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.].

Chalcomoracin is a potent anticancer agent acting through triggering Oxidative stress via a mitophagy- and paraptosis-dependent mechanism.

Chalocomoracin (CMR), one of the major secondary metabolites found in fungus-infected mulberry leaves, is a potent anticancer agent. However, its anticancer mechanism remains elusive. Here, we demonstrated the potent anti-tumor activity and molecular mechanism of CMR both in vitro and in vivo. We showed for the first time that CMR treatment markedly promoted paraptosis along with extensive cytoplasmic vacuolation derived from the endoplasmic reticulum, rather than apoptosis, in PC-3 and MDA-MB-231cell lines. Additional studies revealed that ectopic expression of Myc-PINK1 (PTEN-induced kinase 1), a key regulator of mitophagy, rendered LNCap cells susceptible to CMR-induced paraptosis, suggesting that the mitophagy-dependent pathway plays a crucial role in inducing paraptosis by activating PINK1. CMR treatment directly upregulated PINK1 and downregulated Alix genes in MDA-MB-231 and PC-3 cell lines. Furthermore, mitophagy signaling and paraptosis with cytoplasmic vacuolation could be blocked by antioxidant N-acetylcysteine (NAC), indicating the novel pathway was triggered by reactive oxygen species (ROS) production. An in vivo MDA-MB-231 xenograft tumor model revealed that CMR suppressed tumor growth by inducing vacuolation production through the same signal changes as those observed in vitro. These data suggest that CMR is a potential therapeutic entity for cancer treatment through a non-apoptotic pathway.

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.

A novel HIF-1α-integrin-linked kinase regulatory loop that facilitates hypoxia-induced HIF-1α expression and epithelial-mesenchymal transition in cancer cells.

Here, we described a novel regulatory feedback loop in which hypoxia induces integrin-linked kinase (ILK) expression through a HIF-1α-dependent mechanism and ILK, in turn, stimulates HIF-1α expression through cell type- and cell context-dependent pathways. HIF-1α increased ILK via transcriptional activation. ILK increased HIF-1α levels by promoting mTOR-mediated translation in PC-3 and MCF-7 cells, and by blocking GSK3ß-mediated degradation in LNCaP cells, consistent with the cell line-/cellular context-specific functions of ILK as a Ser473-Akt kinase. We show that ILK can account for the effects of hypoxia on Akt, mTOR, and GSK3ß phosphorylation. Also, ILK can de-repress HIF-1α signaling through the YB-1-mediated inhibition of Foxo3a expression. In concert with HIF-1α, these downstream effectors promote epithelial-mesenchymal transition (EMT) through modulation of Snail and Zeb1. Thus, the ILK-HIF-1α regulatory loop could underlie the maintenance of high HIF-1α expression levels and the promotion of EMT under hypoxic conditions. Finally, we show that the small-molecule ILK inhibitor T315 can disrupt this regulatory loop in vivo and suppress xenograft tumor growth, thereby providing proof-of-concept that targeting ILK represents an effective strategy to block HIF-1α expression and aggressive phenotype in cancer cells.

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 reverses the mesenchymal phenotype of cancer cells by targeting the Akt-MDM2-Foxo3a signaling axis.

In cancer cells, the epithelial-mesenchymal transition (EMT) confers the ability to invade basement membranes and metastasize to distant sites, establishing it as an appealing target for therapeutic intervention. Here, we report a novel function of the master metabolic kinase AMPK in suppressing EMT by modulating the Akt-MDM2-Foxo3 signaling axis. This mechanistic link was supported by the effects of siRNA-mediated knockdown and pharmacologic activation of AMPK on epithelial and mesenchymal markers in established breast and prostate cancer cells. Exposure of cells to OSU-53, a novel allosteric AMPK activator, as well as metformin and AICAR, was sufficient to reverse their mesenchymal phenotype. These effects were abrogated by AMPK silencing. Phenotypic changes were mediated by Foxo3a activation, insofar as silencing or overexpressing Foxo3a mimicked the effects of AMPK silencing or OSU-53 treatment on EMT, respectively. Mechanistically, Foxo3a activation led to the transactivation of the E-cadherin gene and repression of genes encoding EMT-inducing transcription factors. OSU-53 activated Foxo3a through two Akt-dependent pathways, one at the level of nuclear localization by blocking Akt- and IKKß-mediated phosphorylation, and a second at the level of protein stabilization via cytoplasmic sequestration of MDM2, an E3 ligase responsible for Foxo3a degradation. The suppressive effects of OSU-53 on EMT had therapeutic implications illustrated by its ability to block invasive phenotypes in vitro and metastatic properties in vivo. Overall, our work illuminates a mechanism of EMT regulation in cancer cells mediated by AMPK, along with preclinical evidence supporting a tractable therapeutic strategy to reverse mesenchymal phenotypes associated with invasion and metastasis.

Targeting the Warburg effect with a novel glucose transporter inhibitor to overcome gemcitabine resistance in pancreatic cancer cells.

Gemcitabine resistance remains a significant clinical challenge. Here, we used a novel glucose transporter (Glut) inhibitor, CG-5, as a proof-of-concept compound to investigate the therapeutic utility of targeting the Warburg effect to overcome gemcitabine resistance in pancreatic cancer. The effects of gemcitabine and/or CG-5 on viability, survival, glucose uptake and DNA damage were evaluated in gemcitabine-sensitive and gemcitabine-resistant pancreatic cancer cell lines. Mechanistic studies were conducted to determine the molecular basis of gemcitabine resistance and the mechanism of CG-5-induced sensitization to gemcitabine. The effects of CG-5 on gemcitabine sensitivity were investigated in a xenograft tumor model of gemcitabine-resistant pancreatic cancer. In contrast to gemcitabine-sensitive pancreatic cancer cells, the resistant Panc-1 and Panc-1(GemR) cells responded to gemcitabine by increasing the expression of ribonucleotide reductase M2 catalytic subunit (RRM2) through E2F1-mediated transcriptional activation. Acting as a pan-Glut inhibitor, CG-5 abrogated this gemcitabine-induced upregulation of RRM2 through decreased E2F1 expression, thereby enhancing gemcitabine-induced DNA damage and inhibition of cell survival. This CG-5-induced inhibition of E2F1 expression was mediated by the induction of a previously unreported E2F1-targeted microRNA, miR-520f. The addition of oral CG-5 to gemcitabine therapy caused greater suppression of Panc-1(GemR) xenograft tumor growth in vivo than either drug alone. Glut inhibition may be an effective strategy to enhance gemcitabine activity for the treatment of pancreatic cancer.

Prospects on strategies for therapeutically targeting oncogenic regulatory factors by small-molecule agents.

Although the Human Genome Project has raised much hope for the identification of druggable genetic targets for cancer and other diseases, this genetic target-based approach has not improved productivity in drug discovery over the traditional approach. Analyses of known human target proteins of currently marketed drugs reveal that these drugs target only a limited number of proteins as compared to the whole proteome. In contrast to genome-based targets, mechanistic targets are derived from empirical research, at cellular or molecular levels, in disease models and/or in patients, thereby enabling the exploration of a greater number of druggable targets beyond the genome and epigenome. The paradigm shift has made a tremendous headway in developing new therapeutic agents targeting different clinically relevant mechanisms/pathways in cancer cells. In this Prospects article, we provide an overview of potential drug targets related to the following four emerging areas: (1) tumor metabolism (the Warburg effect), (2) dysregulated protein turnover (E3 ubiquitin ligases), (3) protein-protein interactions, and (4) unique DNA high-order structures and protein-DNA interactions. Nonetheless, considering the genetic and phenotypic heterogeneities that characterize cancer cells, the development of drug resistance in cancer cells by adapting signaling circuitry to take advantage of redundant pathways or feedback/crosstalk systems is possible. This "phenotypic adaptation" underlies the rationale of using therapeutic combinations of these targeted agents with cytotoxic drugs.

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.

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.

Antitumor effects of OSU-2S, a nonimmunosuppressive analogue of FTY720, in hepatocellular carcinoma.

UNLABELLED: Accumulating evidence suggests the therapeutic potential of the immunosuppressive agent FTY720 (fingolimod) in hepatocellular carcinoma (HCC). Based on our previous finding that FTY720 mediates apoptosis in HCC cells by activating reactive oxygen species (ROS)-protein kinase Cδ (PKCδ) signaling independent of effects on sphingosine-1-phosphate (S1P) receptors, we embarked on the pharmacological exploitation of FTY720 to develop a nonimmunosuppressive analogue with antitumor activity. This effort led to the development of OSU-2S, which exhibits higher potency than FTY720 in suppressing HCC cell growth through PKCδ activation. In contrast to FTY720, OSU-2S was not phosphorylated by sphingosine kinase 2 (SphK2) in vitro, and did not cause S1P1 receptor internalization in HCC cells or T lymphocyte homing in immunocompetent mice. Although devoid of S1P1 receptor activity, OSU-2S exhibited higher in vitro antiproliferative efficacy relative to FTY720 against HCC cells without cytotoxicity in normal hepatocytes. Several lines of pharmacological and molecular genetic evidence indicate that ROS-PKCδ-caspase-3 signaling underlies OSU-2S-mediated antitumor effects, and that differences in the antitumor activity between FTY720 and OSU-2S were attributable to SphK2-mediated phosphorylation of FTY720, which represents a metabolic inactivation of its antitumor activity. Finally, OSU-2S exhibited high in vivo potency in suppressing xenograft tumor growth in both ectopic and orthotopic models without overt toxicity. CONCLUSION: Using the molecular platform of FTY720, we developed OSU-2S, a novel PKCδ-targeted antitumor agent, which is devoid of S1P1 receptor activity and is highly effective in suppressing HCC tumor growth in vivo. These findings suggest that OSU-2S has clinical value in therapeutic strategies for HCC and warrants continued investigation in this regard.

Energy restriction-mimetic agents induce apoptosis in prostate cancer cells in part through epigenetic activation of KLF6 tumor suppressor gene expression.

Although energy restriction has been recognized as an important target for cancer prevention, the mechanism by which energy restriction-mimetic agents (ERMAs) mediate apoptosis remains unclear. By using a novel thiazolidinedione-derived ERMA, CG-12 (Wei, S., Kulp, S. K., and Chen, C. S. (2010) J. Biol. Chem. 285, 9780-9791), vis-à-vis 2-deoxyglucose and glucose deprivation, we obtain evidence that epigenetic activation of the tumor suppressor gene Kruppel-like factor 6 (KLF6) plays a role in ERMA-induced apoptosis in LNCaP prostate cancer cells. KLF6 regulates the expression of many proapoptotic genes, and shRNA-mediated KLF6 knockdown abrogated the ability of ERMAs to induce apoptosis. Chromatin immunoprecipitation analysis indicates that this KLF6 transcriptional activation was associated with increased histone H3 acetylation and histone H3 lysine 4 trimethylation occupancy at the promoter region. Several lines of evidence demonstrate that the enhancing effect of ERMAs on these active histone marks was mediated through transcriptional repression of histone deacetylases and H3 lysine 4 demethylases by down-regulating Sp1 expression. First, putative Sp1-binding elements are present in the promoters of the affected histone-modifying enzymes, and luciferase reporter assays indicate that site-directed mutagenesis of these Sp1 binding sites significantly diminished the promoter activities. Second, shRNA-mediated knockdown of Sp1 mimicked the repressive effect of energy restriction on these histone-modifying enzymes. Third, ectopic Sp1 expression protected cells from the repressive effect of CG-12 on these histone-modifying enzymes, thereby abolishing the activation of KLF6 expression. Together, these findings underscore the intricate relationship between energy restriction and epigenetic regulation of tumor suppressor gene expression, which has therapeutic relevance to foster novel strategies for prostate cancer therapy.

Histone deacetylase inhibitors stimulate histone H3 lysine 4 methylation in part via transcriptional repression of histone H3 lysine 4 demethylases.

This study investigates the mechanism by which histone deacetylase (HDAC) inhibitors up-regulate histone H3 lysine 4 (H3K4) methylation. Exposure of LNCaP prostate cancer cells and the prostate tissue of transgenic adenocarcinoma of the mouse prostate mice to the pan- and class I HDAC inhibitors (S)-(+)-N-hydroxy-4-(3-methyl-2-phenyl-butyrylamino)-benzamide (AR42), N-(2-aminophenyl)-4-[N-(pyridine-3-yl-methoxycarbonyl)-aminomethyl]-benzamide (MS-275), and vorinostat led to differential increases in H3K4 methylation. Chromatin immunoprecipitation shows that this accumulation of methylated H3K4 occurred in conjunction with decreases in the amount of the H3K4 demethylase RBP2 at the promoter of genes associated with tumor suppression and differentiation, including KLF4 and E-cadherin. This finding, together with the HDAC inhibitor-induced up-regulation of KLF4 and E-cadherin, suggests that HDAC inhibitors could activate the expression of these genes through changes in histone methylation status. Evidence indicates that this up-regulation of H3K4 methylation was attributable to the suppressive effect of these HDAC inhibitors on the expression of RBP2 and other JARID1 family histone demethylases, including PLU-1, SMCX, and LSD1, via the down-regulation of Sp1 expression. Moreover, shRNA-mediated silencing of the class I HDAC isozymes 1, 2, 3, and 8, but not that of the class II isozyme HDAC6, mimicked the drug effects on H3K4 methylation and H3K4 demethylases, which could be reversed by ectopic Sp1 expression. These data suggest a cross-talk mechanism between HDACs and H3K4 demethylases via Sp1-mediated transcriptional regulation, which underlies the complexity of the functional role of HDACs in the regulation of histone modifications.