Jacek Niklinski

MicroRNA-30a inhibits epithelial-to-mesenchymal transition by targeting Snai1 and is downregulated in non-small cell lung cancer.

MicroRNAs (miRNAs) are small non‐coding RNAs which regulate gene expression by base‐pairing to the 3′‐UTR of the target mRNA. Recently, miRNAs have been shown to regulate cancer metastasis, however, central molecular mechanisms of this ability still need to be investigated. Epithelial to mesenchymal transition (EMT), which is characterized especially by repression of E‐cadherin expression and increased cell motility, is an essential component of cancer metastasis and progression. In the present study, we found that Snai1, a known transcriptional repressor of E‐cadherin and modulator of EMT, is post‐transcriptionally targeted by miRNA‐30a in non‐small cell lung cancer (NSCLC). Consistent with this, microRNA‐30a expression was found inversely proportional to the invasive potential of various NSCLC cell lines, correlating positively with E‐cadherin (epithelial marker) and negatively with N‐cadherin (mesenchymal marker) expression. Forced re‐introduction of miR‐30a significantly altered cell morphology, in vitro invasion and migration of invasive cell lines, this being paralleled by a downregulation of Snai1 and upregulation of E‐cadherin expression. Using a chicken embryonic metastasis assay, we found that miR‐30a suppresses in vivo distant metastasis to the lungs and liver. Finally, we screened the expression of miR‐30a in 64 consecutively resected NSCLC patients and found that, in 81% of the patients, expression of miR‐30a was downregulated significantly (p < 0.0001) in tumors compared to corresponding normal tissues. These results suggest that miR‐30a targets Snai1, inhibits invasion and metastasis, and is downregulated in NSCLC.

An Immune Response Enriched 72-Gene Prognostic Profile for Early-Stage Non–Small-Cell Lung Cancer

Purpose: Current staging methods are imprecise for predicting prognosis of early-stage non–small-cell lung cancer (NSCLC). We aimed to develop a gene expression profile for stage I and stage II NSCLC, allowing identification of patients with a high risk of disease recurrence within 2 to 3 years after initial diagnosis. Experimental Design: We used whole-genome gene expression microarrays to analyze frozen tumor samples from 172 NSCLC patients (pT1-2, N0-1, M0) from five European institutions, who had undergone complete surgical resection. Median follow-up was 89 months (range, 1.2-389) and 64 patients developed a recurrence. A random two thirds of the samples were assigned as the training cohort with the remaining samples set aside for independent validation. Cox proportional hazards models were used to evaluate the association between expression levels of individual genes and patient recurrence-free survival. A nearest mean analysis was used to develop a gene-expression classifier for disease recurrence. Results: We have developed a 72-gene expression prognostic NSCLC classifier. Based on the classifier score, patients were classified as either high or low risk of disease recurrence. Patients classified as low risk showed a significantly better recurrence-free survival both in the training set (P < 0.001; n = 103) and in the independent validation set (P < 0.01; n = 69). Genes in our prognostic signature were strongly enriched for genes associated with immune response. Conclusions: Our 72-gene signature is closely associated with recurrence-free and overall survival in early-stage NSCLC patients and may become a tool for patient selection for adjuvant therapy.

Integration of Gene Dosage and Gene Expression in Non-Small Cell Lung Cancer, Identification of HSP90 as Potential Target

Background Lung cancer causes approximately 1.2 million deaths per year worldwide, and non-small cell lung cancer (NSCLC) represents 85% of all lung cancers. Understanding the molecular events in non-small cell lung cancer (NSCLC) is essential to improve early diagnosis and treatment for this disease. Methodology and Principal Findings In an attempt to identify novel NSCLC related genes, we performed a genome-wide screening of chromosomal copy number changes affecting gene expression using microarray based comparative genomic hybridization and gene expression arrays on 32 radically resected tumor samples from stage I and II NSCLC patients. An integrative analysis tool was applied to determine whether chromosomal copy number affects gene expression. We identified a deletion on 14q32.2-33 as a common alteration in NSCLC (44%), which significantly influenced gene expression for HSP90, residing on 14q32. This deletion was correlated with better overall survival (P = 0.008), survival was also longer in patients whose tumors had low expression levels of HSP90. We extended the analysis to three independent validation sets of NSCLC patients, and confirmed low HSP90 expression to be related with longer overall survival (P = 0.003, P = 0.07 and P = 0.04). Furthermore, in vitro treatment with an HSP90 inhibitor had potent antiproliferative activity in NSCLC cell lines. Conclusions We suggest that targeting HSP90 will have clinical impact for NSCLC patients.

Identification of recurrent FGFR3 fusion genes in lung cancer through kinome‐centred RNA sequencing

Oncogenic fusion genes that involve kinases have proven to be effective targets for therapy in a wide range of cancers. Unfortunately, the diagnostic approaches required to identify these events are struggling to keep pace with the diverse array of genetic alterations that occur in cancer. Diagnostic screening in solid tumours is particularly challenging, as many fusion genes occur with a low frequency. To overcome these limitations, we developed a capture enrichment strategy to enable high‐throughput transcript sequencing of the human kinome. This approach provides a global overview of kinase fusion events, irrespective of the identity of the fusion partner. To demonstrate the utility of this system, we profiled 100 non‐small cell lung cancers and identified numerous genetic alterations impacting fibroblast growth factor receptor 3 (FGFR3) in lung squamous cell carcinoma and a novel ALK fusion partner in lung adenocarcinoma.

The anticancer activity of propolis

Propolis and its compounds have been the subject of many studies due to their antimicrobial and antiinflammatory activity; however, it is now known that they also possess antitumor properties. This review aims to summarize the results of studies on the mechanism of activity of propolis and its active compounds such as CAPE and chrysin in the apoptotic process, and their influence on the proliferation of cancer cells. Our review shows that propolis and its presented compounds induce apoptosis pathways in cancer cells. The antiproliferative effects of propolis, CAPE or chrysin in cancer cells are the result of the suppression of complexes of cyclins, as well as cell cycle arrest. The results of in vitro and in vivo studies suggest that propolis, CAPE and chrysin may inhibit tumor cell progression and may be useful as potential chemotherapeutic or chemopreventive anticancer drugs.

Diagnostic and prognostic value of the new tumour marker CYFRA 21-1 in patients with squamous cell lung cancer

We wanted to investigate the diagnostic and prognostic significance of serum CYFRA 21-1, especially in predicting the risk of recurrence in patients with operable squamous cell lung cancer. Serum levels of CYFRA 21-1 were measured using an immunoradiometric assay (CIS bio) in 76 patients with squamous cell lung cancer (64 operable and 12 with unresectable tumours), 22 with other non-small-cell type (12 with adenocarcinoma and 10 with large-cell type) and 45 with nonmalignant lung diseases. Elevated preoperative CYFRA 21-1 levels were identified in 63% of patients with squamous cell type (SqCC), 33% with adenocarcinoma, and 30% with large-cell carcinoma type. The diagnostic specificity of the assay was 96%. Positive CYFRA 21-1 levels were observed in 33% of stage I, 52% of stage II, 76% of stage IIIa and 83% of stage IIIb patients with SqCC type. Statistically significant differences were obtained between stages I and II and between II and IIIa, but not between stages IIIa and IIIb. Recurrence-free survival probability for patients with elevated serum CYFRA 21-1 levels before surgery was 63% (24/38) versus 92% (24/26) for patients with normal serum CYFRA 21-1 levels. However, the difference was not statistically significant when adjusted for the TNM stage (primary tumour, regional lymph node involvement, occurrence of distant metastasis). In 9 of the 10 patients with increased trend for CYFRA 21-1 during follow-up, elevated serum CYFRA 21-1 levels preceded (7) or coincided (2) with the clinical detection of tumour recurrence, providing a predictive value of an increased trend of 90%.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of vascular endothelial growth factor (VEGF) in non-small cell lung cancer (NSCLC): association with p53 gene mutation and prognosis

Vascular endothelial growth factor (VEGF) is a multifunctional cytokine that increases microvascular permeability and directly stimulates endothelial cell growth and angiogenesis. Recent evidence suggests that the genetic regulation of angiogenesis is also of crucial importance and that oncogenes and tumor suppressor genes can regulate it. The aim of this study was to determine the prognostic value of VEGF and its possible association with p53-gene mutation in 89 stage I-IIIa surgically treated NSCLC patients. DNA sequencing of the p53 gene (exons 5-8) showed 40 mutations (45%). Among the 89 NSCLC patients, immunoreactivity for VEGF was weakly, moderately and strongly positive in 35 (39%),36 (40%) and 18 (20%) cases, respectively. A strong, statistically significant association was found between the presence of a p53 gene mutation and expression of VEGF (P<0.001). The positive result of the p53 mutation increased the odds of observing a higher level of VEGF expression approximately 9.5 times (95% confidence interval: [3.44,25.89]). In the univariate analysis of survival, increasing levels of VEGF expression were associated with poor prognosis (P<0.001 for trend). In the multivariate analysis, after adjusting for the presence of a p53-gene mutation, gender, TNM stage and histological type, the prognostic effect of VEGF expression level was marginally non-significant (P=0.077). When the two-category quantification of the VEGF level was considered (low vs. intermediate/high), a marginally significant (P=0.024), unfavorable effect of intermediate/high levels of VEGF expression, independent of the effect of the presence of a p53-gene mutation, was found. In conclusion, we found that the p53 mutation was closely related to VEGF expression. Additionally, we observed that an intermediate/high expression of VEGF might be a useful indicator of prognosis in NSCLC. This latter conjecture, suggested by an analysis of the data, ought however, to be independently verified in further studies.

Prognostic significance of p53 and bcl-2 abnormalities in operable nonsmall cell lung cancer.

The association of p53 abnormalities and bcl-2 protein expression with clinical data and prognosis in 102 patients with resected nonsmall cell lung cancer (NSCLC) was investigated. Deoxyribonucleic acid analysis of exons 5-8 of the p53 gene showed mutations (p53-M) in 47% of resected NSCLC, serum p53 antibodies (p53-Abs) were detected in 25%, p53 protein overexpression (p53-PE) in 54%, and bcl-2 protein overexpression (bcl-2-PE) in 48%. A statistically significant association was found between p53-PE, serum p53-Abs and the presence of a p53 gene alteration. No significant associations were found between results of the p53-M, p53-Abs, bcl-2-PE tests and clinicopathological parameters. In the case of the p53-PE test there were significantly fewer positive results for adenocarcinoma than for squamous cell carcinoma and large cell carcinoma. Survival analysis showed that both p53 abnormalities and negative staining for bcl-2, when analysed separately, were associated with poor overall survival. In a multivariate analysis, only the positive result of the p53-M test remained an independent, statistically significant, unfavourable prognostic factor for survival. When the p53 mutation test was removed from the model, positive results of the p53-PE test and the p53-Abs test became statistically significant, unfavourable prognostic factors. To conclude, among p53 and bcl-2 abnormalities, only p53 gene mutations seem to have a strong and independent effect on prognosis. When deoxyribonucleic acid sequence information is not available, p53 protein expression and the presence of p53 antibodies in serum may be used to obtain important prognostic information.

MicroRNAs expression profiling of eutopic proliferative endometrium in women with ovarian endometriosis

BackgroundThe eutopic endometrium of women with endometriosis, compared with disease-free individuals, contains certain molecular alterations, including the differential expression of microRNA (miRNA). The aim of the study was to compare the expression of the most relevant miRNAs in the eutopic endometrium of women with and without ovarian endometriosis.MethodsA total of 46 regularly menstruating patients, 21 patients with ovarian endometriosis and 25 controls, underwent surgery in the proliferative phase of the cycle. The eutopic endometrium was collected through aspirating biopsy prior to laparoscopy. Only patients with advanced (stage III and IV) histopathologically confirmed ovarian endometriosis were included. TaqMan MicroRNA Array Cards were applied to examine the expression of 667 human miRNAs in 10 patients with endometriosis and 10 controls. Custom-made, low-density real-time PCR arrays were used to confirm the expression of 15 selected molecules in 21 endometriosis patients and 25 disease-free individuals.ResultsOf 667 miRNAs, 2 were highly likely to be upregulated and 13 were downregulated in the eutopic endometrium of patients with endometriosis compared with the controls. Validation using real-time PCR showed that hsa-miR-483-5p (p = 0.012) and hsa-miR-629* (p = 0.02) are significantly downregulated in patients with endometriosis.ConclusionsChanges in the expression of select miRNAs might lead to or be a consequence of an early defect in the physiological activity of the proliferative endometrium, ultimately resulting in the overgrowth of this tissue outside the uterus.

MicroRNAs as novel targets and tools in cancer therapy.

MicroRNAs (miRNAs) are currently experiencing a renewed peak of attention not only as diagnostics but also especially as highly promising novel targets or tools for clinical therapy in several different malignant diseases. Moreover, the recent discovery of competing endogenous RNAs (ceRNAs) as novel miRNA-regulators has contributed exciting insights in this regard. Therefore, this review summarizes and discusses the latest findings on (1) how miRNAs have become therapeutic targets of diverse synthetic antagonists, (2) how novel endogenous regulators of miRNAs such as ceRNAs or pseudogenes could emerge as therapeutics scavenging oncogenic miRNAs and (3) how miRNAs themselves are already, and will increasingly be, used as therapeutics. Recent advances on the importance of miRNA-target affinity and the subcellular localization of miRNAs are also discussed. The potential of these developments in different tumor entities and particular hallmarks of cancer such as metastasis, disease progression, interactions with the tumor microenvironment, or cancer stem cells are equally highlighted.