Amplification of the neu/erbB-2 oncogene in a mouse model of mammary tumorigenesis

Abstract

The neu (c-erbB-2, Her-2) protooncogene is amplified and overexpressed in 20-30% of human breast cancers. Although transgenic mouse models have illustrated the role of Neu in the induction of mammary tumors, Neu expression in these models is driven by a strong viral promoter of questionable relevance to the human disease. To ascertain whether expression of activated Neu under the control of the endogenous promoter in the mammary gland could induce mammary tumors we have generated mice that conditionally express activated Neu under the transcriptional control of the intact endogenous Neu promoter. Expression of oncogenic neu in the mammary gland resulted in accelerated lobulo-alveolar development and formation of focal mammary tumors after a long latency period. However, expression of activated Neu under the normal transcriptional control of the endogenous promoter was not sufficient for the initiation of mammary carcinogenesis. Strikingly, all mammary tumors bear amplified copies (2-22 copies) of the activated neu allele relative to the wild-type allele and express highly elevated levels of neu transcript and protein. Thus, like human erbB-2-positive breast tumors, mammary tumorigenesis in this mouse model requires the amplification and commensurate elevated expression of the neu gene.

Figure 1

Targeting of the conditionally activated neu allele. (A) A schematic representation of the targeting construct and the genomic allele. The 2.5-kb 5′ arm of homology (SphI to Nar1) and the 8-kb 3′ arm of homology (KpnI to SalI) were used to direct the homologous recombination to the wild-type allele, illustrated by the dashed lines. Exon 1 of the endogenous allele was replaced by a loxP (triangle)-flanked PGK-neomycin-HSV poly(A) (Neo) cassette, followed by the neuNT cDNA and a simian virus 40 poly(A) (NeuNT). The loxP-flanked Neo NeuNT cDNA has replaced exon 1 contained within the Nar1/KpnI fragment. The size of the genomic HindIII (H3)-restricted fragment when detected by a probe 5′ to the site of homologous recombination also is depicted. Sp, SphI; N, Nar1; K, KpnI; Sl, SalI. (B) The recombinant allele containing the loxP-flanked Neo NeuNT cassette in place of exon 1 and the corresponding size of the HindIII restriction fragment detected by the external probe are shown. (C) A representative Southern blot of tail DNA from mice that are wild type (WT) and heterozygous for the knock-in (KI) allele.

Figure 2

Generation of MMTV-Cre transgenics. (A) A schematic of the transgenic construct is shown. The MMTV-long terminal repeat (MMTV-LTR) (gray) was used to drive expression of the cre cDNA (gradient fill). The cre cDNA was followed by the HSV polyadenylation [HSV poly(A)] sequence (black). The 480-bp antisense riboprobe is depicted by the arrow and is directed against the HSV poly(A). (B) The RNase protection on the lactating mammary gland (L), virgin mammary gland (V), and testes (T) for the six lines that passed the transgene shows that expression was occurring in the Cre-2, Cre-3, and Cre-7 lines. (C) An RNase protection screening major organs of the Cre-7 line. Note the expression primarily limited to the mammary gland and male accessory reproductive organs. The pgk riboprobe was included as an internal control for equal RNA loading.

Figure 3

Excision of the loxP- flanked neomycin cassette. (A) The recombinant allele is shown before and after Cre recombinase-mediated excision. Before excision the endogenous promoter will not regulate neuNT expression because of the poly(A) in the neomycin cassette. To detect removal of the neomycin cassette, the neuNT cDNA was used as a probe in an EcoRI (E) digest. The size of this fragment is depicted in both the excised and nonexcised forms. (B) A Southern blot shows that excision of the loxP-flanked Neo is limited to the spleen, salivary, and mammary glands by the presence of a 4-kb band in those samples. The wild-type allele also is detected by the neuNT cDNA probe, resulting in a faint band of 6.0 kb and a stronger band of 25 kb.

Figure 4

Digitized micrographic images of whole-mount (A and B) and immunohistochemical (C–E) analysis of the MMTV-Cre Flneo NeuNT mammary gland and tumors. (A) Whole mount of a virgin MMTV-Cre Flneo NeuNT mammary gland at 9 months. The extensive side branching terminating in lobuolalveolar units should be noted. (B) Whole mount of a virgin wild-type control mammary gland at a similar stage of development illustrating a normal duct with few side branches. (C) Immunohistochemical analysis of the same virgin gland shown in A. Note the absence of anti-Neu staining in the acinar structures, which are not dysplastic. Immunohistochemical analysis on the bigenic (D) and a control MMTV-Neu (E) induced tumor shows high levels of Neu expression in these tumors. The contrast in the cytoplasmic and membrane localization of the stain indicated by the arrows in D and E, respectively should be noted. (Magnifications: A and B, × 40; C–E, ×320.)

Figure 5

Overexpression of activated NeuNT in mammary tumors. (A) Using a probe that spans the transmembrane domain of Neu, an RNase protection reveals that MMTV-Cre Flneo NeuNT tumors (TM1–4) overexpress the activated form of neu. The mammary gland from a virgin MMTV-Cre Flneo NeuNT mouse (5801) was included, as was a positive control known to overexpress an activated form of neu (NDL 1–4). The full-length protected fragment is shown at the band labeled WT Neu. The mutant alleles are labeled NDL Neu and NeuNT, corresponding to their activating mutations. Thirty micrograms of RNA was used, except in the control NDL 1–4 tumor sample, where 20 μg was used. pgk was included as an internal control for equal loading of the samples. Adj. Gl., adjacent mammary gland. (B) Blotting for Neu reveals that only the tumors have elevated levels of Neu. Grb-2 was used to control for equal loading of the 120 μg of protein in each sample. MP, multiparious. (C) After immunoprecipitating for Neu, a blot was probed for phosphorylated tyrosine (P-Tyr), which demonstrated that catalytically active Neu was overexpressed in the tumors. Protein (1.3 mg) was used in this immunoprecipitation.

Figure 6

Amplification of neu in mammary tumors. Southern analysis showing the amplification of the recombinant allele (4.5 kb) in respect to the wild-type allele (7.5 kb) in the MMTV-Cre Flneo NeuNT tumors (TM1–6). The TM1 adjacent gland (TM1 Adj. Gl.) also is shown, illustrating that detectable amplification has not occurred in the normal mammary gland. The samples were restricted with HindIII and probed with the external probe using the same strategy described in Fig. 1. Interestingly, a new band has appeared above the wild-type band in TM2, TM3, and TM5, likely a result of the amplification process. This Southern analysis was subjected to a quantitative PhosphorImager analysis where the increase in neuNT copy number was determined relative to the wild-type allele. The results are shown in Table 1.