Kyung-Hee Chun

Targeting of miR34a–NOTCH1 Axis Reduced Breast Cancer Stemness and Chemoresistance

Human breast cancers include cancer stem cell populations as well as nontumorigenic cancer cells. Breast cancer stem cells have self-renewal capability and are resistant to conventional chemotherapy. miRNAs regulate the expression of many target genes; therefore, dysregulation of miRNAs has been associated with the pathogenesis of human diseases, including cancer. However, a role for miRNA dysregulation in stemness and drug resistance has yet to be identified. Members of the miR34 family are reportedly tumor-suppressor miRNAs and are associated with various human cancers. Our results confirm that miR34a expression was downregulated in MCF7/ADR cells compared with MCF7 cells. We hypothesized that this reduction was due to the p53 (TP53) mutation in MCF7/ADR cells. In this study, we found that primary and mature miR34a were suppressed by treatment with p53 RNAi or the dominant-negative p53 mutant in MCF7 cells. Ectopic miR34a expression reduced cancer stem cell properties and increased sensitivity to doxorubicin treatment by directly targeting NOTCH1. Furthermore, tumors from nude mice treated with miR34a were significantly smaller compared with those of mice treated with control lentivirus. Our research suggests that the ectopic expression of miR34a represents a novel therapeutic approach in chemoresistant breast cancer treatment.

Galectin-3 increases the motility of mouse melanoma cells by regulating matrix metalloproteinase-1 expression

Although mounting evidence indicates the involvement of galectin-3 in cancer progression and metastasis, the underlying molecular mechanisms remain largely unknown. In this study, we investigated the effect and possible mechanism of galectin-3 on the migration and invasion of B16F10, a metastatic melanoma cell line, in which galectin-3 and matrix metalloproteinase-1 (MMP-1) were both found to be highly expressed. Knockdown of galectin-3 with specific siRNA reduced migration and invasion, which was associated with reduced expression of MMP-1. To further investigate the underlying mechanism, we examined the effect of galectin-3 knockdown on the activity of AP-1, a transcriptional factor regulating MMP-1 expression. We found that galectin-3 directly interacted with AP-1 and facilitated the binding of this complex to the MMP-1 promoter that drives MMP-1 transcription. Moreover, silencing of galectin-3 inhibited binding of fra-1 and c-Jun to promoter sites of MMP-1 gene. Consistent with these in vitro findings, our in vivo study demonstrated that galectin-3 shRNA treatment significantly reduced the total number of mouse lung metastatic nodules. Taken together, galectin-3 facilitates cell migration and invasion in melanoma in vitro and can induce metastasis in vivo, in part through, regulating the transcription activity of AP-1 and thereby up-regulating MMP-1 expression.

Silencing of galectin-3 changes the gene expression and augments the sensitivity of gastric cancer cells to chemotherapeutic agents.

Galectin‐3 is known to modulate cell proliferation and apoptosis and is highly expressed in human cancers, but its function in gastric cancer is still controversial. Here, we examined the role of galectin‐3 in gastric cancer cells by silencing it with synthetic double‐stranded siRNA. After silencing of galectin‐3, cell numbers decreased and cell shape changed. Galectin‐3 siRNA treatment also induced G1 arrest. DNA microarray analysis was used to assess changes in gene expression following galectin‐3 silencing. We found that silencing of galectin‐3 caused changes in gene expression. RT‐PCR and real‐time PCR were utilized for validation of the changes found in microarray studies. Western blot analysis confirmed changes in the expression of proteins of interest: cyclin D1, survivin, XIAP, XAF, PUMA, and GADD45α. Generally, it tended to increase the expression of several pro‐apoptotic genes, and to decrease the expression of cell cycle progressive genes. We also confirmed that changes in the expression of these genes were caused by galectin‐3 overexpression. Finally, we demonstrated that silencing of galectin‐3 enhanced apoptosis induction with chemotherapeutic agents by further reducing the expression of anti‐apoptotic and/or cell survival molecules such as survivin, cyclin D1, and XIAP, and increasing the expression of pro‐apoptotic XAF‐1. We conclude that galectin‐3 is involved in cancer progression and malignancy by modulating the expression of several relevant genes, and inhibition of galectin‐3 may be an approach to improve chemotherapy of gastric cancers. (Cancer Sci 2009)

Galectin-3 germline variant at position 191 enhances nuclear accumulation and activation of β-catenin in gastric cancer

Mutation of galectin-3 at position 191 (rs4644) substituting proline to histidine (gal-3H64) resulted in the acquisition of resistance to drug-induced apoptosis by breast cancer cells. This study employed gastric cancer cells and patient tissues in attempts to elucidate how and why this mutation in galectin-3 (gal-3H64) enhances cancer progression, compared to wild type galectin-3 (gal-3P64). First, we prepared lenti-virus constructs containing gal-3P64, gal-3H64 and LacZ, and used them to infect galectin-3 null SNU-638 cells. We found that gal-3H64 over-expression increases gastric cancer cell growth more than gal-3P64 or LacZ over-expression. Also, gal-3H64 over-expression conferred more resistance to cisplatin or 5-FU induced cytotoxicity than gal-3P64. Gal-3H64 also enhanced nuclear accumulation of β-catenin as well as increased expression of TCF-4 target genes, such as fascin-1 and c-Myc through the augmented promoter binding activity of TCF-4, than gal-3P64. We also demonstrated stronger staining of β-catenin and galectin-3 in malignant tissues from gastric cancer patients with mutated galectin-3 at position 191 (gal-3 191) (A/A) (H64) and greater localization in the nucleus than in gal-3 191 A/C (P64) cancer patients. Taken together, we elucidated in this study that germline variant of gal-3H64 increases nuclear accumulation of β-catenin and promotes TCF transcriptional activity and enhances more the galectin-3’s role in gastric cancer progression.

STAT3 silencing enhances the efficacy of the HSV.tk suicide gene in gastrointestinal cancer therapy.

Aberrant activation of Signal Transducer and Activator of Transcription 3 (STAT3) signaling has been shown to be associated with uncontrolled cell proliferation and suppression of host-immune surveillance. Conversely, silencing STAT3 can have the dual effects of inhibiting cancer cell proliferation and inducing anti-tumor immune responses. Here, we report on the effects of STAT3 silencing on suicide gene therapy with thymidine kinase (tk). STAT3 silencing by siRNA inhibited the proliferation of AGS human gastric cancer cells through G1 cell cycle arrest, decreased levels of immune-suppressive cytokines, and increased levels of immune-activating cytokines. CT26 mouse colon adenocarcinoma cells, in which STAT3 expression was knocked-down by a STAT3 shRNA-containing lentivirus, grew more slowly in syngenic model Balb/c mice than control CT26 cells. Moreover, we found that STAT3 silencing augmented the efficacy of suicide gene therapy in CT26 cell xenografted mice. When we administrated adenoviruses harboring the herpes simplex virus thymidine kinase gene (Ad5.CMV.HSV.tk) into STAT3-silenced CT26 cell tumors, extensive apoptosis was observed and there was a significant reduction in the size of CT26 cell tumors. STAT3 silencing also enhanced the recruitment and cytotoxic activity of CD3+CD8+ T-cells, and changed the cytokine expression pattern of CT26 cell tumors, reflecting augmentation of anti-cancer immune responses. We conclude that combining suicide gene therapy with STAT3 silencing can result in enhanced anti-cancer effects.

Galectin-3 supports stemness in ovarian cancer stem cells by activation of the Notch1 intracellular domain

Ovarian cancer is the most lethal gynecologic disease because usually, it is lately sensed, easily acquires chemoresistance, and has a high recurrence rate. Recent studies suggest that ovarian cancer stem cells (CSCs) are involved in these malignancies. Here, we demonstrated that galectin-3 maintains ovarian CSCs by activating the Notch1 intracellular domain (NICD1). The number and size of ovarian CSCs decreased in the absence of galectin-3, and overexpression of galectin-3 increased them. Overexpression of galectin-3 increased the resistance for cisplatin and paclitaxel-induced cell death. Silencing of galectin-3 decreased the migration and invasion of ovarian cancer cells, and overexpression of galectin-3 reversed these effects. The Notch signaling pathway was strongly activated by galectin-3 overexpression in A2780 cells. Silencing of galectin-3 reduced the levels of cleaved NICD1 and expression of the Notch target genes, Hes1 and Hey1. Overexpression of galectin-3 induced NICD1 cleavage and increased expression of Hes1 and Hey1. Moreover, overexpression of galectin-3 increased the nuclear translocation of NICD1. Interestingly, the carbohydrate recognition domain of galectin-3 interacted with NICD1. Overexpression of galectin-3 increased tumor burden in A2780 ovarian cancer xenografted mice. Increased expression of galectin-3 was detected in advanced stages, compared to stage 1 or 2 in ovarian cancer patients, suggesting that galectin-3 supports stemness of these cells. Based on these results, we suggest that targeting galectin-3 may be a potent approach for improving ovarian cancer therapy.

A novel miR-34a target, protein kinase D1, stimulates cancer stemness and drug resistance through GSK3/β-catenin signaling in breast cancer.

One of the properties of human breast cancer cells is cancer stemness, which is characterized by self-renewal capability and drug resistance. Protein kinase D1 (PRKD1) functions as a key regulator of many cellular processes and is downregulated in invasive breast cancer cells. In this study, we found that PRKD1 was upregulated in MCF-7-ADR human breast cancer cells characterized by drug resistance. Additionally, we discovered that PRKD1 expression was negatively regulated by miR-34a binding to the PRKD1 3′-UTR. PRKD1 expression increased following performance of a tumorsphere formation assay in MCF-7-ADR cells. We also found that reduction of PRKD1 by ectopic miR-34a expression or PRKD1 siRNA treatment resulted in suppressed self-renewal ability in breast cancer stem cells. Furthermore, we confirmed that the PRKD1 inhibitor CRT0066101 reduced phosphorylated PKD/PKCμ, leading to suppression of breast cancer stemness through GSK3/β-catenin signaling. PRKD1 inhibition also influenced apoptosis initiation in MCF-7-ADR cells. Tumors from nude mice treated with miR-34a or CRT0066101 showed suppressed tumor growth, proliferation, and induced apoptosis. These results provide evidence that regulation of PRKD1, a novel miR-34a target, contributes to overcoming cancer stemness and drug resistance in human breast cancer.

Targeting Notch signaling by γ-secretase inhibitor I enhances the cytotoxic effect of 5-FU in gastric cancer

Abstract Current medication for gastric cancer patients has a low success rate and the patients develop rapid tolerance to these drugs. Therefore, the development of new regimens is desired. In this study, we determined that Notch-signaling-related genes were overexpressed and activated in gastric cancer patients and gastric cancer cell lines. According to recent studies, γ-secretase inhibitors (GSIs), which function as Notch signaling inhibitors, could be used as therapeutic drugs in cancer. We demonstrated that GSI I (cbz-IL-CHO) is the most effective GSI in gastric cancer cells. We also determined the cell survival signaling-related proteins that were affected by GSI I. The levels of phosphorylated AKT were significantly decreased upon GSI I treatment, and constitutively activated myristoylated AKT completely blocked GSI I-induced apoptosis and cell survival, suggesting that inhibition of AKT signaling is critical for GSI I-mediated effects in gastric cancer cells. In order to maximize the effects and safety of GSI I, a combination treatment with GSI I and 5-FU was performed. Inhibition of gastric cancer cell proliferation with the combination treatment was significantly better than that with the single treatment. All phosphorylated forms of AKT, p44/42, JNK, and p38 were drastically changed by the combination treatment. Orthotopically transplanted gastric tumor burdens in mice were reduced using the combined treatment. The outcomes of this study clearly demonstrated the therapeutic potential of GSI I in gastric cancer, as well as the greater efficacy of the combined treatment of GSI I with 5-FU. Therefore, we suggest that further clinical trials examining the potential of combined GSI I and 5-FU treatment in gastric cancer patients be undertaken.

Glucose Deprivation Triggers Protein Kinase C-dependent β-Catenin Proteasomal Degradation

Background: Cellular glucose deprivation induces cell cycle arrest and autophagy through proteasomal degradation of β-catenin. Results: Glucose deprivation-induced β-catenin degradation and autophagy were not affected by GSK3β. Conclusion: Only inhibition of PKCα caused retardation of β-catenin degradation and autophagy. Significance: Cellular glucose deprivation activates PKCα to induce both autophagy and degradation of β-catenin. Autophagy is a conserved process that contributes to cell homeostasis. It is well known that induction mainly occurs in response to nutrient starvation, such as starvation of amino acids and insulin, and its mechanisms have been extensively characterized. However, the mechanisms behind cellular glucose deprivation-induced autophagy are as of now poorly understood. In the present study, we determined a mechanism by which glucose deprivation induced the PKC-dependent proteasomal degradation of β-catenin, leading to autophagy. Glucose deprivation was shown to cause a sub-G1 transition and enhancement of the LC3-II protein levels, whereas β-catenin protein underwent degradation in a proteasome-dependent manner. Moreover, the inhibition of GSK3β was unable to abolish the glucose deprivation-mediated β-catenin degradation or up-regulation of LC3-II protein levels, which suggested GSK3β-independent protein degradation. Intriguingly, the inhibition of PKCα using a pharmacological inhibitor and transfection of siRNA for PKCα was observed to effectively block glucose deprivation-induced β-catenin degradation as well as the increase in LC3-II levels and the accumulation of a sub-G1 population. Together, our results demonstrated a molecular mechanism by which glucose deprivation can induce the GSK3β-independent protein degradation of β-catenin, leading to autophagy.

Tumor Suppressor HIPK2 Regulates Malignant Growth via Phosphorylation of Notch1.

The receptor Notch1 plays an important role in malignant progression of many cancers, but its regulation is not fully understood. In this study, we report that the kinase HIPK2 is responsible for facilitating the Fbw7-dependent proteasomal degradation of Notch1 by phosphorylating its intracellular domain (Notch1-IC) within the Cdc4 phosphodegron motif. Notch1-IC expression was higher in cancer cells than normal cells. Under genotoxic stress, Notch1-IC was phosphorylated constitutively by HIPK2 and was maintained at a low level through proteasomal degradation. HIPK2 phosphorylated the residue T2512 in Notch1-IC. Somatic mutations near this residue rendered Notch1-IC resistant to degradation, as induced either by HIPK2 overexpression or adriamycin treatment. In revealing an important mechanism of Notch1 stability, the results of this study could offer a therapeutic strategy to block Notch1-dependent progression in many types of cancer. Cancer Res; 76(16); 4728-40. ©2016 AACR.