Circulating cell-free miRNAs as biomarker for triple-negative breast cancer.

BACKGROUND: Triple-negative breast cancer (TNBC) accounts for 15-20% of all breast cancer in women globally. This subtype often has early and high recurrence rates resulting in poor survival, partially due to lack of targeted therapies. Therefore, there is an urgent need to identify TNBC-specific biomarkers for early diagnosis and treatment monitoring, and to develop more effective targeted therapy. METHODS: By using miRCURY LNA array platform, we compared the differential miRNA expressions in plasma of patient with TNBC (n=5) and non-TNBC (n=5), as well as healthy controls (n=5). Potential miRNAs were then validated in a large cohort of patients by real-time PCR. RESULTS: Ten putative miRNAs from the microarray data that differentially expressed between non-TNBC and healthy controls were identified. In the screening phase (n=90), we selected five miRNAs (miR-92a-3p, miR-342-3p, miR-16, miR-21 and miR-199a-5p) that could discriminate TNBC from non-TNBC for further validation. Results showed that miR-16, miR-21 and miR-199a-5p were underexpressed in TNBC when compared with non-TNBC, and were further validated in a large cohort (n=252). In addition, post-operative plasma levels of miR-16, miR-21 and miR-199a-5p were significantly restored when compared with pre-operative plasma of TNBC. Plasma miR-199a-5p expression in TNBC had significant difference when compared with non-TNBC and healthy controls, the receiver-operator characteristics curve analysis revealed the highest area under curve (AUC=0.8838) among all. The expression levels were associated with TNM stage and tumour subtypes. CONCLUSIONS: Our data suggest that miR-199a-5p could be a TNBC-specific marker with diagnostic value and provide insights into targeted therapy in the treatment of TNBC.

ADAMTS9 is a functional tumor suppressor through inhibiting AKT/mTOR pathway and associated with poor survival in gastric cancer.

Using genome-wide promoter methylation analysis, we identified a disintegrin-like and metalloprotease with thrombospondin type 1 motif 9 (ADAMTS9) is methylated in cancer. We aim to clarify its epigenetic inactivation, biological function and clinical implication in gastric cancer. ADAMTS9 was silenced in 6 out of 8 gastric cancer cell lines. The loss of ADAMTS9 expression was regulated by promoter hypermethylation and could be restored by demethylation agent. Ectopic expression of ADAMTS9 in gastric cancer cell lines (AGS, BGC823) inhibited cell growth curve in both the cell lines (P<0.0001), suppressed colony formation (P<0.01) and induced apoptosis (P<0.001 in AGS, P<0.01 in BGC823). Moreover, conditioned culture medium from ADAMTS9-transfected cell lines significantly disrupted the human umbilical vein endothelial cell tube formation capacity on Matrigel (P<0.01 in AGS, P<0.001 in BGC823). The in vivo growth of ADAMTS9 cells in nude mice was also markedly diminished after stable expression of ADAMTS9 (P<0.001). On the other hand, ADAMTS9 knockdown promoted cell proliferation (P<0.001). We further revealed that ADAMTS9 inhibited tumor growth by blocking activation of Akt and its downstream target the mammalian target of rapamycin (mTOR). ADAMTS9 also reduced phosphorylation of mTOR downstream targets p70 ribosomal S6 kinase, eIF4E-binding protein and downregulated hypoxia-inducible factor-1α. Therefore, this is the first demonstration that ADAMTS9 is a critical tumor suppressor of gastric cancer progression at least in part through suppression of oncogenic AKT/mTOR signaling. Moreover, promoter methylation of ADAMTS9 was detected in 29.2% (21/72) of primary gastric tumors. Multivariate analysis showed that patients with ADAMTS9 methylation had a poorer overall survival (relative risk (RR)=2.788; 95% confidence interval, 1.474-5.274; P=0.002). Kaplan-Meier survival curves showed that ADAMTS9 methylation was significantly associated with shortened survival in gastric cancer patients (P=0.001, log-rank test). In conclusion, ADAMTS9 acts as a functional tumor suppressor in gastric cancer through inhibiting oncogenic AKT/mTOR signaling pathway. Methylation of ADAMTS9 is an independent prognostic factor of gastric cancer.

Identification of retinoic acid-regulated nuclear matrix-associated protein as a novel regulator of gastric cancer.

BACKGROUND: Retinoic acid-regulated nuclear matrix-associated protein (RAMP) is a WD40 repeat-containing protein that is involved in various biological functions, but little is known about its role in human cancer. This study aims to delineate the oncogenic role of RAMP in gastric carcinogenesis. METHODS: RAMP expression was examined by real-time quantitative RT-PCR, immunohistochemistry and western blotting. Inhibition of RAMP expression was performed by siRNA-mediated knockdown. The functional effects of RAMP on cell kinetics were measured by cell viability assay, colony formation assay and flow cytometry. Cell lines stably expressing RAMP were established to investigate the oncogenic effects of RAMP in vitro. RESULTS: Ramp was readily expressed in all seven gastric cancer cell lines and was significantly increased in human gastric cancer tissues when compared with their adjacent non-cancerous tissues (P<0.001). In keeping with this, expression of RAMP protein was higher in gastric cancer tissues compared with their adjacent non-cancerous tissues, whereas moderate protein expression were noted in intestinal metaplasia. Knockdown of RAMP in gastric cancer cells significantly reduced cell proliferation (P<0.01) and soft agar colony formation (P<0.001), but induced apoptosis and G(2)/M arrest. In additional, knockdown RAMP induced cell apoptosis is dependent on functional accumulation of p53 and p21 and induction of cleaved caspases-9, caspases-3 and PARP. Strikingly, overexpression of RAMP promoted anchorage-independent cell growth in soft agar. CONCLUSION: Our findings demonstrate that RAMP plays an oncogenic role in gastric carcinogenesis. Inhibition of RAMP may be a promising approach for gastric cancer therapy.

MicroRNA-143 targets DNA methyltransferases 3A in colorectal cancer.

BACKGROUND: MicroRNAs (miRNAs) are 19-25-nucleotides regulatory non-protein-coding RNA molecules that regulate the expressions of a wide variety of genes, including some involved in cancer development. In this study, we investigated the possible role of miR-143 in colorectal cancer (CRC). METHODS: Expression levels of human mature miRNAs were examined using real-time PCR-based expression arrays on paired colorectal carcinomas and adjacent non-cancerous colonic tissues. The downregulation of miR-143 was further evaluated in colon cancer cell lines and in paired CRC and adjacent non-cancerous colonic tissues by qRT-PCR. Potential targets of miR-143 were defined. The functional effect of miR-143 and its targets was investigated in human colon cancer cell lines to confirm miRNA-target association. RESULTS: Both real-time PCR-based expression arrays and qRT-PCR showed that miR-143 was frequently downregulated in 87.5% (35 of 40) of colorectal carcinoma tissues compared with their adjacent non-cancerous colonic tissues. Using in silico predictions, DNA methyltranferase 3A (DNMT3A) was defined as a potential target of miR-143. Restoration of the miR-143 expression in colon cell lines decreased tumour cell growth and soft-agar colony formation, and downregulated the DNMT3A expression in both mRNA and protein levels. DNMT3A was shown to be a direct target of miR-143 by luciferase reporter assay. Furthermore, the miR-143 expression was observed to be inversely correlated with DNMT3A mRNA and protein expression in CRC tissues. CONCLUSION: Our findings suggest that miR-143 regulates DNMT3A in CRC. These findings elucidated a tumour-suppressive role of miR-143 in the epigenetic aberration of CRC, providing a potential development of miRNA-based targeted approaches for CRC therapy.

Differential expression of microRNAs in plasma of patients with colorectal cancer: a potential marker for colorectal cancer screening.

OBJECTIVE: MicroRNAs (miRNAs) have been shown to offer great potential in the diagnosis of cancer. We investigated whether plasma miRNAs could discriminate between patients with and without colorectal cancer (CRC). METHODS: This study was divided into three phases: (1) marker discovery using real-time PCR-based miRNA profiling on plasma, corresponding cancerous and adjacent non-cancerous colonic tissues of five patients with CRC, along with plasma from five healthy individuals as controls; (2) marker selection and validation by real-time quantitative RT-PCR on a small set of plasma; and (3) independent validation on a large set of plasma from 90 patients with CRC, 20 patients with gastric cancer, 20 patients with inflammatory bowel disease (IBD) and 50 healthy controls. RESULTS: Of the panel of 95 miRNAs analysed, five were upregulated both in plasma and tissue samples. All the five miRNAs were validated on the plasma of 25 patients with CRC and 20 healthy controls. Both miR-17-3p and miR-92 were significantly elevated in the patients with CRC (p<0.0005). The plasma levels of these markers were significantly reduced after surgery in 10 patients with CRC (p<0.05). Further validation with an independent set of plasma samples (n = 180) indicated that miR-92 differentiates CRC from gastric cancer, IBD and normal subjects. This marker yielded a receiver operating characteristic curve area of 88.5%. At a cut-off of 240 (relative expression in comparison to RNU6B snRNA), the sensitivity was 89% and the specificity was 70% in discriminating CRC from control subjects. CONCLUSION: MiR-92 is significantly elevated in plasma of patients with CRC and can be a potential non-invasive molecular marker for CRC screening.

Clinical and pathological characteristics of Chinese patients with BRCA related breast cancer.

Breast cancers related to BRCA mutations are associated with particular biological features. Here we report the clinical and pathological characteristics of breast cancer in Chinese women with and without BRCA mutations and of carriers of BRCA1 mutations compared to BRCA2 mutations. Two hundred and 26 high-risk Hong Kong Chinese women were tested for BRCA mutations, medical information was obtained from medical records, and risk and demographic information was obtained from personal interviews. In this cohort, 28 (12.4%) women were BRCA mutation carriers and among these carriers, 39.3% were BRCA1 and 60.7% were BRCA2 mutations. Mutation carriers were more likely to have a familial history of breast and ovarian cancer, high-grade cancers, and triple negative (TN) cancers. Prevalence of TN was 48.3% in BRCA carriers and 25.6% in non-carriers and was 67.7% in BRCA1 and 35.3% in BRCA2 carriers. Estrogen receptor (ER) negative cancer was significantly associated with BRCA1 mutations, especially in those under 40 years of age. BRCA-related breast cancer in this Chinese population is associated with family history and adverse pathological/prognostic features, with BRCA2 mutations being more prevalent but BRCA1 carriers having more aggressive and TN cancers. Compared to Caucasian populations, prevalence of BRCA2 mutations and TN cancer in BRCA2 mutation carriers in Chinese population are elevated.

Fibulin 1 is downregulated through promoter hypermethylation in gastric cancer.

Tumour suppressor genes (TSGs) were frequently inactivated through promoter hypermethylation in gastric carcinoma as well as pre-malignant gastric lesions, suggesting that promoter hypermethylation can be used as a marker to define novel TSGs and also biomarkers for early detection of gastric cancer. In an effort to search for such genes aberrantly methylated in gastric cancer development, fibulin 1 (FBLN1) was found as a candidate TSG epigenetically downregulated in gastric cancer. FBLN1 expression was downregulated in all of gastric cancer cell lines used (100%, 7 out of 7) and the primary gastric carcinoma tissues (84%, 86 out of 102) and significantly restored after pharmacological demethylation. Hypermethylation of the FBLN1 promoter was frequently (71%, 5 out of 7) detected in gastric cancer cell lines and primary gastric carcinoma tissues. Ectopic expression of FBLN1 led to the growth inhibition of gastric cancer cells through the induction of apoptosis. In summary, FBLN1 was identified as a novel candidate TSG epigenetically downregulated in gastric cancer.

Genomic characterisation of the severe acute respiratory syndrome coronavirus of Amoy Gardens outbreak in Hong Kong.

Severe acute respiratory syndrome (SARS) is a global health concern. In Hong Kong, two major outbreaks, one hospital based and the other in the Amoy Gardens apartments, were identified. The frequency of diarrhoea, admission to intensive care, and mortality differed significantly between the two outbreaks. We did genomic sequencing for viral isolates from five Amoy Gardens patients. The virus sequence was identical in four of these five patients. The sequence data from one hospital case and the four identical community cases had only three nucleotide differences. Alterations in the SARS coronavirus genome are unlikely to have caused the distinctive clinical features of the Amoy Gardens patients, and these results highlight the importance of non-viral genomic factors in this outbreak.