Binding of FoxM1 to G2/M gene promoters is dependent upon B-Myb.

The promoters of genes which regulate entry into and progress through mitosis are typically induced maximally in G2 by transcription factors that include B-Myb and FoxM1. As FoxM1 gene transcription is a target of B-Myb, we investigated in this study how these transcription factors functionally interact to regulate these G2/M genes. Using a 3T3 cell line containing floxed B-myb alleles (B-myb(F/F)) that could be conditionally deleted by Cre recombinase, we confirmed that B-myb knockout caused both decreased mRNA expression of several G2/M genes, including FoxM1, and delayed entry into mitosis. Although FoxM1 protein expression was actually unaffected by B-myb knockout when quiescent B-myb(F/F) 3T3 cells re-entered the cell cycle upon serum-stimulation, chromatin immunoprecipitation revealed that FoxM1 binding to G2/M promoters was substantially reduced. FoxM1 transcriptional activity requires sequential phosphorylation by Cyclin-dependent kinases and Plk1, which are B-Myb target genes, and we found that phosphorylation at Plk1-specific sites was somewhat reduced upon B-myb knockout. Neither this effect nor nuclear accumulation of FoxM1, which was unaffected by B-myb knockout, was sufficient to account for the dependence on B-Myb for FoxM1 promoter binding, however. More significantly, assays using paired Birc5 (survivin) promoter-luciferase reporters with either wild-type or mutated Myb binding sites showed that FoxM1 was unable to bind and activate the promoter in the absence of B-Myb binding. Our data suggest that B-Myb is required as a pioneer factor to enable FoxM1 binding to G2/M gene promoters and explains how these transcription factors may collaborate to induce mitosis.

Serum-nutrient starvation induces cell death mediated by Bax and Puma that is counteracted by p21 and unmasked by Bcl-x(L) inhibition.

The cyclin-dependent kinase inhibitor p21 (p21WAF1/Cip1) is a multifunctional protein known to promote cell cycle arrest and survival in response to p53-dependent and p53 independent stimuli. We herein investigated whether and how it might contribute to the survival of cancer cells that are in low-nutrient conditions during tumour growth, by culturing isogenic human colorectal cancer cell lines (HCT116) and breast cancer cell lines in a medium deprived in amino acids and serum. We show that such starvation enhances, independently from p53, the expression of p21 and that of the pro-apoptotic BH3-only protein Puma. Under these conditions, p21 prevents Puma and its downstream effector Bax from triggering the mitochondrial apoptotic pathway. This anti-apoptotic effect is exerted from the cytosol but it is unrelated to the ability of p21 to interfere with the effector caspase 3. The survival function of p21 is, however, overcome by RNA interference mediated Bcl-x(L) depletion, or by the pharmacological inhibitor ABT-737. Thus, an insufficient supply in nutrients may not have an overt effect on cancer cell viability due to p21 induction, but it primes these cells to die, and sensitizes them to the deleterious effects of Bcl-x(L) inhibitors regardless of their p53 status.

ERß1 represses FOXM1 expression through targeting ERα to control cell proliferation in breast cancer.

In this study, we investigated the effects of ectopic estrogen receptor (ER)ß1 expression in breast cancer cell lines and nude mice xenografts and observed that ERß1 expression suppresses tumor growth and represses FOXM1 mRNA and protein expression in ERα-positive but not ERα-negative breast cancer cells. Furthermore, a significant inverse correlation exists between ERß1 and FOXM1 expression at both protein and mRNA transcript levels in ERα-positive breast cancer patient samples. Ectopic ERß1 expression resulted in decreased FOXM1 protein and mRNA expression only in ERα-positive but not ERα-negative breast carcinoma cell lines, suggesting that ERß1 represses ERα-dependent FOXM1 transcription. Reporter gene assays showed that ERß1 represses FOXM1 transcription through an estrogen-response element located within the proximal promoter region that is also targeted by ERα. The direct binding of ERß1 to the FOXM1 promoter was confirmed by chromatin immunoprecipitation analysis, which also showed that ectopic expression of ERß1 displaces ERα from the endogenous FOXM1 promoter. Forced expression of ERß1 promoted growth suppression in MCF-7 cells, but the anti-proliferative effects of ERß1 could be overridden by overexpression of FOXM1, indicating that FOXM1 is an important downstream target of ERß1 signaling. Together, these findings define a key anti-proliferative role for ERß1 in breast cancer development through negatively regulating FOXM1 expression.

ATM and p53 regulate FOXM1 expression via E2F in breast cancer epirubicin treatment and resistance.

In this report, we investigated the role and regulation of forkhead box M1 (FOXM1) in breast cancer and epirubicin resistance. We generated epirubicin-resistant MCF-7 breast carcinoma (MCF-7-EPI(R)) cells and found FOXM1 protein levels to be higher in MCF-7-EPI(R) than in MCF-7 cells and that FOXM1 expression is downregulated by epirubicin in MCF-7 but not in MCF-7-EPI(R) cells. We also established that there is a loss of p53 function in MCF-7-EPI(R) cells and that epirubicin represses FOXM1 expression at transcription and gene promoter levels through activation of p53 and repression of E2F activity in MCF-7 cells. Using p53(-/-) mouse embryo fibroblasts, we showed that p53 is important for epirubicin sensitivity. Moreover, transient promoter transfection assays showed that epirubicin and its cellular effectors p53 and E2F1 modulate FOXM1 transcription through an E2F-binding site located within the proximal promoter region. Chromatin immunoprecipitation analysis also revealed that epirubicin treatment increases pRB (retinoblastoma protein) and decreases E2F1 recruitment to the FOXM1 promoter region containing the E2F site. We also found ataxia-telangiectasia mutated (ATM) protein and mRNA to be overexpressed in the resistant MCF-7-EPI(R) cells compared with MCF-7 cells and that epirubicin could activate ATM to promote E2F activity and FOXM1 expression. Furthermore, inhibition of ATM in U2OS cells with caffeine or depletion of ATM in MCF-7-EPI(R) with short interfering RNAs can resensitize these resistant cells to epirubicin, resulting in downregulation of E2F1 and FOXM1 expression and cell death. In summary, our data show that ATM and p53 coordinately regulate FOXM1 via E2F to modulate epirubicin response and resistance in breast cancer.

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

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.

FOXM1 confers acquired cisplatin resistance in breast cancer cells.

The transcription factor Forkhead box M1 (FOXM1) is a key regulator of cell proliferation and is overexpressed in many forms of primary cancers, leading to uncontrolled cell division and genomic instability. To address the role of FOXM1 in chemoresistance, we generated a cisplatin-resistant breast cancer cell line (MCF-7-CIS(R)), which had an elevated level of FOXM1 protein and mRNA expression relative to the parental MCF-7 cells. A close correlation was observed between FOXM1 and the expression of its proposed downstream targets that are involved in DNA repair; breast cancer-associated gene 2 (BRCA2) and X-ray cross-complementing group 1 (XRCC1) were expressed at higher levels in the resistant cell lines compared with the sensitive MCF-7 cells. Moreover, cisplatin treatment induced DNA damage repair in MCF-7-CIS(R) and not in MCF-7 cells. Furthermore, the expression of a constitutively active FOXM1 (DeltaN-FOXM1) in MCF-7 cells alone was sufficient to confer cisplatin resistance. Crucially, the impairment of DNA damage repair pathways through the small interfering RNA knockdown inhibition of either FOXM1 or BRCA2/XRCC1 showed that only the silencing of FOXM1 could significantly reduce the rate of proliferation in response to cisplatin treatment in the resistant cells. This suggests that the targeting of FOXM1 is a viable strategy in circumventing acquired cisplatin resistance. Consistently, the FOXM1 inhibitor thiostrepton also showed efficacy in causing cell death and proliferative arrest in the cisplatin-resistant cells through the downregulation of FOXM1 expression. Taken together, we have identified a novel mechanism of acquired cisplatin resistance in breast cancer cells through the induction of FOXM1.

Sphingosine-1-phosphate protects proliferating endothelial cells from ceramide-induced apoptosis but not from DNA damage-induced mitotic death.

Because of the central role of the endothelium in tissue homeostasis, protecting the vasculature from radiation-induced death is a major concern in tissue radioprotection. Premitotic apoptosis and mitotic death are two prevalent cell death pathways induced by ionizing radiation. Endothelial cells undergo apoptosis after radiation through generation of the sphingolipid ceramide. However, if mitotic death is known as the established radiation-induced death pathway for cycling eukaryotic cells, direct involvement of mitotic death in proliferating endothelial radiosensitivity has not been clearly shown. In this study, we proved that proliferating human microvascular endothelial cells (HMEC-1) undergo two waves of death after exposure to 15 Gy radiation: an early premitotic apoptosis dependent on ceramide generation and a delayed DNA damage-induced mitotic death. The fact that sphingosine-1-phosphate (S1P), a ceramide antagonist, protects HMEC-1 only from membrane-dependent apoptosis but not from DNA damage-induced mitotic death proves the independence of the two pathways. Furthermore, adding nocodazole, a mitotic inhibitor, to S1P affected both cell death mechanisms and fully prevented radiation-induced death. If our results fit with the standard model in which S1P signaling inhibits ceramide-mediated apoptosis induced by antitumor treatments, such as radiotherapy, they exclude, for the first time, a significant role of S1P-induced molecular survival pathway against mitotic death. Discrimination between ceramide-mediated apoptosis and DNA damage-induced mitotic death may give the opportunity to define a new class of radioprotectors for normal tissues in which quiescent endothelium represents the most sensitive target, while excluding malignant tumor containing pro-proliferating angiogenic endothelial cells that are sensitive to mitotic death.