Early modifications of circulating microRNAs levels in metastatic colorectal cancer patients treated with regorafenib.

Biomarkers able to improve the cost/benefit ratio are urgently needed for metastatic colorectal cancer patients that are eligible to receive regorafenib. Here, we measured plasma levels of ten circulating microRNAs (c-miRNAs) and we investigated their early changes during treatment, as well as possible correlation with clinical outcome. Ten literature-selected c-miRNAs were quantified by qRT-PCR on plasma samples collected at baseline (d1) and after 15 days of treatment (d15). C-miRNAs showing significant changes were further analyzed to establish correlations with outcome. A decision tree-based approach was employed to define a c-miRNA signature able to predict the outcome. Results achieved in an exploratory cohort were tested in a validation group. In the exploratory cohort (n = 34), the levels of c-miR-21 (p = 0.06), c-miR-141 (p = 0.04), and c-miR-601 (p = 0.01) increased at d15 compared with d1. A c-miRNA signature involving c-miR-21, c-miR-221, and c-miR-760 predicted response to treatment (p < 0.0001) and was significantly associated to PFS (HR = 10.68; 95% CI 3.2-35.65; p < 0.0001). In the validation cohort (n = 36), the increase in c-miR-21 (p = 0.02) and c-miR-601 (p = 0.02) levels at d15 was confirmed, but the associations with outcome were not. Our data indicate that early changes of c-miRNA levels might be influenced by regorafenib treatment. However, further studies are needed to establish the predictive power of such modifications.

The landscape of BRAF transcript and protein variants in human cancer.

BACKGROUND: The BRAF protein kinase is widely studied as a cancer driver and therapeutic target. However, the regulation of its expression is not completely understood. RESULTS: Taking advantage of the RNA-seq data of more than 4800 patients belonging to 9 different cancer types, we show that BRAF mRNA exists as a pool of 3 isoforms (reference BRAF, BRAF-X1, and BRAF-X2) that differ in the last part of their coding sequences, as well as in the length (BRAF-ref: 76 nt; BRAF-X1 and BRAF-X2: up to 7 kb) and in the sequence of their 3'UTRs. The expression levels of BRAF-ref and BRAF-X1/X2 are inversely correlated, while the most prevalent among the three isoforms varies from cancer type to cancer type. In melanoma cells, the X1 isoform is expressed at the highest level in both therapy-naïve cells and cells with acquired resistance to vemurafenib driven by BRAF gene amplification or expression of the Δ[3-10] splicing variant. In addition to the BRAF-ref protein, the BRAF-X1 protein (the full length as well as the Δ[3-10] variant) is also translated. The expression levels of the BRAF-ref and BRAF-X1 proteins are similar, and together they account for BRAF functional activities. In contrast, the endogenous BRAF-X2 protein is hard to detect because the C-terminal domain is selectively recognized by the ubiquitin-proteasome pathway and targeted for degradation. CONCLUSIONS: By shedding light on the repertoire of BRAF mRNA and protein variants, and on the complex regulation of their expression, our work paves the way to a deeper understanding of a crucially important player in human cancer and to a more informed development of new therapeutic strategies.

Context-dependent miR-204 and miR-211 affect the biological properties of amelanotic and melanotic melanoma cells.

Despite increasing amounts of experimental evidence depicting the involvement of non-coding RNAs in cancer, the study of BRAFV600E-regulated genes has thus far focused mainly on protein-coding ones. Here, we identify and study the microRNAs that BRAFV600E regulates through the ERK pathway.By performing small RNA sequencing on A375 melanoma cells and a vemurafenib-resistant clone that was taken as negative control, we discover miR-204 and miR-211 as the miRNAs most induced by vemurafenib. We also demonstrate that, although belonging to the same family, these two miRNAs have distinctive features. miR-204 is under the control of STAT3 and its expression is induced in amelanotic melanoma cells, where it acts as an effector of vemurafenib's anti-motility activity by targeting AP1S2. Conversely, miR-211, a known transcriptional target of MITF, is induced in melanotic melanoma cells, where it targets EDEM1 and consequently impairs the degradation of TYROSINASE (TYR) through the ER-associated degradation (ERAD) pathway. In doing so, miR-211 serves as an effector of vemurafenib's pro-pigmentation activity. We also show that such an increase in pigmentation in turn represents an adaptive response that needs to be overcome using appropriate inhibitors in order to increase the efficacy of vemurafenib.In summary, we unveil the distinct and context-dependent activities exerted by miR-204 family members in melanoma cells. Our work challenges the widely accepted "same miRNA family = same function" rule and provides a rationale for a novel treatment strategy for melanotic melanomas that is based on the combination of ERK pathway inhibitors with pigmentation inhibitors.

Methods for the Identification of PTEN-Targeting MicroRNAs.

The identification of PTEN-targeting microRNAs usually starts from an in silico bioinformatic prediction and then requires a careful experimental validation that exploits both heterologous and endogenous systems. Here we describe the methods used to carry on these analyses and experiments, examining pitfalls and alternatives for each step. Moreover, we give an overview of the latest high-throughput microRNA target identification techniques which offer a more comprehensive view of the microRNAs that can bind a fundamental tumor suppressor such as PTEN.

Alkaline Phosphatase-Positive Immortal Mouse Embryo Fibroblasts Are Cells in a Transitional Reprogramming State Induced to Face Environmental Stresses.

In this study, we report that immortal mouse embryonic fibroblasts (I-MEFs) have a baseline level of cells positive for alkaline phosphatase (AP(+)) staining. Environmental stresses, including long-lasting growth in the absence of expansion and treatment with drugs, enhance the frequency of AP(+) I-MEFs. By adapting fast red AP staining to the sorting procedure, we separated AP(+) and AP(-) I-MEFs and demonstrated that the differentially expressed genes are consistent with a reprogrammed phenotype. In particular, we found that sestrin 1 is upregulated in AP(+) I-MEFs. We focused on this gene and demonstrated that increased sestrin 1 expression is accompanied by the growth of I-MEFs in the absence of expansion and occurs before the formation of AP(+) I-MEFs. Together with sestrin 1 upregulation, we found that AP(+) I-MEFs accumulated in the G1 phase of the cell cycle, suggesting that the two events are causally related. Accordingly, we found that silencing sestrin 1 expression reduced the frequency and G1 accumulation of AP(+) I-MEFs. Taken together, our data suggested that I-MEFs stressed by environmental changes acquire the AP(+) phenotype and achieve a quiescent state characterized by a new transcriptional network.

Long non-coding RNAs in cancer: implications for personalized therapy.

Long non-coding RNAs (lncRNAs, pseudogenes and circRNAs) have recently come into light as powerful players in cancer pathogenesis and it is becoming increasingly clear that they have the potential of greatly contributing to the spread and success of personalized cancer medicine. In this concise review, we briefly introduce these three classes of long non-coding RNAs. We then discuss their applications as diagnostic and prognostic biomarkers. Finally, we describe their appeal as targets and as drugs, while pointing out the limitations that still lie ahead of their definitive entry into clinical practice.

miRNAs regulate miRNAs: coordinated transcriptional and post-transcriptional regulation.

microRNAs are short single-stranded RNA molecules that negatively regulate gene expression at post-transcriptional level. Recently it has been demonstrated that microRNAs are involved in many physiological processes, including differentiation and development. Moreover, this class of tiny regulators is drammatically involved in many pathological processes, like tumors and genetic diseases. A main issue is how microRNA processing and expression are regulated: thanks to recent findings, we are beginning to get insight into this critical aspect of microRNA biology, but we are far from understanding these processes in all their complexity. In particular, microRNA coordinated expression with other regulatory molecules, such as transcription factors, is still under investigation. We depleted microRNA 221 and 222 and found that the microRNA signature of Human Umbilical Vein Endothelial Cells was affected as a consequence: 9 microRNAs were up-regulated and 23 microRNAs were down-regulated. We propose a model in which a complex network involving coexpressed microRNAs and transcription factors is affected by single microRNA-variations, with the result that microRNA expression profile is in turn modified.

MicroRNAs modulate the angiogenic properties of HUVECs.

MicroRNAs (miRNAs) have recently come into focus as key posttranscriptional modulators of gene expression. In this work, we addressed whether in vitro angiogenesis is an miRNA-regulated process. We performed large-scale analysis of miRNA expression in human umbilical vein endothelial cells (HUVECs) and found that 15 highly expressed miRNAs have the receptors of angiogenic factors as putative targets. In particular, we demonstrated that miR-221 and miR-222 affect c-Kit expression and, as a consequence, the angiogenic properties of its ligand stem cell factor. Interaction between miR-222 and c-Kit is likely to be part of a complex circuit that controls the ability of endothelial cells to form new capillaries.