The circadian gene NPAS2 is a novel prognostic biomarker for breast cancer.

Mounting evidence suggests that neuronal PAS domain protein 2 (NPAS2) and other circadian genes are involved in tumorigenesis and tumor growth, possibly through their control of cancer-related biologic pathways. A missense polymorphism in NPAS2 (Ala394Thr) has been shown to be associated with risk of human tumors including breast cancer. The current study further examined the prognostic significance of NPAS2 in breast cancer by genotyping the Ala394Thr polymorphism and measuring NPAS2 expression. DNA extracted from 348 breast cancer tissue samples was analyzed for NPAS2 genotype using the TaqMan allelic discrimination assay. Of these, 287 also had total RNA available for use in real-time PCR assays to determine NPAS2 expression. NPAS2 genotypes and expression levels were analyzed for associations with prognostic outcomes, as well as correlations with clinical characteristics. A high level of NPAS2 expression was strongly associated with improved disease free survival (AHR = 0.43, 95% CI: 0.21-0.86, P trend = 0.022) and overall survival (AHR = 0.42, 95% CI: 0.19-0.96, P trend = 0.036). In addition, there was a borderline, but nonsignificant association between the NPAS2 genotype corresponding to Thr394Thr and disease free survival (AHR = 1.82, 95% CI: 0.96-3.46). The Ala/Ala, Ala/Thr, and Thr/Thr genotypes were also differentially distributed by tumor severity, as measured by TNM classification (chi (2) (6df, N = 344) = 14.96, P = 0.020). These findings provide the first evidence suggesting prognostic significance of the circadian gene NPAS2 in breast cancer.

Peptide concentrations and mRNA expression of IGF-I, IGF-II and IGFBP-3 in breast cancer and their associations with disease characteristics.

PURPOSE: To measure peptide concentrations and mRNA expression of the IGF Family in breast cancer and to examine their associations with the disease features. EXPERIMENTAL DESIGN: Fresh tumor samples were collected from 348 patients who underwent surgery for breast cancer. Tissue levels of mRNA and peptide of IGF-I, IGF-II, and IGFBP-3 were analyzed with real-time RT-PCR and ELISA, respectively. Cox proportional hazards regression model was used to examine the associations of IGF markers with patient survival. RESULTS: Age was inversely associated with IGF-I, IGF-II and IGFBP-3 at both mRNA and peptide levels. Small tumors, early TNM stages, or low grades were associated with high mRNA expression of IGFs and IGFBP-3. Hormone receptors were positively correlated with IGF-I and IGF-II expression. Survival analysis showed that patients with high expression of one of the IGF-I transcripts, IGF-IA, had lower risk of disease recurrence (HR = 0.47, 95%CI: 0.27-0.81) and death (HR = 0.35, 95%CI: 0.18-0.70) compared to those with low expression. High IGFBP-3 expression was also inversely associated with reduced risk of death (HR = 0.47, 95%CI: 0.23-0.95). Similar associations, however, were not observed when tissue levels of IGF-I peptide or IGFBP-3 protein were analyzed. High IGF-II peptide was related to increased risk of relapse (HR = 1.91, 95%CI: 1.12-3.27). CONCLUSION: Our findings of high mRNA expression of IGFs and IGFBP-3 being associated with less aggressive tumors and favorable prognosis were consistent with previous observations, but were not supported by the measurement of tissue levels of IGF-I peptide and IGFBP-3 protein, suggesting that IGF mRNA expression and tissue levels of IGF peptides are regulated by different mechanisms and assessing these molecules in tumor tissue may have different implications.

Klotho expression in epithelial ovarian cancer and its association with insulin-like growth factors and disease progression.

Klotho is an anti-aging hormone and is able to suppress the action of IGFs. High IGF activities are associated with cancer risk and tumor progression. Klotho's role in cancer is unknown. To evaluate Klotho expression in ovarian cancer and its association with IGFs and ovarian cancer progression, a clinical follow-up study of 189 ovarian cancer patients was conducted. Patients were recruited from a teaching hospital at University of Turin in Italy. All patients were diagnosed with epithelial ovarian cancer and underwent surgery for the disease. Fresh tumor tissue was collected from each patient during surgery. Patient clinical and pathological information was collected, including patient age at surgery, disease stage, tumor grade, tumor histology, residual tumor size, debulking result, post-operative chemotherapeutic agent, treatment response, follow-up time and survival outcome. Expressions of total and secreted Klotho as well as IGFs in tumor tissue were analyzed using quantitative real-time PCR. Survival analysis was performed to evaluate the association of Klotho expression with the risk of disease progression and death using Cox proportional hazards regression model. High expression of secreted Klotho was associated with increased risk of disease progression and death. These associations were independent of patient clinical and pathological characteristics. Expression of secreted Klotho was positively correlated with the expression of IGF-I and IGFBP-3 but not with IGF-II. In conclusion, Klotho expression is associated with epithelial ovarian cancer progression, and the protein may serve as an independent marker for ovarian cancer prognosis. Klotho's role in cancer warrants further elucidation.

Pluripotent factor lin-28 and its homologue lin-28b in epithelial ovarian cancer and their associations with disease outcomes and expression of let-7a and IGF-II.

Lin-28 and lin-28B are RNA-binding proteins which can block microRNA let-7 maturation and affect the differentiation and proliferation of embryonic stem cells. Lin-28 may also regulate the expression of insulin-like growth factor II (IGF-II). As one of the pluripotent factors involved in making induced pluripotent stem cells (iPS), lin-28 is considered a potential therapeutic target for cancer treatment. To further understand the role of lin-28 in cancer, we analysed the expression of lin-28 and its homologue lin-28B in tumour samples, and evaluated their associations with let-7a maturation, IGF-II expression, disease features and outcomes in 211 patients with primary epithelial ovarian cancer. The analysis showed that both lin-28 and lin-28B were positively correlated with primary and pre-let-7a-3; lin-28B, not lin-28, was inversely correlated with mature let-7a. A positive correlation was also observed between lin-28B and IGF-II expression, while no association was found between lin-28B and IGF-I or IGFBP-3. The study further demonstrated that lin-28B expression was associated with the risk of disease progression and death; patients with high lin-28B had shorter progression-free and overall survival than those with low lin-28B. These results seem to support the findings of recent in vitro experiments, showing that lin-28 blocks the process of let-7a maturation. Our study also suggests that lin-28B may promote ovarian cancer progression and serve as an unfavourable prognostic marker for the disease. The correlation between lin-28B and IGF-II indicates that the growth factor may mediate the effect of lin-28B on tumour growth.

Hypermethylation of let-7a-3 in epithelial ovarian cancer is associated with low insulin-like growth factor-II expression and favorable prognosis.

MicroRNAs (miRNA) are endogenous noncoding small RNAs that regulate the activity of mRNAs. Many miRNA genes, including let-7a-3, are located in CpG islands, suggesting possible epigenetic regulation of their expression. Promoter CpG island methylation of tumor suppressor genes is involved in cancer development and progression. Using real-time methylation-specific PCR and real-time reverse transcription-PCR, we analyzed DNA methylation in the let-7a-3 gene and miRNA expression of let-7a in 214 patients with epithelial ovarian cancer to assess the effect of let-7a-3 methylation on the expressions of let-7a as well as a possible target of let-7 regulation, insulin-like growth factor-II (IGF-II). The association of let-7a-3 methylation with patient survival outcomes was also evaluated. let-7a-3 methylation was detected in epithelial ovarian cancer, and the expression of let-7a was slightly affected by the methylation, but the effect was not substantial. The methylation of let-7a-3, however, was inversely correlated with IGF-II expression and positively with insulin-like growth factor binding protein-3 (IGFBP-3) expression. Patients with methylated let-7a-3 seemed to have reduced risk for death compared with those without, and the association was independent of patient age at surgery, tumor grade, disease stage, and IGF-II or IGFBP-3 expression. No association was found for let-7a-3 methylation and disease progression. These results suggest that the let-7a-3 gene is methylated and the methylation may affect IGF-II expression and the survival of ovarian cancer patients. Further investigation of the role of miRNAs and their regulation in cancer is warranted.

IGF-I in epithelial ovarian cancer and its role in disease progression.

Insulin-like growth factor-I (IGF-I) is known to be involved in the development and progression of several types of solid tumors including ovarian cancer. IGF-I levels in local tissue is subject to both endocrine and paracrine/autocrine regulation. To investigate which regulation is more importantly involved in IGF-I action in ovarian cancer regarding tumor progression, we analyzed IGF-I mRNA expression (assuming only from paracrine/autocrine regulation) and peptide concentration (subject to both endocrine and paracrine/autocrine regulation) as well as a genetic polymorphism (CA dinucleotide repeats) in 215 epithelial ovarian cancer patients. Genomic DNA, total RNA and cytosol proteins were extracted from fresh tumor samples. Two alternatively spliced IGF-I transcripts (IGF-IA and IGF-IB) were analyzed using real-time PCR. Cytosol levels of free and total IGF-I were measured with enzyme-linked immunosorbent assay. DNA sizing analysis was performed to determine the CA polymorphism. The study showed that the CA polymorphism had a weak influence on IGF-I expression, but no effect on tumor progression. High levels of free, not total, IGF-I peptide were associated with elevated risk of disease progression (HR = 2.06; 95%CI: 1.22-3.50), and the association was independent of clinicopathologic features of the disease. One of the IGF-I transcripts (IGF-IA) had a similar but less significant impact on disease progression. Women with high IGF-I mRNA and peptide were at greater risk for disease progression compared to those with low in both (HR = 2.13; 95%CI: 1.13-3.95). These findings support the notion that IGF-I is involved in ovarian cancer progression and free IGF-I plays a more important role in the disease. The study also suggests that both endocrine and paracrine/autocrine are involved in the regulation of IGF-I activity in ovarian cancer.