Phosphatase and tensin homologue deleted on chromosome 10 deficiency accelerates tumor induction in a mouse model of ErbB-2 mammary tumorigenesis

Abstract

Loss of the tumor suppressor phosphatase and tensin homologue deleted on chromosome 10 (PTEN) and amplification or elevated expression of ErbB-2 are both involved in human breast cancer. To directly test the importance of these genetic events in mammary tumorigenesis, we have assessed whether mammary-specific disruption of PTEN could cooperate with activation of ErbB-2. Transgenic mice expressing ErbB-2 under the transcriptional control of its endogenous promoter (ErbB-2(KI)) were interbred with mice carrying conditional PTEN alleles and an MMTV/Cre transgene. Loss of one or both PTEN alleles resulted in a dramatic acceleration of mammary tumor onset and an increased occurrence of lung metastases in the ErbB-2(KI) strain. Tumor progression in PTEN-deficient/ErbB-2(KI) strains was associated with elevated ErbB-2 protein levels, which were not due to ErbB-2 amplification or to a dramatic increase in ErbB-2 transcripts. Moreover, the PTEN-deficient/ErbB-2(KI)-derived mouse mammary tumors display striking morphologic heterogeneity in comparison with the homogeneous pathology of the ErbB-2(KI) parental strain. Therefore, inactivation of PTEN would not only have a dramatic effect on ErbB-2-induced mammary tumorigenesis but would also lead to the formation of mammary tumors that, in part, display pathologic and molecular features associated with the basal-like subtype of primary human breast cancer.

Figure 1

PTEN-deficient/ErbB-2^KI mammary tumors exhibit accelerated tumor onset and histologic heterogeneity. A, tumor kinetics. Percentages of mice that were tumor-free were plotted against the age when tumors were first palpable. T₅₀, age at which 50% of the females first developed a tumor. n, number of mice analyzed for each strain as indicated. B, four H&E-stained images illustrating the signature histopathology observed in the bigenic animals: a, ErbB-2 type tumor showing the solid nodular growth of intermediate cells surrounded by a peripheral palisade of cells; b, PTEN-type tumor showing a mixture of gland-like spaces and sheets of cells. The cells have larger nuclei with prominent nucleoli and more abundant cytoplasm that is either pink or red; c, adenomyoepithelial tumor with well-formed, widely spaced glands embedded in a cellular matrix; d, adenosquamous carcinoma. The field illustrates a prominent keratin pearl with concentric lamellae of a cellular keratin surrounded by a poorly organized squamous epithelium.

Figure 2

Determination of PTEN and ErbB-2 status in the PTEN-deficient/ErbB-2^KI mouse models. A, a, PTEN LOH. Southern blot analysis of independent tumor DNAs from the indicated mouse strains, which were digested with SacI and probed with a ³²P-labeled PTEN probe. The two bands represent the wild-type and mutant alleles as indicated. b, total protein lysates from independent mammary tumors (20 μg) were subjected to Western blot analyses with a polyclonal anti-PTEN antibody and a monoclonal anti–β-actin antibody. B, a, ErbB-2 was immunoprecipitated from 1 mg of total tumor lysates. Half of the immunoprecipitate was subjected to two immunoblot analyses with a phosphotyrosine antibody (P-Tyr; top) and the other half with an ErbB-2–specific antibody (middle). Equivalent volumes of total lysates (20 μg) were subjected to immunoblot analysis with a monoclonal anti–β-actin antibody for loading controls (bottom). b, Southern blot analysis showing the presence or absence of activated erbB-2 amplification (4.5 kb) with respect to the erbB2 wild-type allele (7.5 kb). c, relative ErbB-2 mRNA levels were quantified by real-time RT-PCR with primers specific to activated ErbB-2 and normalized against GAPDH. **, P < 0.001, versus FVB and ErbB-2^KI (A). A, amplified transgene; NA, nonamplified; IP, immunoprecipitation; IB, immunoblotting.

Figure 3

PTEN-deficient/ErbB-2^KI mammary tumors are enriched in CK6 and SMA. A, immunohistochemical stains illustrating the distribution of CK6 in ErbB-2 type tumors (a), PTEN-type tumors (b), adenomyoepitheliomas (c), and squamous cell carcinoma (d). Note that the cells overlap with CK5 but do not stain the myoepithelial cells identified by anti-SMA and anti-CK5. B, immunohistochemical stains illustrating the distribution of SMA. a to d, same as in A. Note the marked differences in the staining for SMA and CK6.

Figure 4

PTEN^+/−/ErbB-2^KI mammary tumors display histologic features of the basal-like subtype. PTEN-deficient/ErbB-2^KI mammary tumors express the basal marker CK5. Immunohistochemical stains illustrating the distribution of CK5 in ErbB-2 type tumors (A), PTEN-type tumors (B), adenomyoepitheliomas (C), and three squamous cell carcinomas (D). Note that the distribution of CK5-positive cells is very similar to SMA (Fig. 3B).

Figure 5

PTEN^+/−/ErbB-2^KI mammary tumors exhibit molecular characteristics of the human basal-like subtype. A, unsupervised cluster analysis of seven PTEN^+/−/ErbB-2^KI mammary tumors and 181 human breast cancers using 104 genes from the intersection of human and mouse intrinsic gene lists (40). The horizontal dendogram represents the distance between the tumor samples. Red, human basal-like; pink, human HER2; blue, human luminal; purple, PTEN^+/−/ErbB-2^KI. B, expression of genes well defined as being expressed in luminal, basal-like, or HER2 samples. C, correlation between the individual PTEN^+/−/ErbB-2^KI–derived mammary tumors and the centroid of each human breast cancer subtype, using the cross-species intrinsic gene set. Approximately half of the PTEN-deficient/ErbB-2^KI tumors are closely correlated with the human basal subtype, whereas the other half is more closely correlated with the human HER2 subtype. *, PTEN-deficient/ErbB-2^KI mouse model.