Vascular endothelial growth factor (VEGF) plays a central role in breast cancer development and progression, but the mechanisms that control its expression are poorly understood. Breast cancer tissue microarrays revealed an inverse correlation between the Forkhead transcription factor FOXO3a and VEGF expression. Using the lapatinib-sensitive breast cancer cell lines BT474 and SKBR3 as model systems, we tested the possibility that VEGF expression is negatively regulated by FOXO3a. Lapatinib treatment of BT474 or SKBR3 cells resulted in nuclear translocation and activation of FOXO3a, followed by a reduction in VEGF expression. Transient transfection and inducible expression experiments showed that FOXO3a represses the proximal VEGF promoter whereas another forkhead member, FOXM1, induces VEGF expression. Chromatin immunoprecipitation and oligonucleotide pull-down assays demonstrated that both FOXO3a and FOXM1 bind a consensus Forkhead response element (FHRE) in the VEGF promoter. Upon lapatinib stimulation, activated FOXO3a displaces FOXM1 bound to the FHRE before recruiting histone deacetylase 2 (HDAC2) to the promoter, leading to decreased histones H3 and H4 acetylation, and concomitant transcriptional inhibition of VEGF. These results show that FOXO3a-dependent repression of target genes in breast cancer cells, such as VEGF, involves competitive displacement of DNA-bound FOXM1 and active recruitment of transcriptional repressor complexes.

The phosphoinositide-3 kinase (PI3K)/Akt signal pathway plays a key role in the tumorigenesis of many cancers and in the subsequent development of drug resistance. Using the K562 chronic myelogenous leukemia (CML) cell line and the doxorubicin-resistant derivative lines KD30 and KD225 as models, we observed that enhanced PI3K/Akt activity and the acquisition of chemoresistance correlated unexpectedly with the increased expression and nuclear accumulation of FOXO3a. Moreover, we found that the induction of FOXO3a activity in naïve K562 cells was sufficient to enhance PI3K/Akt activity and to confer resistance to the cytotoxic effects of doxorubicin. Conversely, the knockdown of endogenous FOXO3a expression reduced PI3K/Akt activity and sensitized these cells to doxorubicin. Further chromatin immunoprecipitation and promoter mutation analyses demonstrated that FOXO3a regulates the expression of the PI3K catalytic subunit p110␣ through the activation of a promoter region proximal to a novel untranslated exon upstream from the reported transcription start site of the p110␣ gene PIK3CA. As was the case for FOXO3a, the expression or knockdown of p110␣ was sufficient to amplify or reduce PI3K/Akt activity, respectively. Thus, our results suggest that the chronic activation of FOXO3a by doxorubicin in CML cells can enhance survival through a feedback mechanism that involves enhanced p110␣ expression and hyperactivation of the PI3K/Akt pathway.Chemotherapy is widely used for the treatment of leukemia and other advanced or metastatic cancers. However, its efficacy is often hampered by the development of intrinsic or acquired multidrug resistance (MDR), characterized by simultaneous cross-resistance to anticancer drugs that differ in their chemical structures, modes of action, and cellular targets (35). An understanding of the mechanisms of MDR is important for the development of more effective therapies. At the cellular level, three general mechanisms confer MDR in cancer cells, including decreased hydrophilic drug uptake, increased hydrophobic drug efflux, and enhanced cell survival signals or mechanisms. The first one involves a decrease in the expression or activity of transporters that regulate the uptake of hydrophilic chemotherapeutic drugs, such as folate antagonists, nucleoside analogues, and cisplatin. The second mechanism entails an upregulation of transporters, resulting in an increased energy-dependent efflux of a wide variety of hydrophobic chemotherapeutic agents. Finally, cancer cells often counteract the cytotoxic effects of therapeutic agents by amplifying the activity of proliferation and survival signal pathways, by increasing DNA damage repair, or by altering drug metabolism.Phosphoinositide-3 kinases (PI3Ks) are a family of lipid kinases that serve as mediators of signals generated by many different activated growth factor receptors and adhesion molecules. The class IA PI3Ks are heterodimers composed of a p110 catalytic subunit and a p85 regulatory subunit (25). When activated by growth factors, the p...

BackgroundThe PI3K-Akt signal pathway plays a key role in tumorigenesis and the development of drug-resistance. Cytotoxic chemotherapy resistance is linked to limited therapeutic options and poor prognosis.Methodology/Principal FindingsExamination of FOXO3a and phosphorylated-Akt (P-Akt) expression in breast cancer tissue microarrays showed nuclear FOXO3a was associated with lymph node positivity (p = 0.052), poor prognosis (p = 0.014), and P-Akt expression in invasive ductal carcinoma. Using tamoxifen and doxorubicin-sensitive and -resistant breast cancer cell lines as models, we found that doxorubicin- but not tamoxifen-resistance is associated with nuclear accumulation of FOXO3a, consistent with the finding that sustained nuclear FOXO3a is associated with poor prognosis. We also established that doxorubicin treatment induces proliferation arrest and FOXO3a nuclear relocation in sensitive breast cancer cells. Induction of FOXO3a activity in doxorubicin-sensitive MCF-7 cells was sufficient to promote Akt phosphorylation and arrest cell proliferation. Conversely, knockdown of endogenous FOXO3a expression reduced PI3K/Akt activity. Using MDA-MB-231 cells, in which FOXO3a activity can be induced by 4-hydroxytamoxifen, we showed that FOXO3a induction up-regulates PI3K-Akt activity and enhanced doxorubicin resistance. However FOXO3a induction has little effect on cell proliferation, indicating that FOXO3a or its downstream activity is deregulated in the cytotoxic drug resistant breast cancer cells. Thus, our results suggest that sustained FOXO3a activation can enhance hyperactivation of the PI3K/Akt pathway.Conclusions/SignificanceTogether these data suggest that lymph node metastasis and poor survival in invasive ductal breast carcinoma are linked to an uncoupling of the Akt-FOXO3a signaling axis. In these breast cancers activated Akt fails to inactivate and re-localize FOXO3a to the cytoplasm, and nuclear-targeted FOXO3a does not induce cell death or cell cycle arrest. As such, sustained nuclear FOXO3a expression in breast cancer may culminate in cancer progression and the development of an aggressive phenotype similar to that observed in cytotoxic chemotherapy resistant breast cancer cell models.

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