Prognostic markers that can predict the relapse of localized non–small cell lung cancer (NSCLC) have yet to be defined. We surveyed expression profiles of microRNA (miRNA) in stage I NSCLC to identify patterns that might predict recurrence after surgical resection of this common deadly cancer. Small RNAs extracted from formalin-fixed and paraffin-embedded tissues were hybridized to locked nucleic acid probes against 752 human miRNAs (representing 82% of the miRNAs in the miRBase 13.0 database) to obtain expression profiles for 37 cases with recurrence and 40 cases without recurrence (with clinical follow-up for at least 32 months). Differential expression between the two case groups was detected for 49% of the miRNAs (Wilcoxon rank sum test; P < 0.01). The performance of expression profiles at differentiating the two case groups was assessed by leave-one-out and Monte Carlo cross-validations. In leave-one-out cross-validation using support vector machines- or top-scoring gene pair classifier methods, which looked for six- or two-miRNA-based classifiers, the identified miRNA expression pattern predicted recurrence with an accuracy of 70% and 83%, and hazard ratio of 3.6 [95% confidence interval (95% CI), 1.8–7.1] and 9.0 (95% CI, 4.4–18.2), respectively. Mean accuracy in Monte Carlo cross-validation using 1,000 random 60–17 splits was 69% (95% CI, 68–70) and 72% (95% CI, 71–72), respectively. The specific miRNAs mir-200b*, mir-30c-1*, mir-510, mir-630, mir-657, and mir-146b-3p and mir-124*, mir-585, and mir-708, respectively, represented most commonly among the 1,000 classifiers identified in Monte Carlo cross-validation by the two methods. MiRNAs mir-488, mir-503, and mir-647 were identified as potential reference miRNAs for future studies, based on the stability of their expression patterns across the 77 cases and the two case-groups. Our findings reinforce efforts to profile miRNA expression patterns for better prognostication of stage I NSCLC. Cancer Res; 70(1); 36–45

Purpose: WWOX (WW domain containing oxidoreductase) is a tumor suppressor gene that maps to the common fragile site FRA16D. We showed previously that WWOX is frequently altered in human lung and esophageal cancers. The purpose of this study was to delineate more precisely the role of WWOX in pancreatic carcinogenesis. Experimental Design: We analyzed 15 paired pancreatic adenocarcinoma samples and 9 pancreatic cancer cell lines for WWOX alterations. Colony assay and cell cycle analysis were also performed to evaluate the role of the WWOX as a tumor suppressor gene. Results: Loss of heterozygosity at the WWOX locus was observed in 4 primary tumors (27%). Methylation analysis showed that site-specific promoter hypermethylation was detected in 2 cell lines (22%) and treatment with the demethylating agent 5-aza-2′-deoxycytidine demonstrated an increase in the expression of WWOX. In addition, 2 primary tumor samples (13%) showed promoter hypermethylation including the position of site-specific methylation. Transcripts missing WWOX exons were detected in 4 cell lines (44%) and in 2 tumor samples (13%). Real-time reverse transcription PCR revealed a significant reduction of WWOX expression in all of the cell lines and in 6 primary tumors (40%). Western blot analysis showed a significant reduction of the WWOX protein in all of the cell lines. Furthermore, transfection with WWOX inhibited colony formation of pancreatic cancer cell lines by triggering apoptosis. Conclusion: These results indicate that the WWOX gene may play an important role in pancreatic tumor development.

The WWOX (WW domain containing oxidoreductase) gene at the common fragile site, FRA16D, is altered in many types of cancer, including lung cancer. We have examined the tumor suppressor function of WWOX in preclinical lung cancer models. The WWOX gene was expressed in lung cancer cell lines through recombinant adenovirus (Ad) infection (Ad- WWOX ), and through a drug [ponasterone A, (ponA)]-inducible system. After WWOX restoration in vitro , endogenous Wwox protein-negative cell lines (A549, H460, and H1299) underwent apoptosis through activation of the intrinsic apoptotic caspase cascade in A549 and H460 cells. Ectopic expression of Wwox caused dramatic suppression of tumorigenicity of A549, H460, and H1299 cells in nude mice after Ad- WWOX infection and after ponA induction of Wwox expression in H1299 lung cancer cells. Tumorigenicity and in vitro growth of U2020 (Wwox-positive) lung cancer cells was unaffected by Wwox overexpression. This study confirms that WWOX is a tumor suppressor gene and is highly effective in preventing growth of lung cancer xenografts, whether introduced through viral infection or by induction of a silent WWOX transgene.

The finding of hemizygous or homozygous deletions at band 14 on chromosome 13 in a variety of neoplasms suggests the presence of a tumor-suppressor locus telomeric to the RB1 gene.