The prognostic significance of RUNX2 and miR-10a/10b and their inter-relationship in breast cancer.

BACKGROUND: The major cancer related mortality is caused by metastasis and invasion. It is important to identify genes regulating metastasis and invasion in order to curtail metastatic spread of cancer cells. METHODS: This study investigated the association between RUNX2 and miR-10a/miR-10b and the risk of breast cancer relapse. Expression levels of RUNX2 and miR-10a/b in 108 pairs of tumor and non-tumor tissue of breast cancer were assayed by quantitative PCR analysis and evaluated for their prognostic implications. RESULTS: The median expression levels of RUNX2 and miR-10b in tumor tissue normalized using adjacent non-tumor tissue were significantly higher in relapsed patients than in relapse-free patients. Higher expression of these three genes were significantly correlated with the hazard ratio for breast cancer recurrence (RUNX2: 3.02, 95% CI = 1.50 ~ 6.07; miR-10a: 2.31, 95% CI = 1.00 ~ 5.32; miR-10b: 3.96, 95% CI = 1.21 ~ 12.98). The joint effect of higher expression of all three genes was associated with a hazard ratio of 12.37 (95% CI = 1.62 ~ 94.55) for relapse. In a breast cancer cell line, RUNX2 silencing reduced the expression of miR-10a/b and also impaired cell motility, while RUNX2 overexpression elicited opposite effects. CONCLUSIONS: These findings indicate that higher expression of RUNX2 and miR-10a/b was associated with adverse outcome of breast cancer. Expression levels of RUNX2 and miR-10a/b individually or jointly are potential prognostic factors for predicting breast cancer recurrence. Data from in vitro studies support the notion that RUNX2 promoted cell motility by upregulating miR-10a/b.

miR-149* induces apoptosis by inhibiting Akt1 and E2F1 in human cancer cells.

microRNAs (miRNAs) play vital roles in several biological processes, including apoptosis, by negatively regulating the expression of target genes. The molecular mechanisms of the key survival signal, Akt family, have been widely explored. However, it remains to be ascertained whether Akt1, the predominant isoform in most tissue, is a direct target of miRNA. In this study, we identified Akt1 and E2F1 to be two direct targets of miR-149* and b-Myb to be an indirect target by reporter assays and Western blot analyses. Ectopic expression of miR-149*-induced apoptosis in Be2C, a neuroblastoma cell line, and in HeLa cells. Silencing of Akt1 or E2F1 expression also led to similar apoptotic changes in these two cell lines, suggesting that the pro-apoptotic effects of miR-149* were exerted by repressing Akt1 and E2F1 expressions. Importantly, analysis of primary neuroblastoma samples revealed a significant inverse correlation of miR-149* with E2F1 expressions (P=0.026). Interestingly, using the reporter assays, excess miR-149 introduced by transfection to simulated its preponderance in the in vivo condition, could not overcome the repressive function of miR-149* on the target genes. This implies that the pro-apoptotic function of miR-149* may not be dampened by its predominant cognate, miR-149, in vivo. Our findings not only provided the first evidence that Akt1 is a direct target of miRNA but also demonstrated that miR-149* is a pro-apoptotic miRNA by repressing the expression of Akt1 and E2F1.

c-Myb is an evolutionary conserved miR-150 target and miR-150/c-Myb interaction is important for embryonic development.

Human c-Myb proto-oncogene is highly expressed in hematopoietic progenitors as well as leukemia and certain solid tumor. However, the regulatory mechanisms of its expression and biological functions remain largely unclear. Recently, c-Myb has been shown to be targeted by microRNA-150 (miR-150) which thereby controls B cell differentiation in mice. In this study, we demonstrated that c-Myb is an evolutionary conserved target of miR-150 in human and zebrafish, using reporter assays. Ectopic expression of miR-150 in breast cancer and leukemic cells repressed endogenous c-Myb at both messenger RNA (mRNA) and protein levels. Among several leukemia cell lines, primary leukemia cells, and normal lymphocytes, expression levels of miR-150 inversely correlated with c-Myb. The miR-150 overexpression or c-Myb silencing in zebrafish zygotes led to similar and serious phenotypic defects in zebrafish, and the phenotypic aberrations induced by miR-150 could be reversed by coinjection of c-Myb mRNA. Our findings suggest that c-Myb is an evolutionally conserved target of miR-150 and miR-150/c-Myb interaction is important for embryonic development and possibly oncogenesis.

A conserved telomerase motif within the catalytic domain of telomerase reverse transcriptase is specifically required for repeat addition processivity.

Telomerase is a ribonucleoprotein reverse transcriptase responsible for the maintenance of one strand of the telomere terminal repeats. The catalytic protein subunit of the telomerase complex, known as TERT, possesses a reverse transcriptase (RT) domain that mediates nucleotide addition. The RT domain of TERT is distinguishable from retroviral and retrotransposon RTs in having a sizable insertion between conserved motifs A and B', within the so-called fingers domain. Sequence analysis revealed the existence of conserved residues in this region, named IFD (insertion in fingers domain). Mutations of some of the conserved residues in Saccharomyces cerevisiae TERT (Est2p) abolished telomerase function in vivo, testifying to their importance. Significant effects of the mutations on telomerase activity in vitro were observed, with most of the mutants exhibiting a uniform reduction in activity regardless of primer sequence. Remarkably, one mutant manifested a primer-specific defect, being selectively impaired in extending primers that form short hybrids with telomerase RNA. This mutant also accumulated products that correspond to one complete round of repeat synthesis, implying an inability to effect the repositioning of the DNA product relative to the RNA template that is necessary for multiple repeat addition. Our results suggest that the ability to stabilize short RNA-DNA hybrids is crucial for telomerase function in vivo and that this ability is mediated in part by a more elaborate fingers domain structure.

Concordant and discordant regulation of target genes by miR-31 and its isoforms.

It has been shown that imprecise cleavage of a primary or precursor RNA by Drosha or Dicer, respectively, may yield a group of microRNA (miRNA) variants designated as "isomiR". Variations in the relative abundance of isoforms for a given miRNA among different species and different cell types beg the question whether these isomiRs might regulate target genes differentially. We compared the capacity of three miR-31 isoforms (miR-31-H, miR-31-P, and miR-31-M), which differ only slightly in their 5'- and/or 3'-end sequences, to regulate several known targets and a predicted target, Dicer. Notably, we found isomiR-31s displayed concordant and discordant regulation of 6 known target genes. Furthermore, we validated a predicted target gene, Dicer, to be a novel target of miR-31 but only miR-31-P could directly repress Dicer expression in both MCF-7 breast cancer cells and A549 lung cancer cells, resulting in their enhanced sensitivity to cisplatin, a known attribute of Dicer knockdown. This was further supported by reporter assay using full length 3'-untranslated region (UTR) of Dicer. Our findings not only revealed Dicer to be a direct target of miR-31, but also demonstrated that isomiRs displayed similar and disparate regulation of target genes in cell-based systems. Coupled with the variations in the distribution of isomiRs among different cells or conditions, our findings support the possibility of fine-tuning gene expression by miRNAs.

microRNA signature and expression of Dicer and Drosha can predict prognosis and delineate risk groups in neuroblastoma.

Neuroblastoma is a common childhood tumor and accounts for 15% of pediatric cancer deaths. To investigate the microRNA (miRNA) profile and role of Dicer and Drosha in neuroblastoma, we assessed the expression of 162 human miRNAs, Dicer and Drosha in 66 neuroblastoma tumors by using real-time PCR methods. We found global downregulation of miRNA expression in advanced neuroblastoma and identified 27 miRNAs that can clearly distinguish low- from high-risk patients. Furthermore, expression levels of Dicer or Drosha were low in high-risk neuroblastoma tumors, which accounted for global downregulation of miRNAs in advanced disease and correlated with poor outcome. Notably, for patients with non-MYCN-amplified tumors, low expression of Dicer can serve as a significant and independent predictor of poor outcome (hazard ratio, 9.6; P = 0.045; n = 52). Using plausible neural networks to select a combination of 15 biomarkers that consist of 12 miRNAs' signature, expression levels of Dicer and Drosha, and age at diagnosis, we were able to segregate all patients into four distinct patterns that were highly predictive of clinical outcome. In vitro studies also showed that knockdown of either Dicer or Drosha promoted the growth of neuroblastoma cell lines. Our results reveal that a combination of 15 biomarkers can delineate risk groups of neuroblastoma and serve as a powerful predictor of clinical outcome. Moreover, our findings of growth promotion by silencing Dicer/Drosha implied their potential use as therapeutic targets for neuroblastoma.

The synthetic caged garcinia xanthone cluvenone induces cell stress and apoptosis and has immune modulatory activity.

Several caged Garcinia xanthone natural products have potent bioactivity and a documented value in traditional Eastern medicine. Previous synthesis and structure activity relationship studies of these natural products resulted in the identification of the pharmacophore represented by the structure of cluvenone. In the current study, we examined the anticancer activity of cluvenone and conducted gene expression profiling and pathway analyses. Cluvenone was found to induce apoptosis in T-cell acute lymphoblastic leukemia cells (EC50 = 0.25 µmol/L) and had potent growth-inhibitory activity against the NCI60 cell panel, including those that are multidrug-resistant, with a GI50 range of 0.1 to 2.7 µmol/L. Importantly, cluvenone was approximately 5-fold more potent against a primary B-cell acute lymphoblastic leukemia compared with peripheral blood mononuclear cells from normal donors, suggesting that it has significant tumor selectivity. Comparison of cluvenone's growth-inhibitory profile to those in the National Cancer Institute database revealed that compounds with a similar profile to cluvenone were mechanistically unlike known agents, but were associated with cell stress and survival signaling. Gene expression profiling studies determined that cluvenone induced the activation of mitogen-activated protein kinase and NrF2 stress response pathways. Furthermore, cluvenone was found to induce intracellular reactive oxygen species formation. Lastly, the modulation in the expression of several genes associated with T cell and natural killer cell activation and function by cluvenone suggests a role as an immune-modulator. The current work highlights the potential of cluvenone as a chemotherapeutic agent and provides support for further investigation of these intriguing molecules with regard to mechanism and targets.

Nonparametric estimation of LOH using Affymetrix SNP genotyping arrays for unpaired samples.

Studies of loss of heterozygosity (LOH) play an important role in cancer research. In this paper, we developed a two-step procedure to examine LOH by comparing unpaired tumour and normal samples. In the first step we determined which chromosomes significantly differ between the two sets of samples by using nonparametric procedures. We then used the biplot data visualisation technique and homozygosity intensity estimates to determine the regions of these chromosomes that required further examination. We illustrated our method by examining 22 autosomes in samples of 95 normal controls and 14 acute lymphoblastic leukaemia patients. The genomewide scan of LOH with the Affymetrix Human Mapping 100K Set successfully identified the important tumour suppressor gene, CDKN2A, whose deletion was validated by quantitative polymerase chain reaction in multiple patients of this study.

Human TRIM71 and its nematode homologue are targets of let-7 microRNA and its zebrafish orthologue is essential for development.

Animal microRNAs (miRNAs) are short RNAs that function as posttranscriptional regulators of gene expression by binding to the target mRNAs. Noting that some miRNAs are highly conserved in evolution, we explored the possibility of evolutionary conservation of their targets. We identified human orthologues of experimentally verified let-7 miRNA target genes in Caenorhabditis elegans and used the luciferase reporter system to examine whether these human genes are still the targets of let-7 miRNA. We found that in some cases, the miRNA-target relationship has indeed been conserved in human. Interestingly, human TRIM71, an orthologue of C. elegans let-7-target lin-41 gene, can be repressed by hsa-let-7a and hsa-let-7c. This repression was abolished when both predicted let-7 target sites of TRIM71 were mutated. Moreover, the zebrafish lin-41 orthologue was also repressed by let-7 to a similar degree as was TRIM71. When the expression of zebrafish lin-41 orthologue was silenced by microinjection of RNA interference or morpholino into zebrafish zygotes, retarded embryonic development was observed, providing direct evidence for an essential role of lin-41 in zebrafish development. Taken together, our results suggest that the regulation of TRIM71 expression by let-7 has been evolutionarily conserved and that TRIM71 likely plays an important role in development.

Identification of pulmonary Oct-4+ stem/progenitor cells and demonstration of their susceptibility to SARS coronavirus (SARS-CoV) infection in vitro.

In this study, we report a serum-free culture system for primary neonatal pulmonary cells that can support the growth of octamer-binding transcription factor 4+ (Oct-4+) epithelial colonies with a surrounding mesenchymal stroma. In addition to Oct-4, these cells also express other stem cell markers such as stage-specific embryonic antigen 1 (SSEA-1), stem cell antigen 1 (Sca-1), and Clara cell secretion protein (CCSP) but not c-Kit, CD34, and p63, indicating that they represent a subpopulation of Clara cells that have been implicated as lung stem/progenitor cells in lung injury models. These colony cells can be kept for weeks in primary cultures and undergo terminal differentiation to alveolar type-2- and type-1-like pneumocytes sequentially when removed from the stroma. In addition, we have demonstrated the presence of Oct-4+ long-term BrdU label-retaining cells at the bronchoalveolar junction of neonatal lung, providing a link between the Oct-4+ cells in vivo and in vitro and strengthening their identity as putative neonatal lung stem/progenitor cells. Lastly, these Oct-4+ epithelial colony cells, which also express angiotensin-converting enzyme 2, are the target cells for severe acute respiratory syndrome coronavirus infection in primary cultures and support active virus replication leading to their own destruction. These observations imply the possible involvement of lung stem/progenitor cells, in addition to pneumocytes, in severe acute respiratory syndrome coronavirus infection, accounting for the continued deterioration of lung tissues and apparent loss of capacity for lung repair.