Prognostic value of the MicroRNA regulators Dicer and Drosha in non-small-cell lung cancer: co-expression of Drosha and miR-126 predicts poor survival.

BACKGROUND: Dicer and Drosha are important enzymes for processing microRNAs. Recent studies have exhibited possible links between expression of different miRNAs, levels of miRNA processing enzymes, and cancer prognosis. We have investigated the prognostic impact of Dicer and Drosha and their correlation with miR-126 expression in a large cohort of non-small cell lung cancer (NSCLC) patients. We aimed to find patient groups within the cohort that might have an advantage of receiving adjunctive therapies. METHODS: Dicer expression in the cytoplasm and Drosha expression in the nucleus were evaluated by manual immunohistochemistry of tissue microarrays (TMAs), including tumor tissue samples from 335 patients with resected stages I to IIIA NSCLC. In addition, in situ hybridizations of TMAs for visualization of miR-126 were performed. Kaplan-Meier analysis was performed, and the log-rank test via SPSS v.22 was used for estimating significance levels. RESULTS: In patients with normal performance status (ECOG = 0, n = 197), high Dicer expression entailed a significantly better prognosis than low Dicer expression (P = 0.024). Dicer had no significant prognostic value in patients with reduced performance status (ECOG = 1-2, n = 138). High Drosha expression was significantly correlated with high levels of the microRNA 126 (miR-126) (P = 0.004). Drosha/miR-126 co-expression had a significant negative impact on the disease-specific survival (DSS) rate (P < 0.001). Multivariate analyses revealed that the interaction Dicer*Histology (P = 0.049) and Drosha/miR-126 co-expression (P = 0.033) were independent prognostic factors. CONCLUSIONS: In NSCLC patients with normal performance status, Dicer is a positive prognostic factor. The importance of Drosha as a prognostic factor in our material seems to be related to miR-126 and possibly other microRNAs.

MicroRNA signatures in tumor tissue related to angiogenesis in non-small cell lung cancer.

BACKGROUND: Angiogenesis is regarded as a hallmark in cancer development, and anti-angiogenic treatment is presently used in non-small cell lung cancer (NSCLC) patients. MicroRNAs (miRs) are small non-coding, endogenous, single stranded RNAs that regulate gene expression. In this study we aimed to identify significantly altered miRs related to angiogenesis in NSCLC. METHODS: From a large cohort of 335 NSCLC patients, paraffin-embedded samples from 10 patients with a short disease specific survival (DSS), 10 with a long DSS and 10 normal controls were analyzed. The miRs were quantified by microarray hybridization and selected miRs were validated by real-time qPCR. The impacts of different pathways, including angiogenesis, were evaluated by Gene Set Enrichment Analysis (GSEA) derived from Protein ANalysis THrough Evolutionary Relationship (PANTHER). One of the most interesting candidate markers, miR-155, was validated by in situ hybridization (ISH) in the total cohort (n = 335) and correlation analyses with several well-known angiogenic markers were done. RESULTS: 128 miRs were significantly up- or down-regulated; normal versus long DSS (n = 68) and/or normal versus short DSS (n = 63) and/or long versus short DSS (n = 37). The pathway analysis indicates angiogenesis-related miRs to be involved in NSCLC. There were strong significant correlations between the array hybridization and qPCR validation data. The significantly altered angiogenesis-related miRs of high interest were miR-21, miR-106a, miR-126, miR-155, miR-182, miR-210 and miR-424. miR-155 correlated significantly with fibroblast growth factor 2 (FGF2) in the total cohort (r = 0.17, P = 0.002), though most prominent in the subgroup with nodal metastasis (r = 0.34, P<0.001). CONCLUSIONS: Several angiogenesis-related miRs are significantly altered in NSCLC. Further studies to understand their biological functions and explore their clinical relevance are warranted.

Prognostic impact of MiR-155 in non-small cell lung cancer evaluated by in situ hybridization.

BACKGROUND: In recent years, microRNAs (miRNAs) have been found to play an essential role in tumor development. In lung tumorigenesis, targets and pathways of miRNAs are being revealed, and further translational research in this field is warranted. MiR-155 is one of the miRNAs most consistently involved in various neoplastic diseases. We aimed to investigate the prognostic impact of the multifunctional miR-155 in non-small cell lung cancer (NSCLC) patients. METHODS: Tumor tissue samples from 335 resected stage I to IIIA NSCLC patients were obtained and tissue microarrays (TMAs) were constructed with four cores from each tumor specimen. In situ hybridization (ISH) was used to evaluate the expression of miR-155. RESULTS: There were 191 squamous cell carcinomas (SCCs), 95 adenocarcinomas (ACs), 31 large cell carcinomas and 18 bronchioalveolar carcinomas. MiR-155 expression did not have a significant prognostic impact in the total cohort (P = 0.43). In ACs, high miR-155 expression tended to a significant negative prognostic effect on survival in univariate analysis (P = 0.086) and was an independent prognostic factor in multivariate analysis (HR 1.87, CI 95% 1.01 - 3.48, P = 0.047). In SCC patients with lymph node metastasis, however, miR-155 had a positive prognostic impact on survival in univariate (P = 0.034) as well as in multivariate (HR 0.45, CI 95% 0.21-0.96, P = 0.039) analysis. CONCLUSIONS: The prognostic impact of miR-155 depends on histological subtype and nodal status in NSCLC.

Independent and tissue-specific prognostic impact of miR-126 in nonsmall cell lung cancer: coexpression with vascular endothelial growth factor-A predicts poor survival.

BACKGROUND: Angiogenesis is pivotal in tumor development. Vascular endothelial growth factor-A (VEGF-A) is considered one of the most important angiogenic factors, but lately several microRNAs (miRs) have been associated with vascular development. miR-126 has been related to tumor angiogenesis and in the regulation of VEGF-A. The authors aimed to investigate the prognostic impact of miR-126 and its co-expression with VEGF-A in nonsmall cell lung cancer (NSCLC) patients. METHODS: Tumor tissue samples from 335 resected stage I to IIIA NSCLC patients were obtained and tissue microarrays (TMAs) were constructed with 4 cores from each tumor specimen. VEGF-A expression was evaluated by immunohistochemistry, and in situ hybridization was used to evaluate the expression of miR-126. RESULTS: In the total material, miR-126 was a significant negative prognostic factor in both univariate (P = .005) and multivariate analyses (hazard ratio [HR] 1.8, 95% confidence interval [CI] 1.2-2.8, P = .01). Stratified by histology, miR-126 was only significant in squamous cell carcinomas (univariate: P < .001; multivariate: HR 3.1, CI 95% 1.7-5.6, P<.001). Stratified by lymph node status, miR-126 was significant only in the lymph node-positive subgroup (univariate: P<.001; multivariate: HR 4.1, CI 95% 2.0-8.4, P < .001). High miR-126 expression correlated significantly with high VEGF-A expression (P = .037). The co-expression of miR-126 and VEGF-A had a significant prognostic impact (P = .002), with 5-year survival rates of 68%, 51%, and 42% for low/low (n = 150), mixed combinations (n = 129), and high/high (n = 35) expression, respectively. CONCLUSIONS: miR-126 is a strong and independent negative prognostic factor in NSCLC, and its prognostic impact appears related primarily to histology and nodal status.