Po-Chen Chu

Development of a Novel Class of Glucose Transporter Inhibitors

On the basis of our finding that the antitumor effect of 5-{4-[(1-methylcyclohexyl)methoxy]benzyl}thiazolidine-2,4-dione, a thiazolidinedione peroxisome proliferator-activated receptor (PPAR)γ agonist, was, in part, attributable to its ability to block glucose uptake independently of PPARγ, we used its PPARγ-inactive analogue to develop a novel class of glucose transporter (GLUT) inhibitors. This lead optimization led to compound 30 {5-(4-hydroxy-3-trifluoromethylbenzylidene)-3-[4,4,4-trifluoro-2-methyl-2-(2,2,2-trifluoroethyl)butyl]thiazolidine-2,4-dione} as the optimal agent, which exhibited high antitumor potency through the suppression of glucose uptake (IC(50), 2.5 μM), while not cytotoxic to prostate and mammary epithelial cells. This glucose uptake inhibition was associated with the inhibition of GLUT1 (IC(50), 2 μM). Moreover, the mechanism of antitumor action of compound 30 was validated by its effect on a series of energy restriction-associated cellular responses. Homology modeling analysis suggests that the inhibitory effect of compound 30 on glucose entry was attributable to its ability to bind to the GLUT1 channel at a site distinct from that of glucose.

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 β-transducin repeat-containing protein (β-TrCP) expression represents a cellular response in cancer cells to ERMAs, including CG-12 and 2-deoxyglucose, we demonstrated that this β-TrCP accumulation resulted from decreased Skp2 expression. Evidence indicates that Skp2 targets β-TrCP for degradation via the cyclin-dependent kinase 2-facilitated recognition of the proline-directed phosphorylation motif 412SP. 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 β-TrCP are mutually regulated, providing a feedback mechanism that amplifies the suppressive effect of ERMAs on Skp2. Specifically, cellular accumulation of β-TrCP reduced the expression of Sp1, a β-TrCP substrate, which, in turn, reduced Skp2 gene expression. This Skp2-β-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.

The mRNA-stabilizing Factor HuR Protein Is Targeted by β-TrCP Protein for Degradation in Response to Glycolysis Inhibition

Background: HuR regulates expression of many oncogenic proteins by modulating mRNA stability. Results: Glycolysis inhibition facilitates HuR degradation through a novel β-TrCP-mediated mechanism. Conclusion: This mechanism underlies the complexity in the regulation of HuR turnover under different stress stimuli. Significance: The ability of glycolysis inhibitors to target expression of oncogenic proteins by promoting HuR degradation might foster novel strategies for cancer therapy. 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 β-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 β-TrCP1 and HuR degradation was supported by the ability of ectopically expressed β-TrCP1 to mimic CG-5 to promote HuR degradation and by the protective effect of dominant negative inhibition of β-TrCP1 on HuR ubiquitination and degradation. Substrate targeting of HuR by β-TrCP1 was further verified by coimmunoprecipitation and in vitro GST pull-down assays and by the identification of a β-TrCP1 recognition site. Although HuR does not contain a DSG destruction motif, we obtained evidence that β-TrCP1 recognizes an unconventional motif, 296EEAMAIAS304, 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 β-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.

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.

A triterpenoid from wild bitter gourd inhibits breast cancer cells

The antitumor activity of 3β,7β,25-trihydroxycucurbita-5,23(E)-dien-19-al (TCD), a triterpenoid isolated from wild bitter gourd, in breast cancer cells was investigated. TCD suppressed the proliferation of MCF-7 and MDA-MB-231 breast cancer cells with IC50 values at 72 h of 19 and 23 μM, respectively, via a PPARγ−independent manner. TCD induced cell apoptosis accompanied with pleiotrophic biological modulations including down-regulation of Akt-NF-κB signaling, up-regulation of p38 mitogen-activated protein kinase and p53, increased reactive oxygen species generation, inhibition of histone deacetylases protein expression, and cytoprotective autophagy. Together, these findings provided the translational value of TCD and wild bitter gourd as an antitumor agent for patients with breast cancer.

Regulation of oncogenic KRAS signaling via a novel KRAS-integrin-linked kinase-hnRNPA1 regulatory loop in human pancreatic cancer cells

Integrin-linked kinase (ILK) is a mediator of aggressive phenotype in pancreatic cancer. On the basis of our finding that knockdown of either KRAS or ILK has a reciprocal effect on the other’s expression, we hypothesized the presence of an ILK-KRAS regulatory loop that enables pancreatic cancer cells to regulate KRAS expression. This study aimed to elucidate the mechanism by which this regulatory circuitry is regulated and to investigate the translational potential of targeting ILK to suppress oncogenic KRAS signaling in pancreatic cancer. Interplay between KRAS and ILK and the roles of E2F1, c-Myc and heterogeneous nuclear ribonucleoprotein as intermediary effectors in this feedback loop was interrogated by genetic manipulations through small interfering RNA/short hairpin RNA knockdown and ectopic expression, western blotting, PCR, promoter-luciferase reporter assays, chromatin immunoprecipitation and pull-down analyses. In vivo efficacy of ILK inhibition was evaluated in two murine xenograft models. Our data show that KRAS regulated the expression of ILK through E2F1-mediated transcriptional activation, which, in turn, controlled KRAS gene expression via hnRNPA1-mediated destabilization of the G-quadruplex on the KRAS promoter. Moreover, ILK inhibition blocked KRAS-driven epithelial–mesenchymal transition and growth factor-stimulated KRAS expression. The knockdown or pharmacological inhibition of ILK suppressed pancreatic tumor growth, in part, by suppressing KRAS signaling. These studies suggest that this KRAS-E2F1-ILK-hnRNPA1 regulatory loop enables pancreatic cancer cells to promote oncogenic KRAS signaling and to interact with the tumor microenvironment to promote aggressive phenotypes. This regulatory loop provides a mechanistic rationale for targeting ILK to suppress oncogenic KRAS signaling, which might foster new therapeutic strategies for pancreatic cancer.

The mRNA-Stabilizing Factor HuR Is Targeted by β-TrCP for Degradation in Response to Glycolysis Inhibition

Background: HuR regulates expression of many oncogenic proteins by modulating mRNA stability. Results: Glycolysis inhibition facilitates HuR degradation through a novel β-TrCP-mediated mechanism. Conclusion: This mechanism underlies the complexity in the regulation of HuR turnover under different stress stimuli. Significance: The ability of glycolysis inhibitors to target expression of oncogenic proteins by promoting HuR degradation might foster novel strategies for cancer therapy. 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 β-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 β-TrCP1 and HuR degradation was supported by the ability of ectopically expressed β-TrCP1 to mimic CG-5 to promote HuR degradation and by the protective effect of dominant negative inhibition of β-TrCP1 on HuR ubiquitination and degradation. Substrate targeting of HuR by β-TrCP1 was further verified by coimmunoprecipitation and in vitro GST pull-down assays and by the identification of a β-TrCP1 recognition site. Although HuR does not contain a DSG destruction motif, we obtained evidence that β-TrCP1 recognizes an unconventional motif, 296EEAMAIAS304, 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 β-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.

Pharmacological exploitation of the phenothiazine antipsychotics to develop novel antitumor agents–A drug repurposing strategy

Phenothiazines (PTZs) have been used for the antipsychotic drugs for centuries. However, some of these PTZs have been reported to exhibit antitumor effects by targeting various signaling pathways in vitro and in vivo. Thus, this study was aimed at exploiting trifluoperazine, one of PTZs, to develop potent antitumor agents. This effort culminated in A4 [10-(3-(piperazin-1-yl)propyl)-2-(trifluoromethyl)-10H-phenothiazine] which exhibited multi-fold higher apoptosis-inducing activity than the parent compound in oral cancer cells. Compared to trifluoperazine, A4 demonstrated similar regulation on the phosphorylation or expression of multiple molecular targets including Akt, p38, and ERK. In addition, A4 induced autophagy, as evidenced by increased expression of the autophagy biomarkers LC3B-II and Atg5, and autophagosomes formation. The antitumor activity of A4 also related to production of reactive oxygen species and adenosine monophosphate-activated protein kinase. Importantly, the antitumor utility of A4 was extended in vivo as it, administrated at 10 and 20 mg/kg intraperitoneally, suppressed the growth of Ca922 xenograft tumors. In conclusion, the ability of A4 to target diverse aspects of cancer cell growth suggests its value in oral cancer therapy.

hPuf-A/KIAA0020 Modulates PARP-1 Cleavage upon Genotoxic Stress

Human hPuf-A/KIAA0020 was first identified as a new minor histocompatibility antigen in 2001. Its zebrafish orthologue contains six Pumilio-homology RNA-binding domains and has been shown to participate in the development of eyes and primordial germ cells, but the cellular function of hPuf-A remains unclear. In this report, we showed that hPuf-A predominantly localized in the nucleoli with minor punctate signals in the nucleoplasm. The nucleolar localization of hPuf-A would redistribute to the nucleoplasm after the treatment of RNA polymerase inhibitors (actinomycin D and 5,6-dichlorobenzimidazole riboside) and topoisomerase inhibitors [camptothecin (CPT) and etoposide]. Interestingly, knockdown of hPuf-A sensitized cells to CPT and UV treatment and cells constitutively overexpressing hPuf-A became more resistant to genotoxic exposure. Affinity gel pull-down coupled with mass spectrometric analysis identified PARP-1 as one of the hPuf-A interacting proteins. hPuf-A specifically interacts with the catalytic domain of PARP-1 and inhibits poly(ADP-ribosyl)ation of PARP-1 in vitro. Depletion of hPuf-A increased the cleaved PARP-1 and overexpression of hPuf-A lessened PARP-1 cleavage when cells were exposed to CPT and UV light. Collectively, hPuf-A may regulate cellular response to genotoxic stress by inhibiting PARP-1 activity and thus preventing PARP-1 degradation by caspase-3.

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.