A miR-SNP biomarker linked to an increased lung cancer survival by miRNA-mediated down-regulation of FZD4 expression and Wnt signaling.

Through a new hypothesis-driven and microRNA-pathway-based SNP (miR-SNP) association study we identified a novel miR-SNP (rs713065) in the 3'UTR region of FZD4 gene linked with decreased risk of death in early stage NSCLC patients. We determined biological function and mechanism of action of this FZD4-miR-SNP biomarker in a cellular platform. Our data suggest that FZD4-miR-SNP loci may significantly influence overall survival in NSCLC patients by specifically interacting with miR-204 and modulating FZD4 expression and cellular function in the Wnt-signaling-driven tumor progression. Our findings are bridging the gap between the discovery of epidemiological SNP biomarkers and their biological function and will enable us to develop novel therapeutic strategies that specifically target epigenetic markers in the oncogenic Wnt/FZD signaling pathways in NSCLC.

Assessing the miRNA sponge potential of RUNX1T1 in t(8;21) acute myeloid leukemia.

t(8;21) acute myeloid leukemia (AML) is characterized by a translocation between chromosomes 8 and 21 and formation of a distinctive RUNX1-RUNX1T1 fusion transcript. This translocation places RUNX1T1 under control of the RUNX1 promoter leading to a pronounced upregulation of RUNX1T1 transcripts in t(8;21) AML, compared to normal hematopoietic cells. We investigated the role of highly-upregulated RUNX1T1 under the hypothesis that it acts as competing endogenous RNA (ceRNA) titrating microRNAs (miRNAs) away from their target transcripts and thus contributes to AML formation. Using publicly available t(8;21) AML RNA-Seq and miRNA-Seq data available from The Cancer Genome Atlas (TCGA) project, we obtained a network consisting of 605 genes that may act as ceRNAs competing for miRNAs with the suggested RUNX1T1 miRNA sponge. Among the 605 ceRNA candidates, 121 have previously been implied in cancer development. Players in the integrin, cadherin, and Wnt signaling pathways affected by the RUNX1T1 sponge were overrepresented. Finally, among a set of 21 high interest RUNX1T1 ceRNAs we found multiple genes that have previously been linked to AML formation. In conclusion, our study offers a novel look at the role of the RUNX1-RUNX1T1 fusion transcript in t(8;21) AML beyond previously investigated genetic and epigenetic aberrations.

MicroRNA-mediated target mRNA cleavage and 3'-uridylation in human cells.

MicroRNAs (miRNAs) play an important role in targeted gene silencing by facilitating posttranscriptional and translational repression. However, the precise mechanism of mammalian miRNA-mediated gene silencing remains to be elucidated. Here, we used a stem-loop array reverse-transcription polymerase chain reaction assay to analyse miRNA-induced mRNA recognition, cleavage, posttranscriptional modification, and degradation. We detected endogenous let-7 miRNA-induced and Argonaute-catalysed endonucleolytic cleavage on target mRNAs at various sites within partially paired miRNA:mRNA sequences. Most of the cleaved mRNA 5'-fragments were 3'-oligouridylated by activities of terminal uridylyl transferases (TUTases) in miRNA-induced silencing complexes and temporarily accumulated in the cytosol for 5'-3' degradation or other molecular fates. Some 3'-5' decayed mRNA fragments could also be captured by the miRNA-induced silencing complex stationed at the specific miRNA:mRNA target site and oligouridylated by other TUTases at its proximity without involving Argonaute-mediated RNA cleavage. Our findings provide new insights into the molecular mechanics of mammalian miRNA-mediated gene silencing by coordinated target mRNA recognition, cleavage, uridylation and degradation.

miR-18b overexpression identifies mantle cell lymphoma patients with poor outcome and improves the MIPI-B prognosticator.

Recent studies show that mantle cell lymphoma (MCL) express aberrant microRNA (miRNA) profiles; however, the clinical effect of miRNA expression has not previously been examined and validated in large prospective homogenously treated cohorts. We performed genome-wide miRNA microarray profiling of 74 diagnostic MCL samples from the Nordic MCL2 trial (screening cohort). Prognostic miRNAs were validated in diagnostic MCL samples from 94 patients of the independent Nordic MCL3 trial (validation cohort). Three miRNAs (miR-18b, miR-92a, and miR-378d) were significantly differentially expressed in patients who died of MCL in both cohorts. MiR-18b was superior to miR-92a and miR-378d in predicting high risk. Thus, we generated a new biological MCL International Prognostic Index (MIPI-B)-miR prognosticator, combining expression levels of miR-18b with MIPI-B data. Compared to the MIPI-B, this prognosticator improved identification of high-risk patients with regard to cause-specific, overall, and progression-free survival. Transfection of 2 MCL cell lines with miR-18b decreased their proliferation rate without inducing apoptosis, suggesting that miR-18b may render MCL cells resistant to chemotherapy by decelerating cell proliferation. We conclude that overexpression of miR-18b identifies patients with poor prognosis in 2 large prospective MCL cohorts and adds prognostic information to the MIPI-B. MiR-18b may reduce the proliferation rate of MCL cells as a mechanism of chemoresistance.

The effect of adenovirus-mediated gene expression of FHIT in small cell lung cancer cells.

The candidate tumor suppressor fragile histidine traid (FHIT) is frequently inactivated in small cell lung cancer (SCLC). Mutations in the p53 gene also occur in the majority of SCLC leading to the accumulation of the mutant protein. Here we evaluated the effect of FHIT gene therapy alone or in combination with the mutant p53-reactivating molecule, PRIMA-1(Met)/APR-246, in SCLC. Overexpression of FHIT by recombinant adenoviral vector (Ad-FHIT)-mediated gene transfer in SCLC cells inhibited their growth by inducing apoptosis and when combined with PRIMA-1(Met)/APR-246, a synergistic cell growth inhibition was achieved.

PRIMA-1Met/APR-246 induces apoptosis and tumor growth delay in small cell lung cancer expressing mutant p53.

PURPOSE: Small cell lung cancer (SCLC) is a highly malignant disease with poor prognosis, necessitating the need to develop new and efficient treatment modalities. PRIMA-1(Met) (p53-dependent reactivation of massive apoptosis), also known as APR-246, is a small molecule, which restores tumor suppressor function to mutant p53 and induces cancer cell death in various cancer types. Since p53 is mutated in more than 90% of SCLC, we investigated the ability of PRIMA-1(Met) to induce apoptosis and inhibit tumor growth in SCLC with different p53 mutations. EXPERIMENTAL DESIGN: The therapeutic effect of PRIMA-1(Met)/APR-246 was studied in SCLC cells in vitro using cell viability assay, fluorescence-activated cell-sorting analysis, p53 knockdown studies, and Western blot analyses. The antitumor potential of PRIMA-1(Met)/APR-246 was further evaluated in two different SCLC xenograft models. RESULTS: PRIMA-1(Met)/APR-246 efficiently inhibited the growth of the SCLC cell lines expressing mutant p53 in vitro and induced apoptosis, associated with increased fraction of cells with fragmented DNA, caspase-3 activation, PARP cleavage, Bax and Noxa upregulation and Bcl-2 downregulation in the cells. The growth suppressive effect of PRIMA-1(Met)/APR-246 was markedly reduced in SCLC cell lines transfected with p53 siRNA, supporting the role of mutant p53 in PRIMA-1(Met)/APR-246-induced cell death. Moreover, in vivo studies showed significant antitumor effects of PRIMA-1(Met) after i.v. injection in SCLC mouse models with no apparent toxicity. CONCLUSION: This study is the first to show the potential use of p53-reactivating molecules such as PRIMA-1(Met)/APR-246 for the treatment of SCLC.

Specifically targeted gene therapy for small-cell lung cancer.

Small-cell lung cancer (SCLC) is a highly malignant disease with poor prognosis. Hence, there is great demand for new therapies that can replace or supplement the current available treatment regimes. Gene therapy constitutes a promising strategy and relies on the principle of introducing exogenous DNA into malignant cells causing them to die. Since SCLC is a highly disseminated malignancy, the gene therapeutic agent must be administered systemically, obligating a high level of targeting of tumor tissue and the use of delivery vehicles designed for systemic circulation of the therapeutic DNA. This review describes and discusses the current status of the application of gene therapy in relation to SCLC.

Mechanisms for oncogenic activation of the epidermal growth factor receptor.

The Epidermal growth factor receptor (EGFR) is a membrane spanning glycoprotein, which frequently has been implicated in various cancer types. The mechanisms by which EGFR becomes oncogenic are numerous and are often specific for each cancer type. In some tumors, EGFR is activated by autocrine/paracrine growth factor loops, whereas in others activating mutations promote EGFR signaling. Overexpression and/or amplification of the EGFR gene are prevalent in many cancer types leading to aberrant EGFR signaling. In addition, failure to attenuate receptor signaling by receptor downregulation can also lead to cellular transformation. Heterodimerization of EGFR with ErbB2 inhibits downregulation of EGFR and thereby prolongs growth factor signaling. This also indicates that cross-talk between EGFR and heterologous receptor systems serves as another mechanism for oncogenic activation of EGFR. Because of its role in tumor promotion, the EGFR has been intensely studied as a therapeutic target. There are currently two major mechanisms by which the EGFR is targeted: antibodies binding to the extracellular domain of EGFR and small-molecule tyrosine-kinase inhibitors. However, tumorigenesis is a multi-step process involving several mutations, which might explain why EGFR therapeutics has only been partially successful. This highlights the importance of pinpointing the mechanisms by which EGFR becomes oncogenic in a particular cancer. In this review, each of the above mentioned mechanisms will be discussed, as a detailed molecular and genetic understanding of how EGFR contributes to the malignant phenotype might offer new promise for the design, development and clinical evaluation of future tumor-specific anticancer approaches.

EGFRvIII escapes down-regulation due to impaired internalization and sorting to lysosomes.

EGFRvIII is a mutant variant of the epidermal growth factor receptor (EGFR) found exclusively in various cancer types. EGFRvIII lacks a large part of the extracellular domain and is unable to bind ligands; however, the receptor is constitutively phosphorylated and able to activate downstream signaling pathways. Failure to attenuate signaling by receptor down-regulation could be one of the major mechanisms by which EGFRvIII becomes oncogenic. Using a cell system expressing either EGFR or EGFRvIII with no expression of other EGFR family members and with endogenous levels of key degradation proteins, we have investigated the down-regulation of EGFRvIII and compared it to that of EGFR. We show that, in contrast to EGFR, EGFRvIII is inefficiently degraded. EGFRvIII is internalized, but the internalization rate of the mutated receptor is significantly less than that of unstimulated EGFR. Moreover, internalized EGFRvIII is recycled rather than delivered to lysosomes. EGFRvIII binds the ubiquitin ligase c-Cbl via Grb2, whereas binding via phosphorylated tyrosine residue 1045 seems to be limited. Despite c-Cbl binding, the receptor fails to become effectively ubiquitinylated. Thus, our results suggest that the long lifetime of EGFRvIII is caused by inefficient internalization and impaired sorting to lysosomes due to lack of effective ubiquitinylation.