Tai-Kuang Chao

ALDH1-bright epithelial ovarian cancer cells are associated with CD44 expression, drug resistance, and poor clinical outcome.

The role of aldehyde dehydrogenase 1 (ALDH1) as an ovarian cancer stem cell marker and its clinical significance have rarely been explored. We used an Aldefluor assay to isolate ALDH1-bright (ALDH1(br)) cells from epithelial ovarian cancer cell lines and characterized the properties of the stem cells. ALDH1(br) cells were enriched in ES-2 (1.3%), TOV-21G (1.0%), and CP70 (1.2%) cells. Both ALDH1(br) and ALDH1(low) cells repopulated stem cell heterogeneity, formed spheroids, and grew into tumors in immunocompromised mice, although these processes were more efficient in ALDH1(br) cells. In the ES-2 and CP70 cells, ALDH1(br) cells conferred more chemoresistance, and were more enriched in CD44 (by 1.74-fold and 5.18-fold, respectively) than in CD133 (by 1.39-fold and 1.17-fold, respectively), compared with ALDH1(low) cells. Immunohistochemical staining for ALDH1 on a tissue microarray containing 84 epithelial ovarian cancer samples revealed that patients with higher ALDH1 expression (>50%) had poor overall survival, compared with those with lower ALDH1 (P = 0.004) and yielded an odds ratio of death of 2.43 (95% CI = 1.12 to 5.28) by multivariate analysis. The results did not support ALDH1 alone as an ovarian cancer stem cell marker, but demonstrated that ALDH1 is associated with CD44 expression, chemoresistance, and poor clinical outcome. The use of a combination of ALDH1 with other stem cell markers may help define ovarian cancer stem cells more stringently.

Hypermethylation of the TGF-β target, ABCA1 is associated with poor prognosis in ovarian cancer patients

BackgroundThe dysregulation of transforming growth factor-β (TGF-β) signaling plays a crucial role in ovarian carcinogenesis and in maintaining cancer stem cell properties. Classified as a member of the ATP-binding cassette (ABC) family, ABCA1 was previously identified by methylated DNA immunoprecipitation microarray (mDIP-Chip) to be methylated in ovarian cancer cell lines, A2780 and CP70. By microarray, it was also found to be upregulated in immortalized ovarian surface epithelial (IOSE) cells following TGF-β treatment. Thus, we hypothesized that ABCA1 may be involved in ovarian cancer and its initiation.ResultsWe first compared the expression level of ABCA1 in IOSE cells and a panel of ovarian cancer cell lines and found that ABCA1 was expressed in HeyC2, SKOV3, MCP3, and MCP2 ovarian cancer cell lines but downregulated in A2780 and CP70 ovarian cancer cell lines. The reduced expression of ABCA1 in A2780 and CP70 cells was associated with promoter hypermethylation, as demonstrated by bisulfite pyro-sequencing. We also found that knockdown of ABCA1 increased the cholesterol level and promoted cell growth in vitro and in vivo. Further analysis of ABCA1 methylation in 76 ovarian cancer patient samples demonstrated that patients with higher ABCA1 methylation are associated with high stage (P = 0.0131) and grade (P = 0.0137). Kaplan-Meier analysis also found that patients with higher levels of methylation of ABCA1 have shorter overall survival (P = 0.019). Furthermore, tissue microarray using 55 ovarian cancer patient samples revealed that patients with a lower level of ABCA1 expression are associated with shorter progress-free survival (P = 0.038).ConclusionsABCA1 may be a tumor suppressor and is hypermethylated in a subset of ovarian cancer patients. Hypermethylation of ABCA1 is associated with poor prognosis in these patients.

Promoter hypermethylation of FBXO32 , a novel TGF- β /SMAD4 target gene and tumor suppressor, is associated with poor prognosis in human ovarian cancer

Resistance to TGF-β is frequently observed in ovarian cancer, and disrupted TGF-β/SMAD4 signaling results in the aberrant expression of downstream target genes in the disease. Our previous study showed that ADAM19, a SMAD4 target gene, is downregulated through epigenetic mechanisms in ovarian cancer with aberrant TGF-β/SMAD4 signaling. In this study, we investigated the mechanism of downregulation of FBXO32, another SMAD4 target gene, and the clinical significance of the loss of FBXO32 expression in ovarian cancer. Expression of FBXO32 was observed in the normal ovarian surface epithelium, but not in ovarian cancer cell lines. FBXO32 methylation was observed in ovarian cancer cell lines displaying constitutive TGF-β/SMAD4 signaling, and epigenetic drug treatment restored FBXO32 expression in ovarian cancer cell lines regardless of FBXO32 methylation status, suggesting that epigenetic regulation of this gene in ovarian cancer may be a common event. In advanced-stage ovarian tumors, a significant (29.3%; P<0.05) methylation frequency of FBXO32 was observed and the association between FBXO32 methylation and shorter progression-free survival was significant, as determined by both Kaplan–Meier analysis (P<0.05) and multivariate Cox regression analysis (hazard ratio: 1.003, P<0.05). Reexpression of FBXO32 markedly reduced proliferation of a platinum-resistant ovarian cancer cell line both in vitro and in vivo, due to increased apoptosis of the cells, and resensitized ovarian cancer cells to cisplatin. In conclusion, the novel tumor suppressor FBXO32 is epigenetically silenced in ovarian cancer cell lines with disrupted TGF-β/SMAD4 signaling, and FBXO32 methylation status predicts survival in patients with ovarian cancer.

Kidney-targeting Smad7 gene transfer inhibits renal TGF-β/MAD homologue (SMAD) and nuclear factor κB (NF-κB) signalling pathways, and improves diabetic nephropathy in mice

Aims/hypothesisThe TGF-β/MAD homologue (SMAD) and nuclear factor κB (NF-κB) signalling pathways have been shown to play a critical role in the development of renal fibrosis and inflammation in diabetic nephropathy. We therefore examined whether targeting these pathways by a kidney-targeting Smad7 gene transfer has therapeutic effects on renal lesions in the db/db mouse model of type 2 diabetes.MethodsWe delivered Smad7 plasmids into the kidney of db/db mice using kidney-targeting, ultrasound-mediated, microbubble-inducible gene transfer. The histopathology, ultrastructural pathology and pathways of TGF-β/SMAD2/3-mediated fibrosis and NF-κB-dependent inflammation were evaluated.ResultsIn this mouse model of type 2 diabetes, Smad7 gene therapy significantly inhibited diabetic kidney injury, compared with mice treated with empty vectors. Symptoms inhibited included: (1) proteinuria and renal function impairment; (2) renal fibrosis such as glomerular sclerosis, tubulo-interstitial collagen matrix abundance and renal inflammation, including Inos (also known as Nos2), Il1b and Mcp1 (also known as Ccl2) upregulation, as well as macrophage infiltration; and (3) podocyte and endothelial cell injury as demonstrated by immunohistochemistry and/or electron microscopy. Further study demonstrated that the improvement of type 2 diabetic kidney injury by overexpression of Smad7 was associated with significantly inhibited local activation of the TGF-β/SMAD and NF-κB signalling pathways in the kidney.Conclusions/interpretationOur results clearly demonstrate that kidney-targeting Smad7 gene transfer may be an effective therapy for type 2 diabetic nephropathy, acting via simultaneous modulation of the TGF-β/SMAD and NF-κB signalling pathways.

Drug Screening Identifies Niclosamide as an Inhibitor of Breast Cancer Stem-Like Cells

The primary cause of death from breast cancer is the progressive growth of tumors and resistance to conventional therapies. It is currently believed that recurrent cancer is repopulated according to a recently proposed cancer stem cell hypothesis. New therapeutic strategies that specifically target cancer stem-like cells may represent a new avenue of cancer therapy. We aimed to discover novel compounds that target breast cancer stem-like cells. We used a dye-exclusion method to isolate side population (SP) cancer cells and, subsequently, subjected these SP cells to a sphere formation assay to generate SP spheres (SPS) from breast cancer cell lines. Surface markers, stemness genes, and tumorigenicity were used to test stem properties. We performed a high-throughput drug screening using these SPS. The effects of candidate compounds were assessed in vitro and in vivo. We successfully generated breast cancer SPS with stem-like properties. These SPS were enriched for CD44high (2.8-fold) and CD24low (4-fold) cells. OCT4 and ABCG2 were overexpressed in SPS. Moreover, SPS grew tumors at a density of 103, whereas an equivalent number of parental cells did not initiate tumor formation. A clinically approved drug, niclosamide, was identified from the LOPAC chemical library of 1,258 compounds. Niclosamide downregulated stem pathways, inhibited the formation of spheroids, and induced apoptosis in breast cancer SPS. Animal studies also confirmed this therapeutic effect. The results of this proof-of-principle study may facilitate the development of new breast cancer therapies in the near future. The extension of niclosamide clinical trials is warranted.

Antroquinonol reduces oxidative stress by enhancing the Nrf2 signaling pathway and inhibits inflammation and sclerosis in focal segmental glomerulosclerosis mice.

Oxidative stress, inflammation, and fibrosis are involved in the development and progression of focal segmental glomerulosclerosis (FSGS), a common form of idiopathic nephrotic syndrome that represents a therapeutic challenge because it has a poor response to steroids. Antroquinonol (Antroq), a purified compound, is a major active component of a mushroom, namely Antrodia camphorata, that grows in the camphor tree in Taiwan, and it has inhibitory effects on nitric oxide production and inflammatory reactions. We hypothesized that Antroq might ameliorate FSGS renal lesions by modulating the pathogenic pathways of oxidative stress, inflammation, and glomerular sclerosis in the kidney. We demonstrate that Antroq significantly (1) attenuates proteinuria, renal dysfunction, and glomerulopathy, including epithelial hyperplasia lesions and podocyte injury; (2) reduces oxidative stress, leukocyte infiltration, and expression of fibrosis-related proteins in the kidney; (3) increases renal nuclear factor E2-related factor 2 (Nrf2) and glutathione peroxidase activity; and (4) inhibits renal nuclear factor-κB (NF-κB) activation and decreases levels of transforming growth factor (TGF)-β1 in serum and kidney tissue in a mouse FSGS model. Our data suggest that Antroq might be a potential therapeutic agent for FSGS, acting by boosting Nrf2 activation and suppressing NF-κB-dependent inflammatory and TGF-β1-mediated fibrosis pathways in the kidney.

The expression of six biomarkers in the four most common ovarian cancers : correlation with clinicopathological parameters

This study aimed to evaluate the relationship of fascin‐1, matrix metalloproteinase (MMP)‐2, MMP‐9, cortactin, survivin, and epidermal growth factor receptor (EGFR) expression with clinicopathological parameters for the four most common ovarian surface epithelial carcinomas. Six biomarkers were investigated immunohistochemically using tissue microarrays of 185 specimens including 79 serous cystadenocarcinomas, 47 mucinous cystadenocarcinomas, 45 endometrioid adenocarcinomas, and 14 clear cell carcinomas. The four most common ovarian carcinomas showed significant expression of fascin‐1, cortactin, survivin, and EGFR, but not of MMP‐2 and MMP‐9. In addition, higher immunostaining scores for fascin‐1 in mucinous cystadenocarcinomas correlated with T stage, N stage, American Joint Committee on Cancer AJCC clinical stage, and a poorer survival rate; for cortactin in serous cystadenocarcinomas correlated with T stage; for cortactin in clear cell carcinomas correlated with T and clinical AJCC stages; and for survivin in clear cell carcinomas correlated with T stage and AJCC clinical stage. In addition, higher immunostaining scores for fascin‐1, cortactin, and survivin correlated with poorer tumor differentiation in serous, mucinous, and endometrioid adenocarcinomas. Thus, the expression of fascin‐1, cortactin, and survivin may be helpful in evaluating the aggressiveness of ovarian mucinous, serous, and clear cell adenocarcinoma. Additionally, the expression of fascin‐1 may be an independent prognostic risk factor in mucinous cystadenocarcinoma.

Epigenetic silencing of PTPRR activates MAPK signaling, promotes metastasis and serves as a biomarker of invasive cervical cancer

Epigenetic modifications are a driving force in carcinogenesis. However, their role in cancer metastasis remains poorly understood. The present study investigated the role of DNA methylation in the cervical cancer metastasis. Here, we report evidence of the overexpression of DNA methyltransferases 3B (DNMT3B) in invasive cervical cancer and of the inhibition of metastasis by DNMT3B interference. Using methyl-DNA immunoprecipitation coupled with microarray analysis, we found that the protein tyrosine phosphatase receptor type R (PTPRR) was silenced through DNMT3B-mediated methylation in the cervical cancer. PTPRR inhibited p44/42 MAPK signaling, the expression of the transcription factor AP1, human papillomavirus (HPV) oncogenes E6/E7 and DNMTs. The methylation status of PTPRR increased in cervical scrapings (n=358) in accordance with disease severity, especially in invasive cancer. Methylation of the PTPRR promoter has an important role in the metastasis and may be a biomarker of invasive cervical cancer.

Characterization of LMX-1A as a metastasis suppressor in cervical cancer.

DNA methylation is important in cancer development and is a promising biomarker for cancer detection. An epigenomic approach used in our previous work showed that LMX‐1A is methylation‐silenced in cervical cancer. LMX‐1A, a LIM‐homeobox gene, is known to participate in developmental events; however, there are at present no data on the role of LMX‐1A in cancers. In this study, we characterized the function of this transcription factor by examining cell lines, animal models and human cervical neoplastic tissues, and found that over‐expression of LMX‐1A does not affect cell proliferation or the cell cycle of cervical cancer cell lines but significantly inhibits colony formation and invasion in vitro. Analysis of changes in epithelial–mesenchymal transition (EMT) markers, such as CDH1, CDH2, VIMENTIN, SNAIL, SLUG and TWIST, revealed involvement of the EMT in LMX‐1A‐mediated cancer invasion; this result was validated in a stable transfectant over‐expressing LMX‐1A with RNA interference. Xenograft studies using immunocompromised mice confirmed the suppressor effects of LMX‐1A on tumour formation and distant metastasis in cervical cancer cell lines. LMX‐1A immunohistochemical staining of tissue arrays containing the full spectrum of cervical neoplasms, including normal cervix, low‐grade cervical intra‐epithelial neoplasia (CIN), high‐grade CIN, locally invasive and distant metastatic cancers, demonstrated the critical role of LMX‐1A in invasion and metastasis. Furthermore, we found by analysing TGFβ–BMP signalling that BMP4 and BMP6 are down‐regulated by LMX‐1A. The results of this study suggest that LMX‐1A suppresses cancer invasion and metastasis in cervical cancer through an incomplete EMT. Copyright

Triage of cervical cytological diagnoses of atypical squamous cells by DNA methylation of paired boxed gene 1 (PAX1).

Detection of cervical high‐grade squamous intraepithelial lesions (HSIL) in patients with equivocal cytological abnormalities, such as atypical squamous cells (ASC) of undetermined significance (ASCUS) or inability to exclude high‐grade squamous intraepithelial lesions (ASC‐H) is still a challenge. This study tested the efficacy of PAX1 methylation analysis in the triage of cervical ASCUS and ASC‐H and compared its performance with Hybrid Capture 2 (HC2) HPV test. A hospital‐based case–control study was conducted. Cervical scrapings from patients with ASCUS or ASC‐H were used for the quantitative methylation analysis of PAX1 methylation by MethyLight and HPV testing by HC2. Patients with ASC‐H or ASCUS with repeated abnormal smears underwent colposcopic biopsy and subsequent therapies. Diagnoses were made by histopathology at a follow‐up of 2 years. The efficacies of detecting high‐grade lesions were compared. Fifty‐eight cervical scrapings with cytological diagnosis of ASCUS (n = 41) and ASC‐H (n = 17) were analyzed. One of the 41 (2.4%) ASCUS patients and seven of 17 (41.2%) ASC‐H patients were confirmed to have HSIL. After dichotomy of the PMR, PAX1 methylation rates were significantly higher in ASC developing HSIL compared with those developing reactive atypia (87.5% vs. 12.5%, P < 0.001). Testing PAX1 methylation in cervical swabs of patients with ASC confers better sensitivity (87.5% vs. 62.5%) and specificity (98.0% vs. 86.0%) than HC2 HPV testing. We show for the first time that PAX1 hypermethylation analysis may be a better choice than HC2 in the triage of ASCUS and ASC‐H. Diagn. Cytopathol. 2013.