Seung-Hyun Oh

DOT1L cooperates with the c-Myc-p300 complex to epigenetically derepress CDH1 transcription factors in breast cancer progression

DOT1L has emerged as an anticancer target for MLL-associated leukaemias; however, its functional role in solid tumours is largely unknown. Here we identify that DOT1L cooperates with c-Myc and p300 acetyltransferase to epigenetically activate epithelial–mesenchymal transition (EMT) regulators in breast cancer progression. DOT1L recognizes SNAIL, ZEB1 and ZEB2 promoters via interacting with the c-Myc-p300 complex and facilitates lysine-79 methylation and acetylation towards histone H3, leading to the dissociation of HDAC1 and DNMT1 in the regions. The upregulation of these EMT regulators by the DOT1L-c-Myc-p300 complex enhances EMT-induced breast cancer stem cell (CSC)-like properties. Furthermore, in vivo orthotopic xenograft models show that DOT1L is required for malignant transformation of breast epithelial cells and breast tumour initiation and metastasis. Clinically, DOT1L expression is associated with poorer survival and aggressiveness of breast cancers. Collectively, we suggest that cooperative effect of DOT1L and c-Myc-p300 is critical for acquisition of aggressive phenotype of breast cancer by promoting EMT/CSC.

Targeting the insulin-like growth factor receptor and Src signaling network for the treatment of non-small cell lung cancer

BackgroundTherapeutic interventions in the insulin-like growth factor receptor (IGF-1R) pathway were expected to provide clinical benefits; however, IGF-1R tyrosine kinase inhibitors (TKIs) have shown limited antitumor efficacy, and the mechanisms conveying resistance to these agents remain elusive.MethodsThe expression and activation of the IGF-1R and Src were assessed via the analysis of a publicly available dataset, as well as immunohistochemistry, Western blotting, RT-PCR, and in vitro kinase assays. The efficacy of IGF-1R TKIs alone or in combination with Src inhibitors was analyzed using MTT assays, colony formation assays, flow cytometric analysis, and xenograft tumor models.ResultsThe co-activation of IGF-1R and Src was observed in multiple human NSCLC cell lines as well as in a tissue microarray (n = 353). The IGF-1R and Src proteins mutually phosphorylate on their autophosphorylation sites. In high-pSrc-expressing NSCLC cells, linsitinib treatment initially inactivated the IGF-1R pathway but led a Src-dependent reactivation of downstream effectors. In low-pSrc-expressing NSCLC cells, linsitinib treatment decreased the turnover of the IGF-1R and Src proteins, ultimately amplifying the reciprocal co-activation of IGF-1R and Src. Co-targeting IGF-1R and Src significantly suppressed the proliferation and tumor growth of both high-pSrc-expressing and low-pSrc-expressing NSCLC cells in vitro and in vivo and the growth of patient-derived tissues in vivo.ConclusionsReciprocal activation between Src and IGF-1R occurs in NSCLC. Src causes IGF-1R TKI resistance by acting as a key downstream modulator of the cross-talk between multiple membrane receptors. Targeting Src is a clinically applicable strategy to overcome resistance to IGF-1R TKIs.

Neutral sphingomyelinase 2 modulates cytotoxic effects of protopanaxadiol on different human cancer cells

BackgroundSome of ginsenosides, root extracts from Panax ginseng, exert cytotoxicity against cancer cells through disruption of membrane subdomains called lipid rafts. Protopanaxadiol (PPD) exhibits the highest cytotoxic effect among 8 ginsenosides which we evaluated for anti-cancer activity. We investigated if PPD disrupts lipid rafts in its cytotoxic effects and also the possible mechanisms.MethodsEight ginsenosides were evaluated using different cancer cells and cell viability assays. The potent ginsenoside, PPD was investigated for its roles in lipid raft disruption and downstream pathways to apoptosis of cancer cells. Anti-cancer effects of PPD was also investigated in vivo using mouse xenograft model.ResultsPPD consistently exerts its potent cytotoxicity in 2 cell survival assays using 5 different cancer cell lines. PPD disrupts lipid rafts in different ways from methyl-β-cyclodextrin (MβCD) depleting cholesterol out of the subdomains, since lipid raft proteins were differentially modulated by the saponin. During disruption of lipid rafts, PPD activated neutral sphingomyelinase 2 (nSMase 2) hydrolyzing membrane sphingomyelins into pro-apoptotic intracellular ceramides. Furthermore, PPD demonstrated its anti-cancer activities against K562 tumor cells in mouse xenograft model, confirming its potential as an adjunct or chemotherapeutic agent by itself in vivo.ConclusionsThis study demonstrates that neutral sphingomyelinase 2 is responsible for the cytotoxicity of PPD through production of apoptotic ceramides from membrane sphingomyelins. Thus neutral sphingomyelinase 2 and its relevant mechanisms may potentially be employed in cancer chemotherapies.

The novel IGF-IR/Akt–dependent anticancer activities of glucosamine

BackgroundRecent studies have shown that glucosamine inhibits the proliferation of various human cancer cell lines and downregulates the activity of COX-2, HIF-1α, p70S6K, and transglutaminase 2. Because the IGF-1R/Akt pathway is a common upstream regulator of p70S6K, HIF-1α, and COX-2, we hypothesized that glucosamine inhibits cancer cell proliferation through this pathway.MethodsWe used various in vitro assays including flow cytometry assays, small interfering RNA (siRNA) transfection, western blot analysis, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays, reverse transcription-polymerase chain reaction, and in vivo xenograft mouse model to confirm anticancer activities of glucosamine and to investigate the molecular mechanism.ResultsWe found that glucosamine inhibited the growth of human non-small cell lung cancer (NSCLC) cells and negatively regulated the expression of IGF-1R and phosphorylation of Akt. Glucosamine decreased the stability of IGF-1R and induced its proteasomal degradation by increasing the levels of abnormal glycosylation on IGF-1R. Moreover, picropodophyllin, a selective inhibitor of IGF-1R, and the IGF-1R blocking antibody IMC-A12 induced significant cell growth inhibition in glucosamine-sensitive, but not glucosamine-resistant cell lines. Using in vivo xenograft model, we confirmed that glucosamine prohibits primary tumor growth through reducing IGF-1R signalling and increasing ER-stress.ConclusionsTaken together, our results suggest that targeting the IGF-1R/Akt pathway with glucosamine may be an effective therapeutic strategy for treating some type of cancer.

MEL-18 loss mediates estrogen receptor–α downregulation and hormone independence

The polycomb protein MEL-18 has been proposed as a tumor suppressor in breast cancer; however, its functional relevance to the hormonal regulation of breast cancer remains unknown. Here, we demonstrated that MEL-18 loss contributes to the hormone-independent phenotype of breast cancer by modulating hormone receptor expression. In multiple breast cancer cohorts, MEL-18 was markedly downregulated in triple-negative breast cancer (TNBC). MEL-18 expression positively correlated with the expression of luminal markers, including estrogen receptor-α (ER-α, encoded by ESR1). MEL-18 loss was also associated with poor response to antihormonal therapy in ER-α-positive breast cancer. Furthermore, whereas MEL-18 loss in luminal breast cancer cells resulted in the downregulation of expression and activity of ER-α and the progesterone receptor (PR), MEL-18 overexpression restored ER-α expression in TNBC. Consistently, in vivo xenograft experiments demonstrated that MEL-18 loss induces estrogen-independent growth and tamoxifen resistance in luminal breast cancer, and that MEL-18 overexpression confers tamoxifen sensitivity in TNBC. MEL-18 suppressed SUMOylation of the ESR1 transactivators p53 and SP1, thereby driving ESR1 transcription. MEL-18 facilitated the deSUMOylation process by inhibiting BMI-1/RING1B-mediated ubiquitin-proteasomal degradation of SUMO1/sentrin-specific protease 1 (SENP1). These findings demonstrate that MEL-18 is a SUMO-dependent regulator of hormone receptors and suggest MEL-18 expression as a marker for determining the antihormonal therapy response in patients with breast cancer.

Inonotus Obliquus Suppresses Proliferation of Colorectal Cancer Cells and Tumor Growth in Mice Models by Downregulation of β-Catenin/NF-κB-Signaling Pathways:

Chaga mushroom (Inonotus obliquas) has been used as a folk remedy for several illnesses including gastrointestinal disorders. We recently reported the potent anti-inflammatory effect of chaga extract in experimental colitis. However, its effects on colorectal cancer (CRC) have not been clearly elucidated. We investigated the effects of an aqueous extract of Inonotus obliquus (IOAE) in vitro in HCT116 and DLD1 cell lines and in vivo for adenoma in APCMin/+ mice and colitis-associated colon cancer in AOM/DSS-treated mice. Results show that IOAE suppressed the proliferation of both cell lines, and inhibited the growth of intestinal polyps in APCMin/+ and colon tumors in AOM/DSS-treated mice. IOAE induced mitochondrial intrinsic pathway of apoptosis, autophagy, and S phase cell cycle arrest. IOAE suppressed the expression levels of iNOS and Cox-2 and mRNA levels of pro-inflammatory cytokines (IL-6, IL-1β, TNF-a and IFN-γ) in the intestine of mice models. IOAE suppressed the nuclear levels of β-catenin and inhibited its downstream targets (cyclin D1 and c-Myc) along with CRC oncogene CDK8. IOAE inhibited the expression of NF-κB at cytoplasmic and nuclear levels. Our results demonstrate that IOAE possess potent anti-inflammatory and anti-proliferative properties through downregulation of Wnt/β-catenin and NF-κB pathways. Considering recent anticancer approaches involving natural products with minimal side effects, we advocate that Inonotus obliquus could be a beneficial supplement in prevention of colorectal cancer.

The in vitro and in vivo anti-tumor effect of KO-202125, a sauristolactam derivative, as a novel epidermal growth factor receptor inhibitor in human breast cancer.

Epidermal growth factor receptor (EGFR) is one of the most promising targets for cancer therapy. Here, we show the in vitro and in vivo anticancer effects and associated mechanisms of KO‐202125, one of the synthesized aristolactam analogs, as a novel EGFR inhibitor, in EGFR‐overexpressing cancer cell lines. KO‐202125 showed more effective growth inhibition and apoptosis induction than gefitinib, a representative EGFR inhibitor, in various EGFR‐overexpressing human cancers including estrogen receptor (ER)‐negative MDA‐MB‐231 human breast cancer cells. Epidermal growth factor receptor phosphorylation at Tyr1068 was reduced and, consequently, the association of EGFR with p85 was decreased by KO‐202125 treatment in MDA‐MB‐231 cell lines. This led to inactivation of the PI3K/Akt pathway, and consequently suppression of activation of the Wnt pathway and enhancement of the nuclear import of p27Kip1. KO‐202125 treatment in nude mice injected with MDA‐MB‐231 cells showed inhibition of tumor growth without toxicity. Collectively, our results showed the possibility of KO‐202125 as an effective therapy agent of EGFR‐overexpressing cancer cells through reduced EGFR activity and downregulation of the Akt pathway. (Cancer Sci 2011; 102: 597–604)

Abstract A61: Targeting the c-Met signaling pathway by humanized anti-HGF antibody inhibits irinotecan resistance in colorectal cancer.

Background and Purpose: Preclinical and clinical findings indicate that multiple tumour micro-environmental factors, including growth factors, protect solid tumors from therapeutic interventions. Experimental evidence has clarified some of the resistance mechanisms, which have led to the development of innovative approaches to treat cancer. Irinotecan (CPT-11) is a semi-synthetic derivative of camptothecin, originally isolated from the Chinese/Tibetan ornamental tree Camptothecaacuminata. It is a key component of first- and second-line treatment regimens for metastatic colorectal cancer (CRC). Studies of irinotecan have demonstrated that its efficacy to treat cancer is superior to that of best supportive care. However, de novo or acquired clinical resistance to camptothecins still exists expecting new target. The hepatocyte growth factor (HGF)/c-Met signaling system is known to be implicated in the development and progression of colon cancer, but the relationship between the expressions of HGF or c-MET and clinicopathologic feature remains controversial. Growth Factors derived from the extracellular matrix (ECM) create an environment conducive to tumor growth and invasion. We hypothesized that over-expression of HGF in cancer-associated fibroblast (CAF) is related to tumor metastasis and chemo-resistance. We also hypothesized that HGF deprivation using neutralizing antibody inhibited chemoresistance in colorectal cancer. Results: We measured the levels of mRNA and secreted protein of HGF by real-time PCR and ELISA in fibroblast and CRC cell lines. The mRNA and secreted protein level of HGF were significantly higher in fibroblast than in CRC cell lines. HGF was not detected in conditioned media (CM) from CRC cell cultures established without fibroblasts, but was detected in CM from fibroblast harvested without CRC cells, suggesting that the measured HGF is fibroblast-derived. Fibroblast-derived HGF induced invasion and chemo-resistance of CRC cell lines. We also found elevated levels of HGF in the CM from fibroblast treated with irinotecan. However, anti-HGF antibody removed this resistance to irinitecan. Down regulation of MET expression by a MET-specific siRNA canceled HGF-induced resistance to irinotecan in CRC cells. Fibroblast originated HGF activated c-Met, PI3K/AKT and MAPK signaling in CRC cells. Phosphorylated-MET mediated activation of PI3K/AKT and MAPK rescued HGF effect in CRC cells. Furthermore, fibroblast-derived HGF affected migration and invasion of CRC cells. We further showed that an anti-HGF antibody enhanced anti-tumor activity of irinotecan in vivo, resulting in shrinkage of well-established human colon cancer in mice without enhancing toxicity. Conclusion: We identified HGF as an important determinant of irinotecan resistance and metastasis of CRC. Anti-HGF monoclonal antibody treatment confirmed the importance of this growth factor for chemo-resistance in CRC. These results present new options toward the early diagnosis of chemoresistance and suggest novel combinations of chemotherapy and anti-HGF agents to prevent or significantly delay the onset of therapy resistance. These observations open new avenues toward the diagnosis of chemoresistant tumors and therapies targeting HGF overexpressing cancers. Citation Format: Jong Kyu Woo, Yeong-Su Jang, Ju-Hee Kang, Hwan Mook Kim, Seung-Hyun Oh. Targeting the c-Met signaling pathway by humanized anti-HGF antibody inhibits irinotecan resistance in colorectal cancer.. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr A61. doi:10.1158/1538-7445.CHTME14-A61

Abstract 1716: Targeting the insulin-like growth factor receptor/Insulin receptor and Src signaling network for the treatment of non-small cell lung cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Insulin-like growth factor I receptor (IGF-1R)-mediated signaling plays an important role in the proliferation, survival, and metastasis of cancer cells. The IGF-1R-targeting anticancer agents including monoclonal antibodies and small molecule tyrosine kinase inhibitors (TKIs) have been developed, but their antitumor effects have been marginal and limited in clinical trials. Therefore, the mechanism underlying resistance to the IGF-1R-targeting therapies and the rational combination strategies to overcome potential drug resistance need to be investigated. In this study, we demonstrated the association of Src with the resistance to the IGF-1R TKI in non-small cell lung cancer (NSCLC). We found the co-activation of IGF-1R/IR and Src in various human NSCLC cell lines, The mRNA expression and phosphorylation of IGF-1R and Src were also significantly correlated with each other in NSCLC databases from a public dataset and a tissue microarray (n=353). Next, we found Src can be activated through multiple pathways including EGFR and integrin β3 signaling and function as an alternative kinase for phosphorylation of IGF-1R, especially at Tyr1135/36, but not Tyr1131. Src is activated in lung cancer cells possessing both primary and acquired resistance to an IGF-1R TKI. Consistent with the results, inhibition of Src significantly attenuated activation of IGF-1R/IR and co-targeting of IGF-1R and Src effectively suppressed cell proliferation, colony formation, and tumor growth in vitro and in vivo. Taken together, these results suggest that Src causes resistance to IGF-1R TKIs by functioning as a key downstream modulator of multiple signaling pathways for IGF-1R phosphorylation and thus co-targeting IGF-1R and Src could be an effective therapeutic strategy for NSCLC. Citation Format: Hye-Young Min, Hye Jeong Yun, Hyo-Jong Lee, Jaebeom Cho, Hyun-Ji Jang, Kyung Min Kim, Woo-Young Kim, Seung-Hyun Oh, Diane Liu, J. Jack Lee, Waun Ki Hong, Ignacio I. Wistuba, Ho-Young Lee. Targeting the insulin-like growth factor receptor/Insulin receptor and Src signaling network for the treatment of non-small cell lung cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1716. doi:10.1158/1538-7445.AM2014-1716