Although only 1.5% of the human genome appears to code for proteins, much effort in cancer research has been devoted to this minimal fraction of our DNA. However, the last few years have witnessed the realization that a large class of non-coding RNAs (ncRNAs), named microRNAs, contribute to cancer development and progression by acting as oncogenes or tumor suppressor genes. Recent studies have also shown that epigenetic silencing of microRNAs with tumor suppressor features by CpG island hypermethylation is a common hallmark of human tumors. Thus, we wondered whether there were other ncRNAs undergoing aberrant DNA methylation-associated silencing in transformed cells. We focused on the transcribed-ultraconserved regions (T-UCRs), a subset of DNA sequences that are absolutely conserved between orthologous regions of the human, rat and mouse genomes and that are located in both intra- and intergenic regions. We used a pharmacological and genomic approach to reveal the possible existence of an aberrant epigenetic silencing pattern of T-UCRs by treating cancer cells with a DNA-demethylating agent followed by hybridization to an expression microarray containing these sequences. We observed that DNA hypomethylation induces release of T-UCR silencing in cancer cells. Among the T-UCRs that were reactivated upon drug treatment, Uc.160+, Uc283+A and Uc.346+ were found to undergo specific CpG island hypermethylation-associated silencing in cancer cells compared with normal tissues. The analysis of a large set of primary human tumors (n=283) demonstrated that hypermethylation of the described T-UCR CpG islands was a common event among the various tumor types. Our finding that, in addition to microRNAs, another class of ncRNAs (T-UCRs) undergoes DNA methylation-associated inactivation in transformed cells supports a model in which epigenetic and genetic alterations in coding and non-coding sequences cooperate in human tumorigenesis.Oncogene advance online publication, 30 August 2010; doi:10.1038/onc.2010.361.

INTRODUCTION:: Stage IIIA non-small cell lung cancer (NSCLC) with ipsilateral mediastinal lymph node metastases (N2) is a heterogeneous disease with differing prognoses. In this study, we retrospectively investigated the prognostic value of the expression of 10 molecular markers in 87 patients with stage IIIA pN2 NSCLC treated with radical surgery. METHODS:: Primary tumor tissue microarrays (TMAs) were constructed and sections used for immunohistochemical analysis of epidermal growth factor receptor, ErbB-2, c-kit, cyclooxygenase-2, survivin, bcl-2, cyclin D1, cyclin B1, metalloproteinase (MMP)-2, and MMP-9. Univariate and multivariate analyses and unsupervised hierarchical clustering analysis of clinical pathologic and immunostaining data were performed. RESULTS:: Bcl-2 (p < 0.0001) and cyclin D1 (p = 0.015) were more highly expressed in squamous cell carcinoma (SCC), whereas MMP-2 (p = 0.009), MMP-9 (p = 0.005), and survivin (p = 0.032) had increased expression in other histologic subtypes. In univariate analysis, SCC histology and cyclin D1 expressions were favorable prognostic factors (p = 0.015 and p < 0.0001, respectively); by contrast, MMP-9 expression was associated with worse prognosis (p = 0.042). In multivariate analysis, cyclin D1 was the only positive prognostic factor (p < 0.0001). Unsupervised hierarchical clustering analysis of TMA immunostaining data identified five distinct clusters. They formed two subsets of patients with better (clusters 1 and 2) and worse (clusters 3, 4, and 5) prognoses, and median survival of 51 and 10 months, respectively (p < 0.0001). The better prognosis subset mainly comprised patients with SCC (80%). CONCLUSIONS:: Hierarchical clustering of TMA immunostaining data using a limited set of markers identifies patients with stage IIIA pN2 NSCLC at high risk of recurrence, who may benefit from more aggressive treatment.

Single-nucleotide polymorphisms (SNP) associated with polygenetic disorders, such as breast cancer (BC), can create, destroy, or modify microRNA (miRNA) binding sites; however, the extent to which SNPs interfere with miRNA gene regulation and affect cancer susceptibility remains largely unknown. We hypothesize that disruption of miRNA target binding by SNPs is a widespread mechanism relevant to cancer susceptibility. To test this, we analyzed SNPs known to be associated with BC risk, in silico and in vitro, for their ability to modify miRNA binding sites and miRNA gene regulation and referred to these as target SNPs. We identified rs1982073-TGFB1 and rs1799782-XRCC1 as target SNPs, whose alleles could modulate gene expression by differential interaction with miR-187 and miR-138, respectively. Genome-wide bioinformatics analysis predicted approximately 64% of transcribed SNPs as target SNPs that can modify (increase/decrease) the binding energy of putative miRNA::mRNA duplexes by >90%. To assess whether target SNPs are implicated in BC susceptibility, we conducted a case-control population study and observed that germline occurrence of rs799917-BRCA1 and rs334348-TGFR1 significantly varies among populations with different risks of developing BC. Luciferase activity of target SNPs, allelic variants, and protein levels in cancer cell lines with different genotypes showed differential regulation of target genes following overexpression of the two interacting miRNAs (miR-638 and miR-628-5p). Therefore, we propose that transcribed target SNPs alter miRNA gene regulation and, consequently, protein expression, contributing to the likelihood of cancer susceptibility, by a novel mechanism of subtle gene regulation. Cancer Res; 70(7); 2789-98.

MicroRNAs and heterogeneous ribonucleoproteins (hnRNPs) are posttranscriptional gene regulators that bind mRNA in a sequence-specific manner. Here, we report that loss of miR-328 occurs in blast crisis chronic myelogenous leukemia (CML-BC) in a BCR/ABL dose- and kinase-dependent manner through the MAPK-hnRNP E2 pathway. Restoration of miR-328 expression rescues differentiation and impairs survival of leukemic blasts by simultaneously interacting with the translational regulator poly(rC)-binding protein hnRNP E2 and with the mRNA encoding the survival factor PIM1, respectively. The interaction with hnRNP E2 is independent of the microRNA's seed sequence and it leads to release of CEBPA mRNA from hnRNP E2-mediated translational inhibition. Altogether, these data reveal the dual ability of a microRNA to control cell fate both through base pairing with mRNA targets and through a decoy activity that interferes with the function of regulatory proteins. PAPERFLICK:

Over the past few years, molecular oncology research has revealed that abnormalities in both protein-coding genes (PCGs) and noncoding RNAs (ncRNAs) can be identified in tumors and that the interplay between PCGs and ncRNAs is causally involved in the initiation, progression, and metastases of human cancers. MicroRNAs (miRNAs), which are among the most studied ncRNAs, are small 19- to 25-nucleotide genes involved in the regulation of PCGs and other ncRNAs. With the recent findings of miRNAs' involvement in cancer, RNA inhibition can be used to treat cancer patients in two ways: (1) by using RNA or DNA molecules as therapeutic drugs against messenger RNA of genes involved in the pathogenesis of cancers and (2) by directly targeting ncRNAs that participate in cancer pathogenesis. In this review, we focus on the possible use of miRNAs or compounds interacting with miRNAs as new therapeutic agents in cancer patients.

AIMS: The aim of this case report is to present the pathological, clinical, and ethic problems due to the diagnosis of an adnexal mass in a female patient with an undetected androgen insensitivity syndrome. METHODS: We employed both commonly used pathologic techniques, such as hematoxylin-eosin staining and immunohystochemistry, both fluorescence in situ hybridization on paraffin-embedded tissue sections using probes for chromosomes X and Y. MAIN RESULTS: At pathologic evaluation, a diagnosis of Sertoli cell tumor was made. Fluorescence in situ hybridization showed an XY karyotype of tumor cells. The final diagnosis was bilateral Sertoli cell adenoma in a patient with testicular feminization syndrome. CONCLUSION: Androgen insensitivity syndrome is caused by insensitivity of end organs to androgen caused by a non-functioning receptor. The diagnosis of testicular feminization has been made as late as the ninth decade. In this case, two kinds of problem derived from the pathologist point of view: first, the demonstration of chromosomal sex to confirm the diagnosis, and second, the transfer of diagnosis to the patient and to the clinicians.

Understanding the consequences of miR-145 reintroduction in human breast cancer (BC) could reveal its tumor-suppressive functions and may disclose new aspects of BC biology. Therefore, we characterized the effects of miR-145 re-expression in BC cell lines by using proliferation and apoptosis assays. As a result, we found that miR-145 exhibited a pro-apoptotic effect, which is dependent on TP53 activation, and that TP53 activation can, in turn, stimulate miR-145 expression, thus establishing a death-promoting loop between miR-145 and TP53. We also found that miR-145 can downregulate estrogen receptor-alpha (ER-alpha) protein expression through direct interaction with two complementary sites within its coding sequence. In conclusion, we described a tumor suppression function of miR-145 in BC cell lines, and we linked miR-145 to TP53 and ER-alpha. Moreover, our findings support a view that miR-145 re-expression therapy could be mainly envisioned in the specific group of patients with ER-alpha-positive and/or TP53 wild-type tumors.Cell Death and Differentiation advance online publication, 4 September 2009; doi:10.1038/cdd.2009.117.

MicroRNAs (miRNAs) are noncoding RNAs whose hallmarks are the very short sequences and the ability to repress the translation and/or transcription of target genes. miRNAs can have diverse functions, including regulation of cellular differentiation, proliferation, and embryogenesis. Over the past 5 years, an increasing number of studies have linked different miRNAs with programmed cell death or apoptosis. The principal aim of this chapter is to describe a method that (1) identifies miRNAs involved in apoptosis, using a validated array profiling approach, (2) assesses the direct involvement of candidate miRNAs in apoptosis, and (3) identifies the molecular mechanisms possibly involved in apoptotic response. To disclose the possible molecular targets of miRNAs, we propose the generation of a database created using a list of presumptive miRNA targets and the changes in the transcriptome after ectopic expression of the miRNAs. Our proposed method for doing this is suitable for both discovery of apoptotic pathways that regulate miRNAs and finding new miRNAs able to induce apoptosis.

Recently, microRNAs (miRNAs) have been discovered to have a role in metastasis. Here we describe how miRNAs are involved in advanced stages of tumour progression, stressing their roles as metastasis activators or suppressors, and discuss their possible use in the clinic as predictive markers and as therapeutic strategies for patients with metastases. Furthermore, we develop the concept that the same miRNAs could be involved both in the cancer stem cell phenotype and in the ability of specific cancer cells to produce metastases, thus representing a mechanistic link between the initial and the final steps of tumorigenesis.

After 12 years from its first application, microarray technology has become the reference technique to monitor gene expression of thousands of genes in the same experiment. In the past few years an increasing amount of evidence showed the importance of non-coding RNA (ncRNA) in different human diseases. The microRNAs (miRNAs) are one of the groups of ncRNA. They are small RNA fragments, 19-25 nucleotides long, with a main regulatory function on both protein coding genes and non-coding RNAs. The application of microarray platforms applied to miRNA profiling determined their deregulation in virtually all human diseases that have been studied. We previously developed a custom miRNA microarray platform, and here we describe the protocol we used to work with it including the oligo design strategy, the microarray printing protocol, the target-probe hybridization and the signal detection.

Noncoding RNA (ncRNA) transcripts are thought to be involved in human tumorigenesis. We report that a large fraction of genomic ultraconserved regions (UCRs) encode a particular set of ncRNAs whose expression is altered in human cancers. Genome-wide profiling revealed that UCRs have distinct signatures in human leukemias and carcinomas. UCRs are frequently located at fragile sites and genomic regions involved in cancers. We identified certain UCRs whose expression may be regulated by microRNAs abnormally expressed in human chronic lymphocytic leukemia, and we proved that the inhibition of an overexpressed UCR induces apoptosis in colon cancer cells. Our findings argue that ncRNAs and interaction between noncoding genes are involved in tumorigenesis to a greater extent than previously thought.

BACKGROUND: Colorectal cancer develops through two main genetic instability pathways characterized by distinct pathologic features and clinical outcome. RESULTS: We investigated colon cancer samples (23 characterized by microsatellite stability, MSS, and 16 by high microsatellite instability, MSI-H) for genome-wide expression of microRNA (miRNA) and mRNA. Based on combined miRNA and mRNA gene expression, a molecular signature consisting of twenty seven differentially expressed genes, inclusive of 8 miRNAs, could correctly distinguish MSI-H versus MSS colon cancer samples. Among the differentially expressed miRNAs, various members of the oncogenic miR-17-92 family were significantly up-regulated in MSS cancers. The majority of protein coding genes were also up-regulated in MSS cancers. Their functional classification revealed that they were most frequently associated with cell cycle, DNA replication, recombination, repair, gastrointestinal disease and immune response. CONCLUSION: This is the first report that indicates the existence of differences in miRNA expression between MSS versus MSI-H colorectal cancers. In addition, the work suggests that the combination of mRNA/miRNA expression signatures may represent a general approach for improving bio-molecular classification of human cancer.

The human polyomavirus BK (BKV) is oncogenic in rodents and induces malignant transformation of rodent cells in vitro. Although its role in human tumorigenesis is still debated, BKV represents an excellent model to evaluate molecularly targeted antineoplastic approaches. Here, we have tested whether stable suppression of the T antigen (T-ag) oncogene expression could inhibit the in vitro and in vivo malignant phenotype of BKV-transformed mouse cells. An adenovirus vector system that expresses small hairpin RNAs (shRNAs), which are converted into active small interfering RNAs (siRNA) molecules against the BKV T-ag, was developed. This vector was able to inhibit the expression of BKV T-ag through a highly efficient in vitro and in vivo delivery of the siRNA molecule. In addition, it allowed a stable expression of siRNA for a period of time sufficient to elicit a biological effect. Inhibition of T-ag expression results in reduction of the in vitro growth rate of BKV-transformed cells, which is, at least in part, caused by restoration of p53 activity and induction of apoptosis. In vivo studies proved that adenovirus vectors expressing anti-T-ag siRNA were able to suppress tumorigenicity of BKV-transformed cells. Moreover, adenovirus vector direct treatment of growing tumors resulted in a significant reduction of tumor growth. This study indicates that siRNAs delivery via a viral vector have a potential usefulness as in vivo anticancer tool against viral and cellular oncogenes.

This study was designed to evaluate the activity and tolerability of irinotecan and docetaxel in patients with previously treated non-small cell lung cancer (NSCLC). Eligibility included recurrent or progressive NSCLC, previous chemotherapy, age > or = 18 years, ECOG PS < or = 2. Treatment consisted of irinotecan (160 mg/m2 i.v.), followed by docetaxel (65 mg/m2 i.v.) on day 1 of a 21-day cycle, for a maximum of 6 cycles. Forty patients were enrolled. Median age was 60 years and median ECOG PS was 1. All patients were evaluable for toxicity and 31 (78%) were evaluable for response. A total of 125 cycles was administered (median, 3; range, 1-6). Most common grade 3-4 toxicities were neutropenia (62%), neutropenic fever (22%), and diarrhea (32%). Response rate was 10%; a further 40% of patients achieved stable disease. All responses were observed in patients with ECOG PS < or = 1, age <70 years, and who had received only one prior chemotherapy regimen. Median time to progression was 2.8 months and median survival was 7.4 months. Because of significant toxicity and limited activity, further investigation of irinotecan plus docetaxel in second line NSCLC is not recommended.

MicroRNAs (miRNAs) are a class of small noncoding RNAs that control gene expression by targeting mRNAs and triggering either translation repression or RNA degradation. Their aberrant expression may be involved in human diseases, including cancer. Indeed, miRNA aberrant expression has been previously found in human chronic lymphocytic leukemias, where miRNA signatures were associated with specific clinicobiological features. Here, we show that, compared with normal breast tissue, miRNAs are also aberrantly expressed in human breast cancer. The overall miRNA expression could clearly separate normal versus cancer tissues, with the most significantly deregulated miRNAs being mir-125b, mir-145, mir-21, and mir-155. Results were confirmed by microarray and Northern blot analyses. We could identify miRNAs whose expression was correlated with specific breast cancer biopathologic features, such as estrogen and progesterone receptor expression, tumor stage, vascular invasion, or proliferation index.

It is known that the plasminogen activator inhibitor 1 (PAI-1) protein levels are increased in placentas of preeclamptic subjects. Therefore, we assessed whether polymorphisms related to the transcriptional control of the PAI-1 gene (-675 4G/5G and -844G/A) are associated with mild preeclampsia. We compared 52 women with preeclampsia to 80 women with a normal pregnancy. None of the preeclamptic women suffered from the severe form of preeclampsia. DNA was extracted from blood, and -675 4G/5G and -844G/A genotypes of the PAI-1 gene were determined. Since it has been shown that the presence of factor V Leiden, prothrombin G20210A, and MTHFR C677T gene variants may be associated with preeclampsia, their frequency was also evaluated in our study groups. The factor V Leiden, PT G20210A, and MTHFR C677T gene variants were not associated with preeclampsia. In the case of the -675 4G/5G polymorphism, genotypes 4G/4G and 5G/5G were more prevalent in the preeclamptic and in the control group, respectively. In the case of -844 G/A polymorphism, genotypes A/A and G/G were more prevalent in the preeclamptic and in the control group, respectively. By using the chi(2) test for trend, differences for both genotypes were significant (p = 0.0141 for the -675 genotypes and p = 0.0492 for the -844 genotypes). The frequency of the 4G and 5G alleles of the -675 gene polymorphism was significantly different between preeclamptic and normal women (p = 0.032). Differently, the allelic frequency of the -844 gene polymorphism did not show significant differences between preeclamptic and normal women (p = 0.083). In conclusion, the hypofibrinolytic genotypes 4G/4G and A/A at positions -675 and -844 of the PAI-1 gene are associated with the occurrence of mild preeclampsia independently of thrombophilic mutations of the factor V, prothrombin, and MTHFR genes.

To obtain long-term engraftment and hematopoiesis in myeloablated patients, the cell population used for hematopoietic reconstitution should include a sufficient number of early pluripotent hematopoietic stem cells (HSCs), along with committed cells from the various lineages. For this purpose, the small subset of CD34+ cells purified from different sources must be expanded ex vivo. Since cytokines may induce both proliferation and differentiation, expansion would provide a cell population comprising committed as well as uncommitted cells. Optimization of HSC expansion methods could be obtained by a combination of cytokines able to sustain renewal of pluripotent cells yet endowed with poor differentiation potential. We used variations of the combinations of cytokines described by Brugger et al. [W. Brugger, S. Heimfels, R. J. Berenson, R. Mertelsmann, and L. Kanz (1995) N. Engl. J. Med. 333, 283-287] and Piacibello et al. [W. Piacibello, F. Sanavio, L. Garetto, A. Severino, D. Bergandi, J. Ferrario, F. Fagioli, M. Berger, and M. Aglietta (1997) Blood 89, 2644-2653] to expand UCB CD34+ cells and monitored proliferation rate and phenotype after 14 days of culture. Several hematopoietic lineage-associated surface antigens were evaluated. Our data show that flt3L and thrombopoietin in combination with IL-3, while sustaining a high CD34+ proliferation rate, provide a relatively low enrichment in very early uncommitted CD34+/CD38- cells. Conversely, in the absence of IL-3, they are less effective in inducing proliferation yet significantly increase the number of CD34+/CD38- cells. A combination of the above protocols, applied simultaneously to aliquots of the same sample, would allow expansion of both committed and pluripotent HSC. This strategy may represent a significant improvement for clinical applications.

A major problem with the use of umbilical cord/placental blood (UCB) is the limited blood volume that can be collected from a single donor. In this study, we evaluated a novel system for the collection of UCB and analyzed the kinetics of output of hematopoietic stem cells in the collected blood. Sequential UCB fractions were collected from 48 placentas by gravity following common procedures. When UCB flow was ended, collection was continued using the device. Nucleated cell (NC) density in each fraction was evaluated and the expression of CD34, CD38 and other hematopoietic markers was assessed by flow cytometry. The total collected volume was 60.9 +/- 26.2 ml (mean +/- SD, range 17-141.5). The device yield (volume collected using the device/total volume) was 26.5 +/- 15.1%. No significant difference was observed in NC count in sequential fractions. A significant increase in CD34(+) cell content in sequential fractions and a 2.07 +/- 1.18-fold increase in the percentage of CD34(+) cells in the last versus first fraction were observed. Furthermore, within the CD34(+) population, the percentage of CD38(-) pluripotent stem cells in the first fraction was 3.24 +/- 1.39, while in the last fraction it raised to 34.43 +/- 22.62. Thus, at the end of a collection performed following current procedures, further blood rich in the most primitive progenitor cells can be recovered. Therefore, the optimization and standardization of collection procedures are required to obtain maximal recovery from each placenta and increase the percentage of UCB units suitable for clinical use.