Haojie Huang

Cyclin-dependent kinases regulate epigenetic gene silencing through phosphorylation of EZH2

The Polycomb group (PcG) protein, enhancer of zeste homologue 2 (EZH2), has an essential role in promoting histone H3 lysine 27 trimethylation (H3K27me3) and epigenetic gene silencing. This function of EZH2 is important for cell proliferation and inhibition of cell differentiation, and is implicated in cancer progression. Here, we demonstrate that under physiological conditions, cyclin-dependent kinase 1 (CDK1) and cyclin-dependent kinase 2 (CDK2) phosphorylate EZH2 at Thr 350 in an evolutionarily conserved motif. Phosphorylation of Thr 350 is important for recruitment of EZH2 and maintenance of H3K27me3 levels at EZH2-target loci. Blockage of Thr 350 phosphorylation not only diminishes the global effect of EZH2 on gene silencing, it also mitigates EZH2-mediated cell proliferation and migration. These results demonstrate that CDK-mediated phosphorylation is a key mechanism governing EZH2 function and that there is a link between the cell-cycle machinery and epigenetic gene silencing.

Inhibition of the Androgen Receptor as a Novel Mechanism of Taxol Chemotherapy in Prostate Cancer

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.

CDK1 promotes cell proliferation and survival via phosphorylation and inhibition of FOXO1 transcription factor

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.

FOXO1 Inhibits Runx2 Transcriptional Activity and Prostate Cancer Cell Migration and Invasion

Prostate cancer patients with regional lymph node involvement at radical prostatectomy often experience disease progression to other organs, with the bone as the predominant site. The transcription factor Runx2 plays an important role in bone formation and prostate cancer cell migration, invasion, and metastasis. Here we showed that the forkhead box O (FOXO1) protein, a key downstream effector of the tumor suppressor PTEN, inhibits the transcriptional activity of Runx2 in prostate cancer cells. This inhibition was enhanced by PTEN but diminished by active Akt. FOXO1 bound to Runx2 in vitro and in vivo and suppressed Runx2s activity independent of its transcriptional function. FOXO1 inhibited Runx2-promoted migration of prostate cancer cells, whereas silencing of endogenous FOXO1 enhanced prostate cancer cell migration in a Runx2-dependent manner. Forced expression of FOXO1 also inhibited Runx2-promoted prostate cancer cell invasion. Finally, we found that expression of PTEN and the level of FOXO1 in the nucleus is inversely correlated with expression of Runx2 in a cohort of prostate cancer specimens from patients with lymph node and bone metastasis. These data reveal FOXO1 as a critical negative regulator of Runx2 in prostate cancer cells. Inactivation of FOXO1 due to frequent loss of PTEN in prostate cancer cells may leave the oncogenic activities of Runx2 unchecked, thereby driving promiscuous expression of Runx2 target genes involved in cell migration and invasion and favoring prostate cancer progression.

Androgens Suppress EZH2 Expression Via Retinoblastoma (RB) and p130-Dependent Pathways: A Potential Mechanism of Androgen-Refractory Progression of Prostate Cancer

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.

A Transcription-Independent Function of FOXO1 in Inhibition of Androgen-Independent Activation of the Androgen Receptor in Prostate Cancer Cells

Increasing evidence suggests that aberrant activation of the androgen receptor (AR) plays a pivotal role in the development and progression of androgen depletion-independent prostate cancer (PCa) after androgen deprivation therapy. Here, we show that loss of the PTEN tumor suppressor gene is associated with hyperactivation of the AR in human PCa cell lines. This effect is mediated primarily by its downstream effector FOXO1. In addition to the inhibition of androgenic activation of the AR, forced expression of FOXO1 in PTEN-negative PCa cells also inhibits androgen-independent activation of the AR in a manner independent of FOXO1 transcriptional function. In contrast, silencing of FOXO1 in PTEN-positive cells not only increases the basal activity of the AR in the absence of androgens, it also markedly sensitizes the AR activation by low levels of androgens or nonandrogenic factors such as interleukin-6. FOXO1-mediated inhibition of the AR is partially attenuated by the histone deacetylase (HDAC) inhibitor trichostatin A. Accordingly, FOXO1 interacts with HDAC3 as shown by coimmunoprecipitation assays, and cotransfection of cells with FOXO1 and HDAC3, but not HDAC1 and HDAC2, results in a greater inhibition of AR activity than in cells transfected with FOXO1 or HDAC3 individually. Together, our findings define a novel corepressor function of FOXO1 in inhibition of androgen-independent activation of the AR.

Cyclin D1 promotes anchorage-independent cell survival by inhibiting FOXO-mediated anoikis

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.

Reduced tumor necrosis factor receptor-associated death domain expression is associated with prostate cancer progression

By using LNCaP and its derivative cell lines, we first observed an association between tumor necrosis factor-alpha (TNF-alpha) resistance and hormone independence. Moreover, we found that the expression of tumor necrosis factor receptor-associated death domain (TRADD) was reduced in androgen deprivation-independent cells compared with that in androgen deprivation-dependent cells. TRADD is a crucial transducer for TNF-alpha-induced nuclear factor-kappaB (NF-kappaB) activation. Knocking down TRADD expression in LNCaP cells impaired TNF-alpha-induced NF-kappaB activation and androgen receptor repression, whereas overexpression of TRADD in C4-2B cells restored their sensitivity to TNF-alpha. Finally, we found that androgen deprivation reduces TRADD expression in vitro and in vivo, suggesting that androgen deprivation therapy may promote the development of TNF-alpha resistance by reducing TRADD expression during prostate cancer progression.

Androgens repress expression of the f-box protein Skp2 via p107 dependent and independent mechanisms in LNCaP prostate cancer cells

Androgens control homeostasis of the normal prostate and growth of prostate cancer (PCa) through the androgen receptor (AR) by regulating gene networks involving in cell proliferation, differentiation, and survival. We demonstrated previously that expression of Skp2, a key protein regulating cell entry into the S phase, is inhibited by androgens in an AR‐dependent manner (Oncogene, 2004; 23(12): 2161–2176). However, the underlying mechanism of this regulation is unknown.

Erratum: Cyclin D1 promotes anchorage-independent cell survival by inhibiting FOXO-mediated anoikis (Cell Death and Differentiation (2009) 16 (1408-1417) DOI: 10.1038/cdd.2009.86))

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.