Characterization of mutations in cancer cells provides insights into tumorigenesis and reveals candidates for targeted therapeutics. Most previous studies of genetic aberrations in EC are small with analysis of a limited number of candidate genes. The functional relevance of these alterations in EC is also obscure, hampering the development and implementation of pathway-targeted therapy. Therefore, we performed an integrated analysis to determine the effects of a broad set of candidate mutations on downstream signaling in a large sample set. In agreement with the model of type 1 EC (endometrioid/mucinous) (7
), the EEC in our dataset are characterized by high frequencies of aberrations in the PI3K pathway, KRAS
, and CTNNB1
, whereas TP53
mutations are more frequent in grade 3 EEC. The mixed endometrioid/serous histotype is genetically similar to EEC, suggesting that the endometrioid component is dominant at least in terms of mutational status. MMMT is poorly characterized to date. In addition to the frequent TP53
mutations previously reported (28
), we noted a low rate of aberrations in the PI3K pathway, KRAS
, and CTNNB1
in MMMT, clearly suggesting that MMMT has more in common mutationally with type 2 EC (serous and clear cell), in which TP53
mutation is the most common genetic alteration and is considered an early event (29
Based on frequent aberrations in PIK3CA
, we explored whether mutations in other members of the PI3K pathway are present in EC. Strikingly, the mutation rate of PIK3R1
(20%) was markedly higher in EC than that previously reported for any other lineage demonstrating selective targeting in EC (17
). In the COSMIC database (30
mutations were detected in 36 of 1693 (2%) tumors. PIK3R2
mutation had only been reported in 0.9% (1/108) colon cancers (18
) and in 3 of 817 (0.4%) tumors (30
). Our studies demonstrated PIK3R2
mutations in 5% of EC, with several mutations being demonstrated to exhibit gain of function, establishing PIK3R2
as a novel cancer gene.
Previous reports suggested that KRAS
mutations are mutually exclusive in EC (7
). However, these studies only sequenced exons 9 (helical) and 20 (catalytic domain) of PIK3CA
in small sample sets. The large sample size in this study, the characterization of multiple PI3K pathway members, and our observation that PIK3CA
mutations commonly occur outside of exons 9 and 20 likely accounts for the discrepancy, supporting our observation that KRAS
and PI3K pathway members are coordinately mutated in EC. The concurrent mutations in PI3K pathway members and KRAS
in cell lines support the concept that the mutations occur in a single cell population rather than in independent subclones. This may reflect, in part, a lack of functional redundancy among PI3K pathway and KRAS
mutations in EC that is supported by RPPA demonstrating distinct downstream signaling consequences associated with these mutations. This activation of different pathways could thus cooperate for efficient transformation.
Co-mutations in different components of the PI3K pathway might also cooperate for efficient transformation (31
). Further, we have demonstrated that PTEN protein loss and PIK3CA
mutations have markedly different functional effects on PI3K pathway activation in human breast cancer (22
). Co-mutations in PI3K pathway members in EC occur at frequencies significantly higher than predicted, in contrast to most cancers (19
). PTEN protein loss, regardless of PTEN
mutational status, resulted in PI3K pathway activation. PIK3CA, PIK3R1
, or PIK3R2
mutation was more common in cells where PTEN protein was retained, and these mutations phenocopy the functional effects of PTEN loss on downstream signaling. MMR deficiency, which is an early event in the pathogenesis of EC (21
), might contribute to these co-mutations. However, the mutations in the PI3K pathway members were not typical of MMR aberrations.
The majority of PTEN mutations in EC are heterozygous. PTEN haploinsufficiency has been reported to contribute to development of prostate cancer (32
). However, despite PTEN mutations being heterozygous, almost half of EC demonstrated complete loss of PTEN protein, suggesting that additional mechanisms contribute to PTEN loss. Further, PTEN loss was also seen in a significant fraction of WT PTEN tumors. PTEN protein levels and function are extensively regulated by multiple genomic and epigenetic mechanisms in addition to mRNA and protein modification, emphasizing the importance of this tumor suppressor (25
). Thus multiple mechanisms are likely to converge on regulation of PTEN protein levels and function in EC.
EC exhibits mutations throughout the coding region of PIK3CA
with the exception of the RAS binding domain compatible with interactions with RAS being critical to the function of p110 (33
). Consistent with a recent study, we observed a high frequency of mutations within the ABD domain in PIK3CA
, compared to other tumor lineages (34
). Interestingly, 87% of mutations within the ABD domain were associated with PTEN
heterozygous mutation,suggesting that ABD mutations may selectively interact with PTEN
heterozygous mutations. ABD domain mutations have been suggested to have lower transforming activity and to activate AKT to a lesser degree than catalytic and helical domain mutations (31
). It is possible that co-mutation of the ABD domain with PTEN
increases information flow through the PI3K pathway. Although complete PTEN loss has been reported to elicit senescence in prostate cancer models (35
), it does not appear to do so in EC, as several tumors did exhibit complete PTEN loss. Furthermore, although PTEN loss is often a late event in prostate tumorigenesis, PTEN aberrations occur early during the pathogenesis of EC.
The roles of p85 in PI3K pathway activation and tumorigenesis are complex. Levels of p85α are decreased in a number of tumor lineages (36
). Haploinsufficiency of PIK3R1
can result in PI3K pathway activation, whereas homozygous depletion inhibits the pathway (37
). On the other hand, PIK3R1
knockout mice demonstrated increased PI3K pathway activity and decreased PTEN protein levels, compatible with our data on the effect of WT p85α on PTEN levels (36
). If in excess of p110, p85 could compete with the p85–p110 complex for binding to phosphorylated insulin receptor substrate (IRS) or to other phosphotyrosine-containing proteins, suggesting that free p85 could negatively modulate PI3K signaling (37
). A direct and possibly bidirectional interaction between the N-terminal SH3-Rho-GAP domain of p85 and PTEN has been demonstrated (26
). We showed herein that this p85α-PTEN interaction is associated with increased PTEN stability through decreased ubiquitination. The truncated gain-of-function mutant E160*, which does not bind either p110α or PTEN, binds WT p85α and decreases its binding to PTEN, resulting in PTEN destabilization through ubiquitination.
Overall, the data are most compatible with a model wherein free p85α forms a homodimer that is able to bind PTEN (). This model is supported by evidence that 1) overexpression of p85α did not increase p110α levels, indicating that p85α is not limiting in the cells; 2) p110α is not part of the complex because p110α overexpression inhibited p85α-PTEN interaction, suggesting a mutually exclusive binding; 3) E160* binds to free p85α because overexpression of p110α competed with E160* for binding to p85α; and 4) p85α self-dimerized and this homodimerization was inhibited by overexpression of p110α and E160*. The SH3 and first proline-rich motif have been shown to be sufficient to mediate homodimerization; furthermore, the truncated isoform p55γ, which lacks the N-terminal domains, failed to form dimers (27
). Intermolecular interaction between Rho-GAP domains might also contribute to the p85α homodimer formation (27
). The ability of E160* to bind with WT p85α suggests that SH3 and the first proline-rich domains constitute a major homodimerization interface. However, the failure of the E160*–p85α heterodimer to bind and stabilize PTEN whereas the R348*–p85α heterodimer (or possibly R348* homodimers) binds and stabilizes PTEN, suggests that the Rho-GAP domain likely plays a role in the conformation change in p85α required for binding to PTEN. How the homodimer enhances PTEN stability warrants further investigation, but it is tempting to speculate that the homodimer may provide a combinatorial binding site for PTEN or may facilitate recruitment of other molecules to the complex to stabilize PTEN. Phosphorylation of the C-terminal tail of PTEN inhibits its proteosome-mediated degradation (40
). p85 and unphosphorylated PTEN are part of a high molecular weight complex with formation of the complex associated with diminished AKT activation (38
). In this scenario, it is possible that phosphorylated PTEN adopts a closed conformation that results in lower affinity for the complex (41
). The association of PTEN with p85α is readily observed in EEC in the absence of growth factor addition in contrast to previous studies (26
). This can be potentially due to the frequent presence of other activating mutations in EEC (). In contrast to studies in other systems (38
), we were unable to find evidence for a trimeric (or tetrameric) complex of PTEN, p85α, and p110α by cross-immunoprecipitation in EEC (not presented). Further, the ability of exogenous p110α to decrease p85α homodimers and p85α–PTEN heterodimers suggests that in EEC, the binding of PTEN and p110α to p85α may be mutually exclusive, with p85α monomers interacting with p110α and dimers with PTEN. The spontaneous E160* mutation from EEC will provide a powerful tool to elucidate the mechanisms underlying these processes.
Figure 6 Schematic working model of p85α-mediated PTEN stabilization. A, in cells with wild-type (WT) p85α, in addition to p110α-bound p85α, excess p85α forms homodimers via intermolecular interactions between SH3 and proline-rich (more ...)
Other activating p85α mutations found in EC likely elucidate additional regulatory mechanisms as they do not apparently alter PTEN protein levels but do increase pAKT levels. Three of five activating mutations identified in the Ba/F3 assay reside in the iSH2 domain, which binds the C2 domain of p110 inhibiting the catalytic activity of p110. These activating mutations appear to disrupt iSH2–C2 contact, releasing the inhibitory effect on p110 while retaining the ability to stabilize p110 (24
). One of the novel activating mutations in EC (R503W) is part of an exposed surface composed of basic residues that may bind to negatively charged membrane phosphatidylinositide substrates compatible with an independent mechanism (43
The recurrent R348* activating mutation lacks intact SH2 and iSH2 domains and thus lacks ability to bind p110. It demonstrated lower activity than E160* in multiple repeat Ba/F3 assays. The portion of p85α remaining in the R348* mutant is similar to the minimal domain defined to bind PTEN (26
), a property we confirmed for R348*, which may contribute to its activity. Because the R348* mutation is heterozygous in each of the 5 tumors, p110 might be stabilized by intact p85α expressed from the remaining PIK3R1
allele. Indeed, an artificial p85α mutant unable to bind p110 has been demonstrated to increase kinase activity of a p110α mutant (44
). Interestingly, heterozygous mutations in PTEN
were concurrent in tumors harboring the R348* mutation, suggesting that the activity of this mutant is most clearly manifest in cooperation with other mutants in the PI3K pathway.
Thus, there appear to be multiple mechanisms by which aberrations in PIK3R1 can contribute to tumorigenesis. It is premature to predict the pathophysiological relevance of the types of PIK3R1 mutations on tumor initiation and progression. Possibly the different mutations will contribute to sensitivity to a specific type of PI3K pathway inhibitor. Therefore, the effects of each class of PIK3R1 mutations on tumor initiation and progression warrant further study.
WT p85β demonstrated greater ability to induce Ba/F3 survival than WT p85α. Unlike p85α, p85β did not stabilize PTEN protein in spite of its ability to bind PTEN (unpublished data). It is possible that the two isoforms mediate different functions (45
). Another plausible explanation is that the effects of p85α on PTEN are dominant over p85β when both isoforms are expressed. Indeed, we found that expression of exogenous p85α inhibited binding of p85β to PTEN (unpublished observation), suggesting that PTEN interacts preferentially with p85α. This finding is in contrast to previous studies in other cell lines where p85β appears to be the preferred partner of PTEN (39
) and may represent the unique context of EEC cells. Of note, p85α and p85β share only 30% protein homology in the Rho-GAP domain that contributes to PTEN binding, in contrast to 80% in the SH2 domains. Although p85β possesses similar proline-rich motifs as p85α, whether p85β homodimerizes and/or bind PTEN as efficiently as p85α is unclear and may be context dependent. Therefore, due to the complex effects of p85α and p85β on the PI3K pathway, tumor development is likely to be tissue context dependent and determined by relative levels and activities of p110, p85, PTEN and phosphotyrosine residues.
Germline variants of PIK3R1
(M326I) and PIK3R2
(V727T) were found in our samples, with homozygous mutations occurring in frequencies fitting Hardy-Weinberg equilibrium. Although M326I has been reported to exhibit increased binding to IRS-1, it had no impact on PI3K activity and signaling events (46
). Herein, we found that M326I and V727T had no effect on Ba/F3 survival or AKT phosphorylation. RPPA analysis also showed no difference in downstream signaling between patients with or without these variants (data not shown).
The prevalence of abnormalities in members of the PI3K pathway and KRAS
implicates these pathways as critical drivers of pathogenesis of EC and thus as exciting targets for cancer treatment. Encouragingly, we found that mutations in the PI3K pathway and KRAS
predict in vitro
sensitivity to rapamycin and MEK inhibitor but, like other studies, not to GDC-0941 (47
). Due to feedback loops in the PI3K pathway, rational targeted therapeutic combinations may be required for optimal outcomes. Our data also suggest that EEC cells with both PI3K pathway and KRAS
mutations are relatively more resistant to PI3K pathway inhibitors than those with PI3K pathway aberrations alone, as previously observed (48
). Whether coordinate targeting of the PI3K and RAS pathways will demonstrate higher activity with acceptable toxicity in EC xenografts is under investigation. Further, we found that EC patients with PI3K pathway aberration appear to have a significantly lower recurrence risk (P
= 0.003), suggesting that PI3K pathway mutation status might also predict tumor recurrence.
Currently, the choice of therapy for individual patients with EC is largely empirical. Our data, which include comprehensive characterization of genetic alterations coupled with mechanistic studies, suggest that manipulation of the PI3K and RAS pathways could provide a promising molecular-targeted therapy in EC. The genomic and protein signatures we identified should affect the stratification of patients in trials and hence accelerate the fulfillment of personalized molecular medicine.