PTEN Deficiency in a Luminal ErbB-2 Mouse Model Results in Dramatic Acceleration of Mammary Tumorigenesis and Metastasis
2009; Elsevier BV; Volume: 284; Issue: 28 Linguagem: Inglês
10.1074/jbc.m109.018937
ISSN1083-351X
AutoresBabette Schade, Trisha Rao, Nathalie Dourdin, Robert Lesurf, Michael Hallett, Robert D. Cardiff, William J. Muller,
Tópico(s)HER2/EGFR in Cancer Research
ResumoOverexpression and/or amplification of the ErbB-2 oncogene as well as inactivation of the PTEN tumor suppressor are two important genetic events in human breast carcinogenesis. To address the biological impact of conditional inactivation of PTEN on ErbB-2-induced mammary tumorigenesis, we generated a novel transgenic mouse model that utilizes the murine mammary tumor virus (MMTV) promoter to directly couple expression of activated ErbB-2 and Cre recombinase to the same mammary epithelial cell (MMTV-NIC). Disruption of PTEN in the mammary epithelium of the MMTV-NIC model system dramatically accelerated the formation of multifocal and highly metastatic mammary tumors, which exhibited homogenous pathology. PTEN-deficient/NIC-induced tumorigenesis was associated with an increase in angiogenesis. Moreover, inactivation of PTEN in the MMTV-NIC mouse model resulted in hyperactivation of the phosphatidylinositol 3′-kinase/Akt signaling pathway. However, like the parental strain, tumors obtained from PTEN-deficient/NIC mice displayed histopathological and molecular features of the luminal subtype of primary human breast cancer. Taken together, our findings provide important implications in understanding the molecular determinants of mammary tumorigenesis driven by PTEN deficiency and ErbB-2 activation and could provide a valuable tool for testing the efficacy of therapeutic strategies that target these critical signaling pathways. Overexpression and/or amplification of the ErbB-2 oncogene as well as inactivation of the PTEN tumor suppressor are two important genetic events in human breast carcinogenesis. To address the biological impact of conditional inactivation of PTEN on ErbB-2-induced mammary tumorigenesis, we generated a novel transgenic mouse model that utilizes the murine mammary tumor virus (MMTV) promoter to directly couple expression of activated ErbB-2 and Cre recombinase to the same mammary epithelial cell (MMTV-NIC). Disruption of PTEN in the mammary epithelium of the MMTV-NIC model system dramatically accelerated the formation of multifocal and highly metastatic mammary tumors, which exhibited homogenous pathology. PTEN-deficient/NIC-induced tumorigenesis was associated with an increase in angiogenesis. Moreover, inactivation of PTEN in the MMTV-NIC mouse model resulted in hyperactivation of the phosphatidylinositol 3′-kinase/Akt signaling pathway. However, like the parental strain, tumors obtained from PTEN-deficient/NIC mice displayed histopathological and molecular features of the luminal subtype of primary human breast cancer. Taken together, our findings provide important implications in understanding the molecular determinants of mammary tumorigenesis driven by PTEN deficiency and ErbB-2 activation and could provide a valuable tool for testing the efficacy of therapeutic strategies that target these critical signaling pathways. Genetic alterations in the normal mammary epithelium, including the activation of oncogenes and the loss of function of tumor suppressor genes, have been implicated in the induction of mammary tumors. ErbB-2 (Neu/HER2) is a member of the epidermal growth factor receptor (EGFR) 4The abbreviations used are: EGFRepidermal growth factor receptorPI3Kphosphatidylinositol 3′-kinaseMMTVmurine mammary tumor virusPTENphosphatase and tensin homologue deleted on chromosome tenLOHloss of heterozygosityGsk3βglycogen synthase 3βMINmammary intraepithelial neoplasmKrtcytokeratinBadBcl-associated death promotermTORmammalian target of rapamycin.4The abbreviations used are: EGFRepidermal growth factor receptorPI3Kphosphatidylinositol 3′-kinaseMMTVmurine mammary tumor virusPTENphosphatase and tensin homologue deleted on chromosome tenLOHloss of heterozygosityGsk3βglycogen synthase 3βMINmammary intraepithelial neoplasmKrtcytokeratinBadBcl-associated death promotermTORmammalian target of rapamycin. family of receptor tyrosine kinases, which also includes EGFR, ErbB-3 (HER3), and ErbB-4 (HER4) (1.Olayioye M.A. Neve R.M. Lane H.A. Hynes N.E. Embo J. 2000; 19: 3159-3167Crossref PubMed Google Scholar). Amplification/overexpression of ErbB-2 has been observed in 20–30% of human breast cancers, correlating with poor patient prognosis (2.Slamon D.J. Clark G.M. Wong S.G. Levin W.J. Ullrich A. McGuire W.L. Science. 1987; 235: 177-182Crossref PubMed Scopus (9911) Google Scholar, 3.Andrulis I.L. Bull S.B. Blackstein M.E. Sutherland D. Mak C. Sidlofsky S. Pritzker K.P. Hartwick R.W. Hanna W. Lickley L. Wilkinson R. Qizilbash A. Ambus U. Lipa M. Weizel H. Katz A. Baida M. Mariz S. Stoik G. Dacamara P. Strongitharm D. Geddie W. McCready D. J. Clin. Oncol. 1998; 16: 1340-1349Crossref PubMed Scopus (373) Google Scholar). The importance of ErbB-2 in mammary tumorigenesis has been demonstrated by several transgenic mouse models (4.Ursini-Siegel J. Schade B. Cardiff R.D. Muller W.J. Nat. Rev. Cancer. 2007; 7: 389-397Crossref PubMed Scopus (204) Google Scholar), where engagement of the ErbB-2 receptor results in the recruitment of adaptor proteins, activating primarily the Ras pathway (5.Dankort D.L. Wang Z. Blackmore V. Moran M.F. Muller W.J. Mol. Cell. Biol. 1997; 17: 5410-5425Crossref PubMed Scopus (124) Google Scholar). ErbB-2-induced mammary tumors exhibit elevated ErbB-3 protein levels (6.Siegel P.M. Ryan E.D. Cardiff R.D. Muller W.J. Embo J. 1999; 18: 2149-2164Crossref PubMed Scopus (371) Google Scholar). ErbB-3 mediates activation of the phosphatidylinositol 3′-kinase (PI3K)/Akt pathway by recruiting the p85 regulatory subunit of PI3K (7.Prigent S.A. Gullick W.J. Embo J. 1994; 13: 2831-2841Crossref PubMed Scopus (319) Google Scholar, 8.Soltoff S.P. Carraway 3rd, K.L. Prigent S.A. Gullick W.G. Cantley L.C. Mol. Cell. Biol. 1994; 14: 3550-3558Crossref PubMed Scopus (461) Google Scholar). Additionally, the ErbB-2/ErbB-3 heterodimer is thought to be the most biologically active and pro-tumorigenic receptor complex (9.Alimandi M. Romano A. Curia M.C. Muraro R. Fedi P. Aaronson S.A. Di Fiore P.P. Kraus M.H. Oncogene. 1995; 10: 1813-1821PubMed Google Scholar, 10.Wallasch C. Weiss F.U. Niederfellner G. Jallal B. Issing W. Ullrich A. Embo J. 1995; 14: 4267-4275Crossref PubMed Scopus (354) Google Scholar). These findings suggest that the concerted activation of both Ras and PI3K signaling through this EGFR family heterodimer plays a critical role in mammary tumorigenesis (9.Alimandi M. Romano A. Curia M.C. Muraro R. Fedi P. Aaronson S.A. Di Fiore P.P. Kraus M.H. Oncogene. 1995; 10: 1813-1821PubMed Google Scholar, 10.Wallasch C. Weiss F.U. Niederfellner G. Jallal B. Issing W. Ullrich A. Embo J. 1995; 14: 4267-4275Crossref PubMed Scopus (354) Google Scholar). epidermal growth factor receptor phosphatidylinositol 3′-kinase murine mammary tumor virus phosphatase and tensin homologue deleted on chromosome ten loss of heterozygosity glycogen synthase 3β mammary intraepithelial neoplasm cytokeratin Bcl-associated death promoter mammalian target of rapamycin. epidermal growth factor receptor phosphatidylinositol 3′-kinase murine mammary tumor virus phosphatase and tensin homologue deleted on chromosome ten loss of heterozygosity glycogen synthase 3β mammary intraepithelial neoplasm cytokeratin Bcl-associated death promoter mammalian target of rapamycin. Growth and survival pathways can also become oncogenic through the disruption of tumor suppressors. Loss of the tumor suppressor phosphatase and tensin homologue deleted on chromosome ten (PTEN) due to mutation, loss of heterozygosity (LOH), and epigenetic down-modulation has been reported in about 50% of human cancers, including breast cancer (11.Salmena L. Carracedo A. Pandolfi P.P. Cell. 2008; 133: 403-414Abstract Full Text Full Text PDF PubMed Scopus (876) Google Scholar). PTEN, a lipid phosphatase, directly antagonizes the proto-oncogenic PI3K/Akt pathway by dephosphorylating phosphatidylinositol 3,4,5-trisphosphate to generate phosphatidylinositol 4,5-bisphosphate to attenuate Akt activation (12.Maehama T. Dixon J.E. J. Biol. Chem. 1998; 273: 13375-13378Abstract Full Text Full Text PDF PubMed Scopus (2590) Google Scholar, 13.Stambolic V. Suzuki A. de la Pompa J.L. Brothers G.M. Mirtsos C. Sasaki T. Ruland J. Penninger J.M. Siderovski D.P. Mak T.W. Cell. 1998; 95: 29-39Abstract Full Text Full Text PDF PubMed Scopus (2101) Google Scholar). PTEN-controlled PI3K/Akt signaling regulates various cellular events, including proliferation and survival (14.Furnari F.B. Lin H. Huang H.S. Cavenee W.K. Proc. Natl. Acad. Sci. U.S.A. 1997; 94: 12479-12484Crossref PubMed Scopus (382) Google Scholar, 15.Myers M.P. Pass I. Batty I.H. Van der Kaay J. Stolarov J.P. Hemmings B.A. Wigler M.H. Downes C.P. Tonks N.K. Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 13513-13518Crossref PubMed Scopus (1002) Google Scholar), migration (16.Liliental J. Moon S.Y. Lesche R. Mamillapalli R. Li D. Zheng Y. Sun H. Wu H. Curr. Biol. 2000; 10: 401-404Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar, 17.Suzuki A. Kaisho T. Ohishi M. Tsukio-Yamaguchi M. Tsubata T. Koni P.A. Sasaki T. Mak T.W. Nakano T. J. Exp. Med. 2003; 197: 657-667Crossref PubMed Scopus (197) Google Scholar), and senescence (18.Chen Z. Trotman L.C. Shaffer D. Lin H.K. Dotan Z.A. Niki M. Koutcher J.A. Scher H.I. Ludwig T. Gerald W. Cordon-Cardo C. Pandolfi P.P. Nature. 2005; 436: 725-730Crossref PubMed Scopus (1549) Google Scholar). Furthermore, PTEN-deficiency is associated with resistance to the drug Herceptin (Trastuzumab), a humanized monoclonal antibody targeting ErbB-2 19.Nagata Y. Lan K.H. Zhou X. Tan M. Esteva F.J. Sahin A.A. Klos K.S. Li P. Monia B.P. Nguyen N.T. Hortobagyi G.N. Hung M.C. Yu D. Cancer Cell. 2004; 6: 117-127Abstract Full Text Full Text PDF PubMed Scopus (1530) Google Scholar). Collectively, these observations suggest that cross-talk between ErbB-2 and PTEN disruption may play a critical role in ErbB-2-induced tumorigenesis. To directly assess the role of PTEN in ErbB-2-induced mammary tumorigenesis, we recently interbred conditional PTEN mice to a mouse model expressing a Cre-inducible activated ErbB-2 allele under the transcriptional control of the endogenous ErbB-2 promoter (ErbB-2KI) (20.Dourdin N. Schade B. Lesurf R. Hallett M. Munn R.J. Cardiff R.D. Muller W.J. Cancer Res. 2008; 68: 2122-2131Crossref PubMed Scopus (42) Google Scholar). PTEN deficiency dramatically accelerated ErbB-2-mediated tumorigenesis. About 50% of PTEN-heterozygous/ErbB-2KI tumors displayed LOH at the PTEN locus. PTEN-deficient/ErbB-2KI tumors displayed elevated ErbB-2 protein levels, like the parental strain, but this was not due to genomic amplification or dramatically increased ErbB-2 transcription (20.Dourdin N. Schade B. Lesurf R. Hallett M. Munn R.J. Cardiff R.D. Muller W.J. Cancer Res. 2008; 68: 2122-2131Crossref PubMed Scopus (42) Google Scholar). Strikingly, PTEN-deficient/ErbB-2KI tumors showed morphological heterogeneity with molecular and pathological features of the basal-like and HER2 subtypes of human breast cancer. One limitation of the PTEN-deficient/ErbB-2KI model is that ErbB-2 expression is driven by the endogenous ErbB-2 promoter (21.Andrechek E.R. Hardy W.R. Siegel P.M. Rudnicki M.A. Cardiff R.D. Muller W.J. Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 3444-3449Crossref PubMed Scopus (189) Google Scholar). Thus, initial ErbB-2 transcription in these mammary epithelial cells is low, and therefore, PTEN inactivation occurs before elevated ErbB-2 levels. To directly evaluate the functional consequences of PTEN disruption in a mouse model that places ErbB-2 under transcriptional control of the strong viral MMTV promoter, we have generated mice co-expressing activated ErbB-2 (6.Siegel P.M. Ryan E.D. Cardiff R.D. Muller W.J. Embo J. 1999; 18: 2149-2164Crossref PubMed Scopus (371) Google Scholar) and Cre recombinase from the same bicistronic transcript, due to an internal ribosome entry sequence placed between the two cDNA sequences (MMTV-NIC) (22.Ursini-Siegel J. Hardy W.R. Zuo D. Lam S.H. Sanguin-Gendreau V. Cardiff R.D. Pawson T. Muller W.J. Embo J. 2008; 27: 910-920Crossref PubMed Scopus (110) Google Scholar). This system couples ErbB-2 and Cre recombinase expression within the same mammary epithelial cell (supplemental Fig. 1A). Using this approach we demonstrate that homozygous inactivation of PTEN in the presence of constitutive ErbB-2 expression results in a dramatic acceleration of mammary tumor onset in MMTV-NIC mice. PTEN-deficient/NIC tumor cells exhibit increased capacity to colonize the lung, correlating with elevated angiogenesis in the primary tumors. Furthermore, over 50% of PTEN heterozygous/NIC tumors displayed LOH at the PTEN locus. In contrast to the morphological heterogeneity of the PTEN-deficient/ErbB-2KI tumors, PTEN loss in MMTV-NIC mice results in mammary tumors with a homogenous morphology resembling the parental strain. Unlike the PTEN-deficient/ErbB-2KI model, PTEN-deficient/NIC mammary tumorigenesis is associated with the loss of basal/myoepithelial markers, leading to rapid formation of invasive tumors with histopathological and molecular characteristics of the luminal subtype. Biochemical analysis revealed that loss of PTEN in MMTV-NIC tumors results in hyperactivation of the PI3K/Akt pathway. Consequently, this novel ErbB-2 mouse model may provide valuable insights toward understanding the molecular determinants in mammary tumor progression and represents an attractive tool for testing the efficacy of therapeutic strategies directed against the PI3K/Akt network in combination with receptor tyrosine kinase inhibitors. Mice harboring a bicistronic transcript expressing an oncogenic Neu allele (6.Siegel P.M. Ryan E.D. Cardiff R.D. Muller W.J. Embo J. 1999; 18: 2149-2164Crossref PubMed Scopus (371) Google Scholar) followed by an internal ribosome entry sequence element and Cre recombinase under the control of the MMTV promoter have been described (22.Ursini-Siegel J. Hardy W.R. Zuo D. Lam S.H. Sanguin-Gendreau V. Cardiff R.D. Pawson T. Muller W.J. Embo J. 2008; 27: 910-920Crossref PubMed Scopus (110) Google Scholar). These mice were interbred with Flox-PTEN mice (129/J, The Jackson Laboratory, Bar Harbor, ME) to generate bigenic mice, PTEN+/−/NIC and PTEN−/−/NIC. Genotyping was carried out by PCR as described (21.Andrechek E.R. Hardy W.R. Siegel P.M. Rudnicki M.A. Cardiff R.D. Muller W.J. Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 3444-3449Crossref PubMed Scopus (189) Google Scholar, 23.Li G. Robinson G.W. Lesche R. Martinez-Diaz H. Jiang Z. Rozengurt N. Wagner K.U. Wu D.C. Lane T.F. Liu X. Hennighausen L. Wu H. Development. 2002; 129: 4159-4170Crossref PubMed Google Scholar). Nulliparous females were monitored weekly for mammary tumor formation by physical palpation. All procedures involving mice were conducted in accordance with McGill University Animal Care guidelines. Mammary tumors and lungs were harvested from mice that were tumor-bearing for 4 weeks. Tissue was fixed and embedded as described (5.Dankort D.L. Wang Z. Blackmore V. Moran M.F. Muller W.J. Mol. Cell. Biol. 1997; 17: 5410-5425Crossref PubMed Scopus (124) Google Scholar). Paraffin sections of 5 μm were stained with hematoxylin and eosin. Step sections of lungs (4 × 50 μm) were scanned using a Scanscope XT Digital Slide Scanner (Aperio) and analyzed at 5× magnification using Imagescope software (Aperio) to quantify the total number of lesions per lung. The data are shown as the average number of lesions per lung lobe. Immunohistochemical staining was performed as described (24.White D.E. Kurpios N.A. Zuo D. Hassell J.A. Blaess S. Mueller U. Muller W.J. Cancer Cell. 2004; 6: 159-170Abstract Full Text Full Text PDF PubMed Scopus (352) Google Scholar). Sections were incubated with the primary antibodies Neu (Homemade, 1:100), PTEN (Cell signaling 9559, 1:100), cytokeratin-6 (Krt6; Covance PRB-169P, 1:1000), Krt5 (Covance PRB-160P, 1:1500), Krt14 (Covance PRB-115P, 1:1000), Krt8 (Fitzgerald RDI-PROGP11, 1:1000), smooth muscle actin (Sigma A2547, 1:1000), and Ki67 (Abcam ab15580, 1:100) followed by incubation with Elite anti-mouse, anti-rabbit, or anti-guinea pig IgG secondary antibodies from the Vectastain kit (PK-6102, Vector Laboratories, Burlingame, CA). CD31 staining of was performed on optimal cutting temperature-embedded sections (BD Biosciences 550274, 1:200) as described (25.Maglione J.E. Moghanaki D. Young L.J. Manner C.K. Ellies L.G. Joseph S.O. Nicholson B. Cardiff R.D. MacLeod C.L. Cancer Res. 2001; 61: 8298-8305PubMed Google Scholar). Slides were scanned using a ScanScope XT Digital Slide Scanner, and data were analyzed with Imagescope software using positive pixel count or nuclear algorithms. Tumor lysates were prepared as described (6.Siegel P.M. Ryan E.D. Cardiff R.D. Muller W.J. Embo J. 1999; 18: 2149-2164Crossref PubMed Scopus (371) Google Scholar). ErbB-2 and ErbB-3 immunoprecipitations were performed with 500 μg of lysate using the ErbB-2 (Ab4) monoclonal antibody (Oncogene Research Products, Inc.) and ErbB-3 (C17) polyclonal antibody (Santa Cruz Biotechnology, Inc.), respectively. Immunoblot analyses were performed on 20 μg of lysate as described (6.Siegel P.M. Ryan E.D. Cardiff R.D. Muller W.J. Embo J. 1999; 18: 2149-2164Crossref PubMed Scopus (371) Google Scholar, 26.Dankort D. Maslikowski B. Warner N. Kanno N. Kim H. Wang Z. Moran M.F. Oshima R.G. Cardiff R.D. Muller W.J. Mol. Cell. Biol. 2001; 21: 1540-1551Crossref PubMed Scopus (143) Google Scholar) using the following antibodies: ErbB-2 (Santa Cruz C18, 1:1000), ErbB-3 (Santa Cruz C17, 1:1000), phosphotyrosine (BD Biosciences PY20, 1:1000), PTEN (Cell Signaling 9559, 1:1000), Akt (Cell Signaling 9272, 1:1000), phospho-Akt (Cell Signaling 9275, 1:1000), Erk (Cell Signaling 9102, 1:1000), phospho-Erk (Cell Signaling 9101, 1:1000), Tsc2 (Cell Signaling 3612, 1:1000), phospho-Tsc2 (Cell Signaling 3611, 1:1000), Bcl-associated death promoter (Bad; BD Transduction Laboratories 36420, 1:1000), phospho-Bad (Cell Signaling 5286, 1:1000), glycogen synthase 3β (Gsk3β; Cell Signaling 9315, 1:1000), phospho-Gsk3β (Cell Signaling 9331, 1:1000), and Actin (Sigma A-9718, 1:10000). Horseradish peroxidase-conjugated secondary antibodies (1:10000) were obtained from The Jackson Laboratory. DNA was obtained from tumors and examined for LOH at the PTEN locus as described (21.Andrechek E.R. Hardy W.R. Siegel P.M. Rudnicki M.A. Cardiff R.D. Muller W.J. Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 3444-3449Crossref PubMed Scopus (189) Google Scholar). RNA sample preparations were performed as previously described (27.Schade B. Lam S.H. Cernea D. Sanguin-Gendreau V. Cardiff R.D. Jung B.L. Hallett M. Muller W.J. Cancer Res. 2007; 67: 7579-7588Crossref PubMed Scopus (22) Google Scholar). Total RNA was isolated from five individual (PTEN−/−/NIC) samples. Two RNA pools containing equal amounts of total RNA from five individual MMTV-NIC tumors were generated and functioned as a biological repeat. Labeled aRNA (750 ng) was hybridized against a Universal Mouse Reference RNA (Stratagene) onto Agilent Whole Mouse Genome Oligo Microarrays (G4122A; 44K). Duplicate hybridizations were done for all samples using reverse-dye labeling. All microarray data analysis was carried out in the r statistical programming environment (R Development Core Team, 2008) with Bioconductor (28.Gentleman R.C. Carey V.J. Bates D.M. 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We recently generated a mouse model where expression of activated ErbB-2 and Cre recombinase are coupled within the same mammary epithelial cell (22.Ursini-Siegel J. Hardy W.R. Zuo D. Lam S.H. Sanguin-Gendreau V. Cardiff R.D. Pawson T. Muller W.J. Embo J. 2008; 27: 910-920Crossref PubMed Scopus (110) Google Scholar). To determine the biological significance of heterozygous or homozygous PTEN loss in MMTV-NIC-induced mammary tumorigenesis, we interbred MMTV-NIC animals with mice carrying one or two floxed PTEN alleles (PTEN−/−, PTEN+/−). Thus, both ErbB-2 expression and loss of PTEN will occur simultaneously in each mammary epithelial cell in these bigenic animals. Cohorts of PTEN−/−/NIC, PTEN+/−/NIC, and MMTV-NIC virgin females were generated and monitored for mammary tumor formation by weekly palpation. Homozygous inactivation of PTEN resulted in dramatic acceleration of tumor onset with an average latency of 43 days, whereas heterozygous PTEN loss exhibited a moderate acceleration (126 days) compared with that of 198 days obtained for the parental strain (Fig. 1A, left panel; Table 1). All genotypes developed multifocal mammary tumors with 100% penetrance (Table 1). Four weeks after initial tumor detection, all tumor-bearing animals were sacrificed, the total tumor volume was determined, and lungs were harvested. The average total tumor burden was not significantly elevated in the PTEN-deficient/NIC mice relative to the MMTV-NIC (Fig. 1A, right panel). Conversely, the average tumor volume per tumor was greater in the parental strain compared with the PTEN−/−/NIC (supplemental Fig. 1B). These observations are reflective of the fact that the PTEN−/−/NIC mice developed on average more individual but smaller tumor nodules relative to the MMTV-NIC mice.TABLE 1Phenotypic characteristics of the PTEN-deficient/ErbB-2 mouse modelsStrainPromoter (activated ErbB-2)Average age of mammary tumor formationaValues represent the average age of onset in days of first palpable mammary tumor.Penetrance of tumorsbPercentage of animals that developed mammary tumors.Mammary tumor morphology% of animals with lung lesionscPercentage of tumor-bearing animals that possessed lung lesions; parentheses indicate the numbers of animals with lung lesions over the number of examined animals.% of mammary tumors with PTEN lossdPercentage of tumor-bearing animals showing a reduced amount of PTEN by Southern blot analysis.Days%PTEN+/−/NICMMTV126 (±36)100Multifocal, solid nodular53 (10/19)80PTEN−/−/NICMMTV43 (±12)100Multifocal, solid nodular87.5 (14/16)100MMTV-NICMMTV198 (±43)100Multifocal, solid nodular56 (5/9)NDPTEN+/−/ErbB-2KI (20)Endogenous212 (±92)10050% focal, 50% multifocal heterogeneous morphology35 (6/17)56PTEN−/−/ErbB-2KI (20)Endogenous138 (±125)10060% focal, 40% multifocal heterogeneous morphology0 (0/6)100ErbB-2KI (21)Endogenous419 (±137)83Focal comedo adenocarcinoma6 (2/35)0PTEN+/− (20)NA410 (±101)7560% focal, 40% multifocal heterogeneous morphology18 (2/11)17PTEN−/− (20)NANA0NANANAa Values represent the average age of onset in days of first palpable mammary tumor.b Percentage of animals that developed mammary tumors.c Percentage of tumor-bearing animals that possessed lung lesions; parentheses indicate the numbers of animals with lung lesions over the number of examined animals.d Percentage of tumor-bearing animals showing a reduced amount of PTEN by Southern blot analysis. Open table in a new tab To assess whether PTEN loss affects the ability of tumor cells to colonize the lung in MMTV-NIC mice, lungs from tumor-bearing females were subjected to histological analyses to detect the presence of lung lesions. Overall, the percentage of mice with lung metastases was increased in the PTEN−/−/NIC (87.5%) compared with PTEN+/−/NIC mice (53%) and the parental strain (56%) (Fig. 1B, left panel). Interestingly, we found a significant increase in the average number of lung lesions in the PTEN-deficient/NIC mice (2 metastases/lung lobe) relative to the parental strain (0.6 metastases/lung lobe) (Fig. 1B, right panel). Histological assessment of PTEN-deficient/NIC-derived mammary tumors revealed a morphology similar to that observed in MMTV-NIC tumors. Unlike the morphological heterogeneity observed in the PTEN-deficient/ErbB-2KI mouse model (20.Dourdin N. Schade B. Lesurf R. Hallett M. Munn R.J. Cardiff R.D. Muller W.J. Cancer Res. 2008; 68: 2122-2131Crossref PubMed Scopus (42) Google Scholar), the PTEN-deficient/NIC-derived tumors are composed of large nodular nests with central necrosis that resemble the parental tumors (Fig. 1C). Moreover, a histological survey of adjacent mammary glands from tumor-bearing mice revealed that 81% of the MMTV-NIC adjacent mammary gland structures represent relatively normal ducts/acini, whereas 1% categorize as early mammary intraepithelial neoplasms (MINs), 8% as hyperplasia and 10% as ectasia (supplemental Fig. 1C). In comparison, PTEN-deficient/NIC adjacent mammary glands contained only 38% relatively normal ducts/acini, 8% early MINs, 20% hyperplasia, and 34% ectasia, suggesting that PTEN inactivation may result in increased numbers of pre-neoplastic structures associated with early stages of tumorigenesis (supplemental Fig. 1C). Collectively, these observations suggest that impairment of PTEN has a dramatic impact on both tumor initiation and metastatic potential in ErbB-2-mediated mammary tumorigenesis. Given that PTEN inactivation in our MMTV-driven ErbB-2 mouse model has a significant impact on mammary tumor initiation (Fig. 1A), we next determined whether tumor cell proliferation was affected. Serial tumor sections were subjected to immunohistochemical staining with the proliferative marker Ki67. This survey revealed that PTEN-deficient tumors possessed similar proliferative levels compared with their wild type counterparts (Fig. 2A). However, analysis of adjacent mammary glands from both genotypes displayed a moderate increase in Ki67-positive nuclei within early MINs and hyperplasia of the PTEN-deficient/NIC mice when compared with the parental strain (Fig. 2A). One interesting feature of PTEN-deficient mammary tumors is their increased capacity to metastasize to the lung (Fig. 1B, right panel). To determine whether the enhanced metastatic phenotype was related to increased angiogenesis, we performed CD31 immunohistochemical staining to score for blood vessels on mammary tumors from each genotype. Microvessel density is reflected by the percentage of CD31-positive endothelium per area of tumor epithelium and can be used as an indicator of increased angiogenesis. Immunohistochemical examination of CD31 staining revealed that tumors obtained from PTEN-deficient/NIC females displayed significantly increased angiogenesis relative to MMTV-NIC-derived tumors (Fig. 2B). Our findings suggest that the dramatic effect of PTEN inactivation on ErbB-2-mediated mammary tumorigenesis and metastasis may be due to increased tumor angiogenesis. Histopathological surveys of PTEN-deficient/NIC and MMTV-NIC adjacent mammary glands revealed that early MINs, hyperplasia, and relatively normal ducts/acini, stained intensely for basal/myoepithelial markers including smooth muscle actin, Krt5, Krt14 (34.Lazard D. Sastre X. Frid M.G. Glukhova M.A. Thiery J.P. Koteliansky V.E. Proc. Natl. Acad. Sci. U.S.A. 1993; 90: 999-1003Crossref PubMed Scopus (275) Google Scholar, 35.Da Silva L. Clarke C. Lakhani S.R. J. Clin. Pathol. 2007; 60: 1328-1332Crossref PubMed Scopus (50) Google Scholar, 36.van de Rijn M. Perou C.M. Tibshirani R. Haas P. Kalli
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