BackgroundMicroRNAs (miRs) represent a distinct class of posttranscriptional modulators of gene expression with remarkable stability in sera. Several miRs are oncogenic (oncomiRs) and are deregulated in the pathogenesis of breast cancer and function to inhibit tumor suppressors. Routine blood monitoring of these circulating tumor-derived products could be of significant benefit to the diagnosis and relapse detection of early-stage breast cancer (EBC) patients.MethodsAim of this project was to determine expression of miR-155, miR-19a, miR-181b, miR-24, relative to let-7a in sera of 63 patients with EBC and 21 healthy controls. Longitudinal multivariate data analysis was performed to stochastically model the serum levels of each of the oncomiRs during disease phases: from diagnosis, after surgery, and following chemo/radiotherapy. Moreover, this analysis was utilized to evaluate oncomiR levels in EBC patients subgrouped using current clinical prognostic factors including HER2, Ki-67, and grade III.ResultsEBC patients significantly over-express the oncomiRs at the time of diagnosis. Following surgical resection the serum levels of miR-155, miR-181b, and miR-24 significantly decreased (p = 1.89e-05, 5.41e-06, and 0.00638, respectively) whereas the miR-19a decreased significantly after the therapy (p = 0.00869). Furthermore, in case of high-risk patients serum levels of miR-155, miR-19a, miR-181b, and miR-24 are significantly more abundant in comparison to low-risk group (p = 0.026, 0.02567, 0.0250, and 0.00990) and show a decreasing trend upon therapy.ConclusionsOncomiRs are significantly more abundant in the sera of EBC patients compared to controls at diagnosis. Differences in oncomiR levels reflecting EBC risk were also observed. Testing the oncomiRs may be useful for diagnostic purpose and possibly also for relapse detection in follow-up studies of EBC.

IntroductionTranscriptional regulation of major hematopoietic oncogenes and tumor suppressor genes represents a critical step in tumor formation, tumor aggressiveness, and therapy resistance. [1][2][3] In addition, a posttranscriptional inhibitory mechanism involving microRNA (miRNA) binding to the 3Ј-untranslated region of target mRNAs causes transcript degradation or interferes with the translation initiation and has been linked to tumorigenesis. 4,5 Under physiologic conditions, miRNAs regulate developmental processes and cell fate decisions, and tight regulation of their levels represents an important factor in cell and tissue homeostasis. 6 MiR-155, a well-studied miRNA, regulates hematopoietic cell development as documented by murine gene targeting experiments and also by other studies describing its function during immune B-and T-cell response, in production of cytokines and antibodies and in antigen presentation. 7,8 Next, transgenic miR-155 overexpression in the mouse stimulates B-cell proliferation and frequent development of lymphomas. 9 In humans, miR-155 up-regulation has been repeatedly reported in chronic B-cell lymphocytic leukemia (B-CLL), in its solid indolent form of a small lymphocytic lymphoma [10][11][12] and also in aggressive types, including non-Hodgkin 10,13,14 and Hodgkin lymphomas. 13,15 Deregulation of several microRNAs was repeatedly described in B-CLL. 16,17 B-CLL, the most common adult leukemia, is characterized by clonal accumulation of B celllike mature-appearing elements (Ͼ 5000/L) 18 typically coexpressing the CD5, CD19, CD20, and CD23 surface markers. B-CLL represents a heterogeneous disease, the outcome of which may be predicted by the levels of surface protein CD38, intracellular tyrosine kinase ZAP70, or by a status of IgV H somatic hypermutation. 18,19 Cytogenetic alterations of 2 loci that contain the p53 gene (deletion of 17p) and the ATM gene (deletion of 11q) are associated with poor prognosis, shorter duration of remission, and shortest overall survival, 20 whereas normal karyotype or trisomy 12 is considered intermediate risk and the 13q14 deletion is considered a favorable mark. Subsets of B-CLL patients may progress to non-Hodgkin diffuse large B-cell lymphoma by a mechanism that remains largely unknown. Taken together, miR-155 appears to play a central role in B-cell function, and its up-regulation in lymphoproliferative disorders, including B-CLL, may lead to a block of differentiation and accumulation of lymphoid-like cells.Recent studies brought evidence of a context-dependent transcriptional regulation of the MIR155HG. First, oncogenic properties of miR-155 have been demonstrated in breast cancer cells where MIR155HG is up-regulated by transforming growth factor-␤/ Smad pathway involving a Smad response element at the position Ϫ454 nt from the transcription start site (TSS). 21 This regulatory pathway becomes disabled on inhibition of miR-155, resulting in derepression of miR-155 targets (including the RhoA protein) and in decreased cell migration and invasio...

Oncogenic microRNAs (oncomiRs) accumulate in serum due to their increased stability and thus serve as biomarkers in breast cancer (BC) pathogenesis. Four oncogenic microRNAs (miR-155, miR-19a, miR-181b, and miR-24) and one tumor suppressor microRNA (let-7a) were shown to differentiate between high-and low-risk early breast cancer (EBC) and reflect the surgical tumor removal and adjuvant therapy. Here we applied the longitudinal multivariate data analyses to stochastically model the serum levels of each of the oncomiRs using the RT-PCR measurements in the EBC patients (N = 133) that were followed up 4 years after diagnosis. This study identifies that two of the studied oncomiRs, miR-155 and miR-24, are highly predictive of EBC relapse. Furthermore, combining the oncomiR level with Ki-67 expression further specifies the relapse probability. Our data move further the notion that oncomiRs in serum enable not only monitoring of EBC but also are a very useful tool for predicting relapse independently of any other currently analyzed characteristics in EBC patients. Our approach can be translated into medical practice to estimate individual relapse risk of EBC patients.

PU.1 downregulation within hematopoietic stem and progenitor cells (HSPCs) is the primary mechanism for the development of acute myeloid leukemia (AML) in mice with homozygous deletion of the upstream regulatory element (URE) of PU.1 gene. p53 is a well-known tumor suppressor that is often mutated in human hematologic malignancies including AML and adds to their aggressiveness; however, its genetic deletion does not cause AML in mouse. Deletion of p53 in the PU.1(ure/ure) mice (PU.1(ure/ure)p53(-/-)) results in more aggressive AML with shortened overall survival. PU.1(ure/ure)p53(-/-) progenitors express significantly lower PU.1 levels. In addition to URE deletion we searched for other mechanisms that in the absence of p53 contribute to decreased PU.1 levels in PU.1(ure/ure)p53(-/-) mice. We found involvement of Myb and miR-155 in downregulation of PU.1 in aggressive murine AML. Upon inhibition of either Myb or miR-155 in vitro the AML progenitors restore PU.1 levels and lose leukemic cell growth similarly to PU.1 rescue. The MYB/miR-155/PU.1 axis is a target of p53 and is activated early after p53 loss as indicated by transient p53 knockdown. Furthermore, deregulation of both MYB and miR-155 coupled with PU.1 downregulation was observed in human AML, suggesting that MYB/miR-155/PU.1 mechanism may be involved in the pathogenesis of AML and its aggressiveness characterized by p53 mutation.

Hematopoietic transcription factors GATA-1 and PU.1 bind each other on DNA to block transcriptional programs of undesired lineage during hematopoietic commitment. Murine erythroleukemia (MEL) cells that coexpress GATA-1 and PU.1 are blocked at the blast stage but respond to molecular removal (downregulation) of PU.1 or addition (upregulation) of GATA-1 by inducing terminal erythroid differentiation. To test whether GATA-1 blocks PU.1 in MEL cells, we have conditionally activated a transgenic PU.1 protein fused with the estrogen receptor ligand-binding domain (PUER), resulting in activation of a myeloid transcriptional program. Gene expression arrays identified components of the PU.1-dependent transcriptome negatively regulated by GATA-1 in MEL cells, including CCAAT/enhancer binding protein α (Cebpa) and core-binding factor, β subunit (Cbfb), which encode two key hematopoietic transcription factors. Inhibition of GATA-1 by small interfering RNA resulted in derepression of PU.1 target genes. Chromatin immunoprecipitation and reporter assays identified PU.1 motif sequences near Cebpa and Cbfb that are co-occupied by PU.1 and GATA-1 in the leukemic blasts. Significant derepression of Cebpa and Cbfb is achieved in MEL cells by either activation of PU.1 or knockdown of GATA-1. Furthermore, transcriptional regulation of these loci by manipulating the levels of PU.1 and GATA-1 involves quantitative increases in a transcriptionally active chromatin mark: acetylation of histone H3K9. Collectively, we show that either activation of PU.1 or inhibition of GATA-1 efficiently reverses the transcriptional block imposed by GATA-1 and leads to the activation of a myeloid transcriptional program directed by PU.1.