Haojie Huang

The forkhead box O (FoxO) transcription factors are known to be involved in many physiological and pathological processes including apoptosis, cell cycle arrest, stress resistance, glucose metabolism, cellular differentiation and development, and tumor suppression. The environmental cues, such as growth factors, nutrients, oxidative stress and irradiation, can either positively or negatively modulate FoxO proteins; activities, thereby ensuring distinctive transcription programs in the cell. The potent activities of FoxOs are tightly controlled by multiple mechanisms, which include posttranslational modification such as phosphorylation, acetylation, methylation and ubiquitination, subcellular localization, and direct protein-protein interaction. Mounting evidence suggests that the human FOXO1 protein, a founding member of the FoxO family is likely involved in carcinogenesis, diabetes and other human diseases. Here we give an overview of most recent findings regarding the regulation and function of FoxO1, its potential role in human diseases and useful animal models for functional studies on FoxO1. Prospective ways in which the discoveries from the basic research of FoxO1 can be utilized for drug targeting and development of novel therapeutics for human diseases are also discussed.

Androgens and the androgen receptor are important for both normal prostate development and progression of prostate cancer (PCa). However, the underlying mechanisms are not fully understood. The Polycomb protein enhancer of zeste homolog 2 (EZH2) functions as an epigenetic gene silencer and plays a role in oncogenesis by promoting cell proliferation and invasion. EZH2 has been implicated in human PCa progression, because its expression is often elevated in hormone-refractory PCa. Here, we demonstrated that expression of EZH2 is lower in androgen-sensitive LNCaP PCa cells compared with Rf and C4-2 cells, two androgen-refractory sublines that are derived from LNCaP cells. Androgen ablation by castration increased the level of EZH2 proteins in LNCaP xenografts in mice. In contrast, treatment of LNCaP cells in culture with the synthetic androgen methyltrieolone (R1881) at doses of 1 nm or higher suppressed EZH2 expression. Moreover, our data suggest that androgen repression of EZH2 requires a functional androgen receptor and this effect is mediated through the retinoblastoma protein and its related protein p130. We further showed that androgen treatment not only increases expression of EZH2 target genes DAB2IP and E-cadherin but also affects LNCaP cell migration. Our results reveal that androgens function as an epigenetic regulator in prostatic cells by repression of EZH2 expression through the retinoblastoma protein and p130-dependent pathways. Our findings also suggest that blockade of EZH2 derepression during androgen deprivation therapy may represent an effective tactic for the treatment of androgen-refractory PCa.

Both cell culture and clinical studies show that the androgen receptor (AR) plays a key role in the growth and survival of castration-resistant prostate cancer (CRPC), a lethal form of the disease in the clinic, suggesting that AR remains to be a major target for the treatment of CRPC. Taxol chemotherapy is one of the few therapeutic options for patients with CRPC albeit the underlying mechanism is not fully understood. We have demonstrated recently that Taxol (paclitaxel and its semisynthetic analogue docetaxel) treatment of 22Rv1, a CRPC cell line that expresses the tumor suppressor gene PTEN, inhibits AR transcriptional activity. In contrast, paclitaxel failed to inhibit AR activity in the PTEN-deficient C4-2 CRPC cells. Docetaxel treatment of 22Rv1 xenografts in mice induced mitotic arrest and a decrease in expression of the AR target gene prostate-specific antigen (PSA) mainly in tumor cells adjacent to vascular vessels. Further studies demonstrated that Taxol inhibition of the AR is mediated, at least in part, by Taxol-induced nuclear accumulation of FOXO1, a key downstream effector protein of PTEN and increased association of FOXO1 with the AR. These studies suggest that the status of the functional PTEN/FOXO pathway and the drug bioavailability may be the two key determinants for Taxol chemoresistance of CRPC in the clinic.

Taxol chemotherapy is one of the few therapeutic options for men with castration-resistant prostate cancer (CRPC). However, the working mechanisms for Taxol are not fully understood. Here, we showed that treatment of 22Rv1, a PTEN-positive CRPC cell line, with paclitaxel and its semisynthetic analogue docetaxel decreases expression of the androgen receptor (AR)–activated genes prostate-specific antigen (PSA) and Nkx3.1 but increases expression of the AR repression gene maspin, suggesting that Taxol treatment inhibits AR activity. This was further supported by the observation that the activity of AR luciferase reporter genes was inhibited by paclitaxel. In contrast, paclitaxel treatment failed to inhibit AR activity in the PTEN-null CRPC cell line C4-2. However, pretreatment of C4-2 cells with the phosphoinositide 3-kinase inhibitor LY294002 restored paclitaxel inhibition of the AR. Treatment of 22Rv1 xenografts in mice with docetaxel induced mitotic arrest and a decrease in PSA expression in tumor cells adjacent to vascular vessels. We further showed that paclitaxel induces nuclear accumulation of FOXO1, a known AR suppressive nuclear factor, and increases the association of FOXO1 with AR proteins in the nucleus. FOXO1 knockdown with small interfering RNA attenuated the inhibitory effect of paclitaxel on AR transcriptional activity, expression of PSA and Nkx3.1, and cell survival. These data reveal a previously uncharacterized, FOXO1-mediated, AR-inhibitory effect of Taxol in CRPC cells that may play an important role in Taxol-mediated inhibition of CRPC growth. [Cancer Res 2009;69(21):8386–94]

O-class forkhead box (FOXO) transcription factors are critical regulators of diverse cellular processes, including apoptosis, cell-cycle arrest, DNA damage repair and oxidative stress resistance. Here, we show that FOXO1 and FOXO3a have an essential function in promoting cell detachment-induced anoikis, resistance to which is implicated in cancer development and metastasis. In contrast, the oncoprotein cyclin D1 inhibits anoikis. We further show that cyclin D1 interacts with FOXO proteins and impedes their transcriptional regulatory and anoikis-promoting functions. This effect of cyclin D1 requires its transcription repression domain but is independent of cyclin-dependent kinases CDK4 and CDK6. Moreover, we show that cancer-derived mutants of cyclin D1 are much more stable than wild-type cyclin D1 under anchorage-independent conditions and possess a greater antagonistic effect on FOXO-regulated anoikis and anchorage-independent growth of cancer cells. These data suggest that cyclin D1 may have a critical function in tumorigenesis and cancer metastasis by inhibiting the anoikis-promoting function of FOXO proteins.

The forkhead box O (FOXO) transcription factor FOXO1 functions as a tumor suppressor by regulating expression of genes involved in apoptosis, cell cycle arrest and oxidative detoxification. Here, we demonstrate that cyclin-dependent kinase 1 (CDK1) specifically phosphorylates FOXO1 at serine 249 (S249) in vitro and in vivo. Coimmunoprecipitation assays demonstrate that both endogenous CDK1 and ectopically expressed CDK1 form a protein complex with FOXO1 in prostate cancer (PCa) cells. In vitro protein binding assays reveal that CDK1 interacts directly with FOXO1. Accordingly, overexpression of CDK1 inhibits the transcriptional activity of FOXO1 in PCa cells through S249 phosphorylation on FOXO1. Consistent with the roles of FOXO3a and FOXO4 (two other members of the FOXO family) in cell cycle regulation, forced expression of FOXO1 causes a delay in the transition from G2 to M phase. This effect is blocked completely by overexpression of CDK1 and cyclin B1. Ectopic expression of constitutively active CDK1 also inhibits FOXO1-induced apoptosis in PCa cells. Moreover, we demonstrate that the inhibitory effect of FOXO1 on Ras oncogene-induced colony formation in fibroblasts is diminished by overexpression of CDK1. Given that CDK1 and cyclin B1 are often overexpressed in human cancers including PCa, our findings suggest that aberrant activation of CDK1 may contribute to tumorigenesis by promoting cell proliferation and survival via phosphorylation and inhibition of FOXO1.

The proinflammatory cytokine tumor necrosis factor α (TNFα) inhibits hematopoietic stem cell (HSC) expansion, interferes with HSC self-renewal and compromises the ability of HSC to reconstitute hematopoiesis. We have investigated mechanisms by which TNFα suppresses hematopoiesis using the genomic instability syndrome Fanconi anemia mouse model deficient for the complementation-group-C gene (Fancc). Examination of senescence makers, such as senescence-associated β-galactosidase, HP1-γ, p53 and p16^INK4A shows that TNFα induces premature senescence in bone marrow HSCs and progenitor cells as well as other tissues of Fancc^–/– mice. TNFα-induced senescence correlates with the accumulation of reactive oxygen species (ROS) and oxidative DNA damage. Neutralization of TNFα or deletion of the TNF receptor in Fancc^–/– mice (Fancc^–/–;Tnfr1^–/–) prevents excessive ROS production and hematopoietic senescence. Pretreatment of TNFα-injected Fancc^–/– mice with a ROS scavenger significantly reduces oxidative base damage, DNA strand breaks and senescence. Furthermore, HSCs and progenitor cells from TNFα-treated Fancc^–/– mice show increased chromosomal aberrations and have an impaired oxidative DNA-damage repair. These results indicate an intimate link between inflammatory reactive oxygen species and DNA-damage-induced premature senescence in HSCs and progenitor cells, which may play an important role in aging and anemia.

It has been well established that cyclin-dependent kinases (CDKs) including CDK2 and CDK1 play an essential role in mediating cell-cycle arrest, DNA repair and apoptosis in cells with damaged genomes. Following DNA damage, the function of CDKs is inhibited, and therefore cells are arrested at the G1, S or G2/M phase of the cell-division cycle. It is believed that DNA damage-induced cell cycle arrest is important, because it allows cells to either repair the damage and survive or commit to apoptotic cell death. However, the molecular bases for the two cell fate options are not fully understood. Recently, we provided evidence that CDK2 interacts with and phosphorylates the forkhead transcription factor FOXO1. Importantly, activated CDK2 inhibits the pro-apoptotic function of FOXO1. However, in the presence of high levels of double-strand breaks (DSBs) the CDK2-mediated inhibition of FOXO1 is abrogated due to the ablation of CDK2 activity, and therefore the anti-apoptotic function of FOXO1 is restored. Thus, CDK2 silencing-dependent activation of FOXO1 represents a new mechanism that links DNA strand breakage to cell death.