miRNA Expression In Tumors Research Articles

Acidic extracellular environment affects miRNA expression in tumors in vitro and in vivo.

Hypoxia can control the expression of miRNAs in tumors which play an important role for the control of the malignant behavior. The aim of the study was to analyze whether extracellular acidosis, a common feature of tumors, also has an impact on the miRNA expression in isolated cells as well as in solid tumors. MiRNA expression was analyzed in two rat tumor cell lines (AT1 prostate and Walker-256 mammary carcinomas) by NGS and qPCR. In vivo the same cell lines were implanted subcutaneously and the tumor pH was modulated by inspiratory hypoxia and inhibition of the respiratory chain. In addition, the expression of five genes (Brip1, Ercc6l, Ikbke, Per3, Tlr5) which are potential targets of the miRNAs were analyzed on mRNA level. Screening showed that 38 (AT1) resp. 41 (Walker-256) miRNAs were pH-dependent. Validation by qPCR revealed that only 4 miRNAs were consistently regulated in both cell lines: miR-183, miR-203a, miR-215 and miR-7a. The expression of miR-7a was increased by low pH whereas all others were decreased. In the tumors in vivo all 4 miRNAs were down-regulated. The potential targets showed pH dependency in both cell lines. In conclusion, extracellular acidosis regulates the expression of miRNAs in vitro and in vivo. The expression of targets of these miRNAs were also pH-dependent. The pH may therefore affect the biological behavior of tumors via miRNAs.

Novel MicroRNA-Based Risk Score Identified by Integrated Analyses to Predict Metastasis and Poor Prognosis in Breast Cancer.

The use of biomarkers that allow early therapeutic intervention or intensive follow-up evaluation is expected to be a powerful means for reducing breast cancer mortality. MicroRNAs (miRNAs) are known to play major roles in cancer biology including metastasis. This study aimed to develop a novel miRNA risk score to predict patient survival and metastasis in breast cancer. An integrated unbiased approach was applied to derive a composite risk score for prognosis based on miRNA expression in primary breast tumors in 1051 breast cancer patients from The Cancer Genome Atlas (TCGA). Further analysis of the risk score with metastasis/recurrence was performed using the TCGA data set and validated in a separate patient population using small RNA sequencing. The three-miRNAs risk score (miR-19a, miR-93, and miR-106a) was developed using the TCGA cohort, which predicted poor prognosis (p = 0.0005) independently of known clinical risk factors. The prognostic value was validated in another three following independent cohorts: GSE19536 (p = 0.0009), GSE22220 (p = 0.0003), and the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) (p = 0.0023). The three-miRNAs risk score predicted bone recurrence in TCGA (p = 0.0052), and the findings were validated in another independent population of patients who experienced bone recurrence and age/stage-matched patients without any recurrence. The three-miRNAs risk score enriched multiple metastasis-related gene sets such as angiogenesis and epithelial mesenchymal transition in a gene-set-enrichment analysis. The authors developed the novel miRNA-based risk score, which is a promising biomarker for prediction of worse survival and bone recurrence potential in breast cancer.

MicroRNA-338-3p Suppresses Proliferation of Human Liver Cancer Cells by Targeting SphK2.

Recent studies have revealed abnormal expression of miRNAs in various tumors. Although microRNA-338-3p (miR-338-3p) plays an important role in many types of tumors, its influence on liver cancer (LC) is unknown. In this study, we found that expression of miR-338-3p was decreased in LC cells and tissues. Colony formation and cell proliferation were suppressed by enhanced expression of miR-338-3p in LC cells. Moreover, miR-338-3p targeted sphingosine kinase 2 (SphK2). Silencing of SphK2 had an identical influence as overexpression of miR-338-3p in LC cells. Overexpression of SphK2 without the 3′-untranslated region remarkably enhanced the growth suppression triggered by miR-338-3p in LC cells. These findings indicate that miR-338-3p influences the development of LC by targeting SphK2, suggesting that miR-338-3p can be targeted as an innovative therapeutic strategy for LC.

Bioinformatics and Regression Analyses Manifest Tumor-Specific miRNA Expression Dynamics in Pediatric Embryonal Malignancies.

Pediatric Central Nervous System (CNS) neoplasms are the second most prevalent tumors of childhood. Further on, prognosis of this type of neoplasms still remain poor and the comprehension of the etiology and pathogenesis of the disease still remains scarce. Several reports have identified microRNAs as significant molecules in the development of central nervous system tumors and propose that they might compose key molecules underlying oncogenesis. In a previous study we have identified several miRNAs, common to different subtypes of pediatric embryonal CNS malignancies as well as, we have identified miRNAs that manifest significant dynamics with respect to their expression and the neoplasmatic subtype. Overall, 19 tumor cases from children diagnosed with embryonal brain tumors were investigated. As controls, children who suffered a sudden death underwent autopsy and were not present with any brain malignancy were used (13 samples of varying localization). Our experimental approach included microarrays covering 1211 miRNAs, which appeared to manifest tumor-specific dynamics. In conclusion, it appeared that certain miRNAs are neoplasm specific and in particular, their expression manifests linear dynamics. Thus, the investigation of miRNA expression in pediatric embryonal brain tumors might contribute towards the discovery of tumor-specific miRNA signatures, which could potentially afford the identification of gene-specific biomarkers related to diagnosis, prognosis and patient targeted therapy, as well as help us understand oncogenetic dynamics.

A Panel of MicroRNA Signature as a Tool for Predicting Survival of Patients with Urothelial Carcinoma of the Bladder.

Introduction and Objectives MicroRNA (miRNA) expression is altered in urologic malignancies, including urothelial carcinoma of the bladder (UCB). Individual miRNAs have been shown to modulate multiple signaling pathways that contribute to BC. To identify a panel of miRNA signature that can predict aggressive phenotype from normal nonaggressive counterpart using miRNA expression levels and to assess the prognostic value of this specific miRNA markers in patients with UCB. Methods To determine candidate miRNAs as prognostic biomarkers for dividing aggressive type of UCB, miRNA expression was profiled in patients' samples with an aggressive phenotype or nonaggressive phenotype using 3D-Gene miRNA labeling kit (Toray, Japan). To create a prognostic index model, we used the panel of 9-miRNA signature based on Cancer Genome Atlas (TCGA) data portal (TCGA Data Portal (https://tcgadata.nci.nih.gov/tcga/tcgaHome2.jsp)). miRNA expression data and corresponding clinical data, including outcome and staging information of 84 UCB patients, were obtained. The Kaplan-Meier and log-rank test were performed to quantify the survival functions in two groups. Results Deregulation of nine miRNAs (hsa-miR-99a-5p, hsa-miR-100-5p, hsa-miR-125b-5p, hsa-miR-145-5p, hsa-miR-4324, hsa-miR-34b-5p, hsa-miR-29c-3p, hsa-miR-135a-3p, and hsa-miR-33b-3p) was determined in UCB patients with aggressive phenotype compared with nonaggressive subject. To validate the prognostic power of the nine-signature miRNAs using the TCGA dataset of bladder cancer, the survival status and tumor miRNA expression of all 84 TCGA UCB patients were ranked according to the prognostic score values. Of nine miRNAs, six were associated with high risk (hsa-miR-99a-5p, hsa-miR-100-5p, hsa-miR-125b-5p, hsa-miR-4324, hsa-miR-34b-5p, and hsa-miR-135a-3p) and three were shown to be protective (hsa-miR-145-5p, hsa-miR-29c-3p, and hsa-miR-33b-3p). Patients with the high-risk miRNA signature exhibited poorer OS than patients expressing the low-risk miRNA profile (HR = 7.05, p < 0.001). Conclusions The miRNA array identified nine dysregulated miRNAs from clinical samples. This panel of nine-miRNA signature provides predictive and prognostic value of patients with UCB.

MiR-142-3p is downregulated in aggressive p53 mutant mouse models of pancreatic ductal adenocarcinoma by hypermethylation of its locus

Pancreatic ductal adenocarcinoma (PDAC) is an extremely aggressive disease with poor prognostic implications. This is partly due to a large proportion of PDACs carrying mutations in TP53, which impart gain-of-function characteristics that promote metastasis. There is evidence that microRNAs (miRNAs) may play a role in both gain-of-function TP53 mutations and metastasis, but this has not been fully explored in PDAC. Here we set out to identify miRNAs which are specifically dysregulated in metastatic PDAC. To achieve this, we utilised established mouse models of PDAC to profile miRNA expression in primary tumours expressing the metastasis-inducing mutant p53R172H and compared these to two control models carrying mutations, which promote tumour progression but do not induce metastasis. We show that a subset of miRNAs are dysregulated in mouse PDAC tumour tissues expressing mutant p53R172H, primary cell lines derived from mice with the same mutations and in TP53 null cells with ectopic expression of the orthologous human mutation, p53R175H. Specifically, miR-142-3p is downregulated in all of these experimental models. We found that DNA methyltransferase 1 (Dnmt1) is upregulated in tumour tissue and cell lines, which express p53R172H. Inhibition or depletion of Dnmt1 restores miR-142-3p expression. Overexpression of miR-142-3p attenuates the invasive capacity of p53R172H-expressing tumour cells. MiR-142-3p dysregulation is known to be associated with cancer progression, metastasis and the miRNA is downregulated in patients with PDAC. Here we link TP53 gain-of-function mutations to Dnmt1 expression and in turn miR-142-3p expression. Additionally, we show a correlation between expression of these genes and patient survival, suggesting that they may have potential to be therapeutic targets.

Prediction of radiotherapy response with a 5-microRNA signature-based nomogram in head and neck squamous cell carcinoma.

Radiotherapy is unlikely to benefit all patients with head and neck squamous cell carcinoma (HNSCC). Therefore, novel method is warranted to predict the radiotherapy response. Our study aimed to construct a microRNA (miRNA)‐based nomogram to predict clinical outcomes of patients with HNSCC receiving radiotherapy. We screened out 56 differential miRNAs by analyzing 44 paired tumor and adjacent normal samples miRNA expression profiles from The Cancer Genome Atlas (TCGA). A total of 307 patients with HNSCC receiving adjuvant radiotherapy were randomly divided into a training set (n = 154) and a validation set (n = 153). In the training set, we combined the differential miRNA profiles with clinical outcomes, and LASSO regression model was applied to establish a 5‐miRNA signature. The prediction accuracy of the 5‐miRNA signature was further validated. In addition, target genes of these miRNAs were predicted, and Gene Ontology (GO) analysis as well as KEGG pathway analysis was executed. A 5‐miRNA signature including miR‐99a, miR‐31, miR‐410, miR‐424, and miR‐495 was identified. With a cutoff value of 1.2201 from Youden's index, the training set was divided into high‐risk and low‐risk groups, and the 5‐year overall survival was significantly different (30% vs. 73%, HR 3.65, CI 2.46–8.16; P < 0.0001). Furthermore, our 5‐miRNA signature revealed that only low‐risk group would benefit from radiotherapy. Then, a nomogram combining 5‐miRNA signature with clinical variables to predict radiotherapy response was constructed. The analysis of 108 target genes of these miRNAs revealed some potential mechanisms in HNSCC radiotherapy response for future investigations. In conclusion, the 5‐miRNA signature‐based nomogram is useful in predicting radiotherapy response in HNSCC and might become a promising tool to optimize radiation strategies.

Analysis of circulating extracellular vesicle-associated microRNAs in cortisol-producing adrenocortical tumors.

Circulating microRNAs (miRNA) have been described in patients with adrenocortical tumors, but the expression of miRNAs in non-functioning and cortisol-producing tumors has not been yet compared. Therefore, the objective of this study was to evaluate the expression of plasma extracellular vesicle (EV)-associated microRNAs in patients with non-functioning adrenocortical adenoma (NFA), cortisol-producing adrenocortical adenoma (CPA) and cortisol-producing adrenocortical carcinoma (CP-ACC). Preoperative plasma EV samples of 13 NFAs, 13 CPAs and 9 CP-ACCs were subjected to extracellular vesicle isolation. miRNAs were investigated by targeted quantitative real-time PCR normalized to cel-miR-39 as reference. Five miRNAs have been selected for this analysis based on the previous studies including hsa-miR-22-3p, hsa-miR-27a-3p, hsa-miR-210-3p, hsa-miR-320b and hsa-miR-375. We have observed significant overrepresentation of three miRNAs in both CPA and CP-ACC relative to NFA: hsa-miR-22-3p (p < 0.01 and p < 0.0001, respectively), hsa-miR-27a-3p (p < 0.05 in both comparisons) and hsa-miR-320b (p < 0.05 and p < 0.0001, respectively). Hsa-miR-320b has been significantly overrepresented in CP-ACC relative to CPA (p < 0.01). Hsa-miR-210-3p turned out to be significantly overrepresented only in CP-ACC compared to NFA (p < 0.05). Significant correlation was revealed between circulating miRNA concentrations and urinary free cortisol values for hsa-miR-22-3p, hsa-miR-27a-3p and hsa-miR-320b (p < 0.0001 for all) and cortisol after low-dose dexamethasone test for hsa-miR-22-3p and hsa-miR-320b (p < 0.05). Hsa-miR-27a-3p has been significantly stimulated by low-dose dexamethasone test (p < 0.05). EV-associated miRNAs are differentially expressed in different non-functioning and cortisol-producing adrenocortical tumors.

Comparative Analysis of MicroRNA Expression among Benign and Malignant Tongue Tissue and Plasma of Patients with Tongue Cancer.

Identification of a microRNA (miRNA) pattern to be used as a biomarker for HNSCC is challenging given the heterogeneity of the disease and different methodologies used. To better define the field, we performed a prospective analysis of blood, tumor, and paired benign tissues in tongue squamous cell carcinoma (SCC) patients. Plasma samples were collected prior to surgery, and paired tumor and benign tissue blocks were collected from tongue cancer resections. Circulating free and exosomal miRNA, and paired tumor and benign tissues miRNA were analyzed. TaqMan-based miRNA arrays were used to quantitate the expression of 747 human miRNAs. The comparative Ct method assessed the miRNA profile results, and Student's t-test determined statistical significance between tumor and benign samples. Sixteen of 359 miRNAs detected were differentially expressed between paired tumor and benign tissue. Nine were upregulated, and seven downregulated in tumor tissue. All nine upregulated and six of seven downregulated tumor miRNAs were expressed in circulating exosomes. In contrast, eight of nine upregulated and four of seven downregulated tumor miRNAs were circulating free in the plasma. An aberrantly expressed pattern of miRNA was identified in both tumor and plasma of patients with tongue SCC, suggesting this may be a biomarker for SCC of the oral tongue. Circulating exosomes appear to be a more reliable method for evaluation of circulating tumor-miRNA expression. Further studies with a larger cohort of patients and serial blood samples are needed to validate our findings.

Abstract 1566: Differential expression of Epstein Barr virus miRNAs in triple-negative breast cancer

Abstract Triple-negative breast cancers (TNBC) accounts for up to 20% of breast cancer cases. These tumors are negative for estrogen, progesterone, and HER2 receptors, which are markers used for diagnosis and treatment of other types of breast cancer. Clinically, TNBCs are notoriously aggressive, respond poorly to targeted therapies, have high rates of relapse, as well as poor prognosis. The molecular basis of TNBC oncogenesis is currently unknown, but it is possible that viral factors, such as Epstein-Barr Virus (EBV) miRNAs, play a role. EBV miRNAs have been implicated in the oncogenesis of several forms of cancer, including Burkitt’s lymphoma and nasopharyngeal carcinoma. The objective of this study, therefore, was to determine the differential expression of EBV miRNAs in TNBC tumors as compared to normal breast tissue. We conducted a comprehensive profiling of viral miRNAs in 48 TNBC tumors as compared to 15 control normal breast tissues, utilizing deep sequencing analysis software and publically available deep sequencing data. Four EBV miRNAs (BART18-3p, BART 8-5p, BART15, BART22) were significantly expressed in 11-17% of the TNBC tumors, and not in control normal breast tissue. Four novel putative EBV miRNAs were found to be differentially expressed in TNBC tumors as compared to control normal cells. Two of the novel putative EBV miRNAs were differentially expressed above 125 reads per million (RPM) in 20% and 27% of the TNBC tumors as compared to 0% and 6.6% of normal breast tissues, respectively. One novel putative EBV miRNA was expressed above 125 RPM in 83% of TNBC tumors as compared to 13% of normal breast tissue. One novel putative EBV miRNA was expressed above 125 RPM in 66% of the normal breast tissue as compared to 2% of the TNBCs. These putative EBV miRNAs localize within genomic regions of EBV, including the LMP-2 region. Ongoing work has so far computationally validated one of these miRNAs as a novel EBV miRNA; in vitro validation studies are in progress. This is the first report on the differential expression of EBV miRNAs in TNBC tumors. Since TNBC tumors are extremely heterogeneous, it is intriguing that 20% of TNBC tumors specifically express the same EBV miRNA. Our findings suggest that these differentially expressed EBV miRNAs may potentially play a role in the pathogenesis of TNBC. Citation Format: Alexander Blanchard, Tiffany Walls, Ramon Vidal, Stefan Bonn, Scott Harrison, Perpetua Muganda. Differential expression of Epstein Barr virus miRNAs in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1566. doi:10.1158/1538-7445.AM2017-1566

MiRNA profiling in a case: Control study of African American women with uterine serous carcinoma (USC).

e17116 Background: USC is an aggressive subtype of endometrial cancer associated with worse outcomes in African American patients. We evaluated differences in tumor miRNA expression by race, clinical and tumor characteristics, and survival outcomes. Methods: FFPE tumor tissue from hysterectomy specimens was identified for 29 African American cases. Case matching was performed by computer-based random assignment in a 1:1 ratio with Caucasian controls based on age ( &lt; 70 vs. ≥70 years), stage (FIGO I/II vs. III/IV) and histologic subtype (pure vs. mixed). RNA was extracted from 77 specimens with sufficient tumor cellularity (54 tumors and 23 matched normal endometrium). miRNA array profiling was performed by microRNA Hi-Power Labeling (Hy3/Hy5) and hybridization to miRCURY LNA microRNA Array 7th Gen (Exiqon, Denmark). Analysis was performed with R/Bioconductor using a moderated t-statistic with multiple testing correction. Validation was done using the TCGA dataset. Results: Clinical and treatment characteristics were similar for cases and controls, although use of adjuvant radiation was less common in African Americans (p = 0.03). With a median follow-up of 43 months, 17 patients had recurrent or progressive disease. DFS and OS rates were similar by race (both logrank p &gt; 0.5). Of 968 miRNAs analyzed, 649 were differentially expressed in normal endometrium vs. tumor. When compared by race, histologic subtype, stage or presence of LVI, no differentially expressed miRNAs were identified. In patients with disease recurrence at 3 years, miR-223 was significantly upregulated (fold change 1.5; p = 0.002). In validation using a TCGA dataset of 131 patients with mixed (n = 22) or pure serous (n = 109) histology, increased miR-223 expression ( &gt; median) was associated with worse overall survival (HR 2.47; 95%CI 0.9-6.6). After adjustment for patient age and BMI, upregulation of miR-223 was a significant risk factor for death (adjusted HR 2.94; 95%CI 1.01-8.52). Conclusions: Upregulation of miR-223 was associated with disease recurrence in a cohort of women with uterine serous carcinoma and validated by TCGA data. miRNA profiling did not identify biological differences between African American and Caucasian patients.

Abstract A11: Profiling of miRNAs expression in pediatric brain tumors

Abstract Introduction: The understanding of pediatric CNS tumors biology is essential in the development of disease stratification and in development of less toxic therapeutic agents as well as finding novel markers for early diagnosis. MicroRNAs are short 18-25 nucleotide small non-coding RNA molecules regulate gene expression. Recent studies showed that miRNAs play a significant role in brain tumor biology; and may up-regulated or down-regulated in malignancies, which referred their oncogenic or tumor suppressor effect. MiRNA expression patterns have been linked to clinical outcomes, tumor regulation; such as tumor progression, cell growth, cell death and metastasis. The identification of tumor- specific miRNA signatures may assist in future in the discovery of new biomarkers with diagnostic and prognostic utility. The main objective of the present study is to detect the expression of different miRNAs in different subtypes of pediatric CNS tumors to distinguish between them in discovering biomarkers for early detection in addition to develop novel therapies. Methods: The expression level of 82 miRNAs were detected in 120 cases of pediatric CNS tumors from fixed formalin paraffin embedded tissues (FFPE), divided into four subtypes including; Low Grade Glioma, High Grade Glioma, Ependymoma, and Medulloblastoma using quantitative real time PCR (qRTPCR). Results: Analysis of qRT-PCR data showed significant differences in miRNA expression between tumor subtypes with P value&amp;lt; 0.05. Low expression of (miR-221, miR-9, and miR-181c/d) and over expression in miR-101, miR-222, miR-139, miR-1827 and miR-34c) in Medulloblastoma patients compared to other subtypes. Low expression of miR-10a and overexpression in (miR-10b, and miR-29a) in Ependymoma patients compared to other subtypes. Low expression of miR-26a and over expression in (miR-19a/b, miR-24, miR-27a, miR- 584 and miR-527) in Low Grade Glioma patients compared to other subtypes. Conclusion: MicroRNAs are differentially expressed between different subtypes of pediatric CNS tumors suggesting that they may play a significant role in diagnosis to distinguish between different subtypes. A greater understanding of aberrant miRNA expression in pediatric brain tumors may support in the development of novel therapies. In addition, the characterization of tumor-specific miRNA signatures may play an important role in the discovery of biomarkers with diagnostic or prognostic utility. Note: This abstract was not presented at the conference. Citation Format: Marwa Tantawy, Mariam Elzayat, Dina Yehia, Hala Taha. Profiling of miRNAs expression in pediatric brain tumors. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr A11.

The miRNA landscape of colorectal polyps.

The genomic landscape of adenomas and polyps may help define disease pathways. Expression of miRNAs in adenomas and polyps may importantly contribute to these pathways. We evaluated miRNA expression in 293 polyp-normal colorectal mucosa pairs. Polyps were classified as either adenomatous polyp (AD), hyperplastic polyp (HP), or sessile serrated polyp (SSP). We compared these miRNA expression profiles in polyps to miRNA expression in microsatellite unstable (MSI) and stable (MSS) tumors. A False Discovery Rate of 0.05 based on Benjamini and Hochberg was used to adjust for multiple comparisons. There were 70 miRNAs with differential expression by polyp type with a fold change <0.75 or >1.34 after adjusting for multiple comparisons. The major differences in miRNA expression were observed between AD and SSP and AD and HP, with few differences in expression noted for SSP and HP. AD polyps were more likely to be upregulated from normal colonic mucosa, while SSP and HP were more likely to be downregulated from normal colonic mucosa. MiRNA expression in the SSP and HP tumors almost uniformly go in opposite directions from the MSS tumor miRNA expression and was mixed with MSI tumors. We conclude that different types of polyps have unique miRNA expression profiles. © 2017 Wiley Periodicals, Inc.

Altered expression of apoptosis-regulating miRNAs in salivary gland tumors suggests their involvement in salivary gland tumorigenesis.

Salivary gland tumors comprise a heterogeneous group of lesions with different histological features and diverse clinical pathophysiology. They account for about 3% of all head and neck tumors. Apoptosis plays an important role during morphogenesis of glandular structures, including that of the salivary gland. Recent studies have demonstrated that several microRNAs (miRNAs) are involved in the control of apoptosis. The aim of the present study was to determine the expression of apoptosis-related miRNAs (miR-15a, miR-16, miR-17-5p, miR-20a, miR-21, miR-29, and miR-34) and their target mRNAs in 25 pleomorphic adenomas, 23 mucoepidermoid carcinomas, and 10 non-neoplastic salivary gland samples by real-time RT-PCR. We observed upregulation of miR-15a, miR-16, miR-17-5p, miR-21, miR-29, and miR-34a in pleomorphic adenomas. The expression of miR-21 and miR-34a was upregulated in 91 and 74% of mucoepidermoid carcinomas, respectively. Downregulation of miR-20a was observed in 75% of pleomorphic adenomas and in 57% of mucoepidermoid carcinomas. APAF1, BAX, BCL2, BID, CASP2, CASP8, DIABLO , and TP53 transcripts were upregulated in both tumor types. BAD transcripts were upregulated in pleomorphic adenomas. CASP3 and CASP6 transcripts were upregulated in mucoepidermoid carcinomas. BCL2, CASP2, CASP6, and CASP8 proteins were mostly absent in mucoepidermoid carcinomas but expressed in few cells in pleomorphic adenomas. Our study provides evidence of alterations in the expression of apoptosis-regulating miRNAs in salivary gland tumors, suggesting possible involvement of these microRNAs in salivary gland tumorigenesis.

MiR-9 is overexpressed in spontaneous canine osteosarcoma and promotes a metastatic phenotype including invasion and migration in osteoblasts and osteosarcoma cell lines.

BackgroundMicroRNAs (miRNAs) regulate the expression of networks of genes and their dysregulation is well documented in human malignancies; however, limited information exists regarding the impact of miRNAs on the development and progression of osteosarcoma (OS). Canine OS exhibits clinical and molecular features that closely resemble the corresponding human disease and it is considered a well-established spontaneous animal model to study OS biology. The purpose of this study was to investigate miRNA dysregulation in canine OS.MethodsWe evaluated miRNA expression in primary canine OS tumors and normal canine osteoblast cells using the nanoString nCounter system. Quantitative PCR was used to validate the nanoString findings and to assess miR-9 expression in canine OS tumors, OS cell lines, and normal osteoblasts. Canine osteoblasts and OS cell lines were stably transduced with pre-miR-9 or anti-miR-9 lentiviral constructs to determine the consequences of miR-9 on cell proliferation, apoptosis, invasion and migration. Proteomic and gene expression profiling of normal canine osteoblasts with enforced miR-9 expression was performed using 2D-DIGE/tandem mass spectrometry and RNA sequencing and changes in protein and mRNA expression were validated with Western blotting and quantitative PCR. OS cell lines were transduced with gelsolin (GSN) shRNAs to investigate the impact of GSN knockdown on OS cell invasion.ResultsWe identified a unique miRNA signature associated with primary canine OS and identified miR-9 as being significantly overexpressed in canine OS tumors and cell lines compared to normal osteoblasts. Additionally, high miR-9 expression was demonstrated in tumor-specific tissue obtained from primary OS tumors. In normal osteoblasts and OS cell lines transduced with miR-9 lentivirus, enhanced invasion and migration were observed, but miR-9 did not affect cell proliferation or apoptosis. Proteomic and transcriptional profiling of normal canine osteoblasts overexpressing miR-9 identified alterations in numerous genes, including upregulation of GSN, an actin filament-severing protein involved in cytoskeletal remodeling. Lastly, stable downregulation of miR-9 in OS cell lines reduced GSN expression with a concomitant decrease in cell invasion and migration; concordantly, cells transduced with GSN shRNA demonstrated decreased invasive properties.ConclusionsOur findings demonstrate that miR-9 promotes a metastatic phenotype in normal canine osteoblasts and malignant OS cell lines, and that this is mediated in part by enhanced GSN expression. As such, miR-9 represents a novel target for therapeutic intervention in OS.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2837-5) contains supplementary material, which is available to authorized users.

Abstract A18: Investigating the differential expressions of miRNAs and programmed-death-ligand-1 related to the T-bet positive tumor-infiltrating lymphocytes in axillary-node-negative breast cancer

Abstract Axillary lymph node-negative (ANN) breast cancer (BC) patients generally have a good prognosis; however, 20-30% of ANN patients will experience recurrence. To identify biomarkers that distinguish ANN patients who may benefit from systemic therapy we have been studying characteristics of the breast tumor cells and the tumor microenvironment. We have found that tumor-infiltrating lymphocytes (TILs) that express T-bet (T-box transcription factor) are associated with poor prognosis features such as high grade, hormone receptor negativity and basal subtype. Despite these adverse clinical-pathologic characteristics, T-bet+ TILs cells are associated with a favorable outcome. T-bet is essential for Th1 development and IFN-γ production. To investigate how T-bet+ TILs influence the BC suppression, we are examining molecular differences in T-bet+/high and T-bet-/low tumors including expression of tumor miRNA expression profiles and expression of Programmed-Death-Ligand-1 (PD-L1). Tumors expressing PD-L1 inhibit Programmed-Death-receptor-1-(PD-1)-expressing TILs and evade immune surveillance. Interestingly, PD-L1 is induced by IFNγ, the hallmark cytokine of the T-bet+ TILs, which suggests a potential relationship between the tumor PD-L1 and T-bet+ TILs. Microdissected, formalin-fixed, paraffin-embedded (FFPE) samples of 35 T-bet+/high and 35 T-bet-/low ANN BC tumors are being used to identify the differentially-expressed miRNAs via miRNA sequencing. The sufficiency of the FFPE samples as the miRNA source was confirmed by quantifying miRNA expressions in the 3-5 paired frozen and FFPE samples, and observing the expressions to be highly correlated with both qPCR (r=0.81) and miRNA sequencing (rs=0.73). After identifying the differentially-expressed miRNAs via miRNA sequencing, these miRNAs will be validated via qPCR in both the original and independent BC tumor set. Based on the genes and molecular pathways the validated miRNA(s) may regulate, appropriate in vitro functional assays will be conducted to examine their roles in cellular proliferation, migration and invasion. Furthermore, PD-L1 mRNA expression has been evaluated in 107 ANN tumors and correlations with clinico-pathological characteristics will be determined. Identifying the tumor miRNA(s) associated with T-bet+ TILs and studying their functions may indicate differences between T-bet+/high and T-bet-/low tumors that can be used to improve the prognosis of ANN BC. Characterizing the tumor PD-L1 expression based on T-bet+ TILs and the clinical data may distinguish a subset of ANN BC patients who may benefit from the PD-L1 blockade therapy. Citation Format: Minji Lee, Anna-Marie Mulligan, Shelley Bull, Dushanti Pinnaduwage, Irene Andrulis. Investigating the differential expressions of miRNAs and programmed-death-ligand-1 related to the T-bet positive tumor-infiltrating lymphocytes in axillary-node-negative breast cancer. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr A18.

Abstract 1120: Differential expression of human cytomegalovirus microRNA in triple-negative breast cancer tumors

Abstract Triple-negative breast cancer (TNBC) accounts for 15-20% of the breast cancer cases. These tumors are heterogeneous and aggressive, fail to respond to targeted therapy, have poor prognosis, and a high risk of relapse. The molecular basis of this breast cancer subtype is currently unknown. Viruses, such as human cytomegalovirus (HCMV), Epstein-Barr virus (EBV), and human papillomavirus (HPV), are major suspects in the etiology of breast cancer. Since miRNAs have been implicated in the pathogenesis of breast cancer, including triple-negative breast cancer, we hypothesized that viral miRNAs may play a role in this aggressive disease. The objective of this project, therefore, was to determine the identity, prevalence, sequence variation, and differential expression of viral miRNAs in TNBC tumors as compared to control samples. We conducted a comprehensive profiling of viral miRNAs in 48 TNBC tumors as compared to 15 control normal breast tissues, utilizing deep sequencing analysis software and publically available deep sequencing data. Five novel putative HCMV miRNAs were found to be differentially expressed in TNBC tumors as compared to controls. Two of the putative miRNAs were differentially expressed in 60-79% of the TNBC tumors as compared to 0% of normal controls. Two additional putative miRNAs were expressed in 67-88% of TNBC tumors as compared to 25-31% of normal controls. One putative miRNA was expressed in 67% of TNBC tumors as compared to 88% of normal samples, while 1 putative miRNA in addition to HCMV-US25-1-3p displayed no significant differences. These putative HCMV miRNAs localize within genomic regions of HCMV that encode UL56 DNA packaging terminase subunit 2, single-stranded DNA-binding protein, noncoding RNA4.9, and enveloped glycoprotein M. Ongoing work has so far computationally validated one of these miRNAs as a novel HCMV miRNA. This is the first report on the differential expression of HCMV miRNAs in TNBC tumors. Since TNBC tumors are extremely heterogeneous, it is intriguing that 60-80% of TNBC tumors specifically express the same HCMV miRNA. Our findings suggest that these differentially expressed HCMV miRNAs may potentially play a role in the pathogenesis of TNBC. Citation Format: Tia Hudson, Scott Harrison, Dukka K C, Jan Prins, Perpetua M. Muganda. Differential expression of human cytomegalovirus microRNA in triple-negative breast cancer tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1120.

Abstract IA12: Mechanisms of tumorigenesis due to somatic mutations in DICER1 and DROSHA in childhood kidney cancers

Abstract Wilms tumor is the most common pediatric kidney cancer and the fourth most common childhood cancer overall. Wilms tumor is treated with a combination of surgery, chemotherapy, and radiation, and, while most children are cured, patients suffer adverse late effects of therapy and survival remains poor in those with advanced-stage disease. Known driver mutations (in WT1, WTX, and β-catenin) account for only one-third of Wilms tumor cases. To better define the genomic landscape of Wilms tumor, we performed whole-exome sequencing of 44 Wilms tumors to identify somatic mutations. Through this approach, we identified recurrent somatic mutations in MYCN, DROSHA, and DICER1, which were mutually exclusive with known Wilms tumor driver mutations in WT1 and β-catenin. DROSHA and DICER1 somatic hotspot mutations occur at or near critical metal-binding residues in RNase III domains and partially or completely disrupt enzyme activity. Examination of miRNA expression in tumors, in vitro processing assays, and genomic editing in human cells demonstrate that DICER1 and DROSHA mutations influence miRNA processing through distinct mechanisms. While DICER1 RNase IIIB mutations preferentially impair processing of miRNAs that derive from the 5′ arm of pre-miRNA hairpins, DROSHA RNase IIIB mutations globally inhibit miRNA biogenesis through a dominant-negative mechanism. Despite their distinct mechanisms of action, DROSHA and DICER1 mutations result in decreased expression of several common tumor-suppressing miRNAs, including multiple members of the let-7 and miR-15/16 families. We also identified a patient carrying a germline DICER1 loss-of-function allele who subsequently developed multiple malignancies with distinct DICER1 RNase IIIB somatic hotspot mutations, suggesting that DICER1 can act as both a tumor-suppressor and an oncogene depending on the nature of the mutation. These results provide new insights into the mechanisms through which mutations in miRNA biogenesis components reprogram miRNA expression in human cancer and suggest that mutations causing impairment of miRNA processing define a distinct subclass of Wilms tumors. Citation Format: Kenneth S. Chen, Dinesh Rakheja, Yangjian Liu, Abhay A. Shukla, Joshua T. Mendell, James F. Amatruda. Mechanisms of tumorigenesis due to somatic mutations in DICER1 and DROSHA in childhood kidney cancers. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr IA12.

Abstract A25: microRNAs associated with chemoresistance in gastric cancer patients tumors treated with neoadjuvant chemoterapy

Abstract Abstract: Results of randomized controlled studies conducted to verify the effectiveness of neoadjuvant chemotherapy (NC) for gastric carcinoma (GC) have suggested that this treatment is therapeutically useful for the patients instead of surgery alone. However, in some patients with apparently the same molecular and histological tumor characteristics, the objective clinical response with NC is not achieved, and this lack of response may delay curative surgery and the toxicity induced by chemotherapy may increase surgical complications. Therefore, the main challenge for improving clinical outcomes is to understand the molecular mechanisms that may play essential roles in patients' responses of GC treatments. So, predictive novel molecules identification may be crucial for correct patients' selection. Emerging evidence suggested that some microRNAs (miRNAs) can have prognostic and diagnostic value in many types of cancer. Expression levels of several miRNAs have been found to be correlated with GC prognosis and could determine the clinical response for neoadjuvant chemotherapy. Objective: Our goal was to analyze the association between tumor miRNA expression and the pathological response grade of gastric carcinoma patients treated with neoadjuvant chemotherapy. Methods: A total of 52 paraffined samples were selected from a retrospective database of A.C. Camargo Cancer Center: From these samples, 34 were biopsy tumor samples (collected before chemotherapy) of GC patients treated with NC and 18 were normal gastric tissue (controls). The chemotherapy schemes used in NC were epirubicin, cisplatin and fluoracil, with adaptations using docetaxel, xeloda and oxaliplatin. All patients treated with NC received chemotherapy prior the surgery, and subsequently they were submitted to a complete resection (R0). To determine the tumor response of patients treated of neoadjuvant chemotherapy, we analyzed representative tumor slides stained with hematoxilin and eosine, and evaluated the percentage of viable tumor cells present in the gastric resection after NC. For trial analyses we used TLDA (Taqman® Low Density Array) qRT-PCR to determine the expression of 377 miRNAs. After that, validation of differentially expressed miRNAs was done by qRT-PCR using individual assays. Results: According to the percentage of viable tumor cells seen in microscopic analysis we established a graduation from 0% to 100% of viable tumor cells, being this relationship proportional inversely to treatment response. After evaluating the percentage of viable tumor cells present in gastric resection after NC, we categorized the samples in quartiles. After statistical analysis, the samples with a 100% of viable tumor cells after neoadjuvant treatment, showed us nine downregulated miRNAs. We performed qRT-PCR using individual assays, and confirmed downregulated expression of 3 miRNAs in biopsy tumor samples of patients treated with NC with 100% of viable tumor cells in gastric resection tumor. Conclusion: We found proteomic and expression profiles reports about the relationship of these 3 downregulated miRNAs with some genomic alterations involved in gastric cancer pathways and with some mechanisms of drugs resistance. These miRNA signature may be useful as potential biomarkers to predict response of gastric cancer patients to NC. Citation Format: Michelle Maccarini Barcelos-Baldoni, Claudia Malheiros Coutinho-Camillo, Renato David Puga, Maria Dirlei Ferreira-Begnami. microRNAs associated with chemoresistance in gastric cancer patients tumors treated with neoadjuvant chemoterapy. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr A25.

MiRNA-21 Expression Decreases from Primary Tumors to Liver Metastases in Colorectal Carcinoma.

ObjectiveMetastasis is the major cause of death in colorectal cancer patients. Expression of certain miRNAs in the primary tumors has been shown to be associated with progression of colorectal cancer and the initiation of metastasis. In this study, we compared miRNA expression in primary colorectal cancer and corresponding liver metastases in order to get an idea of the oncogenic importance of the miRNAs in established metastases.MethodsWe analyzed the expression of miRNA-21, miRNA-31 and miRNA-373 in corresponding formalin-fixed paraffin-embedded (FFPE) tissue samples of primary colorectal cancer, liver metastasis and healthy tissues of 29 patients by quantitative real-time PCR.ResultsAll three miRNAs were significantly up-regulated in the primary tumor tissues as compared to healthy colon mucosa of the respective patients (p < 0.01). MiRNA-21 and miRNA-31 were also higher expressed in liver metastases as compared to healthy liver tissues (p < 0.01). No significant difference of expression of miRNA-31 and miRNA-373 was observed between primary tumors and metastases. Of note, miRNA-21 expression was significantly reduced in liver metastases as compared to the primary colorectal tumors (p < 0.01).ConclusionIn the context of previous studies demonstrating increased miRNA-21 expression in metastatic primary tumors, our findings raise the question whether miRNA-21 might be involved in the initiation but not in the perpetuation and growth of metastases.