Hsiao-Ching Chuang

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 γ (PPARγ) agonists mediated PPARγ-independent cyclin D1 degradation, we developed a novel PPARγ-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 3β. Mutational analysis further confirmed the pivotal role of Thr-286 phosphorylation in STG28-induced nuclear export and proteolysis. Of several kinases examined, inhibition of IκB kinase α 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 β-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 β-TrCP. Moreover, β-TrCP expression was up-regulated in response to STG28, and ectopic expression and small interfering RNA-mediated knock-down of β-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 β-TrCP through an unconventional recognition site, 279EEVDLACpT286, reminiscent to that of Wee1. Moreover, we obtained evidence that this β-TrCP-dependent degradation takes part in controlling cyclin D1 turnover when cancer cells undergo glucose starvation, which endows physiological relevance to this novel mechanism.

Thiazolidinediones Mimic Glucose Starvation in Facilitating Sp1 Degradation through the Up-Regulation of β-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 β-catenin and cyclin D1 by up-regulating the E3 ligase SCFβ-TrCP, we hypothesized that β-transducin repeat-containing protein (β-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 β-TrCP. This direct binding was confirmed by in vitro pull-down analysis with bacterially expressed β-TrCP. Although ectopic expression of β-TrCP enhanced the ability of OSU-CG12 to facilitate Sp1 degradation, suppression of endogenous β-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 (727DSGAGS732) that mediates β-TrCP recognition and encompasses a glycogen synthase kinase 3β (GSK3β) phosphorylation motif (SXXXS). Pharmacological and molecular genetic approaches and mutational analyses indicate that extracellular signal-regulated kinase-mediated phosphorylation of Thr739 and GSK3β-mediated phosphorylation of Ser728 and Ser732 were critical for Sp1 degradation. The ability of OSU-CG12 to mimic glucose starvation to activate β-TrCP-mediated Sp1 degradation has translational potential to foster novel strategies for cancer therapy.

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.

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.

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.

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.

Abstract 3505: Energy restriction-mimetic agents (ERMAs) decrease BRCA1 expression levels in triple-negative breast cancer cells: A rationale for combining ERMAs with poly(ADP ribose) polymerase

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Purpose: Triple-negative (ER-negative, PR-negative, HER2/neu-negative) breast cancer (TNBC) is a clinical challenge because of the lack of a specific therapeutic target. Poly (ADP-ribose) polymerase (PARP)-1 is a nuclear enzyme involved in the detection and repair of DNA damage. In DNA repair-defective tumors, inhibition of PARP can cause genomic instability and cell death. Among the PARP inhibitors currently being assessed in clinical trials, recent results showed that, in a randomized phase II clinical trial of TNBC patients, BSI-201 when added to platinum-containing chemotherapy significantly improved the outcome of metastatic breast cancer patients relative to chemotherapy alone. Previously, we reported that the thiazolidinedione family of peroxisome proliferation-activated receptor-γ agonist could induce apoptosis in cancer cells by eliciting cellular responses indicative of energy restriction. Consequently, we used ciglitazone as a scaffold to develop novel energy restriction-mimetic agents (ERMAs) through structural optimization, which has yielded two lead compounds, CG-5 and CG-12. In prostate cancer cells, ERMAs could induce cell death and reduce the expression of BRCA1 protein. BRCA1 helps repair DNA double-stranded breaks through homologous recombination. BRCA1 deficiency has been reported to sensitize cells to PARP inhibition through synthetic lethality. We hypothesize that ERMAs can potentiate the anti-proliferative activity of PARP inhibitors (AZD-2281, AG-014699, ABT-888 and BSI-201) through the down-regulation of BRCA1 expression in TNBC cells. Methods: TNBC cell lines, MDA-MB-468, MDA-MB-231 and Cal-51 (all of which have functional BRCA1) were obtained from ATCC and DSMZ and maintained in low-glucose complete growth medium. Drug effects on cell viability were assessed by MTT assays. The expression of BRCA1, phosphorylated AMP-activated kinase (AMPK), and PARP cleavage in treated cells were examined by western blotting. Results: The ERMAs CG-5 and CG-12 exhibited dose-dependent anti-proliferative activity against all TNBC cell lines tested with IC50 values of about 3 and 4 μM, respectively, after 72 h of treatment. These anti-proliferative effects were attributed to apoptosis as both compounds induced the cleavage of PARP. Moreover, AMPK was activated by both drugs indicative of energy restriction. BRCA1 levels were reduced dose-dependently in all three TNBC cell lines after treatment with CG-5 or CG-12. Conclusion: The ERMAs CG-5 and CG-12 can reduce BRCA1 levels in TNBC cells. These findings provide a rationale for combining the ERMA-mediated ablation of BRCA1 with PARP inhibitors to induce TNBC cell death through synthetic lethality. Further study to address this hypothesis could lead to a new therapeutic approach for TNBC patients for whom available options are limited. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3505. doi:10.1158/1538-7445.AM2011-3505

Abstract 5424: Therapeutic targeting of KRAS mutation-driven tumor progression in colorectal cancer

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA KRAS is the most frequently altered gene in colorectal cancer (CRC), with mutations occurring in 30-40% of colorectal cancer. Although KRAS mutation is associated with poor prognosis and resistance to anti-epidermal growth factor receptor therapy, the mechanism by which it promotes tumor metastasis remains undefined. Our study explored a new hypothesis that targeting the Y-box binding protein-1 (YB-1)-insulin-like growth factor-I receptor (IGF-IR) signaling axis which is downstream of KRAS, with a novel integrin-linked kinase inhibitor, T315, represents a therapeutically relevant strategy to block KRAS mutation-driven tumor progression. In liver metastases from CRC patients, we observed that there was a preponderance (79/108) of over-expression in both YB-1 and IGF-1R by immunohistochemistry. In colon cancer cell lines HCT-116 and SW480, knockdown and over-expression of KRAS affected the protein and mRNA levels of IGF-1R in a dose-dependent manner. But KRAS only affected the protein level and not mRNA of YB-1, thus suggesting that KRAS regulated YB-1 expression at the post-transcriptional level. Manipulation of YB-1 via plasmid overexpression and siRNA affected IGF-1R protein and mRNA levels in a dose-dependent manner, and YB-1 and IGF-1R promoter binding was confirmed via ChIP assay. There was a dose-dependent decrease in YB-1 and IGF-1R protein levels with MEK162, a MEK inhibitor, but not with LY294002m a PI3K inhibitor. This demonstrated that the KRAS regulation of the YB-1- IGFR-1R signaling axis to be via the MEK signaling pathway and not PI3K/Akt. Our novel drug T315 targets YB-1, and was shown to inhibit cell viability and cell migration in a dose-dependent manner. T315 treatment of the colon cancer cells also decreased protein levels of YB-1 and IGF-1R and affected epidermal mesenchymal transition markers such as E-cadherin, vimentin, and snail. These results suggest that a combination of a MEK inhibitor and T315 may be an effective therapeutic strategy to target KRAS mutation driven CRC. Citation Format: Christina Wu, Peng-Chan Lin, Po-Chen Chu, Hsiao-Ching Chuang, Samuel Kulp, Tanios Bekaii-Saab, Ching-Shih Chen. Therapeutic targeting of KRAS mutation-driven tumor progression in colorectal cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5424. doi:10.1158/1538-7445.AM2015-5424