PURPOSE: Foxhead box M1 (FOXM1) expression has been shown to be linked with human papillomavirus (HPV) 16/18–infected cervical cancer. However, the mechanism underlying the induction of FOXM1 in HPV 16/18–infected cancers remains elusive. EXPERIMENTAL DESIGN: The mechanistic actions of FOXM1 induced by the E6/NKX2-1 axis in tumor aggressiveness were elucidated in cellular and animal models. The prognostic value of FOXM1 for overall survival (OS) and relapse-free survival (RFS) in HPV-positive oral and lung cancers was assessed using Kaplan-Meier and Cox regression models. RESULTS: Herein, FOXM1 expression is upregulated by E6-mediated NKX2-1 in HPV-positive cervical, oral, and lung cancer cells. Induction of FOXM1 by E6 through the MZF1/NKX2-1 axis is responsible for HPV-mediated soft agar growth, invasiveness, and stemness through activating Wnt/β-catenin signaling pathway. In a nude mice model, metastatic lung tumor nodules in HPV 18 E6-positive GNM or HPV 16 E6-positive TL-1–injected nude mice were markedly decreased in both cell types with E6 knockdown, FOXM1 knockdown, or treatment with FOXM1 inhibitor (thiostrepton). Among the four subgroup patients, the worst FOXM1 prognostic value for OS and RFS was observed in HPV 16/18–positive patients with tumors with high-expressing FOXM1. CONCLUSIONS: Induction of FOXM1 by E6 oncoprotein through the MZF1/NKX2-1 axis may be responsible for HPV 16/18–mediated tumor progression and poor outcomes in HPV-positive patients.

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Manganese superoxide dismutase (MnSOD) is an antioxidant enzyme responsible for the elimination of superoxide radical. The role of MnSOD in tumor progression in different human cancers is still controversial. In the present study, MnSOD expression in lung cancer cells was explored by knockdown or overexpression using transfection of a short hairpin RNA (shRNA) or an expression vector, respectively, to determine whether MnSOD expression mediates lung cancer cell migration, invasion, and oncogenic potential by increasing FoxM1 and MMP2 expression. Western blotting showed that FoxM1 and MMP2 expression was dependent on MnSOD expression, suggesting that FoxM1 could be upregulated by MnSOD. Three FoxM1 promoters were constructed to verify this activation of FoxM1 by MnSOD and to determine the transcription factors responsible. Luciferase reporter and chromatin immunoprecipitation assays indicated that MnSOD overexpression in lung cancer cells promoted binding of E2F1 and Sp1 to their putative FoxM1 promoter-binding sites and activated FoxM1 reporter activity. MnSOD also enhanced the potential for cell migration, invasion, and anchorage-independent colony growth on soft-agar plates, again via upregulation of FoxM1 and MMP2 expression. In patients with lung cancer, evaluation of MnSOD expression in lung tumors by immunohistochemistry indicated a positive correlation between FoxM1 and MMP2 mRNA expressions. Kaplan-Meier and Cox regression analysis revealed a poorer overall survival (OS) and relapse-free survival (RFS) in patients with MnSOD-positive tumors than with MnSOD-negative tumors. We conclude that MnSOD may promote tumor aggressiveness via upregulation of the FoxM1-MMP2 axis, and that MnSOD expression can independently predict survival and relapse in patients with resected lung adenocarcinoma. Mol Cancer Res; 11(3); 261-71. Ó2012 AACR.

The epithelial-to-mesenchymal (EMT) transition is a prerequisite for conferring metastatic potential during tumor progression. microRNA-30a (miR-30a) expression was significantly lower in aggressive breast cancer cell lines compared with non-invasive breast cancer and non-malignant mammary epithelial cell lines. In contrast, miR-30a overexpression reversed the mesenchymal appearance of cancer cells to result in a cobblestone-like epithelial phenotype. We identified Slug, one of the master regulators of EMT, as a target of miR-30a using in silico prediction. Reporter assays indicated that miR-30a could bind to the 3′-untranslted region of Slug mRNA. Furthermore, we linked miR-30a to increased expression of claudins, a family of tight junction transmembrane proteins. An interaction between Slug and E-box in the claudin promoter sequences was reduced upon miR-30a overexpression, further leading to reduction of filopodia formation and decreased invasiveness/metastasis capabilities of breast cancer cells. Consistently, delivery of miR-30a in xenografted mice decreased tumor invasion and migration. In patients with breast cancer, a significantly elevated risk of the miR-30alow/CLDN2low/FSCNhigh genotype was observed, linking to a phenotypic manifestation of larger tumor size, lymph node metastasis, and advanced tumor stage among patients. In conclusion, the miR-30a/Slug axis inhibits mesenchymal tumor development by interfering with metastatic cancer cell programming and may be a potential target for therapy in breast cancer.

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Magnesium superoxide dismutase (SOD2) has been shown to cause dysfunction of p53 transcriptional activity, whereas, in turn, SOD2 expression is regulated by p53 to modulate lung tumorigenesis. In this study, we found that the level of SOD2 expression in a panel of lung cancer cells was negatively correlated with that of NK2 homeobox 1 (NKX2-1) but was not associated with p53 status. Mechanistic studies indicated that a decrease in NKX2-1 caused by SOD2-activated IKKβ transcription was achieved by derepression of binding of Sp1 to the IKKβ promoter. Immunoprecipitation, glutathione S-transferase pull-down experiments and electrophoretic mobility shift assays demonstrated a direct interaction between NKX2-1 and Sp1, blocking Sp1-mediated IKKβ transcription. SOD2-mediated nuclear factor-kappaB activation, via elevation of IKKβ transcription, promoted anchorage-independent soft-agar growth, invasion and xenograft tumor formation, because of development of the epithelial-to-mesenchymal transition. The expression level of NKX2-1 messenger RNA was negatively associated with the extent of SOD immunostaining and the IKKβ messenger RNA expression level in lung tumors. The extent of SOD2 immunostaining and IKKβ messenger RNA levels may independently predict overall survival and relapse-free survival in lung adenocarcinoma patients. In summary, we found that SOD2 activates nuclear factor-kappaB signaling by increasing IKKβ transcription, which results in progression of lung adenocarcinoma and poorer patient outcomes. We suggest that IKKβ may potentially be targeted to improve outcomes in patients with SOD2-positive tumors.

NKX2-1 plays a dual role in lung adenocarcinoma progression, but the underling mechanism is not fully understood. In the present study, we provide evidence that NKX2-1 directly regulates p53 transcription, and in turn, NKX2-1 elevates the mutant p53/NF-Y complex to up-regulate IKKβ transcription in p53-mutant cells, but NKX2-1-mediated wild-type p53 down-regulates IKKβ transcription via decreased Sp1 binding to IKKβ promoter in p53-WT cells. The IKKβ-mediated p65 nuclear localization and epithelial-to-mesenchymal transition (EMT) modulated by the NKX2-1/p53 axis is responsible for soft-agar growth, invasion, and xenograft tumour formation. Among patients, high-IKKβ mRNA tumours had higher prevalence in p53-mutant or nuclear p65 tumours than their counterparts, but not related with NKX2-1 mRNA expression. However, when tumours were divided into p53-WT and p53-mutant subgroups, NKX2-1 mRNA expression was negatively correlated with IKKβ mRNA in p53-WT subgroup, but positively related with IKKβ mRNA expression in p53-mutant subgroup. Kaplan-Meier and Cox regression analysis indicated that high NKX2-1 mRNA tumours exhibited poorer overall survival and relapse free survival than low NKX2-1 mRNA tumours in p53-WT subgroup, but the opposite was observed in p53-mutant subgroup. Therefore, we suggest that NKX2-1 as a tumour suppressor or a tumour promoter in lung adenocarcinoma progression is dependent on p53 status.

BackgroundThe hOGG1 Ser326Cys polymorphism is associated with lung cancer risk, but there are limited data regarding an association between the APE1 Asp148Glu polymorphism and lung cancer. Biological evidence shows that the hOGG1-Cys allele results in less DNA repair activity; however, this is not associated with p53 mutation in lung cancer. Therefore, we investigated whether an interaction between hOGG1 and APE1 is associated with the frequency of p53 mutation in lung cancer.MethodsWe studied 217 Taiwanese adults with primary lung cancer. DNA polymorphisms of hOGG1 and APE1 were determined by polymerase chain reaction (PCR)-based restriction fragment length polymorphism. Mutations in p53 exons 5–8 were detected by direct sequencing. Multiple logistic regression was used to estimate odds ratios (ORs) and 95% CIs for the risk of p53 mutation associated with polymorphisms of hOGG1 and APE1 in lung cancer.ResultsAs expected, no association between hOGG1 polymorphism and p53 mutation was observed in this population. However, a higher risk of p53 mutation was found in participants with the APE1 Asp/Asp genotype than in those with the APE1-Glu allele (OR, 2.15; 95% CI, 1.19–3.87; P = 0.011). The risk of p53 mutation was also higher in participants with APE1 Asp/Asp plus hOGG1-Cys than in those with APE1-Glu plus hOGG1 Ser/Ser (OR, 3.72; 95% CI, 1.33–10.40; P = 0.012).ConclusionsThese results suggest that the APE1 Asp/Asp genotype and the combination of the APE1 Asp/Asp and hOGG1-Cys variants are associated with increased risk of p53 mutation in non–small cell lung cancer.