The PyV MT oncogene provides an excellent model to allow identification of the important signaling pathways involved in mammary tumorigenesis. Given the potent transforming activity of the PyV MT oncogene in the mammary epithelia of transgenic mice (19
), we sought to exploit this system to elucidate the relative contributions of the PyV MT coupled signaling pathways in PyV MT-mediated mammary tumorigenesis. To elucidate the roles of Shc and PI-3′ kinase in PyV MT-mediated tumorigenesis, we have derived transgenic mice that express mutant PyV MT oncogenes defective in their capacity to bind to these signaling proteins. Consistent with previous studies of MMTV-driven transgenes, analyses of the tissue-specific patterns of expression of both PyV mutant MT cDNAs revealed that the primary site of expression was the mammary gland, with secondary sites noted in the male reproductive tissues and salivary glands (Fig. and Tables and ). In both sets of transgenic strains, the initial phenotype exhibited by female mice was the inability to lactate. Whole-mount analyses of the mammary epithelia of virgin females from either the MT-Y250F or MT-Y315/322F strains showed that they displayed extensive epithelial hyperplasias that were histologically distinct (Fig. ).
The observation that the mammary epithelial hyperplasias derived from the MT-Y315/322F strains exhibit elevated rates of apoptosis (Fig. A) suggests that activation of PI-3′ kinase plays a critical role in promoting cell survival. Moreover, expression of a dominant negative inhibitor of PI-3′ kinase in mammary tumor cells expressing wild-type PyV MT resulted in the rapid induction of programmed cell death (Fig. C). These observations are consistent with the emerging concept that activation of the PI-3′ kinase signaling pathway is involved in the regulation of apoptosis in a number of different cell types. For example, abrogation of growth factor-mediated activation of the PI-3′ kinase signaling pathway through administration of either specific PI-3′ kinase inhibitors or expression of mutant growth factor receptors decoupled from the PI-3′ kinase in a number cell types results in the induction of apoptotic cell death (23
). More recently, it has been demonstrated that activation of the PI-3′ kinase blocks c-Myc- or UVB-induced apoptosis in fibroblasts (23
). In addition, recent studies demonstrate that activation of the Akt kinase, which is immediately downstream of the PI-3′ kinase (15
), can also prevent c-Myc-induced apoptosis (23
). Taken together with our observations, these data suggest that activation of the PI-3′ kinase signaling pathway may be required to promote cell survival.
Although the initial phenotypes observed with the PyV MT mutant strains were mammary epithelial hyperplasias, females derived from several independent strains developed mammary tumors with 100% penetrance. However, in contrast to parental wild-type PyV MT strains, which developed multifocal mammary tumors, the tumors in the mutant PyV MT mutant strains were focal in origin, arising adjacent to hyperplastic mammary tissue (Fig. and ). Moreover, the tumors arose with delayed onset in comparison to the case for wild-type strains (Fig. ). Measurement of associated tyrosine kinase activity of mutant PyV MT species isolated from mammary hyperplasias or tumors revealed that association with and consequent activation of c-Src was unaffected compared to that for wild-type PyV MT (Fig. ). Thus, the increased latency with which mammary tumors arise in both PyV MT mutant transgenic strains is not due to an inability to complex with and activate the Src family of tyrosine kinases. Although recent studies have suggested that tyrosine 322 in PyV MT is also capable of binding PLCγ in certain cell types (47
), we and others have failed to detect evidence of comparable complexes of murine PyV MT and PLCγ in tumors induced by the PyV MT oncogene (3
). Although we cannot formally preclude the potential involvement of other PTB domain- or SH2-containing signaling molecules in these mutant PyV phenotypes, the failure of PyV MT to bind the corresponding associated cellular protein (i.e., Shc in MT-Y250F mice and PI-3′ kinase in MT-Y315/322F transgenic mice) is likely responsible for the delayed onset of mammary tumors observed in these mutant PyV MT strains.
The data generated suggest that expression of the PyV MT mutants is not sufficient for mammary tumorigenesis and requires additional genetic events. In contrast to these observations, it has recently been reported that inactivation of the Shc binding site does not interfere with the ability of PyV to induce a variety of tumors in animals (4
). More recently, another independent group reported that an Shc binding site PyV mutant displayed an altered tumor spectrum (56
). However, in both these reports the incidence of mammary tumors was unaffected by the introduction of the Shc binding site mutant. The difference between these observations and ours may be due to the fact that in the other studies a functional PyV large T antigen is also expressed (4
) and may compensate for inability of PyV MT to associate with and activate Shc. Alternatively, unlike that of other tissues, transformation of the mammary epithelial cell may require activation of Shc. Indeed, it has been demonstrated that the requirement for Src in PyV MT-mediated tumorigenesis is highly dependent on the tissue context (20
). Whatever the explanation, our observations strongly suggest that activation of Shc is required for PyV MT-mediated mammary tumorigenesis.
One important clue to the nature of these additional events derives from observations that in 7% of the tumors arising in the MT-Y250F strains, the mutant PyV MT had reacquired the capacity to bind Shc through somatic mutations occurring in the transgene. DNA sequence analyses of these alterations revealed that restoration of Shc binding can occur either through simple point mutation at the substituted phenylalanine codon or through an in-frame deletion of 18 bp occurring immediately downstream of the phenylalanine codon (Fig. B). Another interesting aspect of these reversions is that they occurred at a much higher frequency in lung metastases (Fig. B). Indeed, of the 11 metastatic lesions examined, 36% possessed either reversion event, suggesting that there is a selective pressure for restoration of Shc binding during PyV MT-mediated metastatic progression. Because Shc can recruit the Grb-2–Sos–Ras complex to PyV MT (14
), the selection for Shc binding during metastasis reflects an essential role for activation of the Ras pathway during metastatic progression. In this regard, we have recently demonstrated that ectopic expression of Grb-2 in the mammary epithelium of the MT-Y250F strains can result in dramatic acceleration of growth of mammary tumors (40
). Alternatively, the requirement for Shc binding may reflect its ability to recruit other signaling pathways. Indeed, it has recently been demonstrated that tyrosine phosphorylation of tyrosine residues 239 and 240 in Shc is involved in the generation of an antiapoptotic signal involving activation of the Myc transcription factor (17
). Determination of the prevalent signaling pathways involved in this phenotype will provide important insight into the molecular basis of metastatic progression.
In contrast to those from MT-Y250F transgenic animals, tumors derived from the MT-Y315/322F strains failed to demonstrate reversion at either tyrosine phosphorylation site in the transgene responsible for binding the p85 subunit of the PI-3′ kinase. It is conceivable that tumorigenesis in these mutant strains requires recruitment of PI-3′ kinase but that this occurs in an indirect manner. Activation of growth factor receptors in an autocrine or paracrine manner during tumor progression in these strains could lead to the indirect recruitment of the PI-3′ kinase signaling pathway. In this regard, we have demonstrated that the ErbB-3 and ErbB-2 EGFR family member which specifically couples to the PI-3′ kinase and Shc signaling pathways (39
) is upregulated during the transition of mammary epithelial hyperplasias to the tumor phenotype in both these mutant strains (Fig. ). Consistent with these observations, other studies have demonstrated that recruitment of PI-3′ kinase by activated growth factors such as platelet-derived growth factor and insulin growth factor-2 is required to provide a survival signal to prevent cells from undergoing apoptosis (35
). However, the identification of potential signaling pathways that may be involved in tumor progression in these mutant MT strains awaits further investigation.
Activation of PI-3′ kinase-coupled growth factor receptors during tumor progression in these various transgenic strains may reflect the requirement for the generation of an antiapoptotic signal (Fig. ). Indeed, in the insulin promoter-SV40 large T antigen transgenic mice there is suppression of apoptotic cell death during tumor progression (35
). In addition to suppressing apoptotic cell death, activation of the PI-3′ kinase by PyV MT may influence other important steps involved in tumor progression. In this regard, we and our collaborators have recently demonstrated that mammary tumor cells expressing the MT-Y315/322F mutant showed a marked decrease in the induction of angiogenic blood supply that was further correlated with a 10-fold decrease in metastatic potential compared to that for a mammary tumor cell line expressing wild-type PyV MT (9
). Consistent with these observations, we have found that only 36% of tumor-bearing female animals carrying the mutant MT-Y315/322F develop metastatic lesions (53a
), whereas 100% of those carrying the parental wild-type MT strains develop lung mestastases (19
). More recently, a transforming homolog of the PI-3′ kinase has been described, suggesting that activation of the PI-3′ kinase can directly result in the induction of tumors (7
). Taken together, these observations suggest that recruitment of the PI-3′ kinase by PyV MT may play multiple roles in tumor progression.
Given that activation of Ras appears to be a common component of receptor tyrosine kinase signaling cascades, the observation that the PyV MT mutant defective in its capacity to associate with Shc–Grb-2 is also impaired in its ability to induce tumors suggests that direct recruitment of the Ras pathway is critical for tumor progression. Indeed, it has been demonstrated that PyV MT requires Ras function to transform fibroblasts in vitro (22
). However, despite the defect in tumor induction, expression of the MT-Y250F mutant is still capable of inducing extensive epithelial hyperplasias. It is conceivable that the mitogenic response of mammary epithelial cells to the PyV MT-Y250F mutant reflects its capacity to signal in a Ras-independent fashion. Indeed, the Raf serine kinase can be activated by c-Src through a Ras-independent mechanism (46
). Alternatively, the MT-Y250F mutant may stimulate cell proliferation by indirectly activating the Ras signaling pathway.
The studies described above have important implications in elucidating the molecular basis for the oncogene-mediated induction of metastatic mammary tumors. Our studies suggest that recruitment of both Shc and PI-3′ kinase signaling molecules to PyV MT plays a critical role in the transition from mammary hyperplasias to metastatic mammary tumors. These observations further argue that mammary tumorigenesis in these transgenic mouse models is dependent on the concerted activation of both cell-proliferative and survival-coupled pathways. The results of this study also have general implications with respect to the roles that growth factor receptor-coupled signal transduction pathways play in tumor progression. Because tumor progression in these mutant PyV MT strains is dependent on the genetic events that complement the defects in these signaling pathways, these transgenic tumor models can serve as a powerful genetic system to dissect the importance of various signaling molecules in mammary tumor progression. Further studies with these mutant PyV MT transgenic mice will provide important insight into the nature of events involved in the progression of mammary epithelial hyperplasias to metastatic mammary tumors.