Retraction: Non-epigenetic function of HDAC8 in regulating breast cancer stem cells by maintaining Notch1 protein stability.

[This retracts the article DOI: 10.18632/oncotarget.6427.].

Targeting breast cancer stem cells by novel HDAC3-selective inhibitors.

Although histone deacetylase (HDAC) inhibitors have been known to suppress the cancer stem cell (CSC) population in multiple types of cancer cells, it remains unclear which HDAC isoforms and corresponding mechanisms contribute to this anti-CSC activity. Pursuant to our previous finding that HDAC8 regulates CSCs in triple-negative breast cancer (TNBC) cells by targeting Notch1 stability, we investigated related pathways and found HDAC3 to be mechanistically linked to CSC homeostasis by increasing ß-catenin expression through the Akt/GSK3ß pathway. Accordingly, we used a pan-HDAC inhibitor, AR-42 (1), as a scaffold to develop HDAC3-selective inhibitors, obtaining the proof-of-concept with 18 and 28. These two derivatives exhibited high potency and isoform selectivity in HDAC3 inhibition. Equally important, they showed in vitro and/or in vivo efficacy in suppressing the CSC subpopulation of TNBC cells via the downregulation of ß-catenin.

Pharmacological strategies to target oncogenic KRAS signaling in pancreatic cancer.

The clear importance of mutated KRAS as a therapeutic target has driven the investigation of multiple approaches to inhibit oncogenic KRAS signaling at different molecular levels. However, no KRAS-targeted therapy has reached the clinic to date, which underlies the intrinsic difficulty in developing effective, direct inhibitors of KRAS. Thus, this article provides an overview of the history and recent progress in the development of pharmacological strategies to target oncogenic KRAS with small molecule agents. Mechanistically, these KRAS-targeted agents can be classified into the following four categories. (1) Small-molecule RAS-binding ligands that prevent RAS activation by binding within or outside the nucleotide-binding motif. (2) Inhibitors of KRAS membrane anchorage. (3) Inhibitors that bind to RAS-binding domains of RAS-effector proteins. (4) Inhibitors of KRAS expression. The advantage and limitation of each type of these anti-KRAS agents are discussed.

Non-epigenetic function of HDAC8 in regulating breast cancer stem cells by maintaining Notch1 protein stability.

Here, we report a novel non-epigenetic function of histone deacetylase (HDAC) 8 in activating cancer stem cell (CSC)-like properties in breast cancer cells by enhancing the stability of Notch1 protein. The pan-HDAC inhibitors AR-42 and SAHA, and the class I HDAC inhibitor depsipeptide, suppressed mammosphere formation and other CSC markers by reducing Notch1 expression in MDA-MB-231 and SUM-159 cells. Interrogation of individual class I isoforms (HDAC1-3 and 8) using si/shRNA-mediated knockdown, ectopic expression and/or pharmacological inhibition revealed HDAC8 to be the primary mediator of this drug effect. This suppression of Notch1 in response to HDAC8 inhibition was abrogated by the proteasome inhibitor MG132 and siRNA-induced silencing of Fbwx7, indicating Notch1 suppression occurred through proteasomal degradation. However, co-immunoprecipitation analysis indicated that HDAC8 did not form complexes with Notch1 and HDAC inhibition had no effect on Notch1 acetylation. In a xenograft tumor model, the tumorigenicity of breast cancer cells was decreased by HDAC8 knockdown. These findings suggest the therapeutic potential of HDAC8 inhibition to suppress Notch1 signaling in breast cancer.

Exploitation of the ability of γ-tocopherol to facilitate membrane co-localization of Akt and PHLPP1 to develop PHLPP1-targeted Akt inhibitors.

Previously, we reported that Akt inactivation by γ-tocopherol (2) in PTEN-negative prostate cancer cells resulted from its unique ability to facilitate membrane co-localization of Akt and PHLPP1 (PH domain leucine-rich repeat protein phosphatase isoform 1), a Ser473-specific Akt phosphatase, through pleckstrin homology (PH) domain binding. This finding provided a basis for exploiting 2 to develop a novel class of PHLPP1-targeted Akt inhibitors. Here, we used 3 (γ-VE5), a side chain-truncated 2 derivative, as a scaffold for lead optimization. The proof-of-concept of this structural optimization was obtained by 20, which exhibited higher antitumor efficacy than 3 in PTEN-negative cancer cells through PHLPP1-facilitated Akt inactivation. Like 3, 20 preferentially recognized the PH domains of Akt and PHLPP1, as its binding affinities for other PH domains, including those of ILK and PDK1, were an order-of-magnitude lower. Moreover, 20 was orally active in suppressing xenograft tumor growth in nude mice, which underlines the translational potential of this new class of Akt inhibitor in PTEN-deficient cancers.

AMPK as a potential anticancer target - friend or foe?

Adenosine monophosphate-activated protein kinase (AMPK) is a key player in maintaining energy homeostasis in response to metabolic stress. Beyond diabetes and metabolic syndrome, there is a growing interest in the therapeutic exploitation of the AMPK pathway in cancer treatment in light of its unique ability to regulate cancer cell proliferation through the reprogramming of cell metabolism. Although many studies support the tumor-suppressive role of AMPK, emerging evidence suggests that the metabolic checkpoint function of AMPK might be overridden by stress or oncogenic signals so that tumor cells use AMPK activation as a survival strategy to gain growth advantage. These findings underscore the complexity in the cellular function of AMPK in maintaining energy homeostasis under physiological versus pathological conditions. Thus, this review aims to provide an overview of recent findings on the functional interplay of AMPK with different cell metabolic and signaling effectors, particularly histone deacetylases, in mediating downstream tumor suppressive or promoting mechanisms in different cell systems. Although AMPK activation inhibits tumor growth by targeting multiple signaling pathways relevant to tumorigenesis, under certain cellular contexts or certain stages of tumor development, AMPK might act as a protective response to metabolic stresses, such as nutrient deprivation, low oxygen, and low pH, or as downstream effectors of oncogenic proteins, including androgen receptor, hypoxia-inducible factor-1α, c-Src, and MYC. Thus, investigations to define at which stage(s) of tumorigenesis and cancer progression or for which genetic aberrations AMPK inhibition might represent a more relevant strategy than AMPK activation for cancer treatment are clearly warranted.

Functional Role of mTORC2 versus Integrin-Linked Kinase in Mediating Ser473-Akt Phosphorylation in PTEN-Negative Prostate and Breast Cancer Cell Lines.

Although the rictor-mTOR complex (mTORC2) has been shown to act as phosphoinositide-dependent kinase (PDK)2 in many cell types, other kinases have also been implicated in mediating Ser473-Akt phosphorylation. Here, we demonstrated the cell line specificity of integrin-linked kinase (ILK) versus mTORC2 as PDK2 in LNCaP and PC-3 prostate and MDA-MB-468 breast cancer cells, of which the PTEN-negative status allowed the study of Ser473-Akt phosphorylation independent of external stimulation. PC-3 and MDA-MB-468 cells showed upregulated ILK expression relative to LNCaP cells, which expressed a high abundance of mTOR. Exposure to Ku-0063794, a second-generation mTOR inhibitor, decreased Ser473-Akt phosphorylation in LNCaP cells, but not in PC-3 or MDA-MB-468 cells. In contrast, treatment with T315, a novel ILK inhibitor, reduced the phosphorylation of Ser473-Akt in PC-3 and MDA-MB-468 cells without affecting that in LNCaP cells. This cell line specificity was verified by comparing Ser473-Akt phosphorylation status after genetic knockdown of rictor, ILK, and other putative Ser-473-Akt kinases. Genetic knockdown of rictor, but not ILK or the other kinases examined, inhibited Ser473-Akt phosphorylation in LNCaP cells. Conversely, PC-3 and MDA-MB-468 cells were susceptible to the effect of ILK silencing on Ser473-Akt phosphorylation, while knockdown of rictor or any of the other target kinases had no appreciable effect. Co-immunoprecipitation analysis demonstrated the physical interaction between ILK and Akt in PC-3 cells, and T315 blocked ILK-mediated Ser473 phosphorylation of bacterially expressed Akt. ILK also formed complexes with rictor in PC-3 and MDA-MB-468 cells that were disrupted by T315, but such complexes were not observed in LNCaP cells. In the PTEN-functional MDA-MB-231 cell line, both T315 and Ku-0063794 suppressed EGF-induced Ser473-Akt phosphorylation. Inhibition of ILK by T315 or siRNA-mediated knockdown suppressed epithelial-mesenchymal transition in MDA-MB-468 and PC-3 cells. Thus, we hypothesize that ILK might bestow growth advantage and metastatic potential in the course of tumor progression.

Vitamin E facilitates the inactivation of the kinase Akt by the phosphatase PHLPP1.

Vitamin E is a fat-soluble vitamin with antioxidant properties. Tocopherols are the predominant form of vitamin E found in the diet and in supplements and have garnered interest for their potential cancer therapeutic and preventive effects, such as the dephosphorylation of Akt, a serine/threonine kinase with a pivotal role in cell growth, survival, and metabolism. Dephosphorylation of Akt at Ser473 substantially reduces its catalytic activity and inhibits downstream signaling. We found that the mechanism by which α-tocopherol and γ-tocopherol facilitate this site-specific dephosphorylation of Akt was mediated through the pleckstrin homology (PH) domain-dependent recruitment of Akt and PHLPP1 (PH domain leucine-rich repeat protein phosphatase, isoform 1) to the plasma membrane. We structurally optimized these tocopherols to obtain derivatives with greater in vitro potency and in vivo tumor-suppressive activity in two prostate xenograft tumor models. Binding affinities for the PH domains of Akt and PHLPP1 were greater than for other PH domain-containing proteins, which may underlie the preferential recruitment of these proteins to membranes containing tocopherols. Molecular modeling revealed the structural determinants of the interaction with the PH domain of Akt that may inform strategies for continued structural optimization. By describing a mechanism by which tocopherols facilitate the dephosphorylation of Akt at Ser473, we provide insights into the mode of antitumor action of tocopherols and a rationale for the translational development of tocopherols into novel PH domain-targeted Akt inhibitors.

The mRNA-stabilizing factor HuR protein is targeted by ß-TrCP protein for degradation in response to glycolysis inhibition.

The mRNA-stabilizing protein HuR acts a stress response protein whose function and/or protein stability are modulated by diverse stress stimuli through posttranslational modifications. Here, we report a novel mechanism by which metabolic stress facilitates proteasomal degradation of HuR in cancer cells. In response to the glucose transporter inhibitor CG-5, HuR translocates to the cytoplasm, where it is targeted by the ubiquitin E3 ligase ß-TrCP1 for degradation. The cytoplasmic localization of HuR is facilitated by PKCα-mediated phosphorylation at Ser-318 as the Ser-318 → alanine substitution abolishes the ability of the resulting HuR to bind PKCα and to undergo nuclear export. The mechanistic link between ß-TrCP1 and HuR degradation was supported by the ability of ectopically expressed ß-TrCP1 to mimic CG-5 to promote HuR degradation and by the protective effect of dominant negative inhibition of ß-TrCP1 on HuR ubiquitination and degradation. Substrate targeting of HuR by ß-TrCP1 was further verified by coimmunoprecipitation and in vitro GST pull-down assays and by the identification of a ß-TrCP1 recognition site. Although HuR does not contain a DSG destruction motif, we obtained evidence that ß-TrCP1 recognizes an unconventional motif, (296)EEAMAIAS(304), in the RNA recognition motif 3. Furthermore, mutational analysis indicates that IKKα-dependent phosphorylation at Ser-304 is crucial to the binding of HuR to ß-TrCP1. Mechanistically, this HuR degradation pathway differs from that reported for heat shock and hypoxia, which underlies the complexity in the regulation of HuR turnover under different stress stimuli. The ability of glycolysis inhibitors to target the expression of oncogenic proteins through HuR degradation might foster novel strategies for cancer therapy.

Targeting the oncogenic E3 ligase Skp2 in prostate and breast cancer cells with a novel energy restriction-mimetic agent.

Substantial evidence supports the oncogenic role of the E3 ubiquitin ligase S-phase kinase-associated protein 2 (Skp2) in many types of cancers through its ability to target a broad range of signaling effectors for ubiquitination. Thus, this oncogenic E3 ligase represents an important target for cancer drug discovery. In this study, we report a novel mechanism by which CG-12, a novel energy restriction-mimetic agent (ERMA), down-regulates the expression of Skp2 in prostate cancer cells. Pursuant to our previous finding that upregulation of ß-transducin repeat-containing protein (ß-TrCP) expression represents a cellular response in cancer cells to ERMAs, including CG-12 and 2-deoxyglucose, we demonstrated that this ß-TrCP accumulation resulted from decreased Skp2 expression. Evidence indicates that Skp2 targets ß-TrCP for degradation via the cyclin-dependent kinase 2-facilitated recognition of the proline-directed phosphorylation motif (412)SP. This Skp2 downregulation was attributable to Sirt1-dependent suppression of COP9 signalosome (Csn)5 expression in response to CG-12, leading to increased cullin 1 neddylation in the Skp1-cullin1-F-box protein complex and consequent Skp2 destabilization. Moreover, we determined that Skp2 and ß-TrCP are mutually regulated, providing a feedback mechanism that amplifies the suppressive effect of ERMAs on Skp2. Specifically, cellular accumulation of ß-TrCP reduced the expression of Sp1, a ß-TrCP substrate, which, in turn, reduced Skp2 gene expression. This Skp2-ß-TrCP-Sp1 feedback loop represents a novel crosstalk mechanism between these two important F-box proteins in cancer cells with aberrant Skp2 expression under energy restriction, which provides a proof-of-concept that the oncogenic Csn5/Skp2 signaling axis represents a "druggable" target for this novel ERMA.

Differential anti-proliferative activities of poly(ADP-ribose) polymerase (PARP) inhibitors in triple-negative breast cancer cells.

Despite recent advances in the clinical evaluation of various poly(ADP-ribose) polymerase (PARP) inhibitors in triple-negative breast cancer (TNBC) patients, data defining potential anti-tumor mechanisms beyond PARP inhibition for these agents are lacking. To address this issue, we investigated the effects of four different PARP inhibitors (AG-014699, AZD-2281, ABT-888, and BSI-201) in three genetically distinct TNBC cell lines (MDA-MB-468, MDA-MB-231, and Cal-51). Assays of cell viability and colony formation and flow cytometric analysis were used to determine effects on cell growth and cell cycle progression. PARP-dependent and -independent signaling mechanisms of each PARP inhibitor were investigated by western blotting and shRNA approaches. Potential synergistic interactions between PARP inhibitors and cisplatin in suppressing TNBC cell viability were assessed. These PARP inhibitors exhibited differential anti-tumor activities, with the relative potencies of AG-014699 > AZD-2281 > ABT-888 > BSI-201. The higher potencies of AG-014699 and AZD-2281 were associated with their effects on G(2)/M arrest and DNA damage as manifested by γ-H2AX formation and, for AG-014699, its unique ability to suppress Stat3 phosphorylation. Abilities of individual PARP inhibitors to sensitize TNBC cells to cisplatin varied to a great extent in a cell context- and cell line-specific manner. Differential activation of signaling pathways suggests that the PARP inhibitors currently in clinical trials have different anti-tumor mechanisms beyond PARP inhibition and these PARP-independent mechanisms warrant further investigation.

Identification and characterization of a novel integrin-linked kinase inhibitor.

Integrin-linked kinase (ILK) represents a relevant target for cancer therapy in light of its role in promoting oncogenesis and tumor progression. Through the screening of an in-house focused compound library, we identified N-methyl-3-(1-(4-(piperazin-1-yl)phenyl)-5-(4'-(trifluoromethyl)-[1,1'-biphenyl]-4-yl)-1H-pyrazol-3-yl)propanamide (22) as a novel ILK inhibitor (IC(50), 0.6 µM), which exhibited high in vitro potency against a panel of prostate and breast cancer cell lines (IC(50), 1-2.5 µM), while normal epithelial cells were unaffected. Compound 22 facilitated the dephosphorylation of Akt at Ser-473 and other ILK targets, including glycogen synthase kinase-3ß and myosin light chain. Moreover, 22 suppressed the expression of the transcription/translation factor YB-1 and its targets HER2 and EGFR in PC-3 cells, which could be rescued by the stable expression of constitutively active ILK. Evidence indicates that 22 induced autophagy and apoptosis, both of which were integral to its antiproliferative activity. Together, this broad spectrum of mechanisms underlies the therapeutic potential of 22 in cancer treatment, which is manifested by its in vivo efficacy as a single oral agent in suppressing PC-3 xenograft tumor growth.

Novel mechanism by which histone deacetylase inhibitors facilitate topoisomerase IIα degradation in hepatocellular carcinoma cells.

UNLABELLED: Histone deacetylase (HDAC) inhibitors exhibit a unique ability to degrade topoisomerase (topo)IIα in hepatocellular carcinoma (HCC) cells, which contrasts with the effect of topoII-targeted drugs on topoIIß degradation. This selective degradation might foster novel strategies for HCC treatment in light of the correlation of topoIIα overexpression with the aggressive tumor phenotype and chemoresistance. Here we report a novel pathway by which HDAC inhibitors mediate topoIIα proteolysis in HCC cells. Our data indicate that HDAC inhibitors transcriptionally activated casein kinase (CK)2α expression through increased association of acetylated histone H3 with the CK2α gene promoter. In turn, CK2 facilitated the binding of topoIIα to COP9 signalosome subunit (Csn)5 by way of topoIIα phosphorylation. Furthermore, we identified Fbw7, a Csn5-interacting F-box protein, as the E3 ligase that targeted topoIIα for degradation. Moreover, knockdown of CK2α, Csn5, or Fbw7 reversed HDAC inhibitor-induced topoIIα degradation. Mutational analysis indicates that the (1361) SPKLSNKE(1368) motif plays a crucial role in regulating topoIIα protein stability. This motif contains the consensus recognition sites for CK2 (SXXE), glycogen synthase kinase (GSK)3ß (SXXXS), and Fbw7 (SPXXS). This study also reports the novel finding that topoIIα may be a target of GSK3ß phosphorylation. Evidence suggests that CK2 serves as a priming kinase, through phosphorylation at Ser1365, for GSK3ß-mediated phosphorylation at Ser1361. This double phosphorylation facilitated the recruitment of Fbw7 to the phospho-degron (1361) pSPKLpS(1365) of topoIIα, leading to its ubiquitin-dependent degradation. CONCLUSION: This study shows a novel pathway by which HDAC inhibitors facilitate the selective degradation of topoIIα, which underlies the complexity of the functional role of HDAC in regulating tumorigenesis and aggressive phenotype in HCC cells.

alpha-Tocopheryl succinate as a scaffold to develop potent inhibitors of breast cancer cell adhesion.

This study is aimed at the pharmacological exploitation of alpha-tocopheryl succinate (1) to develop potent antiadhesion agents. Considering the structural cooperativity between the phytyl chain and the carboxylic terminus in determining the antiadhesion activity, our structural optimization led to compound 5 ([2-(4,8-dimethyl-non-1-enyl)-2,5,7,8-tetramethyl-chroman-6-yloxy]-acetic acid), which exhibited an-order-of-magnitude higher potency than 1 in blocking the adhesion of 4T1 metastatic breast cancer cells to extracellular matrix proteins (IC(50), 0.6 microM versus 10 microM). Evidence indicates that the ability of compound 5 to block cell adhesion and migration was attributable to its effect on disrupting focal adhesion and actin cytoskeletal integrity by facilitating the degradation of focal adhesion kinase. Interactions between tumor cells and the ECM in the tumor microenvironment have been increasingly recognized as critical modulators of the metastatic potential of tumor cells. Consequently, the ability of compound 5 to block such interactions provides a unique pharmacological tool to shed light onto mechanisms that govern cell adhesion and tumor metastasis.

alpha-Tocopheryl succinate and derivatives mediate the transcriptional repression of androgen receptor in prostate cancer cells by targeting the PP2A-JNK-Sp1-signaling axis.

As part of our effort to understand the mechanism underlying alpha-tocopheryl succinate [vitamin E succinate (VES)]-mediated antitumor effects, we investigated the signaling pathway by which VES suppresses androgen receptor (AR) expression in prostate cancer cells. VES and, to a greater extent, its truncated derivative TS-1 mediated transcriptional repression of AR in prostate cancer cells but not in normal prostate epithelial cells; a finding that underscores the differential susceptibility of normal versus malignant cells to the antiproliferative effect of these agents. This AR repression was attributable to the ability of VES and TS-1 to facilitate the proteasomal degradation of the transcription factor Sp1. This mechanistic link was corroborated by the finding that proteasome inhibitors or ectopic expression of Sp1 protected cells against drug-induced AR ablation. Furthermore, evidence suggests that the destabilization of Sp1 by VES and TS-1 resulted from the inactivation of Jun N-terminal kinases (JNKs) as a consequence of increased phosphatase activity of protein phosphatase 2A (PP2A). Stable transfection of LNCaP cells with the dominant-negative JNK1 plasmid mimicked drug-induced Sp1 repression, whereas constitutive activation of JNK kinase activity or inhibition of PP2A activity by okadaic acid protected Sp1 from VES- and TS-1-induced degradation. From a mechanistic perspective, the ability of VES and TS-1 to activate PP2A activity underscores their broad spectrum of effects on multiple signaling mechanisms, including those mediated by Akt, mitogen-activated protein kinases, nuclear factor kappaB, Sp1 and AR. This pleiotropic effect in conjunction with low toxicity suggests the translational potential for developing TS-1 into potent PP2A-activating agents for cancer therapy.

Thiazolidinediones mimic glucose starvation in facilitating Sp1 degradation through the up-regulation of beta-transducin repeat-containing protein.

This study investigated the mechanism by which the transcription factor Sp1 is degraded in prostate cancer cells. We recently developed a thiazolidinedione derivative, (Z)-5-(4-hydroxy-3-trifluoromethylbenzylidene)-3-(1-methylcyclohexyl)-thiazolidine-2,4-dione (OSU-CG12), that induces Sp1 degradation in a manner paralleling that of glucose starvation. Based on our finding that thiazolidinediones suppress beta-catenin and cyclin D1 by up-regulating the E3 ligase SCF(beta-TrCP), we hypothesized that beta-transducin repeat-containing protein (beta-TrCP) targets Sp1 for proteasomal degradation in response to glucose starvation or OSU-CG12. Here we show that either treatment of LNCaP cells increased specific binding of Sp1 with beta-TrCP. This direct binding was confirmed by in vitro pull-down analysis with bacterially expressed beta-TrCP. Although ectopic expression of beta-TrCP enhanced the ability of OSU-CG12 to facilitate Sp1 degradation, suppression of endogenous beta-TrCP function by a dominant-negative mutant or small interfering RNA-mediated knockdown blocked OSU-CG12-facilitated Sp1 ubiquitination and/or degradation. Sp1 contains a C-terminal conventional DSG destruction box ((727)DSGAGS(732)) that mediates beta-TrCP recognition and encompasses a glycogen synthase kinase 3beta (GSK3beta) phosphorylation motif (SXXXS). Pharmacological and molecular genetic approaches and mutational analyses indicate that extracellular signal-regulated kinase-mediated phosphorylation of Thr739 and GSK3beta-mediated phosphorylation of Ser728 and Ser732 were critical for Sp1 degradation. The ability of OSU-CG12 to mimic glucose starvation to activate beta-TrCP-mediated Sp1 degradation has translational potential to foster novel strategies for cancer therapy.

A novel mechanism by which thiazolidinediones facilitate the proteasomal degradation of cyclin D1 in cancer cells.

This study identifies a novel mechanism by which thiazolidinediones mediate cyclin D1 repression in prostate cancer cells. Based on the finding that the thiazolidinedione family of peroxisome proliferator-activated receptor gamma (PPARgamma) agonists mediated PPARgamma-independent cyclin D1 degradation, we developed a novel PPARgamma-inactive troglitazone derivative, STG28, with high potency in cyclin D1 ablation. STG28-mediated cyclin D1 degradation was preceded by Thr-286 phosphorylation and nuclear export, which however, were independent of glycogen synthase kinase 3beta. Mutational analysis further confirmed the pivotal role of Thr-286 phosphorylation in STG28-induced nuclear export and proteolysis. Of several kinases examined, inhibition of IkappaB kinase alpha blocked STG28-mediated cytoplasmic sequestration and degradation of cyclin D1. Pulldown of ectopically expressed Cul1, the scaffold protein of the Skp-Cullin-F-box E3 ligase, in STG28-treated cells revealed an increased association of cyclin D1 with beta-TrCP, whereas no specific binding was noted with other F-box proteins examined, including Skp2, Fbw7, Fbx4, and Fbxw8. This finding represents the first evidence that cyclin D1 is targeted by beta-TrCP. Moreover, beta-TrCP expression was up-regulated in response to STG28, and ectopic expression and small interfering RNA-mediated knock-down of beta-TrCP enhanced and protected against STG28-facilitated cyclin D1 degradation, respectively. Because cyclin D1 lacks the DSG destruction motif, mutational and modeling analyses indicate that cyclin D1 was targeted by beta-TrCP through an unconventional recognition site, (279)EEVDLACpT(286), reminiscent to that of Wee1. Moreover, we obtained evidence that this beta-TrCP-dependent degradation takes part in controlling cyclin D1 turnover when cancer cells undergo glucose starvation, which endows physiological relevance to this novel mechanism.

A novel polyacetylene significantly inhibits angiogenesis and promotes apoptosis in human endothelial cells through activation of the CDK inhibitors and caspase-7.

A novel bioactive polyacetylene compound, 1,2-dihydroxy-5(E)-tridecene-7,9,11-triyne (compound 1), was identified from the Bidens pilosa extract using an ex vivo primary human umbilical vein endothelium cell (HUVEC) bioassay-guided fractionation protocol. Our results demonstrate that compound 1 (at 2.5 microg/mL) possessed significant anti-angiogenic effects, as manifested by an inhibition of HUVEC proliferation, migration, and the formation of tube-like structures in collagen gel. Moreover, compound 1 induced HUVECs to undergo cell death in a concentration- and time-dependent manner. The mechanisms underlying these pharmacological effects include reduced expression of cell cycle mediators such as CDK4, cyclins D1 and A, retinoblastoma (Rb) and vascular endothelial growth factor receptor 1 (VEGFR-1), and promotion of caspase-mediated activation of CDK inhibitors p21(Cip1) and p27(Kip). Moreover, apoptotic induction in HUVECs mediated by compound 1 was found to be in part through overexpression of FasL protein, down-regulation of anti-apoptotic Bcl-2, and activation of caspase-7 and poly(ADP-ribose) polymerase. This study demonstrates the potent anti-angiogenic and apoptotic activities of compound 1, suggesting that phytocompounds such as polyacetylenes deserve more attention regarding their potential as candidates for anti-angiogenic therapeutics.

Histone deacetylase 3 binds to and regulates the GCMa transcription factor.

Human GCMa transcription factor regulates expression of syncytin, a placental fusogenic protein mediating trophoblastic fusion. Recently, we have demonstrated that CBP-mediated GCMa acetylation underlies the activated cAMP/PKA signaling pathway that stimulates trophoblastic fusion. Because protein acetylation is a reversible modification governed by histone acetyltransferases (HATs) and histone deacetylase (HDACs), in this study we investigated the key HDACs responsible for deacetylation of GCMa and thus the reduction in GCMa activity to avoid unwanted fusion events that may have adverse effects on placental morphogenesis. We herein demonstrate that the HDAC inhibitor, trichostatin A (TSA), increases the level of acetylated GCMa and that HDAC1, 3, 4 and 5 interact with and deacetylate GCMa. Glutathione S-transferase (GST) pull-down assays further verified direct interaction between GCMa and HDAC3 or CBP and HDAC3. HDAC3 counteracts the transcriptional coactivator activity of CBP and the enhancement effect of CBP on GCMa-mediated transcriptional activation. Correlatively, we found in placental cells that HDAC3 associates with the proximal GCMa-binding site (pGBS) in the syncytin promoter and dissociates from pGBS in the presence of forskolin, which stimulates the association of CBP and GCMa with pGBS. Our studies support that trophoblastic fusion in placental morphogenesis depends on the regulation of GCMa activity by HAT and HDAC.

Stimulation of GCMa transcriptional activity by cyclic AMP/protein kinase A signaling is attributed to CBP-mediated acetylation of GCMa.

Human GCMa is a zinc-containing transcription factor primarily expressed in placenta. GCMa regulates expression of syncytin gene, which encodes for a placenta-specific membrane protein that mediates trophoblastic fusion and the formation of syncytiotrophoblast layer required for efficient fetal-maternal exchange of nutrients and oxygen. The adenylate cyclase activator, forskolin, stimulates syncytin gene expression and cell fusion in cultured placental cells. Here we present evidence that cyclic AMP (cAMP) signaling pathway activates the syncytin gene expression by regulating GCMa activity. We found that forskolin and protein kinase A (PKA) enhances GCMa-mediated transcriptional activation. Furthermore, PKA treatment stimulates the association of GCMa with CBP and increases GCMa acetylation. CBP primarily acetylates GCMa at lysine367, lysine406, and lysine409 in the transactivation domain (TAD). We found that acetylation of these residues is required to protect GCMa from ubiquitination and increases the TAD stability with a concomitant increase in transcriptional activity, supporting the importance of acetylation in PKA-dependent GCMa activation. Our results reveal a novel regulation of GCMa activity by cAMP-dependent protein acetylation and provide a molecular mechanism by which cAMP signaling regulates trophoblastic fusion.