Dysregulated miR-671-5p / CDR1-AS / CDR1 / VSNL1 axis is involved in glioblastoma multiforme.

MiR-671-5p is encoded by a gene localized at 7q36.1, a region amplified in human glioblastoma multiforme (GBM), the most malignant brain cancer. To investigate whether expression of miR-671-5p were altered in GBM, we analyzed biopsies from a cohort of forty-five GBM patients and from five GBM cell lines. Our data show significant overexpression of miR-671-5p in both biopsies and cell lines. By exploiting specific miRNA mimics and inhibitors, we demonstrated that miR-671-5p overexpression significantly increases migration and to a less extent proliferation rates of GBM cells. Through a combined in silico and in vitro approach, we identified CDR1-AS, CDR1, VSNL1 as downstream miR-671-5p targets in GBM. Expression of these genes significantly decreased both in GBM biopsies and cell lines and negatively correlated with that of miR-671-5p. Based on our data, we propose that the axis miR-671-5p / CDR1-AS / CDR1 / VSNL1 is functionally altered in GBM cells and is involved in the modification of their biopathological profile.

Specific alterations of the microRNA transcriptome and global network structure in colorectal cancer after treatment with MAPK/ERK inhibitors.

The mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway has a master control role in various cancer-related biological processes as cell growth, proliferation, differentiation, migration, and apoptosis. It also regulates many transcription factors that control microRNAs (miRNAs) and their biosynthetic machinery. To investigate on the still poorly characterised global involvement of miRNAs within the pathway, we profiled the expression of 745 miRNAs in three colorectal cancer (CRC) cell lines after blocking the pathway with three different inhibitors. This allowed the identification of two classes of post-treatment differentially expressed (DE) miRNAs: (1) common DE miRNAs in all CRC lines after treatment with a specific inhibitor (class A); (2) DE miRNAs in a single CRC line after treatment with all three inhibitors (class B). By determining the molecular targets, biological roles, network position of chosen miRNAs from class A (miR-372, miR-663b, miR-1226*) and class B (miR-92a-1*, miR-135b*, miR-720), we experimentally demonstrated that they are involved in cell proliferation, migration, apoptosis, and globally affect the regulation circuits centred on MAPK/ERK signaling. Interestingly, the levels of miR-92a-1*, miR-135b*, miR-372, miR-720 are significantly higher in biopsies from CRC patients than in normal controls; they also are significantly higher in CRC patients with mutated KRAS than in those with wild-type genotypes (Wilcoxon test, p < 0.05): the latter could be a downstream effect of ERK pathway overactivation, triggered by KRAS mutations. Finally, our functional data strongly suggest the following miRNA/target pairs: miR-92a-1*/PTEN-SOCS5; miR-135b*/LATS2; miR-372/TXNIP; miR-663b/CCND2. Altogether, these results contribute to deepen current knowledge on still uncharacterized features of MAPK/ERK pathway, pinpointing new oncomiRs in CRC and allowing their translation into clinical practice and CRC therapy.

Specific alterations of microRNA transcriptome and global network structure in colorectal carcinoma after cetuximab treatment.

The relationship between therapeutic response and modifications of microRNA (miRNA) transcriptome in colorectal cancer (CRC) remains unknown. We investigated this issue by profiling the expression of 667 miRNAs in 2 human CRC cell lines, one sensitive and the other resistant to cetuximab (Caco-2 and HCT-116, respectively), through TaqMan real-time PCR. Caco-2 and HCT-116 expressed different sets of miRNAs after treatment. Specifically, 21 and 22 miRNAs were differentially expressed in Caco-2 or HCT-116, respectively (t test, P < 0.01). By testing the expression of differentially expressed miRNAs in CRC patients, we found that miR-146b-3p and miR-486-5p are more abundant in K-ras-mutated samples with respect to wild-type ones (Wilcoxon test, P < 0.05). Sixty-seven percent of differentially expressed miRNAs were involved in cancer, including CRC, whereas 19 miRNA targets had been previously reported to be involved in the cetuximab pathway and CRC. We identified 25 transcription factors putatively controlling these miRNAs, 11 of which have been already reported to be involved in CRC. On the basis of these data, we suggest that the downregulation of let-7b and let-7e (targeting K-ras) and the upregulation of miR-17* (a CRC marker) could be considered as candidate molecular markers of cetuximab resistance. Global network functional analysis (based on miRNA targets) showed a significant overrepresentation of cancer-related biological processes and networks centered on critical nodes involved in epidermal growth factor receptor internalization and ubiquitin-mediated degradation. The identification of miRNAs, whose expression is linked to the efficacy of therapy, should allow the ability to predict the response of patients to treatment and possibly lead to a better understanding of the molecular mechanisms of drug response.

Expression profile and specific network features of the apoptotic machinery explain relapse of acute myeloid leukemia after chemotherapy.

BACKGROUND: According to the different sensitivity of their bone marrow CD34+ cells to in vitro treatment with Etoposide or Mafosfamide, Acute Myeloid Leukaemia (AML) patients in apparent complete remission (CR) after chemotherapy induction may be classified into three groups: (i) normally responsive; (ii) chemoresistant; (iii) highly chemosensitive. This inversely correlates with in vivo CD34+ mobilization and, interestingly, also with the prognosis of the disease: patients showing a good mobilizing activity are resistant to chemotherapy and subject to significantly higher rates of Minimal Residual Disease (MRD) and relapse than the others. Based on its known role in patients' response to chemotherapy, we hypothesized an involvement of the Apoptotic Machinery (AM) in these phenotypic features. METHODS: To investigate the molecular bases of the differential chemosensitivity of bone marrow hematopoietic stem cells (HSC) in CR AML patients, and the relationship between chemosensitivity, mobilizing activity and relapse rates, we analyzed their AM expression profile by performing Real Time RT-PCR of 84 AM genes in CD34+ pools from the two extreme classes of patients (i.e., chemoresistant and highly chemosensitive), and compared them with normal controls. RESULTS: The AM expression profiles of patients highlighted features that could satisfactorily explain their in vitro chemoresponsive phenotype: specifically, in chemoresistant patients we detected up regulation of antiapoptotic BIRC genes and down regulation of proapoptotic APAF1, FAS, FASL, TNFRSF25. Interestingly, our analysis of the AM network showed that the dysregulated genes in these patients are characterized by high network centrality (i.e., high values of betweenness, closeness, radiality, stress) and high involvement in drug response. CONCLUSIONS: AM genes represent critical nodes for the proper execution of cell death following pharmacological induction in patients. We propose that their dysregulation (either due to inborn or de novo genomic mutations selected by treatment) could cause a relapse in apparent CR AML patients. Based on this, AM profiling before chemotherapy and transplantation could identify patients with a predisposing genotype to MRD and relapse: accordingly, they should undergo a different, specifically tailored, therapeutic regimen and should be carefully checked during the post-treatment period.

The apoptotic machinery as a biological complex system: analysis of its omics and evolution, identification of candidate genes for fourteen major types of cancer, and experimental validation in CML and neuroblastoma.

BACKGROUND: Apoptosis is a critical biological phenomenon, executed under the guidance of the Apoptotic Machinery (AM), which allows the physiologic elimination of terminally differentiated, senescent or diseased cells. Because of its relevance to BioMedicine, we have sought to obtain a detailed characterization of AM Omics in Homo sapiens, namely its Genomics and Evolution, Transcriptomics, Proteomics, Interactomics, Oncogenomics, and Pharmacogenomics. METHODS: This project exploited the methodology commonly used in Computational Biology (i.e., mining of many omics databases of the web) as well as the High Throughput biomolecular analytical techniques. RESULTS: In Homo sapiens AM is comprised of 342 protein-encoding genes (possessing either anti- or pro-apoptotic activity, or a regulatory function) and 110 MIR-encoding genes targeting them: some have a critical role within the system (core AM nodes), others perform tissue-, pathway-, or disease-specific functions (peripheral AM nodes). By overlapping the cancer type-specific AM mutation map in the fourteen most frequent cancers in western societies (breast, colon, kidney, leukaemia, liver, lung, neuroblastoma, ovary, pancreas, prostate, skin, stomach, thyroid, and uterus) to their transcriptome, proteome and interactome in the same tumour type, we have identified the most prominent AM molecular alterations within each class. The comparison of the fourteen mutated AM networks (both protein- as MIR-based) has allowed us to pinpoint the hubs with a general and critical role in tumour development and, conversely, in cell physiology: in particular, we found that some of these had already been used as targets for pharmacological anticancer therapy. For a better understanding of the relationship between AM molecular alterations and pharmacological induction of apoptosis in cancer, we examined the expression of AM genes in K562 and SH-SY5Y after anticancer treatment. CONCLUSION: We believe that our data on the Apoptotic Machinery will lead to the identification of new cancer genes and to the discovery of new biomarkers, which could then be used to profile cancers for diagnostic purposes and to pinpoint new targets for pharmacological therapy. This approach could pave the way for future studies and applications in molecular and clinical Medicine with important perspectives both for Oncology as for Regenerative Medicine.