Characterization of NOL7 gene point mutations, promoter methylation, and protein expression in cervical cancer.

NOL7 is a putative tumor suppressor gene localized to 6p23, a region with frequent loss of heterozygosity in a number of cancers, including cervical cancer (CC). We have previously demonstrated that reintroduction of NOL7 into CC cells altered the angiogenic phenotype and suppressed tumor growth in vivo by 95%. Therefore, to understand its mechanism of inactivation in CC, we investigated the genetic and epigenetic regulation of NOL7. NOL7 mRNA and protein levels were assessed in 13 CC cell lines and 23 consecutive CC specimens by real-time quantitative polymerase chain reaction, western blotting, and immunohistochemistry. Methylation of the NOL7 promoter was analyzed by bisulfite sequencing and mutations were identified through direct sequencing. A CpG island with multiple CpG dinucleotides spanned the 5' untranslated region and first exon of NOL7. However, bisulfite sequencing failed to identify persistent sites of methylation. Mutational sequencing revealed that 40% of the CC specimens and 31% of the CC cell lines harbored somatic mutations that may affect the in vivo function of NOL7. Endogenous NOL7 mRNA and protein expression in CC cell lines were significantly decreased in 46% of the CC cell lines. Finally, immunohistochemistry demonstrated strong NOL7 nucleolar staining in normal tissues that decreased with histologic progression toward CC. NOL7 is inactivated in CC in accordance with the Knudson 2-hit hypothesis through loss of heterozygosity and mutation. Together with evidence of its in vivo tumor suppression, these data support the hypothesis that NOL7 is the legitimate tumor suppressor gene located on 6p23.

The RB tumor suppressor positively regulates transcription of the anti-angiogenic protein NOL7.

The expression of the angiogenic phenotype is regulated by a balance of pro-angiogenic and anti-angiogenic factors released into the tumor microenvironment. Nuclear protein 7 (NOL7), a novel tumor suppressor, acts as a master regulator of angiogenesis by downregulating pro-angiogenic factors and upregulating anti-angiogenic factors. Using cervical cancer as a model of investigation, we have previously shown that loss of NOL7 mRNA and protein expression is observed as early as the premalignant phase. Analysis of the gene failed to identify tumor-specific promoter methylation or coding region mutations, suggesting that NOL7 loss may be mediated by aberrant expression of its upstream regulators. In this study, we show that the RB tumor suppressor gene (RB) positively regulates NOL7 at the transcriptional level by recruiting transcription factors and transcription machinery proteins to its promoter region. Conversely, the loss of RB represses NOL7 transcription by inhibiting assembly of these proteins. This loss of NOL7 expression is also observed in RB-deficient human malignancies. Together, this work further characterizes the transcriptional activator function of RB and defines a potential role for RB in regulating angiogenesis through activation of NOL7. Current anti-angiogenic therapies lack long-term efficacy, as they are unable to target the diverse angiogenic signals generated by tumors. Our data provide evidence to support the hypothesis that reactivation of pRB can potentially modulate the expression of the angiogenic phenotype through regulation of NOL7. Therefore, this knowledge may be employed to design more comprehensive and effective therapies.

Identification and characterization of the human NOL7 gene promoter.

NOL7 is a candidate tumor suppressor gene that localizes to 6p23, a chromosomal region frequently associated with loss of heterozygosity in a number of malignancies including cervical cancer (CC). Re-expression of NOL7 in CC cells suppresses in vivo tumor growth by 95% and alters the angiogenic phenotype by modulating the expression of VEGF and TSP1. Here, we describe the determination of two NOL7 transcriptional start sites (TSS), the cloning of its regulatory promoter region, and the identification of transcription factors that regulate its expression. Using 5' Rapid amplification of complementary DNA ends (RACE), two transcriptional start sites were identified. Deletion analysis determined that the essential elements required for the optimal promoter activity of NOL7 were 560 bp upstream of its translation start site. In silico analysis suggested that the promoter region contained potential binding sites for the SP1, c-Myc and RXRalpha transcription factors as well as an overall GC content of greater than 60%. Chromatin immunoprecipitation (ChIP) confirmed that SP1, c-Myc and RXRalpha bound to the NOL7 promoter region. Finally, we demonstrate that NOL7 expression was positively regulated by c-Myc and RXRalpha. These results demonstrate that the NOL7 promoter region possesses each of the key elements of a TATA-less promoter. In addition, the positive regulation of NOL7 by c-Myc and RXRalpha provides additional mechanistic insights into the potential role of NOL7 in CC and other malignancies.

Dual inhibition of vascular endothelial growth factor receptor and epidermal growth factor receptor is an effective chemopreventive strategy in the mouse 4-NQO model of oral carcinogenesis.

Despite recent therapeutic advances, several factors, including field cancerization, have limited improvements in long-term survival for oral squamous cell carcinoma (OSCC). Therefore, comprehensive treatment plans must include improved chemopreventive strategies. Using the 4-nitroquinoline 1-oxide (4-NQO) mouse model, we tested the hypothesis that ZD6474 (Vandetanib, ZACTIMA) is an effective chemopreventive agent. CBA mice were fed 4-NQO (100 µg/mL) in their drinking water for 8 weeks and then randomized to no treatment or oral ZD6474 (25 mg/kg/d) for 24 weeks. The percentage of animals with OSCC was significantly different between the two groups (71% in control and 12% in the ZD6474 group; P ≤ 0.001). The percentage of mice with dysplasia or OSCC was significantly different (96% in the control and 28% in the ZD6474 group; P ≤ 0.001). Proliferation and microvessel density scores were significantly decreased in the ZD6474 group (P ≤ 0.001 for both). Although proliferation and microvessel density increased with histologic progression in control and treatment cohorts, epidermal growth factor receptor and vascular endothelial growth factor receptor-2 phosphorylation was decreased in the treatment group for each histologic diagnosis, including mice harboring tumors. OSCC from ZD6474-treated mice exhibited features of epithelial to mesenchymal transition, as shown by loss E-cadherin and gain of vimentin protein expression. These data suggest that ZD6474 holds promise as an OSCC chemopreventive agent. They further suggest that acquired resistance to ZD6474 may be mediated by the expression of an epithelial to mesenchymal transition phenotype. Finally, the data suggests that this model is a useful preclinical platform to investigate the mechanisms of acquired resistance in the chemopreventive setting.