Seung-Woo Hong

Increased expression of ATG10 in colorectal cancer is associated with lymphovascular invasion and lymph node metastasis.

Background Autophagy has paradoxical and complex functions in cancer development, and autophagy-related genes (ATG) are key regulators in autophagy. Until now, more than 30 different ATG proteins have been identified in yeast, and their mammalian counterparts also have been reported. Although the roles of a few ATG proteins in cancer have been characterized, the role of ATG10 is almost completely unknown. Methodology/Principal Findings To investigate the clinicopathological role of ATG10 in colorectal cancer, we analyzed ATG10 expression in colorectal cancer tissues and cell lines. Protein expression analysis showed that ATG10 is highly increased in colorectal cancer (tissue - 18/37 cases, 48%; cell line –8/12 cell lines, 66%). Immunohistochemical analysis with clinicopathological features indicated a strong association of the up-regulation of ATG10 with tumor lymph node metastasis (pu200a=u200a0.005) and invasion (p<0.001). Moreover, both 5-year disease free survival and overall survival rates of patients bearing tumors that did not express ATG10 were significantly higher than those of patients bearing ATG10-expressing tumors (pu200a=u200a0.012). Conclusion/Significance Increased expression of ATG10 in colorectal cancer is associated with lymphovascular invasion and lymph node metastasis indicating that ATG10 may be a potential prognostic maker in colorectal cancer.

Depletion of ascorbic acid impairs NK cell activity against ovarian cancer in a mouse model

Ascorbic acid (Vitamin C) administration has been used to prevent infectious diseases in public or as a therapeutic agent by the physicians in treatment of several diseases. Ascorbic acid is also involved in immune cell functions and immune responses, although the mechanisms by which it exerts effects on immune cells against cancer cells are not fully understood at the normal plasma level. In this study, we used the mice lacking l-gulono-γ-lactone oxidase (Gulo), the enzyme required for the biosynthesis of ascorbic acid, to characterize the effects of ascorbic acid on NK cell cytotoxicity against ovarian cancer cells, MOSECs (murine ovarian surface epithelial cells). Gulo(-/-) mice depleted of ascorbic acid survived for a shorter time than the normal control or Gulo(-/-) mice supplemented with ascorbic acid after tumor challenge regardless of treatment with IL-2. CD69 and NKG2D expression was clearly reduced in NK cells isolated from mice depleted of ascorbic acid as compared to that in the normal control and the mice supplemented with ascorbic acid. We also observed that IFN-γ secretion by NK cells isolated from Gulo(-/-) mice depleted of ascorbic acid was decreased after NK cells were co-cultured with MOSECs. Furthermore, the mRNA expression of perforin and granzyme B genes was also significantly decreased in NK cells isolated from mice depleted of ascorbic acid. Taken together, our results suggest that ascorbic acid at the normal plasma concentration has an essential role in maintaining the NK cytotoxicity against cancer cells.

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.

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.

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.

SAHA, an HDAC inhibitor, overcomes erlotinib resistance in human pancreatic cancer cells by modulating E-cadherin

Pancreatic cancer is one of the most lethal cancers and remains a major unsolved health problem. Less than 20xa0% of patients are surgical candidates, and the median survival for non-resected patients is approximately 3 to 4xa0months. Despite the existence of many conventional cancer therapies, few targeted therapies have been developed for pancreatic cancer. Combination therapy using erlotinib and gemcitabine is an approved standard chemotherapy for advanced pancreatic cancer, but it has marginal therapeutic benefit. To try to improve the therapeutic outlook, we studied the efficacy of another combination treatment and the relevance to E-cadherin in human pancreatic cancer cells. We treated two human pancreatic cancer cell lines with the histone deacetylase inhibitor (HDACi) SAHA. Interestingly, in these Panc-1 and Capan1 cells, we observed that the expression levels of E-cadherin and phosphorylated EGFR were gradually upregulated after treatment with SAHA. Furthermore, these cells underwent induced cell death after exposure to the combination treatment of SAHA and erlotinib. In Panc-1 cells, overexpression of E-cadherin activated the phosphorylation of EGFR and increased the cell sensitivity to erlotinib. In Capan1 cells, knocking down E-cadherin decreased the expression of phosphorylated EGFR, and these cells did not respond to erlotinib. Therefore, we demonstrated the efficacy of the combined treatment with SAHA and erlotinib in human pancreatic cancer cells, and we determined that the increased efficacy was due, at least in part, to the effects of SAHA on the expression of E-cadherin. Our studies suggest that E-cadherin may be a potent biomarker for pancreatic cancer.

SH003 selectively induces p73‑dependent apoptosis in triple‑negative breast cancer cells

Triple-negative breast cancer (TNBC) is a breast cancer subtype that has an aggressive phenotype, is highly metastatic, has limited treatment options and is associated with a poor prognosis. In addition, metastatic TNBC has no preferred standard chemotherapy due to resistance to anthracyclines and taxanes. The present study demonstrated that a herbal extract, SH003, reduced cell viability and induced apoptosis in TNBC without cell cytotoxicity. Cell viability was examined using trypan blue exclusion and colony formation assays, which revealed a decrease in the cell viability. Additionally, apoptosis was determined using flow cytometry and a sub‑G1 assay, which revealed an increase in the proportion of cells in the sub‑G1 phase. The present study investigated the anticancer effect of SH003 in the Hs578T, MDA‑MB‑231 and ZR‑751 TNBC cell lines, and in the MCF7 and T47D non‑TNBC cell lines. Western blot analysis revealed that the expression levels of poly‑ADP‑ribose polymerase (PARP) cleavage protein in cells treated with SH003 were increased dose‑dependent manner, indicating that SH003 induced apoptosis via a caspase‑dependent pathway. Pre‑treatment with the caspase inhibitor Z‑VAD reduced SH003‑induced apoptosis was examined using trypan blue exclusion. Moreover, SH003 treatment enhanced the p73 levels in MDA‑MB‑231 cells but not in MCF7 cells. Transfection of p73 small interfering RNA (siRNA) in MDA‑MB0231 cells revealed that the apoptotic cell death induced by SH003 was significantly impaired in comparison with scramble siRNA transfected MDA‑MB‑231 cells. This was examined using trypan blue exclusion and flow cytometry analysis (sub‑G1). In addition, SH003 and paclitaxel exhibited synergistic anticancer effects on TNBC cells. The results indicate that SH003 exerts its anticancer effect via p73 protein induction and exhibits synergistic anticancer effects when combined with paclitaxel.

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 C276: Ruxolitinib induces apoptotic cell death through the suppression of pJAK1 in human colon cancer cells.

Janus kinase (JAK) is one of the main upstream activators of signal transducers and activators of transcription (STAT) that are constitutively activated in various malignancies and are associated with cell growth, survival, and carcinogenesis. Here, we investigated to role of JAKs in colorectal cancer in order to develop effective therapeutic targets for ruxolitinib, which is the first JAK1/2 inhibitor to be approved by FDA. After examining the basal expression levels of phospho-JAK1 and phospho-JAK2, we measured the effects of ruxolitinib on the phosphorylation of JAK1/2 using western blot analysis. Cell viability was determined using the trypan blue exclusion assay. The cell death mechanism was identified by the activation of caspase 3 using western blot and annexin V staining. The basal levels of phospho-JAK1 and phospho-JAK2 were cancer cell type dependent. Colorectal cancer cell lines that phosphorylate both JAK1 and JAK2 include DLD-1 and RKO. Ruxolitinib inactivates both JAK1 and JAK2 in DLD-1 cells but inactivates only JAK1 in RKO cells. Cell death was proportional to the inactivation of JAK1 but not JAK2. Ruxolitinib causes caspase-dependent cell death, which is prevented by treatment with z-VAD. The inhibition of JAK1 phosphorylation seemed sufficient to allow ruxolitinib-mediated apoptosis. JAK1 is a key molecule that is involved in colon cancer cell survival and the inhibition of JAK1 by ruxolitinib results in caspase-dependent apoptosis in colorectal cancer cells. The use of selective JAK1 inhibitors could be an attractive therapy against colorectal cancer, but further clinical investigations are needed to test this possibility. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C276. Citation Format: Ho Jung An, Eun Kyoung Choi, Jin-Sun Kim, Seung-Woo Hong, Jai-Hee Moon, Jae-Sik Shin, Seung-Hee Ha, Kyu-Pyo Kim, Yong Sang Hong, Jae-Lyun Lee, Eun Kyung Choi, Jung Shin Lee, Dong-Hoon Jin, Tae Won Kim. Ruxolitinib induces apoptotic cell death through the suppression of pJAK1 in human colon cancer cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C276.

Abstract B30: Identification of new regulator in p53-mediated cellular senescence

The downstream events and target genes of p53 in senescence responses are not fully understood. Here, we identify a novel regulator, A-core gene, in p53-mediated cellular senescence. Overexpression of A-core in p53-deficient mouse embryonic fibroblasts (MEFs) accelerates senescence, whereas A-core gene knockdown by small interfering RNA (siRNA) leads to escape from p53-mediated senescence in p53-expressing MEFs. A-core overexpression also increased intracellular levels of reactive oxygen species (ROS). The ROS inhibitor N-acetyl-L-cysteine rescued A-core overexpression-induced senescence. Surprisingly, the elevated levels of ROS that accompanied A-core overexpression were paralleled by an increase in p21 expression. siRNA-mediated knockdown of p21 in A-core overexpressing MEFs abrogated the A-core-dependent increase in ROS levels, indicating that A-core acts through p21 induction and subsequent ROS elevation to trigger senescence. In addition, expression of the cell proliferation-related proteins cyclin E and B was decreased after A-core expression. Collectively, these results suggest that A-core is a downstream target of p53 that is sufficient to induce p21 expression and ROS production, and is necessary for p53-mediated senescence.nnCitation Format: Jae-Sik Shin, Seung-Woo Hong, Dae-Hee Lee, Jai-Hee Moon, Jeong Eun Kim, Yong Sang Hong, Kyu-pyo Kim, Jae-Lyun Lee, Eun Kyung Choi, Jung Shin Lee, Dong-Hoon Jin, Tae Won Kim. Identification of new regulator in p53-mediated cellular senescence. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr B30.