Counterbalance between RB inactivation and miR-17-92 overexpression in reactive oxygen species and DNA damage induction in lung cancers.

Small-cell lung cancer (SCLC) is a highly aggressive disease that exhibits rapid growth and genetic instability. We found earlier frequent overexpression of the miR-17-92 microRNA cluster, and showed that SCLC cells were addicted to continued expressions of miR-17-5p and miR-20a, major components of this microRNA cluster. In this study, we identified the frequent presence of constitutively phosphorylated H2AX (gamma-H2AX), which reflects continuing DNA damage, preferentially in SCLC. Knockdown of RB induced gamma-H2AX foci formation in non-small cell lung cancer (NSCLC) cells with wild-type RB, in association with growth inhibition and reactive oxygen species (ROS) generation, which was canceled by overexpression of miR-17-92. Conversely, induction of gamma-H2AX was observed in a miR-17-92-overexpressing SCLC cell line with miR-20a antisense oligonucleotides. These findings suggest that miR-17-92 overexpression may serve as a fine-tuning influence to counterbalance the generation of DNA damage in RB-inactivated SCLC cells, thus reducing excessive DNA damage to a tolerable level and consequently leading to genetic instability. Therefore, miR-17-92 may be an excellent therapeutic target candidate to elicit excessive DNA damage in combination with DNA-damaging chemotherapeutics.

Relationship between expression of 5-fluorouracil metabolic enzymes and 5-fluorouracil sensitivity in esophageal carcinoma cell lines.

5-Fluorouracil (5-FU) is a key drug in the treatment of esophageal squamous cell carcinoma (ESCC). Gene expression of 5-FU metabolic enzymes such as thymidylate synthase (TS), thymidine phosphorylase (TP), dihydropyrimidine dehydrogenase (DPD) and orotate phosphoribosyl transferase (OPRT), has recently been investigated in order to predict the 5-FU sensitivity of several cancers. We examined the relationship between such gene expression and 5-FU sensitivity in 25 ESCC cell lines. TS, DPD, TP and OPRT mRNA levels were assessed by real-time polymerase chain reaction. The 50% inhibitory concentrations (IC50) of 5-FU in 25 ESCC cell lines were determined by cell proliferation assay. IC50 values for 5-FU ranged from 1.00 to 39.81 micromol/L. There were significant positive correlations between IC50 and TS mRNA expression (R(2) = 0.5781, P < 0.0001) and DPD mRNA expression (R(2) = 0.3573, P = 0.0016). There were no correlations between IC50 and TP or OPRT mRNA expression. TS and DPD mRNA expression levels may be useful indicators in predicting the anti-tumor activity of 5-FU in ESCC.

CLCP1 interacts with semaphorin 4B and regulates motility of lung cancer cells.

We previously established a highly metastatic subline, LNM35, from the NCI-H460 lung cancer cell line, and demonstrated upregulation of a novel gene, CLCP1 (CUB, LCCL-homology, coagulation factor V/VIII homology domains protein), in LNM35 and lung cancer specimens. In this study, we focused on the potential roles of that gene in cancer metastasis. First, we established stable LNM35 RNAi clones, in which CLCP1 expression was suppressed by RNAi, and found that their motility was significantly reduced, although growth rates were not changed. Next, in vitro selection of a phage display library demonstrated that a phage clone displaying a peptide similar to a sequence within the Sema domain of semaphorin 4B (SEMA4B) interacted with LNM35. Immunoprecipitation experiments confirmed interaction of CLCP1 with SEMA4B, regulation of CLCP1 protein by ubiquitination and proteasome degradation enhanced in the presence of SEMA4B. These results are the first to indicate that CLCP1 plays a role in cell motility, whereas they also showed that at least one of its ligands is SEMA4B and that their interaction mediates proteasome degradation by CLCP1. Although the physiological role of the interaction between CLCP1 and SEMA4B remains to be investigated, this novel gene may become a target of therapy to inhibit metastasis of lung cancers.

Decreased expression of NDRG1 is correlated with tumor progression and poor prognosis in patients with esophageal squamous cell carcinoma.

NDRG1 (N-myc downstream regulated gene-1) was reported to be necessary for p53-mediated apoptosis and to be regulated by PTEN (phosphatase and tensin homolog). In several cancers, it was suggested to be a tumor suppressor gene. Its significance in esophageal squamous cell carcinoma (ESCC) has not been studied. The objective of this study was to clarify the relation between clinicopathological and biologic factors in esophageal carcinoma and to determine the prognostic significance of the expression of NDRG1. Expression of NDRG1 mRNA was quantified by real-time reverse transcription polymerase chain reaction using a Lightcycler in 47 esophageal ESCC specimens. The data were analyzed with reference to clinicopathological factors. Among the esophageal cancer tissues, NDRG1 mRNA expression was significantly lower in tumors of more advanced pathological stage (0-I vs. II-IV; P = 0.0027) and local tumor invasion (T1-2 vs. T3-4; P = 0.0136). Patients who had low NDRG1 mRNA expression had a significantly shorter survival after surgery compared with patients who had high NDRG1 mRNA expression (log-rank test, P = 0.0478). Impaired NDRG1 expression may lead to more aggressive invasion of ESCC.