Constitutively nuclear FOXO3a localization predicts poor survival and promotes Akt phosphorylation in breast cancer

Abstract

Background: The 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 findings: Examination 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/significance: Together 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.

Figure 1. Representative expression patterns of FOXO3a and P-Akt in tissue microarray.

Tumour tissue samples obtained from breast cancer patients that had been formalin-fixed and paraffin-embedded were immunohistochemically stained with FOXO3a and P-Akt (Thr308) antibodies using the streptavidin-biotin-peroxidase technique. A) Representative FOXO3a staining patterns in both tumour and non-tumour cases (magnification ×170). B) Two representative tumour cases showing corresponding FOXO3a and P-Akt staining patterns (magnification ×170). Case 1 shows high cytoplasmic pAkt staining and strong cytoplasmic and nucleus FOXO3a staining. Case 2 shows weak pAkt and FOXO3a staining.

Figure 2. Survival Analysis with nuclear FOXO3a staining in invasive ductal carcinoma cases.

Of the 120 cancer samples assessed, there were 94 cases of invasive ductal carcinoma. The correlation between nuclear FOXO3a expression and survival was studied using Kaplan Meier analysis (P = 0.014) and was considered significant at p<0.05.

Figure 3. Effects of tamoxifen on cell proliferation of a panel of breast carcinoma cell lines.

MCF-7(LCC1), LCC2, LCC9, R27 and BT474 cells were treated with 0 to 10 µM of tamoxifen for 6 days. Cell proliferation was determined by SRB assay. Points, mean of three independent experiments; bars, SD.

Figure 4. Effects of doxorubicin on cell proliferation of a panel of breast carcinoma cell lines.

A) MCF-7 and the derived MCF-7 Dox^R cells were treated with 0 to 10 µM of doxorubicin for 0, 24, and 48 h. B) MCF-7, MCF-7 Dox^R, MDA-MB-231, BT474, and LCC9 cells were treated with 1 µM of doxorubicin for 0, 24, and 48 h. C) MCF-7(LCC1), LCC2, LCC9, R27 and BT474 cells were treated with 1 µM of doxorubicin for 0, 24, and 48 h. Cell proliferation was determined by SRB assay. Points, mean of three independent experiments; bars, SD.

Figure 5. Expression of total and phosphorylated Akt and FOXO3a in a panel of tamoxifen and/or doxorubicin sensitive and resistant breast carcinoma cell lines.

The tamoxifen sensitive MCF-7(LCC1), and resistant LCC2, LCC9, R27 and BT474 cells as well as the doxorubicin sensitive MCF-7 and resistant MCF-7-Dox^R and MDA-MB-231 cells were cultured in normal growth medium and used for Western blot analysis for P-Akt (Ser473), total Akt, P-FOXO3a (Thr32), total FOXO3a and β-tubulin.

Figure 6. Nuclear location of FOXO3a in a panel of tamoxifen and/or doxorubicin sensitive and resistant breast carcinoma cell lines.

The tamoxifen sensitive MCF-7(LCC1), and resistant LCC2, LCC9, R27 and BT474 cells as well as the doxorubicin sensitive MCF-7 and resistant MCF-7-DoxR and MDA-MB-231 cells were cultured in normal growth medium and used for studies of the subcellular localization of FOXO3a. A) Cytoplasmic and nuclear protein lysates were prepared and protein expression levels were analyzed by Western blotting using antibodies against specific antibodies against FOXO3a, lamin B1 and and β-tubulin, B) The cells were cultured on sterile coverslips, before being fixed in 4% formaldehyde. FOXO3a expression was visualized with a specific rabbit polyclonal antibody against FOXO3a followed by the addition of ALEX488 (green) labeled anti-rabbit antisera. DAPI (blue) were also applied to visualize the nuclei.

Figure 7. Doxorubicin treatment causes a nuclear relocation of FOXO3a expression.

The doxorubicin sensitive MCF-7 and resistant MCF-7-Dox^R and MDA-MB-231 cells were treated with 1 µM doxorubicin. A) Cytoplasmic and nuclear protein lysates were prepared at the times indicated after doxorubicin treatment and protein expression levels were analyzed by Western blotting using antibodies against specific antibodies against FOXO3a, lamin B1 and and β-tubulin, B) The cells were cultured on sterile coverslips and treated for 16 h with or without 1 µM doxorubicin, before being fixed in 4% formaldehyde. FOXO3a expression was visualized with a specific rabbit polyclonal antibody against FOXO3a followed by the addition of ALEX488 (green) labeled anti-rabbit antisera. DAPI (blue) were also applied to visualize the nuclei.

Figure 8. Overexpressed active FOXO3a results in cell proliferation arrest and Akt phosphorylation in drug sensitive breast cancer cell line MCF-7.

MCF-7 cells were transiently transfected with the constitutively active FOXO3a(A3) or control vector, and the transfected cells analysed by A) Western blot analysis for FOXO3a, P-Akt (Ser473), Akt and β-actin. B) The transfected cells were also examined by qRT-PCR analysis for FOXO3a and PIK3CA mRNA expression. TBP mRNA was used as an internal control. C) SRB assays were performed on these transfected cells, indicating that the ectopic expression of FOXO3a(A3) decreases the cell proliferation rate.

Figure 9. Knockdown of FOXO3a expression decreases Akt phosphorylation and PIK3CA mRNA expression.

BT474 cells were transiently transfected with FOXO3a or control shRNA, and 24 h after transfection cells were analysed by A) Western blot using specific antibodies P-FOXO3a (Thr32), FOXO3a, P-Akt (Ser473), Akt and Actin as indicated and by B) qRT-PCR.

Figure 10. FOXO3a induces Akt phosphorylation, PIK3CA and IGFR1 gene expression in the drug resistant MDA-MB-231 breast carcinoma cells.

MDA-MB-231-FOXO3a(A3):ER and MDA-MB-231 cells were treated with 200 nM 4-OHT for the indicated times. A) Total, cytoplasmic and nuclear extracts were prepared at the times indicated, separated on polyacrylamide gels, and subjected to immunoblotting with specific antibodies. The expression levels of FOXO3a, P-FOXO3a (Thr-32), p110α (PIK3CA), P-Akt (Ser473), Akt, and lamin B1 were analyzed by Western blotting. B) Total RNA was extracted from these cells and analyzed for PIK3CA, and IGFR1, mRNA expression using qRT-PCR as described in the text and normalized to the level of L19 RNA. All data shown represent the averages of data from three experiments, and the error bars show the standard deviations.

Figure 11. FOXO3a induction does not result in proliferative arrest in the drug resistant MDA-MB-231 breast carcinoma cells.

A) MDA-MB-231-FOXO3a(A3):ER cells were cultured on sterile coverslips and treated for 16 h with or without 200 nM 4-OHT as in Fig. 10, before being fixed in 4% formaldehyde. FOXO3a(A3):ER was visualized with a rabbit polyclonal antibody against ERα followed by the addition of ALEX488 (green) labeled anti-rabbit antisera. DAPI (blue) were also applied to visualize the nuclei. B) SRB assays were performed on these cytotoxic resistant MDA-MB-231 cells, indicating that the induction of FOXO3a(A3) has little effects on the cell proliferation rate of the drug resistant breast carcinoma cells.

Figure 12. Overexpressed active FOXO3a has little effect on cell proliferation and drug sensitivity of the drug resistant breast cancer cell line MCF-7-Dox^R.

MCF-7-Dox^R cells were transiently transfected with the constitutively active FOXO3a(A3) or control vector pcDNA3, A) The transfected cells were analysed by Western blot analysis for FOXO3a and β-actin expression. B) The cells were treated with 0 to 10 µM of doxorubicin for 0, 24, 48 and 72 h and SRB assays were performed on these transfected MCF-7-Dox^R cells after 48 h. The results indicated that the induction of FOXO3a(A3) has little effect on the cell proliferation rate of the drug resistant breast carcinoma cells and in response to doxorubicin treatment.

Figure 13. Induction of FOXO3a can confer resistance to the MDA-MB-231 cells.

MDA-MB-231-FOXO3a(A3):ER cells were cultured with 200 nM 4-OHT and collected at 0, 4, 8, 24 and 48 h. A) Total cytoplasmic and nuclear extracts were prepared at the times indicated, separated on polyacrylamide gels, and subjected to immunoblotting with FOXO3a, P-FOXO3a(Thr-32), actin and lamin B1 antibodies. B) SRB assays were performed on the untreated and 4-OHT-treated MDA-MB-231 cells after 48 h of treatment with various concentrations of doxorubicin. The results indicated that the induction of FOXO3a (A3) can protect the drug resistant breast carcinoma MDA-MB-231 cells from the effects of doxorubicin.