Ayelet Chajut

Serum MicroRNAs Are Promising Novel Biomarkers

Background Circulating nucleic acids (CNAs) offer unique opportunities for early diagnosis of clinical conditions. Here we show that microRNAs, a family of small non-coding regulatory RNAs involved in human development and pathology, are present in bodily fluids and represent new effective biomarkers. Methods and Results After developing protocols for extracting and quantifying microRNAs in serum and other body fluids, the serum microRNA profiles of several healthy individuals were determined and found to be similar, validating the robustness of our methods. To address the possibility that the abundance of specific microRNAs might change during physiological or pathological conditions, serum microRNA levels in pregnant and non pregnant women were compared. In sera from pregnant women, microRNAs associated with human placenta were significantly elevated and their levels correlated with pregnancy stage. Conclusions and Significance Considering the central role of microRNAs in development and disease, our results highlight the medically relevant potential of determining microRNA levels in serum and other body fluids. Thus, microRNAs are a new class of CNAs that promise to serve as useful clinical biomarkers.

MicroRNAs accurately identify cancer tissue origin

MicroRNAs (miRNAs) belong to a class of noncoding, regulatory RNAs that is involved in oncogenesis and shows remarkable tissue specificity. Their potential for tumor classification suggests they may be used in identifying the tissue in which cancers of unknown primary origin arose, a major clinical problem. We measured miRNA expression levels in 400 paraffin-embedded and fresh-frozen samples from 22 different tumor tissues and metastases. We used miRNA microarray data of 253 samples to construct a transparent classifier based on 48 miRNAs. Two-thirds of samples were classified with high confidence, with accuracy >90%. In an independent blinded test-set of 83 samples, overall high-confidence accuracy reached 89%. Classification accuracy reached 100% for most tissue classes, including 131 metastatic samples. We further validated the utility of the miRNA biomarkers by quantitative RT-PCR using 65 additional blinded test samples. Our findings demonstrate the effectiveness of miRNAs as biomarkers for tracing the tissue of origin of cancers of unknown primary origin.

Identification of a Novel Hypoxia-Inducible Factor 1-Responsive Gene, RTP801, Involved in Apoptosis

ABSTRACT Hypoxia is an important factor that elicits numerous physiological and pathological responses. One of the major gene expression programs triggered by hypoxia is mediated through hypoxia-responsive transcription factor hypoxia-inducible factor 1 (HIF-1). Here, we report the identification and cloning of a novel HIF-1-responsive gene, designated RTP801. Its strong up-regulation by hypoxia was detected both in vitro and in vivo in an animal model of ischemic stroke. When induced from a tetracycline-repressible promoter, RTP801 protected MCF7 and PC12 cells from hypoxia in glucose-free medium and from H2O2-triggered apoptosis via a dramatic reduction in the generation of reactive oxygen species. However, expression of RTP801 appeared toxic for nondividing neuron-like PC12 cells and increased their sensitivity to ischemic injury and oxidative stress. Liposomal delivery of RTP801 cDNA to mouse lungs also resulted in massive cell death. Thus, the biological effect of RTP801 overexpression depends on the cell context and may be either protecting or detrimental for cells under conditions of oxidative or ischemic stresses. Altogether, the data suggest a complex type of involvement of RTP801 in the pathogenesis of ischemic diseases.

Diagnostic Assay Based on hsa-miR-205 Expression Distinguishes Squamous From Nonsquamous Non–Small-Cell Lung Carcinoma

PURPOSE Recent advances in treatment of lung cancer require greater accuracy in the subclassification of non-small-cell lung cancer (NSCLC). Targeted therapies which inhibit tumor angiogenesis pose higher risk for adverse response in cases of squamous cell carcinoma. Interobserver variability and the lack of specific, standardized assays limit the current abilities to adequately stratify patients for such treatments. In this study, we set out to identify specific microRNA biomarkers for the identification of squamous cell carcinoma, and to use such markers for the development of a standardized assay. PATIENTS AND METHODS High-throughput microarray was used to measure microRNA expression levels in 122 adenocarcinoma and squamous NSCLC samples. A quantitative real-time polymerase chain reaction (qRT-PCR) platform was used to verify findings in an independent set of 20 NSCLC formalin-fixed, paraffin-embedded (FFPE) samples, and to develop a diagnostic assay using an additional set of 27 NSCLC FFPE samples. The assay was validated using an independent blinded cohort consisting of 79 NSCLC FFPE samples. RESULTS We identified hsa-miR-205 as a highly specific marker for squamous cell lung carcinoma. A microRNA-based qRT-PCR assay that measures expression of hsa-miR-205 reached sensitivity of 96% and specificity of 90% in the identification of squamous cell lung carcinomas in an independent blinded validation set. CONCLUSION Hsa-miR-205 is a highly accurate marker for lung cancer of squamous histology. The standardized diagnostic assay presented here can provide highly accurate subclassification of NSCLC patients.

Identification of a novel stress-responsive gene Hi95 involved in regulation of cell viability.

cDNA microarray hybridization was used in an attempt to identify novel genes participating in cellular responses to prolonged hypoxia. One of the identified novel genes, designated Hi95 shared significant homology to a p53-regulated GADD family member PA26. In addition to its induction in response to prolonged hypoxia, the increased Hi95 transcription was observed following DNA damage or oxidative stress, but not following hyperthermia or serum starvation. Whereas induction of Hi95 by prolonged hypoxia or by oxidative stress is most likely p53-independent, its induction in response to DNA damaging treatments (γ- or UV-irradiation, or doxorubicin) occurs in a p53-dependent manner. Overexpression of Hi95 full-length cDNA was found toxic for many types of cultured cells directly leading either to their apoptotic death or to sensitization to serum starvation and DNA damaging treatments. Unexpectedly, conditional overexpression of the Hi95 cDNA in MCF7-tet-off cells resulted in their protection against cell death induced by hypoxia/glucose deprivation or H2O2. Thus, Hi95 gene seems to be involved in complex regulation of cell viability in response to different stress conditions.

Accurate Classification of Non–Small Cell Lung Carcinoma Using a Novel MicroRNA-Based Approach

Purpose: Advances in targeted lung cancer therapy now demand accurate classification of non–small cell lung cancer (NSCLC). MicroRNAs (miRNA) are recently discovered short, noncoding genes that play essential roles in tissue differentiation during normal development and tumorigenesis. For example, hsa-miR-205 is a miRNA that is highly expressed in lung squamous cell carcinomas (SqCC) but not in lung adenocarcinomas. The differential expression of miRNAs could be exploited to distinguish these tumor types. Experimental Design: One hundred and two resected NSCLCs were classified as SqCC or adenocarcinoma based on their histologic features and immunohistochemical profiles. Corresponding preoperative biopsies/aspirates that had been originally diagnosed as poorly differentiated NSCLCs were available for 21 cases. A quantitative reverse transcription-PCR diagnostic assay that measures the expression level of hsa-miR-205 was used to classify the carcinomas as SqCC or adenocarcinoma based solely on expression levels. The two sets of diagnoses were compared. Results: Using standard pathologic methods of classification (i.e., microscopy and immunohistochemistry), 52 resected lung carcinomas were classified as SqCCs and 50 as adenocarcinomas. There was 100% concordance between the diagnoses established by conventional and miRNA-based methods. MiRNA profiling also correctly classified 20 of the 21 preoperative biopsy specimens. Conclusions: MiRNA profiling is a highly reliable strategy for classifying NSCLCs. Indeed, classification is consistently accurate even in small biopsies/aspirates of poorly differentiated tumors. Confirmation of its reliability across the full range of tumor grades and specimen types represents an important step toward broad application. Clin Cancer Res; 16(2); 610–9

hsa-miR-29c* Is Linked to the Prognosis of Malignant Pleural Mesothelioma

The inability to forecast outcomes for malignant mesothelioma prevents clinicians from providing aggressive multimodality therapy to the most appropriate individuals who may benefit from such an approach. We investigated whether specific microRNAs (miR) could segregate a largely surgically treated group of mesotheliomas into good or bad prognosis categories. A training set of 44 and a test set of 98 mesothelioma tumors were analyzed by a custom miR platform, along with 9 mesothelioma cell lines and 3 normal mesothelial lines. Functional implications as well as downstream targets of potential prognostic miRs were investigated. In both the training and test sets, hsa-miR-29c* was an independent prognostic factor for time to progression as well as survival after surgical cytoreduction. The miR was expressed at higher levels in epithelial mesothelioma, and the level of this miR could segregate patients with this histology into groups with differing prognosis. Increased expression of hsa-miR-29c* predicted a more favorable prognosis, and overexpression of the miR in mesothelioma cell lines resulted in significantly decreased proliferation, migration, invasion, and colony formation. Moreover, major epigenetic regulation of mesothelioma is mediated by hsa-miR-29c* and was shown through downregulation of DNA methyltransferases as well as upregulation of demethylating genes. A single miR has the potential to be a prognostic biomarker in mesothelioma, and validation of these findings as well as investigation of its downstream targets may give insight for potential therapies in the future.

hsa-miR-191 Is a Candidate Oncogene Target for Hepatocellular Carcinoma Therapy

Hepatocellular carcinoma (HCC) is generally a fatal disease due to a paucity of effective treatment options. The identification of oncogenic microRNAs that exert pleiotropic effects in HCC cells may offer new therapeutic targets. In this study, we have identified the human microRNA miR-191 as a potential target for HCC therapy. Inhibition of miR-191 decreased cell proliferation and induced apoptosis in vitro and significantly reduced tumor masses in vivo in an orthotopic xenograft mouse model of HCC. Additionally, miR-191 was found to be upregulated by a dioxin, a known liver carcinogen, and was found to be a regulator of a variety of cancer-related pathways. Our findings offer a preclinical proof of concept for miR-191 targeting as a rational strategy to pursue for improving HCC treatment.

Pro-tumorigenic Effects of miR-31 Loss in Mesothelioma

The human genome encodes several hundred microRNA (miRNA) genes that produce small (21–23n) single strand regulatory RNA molecules. Although abnormal expression of miRNAs has been linked to cancer progression, the mechanisms of this dysregulation are poorly understood. Malignant mesothelioma (MM) of pleura is an aggressive and highly lethal cancer resistant to conventional therapies. We and others previously linked loss of the 9p21.3 chromosome in MM with short time to tumor recurrence. In this study, we report that MM cell lines derived from patients with more aggressive disease fail to express miR-31, a microRNA recently linked with suppression of breast cancer metastases. We further demonstrate that this loss is due to homozygous deletion of the miR-31-encoding gene that resides in 9p21.3. Functional assessment of miR-31 activity revealed its ability to inhibit proliferation, migration, invasion, and clonogenicity of MM cells. Re-introduction of miR-31 suppressed the cell cycle and inhibited expression of multiple factors involved in cooperative maintenance of DNA replication and cell cycle progression, including pro-survival phosphatase PPP6C, which was previously associated with chemotherapy and radiation therapy resistance, and maintenance of chromosomal stability. PPP6C, whose mRNA is distinguished with three miR-31-binding sites in its 3′-untranslated region, was consistently down-regulated by miR-31 introduction and up-regulated in clinical MM specimens as compared with matched normal tissues. Taken together, our data suggest that tumor-suppressive propensity of miR-31 can be used for development of new therapies against mesothelioma and other cancers that show loss of the 9p21.3 chromosome.

Validation of a microRNA-based qRT-PCR test for accurate identification of tumor tissue origin

Identification of the tissue of origin of a tumor is vital to its management. Previous studies showed tissue-specific expression patterns of microRNA and suggested that microRNA profiling would be useful in addressing this diagnostic challenge. MicroRNAs are well preserved in formalin-fixed, paraffin-embedded (FFPE) samples, further supporting this approach. To develop a standardized assay for identification of the tissue origin of FFPE tumor samples, we used microarray data from 504 tumor samples to select a shortlist of 104 microRNA biomarker candidates. These 104 microRNAs were profiled by proprietary quantitative reverse transcriptase polymerase chain reaction (qRT–PCR) on 356 FFPE tumor samples. A total of 48 microRNAs were chosen from this list of candidates and used to train a classifier. We developed a clinical test for the identification of the tumor tissue of origin based on a standardized protocol and defined the classification criteria. The test measures expression levels of 48 microRNAs by qRT–PCR, and predicts the tissue of origin among 25 possible classes, corresponding to 17 distinct tissues and organs. The biologically motivated classifier combines the predictions generated by a binary decision tree and K-nearest neighbors (KNN). The classifier was validated on an independent, blinded set of 204 FFPE tumor samples, including nearly 100 metastatic tumor samples. The test predictions correctly identified the reference diagnosis in 85% of the cases. In 66% of the cases the two algorithm predictions (tree and KNN) agreed on a single-tissue origin, which was identical to the reference diagnosis in 90% of cases. Thus, a qRT–PCR test based on the expression profile of 48 tissue-specific microRNAs allows accurate identification of the tumor tissue of origin.