Jai-Hee Moon

YM155 Induces EGFR Suppression in Pancreatic Cancer Cells

YM155, which inhibits the anti-apoptotic protein survivin, is known to exert anti-tumor effects in various cancers, including prostate and lung cancer. However, there are few reports describing the inhibitory effect of YM155 on human pancreatic cancers that highly express survivin. Here, we tested the effects of YM155 on a variety of cancer cell lines, including pancreatic cancer cells. We found that YM155 exerts an anti-proliferative effect in pancreatic cancer cells, inducing cell death through suppression of XIAP (X-linked inhibitor of apoptosis) as well as survivin without affecting the anti-apoptotic proteins Bcl-xL or Mcl-1. YM155 also inhibited tumor growth in vivo, reducing the size of pancreatic cancer cell line MIAPaCa-2 xenografts by 77.1% on day 31. Western blot analyses further showed that YM155 downregulated phosphoinoside 3-kinase (PI3K) expression and reduced the levels of phosphorylated (activated) extracellular signal-regulated kinase (ERK) and STAT3 (signal transducer and activator of transcription 3) in PANC-1 cells. Interestingly, we also found that YM155 downregulated the epidermal growth factor receptor (EGFR) in various cancer cell lines and induced the EGFR phosphorylation and ubiquitination of EGFR in PANC-1 cells. YM155 also modestly promoted the ubiquitination of survivin and XIAP. Therefore, YM155 acts through modulation of EGFR and survivin expression to subsequently reduce survival. We suggest that YM155 has potential as a therapeutic agent in the treatment of pancreatic cancer.

The MEK1/2 inhibitor AS703026 circumvents resistance to the BRAF inhibitor PLX4032 in human malignant melanoma cells.

Background:Although inhibitors of the proto-oncogene BRAF have shown excellent antitumor activity against malignant melanoma, their efficacy is limited by the development of acquired drug resistance, a process in which reactivation of MAP kinase (MEK) is known to play an important role. In this study, we evaluated the efficacy of AS703026, a new MEK inhibitor, in BRAF inhibitor–resistant melanoma cell lines. Methods:Two melanoma cells lines, RPMI-7951 and SK-MEL5, harboring an activating mutation of BRAF (V600E) were treated with the BRAF inhibitor PLX4032 to select a BRAF inhibitor–resistant cell line for further study. Cell viability assay was determined with MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay and trypan blue exclusion method; apoptosis assay was performed by annexin-V staining. Knockdown of BRAF was investigated by small interfering RNA. Results:RPMI-7951 cells exhibited an increased sensitivity to combined treatment with PLX4032 and AS703026 compared to either drug alone. Consistent with this, the combination of PLX4032 and AS703026 significantly induced apoptosis, whereas each drug used alone did not, as demonstrated by a flow cytometric analysis of annexin-V/propidium iodide–stained cells and Western blot analysis of cleaved caspase-3. Notably, immunoblot analyses also showed a depletion of phosphorylated-ERK with combined drug treatment. In addition, AS703026 synergized with small interfering RNA–mediated downregulation of BRAF to produce results similar to those of combined treatment with PLX4032 and AS703026. Conclusions:Our results suggest that combined treatment with AS703026 and a BRAF inhibitor overcomes the resistance to BRAF inhibitors in malignant melanoma cells harboring a mutant form of BRAF.

Ring Finger Protein 149 Is an E3 Ubiquitin Ligase Active on Wild-type v-Raf Murine Sarcoma Viral Oncogene Homolog B1 (BRAF)

Background: BRAF is a downstream effector kinase of Ras. Results: RNF149, a RING finger domain-containing E3 ubiquitin ligase, is one of the several proteins shown to interact with wild-type BRAF by tandem affinity purification. Conclusion: RNF149 induces ubiquitination and subsequent proteasomal degradation of wild-type but not mutant BRAF. Significance: This is the first ubiquitin ligase shown to degrade wild-type BRAF in a proteasome-dependent manner. Members of the RAF family (ARAF, BRAF, and CRAF/RAF-1) are involved in a variety of cellular activities, including growth, survival, differentiation, and transformation. An oncogene encodes BRAF, the function of which is linked to MEK activation. BRAF is the most effective RAF kinase in terms of induction of MEK/ERK activity. However, the mechanisms involved in BRAF regulation remain unclear. In the present work, we used a tandem affinity purification approach to show that RNF149 (RING finger protein 149) interacts with wild-type BRAF. The latter protein is a RING domain-containing E3 ubiquitin ligase involved in control of gene transcription, translation, cytoskeletal organization, cell adhesion, and epithelial development. We showed that RNF149 bound directly to the C-terminal kinase-containing domain of wild-type BRAF and induced ubiquitination, followed by proteasome-dependent degradation, of the latter protein. Functionally, RNF149 attenuated the increase in cell growth induced by wild-type BRAF. However, RNF149 did not bind to mutant BRAF or induce ubiquitination thereof. Thus, we show that RNF149 is an E3 ubiquitin ligase active on wild-type BRAF.

Combined treatment with vitamin C and sulindac synergistically induces p53- and ROS-dependent apoptosis in human colon cancer cells.

Sulindac has anti-neoplastic properties against colorectal cancers; however, its use as a chemopreventive agent has been limited due to toxicity and efficacy concerns. Combinatorial treatment of colorectal cancers has been attempted to maximize anti-cancer efficacy with minimal side effects by administrating NSAIDs in combination with other inhibitory compounds or drugs such as l-ascorbic acid (vitamin C), which is known to exhibit cytotoxicity towards various cancer cells at high concentrations. In this study, we evaluated a combinatorial strategy utilizing sulindac and vitamin C. The death of HCT116 cells upon combination therapy occurred via a p53-mediated mechanism. The combination therapeutic resistance developed in isogenic p53 null HCT116 cells and siRNA-mediated p53 knockdown HCT116 cells, but the exogenous expression of p53 in p53 null isogenic cells resulted in the induction of cell death. In addition, we investigated an increased level of intracellular ROS (reactive oxygen species), which was preceded by p53 activation. The expression level of PUMA (p53-upregulated modulator of apoptosis), but not Bim, was significantly increased in HCT116 cells in response to the combination treatment. Taken together, our results demonstrate that combination therapy with sulindac and vitamin C could be a novel anti-cancer therapeutic strategy for p53 wild type colon cancers.

A novel small-molecule IAP antagonist, AZD5582, draws Mcl-1 down-regulation for induction of apoptosis through targeting of cIAP1 and XIAP in human pancreatic cancer

Inhibitor of apoptosis proteins (IAPs) plays an important role in controlling cancer cell survival. IAPs have therefore attracted considerable attention as potential targets in anticancer therapy. In this study, we investigated the anti-tumor effect of AZD5582, a novel small-molecule IAP inhibitor, in human pancreatic cancer cells. Treating human pancreatic cancer cells with AZD5582 differentially induced apoptosis, dependent on the expression of p-Akt and p-XIAP. Moreover, the knockdown of endogenous Akt or XIAP via RNA interference in pancreatic cancer cells, which are resistant to AZD5582, resulted in increased sensitivity to AZD5582, whereas ectopically expressing Akt or XIAP led to resistance to AZD5582. Additionally, AZD5582 targeted cIAP1 to induce TNF-α-induced apoptosis. More importantly, AZD5582 induced a decrease of Mcl-1 protein, a member of the Bcl-2 family, but not that of Bcl-2 and Bcl-xL. Interestingly, ectopically expressing XIAP and cIAP1 inhibited the AZD5582-induced decrease of Mcl-1 protein, which suggests that AZD5582 elicits Mcl-1 decrease for apoptosis induction by targeting of XIAP and cIAP1. Taken together, these results indicate that sensitivity to AZD5582 is determined by p-Akt-inducible XIAP phosphorylation and by targeting cIAP1. Furthermore, Mcl-1 in pancreatic cancer may act as a potent marker to analyze the therapeutic effects of AZD5582.

Targeting FGFR Pathway in Human Hepatocellular Carcinoma: Expressing pFGFR and pMET for Antitumor Activity

The MET receptor tyrosine kinase, the receptor for hepatocyte growth factor (HGF), has been implicated in cancer growth, invasion, migration, angiogenesis, and metastasis in a broad variety of human cancers, including human hepatocellular carcinoma (HCC). Recently, MET was suggested to be a potential target for the personalized treatment of HCC with an active HGF–MET signaling pathway. However, the mechanisms of resistance to MET inhibitors need to be elucidated to provide effective treatment. Here, we show that HCC cells exhibit different sensitivities to the MET inhibitor PHA665752, depending on the phosphorylation status of FGFR. Treatment of cells expressing both phospho-FGFR and phospho-MET with the inhibitor PHA665752 did not cause growth inhibition and cell death, whereas treatment with AZD4547, a pan-FGFR inhibitor, resulted in decreased colony formation and cleavage of caspase-3. Moreover, silencing of endogenous FGFR1 and FGFR2 by RNAi of HCC cells expressing phospho-FGFR, phospho-FGFR2, and phospho-MET overcame the resistance to PHA665752 treatment. Treatment of primary cancer cells from patients with HCC expressing both phospho-FGFR and phospho-MET with PHA665752 did not induce cell death, whereas AZD4547 treatment induced cell death through the cleavage of caspase-3. In addition, treatment of cells resistant to PHA665752 with AZD4547 abrogated the activation of downstream effectors of cell growth, proliferation, and survival. On the basis of these results, we conclude that the FGFR pathway is critical for HCC survival, and that targeting this pathway with AZD4547 may be beneficial for the treatment of patients with HCC-expressing phospho-FGFR and phospho-MET. Mol Cancer Ther; 14(11); 2613–22. ©2015 AACR.

Cellular Inhibitor of Apoptosis Protein 1 (cIAP1) Stability Contributes to YM155 Resistance in Human Gastric Cancer Cells

Background: YM155, a survivin suppressant, has been shown anticancer efficacy in various cancer cells. Results: Human gastric cancer cells differentially displayed the sensitivity to YM155 dependent to degradation of cIAP1. Conclusion: Cellular inhibitor of apoptosis protein 1 (cIAP1) interacts with survivin to control their mutual stability and determine sensitivity to YM155. Significance: The regulation of cIAP1 enhances the efficacy for YM155 treatment in gastric cancer. YM155, which blocks the expression of survivin, a member of the inhibitor of apoptosis (IAP) family, induces cell death in a variety of cancer types, including prostate, bladder, breast, leukemia, and non-small lung cancer. However, the mechanism underlying gastric cancer susceptibility and resistance to YM155 is yet to be specified. Here, we demonstrate that cIAP1 stability dictates resistance to YM155 in human gastric cancer cells. Treatment of human gastric cancer cells with YM155 differentially induced cell death dependent on the stability of cIAP1 as well as survivin. Transfection with cIAP1 expression plasmids decreased cell sensitivity to YM155, whereas knockdown of endogenous cIAP1 using RNA interference enhanced sensitivity to YM155. In addition, double knockdown of survivin and cIAP1 significantly induced cell death in the YM155-resistant cell line, MKN45. We also showed that YM155 induced autoubiquitination and proteasome-dependent degradation of cIAP1. Surprisingly, survivin affected the stability of cIAP1 through binding, contributing to cell sensitivity to YM155. Thus, our findings reveal that YM155 sensitizes human gastric cancer cells to apoptotic cell death by degrading cIAP1, and furthermore, cIAP1 in gastric cancer cells may act as a PD marker for YM155 treatment.

Iris Nertschinskia ethanol extract differentially induces cytotoxicity in human breast cancer cells depending on AKT1/2 activity.

Recently, we reported that an ethanol extract of Iris nertschinskia induces p53-dependent apoptosis in the MCF7 human breast cancer cell line. However, the detailed mechanisms were not fully explored. Here, we demonstrate another aspect of the activity of I. nertschinskia in breast cancer cells. We compared the response to an ethanol extract of I. nertschinskia in two different human breast cancer cell lines, Hs578Tand MDA-MB231, respectively with relatively low and high AKT1/2 activity by trypan blue exclusion assay and FACS analysis. Knockdown of endogenous AKT1 or AKT2 in breast cancer cells by RNA interference determined the sensitivity to I. nertschinskia ethanol extract compared to control cells. The I. nertschinskia ethanol extract induced cell death in a manner that depended on the level of phosphorylated AKT1/2 protein and was associated with a significant increase in the sub-G1 cell population, indicative of apoptosis. Our results indicate that an ethanol extract of I. nertschinskia differentially induces cell death in breast cancer cells depending on their level of phosphorylated AKT1/2.

Human breast cancer cells display different sensitivities to ABT-263 based on the level of survivin

ABT-263 (navitoclax), a Bcl-2 family protein inhibitor, was clinically tested as an anti-cancer agent. However, the clinical trials were limited given the occurrence of resistance to monotherapy in breast cancer cells. Our study investigates the mechanisms for overcoming navitoclax resistance by combining it with an mTOR inhibitor to indirectly target survivin. The apoptotic effects of navitoclax occurred in MDA-MB-231 breast cancer cells in a time- and dose-dependent fashion, but MCF-7 cells were resistant to navitoclax treatment. The expression of Bcl-2 family genes was not altered by navitoclax, but the expression of survivin, a member of the inhibitors of apoptosis proteins (IAP) family, was downregulated, which increased death signaling in MDA-MB-231 cells. In MCF-7 cells, a navitoclax-resistant cell line, combined treatment with navitoclax and everolimus synergistically reduced survivin expression and induced cell death. These data indicate that navitoclax induces cell death in MDA-MB-231 cells but not in MCF-7 cells. Decreased survivin expression in MDA-MB-231 cells may be a primary pathway for death signaling. Combined navitoclax and everolimus treatment induces cell death by reducing the stability of survivin in MCF-7 cells. Given that survivin-targeted therapy overcomes resistance to navitoclax, this strategy could be used to treat breast cancer patients.

Abstract 5171: Design and development of a tankyrase inhibitor STP06-1002 as an anticancer therapeutic agent

Background The Wnt/β-catenin signaling pathway plays a pivotal role in numerous biological processes and its dysregulation has been implicated in diverse oncolytic initiation. Aberrant overactivation of the Wnt/β-catenin pathway due to overexpression and accumulation of the β-catenin has been often observed in colorectal cancer (CRC). As a member of PARP family (PARP5 isoform), tankyrase (TNKS) regulates stability of the β-catenin destruction complex through Axin poly-ADP ribosylation maintaining homeostasis of level of the β-catenin. Axin is a rate-limiting component of the destruction complex and inhibition of tankyrase stabilizes Axin level to prevent subsequently CRC development by downregulation of the Wnt target genes. We herein report an orally-active tankyrase inhibitor STP06-1002 with excellent cellular potency, good ADME properties, and in vivo anti-tumor efficacy with moderate safety. Results The novel tankyrase inhibitor STP06-1002 shows good inhibition activities with IC50 29.9 nM (TNKS1) and 3.7 nM (TNKS2) and its excellent cellular potency with IC50 6.7 nM (TCF/LEF). STP06-1002 also shows excellent selectivity against PARP1 isoform with IC50 >10 μM. STP06-1002 has good ADME properties, particularly low CYP450 inhibition & induction in CYP3A and PXR, suggesting no DDI issues. It also displays good in vivo pharmacokinetic profiles in rats (B.A. [F] >60%) and dogs (27%). No significant toxicity issues are observed from cytotoxicity studies, hERG assay and Ames test. The off-target studies in both Pan Kinase panel (against 75 cancer-focused kinases) and Lead Profiling Screen® (against 68 receptors and ion channels) prove its high selectivity. In the xenograft efficacy studies for in vivo proof-of-concept, the dose-dependent effect on Colo320DM (Wnt-dependent, KRas wild type) shows tumor growth inhibition with 45% and DLD-1 (Wnt-dependent, KRas mutant) with 60%. In the case of Wnt-independent cell lines HCT116 and RKO as negative controls, both did not show any tumor growth inhibition effects. Dose range finding study of rodent (rat) resulted in maximum tolerated dose of (MTD) 200 mg/kg/day, and the corresponding of non-rodent (dog) MTD of 30 mg/kg/day. Taking these promising preclinical results of optimal in vivo efficacy dose, MTD and HSTD (50 mg/kg/day) into account, we suggest safety margin of the STP06-1002 as 6 to 11 folds applicable to clinical human dosages. Discussion The preclinical studies of STP06-1002 show successfully its potentials as an anticancer therapeutic agent. Based on the excellent biological and preclinical profiles, STP06-1002 will be a suitable candidate as an orally-active novel TNKS inhibitor and move toward phase I clinical trials with strong biomarker-based strategies. Citation Format: Kyungjin Kim, Uk-Il Kim, Hyung Tae Bang, Jihye Yoon, Jin Ha Hwang, Jung-Nyoung Heo, Kwang-Rok Kim, Hwan Jung Lim, Jai-Hee Moon, Eun Young Lee, Seul Lee, Dong-Hoon Jin. Design and development of a tankyrase inhibitor STP06-1002 as an anticancer therapeutic agent [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5171. doi:10.1158/1538-7445.AM2017-5171