A High-Content Screening of Anticancer Compounds Suggests the Multiple Tyrosine Kinase Inhibitor Ponatinib for Repurposing in Neuroblastoma Therapy.

Novel druggable targets have been discovered in neuroblastoma (NB), paving the way for more effective treatments. However, children with high-risk NB still show high mortality rates prompting for a search of novel therapeutic options. Here, we aimed at repurposing FDA-approved drugs for NB treatment by performing a high-content screening of a 349 anticancer compounds library. In the primary screening, we employed three NB cell lines, grown as three-dimensional (3D) multicellular spheroids, which were treated with 10 µmol/L of the library compounds for 72 hours. The viability of 3D spheroids was evaluated using a high-content imaging approach, resulting in a primary hit list of 193 compounds. We selected 60 FDA-approved molecules and prioritized drugs with multi-target activity, discarding those already in use for NB treatment or enrolled in NB clinical trials. Hence, 20 drugs were further tested for their efficacy in inhibiting NB cell viability, both in two-dimensional and 3D models. Dose-response curves were then supplemented with the data on side effects, therapeutic index, and molecular targets, suggesting two multiple tyrosine kinase inhibitors, ponatinib and axitinib, as promising candidates for repositioning in NB. Indeed, both drugs showed induction of cell-cycle block and apoptosis, as well as inhibition of colony formation. However, only ponatinib consistently affected migration and inhibited invasion of NB cells. Finally, ponatinib also proved effective inhibition of tumor growth in orthotopic NB mice, providing the rationale for its repurposing in NB therapy. Mol Cancer Ther; 17(7); 1405-15. ©2018 AACR.

Loss of ZFP36 expression in colorectal cancer correlates to wnt/ ß-catenin activity and enhances epithelial-to-mesenchymal transition through upregulation of ZEB1, SOX9 and MACC1.

The mRNA-destabilizing protein ZFP36 has been previously described as a tumor suppressor whose expression is lost during colorectal cancer development. In order to evaluate its role in this disease, we restored ZFP36 expression in different cell contexts, showing that the presence of this protein impairs the epithelial-to-mesenchymal transition (EMT) and induces a higher susceptibility to anoikis. Consistently, we found that ZFP36 inhibits the expression of three key transcription factors involved in EMT: ZEB1, MACC1 and SOX9. Finally, we observed for the first time that its expression negatively correlates with the activity of Wnt/ß-catenin pathway, which is constitutively activated in colorectal cancer. This evidence provides a clue on the mechanism leading to the loss of ZFP36 in CRC.

An integrative approach for the identification of prognostic and predictive biomarkers in rectal cancer.

INTRODUCTION: Colorectal cancer is the third most common cancer in the world, a small fraction of which is represented by locally advanced rectal cancer (LARC). If not medically contraindicated, preoperative chemoradiotherapy, represent the standard of care for LARC patients. Unfortunately, patients shows a wide range of response rates in which approximately 20% has a complete pathological response, whereas in 20 to 40% the response is poor or absent. RESULTS: The following specific gene signature, able to discriminate responders' patients from non-responders, were founded: AKR1C3, CXCL11, CXCL10, IDO1, CXCL9, MMP12 and HLA-DRA. These genes are mainly involved in immune system pathways and interact with drugs traditionally used in the adjuvant treatment of rectal cancer. DISCUSSION: The present study suggests that new ideas for therapy could be found not only limited to studying genes differentially expressed between the two groups of patients but deepening the mechanisms, associated to response, in which they are involved. METHODS: Gene expression studies performed by: Agostini et al., Rimkus et al. and Kim et al. have been merged through a meta-analysis of the raw data. Gene expression data-sets have been processed using A-MADMAN. Common differentially expressed gene (DEG) were identified through SAM analysis. To further characterize the identified DEG we deeply investigated its biological role using an integrative computational biology approach.

Circulating miR-182 is a biomarker of colorectal adenocarcinoma progression.

MiR-182 expression was evaluated by qRT-PCR and in situ hybridization in 20 tubular adenomas, 50 colorectal carcinoma (CRC), and 40 CRC liver metastases. Control samples obtained from patients with irritable bowel syndrome, or tumor-matched normal colon mucosa were analyzed (n=50). MiR-182 expression increased progressively and significantly along with the colorectal carcinogenesis cascade, and in CRC liver metastases. The inverse relation between miR-182 and the expression of its target gene ENTPD5 was investigated by immunohistochemical analysis. We observed that normal colocytes featured a strong ENTPD5 cytoplasmic expression whereas a significantly and progressively lower expression was present along with dedifferentiation of the histologic phenotype. Plasma samples from 51 CRC patients and controls were tested for miR-182 expression. Plasma miR-182 concentrations were significantly higher in CRC patients than in healthy controls or patients with colon polyps at endoscopy. Moreover, miR-182 plasma levels were significantly reduced in post-operative samples after radical hepatic metastasectomy compared to preoperative samples. Our results strengthen the hypothesis of a central role of miR-182 dysregulation in colon mucosa transformation, demonstrate the concomitant progressive down-regulation of ENTPD5 levels during colon carcinogenesis, and indicate the potential of circulating miR-182 as blood based biomarker for screening and monitoring CRC during the follow-up.

Human miRNome profiling in colorectal cancer and liver metastasis development.

Qualitative alterations or abnormal expression of microRNAs (miRNAs) in colorectal cancer has mainly been demonstrated in primary tumors. The miRNA expression profiles in 78 samples from 46 patients were analyzed to identify changes in miRNA expression level among normal colon mucosa, primary tumor and liver metastasis samples. Using this dataset, we describe the interplay of miRNA groups in regulating pathways that are important for tumor development. Here we describe in details the contents and quality controls for the miRNA expression and clinical data associated with the study published by Pizzini and colleagues in the BMC Genomics in 2013 (Pizzini et al., 2013). Data are deposited in GEO database as GSE35834 series.

An integrative framework identifies alternative splicing events in colorectal cancer development.

Alternative splicing (AS) is a common mechanism which creates diverse RNA isoforms from a single gene, potentially increasing protein variety. Growing evidence suggests that this mechanism is closely related to cancer progression. In this study, whole transcriptome analysis was performed with GeneChip Human exon 1.0 ST Array from 80 samples comprising 23 normal colon mucosa, 30 primary colorectal cancer and 27 liver metastatic specimens from 46 patients, to identify AS events in colorectal cancer progression. Differentially expressed genes and exons were estimated and AS events were reconstructed by combining exon-level analyses with AltAnalyze algorithms and transcript-level estimations (MMBGX probabilistic method). The number of AS genes in the transition from normal colon mucosa to primary tumor was the most abundant, but fell considerably in the next transition to liver metastasis. 206 genes with probable AS events in colon cancer development and progression were identified, that are involved in processes and pathways relevant to tumor biology, as cell-cell and cell-matrix interactions. Several AS events in VCL, CALD1, B3GNT6 and CTHRC1 genes, differentially expressed during tumor development were validated, at RNA and at protein level. Taken together, these results demonstrate that cancer-specific AS is common in early phases of colorectal cancer natural history.

Impact of microRNAs on regulatory networks and pathways in human colorectal carcinogenesis and development of metastasis.

BACKGROUND: Qualitative alterations or abnormal expression of microRNAs (miRNAs) in colon cancer have mainly been demonstrated in primary tumors. Poorly overlapping sets of oncomiRs, tumor suppressor miRNAs and metastamiRs have been linked with distinct stages in the progression of colorectal cancer. To identify changes in both miRNA and gene expression levels among normal colon mucosa, primary tumor and liver metastasis samples, and to classify miRNAs into functional networks, in this work miRNA and gene expression profiles in 158 samples from 46 patients were analysed. RESULTS: Most changes in miRNA and gene expression levels had already manifested in the primary tumors while these levels were almost stably maintained in the subsequent primary tumor-to-metastasis transition. In addition, comparing normal tissue, tumor and metastasis, we did not observe general impairment or any rise in miRNA biogenesis. While only few mRNAs were found to be differentially expressed between primary colorectal carcinoma and liver metastases, miRNA expression profiles can classify primary tumors and metastases well, including differential expression of miR-10b, miR-210 and miR-708. Of 82 miRNAs that were modulated during tumor progression, 22 were involved in EMT. qRT-PCR confirmed the down-regulation of miR-150 and miR-10b in both primary tumor and metastasis compared to normal mucosa and of miR-146a in metastases compared to primary tumor. The upregulation of miR-201 in metastasis compared both with normal and primary tumour was also confirmed. A preliminary survival analysis considering differentially expressed miRNAs suggested a possible link between miR-10b expression in metastasis and patient survival. By integrating miRNA and target gene expression data, we identified a combination of interconnected miRNAs, which are organized into sub-networks, including several regulatory relationships with differentially expressed genes. Key regulatory interactions were validated experimentally. Specific mixed circuits involving miRNAs and transcription factors were identified and deserve further investigation. The suppressor activity of miR-182 on ENTPD5 gene was identified for the first time and confirmed in an independent set of samples. CONCLUSIONS: Using a large dataset of CRC miRNA and gene expression profiles, we describe the interplay of miRNA groups in regulating gene expression, which in turn affects modulated pathways that are important for tumor development.

A ten markers panel provides a more accurate and complete microsatellite instability analysis in mismatch repair-deficient colorectal tumors.

UNLABELLED: Tumour microsatellite instability (MSI) is useful in identifying patients with hereditary non-polyposis colorectal cancer (HNPCC) with defective DNA mismatch repair (MMR) genes. A reference Bethesda panel has limitations resulting from the inclusion of dinucleotide markers, which are less sensitive and specific for detection of tumours with MMR deficiencies. We developed a multiplex PCR assay with additional four mononucleotide markers and one dinucleotide marker (NR-21, NR-24, BAT-40, TGF-BetaR and D18S58) for a rapid and proper classification of MSI-H, MSI-L and MSS colorectal cancers. Two tetranucleotide markers were added to identify sample mix-ups and/or contamination. RESULTS: all the 44 cases test cases were in agreement with previous classification except for three cases: one case MSI-H-Bethesda unstable only for dinucleotides markers shifted to MSI-L category and two cases MSI-L-Bethesda unstable for mononucleotide markers shifted to MSI-H category. Immunohistochemistry analysis revealed that these two MSI-H cases did not expressed hMLH1 and they were found to be methylated at the MLH1 promoter, while the first one that shifted to MSI-L showed MMR protein expression. CONCLUSION: a complete panel of ten markers including four dinucleotide and six mononucleotide microsatellites allows accurate evaluation of tumor MSI status.