Hsiao-Yen Hsieh

MicroRNA-30a increases tight junction protein expression to suppress the epithelial-mesenchymal transition and metastasis by targeting Slug in breast cancer

The epithelial-to-mesenchymal (EMT) transition is a prerequisite for conferring metastatic potential during tumor progression. microRNA-30a (miR-30a) expression was significantly lower in aggressive breast cancer cell lines compared with non-invasive breast cancer and non-malignant mammary epithelial cell lines. In contrast, miR-30a overexpression reversed the mesenchymal appearance of cancer cells to result in a cobblestone-like epithelial phenotype. We identified Slug, one of the master regulators of EMT, as a target of miR-30a using in silico prediction. Reporter assays indicated that miR-30a could bind to the 3′-untranslted region of Slug mRNA. Furthermore, we linked miR-30a to increased expression of claudins, a family of tight junction transmembrane proteins. An interaction between Slug and E-box in the claudin promoter sequences was reduced upon miR-30a overexpression, further leading to reduction of filopodia formation and decreased invasiveness/metastasis capabilities of breast cancer cells. Consistently, delivery of miR-30a in xenografted mice decreased tumor invasion and migration. In patients with breast cancer, a significantly elevated risk of the miR-30alow/CLDN2low/FSCNhigh genotype was observed, linking to a phenotypic manifestation of larger tumor size, lymph node metastasis, and advanced tumor stage among patients. In conclusion, the miR-30a/Slug axis inhibits mesenchymal tumor development by interfering with metastatic cancer cell programming and may be a potential target for therapy in breast cancer.

Cyproheptadine, an antihistaminic drug, inhibits proliferation of hepatocellular carcinoma cells by blocking cell cycle progression through the activation of P38 MAP kinase

BackgroundHepatocellular carcinoma (HCC) is a major cause of cancer deaths worldwide. However, current chemotherapeutic drugs for HCC are either poorly effective or expensive, and treatment with these drugs has not led to satisfactory outcomes. In a 2012 case report, we described our breakthrough finding in two advanced HCC patients, of whom one achieved complete remission of liver tumors and the other a normalized α-fetoprotein level, along with complete remission of their lung metastases, after the concomitant use of thalidomide and cyproheptadine. We assumed the key factor in our effective therapy to be cyproheptadine. In this study, we investigated the antiproliferative effects and molecular mechanisms of cyproheptadine.MethodsThe effect of cyproheptadine on cell proliferation was examined in human HCC cell lines HepG2 and Huh-7. Cell viability was assayed with Cell Counting Kit-8; cell cycle distribution was analyzed by flow cytometry. Mechanisms underlying cyproheptadine-induced cell cycle arrest were probed by western blot analysis.ResultsCyproheptadine had a potent inhibitory effect on the proliferation of HepG2 and Huh-7 cells but minimal toxicity in normal hepatocytes. Cyproheptadine induced cell cycle arrest in HepG2 cells in the G1 phase and in Huh-7 cells at the G1/S transition. The cyproheptadine-induced G1 arrest in HepG2 cells was associated with an increased expression of HBP1 and p16, whereas the G1/S arrest in Huh-7 cells was associated with an increase in p21 and p27 expression and a dramatic decrease in the phosphorylation of the retinoblastoma protein. Additionally, cyproheptadine elevated the percentage of Huh-7 cells in the sub-G1 population, increased annexin V staining for cell death, and raised the levels of PARP and its cleaved form, indicating induction of apoptosis. Finally, cyproheptadine-mediated cell cycle arrest was dependent upon the activation of p38 MAP kinase in HepG2 cells and the activation of both p38 MAP kinase and CHK2 in Huh-7 cells.ConclusionsOur results demonstrate that a non-classical p38 MAP kinase function, regulation of cell cycle checkpoints, is one of the underlying mechanisms promoted by cyproheptadine to suppress the proliferation of HCC cells. These results provide evidence for the drug’s potential as a treatment option for liver cancer.

Methylomics analysis identifies ZNF671 as an epigenetically repressed novel tumor suppressor and a potential non-invasive biomarker for the detection of urothelial carcinoma.

The molecular mechanism underlying the lethal phenomenon of urothelial carcinoma (UC) tumor recurrence remains unresolved. Here, by methylation microarray, we identified promoter methylation of the zinc-finger protein gene, ZNF671 in bladder UC tumor tissue samples, a finding that was independently validated by bisulphite pyrosequencing in cell lines and tissue samples. Subsequent assays including treatment with epigenetic depressive agents and in vitro methylation showed ZNF671 methylation to result in its transcriptional repression. ZNF671 re-expression in UC cell lines, via ectopic expression, inhibited tumor growth and invasion, in possible conjunction with downregulation of cancer stem cell markers (c-KIT, NANOG, OCT4). Clinically, high ZNF671 methylation in UC tumor tissues (n=96; 63 bladder, 33 upper urinary tract) associated with tumor grade and poor locoregional disease-free survival. Quantitative MSP analysis in a training (n=97) and test (n=61) sets of voided urine samples from bladder UC patients revealed a sensitivity and specificity of 42%-48% and 89%-92.8%, respectively, for UC cancer detection. Moreover, combining DNA methylation of ZNF671 and 2 other genes (IRF8 and sFRP1) further increased the sensitivity to 96.2%, suggesting a possible three-gene UC biomarker. In summary, ZNF671, an epigenetically silenced novel tumor suppressor, represents a potential predictor for UC relapse and non-invasive biomarker that could assist in UC clinical decision-making.

Epigenetic silencing of the NR4A3 tumor suppressor, by aberrant JAK/STAT signaling, predicts prognosis in gastric cancer

While aberrant JAK/STAT signaling is crucial to the development of gastric cancer (GC), its effects on epigenetic alterations of its transcriptional targets remains unclear. In this study, by expression microarrays coupled with bioinformatic analyses, we identified a putative STAT3 target gene, NR4A3 that was downregulated in MKN28 GC daughter cells overexpressing a constitutively activated STAT3 mutant (S16), as compared to an empty vector control (C9). Bisulphite pyrosequencing and demethylation treatment showed that NR4A3 was epigenetically silenced by promoter DNA methylation in S16 and other GC cell lines including AGS cells, showing constitutive activation of STAT3. Subsequent experiments revealed that NR4A3 promoter binding by STAT3 might repress its transcription. Long-term depletion of STAT3 derepressed NR4A3 expression, by promoter demethylation, in AGS GC cells. NR4A3 re-expression in GC cell lines sensitized the cells to cisplatin, and inhibited tumor growth in vitro and in vivo, in an animal model. Clinically, GC patients with high NR4A3 methylation, or lower NR4A3 protein expression, had significantly shorter overall survival. Intriguingly, STAT3 activation significantly associated only with NR4A3 methylation in low-stage patient samples. Taken together, aberrant JAK/STAT3 signaling epigenetically silences a potential tumor suppressor, NR4A3, in gastric cancer, plausibly representing a reliable biomarker for gastric cancer prognosis.

The investigation of a traditional Chinese medicine, Guizhi Fuling Wan (GFW) as an intravesical therapeutic agent for urothelial carcinoma of the bladder

BackgroundThe high risk of recurrence faced by patients with bladder cancer has necessitated the administration of supplemental intravesical chemotherapy; however, such treatments often result in severe side effects. As a result, novel intravesical agents with enhanced efficacy and minimal toxicity are urgently required for the treatment of bladder cancer.MethodsGuizhi Fuling Wan (GFW) is a traditional Chinese medicine shown to inhibit the growth of hepatocellular carcinoma. This study evaluated the growth inhibition of GFW using normal human urothelial cells and bladder cancer cells; the efficacy of GFW treatment was further compared with mitomycin C, epirubicin, and cisplatin. We also examined the progression of cell cycle and apoptosis in bladder cancer cells in response to GFW treatment. CCK-8 was employed to analyze cell viability and flow cytometry was used to study the cell cycle and apoptosis. The mechanisms underlying GFW-induced cell cycle arrest were determined by Western blot analysis.ResultsOur data demonstrate the potent inhibitory effect of GFW in the proliferation of bladder cancer cell lines, BFTC 905 and TSGH 8301. GFW presented relatively high selectivity with regard to cancer cells and minimal toxicity to normal urothelial cells. Our results also demonstrate that GFW interferes with cell cycle progression through the activation of CHK2 and P21 and induces apoptosis in these bladder cancer cells.ConclusionsOur results provide experimental evidence to support GFW as a strong candidate for intravesicle chemotherapy against bladder cancer.

Age-dependent Association Between Dickkopf-1 and Calcium-containing Urolithiasis

OBJECTIVE To evaluate the serum Dickkopf-1 (DKK1) level in patients with calcium-containing upper urinary tract stones (Ca-UUTS). METHODS The study retrospectively enrolled 184 patients with Ca-UUTS and 46 age-matched controls. The serum DKK1 level and urine calcium/creatinine ratio were detected in both groups. RESULTS The mean serum DKK1 level in the controls was 321.7 ± 284.1 pg/mL, which was significantly lower than that of the patients with calcium oxalate and calcium phosphate (CaOx + CaP), CaOx, and CaP stones (687.8 ± 600.2, 640.5 ± 721.5, and 857.9 ± 913.2 pg/mL, respectively). The mean urine calcium/creatinine ratio, an indicator of hypercalciuria, was higher in the Ca-UUTS patients with CaOx + CaP (0.10 ± 0.06), CaOx (0.13 ± 0.07), and CaP (0.12 ± 0.07) stones than in the controls (0.08 ± 0.04). Statistical significance was noted only in the patients with CaOx (P = .005) and CaP (P = .037) stones. A significant positive association was found between the serum DKK1 level and age in the control group but not in the Ca-UUTS patients. In subjects aged younger than 50 years, the serum DKK1 level in the Ca-UUTS group was significantly higher than in the control group (605.3 ± 514.4 vs 274 ± 229.8 pg/mL, P = .0003). The serum DKK1 level was not associated with stone size. CONCLUSION Serum DKK1, an inhibitor of the Wnt signaling pathway, was positively associated with the formation of Ca-UUTS, especially in patients aged younger than 50 years.

Guizhi Fuling Wan as a Novel Agent for Intravesical Treatment for Bladder Cancer in a Mouse Model

Alternative intravesical agents are required to overcome the side effects currently associated with the treatment of bladder cancer. This study used an orthotopic bladder cancer mouse model to evaluate Guizhi Fuling Wan (GFW) as an intravesical agent. The effects of GFW were compared with those of mitomycin-C (Mito-C) and bacille Calmette-Guérin (BCG). We began by evaluating the response of the mouse bladder cancer cell line MB49 to GFW treatment, with regard to cell viability, cell cycle progression and apoptosis. MB49 cells were subsequently implanted into the urothelial walls of the bladder in female C57BL/6 mice. The success of the model was confirmed by the appearance of hematuria and tumor growth in the bladder. Intravesical chemotherapy was administered in accordance with a published protocol. In vitro data revealed that GFW arrested MB49 cell cycle in the G0/G1 phase, resulting in the suppression of cell proliferation and induced apoptosis. One possible mechanism underlying these effects is an increase in intracellular reactive oxygen species (ROS) levels leading to the activation of ataxia telangiectasia-mutated (ATM)/checkpoint kinase 2 (CHK2) and ATM/P53 pathways, thereby mediating cell cycle progression and apoptosis, respectively. This mouse model demonstrates the effectiveness of GFW in the tumor growth, with results comparable to those achieved by using BCG and Mito-C. Furthermore, GFW was shown to cause only mild hematuria. The low toxicity of the compound was confirmed by a complete lack of lesions on bladder tissue, even after 10 consecutive treatments using high concentrations of GFW. These results demonstrate the potential of GFW for the intravesical therapy of bladder cancer.

Abstract 4561: The antihistamine cyproheptadine induces cell apoptosis through inhibition of β-catenin signaling pathways in urothelial carcinoma

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Recent studies have revealed that the cyproheptadine is a novel therapeutic agent for the treatment of malignancy, such as myeloma, leukemia and HCC. However, its effect on urothelial carcinoma cell is still unknown. The aim of this study was to investigate the effect and underline mechanism of cyproheptadine on UC. We used cell viability assay to investigate the drug effect on two UC cell lines, TSGH8301 and BFTC905, and found the IC50 which was about 55 μM. After treatment of cyproheptadine, cell cycle and apoptosis status of the UC cells were determined by flow cytometry and indicated cell cycle arrest in G0/G1 phase and induction of apoptosis. We further investigated the mechanism of the drug effect using western blot analysis and found that cyproheptadine induced downregulation of cyclin-D1 and c-Myc expression through the inhibition of β-catenin signaling pathway in UC cells. More importantly, Inhibition of β-catenin signaling pathway was related to downregulation of CCRK protein in TSGH8301 cell line, but through activation of IDAX protein leading to inhibition of β-catenin signaling pathway in BFTC905 cell line. These novel results demonstrated that cyproheptadine suppressed the β-catenin signaling pathway and induced UC cell apoptosis. Citation Format: Hsiao-Yen Hsieh, Yuan-Hung Wang, Cheng-Huang Shen, Shiu-Yi Chen, Michael Wy Chan, Cheng-Da Hsu. The antihistamine cyproheptadine induces cell apoptosis through inhibition of β-catenin signaling pathways in urothelial carcinoma. [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 4561. doi:10.1158/1538-7445.AM2014-4561

Abstract 3534: Epigenetic control of the tumor suppressive microRNA miR-34a in bladder cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Bladder cancer is the sixth most common cancer in the world and the incidence is particularly high in southwestern Taiwan. One of the hypotheses suggests that cancers arise from cancer stem-like cell (CSC) with surface antigen CD44. MicroRNAs (miRNAs) are endogenous, non-protein-coding RNAs that can regulate the expression of their target mRNAs. Up to 90% of human genes can be regulated by this mechanism. However, the role of miRNAs in the regulation of CSC in bladder cancer is not fully explored. Bioinformatics prediction identified that miR-34a regulates the expression of CD44. In this study, we aimed to investigate how miR-34a are regulated in bladder cancer. Expression analysis showed that miR-34a was down-regulated in UMUC3 bladder cancer cells. COBRA assay and bisulphite pyrosequencing demonstrated extensive DNA methylation of miR-34a in this cell. Treatment of DNMT inhibitor, 5-aza restored miR-34a expression in UMUC3 cells thus suggesting that miR-34a is epigenetically silenced in UMUC3 cells. Over-expression of miR-34a resulted in a reduction of chemo-resistance to cisplatin in UMUC3 cells. This may be due to a down-regulation of CD44 and another predicted miR-34a target, c-myc in this cell. Recently, additional mechanism namely, competitive endogenous RNA (ceRNA) hypothesis in controlling the function of miRNAs are demonstrated. To test this hypothesis in bladder cancer, we overexpressed 3′UTR of CD44 in TSGH8301 bladder cancer cells which expressed miR-34a, and resulted in an up regulation of another target of miR-34a, c-myc in this cells. Since amplification of chromosome 8q24 where c-myc resides is frequently observed in bladder cancer, we investigated the relationship between expression of c-myc and CD44 in a publicly available expression array data set (GDS1479) using 60 bladder cancer patients. Interestingly, a positive correlation was found between the expression of c-myc and CD44 (P=0.0016) in this data set. In conclusion, aberrant promoter methylation and ceRNA mechanism may contribute to attenuate the function of miR-34a in bladder cancer. The tumor suppressive role of miR-34a in controlling CSC phenotype in bladder cancer deserves further investigation. Citation Format: Wen-Yu Huang, Pi-Che Chen, Chia-Ming Yeh, Frank H.C. Cheng, Hsiao-Yen Hsieh, Cheng-Huang Shen, Cheng-Da Hsu, Michael WY Chan. Epigenetic control of the tumor suppressive microRNA miR-34a in bladder 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 3534. doi:10.1158/1538-7445.AM2014-3534