2309

FOXO3

forkhead box O3;FOXO3;forkhead box O3

forkhead box O3A|forkhead box protein O3|forkhead homolog (rhabdomyosarcoma) like 1|forkhead in rhabdomyosarcoma-like 1|forkhead, Drosophila, homolog of, in rhabdomyosarcoma-like 1

6q21

protein-coding

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.

PURPOSE: To explore the mechanisms underlying clear-cell renal cell carcinoma (ccRCC) metastasis using transcriptional profiling and bioinformatics analysis of ccRCC samples, and to elucidate the role of FOXO3a in ccRCC metastasis. EXPERIMENTAL DESIGN: Gene expression profiling was performed using four primary metastatic and five primary nonmetastatic ccRCC samples. The mRNA and protein levels of FOXO3a in ccRCC samples were investigated by real-time reverse transcription PCR and immunohistochemistry, respectively. The association between metastasis-free survival of patients with ccRCC and FOXO3a mRNA levels was analyzed. Biologic functions of FOXO3a in renal cancer cell lines were investigated. The influence of FOXO3a on tumor metastasis was also studied in vivo orthotopic xenograft tumor model. Finally, the mechanism by which FOXO3a attenuation could increase invasion and migration of tumor cells was explored. RESULTS: Bioinformatics analysis of the profiling data identified FOXO3a as a key factor in ccRCC metastasis. FOXO3a expression was decreased in primary metastatic ccRCC samples. Patients with low FOXO3a mRNA levels had poor metastasis-free survival (P = 0.003). Knocking down FOXO3a induced tumor cell invasion and migration in the nonmetastatic ccRCC cells. Induced FOXO3a overexpression in SN12-PM6 cells could inhibit tumor metastasis in vivo. Downregulation of FOXO3a increased SNAIL1 expression, thereby activating the epithelial-mesenchymal transition (EMT) of RCC cell lines. CONCLUSIONS: The loss of FOXO3a induced EMT of tumor cells by upregulating SNAIL1, which promoted tumor cells metastasis in vitro and in vivo. Thus, FOXO3a could be considered as an independent prognostic factor in ccRCC metastasis and could be a marker of occult metastases.

The highly aggressive character of melanoma makes it an excellent model for probing the mechanisms underlying metastasis, which remains one of the most difficult challenges in treating cancer. We find that miR-182, member of a miRNA cluster in a chromosomal locus (7q31-34) frequently amplified in melanoma, is commonly up-regulated in human melanoma cell lines and tissue samples; this up-regulation correlates with gene copy number in a subset of melanoma cell lines. Moreover, miR-182 ectopic expression stimulates migration of melanoma cells in vitro and their metastatic potential in vivo, whereas miR-182 down-regulation impedes invasion and triggers apoptosis. We further show that miR-182 over-expression promotes migration and survival by directly repressing microphthalmia-associated transcription factor-M and FOXO3, whereas enhanced expression of either microphthalmia-associated transcription factor-M or FOXO3 blocks miR-182's proinvasive effects. In human tissues, expression of miR-182 increases with progression from primary to metastatic melanoma and inversely correlates with FOXO3 and microphthalmia-associated transcription factor levels. Our data provide a mechanism for invasion and survival in melanoma that could prove applicable to metastasis of other cancers and suggest that miRNA silencing may be a worthwhile therapeutic strategy.