Our results reveal that NEDD9 is a cancer cell-intrinsic protein that contributes to mammary tumor development in the MMTV-PyVmT mouse model. In reference to other genetic modifiers that have been tested in the MMTV-PyVmT model, the effect of Nedd9
null status is not as dramatic as that seen with deletion of its binding partner FAK (40
), but comparable to or greater than that observed in mice null for the important breast cancer signaling proteins PAR1 (41
), MEKK1 (42
), CD44 (43
), and others. These results suggest that Nedd9
expression may be an important modulator of breast cancer incidence in humans.
Although our data are compatible with the pro-oncogenic role identified for Nedd9
overexpression in glioblastoma, melanoma, and lung cancers (14
), they differ in important ways. In particular, lack of Nedd9
reduces proliferation from very early stages of tumor development, rather than acting at the point of invasion and metastasis, as suggested by other studies. This reduced rate of tumor onset likely reflects an important role for Nedd9
in maintaining activation not only of its direct partners, FAK and SRC, but also the key Ras effectors AKT, SHCA, and ERK1/2. The striking correlation of the degree to which these pathways are activated with the time of tumor detection argues that supporting this activation is a critical early action of NEDD9. In this context, the novel interaction between NEDD9 and SHC we report here provides a direct means by which NEDD9 supports Ras/Raf pathway activation. Together with the fact that an earlier study has demonstrated that NEDD9-dependent tumor promotion has previously been shown to be partly dependent on Ras/Raf pathway activation (14
), these data suggest close coordination rather than strict unidirectional epistasis explains the relation of NEDD9 and Ras signaling in tumor growth.
Given our results, the opposite suggestion by Minn et al, that down-regulation of Nedd9
is part of a signature for breast cancer metastasis (20
), may reflect the fact that Nedd9
is required at early stages in the tumor process, but downregulated after metastasis. Indeed, MMTV-PyVmT;Nedd9-/-
mammary tumor cells lines are less migratory than MMTV-PyVmT;Nedd9+/+
cells, in agreement with our earlier observations that NEDD9 positively regulates cell migration and matrix metalloproteinase expression in transformed breast adenocarcinoma cells (12
), but in opposition to the report that NEDD9 negatively regulates migration in normal mammary epithelial cells (19
). Alternatively, the different findings may reflect a distinct feature of the TGF-β model employed in the Minn study. NEDD9 directly binds and influences the function of TGF-β effectors including SMAD3 (44
) and other SMADs (45
), making it plausible that NEDD9 might act differently in a TGF-β—driven tumor. Epithelial-mesenchymal transition (EMT) is an important consequence of increased TGF-β signaling in tumor metastasis. In support of the present study, we have examined a number of hallmarks of TGF-β-induced EMT in Nedd9-/-
tumors and tumor-derived cell lines, including upregulation of vimentin and downregulation of E-cadherin. No significant differences were detected in the MMTV-PyVmT model (unpublished results). Finally, the difference between our data and those reported by Minn may reflect a difference observed at the mRNA level that does not affect NEDD9 protein levels, as we and others have shown that NEDD9 undergoes significant post-translational regulation during cell division and cell death (9
). More study of these issues is required.
Interestingly, our data indicate that continued expression of the NEDD9 paralog BCAR1/p130Cas is unable to compensate for Nedd9
null status, even though transgenic overexpression of p130Cas activates SRC and AKT and promotes mammary tumor progression in a mouse model (47
). A possible explanation lies in the typically non-dynamic expression level of p130Cas under different growth conditions. In contrast, NEDD9 is strikingly up-regulated in highly proliferating cells (17
) and may be required to support the proliferation of such cells. Levels of p130Cas were constant in tumors derived from Nedd9-/-
mice (), suggesting that even under strong selective conditions, upregulation of this protein was either non-tumor promoting or impossible in a Nedd9-/-
background. Another possibility is that Nedd9
induces additional signaling pathways that are not influenced by p130Cas, which contribute specifically to tumor growth. In interesting contrast, p130Cas-/-
status is embryonally lethal (day 11.5) (48
), while Nedd9-/-
status is not associated with known pre-natal defects, emphasizing the different requirements for signaling proteins in tumors versus normal development.
Large-scale studies of the breast cancer and other cancer genomes have begun to unearth a large number of potential pro-oncogenic mutations targeting genes previously not known to be important for cancer. These genes are not identical in different cancer types, or in a given type of cancer arising in different individuals. In parallel, systems biology studies of large biological networks (49
) have suggested that activity of cancer signaling pathways can be enhanced by inappropriate activation of proteins acting at various points within a pathway, and that such activation can arise not only from mutations, but also from epigenetic, post-transcriptional, or post-translational events that alter the signaling landscape of a cell. Together, these findings suggest that proteins that broadly influence pathway activation status may have important actions in conditioning cancer initiation and progression. Since NEDD9 is not only coupled to the cell adhesion and migration machinery (9
), but also contributes to normal mitotic progression (50
) and mediates proliferative and survival signaling (46
), we believe it is likely that the dynamic upregulation of NEDD9 may represent convenient means for cancer signaling networks to coordinately activate multiple pathways useful for tumor growth.