The let-7 microRNA binding site variant in KRAS as a predictive biomarker for head and neck cancer patients with lymph node metastasis.

BACKGROUND: The let-7 family of microRNAs regulate multiple oncogenes including the KRAS gene and has been shown to play a critical role in carcinogenesis. In this study, we aimed to investigate polymorphic alterations of the let-7 miRNA binding site (rs61764370) in the 3'UTR region of the KRAS gene as a predictive biomarker for head and neck cancer (HNC) and to evaluate its association with clinicopathological parameters. MATERIAL AND METHODS: The frequency of the KRAS-LCS6 variant in 216 Turkish HNC' patients and 85 healthy individuals were evaluated. After extracting DNA from whole blood, the variant allele was analyzed by polymerase chain reaction and restriction fragment length polymorphism method. Genotype and allele frequencies were evaluated using the De-Finetti case-control program. RESULTS: 85.6 % of the patients were wild type, 13 % heterozygous and 1.4 % homozygous variant. Although the KRAS-LCS6 variant was not associated with the risk of HNC (p > 0.05), G homozygous variant allele was found to be significantly associated with HNC patients having lymph node metastasis [T vs G: OR(%95 CI)= 2.370 (1.03-5.41), p = 0.03, χ2 = 4.38]. It was found statistical significance between genotype frequencies and smoker patients [TT vs TG: OR(%95 CI)= 0.357 (0.13-0.97), p = 0.03, χ2 = 4.32] by using De-Finetti analysis. Statistical significance was observed between KRAS-LCS6 genotype frequencies and gender, smoking, alcohol, early/late-stage, lymph node metastasis according to univariate analysis and Cox proportional hazards regression model (p < 0.05). CONCLUSION: This is the first study to reveal the relationship between KRAS-LCS6 variant and lymph node metastasis in HNC. The LCS6 variant of the KRAS gene may be a candidate predictor risk biomarker for lymph node metastasis in HNC.

Epigenetic Silencing of SOX15 Is Controlled by miRNAs rather than Methylation in Papillary Thyroid Cancer.

METHODS: In this study, qRT-PCR was used to investigate the expression levels of the SOX15 gene and of miR-182, miR-183, miR-375, and miR-96 in thyroid tumors and adjacent noncancerous tissues. We also investigated the methylation status of the SOX15 promoter by methylation-specific PCR in tumors and adjacent noncancerous tissues. RESULTS: We observed a statistically significant downregulation of SOX15 expression in tumors compared to noncancerous tissue samples. The methylation levels of tumors and matched noncancerous tissues were similar, but miR-182, miR-183, and miR-375 expression levels were elevated in tumor tissues compared to noncancerous tissue samples. CONCLUSIONS: Our results indicate that SOX15 gene expression is associated with the pathogenesis of papillary thyroid carcinoma (PTC), and the epigenetic control of the SOX15 gene is regulated by miRNAs rather than by promoter methylation.

No Tumor Suppressor Role for LKB1 in Prostate Cancer.

To elucidate the pathogenesis of prostate diseases, following in silico analysis, the LKB1 gene was selected for further investigation. The LKB1 gene has been associated with poor prognosis and is frequently mutated in different types of cancers. In this study, 50 benign prostatic hyperplasia (BPH) and 57 prostate cancer (PCa) tissues, including matched normal tissue for the patients, were analyzed by qRT-PCR and DNA sequencing for LKB1 expression and the mutation profile, respectively. Expression of LKB1 was increased in 60.7% of the PCa tissues compared with noncancerous tissue samples (p ≤ 0.001). However, LKB1 expression was lower when compared with normal tissues in BPH (p = 0.920). Four coding sequence alterations were detected in BPH. Three silent mutations were located in codons 9, 32, and 275 and a missense mutation was observed in codon 384. Six alterations were identified in the intronic regions of the LKB1 gene in both PCa and BPH. Five mutations were observed in both patient groups. A new alteration in intron 6 was observed in a patient with PCa. The LKB1 gene may be associated with benign transformations rather than the tumors in prostate pathogenesis when its expression and mutation status are considered. However, the mechanism of LKB1 in PCa needs further studies.

ZNF703 is an important player in head and neck cancer.

OBJECTIVES: Despite advances in treatment, head and neck squamous cell carcinoma (HNSCC) remains difficult to treat and the overall survival rate has only modestly improved over the past years. Therefore, there is a need to understand the molecular mechanism of HNSCC. Zinc finger protein 703 (ZNF703) is an oncogenic transcription factor, and ZNF703 gene expression is altered in many cancers as a result of chromosome 8p12 amplification. The purpose of this study was to investigate the expression pattern of ZNF703 in HNSCC in association with CCND1 expression and Akt phosphorylation. DESIGN: Prospective study. SETTING: University hospital. PARTICIPANTS: One hundred and five patients with HNSCC. METHODS: Fifty HNSCC tumour and non-cancerous tissue samples were investigated by qRT-PCR and Western blotting. RESULTS: ZNF703 gene expression was increased in 22.9% of tumour tissues compared with its normal counterparts. The results were correlated with clinicopathological features, copy number variation and survival data. CONCLUSION: ZNF703 over-expression is associated with copy number variation and this over-expression may activate PI3K/Akt signalling pathway in HNSCC.

Downregulation of TCEAL7 expression induces CCND1 expression in non-small cell lung cancer.

Transcription Elongation Factor A-like 7 (TCEAL7) was first reported as a candidate tumor suppressor gene because of its inactivation in ovarian cancer as a result of promoter methylation. Down-regulation of the TCEAL7 gene expression was also associated with other cancers such as endometrial, breast, brain, prostate, gastric cancers, glioblastoma and linked to tumor phenotypes and clinical outcomes. However, there is no report in the literature investigating the role of TCEAL7 in non-small cell lung cancer. Cyclin D1 is an important molecule in the transition from G1 to S phase of the cell cycle, and is frequently deregulated in cancers. Cylin D1 (CCND1) gene is amplified or overexpressed in a variety of tumors. In our previous study we reported that CCND1 over-expression was not associated with amplification in non-small cell lung cancer. Recently, it has been reported that TCEAL7 regulates CCND1 expression through myc-binding E-box sequences. The aim of this study was to investigate the expression of TCEAL7 gene in non-small cell lung cancer and to determine its effect on the CCND1 expression level. For this purpose, expression levels of TCEAL7 and CCND1 genes were investigated in 50 patients with non-small cell lung cancer by quantitative real time polymerase chain reaction (qRT-PCR). TCEAL7 was under-expressed (68%) in non-small cell lung cancer tumor tissues while CCND1 was over-expressed (42%). The TCEAL7 levels negatively correlated with increased CCND1 expression (p = 0.002).

Detection of RB1 Gene Copy Number Variations Using a Multiplex Ligation-Dependent Probe Amplification Method.

Multiplex ligation-dependent probe amplification (MLPA) is based on simultaneous multiplex PCR of specific probes that hybridize to multiple different target DNA regions. The method can identify copy number changes, gross gene rearrangements, methylation patterns or even point mutations. MLPA has been a reliable approach to identify copy number changes in the clinical and research settings and is widely used for the screening and investigation of copy number variations and genomic aberrations of interest in various diseases. In this chapter the analysis of the copy number changes in the RB1 gene locus by MLPA is described.

The EMSY Gene Collaborates with CCND1 in Non-Small Cell Lung Carcinogenesis.

Background: Lung cancer is the leading cause of cancer deaths. The main risk factor is smoking but the risk is also associated with various genetic and epigenetic components in addition to environmental factors. Increases in the gene copy numbers due to chromosomal amplifications constitute a common mechanism for oncogene activation. A gene-dense region on chromosome 11q13 which harbors four core regions that are frequently amplified, has been associated with various types of cancer. The important cell cycle regulatory protein cyclin D1 (CCND1) is an essential driver of the first core region of the Chr11q13 amplicon. Deregulation of CCND1 has been associated with different kinds of human malignancies including lung cancer. The EMSY (c11orf30) gene has been proposed as the possible driver of the fourth core of the 11q13 amplicon and its amplification has been associated with breast and ovarian cancers. There is no report in the literature investigating the EMSY gene in lung cancer. Methods: In this study, expression levels of the EMSY and CCND1 genes were investigated in 85 patients with non small cell lung cancer by Real Time PCR. Results: Expression of the EMSY and CCND1 genes were increased in 56 (65.8%) and 50 (58.8%) of the patients, respectively. Both genes showed a higher expression in the tumors when compared to normal tissues. A strong correlation was present between the expression rates of both genes (p<0.001). Patients with adenocarcinoma had higher expression levels of both genes (p=0.02). Conclusion: We conclude that EMSY and CCND1 work in collaboration and contribute to the pathogenesis of lung cancer.

Copy number profiling of tumor suppressor genes in head and neck cancer.

BACKGROUND: Sensitive and reliable new biomarkers are needed in head and neck cancer to predict the outcome and for therapy that is more effective. Copy number alterations are frequent and play a critical role in cancer. METHODS: Copy number alterations of 24 tumor suppressor genes in head and neck cancer were analyzed simultaneously in matched tumor and normal samples from 93 patients using multiplex ligation-dependent probe amplification (MLPA). RESULTS: Chromosomes 3p and 9p displayed the most common alterations. The gene displaying most frequent losses was the mutL homolog 1 (MLH1) gene, followed by the cyclin-dependent kinase inhibitor 2A (CDKN2A) and CDKN2B genes. A significant correlation was observed between the CDKN2A and CDKN2B genes. The tissue inhibitor of metalloproteinase (TIMP)3 gene alterations were observed in 8 tumors. CONCLUSION: Our data confirm previous observations and suggest that losses of the MLH1 and CDKN2 genes and alterations of the TIMP3 gene play an important role in head and neck carcinogenesis. © 2016 Wiley Periodicals, Inc. Head Neck 39: 341-346, 2017.

ZNF703 Overexpression may act as an oncogene in non-small cell lung cancer.

Despite therapeutic advances, lung cancer remains one of the most common causes of cancer-related deaths worldwide. The ZNF703 gene has been identified as the driver of the 8p11-12 region and its amplification or overexpression has been associated with several types of cancers. It has also been shown that ZNF703 overexpression can activate the Akt/mTOR signaling pathway. The aim of our study was to investigate the role of the ZNF703 gene in association with Akt/mTOR activation in non-small cell lung cancer (NSCLC). Expression levels in tumors and matched noncancerous tissue samples from 47 patients were analyzed by qRT-PCR and the Akt phosphorylation levels were investigated by Western blotting. Our results show that ZNF703 is up-regulated in 63.4% of NSCLC tumor samples. Althogh the correlation did not reach a statistically significant level Akt phosphorylation was increased in tumor tissues expressing high levels of ZNF703. The role of the ZNF703 gene has not been investigated in NSCLC. Our data show that ZNF703 may contribute to tumor development in NSCLC by activating the Akt/mTOR pathway.

MicroRNA Expression Profiles in Papillary Thyroid Carcinoma, Benign Thyroid Nodules and Healthy Controls.

MicroRNAs (miRNAs) represent a class of short endogenous non-coding RNAs that negatively regulate gene expression at the post-transcriptional level in many biological processes, including proliferation, differentiation, stress response and apoptosis. In this study we analyzed a set of seven miRNA molecules in sera of patients with papillary thyroid cancer, multinodular goiter and healthy controls to identify miRNA molecules that may have utility as markers for PTC. MiR-21 serum levels in the preoperative PTC and MG groups were significantly higher than the control group. Likewise, postoperative levels of miR-151-5p, miR-221 and miR-222 were significantly lower in patients with PTC. When serum miRNA levels were evaluated according to stage, postoperative levels of miR-151-5p and miR-222 were significantly lower in patients with advanced stages of the disease. The miRNA levels were also found associated with the size of the primary tumor. Our data imply that specific miRNA molecules which are differentially expressed in thyroid tumors may play role in the development of papillary thyroid carcinoma.

Analysis of circulating microRNAs during adjuvant chemotherapy in patients with luminal A breast cancer.

Breast cancer is a complex disease displaying different profiles involving genetic as well as epigenetic factors. MicroRNAs (miRNAs) are small, non-coding RNAs that regulate gene expression. Recent studies demonstrated that miRNAs may display great potential for the development of novel biomarkers and therapeutic targets. In the present study, the levels of miR-21 and miR-145 were analyzed in the peripheral blood of 52 patients with luminal A breast cancer. miRNA expression was determined in serum samples from matched pre- and post-treatment patients with breast cancer by quantitative polymerase chain reaction. There were no statistically significant differences in miR-145 and miR-21 levels between pre- and post-treatment samples. In addition, the miRNA levels were not found to be associated with the clinical parameters.

Global quantification of heterochromatin-associated histone methylations in cell lines with differential sensitivity to ionizing radiation.

Histone modifications are involved in the DNA damage response (DDR). Here, by utilizing an ELISA immunoassay we assessed the methylation at H3K9 (H3K9me2 and H3K9me3) in two cell lines with differential sensitivity to radiation-induced apoptosis, HeLa (sensitive) and MCF-7 (resistant). We found that DNA damage induction by γ-irradiation leads to considerable accumulation (up to 5-fold) of H3K9me2 and H3K9me3, but not of H4K20me3 (control modification) in MCF-7 cells (p<0.05). Interestingly, a lower dose (2 Gy) was more effective than 5 Gy. In HeLa cells a smaller effect (approx. 1.5-1.8-fold) was evident only at 5 Gy. In conclusion, our findings reveal that DNA damage leads to specific accumulation of H3K9me2 and H3K9me3 in a cell-type specific manner.

Exosomal lncRNA-p21 levels may help to distinguish prostate cancer from benign disease.

Exosomes are membranous vesicles containing various biomolecules including lncRNAs which are involved in cellular communication and are secreted from many cells including cancer cells. In our study, investigated the exosomal GAS5 and lincRNA-p21 lncRNA levels in urine samples from 30 patients with prostate cancer (PCa) and 49 patients with benign prostatic hyperplasia. Quantification of lncRNA molecules was performed by real-time PCR. We observed a significant difference in the exosomal lincRNA-p21 levels between PCa and BPH patients whereas the GAS5 levels did not reveal a difference. Our data suggest that the discriminative potential of exosomal lincRNA-p21 levels may help to improve the diagnostic prediction of the malignant state for patients with PCa.

Assessment of circulating serum DNA integrity in colorectal cancer patients.

UNLABELLED: Background /Aim: Studies evaluating the integrity of cell-fee DNA (cfDNA) in colorectal cancer (CRC) have led to inconsistent results. Herein, we analyzed the utility of two different DNA integrity indexes (ACTB(384)/ACTB(106) and ALU(247)/ALU(115)) to assess cfDNA fragmentation in Turkish CRC patients. The correlation of circulating nucleosomes (cNUC) with the fragment sizes was also evaluated. MATERIALS AND METHODS: Seventy two CRC patients and 42 CRC-free control individuals were enrolled in the study. cfDNA was analyzed by quantitative polymerase chain reaction (qPCR). RESULTS: While with ALU(247)/ALU(115)there was a small difference between the groups, an approximately 3-fold (median values=0.12 and 0.34) lower integrity was found in the patients using the ACTB(384)/ACTB(106) ratio (p=0.06). The correlation between cNUC and qPCR threshold cycles was much higher for shorter fragments. CONCLUSION: Lower DNA integrity and the predominance of mononuclesomes in serum reveal high fragmentation of cfDNA in CRC patients.

LncRNAs and neoplasia.

Long noncoding RNAs are emerging as new mediators of tumorigenesis by virtue of their various functions and their capacity to induce different mechanisms as a result of their wide spectrum of interactions. They play critical roles in a broad range of cellular processes including regulation of gene expression, imprinting, chromatin modification, transcription and posttranslational processing. Expression and activity of lncRNAs are deregulated in several types of human cancer. Impairment of lncRNA activity may affect key components of the cellular gene regulatory networks and is associated with deregulation of a large number of cellular oncogenic pathways. LncRNAs are also being evaluated as diagnostic and prognostic biomarkers and may provide targets for potential therapeutic applications. An improved understanding of the roles played by lncRNAs in cancer will lead to more effective therapeutic strategies. In this review we summarize the current knowledge on lncRNAs and their function as mediators of tumor development.

Amplification of chromosome 8 genes in lung cancer.

Chromosomal alterations are frequent events in lung carcinogenesis and usually display regions of focal amplification containing several overexpressed oncogenes. Although gains and losses of chromosomal loci have been reported copy number changes of the individual genes have not been analyzed in lung cancer. In this study 22 genes were analyzed by MLPA in tumors and matched normal tissue samples from 82 patients with non-small cell lung cancer. Gene amplifications were observed in 84% of the samples. Chromosome 8 was found to harbor the most frequent copy number alterations. The most frequently amplified genes were ZNF703, PRDM14 and MYC on chromosome 8 and the BIRC5 gene on chromosome 17. The frequency of deletions were much lower and the most frequently deleted gene was ADAM9. Amplification of the ZNF703, PRDM14 and MYC genes were highly correlated suggesting that the genes displaying high copy number changes on chromosome 8 collaborate during lung carcinogenesis.

Androgen Stimulation of PCA3 and miR-141 and Their Release from Prostate Cancer Cells.

OBJECTIVE: Prostate cancer antigen 3 (PCA3) and microRNA-141 (miR-141) are emerging molecules in prostate cancer (PCa) pathogenesis and have been shown to be involved in androgen signaling. In this original research, we designed an experimental cell model with androgen-sensitive LNCaP cells to comparatively assess the extent of androgen responsiveness of PCA3-mRNA and miR-141 along with prostate specific antigen (PSA)mRNA and their release into culture medium. These molecules were also measured in the plasma of the patients with early PCa which is considered to be analogous to androgenresponsive cells. MATERIALS AND METHODS: In this experimental study, LNCaP cells were exposed to androgen ablation for 48 hours and treated then with dihydrotestosterone (DHT) for 24 hours. Expression of all three RNA molecules in cells, culture medium or plasma was measured by quantitative polymerase chain reaction (qPCR). RESULTS: Our results show that DHT differentially affects the expression of these molecules. PCA3 was the most evidently induced molecule (up to 400-fold, p<0.001), while the effect was moderate for PSA-mRNA (up to 30-fold, p<0.001). In contrast, the stimulation of miR-141 was much weaker (up to 1.5-fold, p>0.05). With regard to the release into culture medium, a similar picture was observed except for PCA3. PCA3 was below the detection level despite its high stimulation. DHT treatment led to a significant release of PSA-mRNA (up to 12-fold). Similar to its induction pattern in LNCaP cells, miR-141 was released at a limited quantity into the medium (up to 1.7- fold, p=0.07). In plasma, only PCA3 differed significantly between the patients and healthy subjects (p=0.001). CONCLUSION: Our findings indicate that PCa-related RNA molecules respond differentially to androgen stimulation suggesting differential regulation by androgens.

Novel mutations and role of the LKB1 gene as a tumor suppressor in renal cell carcinoma.

The tumor suppressor LKB1 gene is a master kinase and inhibits mammalian target of rapamycin (mTOR) by activating AMP-activated protein kinase (AMPK) and AMPK-related kinases. LKB1 is a critical intermediate in the mTOR signaling pathway, and mutations of the LKB1 gene have been implicated in the development of different tumor types. Recent evidence indicates that LKB1 alterations contribute to cancer progression and metastasis by modulating vascular endothelial growth factor (VEGF) production. The Ras homolog enriched in brain (RHEB) protein is a component of the mTOR pathway and functions as a positive regulator of mTOR. However, the mechanisms and effectors of RHEB in mTOR signaling are not well known. In this study, we analyzed the expression of RHEB and HIF1α genes in correlation with LKB1 gene mutations. All coding exons and exon/intron boundaries of the LKB1 gene were analyzed by direct sequencing in 77 renal cell carcinoma (RCC) tumors and 62 matched noncancerous tissue samples. In 51.6 % of the patients, ten different mutations including four novel mutations in the coding sequences and six single nucleotide substitutions in the introns were observed. Rheb and HIF1α expression levels were not statistically different between the tumor and corresponding noncancerous tissue samples. However, expression of the Rheb gene was upregulated in the tumor samples carrying the intron 2 (+24 G→T) alteration. Association between the gene expression and tissue protein levels was also analyzed for HIF1α in a subgroup of patients, and a high correlation was confirmed. Our results indicate that the LKB1 gene is frequently altered in RCC and may play a role in RCC progression.

Abundant circulating microRNAs in breast cancer patients fluctuate considerably during neoadjuvant chemotherapy.

Previous studies have revealed the aberrant expression of a number of microRNAs (miRNA/miRs) in the blood circulation of patients with breast cancer (BC). The aim of the present study was to assess the effect of neoadjuvant chemotherapy on the levels of a panel of BC-associated miRNAs, which are at relatively low (let-7, miR-10b, miR-34, miR-155, miR-200c and miR-205) or abundant (miR-21, miR-195 and miR-221) levels in the circulation. Patients with primary operable or locally advanced BC were enrolled in the study. The plasma levels of the miRNAs at baseline and at the fourth cycle of treatment were compared. Patients with stage II disease exhibited higher basal miRNAs levels than those with higher stages. The difference was most evident for miR-155 and miR-21 (P=0.05). From the initial to the fourth cycle of chemotherapy, the miRNA levels changed substantially. In samples in which the miRNA levels generally declined, a marked decrease (≤15,500-fold) was evident for the abundant miRNAs. Notably, the occurrence of a decrease in miRNA levels was more frequent in patients with smaller tumor sizes (P<0.05 for miR-21 and miR-195). This proof-of-concept study provides evidence that highly expressed miRNAs are affected most frequently by chemotherapy, particularly in patients with early stage tumors. This information may be valuable in assessing the response of the patients to therapy.

Long non-coding RNAs with low expression levels in cells are enriched in secreted exosomes.

Long non-coding RNAs (lncRNAs) are involved in regulating chromatin modifications, gene transcription, mRNA translation, and protein function. We recently reported a high variation in the basal expression levels of a panel of lncRNAs in HeLa and MCF-7 cells and their differential response to DNA damage induction. Here, we hypothesized that lncRNA molecules with different cellular expression may have a differential abundance in secreted exosomes, and their exosome levels would reflect cellular response to DNA damage. MALAT1, HOTAIR, lincRNA-p21, GAS5, TUG1, CCND1-ncRNA in exosomes secreted from cultured cells were characterized. A different expression pattern of lncRNAs in exosomes was seen compared to cells. RNA molecules with relative low expression levels (lincRNA-p21, HOTAIR, ncRNA-CCND1) were highly enriched in exosomes. TUG1 and GAS5 levels were moderately elevated in exosomes, whereas MALAT1--which was the most abundant molecule in cells--was present at levels comparable to its cellular levels. lincRNA-p21 and ncRNA-CCND1 were the main molecules; exosome levels of them best reflect the change of their cellular levels upon exposure of the cells to bleomycin-induced DNA damage. In conclusion, we provide evidence that lncRNAs have a differential abundance in exosomes, indicating a selective loading.