Rachana Agarwal

MicroRNA-21 is overexpressed in human cholangiocarcinoma and regulates programmed cell death 4 and tissue inhibitor of metalloproteinase 3†

Cholangiocarcinomas (CCAs) are aggressive cancers, with high mortality and poor survival rates. Only radical surgery offers patients some hope of cure; however, most patients are not surgical candidates because of late diagnosis secondary to relatively poor accuracy of diagnostic means. MicroRNAs (miRs) are involved in every cancer examined, but they have not been evaluated in primary CCA. In this study, miR arrays were performed on five primary CCAs and five normal bile duct specimens (NBDs). Several miRs were dysregulated and miR‐21 was overexpressed in CCAs. miR‐21 differential expression in these 10 specimens was verified by quantitative reverse transcriptase polymerase chain reaction (qRT‐PCR). To validate these findings, qRT‐PCR for miR‐21 was then performed on 18 additional primary CCAs and 12 normal liver specimens. MiR‐21 was 95% sensitive and 100% specific in distinguishing between CCA and normal tissues, with an area under the receiver operating characteristic curve of 0.995. Inhibitors of miR‐21 increased protein levels of programmed cell death 4 (PDCD4) and tissue inhibitor of metalloproteinases 3 (TIMP3). Notably, messenger RNA levels of TIMP3 were significantly lower in CCAs than in normals. Conclusions: MiR‐21 is overexpressed in human CCAs. Furthermore, miR‐21 may be oncogenic, at least in part, by inhibiting PDCD4 and TIMP3. Finally, these data suggest that TIMP3 is a candidate tumor suppressor gene in the biliary tree. (HEPATOLOGY 2009.)

A multicenter, double-blinded validation study of methylation biomarkers for progression prediction in Barrett’s esophagus

Esophageal adenocarcinoma risk in Barretts esophagus (BE) is increased 30- to 125-fold versus the general population. Among all BE patients, however, neoplastic progression occurs only once per 200 patient-years. Molecular biomarkers are therefore needed to risk-stratify patients for more efficient surveillance endoscopy and to improve the early detection of progression. We therefore performed a retrospective, multicenter, double-blinded validation study of eight BE progression prediction methylation biomarkers. Progression or nonprogression were determined at 2 years (tier 1) and 4 years (tier 2). Methylation was assayed in 145 nonprogressors and 50 progressors using real-time quantitative methylation-specific PCR. Progressors were significantly older than nonprogressors (70.6 versus 62.5 years; P < 0.001). We evaluated a linear combination of the eight markers, using coefficients from a multivariate logistic regression analysis. Areas under the ROC curve (AUC) were high in the 2-year, 4-year, and combined data models (0.843, 0.829, and 0.840; P < 0.001, <0.001, and <0.001, respectively). In addition, even after rigorous overfitting correction, the incremental AUCs contributed by panels based on the 8 markers plus age versus age alone were substantial (Delta-AUC = 0.152, 0.114, and 0.118, respectively) in all 3 models. A methylation biomarker-based panel to predict neoplastic progression in BE has potential clinical value in improving both the efficiency of surveillance endoscopy and the early detection of neoplasia.

Hypomethylation of Noncoding DNA Regions and Overexpression of the Long Noncoding RNA, AFAP1-AS1, in Barrett’s Esophagus and Esophageal Adenocarcinoma

BACKGROUND & AIMS Alterations in methylation of protein-coding genes are associated with Barretts esophagus (BE) and esophageal adenocarcinoma (EAC). Dysregulation of noncoding RNAs occurs during carcinogenesis but has never been studied in BE or EAC. We applied high-resolution methylome analysis to identify changes at genomic regions that encode noncoding RNAs in BE and EAC. METHODS We analyzed methylation of 1.8 million CpG sites using massively parallel sequencing-based HELP tagging in matched EAC, BE, and normal esophageal tissues. We also analyzed human EAC (OE33, SKGT4, and FLO-1) and normal (HEEpic) esophageal cells. RESULTS BE and EAC exhibited genome-wide hypomethylation, significantly affecting intragenic and repetitive genomic elements as well as noncoding regions. These methylation changes targeted small and long noncoding regions, discriminating normal from matched BE or EAC tissues. One long noncoding RNA, AFAP1-AS1, was extremely hypomethylated and overexpressed in BE and EAC tissues and EAC cells. Its silencing by small interfering RNA inhibited proliferation and colony-forming ability, induced apoptosis, and reduced EAC cell migration and invasion without altering the expression of its protein-coding counterpart, AFAP1. CONCLUSIONS BE and EAC exhibit reduced methylation that includes noncoding regions. Methylation of the long noncoding RNA AFAP1-AS1 is reduced in BE and EAC, and its expression inhibits cancer-related biologic functions of EAC cells.

Dynamic changes in the expression of microRNA-31 during inflammatory bowel disease-associated neoplastic transformation

Background: Patients with inflammatory bowel disease (IBD) are at increased risk of developing colorectal cancer. Aberrant microRNA (miR) expression has been linked to carcinogenesis; however, no reports document a relationship between IBD‐related neoplasia (IBDN) and altered miR expression. In the current study we sought to identify specific miR dysregulation along the normal–inflammation–cancer axis. Methods: miR microarrays and quantitative reverse‐transcriptase polymerase chain reaction (RT‐PCR) were used to detect dysregulated miRs. Receiver operating characteristic curve analysis was employed to test for potential usefulness of miR‐31 as a disease marker of IBDNs. In silico prediction analysis, Western blot, and luciferase activity measurement were employed for target identification. Results: Several dysregulated miRs were identified between chronically inflamed mucosae and dysplasia arising in IBD. MiR‐31 expression increases in a stepwise fashion during progression from normal to IBD to IBDN and accurately discriminated IBDNs from normal or chronically inflamed tissues in IBD patients. Finally, we identified factor inhibiting hypoxia inducible factor 1 as a direct target of miR‐31. Conclusions: Our study reveals specific miR dysregulation as chronic inflammation progresses to dysplasia. MiR‐31 expression levels increase with disease progression and accurately discriminates between distinct pathological entities that coexist in IBD patients. The novel effect of miR‐31 on regulating factor inhibiting hypoxia inducible factor 1 expression provides a new insight on the pathogenesis of IBDN. (Inflamm Bowel Dis 2011;)

MicroRNA-192 and -215 are upregulated in human gastric cancer in vivo and suppress ALCAM expression in vitro.

The dismal outcome of gastric cancer patients highlights the need for diagnostic biomarkers and effective therapeutic targets, such as microRNAs. We sought to discover microRNAs involved in gastric cancer, and to elucidate their downstream target mechanisms. Both cultured gastric epithelial cells (HFE145 and NCI-N87) and primary human gastric tissues (31 non-neoplastic stomach (NS) and 25 gastric carcinomas (GC)) were studied. MicroRNA microarrays and quantitative RT–PCR were applied to discover and verify differentially expressed microRNAs. in vitro cell migration and invasion, cell proliferation, cell cycle and apoptosis assays were executed to elucidate biological effects of microRNA-192 and -215. Western blotting and luciferase assays were performed to confirm direct messenger RNA targeting by microRNA-192 and -215. MicroRNA microarray analyses revealed that 25 and 20 microRNAs were upregulated and downregulated in GC vs NS, respectively. Expression levels of both microRNA-192 and -215 were significantly higher in GC than in NS (P<0.05). Luciferase assays suggested that microRNA-215 inhibits activated leukocyte cell adhesion molecule (ALCAM) expression at the posttranscriptional level. In addition, expression levels of ALCAM were significantly lower in GC than in NS. Mimics and inhibitors, respectively, of microRNA-192 or -215 exerted no effect on cell cycle or apoptosis in the immortalized normal gastric cell line HFE145 or the gastric cancer cell line NCI-N87. However, mimics of microRNA-192 or -215 significantly increased growth rates in HFE145 cells, whereas inhibitors of microRNA-192 or -215 caused significant decreases in growth rates in NCI-N87 cells. ALCAM knockdown by an ALCAM-specific siRNA significantly increased cell growth in HFE145 cells. Both transfection of mimics of microRNA-192 or -215 and ALCAM knockdown by an ALCAM-specific siRNA significantly increased the migration of HFE145 cells. In conclusion, in gastric cancer, both microRNA-192 and -215 are overexpressed in vivo and exert cell growth and migration-promoting effects in vitro, thus representing potential microRNAs with a role in cancer in the human stomach.

Hypermethylation of tachykinin-1 is a potential biomarker in human esophageal cancer.

Purpose: Our aim was to investigate whether and at what stage hypermethylation of the tachykinin-1 (TAC1) gene is associated with human esophageal neoplastic transformation. Experimental Design:TAC1 promoter hypermethylation was examined by real-time methylation-specific PCR in 258 human esophageal specimens and 126 plasma samples from patients or tissues at various stages of neoplastic evolution. Results:TAC1 hypermethylation in tissue samples showed highly discriminative receiver-operator characteristic curve profiles, clearly distinguishing esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC) from normal esophagus (P < 0.0001). Both frequencies and normalized methylation values of TAC1 tissue methylation were significantly higher in Barretts metaplasia (BE), dysplastic Barretts esophagus, EAC, and ESCC than in normal esophagus (P < 0.01). The frequency of TAC1 hypermethylation increased dramatically and early during neoplastic progression, from 7.5% in normal esophagus to 55.6% in BE from patients with Barretts metaplasia alone, 57.5% in dysplastic Barretts esophagus, and 61.2% in EAC. There was a significant relationship between TAC1 hypermethylation and BE segment length, a known clinical risk factor for neoplastic progression. Twelve (50%) of 24 ESCC exhibited TAC1 hypermethylation. Overall patient survival correlated significantly with TAC1 methylation status in ESCC patients (mean survival, 22 versus 110 months; P = 0.0102, log-rank test), but not in EAC patients. Both mean normalized methylation values and frequency of TAC1 hypermethylation in plasma samples were significantly higher in EAC patients than in control subjects. Treatment of KYSE220 ESCC and BIC EAC cells with 5-aza-2′-deoxycytidine reduced TAC1 methylation and increased TAC1 mRNA expression. Conclusions:TAC1 promoter hypermethylation is a common event in both major histologic types of human esophageal carcinoma, occurs early, correlates with other progression risk factors in esophageal adenocarcinogenesis, and is a tissue biomarker of a poor prognosis in ESCC. Circulating methylated TAC1 promoter DNA also offers potential as a biomarker for the diagnosis of EAC.

Silencing of Claudin-11 Is Associated with Increased Invasiveness of Gastric Cancer Cells

Background Claudins are membrane proteins that play critical roles in tight junction (TJ) formation and function. Members of the claudin gene family have been demonstrated to be aberrantly regulated, and to participate in the pathogenesis of various human cancers. In the present study, we report that claudin-11 (CLDN11) is silenced in gastric cancer via hypermethylation of its promoter region. Methodology/Principal Findings Levels of CLDN11 methylation and mRNA expression were measured in primary gastric cancer tissues, noncancerous gastric mucosae, and cell lines of gastric origin using quantitative methylation-specific PCR (qMSP) and quantitative reverse transcriptase-PCR (qRT-PCR), respectively. Analyses of paired gastric cancers and adjacent normal gastric tissues revealed hypermethylation of the CLDN11 promoter region in gastric cancers, and this hypermethylation was significantly correlated with downregulation of CLDN11 expression vs. normal tissues. The CLDN11 promoter region was also hypermethylated in all gastric cancer cell lines tested relative to immortalized normal gastric epithelial cells. Moreover, CLDN11 mRNA expression was inversely correlated with its methylation level. Treatment of CLDN11-nonexpressing gastric cancer cells with 5-aza-2′-deoxycytidine restored CLDN11 expression. Moreover, siRNA-mediated knockdown of CLDN11 expression in normal gastric epithelial cells increased their motility and invasiveness. Conclusions/Significance These data suggest that hypermethylation of CLDN11, leading to downregulated expression, contributes to gastric carcinogenesis by increasing cellular motility and invasiveness. A further understanding of the mechanisms underlying the role of claudin proteins in gastric carcinogenesis will likely help in the identification of novel approaches for diagnosis and therapy of gastric cancer.

Claudin-3 and Claudin-4 expression in serous papillary, clear cell, and endometrioid endometrial cancer

OBJECTIVE Tight junction (TJ) proteins claudin-3 and claudin-4 may be differentially expressed in uterine serous papillary carcinoma (USPC), a rare form of endometrial cancer characterized by a particularly poor prognosis. Our aim was to determine the expression pattern and prognostic relevance of claudin-3 and claudin-4 in a large cohort of endometrial cancer patients of diverse histological type and stage. METHODS Claudin-3 and claudin-4 expression was studied in a cohort of 287 patients with endometrial cancer including 137 cases of USPC or clear-cell histology using immunohistochemistry. Patients were completely surgically staged. Outcome data is available on all 287 patients. RESULTS The rate of claudin-3 and claudin-4 expression was significantly higher in USPC and clear-cell endometrial cancer compared to endometrioid endometrial cancer (claudin-3: 78% and 61% versus 38%, p<.0001; claudin-4: 56% and 44% versus 9%, p<.0001). Furthermore, expression of both TJ proteins was significantly associated with poor clinical outcome (claudin-3, DFS RR 1.70, p=.0087, OS RR 1.62, p=.0247; claudin-4, DFS RR 2.66, p<0.0001, and OS RR 2.50, p<0.0001). However, both markers did not maintain prognostic independence in multivariate analyses, as their expression was tightly associated with more advanced disease stages (p<.0001 for both), and higher nuclear grade (p<.0001 for both). CONCLUSION These clinical observations confirm the hypothesis based on preclinical evidence that increased expression of claudin-3 and claudin-4 may contribute to the aggressive phenotype of endometrial cancer of serous papillary or clear-cell histology and suggest their potential utility as diagnostic biomarkers and possible targets for therapeutic intervention.

Hypermethylation of the nel-like 1 gene is a common and early event and is associated with poor prognosis in early-stage esophageal adenocarcinoma

The nel-like1 (NELL1) gene maps to chromosome 11p15, which frequently undergoes loss of heterozygosity in esophageal adenocarcinoma (EAC). NELL1 promoter hypermethylation was examined by real-time methylation-specific polymerase chain reaction in 259 human esophageal tissues. Hypermethylation of this promoter showed highly discriminative receiver–operator characteristic curve profiles, clearly distinguishing esophageal squamous cell carcinoma (ESCC) and EAC from normal esophagus (NE) (P<0.001). NELL1 normalized methylation values were significantly higher in Barretts metaplasia (BE), dysplastic Barretts (D) and EAC than in NE (P<0.0000001). NELL1 hypermethylation frequency was zero in NE but increased early during neoplastic progression, to 41.7% in BE from patients with Barretts alone, 52.5% in D and 47.8% in EAC. There was a significant correlation between NELL1 hypermethylation and BE segment length. Three (11.5%) of 26 ESCCs exhibited NELL1 hypermethylation. Survival correlated inversely with NELL1 hypermethylation in patients with stages I–II (P=0.0264) but not in stages III–IV (P=0.68) EAC. Treatment of KYSE220 ESCC and BIC EAC cells with 5-aza-2′-deoxycytidine reduced NELL1 methylation and increased NELL1 mRNA expression. NELL1 mRNA levels in EACs with an unmethylated NELL1 promoter were significantly higher than those in EACs with a methylated promoter (P=0.02). Promoter hypermethylation of NELL1 is a common, tissue-specific event in human EAC, occurs early during Barretts-associated esophageal neoplastic progression, and is a potential biomarker of poor prognosis in early-stage EAC.

Novel Candidate Colorectal Cancer Biomarkers Identified by Methylation Microarray-Based Scanning

DNA hypermethylation is a common epigenetic abnormality in colorectal cancers (CRCs) and a promising class of CRC screening biomarkers. We conducted a genome-wide search for novel neoplasia-specific hypermethylation events in the colon. We applied methylation microarray analysis to identify loci hypermethylated in 17 primary CRCs relative to eight non-neoplastic colonic mucosae (NCs) from neoplasia-free subjects. These CRC-associated hypermethylation events were then individually evaluated for their ability to discriminate neoplastic from non-neoplastic cases, based on real-time quantitative methylation-specific PCR (qMSP) assays in 113 colonic tissues: 51 CRCs, nine adenomas, 19 NCs from CRC patients (CRC-NCs), and 34 NCs from neoplasia-free subjects (control NCs). A strict microarray data filtering identified 169 candidate CRC-associated hypermethylation events. Fourteen of these 169 loci were evaluated using qMSP assays. Ten of these 14 methylation events significantly distinguished CRCs from age-matched control NCs (P<0.05 by receiver operator characteristic curve analysis); methylation of visual system homeobox 2 (VSX2) achieved the highest discriminative accuracy (83.3% sensitivity and 92.3% specificity, P<1×10(-6)), followed by BEN domain containing 4 (BEND4), neuronal pentraxin I (NPTX1), ALX homeobox 3 (ALX3), miR-34b, glucagon-like peptide 1 receptor (GLP1R), BTG4, homer homolog 2 (HOMER2), zinc finger protein 583 (ZNF583), and gap junction protein, gamma 1 (GJC1). Adenomas were significantly discriminated from control NCs by hypermethylation of VSX2, BEND4, NPTX1, miR-34b, GLP1R, and HOMER2 (P<0.05). CRC-NCs were significantly distinguished from control NCs by methylation of ALX3 (P<1×10(-4)). In conclusion, systematic methylome-wide analysis has identified ten novel methylation events in neoplastic and non-neoplastic colonic mucosae from CRC patients. These potential biomarkers significantly discriminate CRC patients from controls. Thus, they merit further evaluation in stool- and circulating DNA-based CRC detection studies.