Kwok-Ming Yao

Raf/MEK/MAPK signaling stimulates the nuclear translocation and transactivating activity of FOXM1c.

The forkhead box (FOX) transcription factor FOXM1 is ubiquitously expressed in proliferating cells. FOXM1 expression peaks at the G2/M phase of the cell cycle and its functional deficiency in mice leads to defects in mitosis. To investigate the role of FOXM1 in the cell cycle, we used synchronized hTERT-BJ1 fibroblasts to examine the cell cycle-dependent regulation of FOXM1 function. We observed that FOXM1 is localized mainly in the cytoplasm in cells at late-G1 and S phases. Nuclear translocation occurs just before entry into the G2/M phase and is associated with phosphorylation of FOXM1. Consistent with the dependency of FOXM1 function on mitogenic signals, nuclear translocation of FOXM1 requires activity of the Raf/MEK/MAPK signaling pathway and is enhanced by the MAPK activator aurintricarboxylic acid. This activating effect was suppressed by the MEK1/2 inhibitor U0126. In transient reporter assays, constitutively active MEK1 enhances the transactivating effect of FOXM1c, but not FOXM1b, on the cyclin B1 promoter. RT-PCR analysis confirmed that different cell lines and tissues predominantly express the FOXM1c transcript. Mutations of two ERK1/2 target sequences within FOXM1c completely abolish the MEK1 enhancing effect, suggesting a direct link between Raf/MEK/MAPK signaling and FOXM1 function. Importantly, inhibition of Raf/MEK/MAPK signaling by U0126 led to suppression of FOXM1 target gene expression and delayed progression through G2/M, verifying the functional relevance of FOXM1 activation by MEK1. In summary, we provide the first evidence that Raf/MEK/MAPK signaling exerts its G2/M regulatory effect via FOXM1c.

Over-expression of FoxM1 stimulates cyclin B1 expression.

FoxM1 (previously named WIN, HFH‐11 or Trident) is a Forkhead box (Fox) transcription factor widely expressed in proliferating cells. Various findings, including a recent analysis of FoxM1 knockout mice, suggest that FoxM1 is required for normal S–M coupling during cell cycle progression. To study the regulatory role of FoxM1 and its downstream regulatory targets, three stably transfected HeLa lines that display doxycycline (dox)‐inducible FoxM1 expression were established. Over‐expression of FoxM1 by dox induction facilitates growth recovery from serum starvation. Quantitation of cyclin B1 and D1 levels using flow cytometric, Western and Northern analyses reveals that elevated FoxM1 levels lead to stimulation of cyclin B1 but not cyclin D1 expression. Transient reporter assays in the dox‐inducible lines and upon co‐transfection with a constitutive FoxM1 expression plasmid suggest that FoxM1 can activate the cyclin B1 promoter.

The Forkhead Box M1 Protein Regulates the Transcription of the Estrogen Receptor α in Breast Cancer Cells

In this study, we have identified the Forkhead transcription factor FoxM1 as a physiological regulator of estrogen receptor α (ERα) expression in breast carcinoma cells. Our survey of a panel of 16 different breast cell lines showed a good correlation (13/16) between FoxM1 expression and expression of ERα at both protein and mRNA levels. We have also demonstrated that ectopic expression of FoxM1 in two different estrogen receptor-positive breast cancer cell lines, MCF-7 and ZR-75–30, led to up-regulation of ERα expression at protein and transcript levels. Furthermore, treatment of MCF-7 cells with the MEK inhibitor U0126, which blocks ERK1/2-dependent activation of FoxM1, also repressed ERα expression. Consistent with this, silencing of FoxM1 expression in MCF-7 cells using small interfering RNA resulted in the almost complete abrogation of ERα expression. We also went on to show that FoxM1 can activate the transcriptional activity of human ERα promoter primarily through two closely located Forkhead response elements located at the proximal region of the ERα promoter. Chromatin immunoprecipitation and biotinylated oligonucleotide pulldown assays have allowed us to confirm these Forkhead response elements as important for FoxM1 binding. Further co-immunoprecipitation experiments showed that FoxO3a and FoxM1 interact in vivo. Together with the chromatin immunoprecipitation and biotinylated oligonucleotide pulldown data, the co-immunoprecipitation results also suggest the possibility that FoxM1 and FoxO3a cooperate to regulate ERα gene transcription.

FoxM1c Counteracts Oxidative Stress-induced Senescence and Stimulates Bmi-1 Expression

The Forkhead box transcription factor FoxM1 is expressed in proliferating cells. When it was depleted in mice and cell lines, cell cycle defects and chromosomal instability resulted. Premature senescence was observed in embryonic fibroblasts derived from FoxM1 knock-out mice, but the underlying cause has remained unclear. To investigate whether FoxM1 can protect cells against stress-induced premature senescence, we established NIH3T3 lines with doxycycline-inducible overexpression of FoxM1c. Treatment of these lines with sublethal doses (20 and 100 μm) of H2O2 induced senescence with senescence-associated β-galactosidase expression and elevated levels of p53 and p21. Induction of FoxM1c expression markedly suppressed senescence and expression of p53 and p21. Consistent with down-regulation of the p19Arf-p53 pathway, p19Arf levels decreased while expression of the Polycomb group protein Bmi-1 was induced. That Bmi-1 is a downstream target of FoxM1c was further supported by the dose-dependent induction of Bmi-1 by FoxM1c at both the protein and mRNA levels, and FoxM1 and Bmi-1 reached maximal levels in cells at the G2/M phase. Depletion of FoxM1 by RNA interference decreased Bmi-1 expression. Using Bmi-1 promoter reporters with wild-type and mutated c-Myc binding sites and short hairpin RNAs targeting c-Myc, we further demonstrated that FoxM1c activated Bmi-1 expression via c-Myc, which was recently reported to be regulated by FoxM1c. Our results reveal a functional link between FoxM1c, c-Myc, and Bmi-1, which are major regulators of tumorigenesis. This link has important implications for the regulation of cell proliferation and senescence by FoxM1 and Bmi-1.

Bridge-1, a Novel PDZ-Domain Coactivator of E2A-Mediated Regulation of Insulin Gene Transcription

ABSTRACT Proteins in the E2A family of basic helix-loop-helix transcription factors are important in a wide spectrum of physiologic processes as diverse as neurogenesis, myogenesis, lymphopoeisis, and sex determination. In the pancreatic β cell, E2A proteins, in combination with tissue-specific transcription factors, regulate expression of the insulin gene and other genes critical for β-cell function. By yeast two-hybrid screening of a cDNA library prepared from rat insulinoma (INS-1) cells, we identified a novel protein, Bridge-1, that interacts with E2A proteins and functions as a coactivator of gene transcription mediated by E12 and E47. Bridge-1 contains a PDZ-like domain, a domain known to be involved in protein-protein interactions. Bridge-1 is highly expressed in pancreatic islets and islet cell lines and the expression pattern is primarily nuclear. The interaction of Bridge-1 with E2A proteins is further demonstrated by coimmunoprecipitation of in vitro-translated Bridge-1 with E12 or E47 and by mammalian two-hybrid studies. The PDZ-like domain of Bridge-1 is required for interaction with the carboxy terminus of E12. In both yeast and mammalian two-hybrid interaction studies, Bridge-1 mutants lacking an intact PDZ-like domain interact poorly with E12. An E12 mutant (E12ΔC) lacking the carboxy-terminal nine amino acids shows impaired interaction with Bridge-1. Bridge-1 has direct transactivational activity, since a Gal4 DNA-binding domain–Bridge-1 fusion protein transactivates a Gal4CAT reporter. Bridge-1 also functions as a coactivator by enhancing E12- or E47-mediated activation of a rat insulin I gene minienhancer promoter-reporter construct in transient-transfection experiments. Substitution of the mutant E12ΔC for E12 reduces the coactivation of the rat insulin I minienhancer by Bridge-1. Inactivation of endogenous Bridge-1 in insulinoma (INS-1) cells by expression of a Bridge-1 antisense RNA diminishes rat insulin I promoter activity. Bridge-1, by utilizing its PDZ-like domain to interact with E12, may provide a new mechanism for the coactivation and regulation of transcription of the insulin gene.

Signaling mechanisms of melatonin in antiproliferation of hormone-refractory 22Rv1 human prostate cancer cells: Implications for prostate cancer chemoprevention

Abstract:  There is an unmet clinical demand for safe and effective pharmaceuticals/nutraceuticals for prostate cancer prevention and hormone‐refractory prostate cancer treatment. Previous laboratory and human studies of our laboratory demonstrated an association between the antiproliferative action of melatonin and melatonin MT1 receptor expression in prostate cancer. The aim of this study was to determine, using a pharmacological approach, the signaling mechanisms of melatonin in hormone‐refractory 22Rv1 human prostate cancer cell antiproliferation. Both immunoreactive MT1 and MT2 subtypes of G protein‐coupled melatonin receptor were expressed in 22Rv1 cells. Melatonin inhibited, concentration dependently, cell proliferation, upregulated p27Kip1 gene transcription and protein expression, and downregulated activated androgen signaling in 22Rv1 cells. While the effects of melatonin were mimicked by 2‐iodomelatonin, a high‐affinity nonselective MT1 and MT2 receptor agonist, melatonin effects were blocked by luzindole, a nonselective MT1 and MT2 receptor antagonist, but were unaffected by 4‐phenyl‐2‐propionamidotetraline, a selective MT2 receptor antagonist. Importantly, we discovered that the antiproliferative effect of melatonin exerted via MT1 receptor on p27Kip1 gene and protein upregulation is mediated by a novel signaling mechanism involving co‐activation of protein kinase C (PKC) and PKA in parallel. Moreover, we also showed that a melatonin/MT1/PKC mechanism is involved in melatonin‐induced downregulation of activated androgen signal transduction in 22Rv1 cells. Taken together with the known molecular mechanisms of prostate cancer progression and transition to androgen independence, our data provide strong support for melatonin to be a promising small‐molecule useful for prostate cancer primary prevention and secondary prevention of the development and progression of hormone refractoriness.

Activation of AMPK inhibits cervical cancer cell growth through AKT/FOXO3a/FOXM1 signaling cascade

BackgroundAlthough advanced-stage cervical cancer can benefit from current treatments, approximately 30% patients may fail after definitive treatment eventually. Therefore, exploring alternative molecular therapeutic approaches is imperatively needed for this disease. We have recently shown that activation of AMP-activated protein kinase (AMPK), a metabolic sensor, hampers cervical cancer cell growth through blocking the Wnt/β-catenin signaling activity. Here, we report that activated AMPK (p-AMPK) also inhibits cervical cancer cell growth by counteracting FOXM1 function.MethodsEffect of the activation of AMPK on FOXM1 expression was examined by hypoxia and glucose deprivation, as well as pharmacological AMPK activators such as A23187, AICAR and metformin. RT Q-PCR and Western blot analysis were employed to investigate the activities of AMPK, FOXM1 and AKT/FOXO3a signaling.ResultsConsistent with our previous findings, the activation of AMPK by either AMPK activators such as AICAR, A23187, metformin, glucose deprivation or hypoxia significantly inhibited the cervical cancer cell growth. Importantly, we found that activated AMPK activity was concomitantly associated with the reduction of both the mRNA and protein levels of FOXM1. Mechanistically, we showed that activated AMPK was able to reduce AKT mediated phosphorylation of p-FOXO3a (Ser253). Interestingly, activated AMPK could not cause any significant changes in FOXM1 in cervical cancer cells in which endogenous FOXO3a levels were knocked down using siRNAs, suggesting that FOXO3a is involved in the suppression of FOXM1.ConclusionTaken together, our results suggest the activated AMPK impedes cervical cancer cell growth through reducing the expression of FOXM1.

Identification of cis-elements for ethylene and circadian regulation of the Solanum melongena gene encoding cysteine proteinase

We have previously shown that the expression of SmCP which encodes Solanum melongena cysteine proteinase is ethylene-inducible and is under circadian control. To understand the regulation of SmCP, a 1.34-kb SmCP 5′-flanking region and its deletion derivatives were analyzed for cis-elements using GUS and luc fusions and by invitro binding assays. Analysis of transgenic tobacco transformed with SmCP promoter-GUS constructs confirmed that the promoter region −415/+54 containing Ethylene Responsive Element ERE(−355/−348) conferred threefold ethylene-induction of GUS expression, while −827/+54 which also contains ERE(−683/−676), produced fivefold induction. Using gel mobility shift assays, we demonstrated that each ERE binds nuclear proteins from both ethephon-treated and untreated 5-week-old seedlings, suggesting that different transcriptions factors bind each ERE under varying physiological conditions. Binding was also observed in extracts from senescent, but not young, fruits. The variation in binding at the EREs in fruits and seedlings imply that organ-specific factors may participate in binding. Analysis of transgenic tobacco expressing various SmCP promoter-lucconstructs containing wild-type or mutant Evening Elements (EEs) confirmed that both conserved EEs at −795/−787 and −785/−777 are important in circadian control. We confirmed the binding of total nuclear proteins to EEs in gel mobility shift assays and in DNase I footprinting. Our results suggest that multiple proteins bind the EEs which are conserved in plants other thanArabidopsisand that functional EEs and EREs are present in the 5′-flanking region of a gene encoding cysteine proteinase.

Identification and Characterization of Human VCY2-Interacting Protein: VCY2IP-1, a Microtubule-Associated Protein-Like Protein

Abstract VCY2 is a testis-specific protein that locates in a frequently deleted azoospermia factor c region on chromosome Yq. Although its genomic structure has been characterized, the function of VCY2 is still unknown. To gain insight regarding the likely function of VCY2, we investigated the proteins that interact with VCY2 using the yeast two-hybrid system. We identified a novel VCY2 interaction partner, named VCY2IP-1, that encodes an open reading frame of 1059 amino acids. The amino acid sequence of VCY2IP-1 shows 59.3% and 41.9% homology to two human microtubule-associated proteins (MAPs), MAP1B and MAP1A, respectively. VCY2IP-1 has an extensive homology to the N-terminus and C-terminus regions of MAP1B and MAP1A, placing it within a large family of MAPs. We mapped VCY2IP-1 to chromosome 19p13.11. The VCY2IP-1 gene spans 15 kilobases (kb) and consists of seven exons. Northern blot analysis identified a single, intense band of approximately 3.2-kb VCY2IP-1 transcript, predominantly expressed in human testis. In situ hybridization of human testicular sections showed the localization of VCY2IP-1 transcripts in germ cells, and reverse transcription-polymerase chain reaction analysis demonstrated the presence of VCY2 and VCY2IP-1 transcripts in human ejaculated spermatozoa. Our expression data support the involvement of VCY2 and VCY2IP-1 in spermatogenesis. Based on the high homology of VCY2IP-1 with MAPs, we propose the involvement of VCY2 in the cytoskeletal network via interaction with VCY2IP-1.

Signal transduction of receptor-mediated antiproliferative action of melatonin on human prostate epithelial cells involves dual activation of Gαs and Gαq proteins

Abstract:  Melatonin has been shown to inhibit the proliferation of malignant and transformed human prostate epithelial cells by transcriptional up‐regulation of p27Kip1 expression via MTNR1A receptor‐mediated activation of protein kinase A (PKA) and protein kinase C (PKC) in parallel. Given that melatonin MTNR1A receptor is a G protein‐coupled receptor, this study was conducted to identify the specific G proteins that mediate the antiproliferative action of melatonin on human prostate epithelial cells. In 22Rv1 and RWPE‐1 cells, knockdown of either Gαs or Gαq, but not Gαi2 expression by RNA interference, abrogated the effects of melatonin on p27Kip1 and cell proliferation. Conversely, cellular overexpression of activated mutants of Gαs and Gαq in 22Rv1 and RWPE‐1 cells mimicked the effects of melatonin on prostate epithelial cell antiproliferation by increasing p27Kip1 expression through downstream activation of PKA and PKC in parallel. Moreover, melatonin or 2‐iodomelatonin induced elevation of adenosine‐3′,5′‐cyclic monophosphate (cAMP) in 22Rv1 and RWPE‐1 cells. The effects of 2‐iodomelatonin on cAMP were blocked by the nonselective MTNR1A/MTNR1B receptor antagonist luzindole but were not affected by the selective MTNR1B receptor antagonist 4‐phenyl‐2‐propionamidotetraline (4‐P‐PDOT). Furthermore, knockdown of Gαs mitigated the stimulatory effects of 2‐iodomelatonin on cAMP. Collectively, the data demonstrated, for the first time, functional coupling of MTNR1A receptor to Gαs in cancerous or transformed human cells expressing endogenous melatonin receptors. Our results also showed that dual activation of Gαs and Gαq proteins is involved in the signal transduction of MTNR1A receptor‐mediated antiproliferative action of melatonin on human prostate epithelial cells.