There has recently been great emphasis on somatic hotspot mutations in cancer as these invariant mutations represent ideal candidates for targeted therapies. The discovery of the AKT1 E17K mutation in human cancers, albeit at low frequency, is of particular interest due to Akt’s known importance in acting as a central mediator of signal transduction pathways that are altered in human malignancies. Although many human cancer cell lines exist with somatic mutations that represent the spectra and types found in primary human tissue samples, currently, there are no available cell lines with a naturally occurring AKT1 E17K mutation, though Mills and colleagues recently reported two human melanoma cell lines with AKT3 E17K mutations (Davies et al., 2008
). To further study the effects of AKT1 E17K when expressed in a physiologic manner, we have created an isogenic set of nontumorigenic human breast epithelial cell lines with and without the AKT1 E17K mutation. The use of nontumorigenic cell lines has several advantages over human cancer cell lines. First, the MCF10A cell line is genetically stable as assessed by chromosomal instability analysis with FISH (Yoon et al., 2002
), microsatellite and karyotype analyses (unpublished data), sequencing of p53 (Abukhdeir et al., 2006
; Konishi et al., 2007a
), and copy number variation analysis (manuscript submitted). This provides stronger support that gene targeted knock in clones derived from MCF10A are truly isogenic. Second, the ability to study the effects of a single oncogenic mutation can be easily observed using the various assays we have employed in past and present studies. Indeed, our previous work with mutant PIK3CA knock in clones emphasizes this fact. Third, knock in of AKT1 E17K into breast cancer cell lines would likely not yield any definitive data, as most breast cancer cell lines would be expected to already have activation of the PI3K pathway, as evidenced by the high frequency of PIK3CA mutations found in these cell lines (Bachman et al., 2004
). Thus, for these reasons, MCF10A cells enable us to definitively address the phenotypic consequences of introducing oncogenic mutations via gene targeting.
Somewhat surprisingly, knock in of mutant AKT1 failed to transform cells in vitro, in contrast to observations with overexpressed mutant AKT1 (Carpten et al., 2007
). This finding is similar to what we and others have observed with knock in of mutant KRAS and may reflect differences between knock in and transgenic overexpression as well as differences between cell types in their susceptibility to transformation (Arena et al., 2007b
; Konishi et al., 2007a
). Although only biochemical evidence of Akt activation was seen as evidenced by a slight increase in Akt phosphorylation under conditions of no EGF, there are limitations to the present study. Notably, it should be pointed out that our results are derived from a single cell line, MCF10A. Therefore the effects of AKT1 E17K with MCF10A cells, which are ER negative, may be masking a true phenotype since the AKT1 E17K mutation has only been seen in ER positive breast cancers. Arguing against this is the fact that knock in of mutant PIK3CA has dramatic effects in vitro, despite the fact that the majority of PIK3CA oncogenic mutations are also found in ER positive breast tumors. In addition, physiologic expression of ER in AKT1 E17K cells did not demonstrate any overt difference compared to ER expressing MCF10A cells with wild type AKT1. Thus, it may be that AKT1 E17K requires additional genetic events in other pathways to realize its full oncogenic potential. In support of this view is the fact that mutant PIK3CA knock in cells were shown to unexpectedly activate the MAP kinase pathway, a phenotype not observed with AKT1 E17K knock in cell lines. Thus, mutant PIK3CA activates multiple pathways simultaneously likely accounting for its transforming properties in vitro. Engineering additional oncogenic events within the AKT1 E17K knock in cell lines will allow us to address this readily testable hypothesis.
Given the lack of naturally occurring AKT1 E17K cell lines, we feel that these knock in clones will be a valuable resource for understanding the biological and clinical implications of mutant AKT1, as well as other PI3K pathway mutations and alterations found in human cancers. Although the prognostic implications of PI3K pathway mutations in breast cancer are controversial and still emerging, there has been a suggestion from the available data that AKT1 mutant cancers may have a more favorable prognosis (Stemke-Hale et al., 2008
). Certainly as a class effect, AKT1 mutant cancers largely belong to the more-favorable hormone receptor-positive subset of breast cancer (Carpten et al., 2007
; Stemke-Hale et al., 2008
). A previous study of primary human breast cancers found that cancers with AKT1 mutations or PTEN mutations/loss, displayed high levels of in vivo Akt, mTOR, and p70S6 Kinase phosphorylation, as compared to tumors with PIK3CA mutations (Stemke-Hale et al., 2008
). This result was surprising given the current body of knowledge of the PI3K pathway, and in light of in vitro data from traditional oncogene overexpression experiments as well as our own PIK3CA knock in cells. In contrast, our data suggest that the Akt1 mutant is a weaker activator of the PI3K pathway than mutant PIK3CA. However, our data do support the notion that not all PI3K pathway mutations are equivalent, a concept that has recently emerged in breast cancers as Perez-Tenorio et al. and Stemke-Hale et al. have demonstrated PIK3CA mutations with concordant PTEN loss (Perez-Tenorio et al., 2007
; Stemke-Hale et al., 2008
). During review of this manuscript, Fine et al. and Vasudevan et al. presented studies suggesting that differences in P-REX2a and PDK-1 activity may account for the variability seen in PI3K pathway signaling depending on the genetic lesions present within cancer cells (Fine et al., 2009
; Vasudevan et al., 2009
). In our isogenic system, PIK3CA mutations appear to generate a stronger proliferative signal than the AKT1 E17K mutation, conferring growth factor independence and altering the threshold for mTOR activation. In addition, mutant PIK3CA stimulates mTOR-independent growth even under EGF-free conditions, suggesting that mutant PIK3CA provides a stronger oncogenic signal, either via quantitative increases in Akt1 phosphorylation, activation of other Akt isoforms, crosstalk with the MAP Kinase pathway, and/or other Akt1-independent mechanisms.
The results presented here also have potential clinical implications involving pathway inhibitors that are being rapidly studied in clinical trials. In keeping with the quantitative and qualitative differences in signaling, PIK3CA mutations but not AKT1 E17K, increased sensitivity to the PI3K inhibitor LY294002 and the mTOR inhibitor rapamycin under appropriate growth conditions. It will be of interest to determine in larger clinical studies whether these mutations predict for differential sensitivity to various PI3K, MAP Kinase and mTOR inhibitors currently in clinical trials.