(Received for publication, September 28, 1995; and in revised form, December 4, 1995)
From the
Expression of the activated neu oncogene in transgenic mice has been associated with both the synchronous (single-step) and the stochastic (multistep) transformation of the mammary epithelium. To determine the basis for these conflicting observations, additional strains of transgenic mice carrying the activated neu oncogene under the transcriptional control of the mouse mammary tumor virus promoter/enhancer were produced. Activated neu transgene expression, as measured by in situ hybridization and ribonuclease protection assays, resulted in rapid conversion of the normal mammary epithelium to malignant phenotype in three independent strains of mice. Expression of the transgene in male mice led to epithelial hyperplasia of the epididymis and male infertility but not malignancy. These results indicate that tissue context is an important parameter in malignant progression and that expression of appropriate levels of activated neu is sufficient for rapid production of mammary tumors in transgenic mice.
The neu oncogene was initially isolated from chemically induced rat neuroblastomas and was shown to encode a 185-kDa transmembrane protein that bears close homology to epidermal growth factor receptor(1) . Sequence analyses of cDNA clones isolated from these neuroblastomas revealed that activation of neu occurred through a single amino acid change in the transmembrane portion of the protein(2) . In addition, the human homologue of neu oncogene (c-erbB-2) was shown to be amplified and expressed in many human primary breast cancers(3, 4, 5) , and its amplification inversely correlated with patient survival(6, 7) . Although amplification of neu has been consistently observed in a large proportion of human primary human breast cancers, other groups have reported no correlation between amplification status of neu and clinical prognosis(8, 9, 10, 11) . However, overexpression rather than mutation of neu may be the primary mechanism contributing to breast cancer since examination of primary breast cancer biopsies has yet to reveal comparable activating mutations(7, 12) .
Given the limitations of these
studies, a number of laboratories have turned to the transgenic mouse
to directly assess the oncogenic potential of neu in the
mammary epithelium. Initially, this was accomplished by linking the
mouse mammary tumor virus (MMTV) ()promoter/enhancer to a
cDNA encoding the activated neu oncogene and introducing this
fusion gene into the germ line of mice(13) . In one strain of
MMTV/activated neu transgenic mice, the early onset of
transgene expression was initially associated with poor lactation.
Later, tumors involving all mammary glands in every transgenic mouse,
both male and female, appeared synchronously. Histological examination
of these tumors revealed a complete absence of normal mammary
epithelium. Both the simultaneous occurrence and multifocal nature of
these tumors argued that expression of activated neu alone was
sufficient for rapid transformation of the mammary epithelium. By
contrast, another group (14) has reported that in four other
transgenic strains, expression of a similar MMTV/activated neu fusion gene resulted in the stochastic appearance of mammary
tumors. Because these tumors arose next to normal epithelium, which
also expressed the transgene, additional genetic events were thought to
be required for malignant transformation of neu-expressing
cells.
Given the limited number of MMTV/activated neu strains examined and the considerable variation in both the spatial and temporal expressions among different transgenic strains bearing identical transgenes(13, 15) , we decided to produce several other independent transgenic strains carrying MMTV/activated neu fusion genes. We also altered the temporal expression of the activated neu product during mammary epithelial differentiation to establish whether the state of epithelial differentiation could influence the transforming properties of the activated neu transgene. These goals were accomplished by coupling the neu transgene to a truncated version of the MMTV long terminal repeats (LTR) that is transcriptionally active only after the induction of pregnancy(16) . Expression of the activated neu oncogene in three independent transgenic strains carrying either the intact or truncated MMTV/activated neu fusion gene resulted in the synchronous appearance of multifocal mammary tumors involving all mammary glands. In situ hybridization with transgene-specific probes revealed that acquisition of the transformed phenotype was closely associated with high levels of transgene expression. These observations support the contention that expression of activated neu requires few, if any, additional genetic events to transform the primary epithelial cell.
To identify transgenic progeny,
genomic DNA was extracted from a 1.5-cm tail using the protocol
described by Muller et al.(13) . The nucleic acid
pellet was resuspended in 100 µl of distilled water at an
approximate DNA concentration of 1 µg/ml. Fifteen µl of the DNA
solution was digested with 30 units of BamHI for 90 min. After
gel electrophoresis and Southern blot transfer(19) , the Gene
Screen filters (DuPont NEN) were hybridized with a neu cDNA
probe radiolabeled with [-
P]dCTP by random
priming(20) .
Western analyses were conducted as described by Kanner et al.(23) . Briefly, 50 µg of tumor extract was electrophoresed through 8% sodium dodecyl sulfate-polyacrylamide gels and then transferred to nitrocellulose and reacted with 2 µg/ml of neu polyclonal antibody (Ab-3; Oncogene Sciences, Cambridge, MA). The proteins were visualized using the enhanced chemiluminescence (ECL) detection system (Amersham Corp.).
Figure 4:
Photomicrographs of hematoxylin and eosin
stained slides. The organization and distribution of mammary cells are
compared in the following: the mammary fat pad of a nontransgenic FVB
mouse control demonstrating well spaced ducts lined by a single layer
of epithelial cells (87) (A); the mammary fat pad of an
activated neu transgenic animal demonstrating a dilated
collecting duct with adjacent areas of dysplastic cystic alveoli and
solid neoplastic alveolar development (NT163, 173 days of age)
(87
) (B); the solid neoplastic area of the gland in panel B enlarged to demonstrate dysplastic cells filling the
mammary lobules but without growth through the basement membrane,
thereby fulfilling the criteria of carcinoma in situ (350
) (C); and a typical malignant tumor showing
the nuclear pleomorphism and irregular, invasive growth pattern of a
mammary adenocarcinoma found in animals with the activated neu transgene (NT163, 173 days of age) (350
) (D).
Figure 1: Structure of the transgenes. A and B, the unshaded region represents sequences within the pBR322 vector backbone. The striped region corresponds to the MMTV LTR sequences, and the filled region corresponds to an inert fragment derived from the original PA9 clone (17) . The boundaries of the activated neu cDNA are shown by the appropriate arrows. The transgenic strains generated with each of these constructs are shown above the plasmid diagrams. Relevant restriction endonuclease sites are also identified in the figure.
The tissue specificity of transgene expression for female and male transgene carriers derived from the CRNT128 line was assessed by RNase protection assay. Ten µg of total RNA derived from a variety of organs were hybridized with a transgene-specific probe. The antisense probe used in this analysis was directed to the SV40 component of the transgene and yields a 784-nucleotide protected fragment (see Fig. 2B). As shown in Fig. 2, expression of the transgene was detected primarily in the mammary tumors of female carriers and in mammary tumors, salivary glands, and epididymides of male carriers. After longer exposure of the autoradiogram, lower levels of transgene expression were also noted in a variety of other organs including brain, spleen, thymus, lung, and kidney (data not shown).
Figure 2: Tissue specificity of transgene expression. RNA transcripts corresponding to the pMMTV-neu CRNT transgene in various organs of the CRNT128 transgenic strain. The antisense probe used in this analysis is derived from the plasmid pASV (13) and is shown in the map below the autoradiograph. Also shown is the map of the SV40-derived portion of the transgene. The origins of the sequences in the transgene and the probe are indicated in the figure. Numbers in the diagrams refer to the SV40 nucleotide sequence. Expression of the MMTV/neu transgene yields a 784-nucleotide protected fragment and is indicated by the arrow. The female carrier (CRNT439) is a multparous individual at 135 days of age, and the male carrier (CRNT440) was sacrificed at 220 days of age.
Two other female founder strains (NT163 and CRNT572) were also assessed for their capacity to express the transgene. As shown in Table 1, NT163 and CRNT572 female founder animals expressed high levels of the transgene in the mammary gland. In contrast to the CRNT128 strain, neither of these founder animals expressed the transgene in any other tissue examined. The remaining male NT222 founder animal failed to express the transgene.
To ensure that these transcripts encoded Neu protein, Western analyses were conducted on tumor protein extracts using a specific polyclonal antisera (Ab-3; Oncogene Sciences). As illustrated in Fig. 3, a prominent 185-kDa species comigrating with the NIH 3T3 cell line expressing activated neu (SPBNT-5) was observed in tumor extracts derived from CRNT572 and CRNT128 animals. Lower amounts of Neu were detected in normal mammary glands derived from nontransgenic FVB females after longer exposure of the autoradiogram. These results indicated that the MMTV/activated neu strains express elevated levels of the activated Neu product in the mammary epithelium.
Figure 3: MMTV/activated neu tumors express elevated levels of the activated Neu protein. Western analyses of tumor extracts (CRNT572, 115 days of age, and CRNT128, 132 days of age) or normal mammary gland extracts derived from nontransgenic FVB/N female mouse reacted with a neu-specific antibody (Ab-3, Oncogene Science). Also included are extracts derived from NIH 3T3 cells (SPBNT-5) expressing the activated neu product under SV40 transcriptional control and the parental NIH 3T3 cell. The 185-kDa Neu protein is illustrated by an arrow.
Figure 5:
In situ hybridization of
MMTV/activated neu mammary tissue. A,
transillumination photograph of a section of mammary tissue derived
from a multiparous CRNT128 female (200) stained with hematoxylin
and eosin. B, sequential section of CRNT128 mammary tissue
hybridized to [
-
S]UTP labeled antisense
SV40 probe (see Fig. 2) (200
). The tumorous tissue
covered with silver grains is the same mammary duct as in panel
A, and this section is immediately adjacent to that shown in panel A. Note the lack of hybridization over the normal
epithelium (see arrow). C, sequential section of
CRNT128 (200
) tissue hybridized to a labeled sense control
probe. No significant hybridization was
observed.
Although we were unsuccessful in propagating the CRNT572 strain, female transgenic progenies were obtained from the CRNT128 founder animal. These transgene carriers also developed multifocal mammary tumors that involved the entire mammary epithelium following weaning of their first litters. Consistent with this histological evaluation, these tumors grew when transplanted into syngeneic recipients. Both the rapid kinetics by which these tumors appear and their global involvement suggest that expression of activated neu in the mammary gland results in rapid conversion of the epithelium to the malignant phenotype.
Male transgenic carriers derived from the CRNT128 strain also developed mammary tumors with 100% penetrance, albeit with delayed kinetics (Table 1). In addition, these males had bilateral epididymal hypertrophy and were infertile. Consistent with previous observations (13, 14) , these epididymides exhibited extensive epithelial hyperplasia (data not shown).
Here, we present further evidence that mammary gland-specific expression of the transforming allele of the neu oncogene results in the induction of multifocal mammary adenocarcinomas. Three independently derived transgenic lines expressing activated neu eventually developed multifocal adenocarcinomas. Multiple mammary tumors expressing high levels of the transgene arose synchronously in both transgenic male and female carriers. Histological evaluation of tissue adjacent to tumors revealed only a sparse amount of normal tissue. The normal tissue adjacent to the tumors did not express activated neu as assessed by in situ hybridization analyses (Fig. 5B). Both the rapid tumor kinetics and multifocal tumor phenotype exhibited by these transgenic strains supports the hypothesis that expression of activated neu leads to rapid and uniform transformation of the mammary epithelium.
Interestingly, two of the founder strains derived by microinjection of the activated neu construct in which the MMTV LTR was truncated at the ClaI site (CRNT128 and CRNT572), did not exhibit mammary epithelial abnormalities until after weaning of their first litter. While we were not able to assess the levels of transgene expression in these strains prior to tumor development, previous experience with transgenic mice bearing a truncated MMTV LTR driving the expression of the murine int-2 gene indicated that transgene expression is not detectable in virgin mammary epithelium and does not commence until the animals have become pregnant(16) . Consistent with these observations, it has recently been reported that cis-acting sequences that are removed in these truncated LTR are required for optimal hormonal response by the MMTV LTR promoter/enhancer(27) . By contrast, transgenic mice possessing an intact MMTV LTR driving the expression of the activated neu gene express the transgene early in mammary gland development and exhibit early onset of mammary epithelial abnormalities (NT163; (13) ).
Other strains of MMTV/activated neu mice develop tumors in a stochastic fashion(13, 14) . In the former report(13) , the stochastic appearance of tumors correlated with increased expression of activated neu (as assessed by in situ hybridization) in tumor tissue by comparison with the adjacent normal mammary epithelium (NK line). By contrast, Bouchard et al. (14) reported that by Northern blot analyses, the normal epithelium derived from several transgenic strains expressed comparable levels of activated neu transcript in adjacent normal and tumor tissue. Although unpublished observations made by the authors suggested that expression of activated neu in the normal epithelium could also be detected at the level of the single cell by in situ hybridization, it was not clear whether quantitative differences of expression were observed between normal and transformed tissue. Conceivably, the differences between the observations made here and elsewhere (13) and those made by Bouchard et al. (14) are simply due to variation in the levels of expression of the active neu tyrosine kinase activity in the mammary epithelium of the different MMTV neu strains. Moreover, Lucchini et al.(28) have recently reported multifocal mammary tumors arising in transgenic mice carrying an identical MMTV/activated neu transgene. Direct comparison of the levels of neu kinase activity in the mammary epithelium of the stochastic and rapid tumor progression strains should allow this point to be addressed.
The behavior of the activated neu oncogene in the mammary epithelium contrasts with the observations made by a number of laboratories with transgenic mice carrying other activated oncogenes. For example, expression of c-myc, c-fos, or v-Ha-ras, in a number of tissue types requires the complementary action of other genes to convert the primary cell to the malignant phenotype(17, 29, 30, 31, 32) . It has been suggested that the rapid tumor progression phenotype observed in the original TG.NF MMTV/activated neu strain might be the consequence of the integration site(33) . However, three additional strains develop similar phenotypes. This strongly implies that the rapid tumor progression observed is the direct consequence of elevated expression of activated neu.
The potent tissue-specific transforming activity of activated neu in the mammary epithelium differs from the observations made with transgenic mice expressing the unactivated neu allele in the mammary epithelium(34) . In those studies, focal mammary tumors appeared next to transgene expressing normal epithelium only after long onset(34) . Perhaps the level of neu tyrosine kinase activity in the MMTV/unactivated neu lines is below a critical threshold required to convert the cell to the malignant phenotype, and cellular transformation occurs only when this threshold is exceeded. Consistent with this hypothesis, both in vitro kinase assays with neu-specific antibodies on protein extracts derived from the MMTV/unactivated neu tumors demonstrate that the neu-induced tumors express a much higher neu-associated tyrosine kinase activity by comparison with the adjacent mammary epithelium(34) . The levels of active neu tyrosine kinase may also be important in the genesis of human breast cancer since a large proportion of human breast tumors express relatively high level of the Neu protein by comparison with the adjacent normal mammary epithelium(6, 7) .
Expression of the polyoma virus middle T associated tyrosine kinase in the mammary epithelium also results in the rapid conversion of the entire mammary epithelium to the malignant phenotype(35) . Conceivably, the powerful tissue-specific transforming activities of both the activated neu and polyoma virus middle T oncogene might be explained by the fact that their tyrosine kinase activities are refractory to normal cellular regulation. Thus, as a consequence, these activated oncogenes can constitutively signal cell proliferation. Whether both of these potent tyrosine kinases signal cellular proliferation through a common signal transduction pathway in the mammary epithelium awaits further analyses.