The bulk of oncogenic mutations of p110α cluster in two hot spots: in an acid cluster in the helical domain (E542, E545 and Q546) and in the C-terminus of the kinase domain (H1047). Both helical domain and kinase domain mutants of p110α cause increased lipid kinase activity in cells, but by different mechanisms (2
). The distinct mechanisms by which the helical domain and kinase domain mutants activate p85/p110 dimers might lead to distinct patterns of PI[3,4,5]P3 production in cells.
When we directly compared the effect of p110α helical domain versus kinase domain mutations in cell lines with otherwise identical genetic backgrounds, expression of the helical domain mutation lead to increased chemotaxis in vitro, and increased activity during in vivo metastasis assays. Cells expressing the helical domain mutant showed a small but significant increase in tumor growth rate as compared to cells expressing the kinase domain mutant; both mutant cell lines produced tumors much faster than cells expressing wild type p110α. However, the presence of tumor cells in the blood of animals with helical domain mutants was nearly 3-fold higher than in animals with kinase domain tumors. Similarly, metastasis to the lung was much faster upon tail vein injection of helical domain opposed to kinase domain cells. These differences did not correspond to marked differences in Akt activation in the two cell lines, consistent with the idea that site specific PI 3-kinase activity might be important in defining the phenotype of these mutants in vivo.
Several previous studies have compared signaling by overexpressed helical domain and kinase domain mutants of p110α. Overexpression of either N-terminally tagged mutant in NIH 3T3 cells or mammary epithelial cells led to increased Akt activation, growth in soft agar, disruption of mammary acinar morphogenesis in 3D culture, and tumor formation in a xenograft model(7
). Similar results were seen in Ba/F3 mouse pro-B cells (10
). In these studies, the phenotype produced by overexpression of either the helical domain or kinase domain mutants were similar. A concern in some of these studies is the use of N-terminal epitope tags, which stabilize p110α independently of binding to p85α and might obscure differences between the mutants (24
). The C-terminal tag used in this study does not stabilize p110α, although it is possible that it could still have some unforeseen effect on signaling in vivo
. Alternatively, Samuels et al
. used a genetic strategy to silence the expression of helical domain or kinase domain mutants in human cell lines expressing a single copy of the mutant allele (DLD1 and HCT116 cells, respectively) (12
). The helical and kinase domain mutant lines both led to increased levels of tumor formation and metastasis in a xenograft model, as compared to the cells in which the mutant allele was ablated. However, a direct comparison of the in vivo
phenotypes of the helical versus
kinase domain mutants was difficult, given that different cells lines were used. In a more recent study, knock-in of helical versus
kinase domain mutations led to the activation of a similar range of downstream activators in MCF-10A cells (25
). We also failed to see differences between the helical domain and kinase domain mutants in responses such as protrusion or motility in the Boyden chamber, but we did see differences in complex behaviors such as in vitro
chemotaxis in an EGF gradient and metastatic behavior in vivo
In contrast, clear differences between the helical domain and kinase domain mutants were seen in studies using retroviral expression of untagged p110α mutants in chicken fibroblasts; this method depends on endogenous p85 for stabilization of p110α, and should not lead to overexpression. Although both mutations lead to increased PI 3-kinase activity, expression of the kinase domain mutation led to more robust Akt activation and foci formation in chicken fibroblasts, and tumor production in the chicken embryo chorioallantoic membrane assay (11
). These studies are not consistent with our data, which show similar rates of tumor growth but increased metastatic behavior for the helical domain relative to the kinase domain mutants. However, multiple differences in the systems used (stromal factors, interactions with macrophages and other inflammatory cells, cell type and species-specific differences) could explain the different results.
Our data would suggest that in human breast cancer cells identical in other respects, the presence of helical domain mutants of p110α would predict a more aggressive metastatic phenotype. The clinical evidence in support of this hypothesis is mixed. A number of studies have suggested that mutations in p110α correlate with more severe disease in breast, colon, and endometrial cancer, but these studies did not separately compare helical domain versus
kinase domain mutants (27
). Helical domain mutants were found to predominate, relative to kinase domain mutations, in aggressive lobular breast carcinoma (33
), and were found to be independently associated with poor prognosis in these tumors (34
). Other studies found an association of kinase domain mutants, but not helical domains, with poor prognosis in breast cancer and endometrial cancer (35
). Finally, as mentioned earlier, kinase domain mutants were found at a higher rate than helical or C2 domain mutants (67% versus
19%) in tumors with a PTEN loss gene signature, which correlates with poor prognosis in a number of tumor types (13
The relative effect of different p110α mutations on patient prognosis is likely to be complex, and will undoubtedly also be influenced by the presence of other oncogenic mutations in a given tumor. For example, recent data from Vasudevan et al. suggest that in some breast cancer lines expressing helical domain mutants of p110α, adaptations have occurred such that activation of Akt is minimal, and anchorage independent growth relies on activation of PDK1 and SGK3 (39
). In MDA-MB-231 cells, the Ras/Erk pathway is activated by mutations in both K-Ras and B-Raf (http://www.sanger.ac.uk/perl/genetics/CGP/cosmic?action=sample&id=905960
). K-Ras associates with distinct non-raft regions of the plasma membrane (6
). Given that helical domain mutants of p110α show increased activity in the presence of oncogenic Ras, whereas kinase domain mutants do not (2
), the presence of constitutively active K-Ras in MDA-MB-231 cells could lead to a localized activation of helical domain that would not occur in cells expressing kinase domain mutants of p85/p110α. It will be important to determine how such differential targeting regulates chemotaxis and metastatic behavior in breast cancer lines expressing mutant p110α.