells of themammary glands in Tgfbr2MGKO mice was determined by lasercapture microdissection followed by genomic DNA extraction and

Figure 1. Cre expression and recombination of theROSA26r locus and Tgfbr2 in mammary glandepithelial cells of Tgfbr2MGKO mice. A, lac-Zexpression in whole mounts of 6-week-old mammaryglands from Tgfbr2flox/flox /Rosa26r and Tgfbr2MGKO/Rosa26r mice. A photomicrograph of a section froma Tgfbr2MGKO/Rosa26r whole mount where bluestaining represents epithelial cells which haveundergone recombination. B, laser capturemicrodissection of Tgfbr2flox/flox and Tgfbr2MGKO

mammary gland tissue before and after dissection.C, recombination of Tgfbr2 in captured genomicDNA by PCR analysis. Tgfbr2 genomic locustargeted for recombination. Black bars , position ofPCR primers. Black arrowheads , LoxP sites.

Type II TGF-bb Receptor Knockout in Mammary Epithelia

www.aacrjournals.org 2297 Cancer Res 2005; 65: (6). March 15, 2005



PCR. The 241-bp PCR product indicative of the recombined allele isdetected only in Tgfbr2MGKO mice, but not in Tgfbr2flox/flox mice(Fig. 1B and C). Whereas these studies were done in a mixedgenetic background, similar results have been obtained in a pureFVB background (data not shown).Tgfbr2MGKO Mice Exhibit Lobular-Alveolar Hyperplasia.

After demonstrating recombination of Tgfbr2 in the mammaryepithelium of Tgfbr2MGKO mice, we analyzed the developmentalphenotype of the mammary gland. By whole mount preparationsand sectioning of the inguinal glands we observed that a majorityof the mice exhibit lobular-alveolar hyperplasia between 11 and 19weeks of age when compared with age-matched Tgfbr2flox/flox

mice (Fig. 2A). Interestingly, this phenotype seems to regress after20 weeks of age. Tgfbr2MGKO mice exhibit a higher rate of apoptosiswhen compared with Tgfbr2flox.flox mice as detected in mammarygland sections by terminal deoxynucleotidyl transferase–mediatednick end labeling analysis when an equal number of cells of eachgenotype were counted (Fig. 2B). The significant increase in therate of apoptosis in Tgfbr2MGKO mice could contribute to theregression of the hyperplastic phenotype, and additional studies areunder way investigating this possibility.Conditional Loss of Tgfbr2 in the Mammary Epithelium

Shortens the Latency of Tumor Formation. To determine theeffects of complete loss of TGF-h signaling in the mammary gland

on mammary tumor metastasis, we crossed the Tgfbr2MGKO micewith the MMTV-PyVmT mice, both in the FVB background andfollowed the mice until they reached 100 days of age. Conditionalloss of ThRII in the mammary gland shortened the median latencyof tumor formation from 71 to 47.5 days (Fig. 3A). Both thePyVmT/Tgfbr2flox/flox and PyVmT/Tgfbr2MGKO mice develop highlyinvasive mammary adenocarcinomas with no apparent histologicdifferences in the primary tumors, and there is no detectable effectof knockout of Tgfbr2 on the number or size of primary mammarycarcinomas (data not shown).Conditional Loss of Tgfbr2 in the Mammary Epithelium

Increases Pulmonary Metastases in MMTV-PyVmT TransgenicMice. Our current understanding of the role of TGF-h signaling inmetastasis would predict that knockout of Tgfbr2 in the mammaryepithelium would decrease the incidence of pulmonary metastasesinMMTV-PyVmTmice. However, the growth of metastatic lesions inthe PyVmT/Tgfbr2MGKO mice was strikingly increased whencompared with the PyVmT/Tgfbr2flox/flox mice. As stated previously,PyVmT/Tgfbr2flox/flox mice and PyVmT/Tgfbr2MGKO were followeduntil the mice reached 100 days of age. At this point, the mice weresacrificed and the full necropsies were done. However, to rule out thepossibility that the increase in metastatic burden was simply theresult of enhanced rate of primary tumorigenesis in the PyVmT/Tgfbr2MGKO mice, mice of both genotypes were followed for 45 days

Figure 2. Mammary gland phenotype ofTgfbr2flox/flox and Tgfbr2MGKO mice.A, representative whole mounts of16-week-old mammary glands from virginTgfbr2flox/flox and Tgfbr2MGKO mice.Graphical representation of the distributionof Tgfbr2MGKO virgin females exhibitinglobular-alveolar hyperplasia or nohyperplasia (n). B, quantification ofapoptotic cells TUNEL analysis.Representative photomicrographs ofTgfbr2flox/flox and Tgfbr2MGKO mammarygland sections stained for apoptotic cells;5,000 nuclei from each genotype werecounted. Graphical representationof % apoptotic cells in Tgfbr2flox/flox andTgfbr2MGKO mammary gland sections.

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after the first palpable tumor was noted. At either time point, therewas a dramatic increase in the number and size of pulmonarymetastases present in the PyVmT/Tgfbr2MGKO mice. The massivenumber of pulmonary metastases in the PyVmT/Tgfbr2MGKO micemade it impossible to accurately count surface metastases;therefore, wet lung weights were recorded and used as a measureof metastatic burden. PyVmT/Tgfbr2flox/flox mice sacrificed 45 daysafter the first palpable tumor appeared had a mean wet lung weightof 0.169F 0.018 g compared with PyVmT/Tgfbr2MGKO mice that hada mean wet lung weight of 0.314F 0.082 g (P = 0.0049, n = 5 for eachgenotype). Similarly, PyVmT/Tgfbr2flox/flox mice sacrificed at100 days of age had a mean wet lung weight of 0.182 F 0.056 gcompared with PyVmT/Tgfbr2MGKO mice that had a mean wet lungweight of 0.394F 0.196 g (P = 0.0371, n = 10 for PyVmT/Tgfbr2MGKO

mice and n = 5 for PyVmT/Tgfbr2flox/flox mice). This increase inpulmonary metastases in the PyVmT/Tgfbr2MGKO mice was alsoevident upon examination of histologic sections of the lungs(Fig. 3B). Whereas there was no difference in the histology of themetastases with knockout of Tgfbr2 , there was a remarkabledifference in the size of lesions present in each section.To rule out the possibility that the metastatic tumors arose from

primary tumors in which there was no recombination, we isolatedDNA from several mammary tumors, pulmonary metastases, and

primary tumor cell cultures from both PyVmT/Tgfbr2flox/flox andPyVmT/Tgfbr2MGKO mice. Southern blot analyses of these samplesreveal extensive recombination in all of the PyVmT/Tgfbr2MGKO

samples (Fig. 3C). The small amount of nonrecombined DNA islikely due to stromal and hematopoietic cells present in the tumortissue. Immunostaining for phosphorylated Smad2 (p-Smad2) wasdone on sections of primary mammary tumors from PyVmT/Tgfbr2flox/flox and PyVmT/Tgfbr2MGKO as an indicator of TGF-hsignaling in vivo (Fig. 4A and B). Most PyVmT/Tgfbr2flox/flox tumorcells show nuclear staining for p-Smad2 (Fig. 4A), whereas PyVmT/Tgfbr2MGKO tumor cells lacked such staining (Fig. 4B). Immunos-taining for p-Smad2 was also done on sections of pulmonarymetastases from PyVmT/Tgfbr2flox/flox and PyVmT/Tgfbr2MGKO

mice. Interestingly, whereas primary mammary tumors fromPyVmT/Tgfbr2flox/flox mice display intense, homogenous p-Smad2staining, the lung metastases from PyVmT/Tgfbr2flox/flox micedisplay variable staining. In fact, the majority of the lungmetastases display a decreased proportion of cells staining positivefor nuclear p-Smad2 compared with the primary tumors (Fig. 4Cand D). As expected, the epithelial cells of primary mammarytumors and lung metastases from PyVmT/Tgfbr2MGKO display asignificant loss of p-Smad2 staining; however, fibroblasts and otherhost immune cells clearly stain positive (Fig. 4E and F). These data

Figure 3. Effects of conditional knockout ofTgfbr2 on mammary tumor formation andpulmonary metastases in MMTV-PyVmTmice. A, MMTV-PyVmT/Tgfbr2MGKO micedevelop mammary tumors with a significantlyshorter median latency compared withMMTV-PyVmT/Tgfbr2flox/flox. Dashed lines ,95% confidence intervals. B, representativephotomicrographs of an H&E-stainedsections of MMTV-PyVmT/Tgfbr2flox/flox

lungs showing pulmonary metastases (top )and MMTV-PyVmT/Tgfbr2MGKO lungs withmetastases (bottom ). Forty-five days afterthe first palpable tumors appeared,MMTV-PyVmT/Tgfbr2flox/flox mice had amean wet lung weight of 0.169 g comparedwith MMTV-PyVmT/Tgfbr2MGKO mice thathad a mean wet lung weight of 0.314 g(P = 0.0049). C, Southern blot analyses ofDNA isolated from mammary tumors (MT )and pulmonary metastases (LM ) ofMMTV-PyVmT/Tgfbr2flox/flox (lanes 1 -2) andMMTV-PyVmT/Tgfbr2MGKO (lanes 3-6) andmammary tumor cell lines established fromMMTV-PyVmT (lane a), MMTV-PyVmT/Tgfbr2flox/flox (lane b ), and MMTV-PyVmT/Tgfbr2MGKO (lane c ) mice.





clearly indicate that TGF-h signaling in the mammary epitheliumfunctions to suppress early tumorigenesis and growth of metastasisin the PyVmT transgenic mouse line.PyVmT/Tgf br2MGKO Tumor Cell Lines Are Resistant to

Transforming Growth Factor–B Growth Inhibitory Effects. Weestablished cultures from primary mammary tumors of MMTV-PyVmT/Tgfbr2flox/flox , MMTV-PyVmT/Tgfbr2MGKO and from thelung metastases of MMTV-PyVmT/Tgfbr2flox/flox and MMTV-PyVmT/Tgfbr2MGKO mice [primary floxed tumor (PF), primaryknockout tumor (PK), lung metastases from a floxed mouse (LF)and lung metastases from a knockout mouse (LK), respectively].Sequential cell count experiments using these cell lines indicatethat the PF cells are still sensitive to TGF-h’s antiproliferativeeffects. As anticipated, the PK cells are no longer responsive toTGF-h demonstrating that Tgfbr2 has been successfully deleted(Fig. 5Aa). Similarly, neither the LK nor LF cell lines are sensitive toTGF-hs growth inhibitory effects (Fig. 5Ab). Reverse transcription-PCR analysis of all cell lines using primers specific for exons 2 to 3expression indicates a complete loss of Tgfbr2 expression in the PKtumor line and decreased expression in the LF and LK cell lines(Fig. 5B). Primers specific for exons 2 to 3 were used because exon

2 of Tgfbr2 is the region floxed by loxP sites and thus excised bythe addition of Cre. This decreased level of expression instead of acomplete loss of expression of Tgfbr2 expression in LK cells couldbe due to the presence a few remaining fibroblasts in the culture.The same may be true for the LF cell line, or it is also possible that,similar to human breast cancers, TGF-h type II receptor levelswere down-regulated and not completely lost. As anticipated thePF tumor line displays high levels of Tgfbr2 expression (Fig. 5B).When the cell lines are treated with TGF-h, the PF line showsstrong induction of p-Smad2 whereas the PK, LF, and LK cell linesshowed no increase in p-Smad2 and total Smad2 levels remainedunchanged (Fig. 5C).All of the data indicate a loss of TGF-h responsiveness not only

in the PK cells but also in the LF and LK cells and show that TGF-hsignaling in carcinoma cells is not required for mammary tumormetastasis. The data also suggest that early loss of TGF-h signalingin the mammary epithelium promotes mammary tumor develop-ment and metastasis. Furthermore, we have found that PF and PKlines undergo no morphologic changes or epithelial to mesenchy-mal transition upon treatment with TGF-h (Fig. 5D). We alsoexamined protein from four primary mammary tumors from

Figure 4. Immunostaining for p-Smad2 in primarymammary tumors and pulmonary metastasesfrom PyVmT/Tgfbr2flox/flox and PyVmT/Tgfbr2MGKO

mice. A, PyVmT/Tgfbr2flox/flox primary mammarytumor. B, PyVmT/Tgfbr2MGKO primary mammarytumor. Arrows , presence of stromal cells thatstain positive for p-Smad2. C, PyVmT/Tgfbr2flox/flox

lung metastases. D, PyVmT/Tgfbr2flox/flox

lung metastases. E, PyVmT/Tgfbr2MGKO lungmetastases. F, PyVmT/Tgfbr2MGKO lungmetastases. Arrows , presence of host cells thatstain positive for p-Smad2. Bar , 250 Am(A , B , D , F ) and 500 Am (C and E).

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MMTV-PyVmT/Tgfbr2flox/flox , MMTV-PyVmT/Tgfbr2MGKO mice todetermine loss of TGF-h signaling and found that tumors fromMMTV-PyVmT/Tgfbr2MGKO have decreased expression of p-Smad3(Supplementary Fig. 1). However, there are no changes in the levelsof mitogen-activated protein kinase, p-mitogen-activated proteinkinase, AKT, p-AKT or cyclin D1 (Supplementary Fig. 1). Inaddition, there are no differences detected in the expression ofTGF-h1, TGF-h2, or TGF-h3 mRNA in whole tumor extracts fromMMTV-PyVmT/Tgfbr2flox/flox and PyVmT/Tgfbr2MGKO mice (Sup-plementary Fig. 1).

Discussion

In this study, we conditionally knocked out Tgfbr2 in themammary epithelium to derive Tgfbr2MGKO mice. These miceexhibit varying degrees of lobular-alveolar hyperplasia, and sponta-neous tumor development studies are currently under way. Whentumor formation is induced by the PyVmT transgene, loss of Tgfbr2in the mammary epithelium resulted in formation of mammarytumors with a significantly shortened median latency whencompared with PyVmT mice. Contrary to expectations, MMTV-PyVmT/Tgfbr2MGKO mice developed significantly more pulmonarymetastases compared with MMTV-PyVmT/Tgfbr2flox/flox mice.

We were able to detect very high levels of recombination of Tgfbr2not only in the primary mammary tumors arising from MMTV-PyVmT/Tgfbr2MGKO mice but also in the pulmonary metastases.Therefore, it is highly unlikely that metastases of the primarymammary tumors were from cells in which recombination had notoccurred. The extent of abrogation of the TGF-h signaling pathwaywas also confirmed although p-Smad2 immunostaining. Interest-ingly, we derived evidence for diminished TGF-h signaling in vitroand in vivo in lung metastases of PyVmT/Tgfbr2flox/flox mice. Theseresults imply that decrease of TGF-h signaling occurs spontaneouslyand frequently in the metastatic process of PyVmT/Tgfbr2flox/flox

mice. Thus, all of the results generated in this study are consistentwith Tgfbr2 acting as a tumor suppressor in the mammary gland.Furthermore, these data show that mammary tumor metastasis canclearly occur in the absence of TGF-h signaling and in fact isenhanced by the loss of TGF-h signaling at least in this model.Our current findings are in agreement with other recent studies

done using colon and prostate mouse models in which disruptionof TGF-h signaling results in enhanced tumor progression (22, 23).Whereas our study contradicts the reports that TGF-h signalingpromotes mammary tumor metastasis (reviewed in ref. 24), it isimportant to analyze the differences in the model systems. Severalstudies to date clearly show systemic inhibition of TGF-h signaling

Figure 5. PyVmT/Tgfbr2MGKO mammarytumor cells are unresponsive to TGF-h1treatment. A, sequential cell countexperiment. Left, PyVmT/Tgfbr2flox/flox

mammary tumor cells (n),PyVmT/Tgfbr2flox/flox mammary tumor cellline + TGF-h1(o), PyVmT/Tgfbr2MGKO

mammary tumor cell line (E), and PyVmT/Tgfbr2MGKO mammary tumor cell line +TGF-h1(d). Right , PyVmT/Tgfbr2flox/flox

lung metastases cell line (n),PyVmT/Tgfbr2flox/flox lung metastases cellline +TGF-h1(o), PyVmT/Tgfbr2MGKO lungmetastases cell line (E), andPyVmT/Tgfbr2MGKO lung metastasescell line +TGF-h1(d). B, reversetranscription-PCR analysis ofPyVmT/Tgfbr2flox/flox mammary tumorcell line (lane 1), PyVmT/Tgfbr2MGKO

mammary tumor cell line (lane 2 ), PyVmT/Tgfbr2flox/flox lung metastases cell line(lane 3), PyVmT/Tgfbr2MGKO mammarytumor cell line (lane 4), reference RNA(lane 5), negative control (no reversetranscriptase, lane 6). C, Western blotanalysis of whole cell lysates isolated fromPyVmT/Tgfbr2flox/flox mammary tumor cells(PF), PyVmT/Tgfbr2MGKO mammary tumorcells (PK), PyVmT/Tgfbr2flox/flox lungmetastases cells (LF), andPyVmT/Tgfbr2MGKO lung metastasescells (LK). D, photomicrographs ofPyVmT/Tgfbr2flox/flox andPyVmT/Tgfbr2MGKO mammarytumors cells treated for 48 hours with andwithout TGF-h. TGF-h treatment doesnot induce a morphologic change ineither cell line.





suppresses mammary tumor metastasis (12, 13). Systemic inhibi-tion of TGF-h signaling affects activity of the immune system andstroma cells. Several studies point to the importance of theimmune system in the regulation of tumor growth and metastasis(reviewed in ref. 25). Importantly, blockade of TGF-h signalingspecifically in T cells has been shown to play a role in theenhancement of antitumor immunity and protection againstformation of metastases (26, 27). However, our unique modelsystem allows us to precisely study the effects of abrogation ofTGF-h signaling specifically on the mammary epithelium and thusmammary tumor formation and metastasis. It is also possible thatthe timing of the loss of TGF-h signaling may determine whetheror not TGF-h functions as a tumor suppressor or tumor promoter.For example, in human colon cancers loss of ThRII is correlatedwith the progression of adenomas to invasive carcinomas, but laterin tumorigenesis loss of ThRII in colon carcinomas with micro-satellite instability is correlated with a better prognosis (28, 29).Therefore, loss or attenuation of TGF-h signaling later in mammarytumorigenesis may decrease the frequency of metastatic spread.Finally, loss of Tgfbr2 in mammary tumor cells may lead toincreased genomic instability (30) and this could represent anothermechanism by which loss of TGF-h signaling promotes metastasis.

Here we report that early loss of TGF-h signaling in the mouse

mammary gland in the context of expression of the MMTV-PyVmT

transgene results in shortened median tumor latency and, more

importantly, an increased incidence of metastasis. Our studies

suggest that the effects of systemic inhibition of TGF-h may largely

be on host cells and not tumor cells. Thus, these results support the

model of epithelial TGF-h signaling in mammary tumors being

suppressive for both tumor development and metastasis by clearly

demonstrating that early loss of Tgfbr2 results in more rapid

primary tumor formation and a marked increase in pulmonary

metastases.

Acknowledgments

Received 9/10/2004; revised 11/16/2004; accepted 12/29/2004.Grant support: Susan G. Komen Breast Cancer Foundation grant DISS0402357

(E. Forrester), Public Health Service grants CA42572 and CA85492 (H.L. Moses),Vanderbilt-Ingram Cancer Center grant CA68485, and TJ Martell Foundation.

The costs of publication of this article were defrayed in part by the payment of pagecharges. These articles must therefore be marked advertisement in accordance with 18U.S.C. Section 1734 solely to indicate this fact.

We thank Philippe Soriano (Fred Hutchinson Cancer Research Center, Seattle, WA)for generously providing the ROSA26r mice (C57/B6), Dr. Yu Shyr and BasharShakhtour for statistical analysis of our data, and Dr. Carlos Arteaga for generouslyproviding us with the MMTV-PyVmT mice and for reviewing this article.

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Cancer Research




2005;65:2296-2302. Cancer Res Elizabeth Forrester, Anna Chytil, Brian Bierie, et al. Tumor Formation and MetastasisDevelopment and Polyomavirus Middle T Antigen InducedGene in Mammary Epithelia on Mammary Gland

ReceptorβTGF-Effect of Conditional Knockout of the Type II

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