In this study, we demonstrated that 14-3-3ζ overexpression significantly associated with disease recurrence and poor survival in breast cancer patients. Overexpression of 14-3-3ζ induced anchorage independent growth and conferred a survival advantage under stress conditions, whereas knockdown of 14-3-3ζ reduced tumor growth and sensitized cells to chemotherapeutic treatment, suggesting that 14-3-3ζ promotes malignancy through regulation of cancer cell survival. Our findings linked a clinically relevant prognostic marker (14-3-3ζ) with biological outcome (apoptosis resistance) and allow for identification of patients who may be resistant to standard chemotherapies and require more aggressive treatment strategies.
The highly significant association between 14-3-3ζ overexpression and decreased disease-free survival of breast cancer patients suggest the clinical potential of 14-3-3ζ as a marker of cancer recurrence. One of the major challenges in breast cancer treatment is to identify patients at high risk who would benefit from otherwise potentially unnecessary and toxic systemic therapy. Our data demonstrated that the addition of 14-3-3ζ expression status to other well-known breast cancer prognostic factors stratified a subgroup of patients at a high risk for disease recurrence with distant metastasis who need more intensive or alternative adjuvant therapy. Consistent with our findings, 14-3-3ζ overexpression was also found to be associated with decreased survival in lung cancer patients (25
). Notably, overexpression of 14-3-3ζ in our patient population (>40%) was a more frequent event than alterations of currently established molecular markers of poor patient outcome in breast cancer patients (e.g., <30% for ErbB2/HER2 and 15% for urokinase plasminogen activator) (26
). We further determined that increased expression of 14-3-3ζ was associated with ErbB2 overexpression and stage 3 breast disease. These associations indicate that overexpression of 14-3-3ζ may cooperate with known oncogenes leading to more aggressive breast cancers. Our studies also demonstrated that gene amplification and chromosome 8 polysomy could account for up to 80% of 14-3-3ζ protein overexpression in breast tumors. Similarly, amplification of 14-3-3 genes have been discovered in urothelial carcinomas (27
), suggesting amplification may be a common mechanism leading to 14-3-3ζ overexpression in multiple cancers. Mechanisms independent of increased gene copy number, such as modulation of 14-3-3ζ gene transcription, protein translation, or RNA and protein stability may also contribute to increased 14-3-3ζ expression. Although a larger cohort study will be needed to validate these findings before being applied in a clinical setting, our novel findings indicated 14-3-3ζ as an important molecular marker for disease recurrence in breast cancer patients.
Our data demonstrated that 14-3-3ζ overexpression provided an increased survival advantage in response to anchorage independent growth and low serum conditions in breast epithelial and breast cancer cells. In contrast, inhibiting 14-3-3ζ expression sensitized breast cancer cells to chemotherapeutics in vitro
and transient blockade of 14-3-3ζ reduced the growth of breast tumor xenografts in vivo. These data suggest that 14-3-3ζ may serve as a therapeutic target for chemo-sensitization and effective tumor inhibition. Interestingly, reducing 14-3-3ζ expression in lung cancer cells sensitized the cells to ionizing radiation, cisplatin, and anoikis (25
), whereas, high expression of 14-3-3ζ in breast cancer cells predicted poor response to tamoxifen treatment (30
). We have recently found that 14-3-3ζ down-regulates the tumor suppressor gene p53 via activation of Akt and Mdm2 mediated p53 protein degradation, conferring anoikis resistance in mammary epithelial cells (11
). Additionally, 14-3-3 is known to modulate multiple survival signals including Akt downstream signals. Therefore, targeting 14-3-3ζ or targeting downstream pathways regulated by 14-3-3ζ may sensitize cells to apoptosis and serve as effective anti-cancer strategies in patients whose tumors overexpress 14-3-3ζ.
Our data suggest overexpression of 14-3-3ζ may have an oncogenic role in breast cancer. In contrast, 14-3-3σ can act as a tumor suppressor and its expression is lost in many breast cancers (31
). 14-3-3σ also inhibits Akt activity after DNA damage (32
) while our data have shown that 14-3-3ζ overexpression increases Akt activation (11
). Thus, different 14-3-3 isoforms may have specific, non-overlapping and even opposing biological functions. Although silencing 14-3-3ζ, without altering 14-3-3β levels, decreased transformation and tumorigenesis in breast cancer cells, our data do not exclude the possibility that overexpression of other canonical 14-3-3 isoforms may also contribute to tumorigenicity. It is known that 14-3-3 proteins can form homodimers or heterodimers with other isoforms (33
). In the context of cancer, overexpression of 14-3-3ζ may interfere with the balance of various dimmer compositions within the cell. This would lead to substrate bias for 14-3-3ζ dimers, which may explain why increasing 14-3-3ζ, even in the presence of other 14-3-3 proteins, leads to enhanced transformation. It may also explain why decreasing only 14-3-3ζ with siRNA, even in the presence of other 14-3-3 proteins, dramatically decreases tumorigenic properties in vitro
and in vivo
. Our findings call for the development of 14-3-3ζ-targeting therapies for a more effective, personalized cancer care of patients whose tumors overexpress 14-3-3ζ and are at high risk of having disease recurrence with metastasis.