Dong-Hui Shin

Anticancer effects of wogonin in both estrogen receptor-positive and -negative human breast cancer cell lines in vitro and in nude mice xenografts.

Wogonin is a plant monoflavonoid which has been reported to inhibit cell growth and/or induce apoptosis in various tumors. Herein, we investigated the in vitro and in vivo anticancer effects and associated mechanisms of wogonin in human breast cancer. Effects of wogonin were examined in estrogen receptor (ER)‐positive and ‐negative human breast cancer cells in culture for proliferation, cell cycle progression, and apoptosis. The in vivo effect of oral wogonin was examined on tumor xenograft growth in athymic nude mice. The molecular changes associated with the biological effects of wogonin were analyzed by immunoblotting. Cell growth was attenuated by wogonin (50–200 μM), independently of its ER status, in a time‐ and concentration‐dependent manner. Apoptosis was enhanced and accompanied by upregulation of PARP and Caspase 3 cleavages as well as proapoptotic Bax protein. Akt activity was suppressed and reduced phosphorylation of its substrates, GSK‐3β and p27, was observed. Suppression of Cyclin D1 expression suggested the downregulation of the Akt‐mediated canonical Wnt signaling pathway. ER expression was downregulated in ER‐positive cells, while c‐ErbB2 expression and its activity were suppressed in ER‐negative SK‐BR‐3 cells. Wogonin feeding to mice showed inhibition of tumor growth of T47D and MDA‐MB‐231 xenografts by up to 88% without any toxicity after 4 weeks of treatment. As wogonin was effective both in vitro and in vivo, our novel findings open the possibility of wogonin as an effective therapeutic and/or chemopreventive agent against both ER‐positive and ‐negative breast cancers, particularly against the more aggressive and hormonal therapy‐resistant ER‐negative types.

Mel-18 Negatively Regulates INK4a/ARF-Independent Cell Cycle Progression via Akt Inactivation in Breast Cancer

Mel-18, a polycomb group (PcG) protein, has been suggested as a tumor suppressor in human breast cancer. Previously, we reported that Mel-18 has antiproliferative activity in breast cancer cells. However, its functional mechanism has not been fully elucidated. Here, we investigated the role of Mel-18 in human breast cancer. We saw an inverse correlation between Mel-18 and phospho-Akt, which were expressed at low and high levels, respectively, in primary breast tumor tissues from 40 breast cancer patients. The effect of Mel-18 on cell growth was examined in two breast cancer cell lines, SK-BR-3 and T-47D, which express relatively low and high levels of endogenous Mel-18, respectively. On Mel-18 overexpression in SK-BR-3 cells, cell growth was attenuated and G(1) arrest was observed. Likewise, suppression of Mel-18 by antisense expression in T-47D cells led to enhanced cell growth and accelerated G(1)-S phase transition. In these cells, cyclin-dependent kinase (Cdk)-4 and Cdk2 activities were affected by Mel-18, which were mediated by changes in cyclin D1 expression and p27(Kip1) phosphorylation at Thr(157), but not by INK4a/ARF genes. The changes were both dependent on the phosphatidylinositol 3-kinase/Akt signaling pathway. Akt phosphorylation at Ser(473) was reduced by Mel-18 overexpression in SK-BR-3 cells and enhanced by Mel-18 suppression in T-47D cells. Akt-mediated cytoplasmic localization of p27(Kip1) was inhibited by Mel-18 in SK-BR-3 cells. Moreover, Mel-18 overexpression showed reduced glycogen synthase kinase-3beta phosphorylation, beta-catenin nuclear localization, T-cell factor/lymphoid enhancer factor promoter activity, and cyclin D1 mRNA level. Taken together, we established a linear relationship between Mel-18-->Akt-->G(1) phase regulators.

The antitumor effect of LJ-529, a novel agonist to A3 adenosine receptor, in both estrogen receptor–positive and estrogen receptor–negative human breast cancers

Agonists to A3 adenosine receptor (A3AR) have been reported to inhibit cell growth and/or induce apoptosis in various tumors. We tested the effect of a novel A3AR agonist generically known as LJ-529 in breast cancer cells. Anchorage-dependent cell growth and in vivo tumor growth were attenuated by LJ-529, independently of its estrogen receptor (ER) α status. In addition, apoptosis was induced as evidenced by the activation of caspase-3 and c–poly(ADP)ribose polymerase. Furthermore, the Wnt signaling pathway was down-regulated and p27kip was induced by LJ-529. In ER-positive cells, the expression of ER was down-regulated by LJ-529, which might have additionally contributed to attenuated cell proliferation. In ER-negative, c-ErbB2-overexpressing SK-BR-3 cells, the expression of c-ErbB2 and its downstream extracellular signal-regulated kinase pathway were down-regulated by LJ-529. However, such effect of LJ-529 acted independently of its receptor because no A3AR was detected by reverse transcription-PCR in all four cell lines tested. In conclusion, our novel findings open the possibility of LJ-529 as an effective therapeutic agent against both ER-positive and ER-negative breast cancers, particularly against the more aggressive ER-negative, c-ErbB2-overexpressing types. [Mol Cancer Ther 2006;5(3):685–92]

Loss of Mel-18 enhances breast cancer stem cell activity and tumorigenicity through activating Notch signaling mediated by the Wnt/TCF pathway

Mel‐18 has been proposed as a negative regulator of Bmi‐1, a cancer stem cell (CSC) marker, but it is still unclear whether Mel‐18 is involved in CSC regulation. Here, we examined the effect of Mel‐18 on the stemness of human breast CSCs. In Mel‐18 small hairpin RNA (shRNA)‐transduced MCF‐7 cells, side population (SP) cells and breast CSC surface marker (CD44+/CD24–/ESA+)‐expressing cells, which imply a CSC population, were enriched. Moreover, the self‐renewal of CSCs was enhanced by Mel‐18 knockdown, as measured by the ability for tumorsphere formation in vitro and tumor‐initiating capacity in vivo. Similarly, Mel‐18 overexpression inhibited the number and self‐renewal activity of breast CSCs in SK‐BR‐3 cells. Furthermore, our data showed that Mel‐18 blockade up‐regulated the expression of the Wnt/TCF target Jagged‐1, a Notch ligand, and consequently activated the Notch pathway. Pharmacologic inhibition of the Notch and Wnt pathways abrogated Mel‐18 knockdown‐mediated tumorsphere formation ability. Taken together, our findings suggest that Mel‐18 is a novel negative regulator of breast CSCs that inhibits the stem cell population and in vitro and in vivo self‐renewal through the inactivation of Wnt‐mediated Notch signaling.—Won, H.‐Y., Lee, J.‐Y., Shin, D.‐H., Park, J.‐H., Nam, J.‐S., Kim, H.‐C., Kong, G. Loss of Mel‐18 enhances breast cancer stem cell activity and tumorigenicity through activating Notch signaling mediated by the Wnt/TCF pathway. FASEB J. 26, 5002–5013 (2012). www.fasebj.org

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.

Constitutive overexpression of Id-1 in mammary glands of transgenic mice results in precocious and increased formation of terminal end buds, enhanced alveologenesis, delayed involution.

Inhibitor of differentiation‐1 (Id‐1) has been shown to play an essential role in cell proliferation, invasion, migration, and anti‐apoptosis. However, the effect of Id‐1 in mammary gland development remains unknown. Here, we generated MMTV‐Id‐1 transgenic mice to study the role of Id‐1 in mammary gland development. In virgin mice, Id‐1 overexpression led to precocious development and delayed regression of terminal end buds (TEBs) compared with wild‐type mice. The number of BrdU‐positive cells and the expression of Wnt signaling molecules, β‐catenin and cyclin D1, which regulate ductal extension and TEB formation in virgin, were statistically higher in Id‐1 transgenic mice than in wild‐type mice. Id‐1 also had an effect on the formation and proliferation of lobuloalveolar structures during early and mid‐pregnancy. Id‐1 transgenic mice had more lobulated and prominent alveolar budding than wild‐type mice and had significantly greater counts of lobuloalveolar structures in early pregnancy. The expression of BrdU, β‐catenin, and cyclin D1 was also predominantly increased in Id‐1 transgenic mice. Moreover, Id‐1 transgenic mice showed delayed involution. Id‐1 regulated the expression levels of anti‐apoptotic Bcl‐2 and pro‐apoptotic Bax, and resulted in delay of apoptotic peak during postlactational involution. We also found that Id‐1 was able to modulate expression of the regulators of Wnt/β‐catenin signaling such as phospho‐Akt, BMP2, FGF3, and RAR‐β in tubuloalveolar development of mammary glands. Taken together, our results suggest that Id‐1 plays a pivotal role in mammary gland development through Wnt signaling‐mediated acceleration of precocity and alveologenesis and Bcl‐2 family members‐mediated delay of involution. J. Cell. Physiol. 226: 1340–1352, 2011.

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 A040: Id-1 increases mammary basal stem cell activity and tumorigenesis by activating the Wnt/c-Myc pathway

Inhibitor of differentiation/DNA binding (Id)-1 is a crucial regulator of mammary development and breast cancer progression. Here, we demonstrate that Id-1 induces mammary tumorigenesis by increasing normal and malignant mammary stem cell (MaSC) activities. MaSC expansion and increased self-renewal and in vivo regenerative capacity of MaSCs were observed in the mammary glands of MMTV-Id-1 transgenic mice. Id-1 maintains the MaSC-enriched basal cells, but not the luminal cell lineage, in the mammary glands. Furthermore, Id-1 induces ductal hyperplasia and mammary tumors with high expression of basal markers in MMTV-Id-1 transgenic mice. It also increases breast cancer stem cell populations and activity in human breast cancer lines. In experiments with mouse primary mammary epithelial cells and human cancer cells, the effects of Id-1 on normal and cancer stem cell activity were shown to be mediated by the Wnt/c-Myc pathway. Collectively, these findings indicate that Id-1 regulates mammary basal stem cells by activating the Wnt/c-Myc pathway, thereby contributing to basal marker-positive breast tumor development. Citation Format: Dong-Hui Shin, Jeong-Yeon Lee, Ki-Seok Jang, Kyueng-Whan Min, Si-Hyong Jang, Seung Hyun Oh, Gu Kong. Id-1 increases mammary basal stem cell activity and tumorigenesis by activating the Wnt/c-Myc pathway. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A040.