We have previously shown that PTEN ATP-binding motif mutations alter cellular proliferation, apoptosis and anchorage-dependent growth (9
). Our results here demonstrate that PTEN ATP-binding mutations can lead to both qualitative and quantitative impairment of the tumor-suppressive function of PTEN. Mutations within the ATP-binding motifs not only change PTEN subcellular localization, but also lead to deregulated PTEN expression and activity.
The phosphatase activity of PTEN is important to its role in cell cycle arrest. Our current experiments show that all three ATP-binding motif mutations have abrogated phosphatase activity accompanied by unchanged cyclin D1 levels. Considering both ATP-binding motifs are located within the phosphatase domain of PTEN (residues 1–185), especially for PTENK125E
, which is located within the catalytic core motif of PTEN (HCXXGXXR; residues 123–130), it is reasonable to postulate that these mutations impair PTEN's phosphatase activity and thus diminish PTEN's tumor-suppressive function (15
The human breast carcinoma MCF-7 cell line was the main cell line used in our in vitro
studies. We chose the MCF-7 cell line (which contains both WT PTEN
and WT p53
) for two major reasons: (i) we wanted to further investigate the interplay between nuclear PTEN and p53 in breast cancer cells and (ii) more importantly, we wanted to eventually examine mutant PTEN
in a heterozygous state that mimics the in vivo
situation in CS breast cancer and sporadic primary breast carcinomas (16
). PTEN has been reported to upregulate p53 protein levels and transcriptional activity. In the nucleus, PTEN physically interacts with p53 through its C2 domain and forms a PTEN/p53 complex to stabilize the latter (17
). Because the ATP-binding mutations are within PTEN's phosphatase domain instead of its C2 domain, it is unlikely that this effect is through the disruption of direct binding between PTEN and p53 in the nucleus.
One of the main pathways for p53 degradation is through MDM2-induced ubiquitination. In our study, however, a significant decrease in the P-MDM2 signal was observed in MCF-7 cells with ectopic expression of the PTEN ATP-binding mutants, suggesting that the downregulation of p53 in the ATP-binding mutants may be mediated via a phosphatase-independent and MDM2-independent manner. In the nucleus, PTEN can form a complex with p300 and plays a role in maintenance of high p53 acetylation, which is important in the control of p53 protein stability and DNA binding (18
). The accumulated PTEN ATP-binding mutants in the nucleus may interfere with PTENWT forming a complex with p300 for p53 acetylation, and therefore reducing both the stability and the transcriptional activity of p53. Further detailed work will be required in the future to elucidate the precise mechanism of how PTEN ATP-binding mutations/mutants participate in the downregulation of p53 in the nucleus.
As a transcription factor, p53 regulates genes involving apoptosis, cell cycle and oxidative stress. In agreement with the reduced p53 protein levels, repressed p53 transcriptional activity was demonstrated by luciferase assay in cells overexpressing PTEN mutants. Also consistent, we found that transcript levels of a key downstream effector of p53, BAX
, were significantly suppressed in the context of PTEN ATP-binding mutants. Loss of BAX
transcription likely boosts proliferative and survival signals consistent with our previously published data (9
). All in all, this may suggest a fine balance of nuclear PTEN and p53 and that the regulation of this balance is important for normal cell function (Fig. E).
The balance of nuclear to cytoplasmic PTEN is important for its tumor-suppressor function. PTEN must associate with the plasma membrane to maintain appropriate steady-state levels of phosphatidylinositol 3,4,5-triphosphate (20
). At the same time, nuclear PTEN is a key factor for proper cell cycling and chromosomal integrity. Shen et al
) reported increased spontaneous DNA DSBs in Pten
-deleted mouse embryonic ﬁbroblasts. They also found that a protein from a CS patient with a PTEN R189X mutation does not bind to CENP-C, indicating that PTEN associates with CENP-C in a phosphatase-independent manner (through its C-terminal). These observations together suggest that PTEN's C-terminal domain associates with centrosome proteins to maintain chromosome integrity.
Similar to their results, in our study, we found that MCF-7 cells overexpressing PTEN ATP-binding mutations exhibited spontaneous DNA DSBs. Because all three analyzed mutations contain an intact C-terminal of PTEN, it is unlikely that the spontaneous DNA DSBs caused by these mutations are due to the loss of binding between the mutant PTEN and CENP-C.
Unlike the previous observations, in our study, the DSBs only accumulate with aging of the cells. Oxidative stress plays an important role in breast tumorigenesis through promoting the survival of mammary tumor cells and inducing DNA damages (22
). ROS is involved in the pathogenesis of aging and cancer, and the escalated ROS generation provides an endogenous source of DNA-damaging agents that promote genetic instability (24
). PTEN deletion has been reported to be a key player in ROS-induced oxidative damage of DNA (25
). Notably, unlike PTENWT
, PTEN-containing ATP-binding mutations are unable to inhibit ROS production, as presented by significantly higher ROS activation (Fig. ) and consequent elevation of SOD1, suggesting that their anti-oxidative abilities are impaired.
p53 protects the genome from oxidation by ROS through the upregulation of several genes with antioxidant products, associated with a decrease in intracellular ROS (26
). The downregulation of p53 results in an increase in intracellular ROS and excessive oxidation of DNA, increased mutation rate and genomic instability (27
). TP53INP1 is a major player in p53-driven oxidative stress response (14
). Our observation that cells overexpressing PTEN with ATP-binding mutations have decreased p53 protein levels, impaired p53 transcriptional activity and reduced TP53INP1 transcription is consistent with the hypothesis that such mutations result in loss of PTEN's protective effect against ROS-induced oxidative damage, at least as cells age, and therefore may contribute to the oxidative damage of DNA in that setting.
PTEN stability is regulated by ubiquitination, oxidation and phosphorylation (28
). In our study we found PTENK62R
is unstable, and we also found patients harboring germline mutation of PTENY68H
have decreased PTEN protein levels than the WT controls. Our experiment confirmed that PTENK62R is degraded mainly through the proteasome pathway which underlines the quantitative impairment of the ATP-binding mutations of PTEN.
Our findings therefore beg one question: do these three mutations (K62R, Y65C and K125E) and their consequences promote breast tumorigenesis? K125E, lying within ATP-binding motif B (residues 122–136), is within the core catalytic domain of PTEN (residues 124–129), and it is only one amino acid away from the important C124 residue which confers PTEN's lipid and protein phosphatase activities. We found that K125E mutant cells have the most robust proliferation (9
), fastest clonogenic formation ability (9
) and the lowest p53 transcriptional activity (as presented by the luciferase assay and BAX
mRNA levels; Fig. ). In contrast, K62R and Y65C, both located within the ATP-binding motif A (residues 60–73), appear to behave more similar to each other than to K125E. K125E is almost certainly exerting its effect in at least a dual manner: as a phosphatase dead (most likely) trapping mutant (dominant negative) and by nuclear mislocalization. We therefore believe that the effect of these ATP motif mutants is an end result of a balance between quantitative (dosage effect) and qualititative (e.g. mislocalization, phosphatase affected, etc.) problems.
In conclusion, we provide evidence that a mechanism for tumorigenesis of cells harboring germline or somatic mutations within PTEN ATP-binding motifs is through the quantitative and qualitative impairment of PTEN tumor-suppressor function. Moreover, our study provides novel insights on oxidative stress which may contribute to breast carcinogenesis in patients with germline or somatic PTEN mutations that result in PTEN nuclear predominance and may help explain why cells become more susceptible to cancer by accumulating DSBs due to loss of PTEN protection against ROS with age. In addition, targeting oxidative stress may help in the development of a therapeutic strategy for patients harboring such mutations or mutations with similar functional effects.