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Background: Metabolic reprogramming is a well-known marker of cancer, and it represents an early event during hepatocellular carcinoma (HCC) development. The recent approval of several molecular targeted agents has revolutionized the management of advanced HCC patients. Nevertheless, the lack of circulating biomarkers still affects patient stratification to tailored treatments. In this context, there is an urgent need for biomarkers to aid treatment choice and for novel and more effective therapeutic combinations to avoid the development of drug-resistant phenotypes. This study aims to prove the involvement of miR-494 in metabolic reprogramming of HCC, to identify novel miRNA-based therapeutic combinations and to evaluate miR-494 potential as a circulating biomarker., Methods: Bioinformatics analysis identified miR-494 metabolic targets. QPCR analysis of glucose 6-phosphatase catalytic subunit (G6pc) was performed in HCC patients and preclinical models. Functional analysis and metabolic assays assessed G6pc targeting and miR-494 involvement in metabolic changes, mitochondrial dysfunction, and ROS production in HCC cells. Live-imaging analysis evaluated the effects of miR-494/G6pc axis in cell growth of HCC cells under stressful conditions. Circulating miR-494 levels were assayed in sorafenib-treated HCC patients and DEN-HCC rats., Results: MiR-494 induced the metabolic shift of HCC cells toward a glycolytic phenotype through G6pc targeting and HIF-1A pathway activation. MiR-494/G6pc axis played an active role in metabolic plasticity of cancer cells, leading to glycogen and lipid droplets accumulation that favored cell survival under harsh environmental conditions. High miR-494 serum levels associated with sorafenib resistance in preclinical models and in a preliminary cohort of HCC patients. An enhanced anticancer effect was observed for treatment combinations between antagomiR-494 and sorafenib or 2-deoxy-glucose in HCC cells., Conclusions: MiR-494/G6pc axis is critical for the metabolic rewiring of cancer cells and associates with poor prognosis. MiR-494 deserves attention as a candidate biomarker of likelihood of response to sorafenib to be tested in future validation studies. MiR-494 represents a promising therapeutic target for combination strategies with sorafenib or metabolic interference molecules for the treatment of HCC patients who are ineligible for immunotherapy., (© 2023. The Author(s).)

Background: In the last years, several efforts have been made to classify colorectal cancer (CRC) into well-defined molecular subgroups, representing the intrinsic inter-patient heterogeneity, known as Consensus Molecular Subtypes (CMSs)., Methods: In this work, we performed a meta-analysis of CRC patients stratified into four CMSs. We identified a negative correlation between a high level of anaplastic lymphoma kinase (ALK) expression and relapse-free survival, exclusively in CMS1 subtype. Stemming from this observation, we tested cell lines, patient-derived organoids and mice with potent ALK inhibitors, already approved for clinical use., Results: ALK interception strongly inhibits cell proliferation already at nanomolar doses, specifically in CMS1 cell lines, while no effect was found in CMS2/3/4 groups. Furthermore, in vivo imaging identified a role for ALK in the dynamic formation of 3D tumor spheroids. Consistently, ALK appeares constitutively phosphorylated in CMS1, and it signals mainly through the AKT axis. Mechanistically, we found that CMS1 cells display several copies of ALKAL2 ligand and ALK-mRNAs, suggesting an autocrine loop mediated by ALKAL2 in the activation of ALK pathway, responsible for the invasive phenotype. Consequently, disruption of ALK axis mediates the pro-apoptotic action of CMS1 cell lines, both in 2D and 3D and enhanced cell-cell adhesion and e-cadherin organization. In agreement with all these findings, the ALK signature encompassing 65 genes statistically associated with worse relapse-free survival in CMS1 subtype. Finally, as a proof of concept, the efficacy of ALK inhibition was demonstrated in both patient-derived organoids and in tumor xenografts in vivo., Conclusions: Collectively, these findings suggest that ALK targeting may represent an attractive therapy for CRC, and CMS classification may provide a useful tool to identify patients who could benefit from this treatment. These findings offer rationale and pharmacological strategies for the treatment of CMS1 CRC., (© 2022. The Author(s).)

Background: Breast cancer circulating biomarkers include carcinoembryonic antigen and carbohydrate antigen 15-3, which are used for patient follow-up. Since sensitivity and specificity are low, novel and more useful biomarkers are needed. The presence of stable circulating microRNAs (miRNAs) in serum or plasma suggested a promising role for these tiny RNAs as cancer biomarkers. To acquire an absolute concentration of circulating miRNAs and reduce the impact of preanalytical and analytical variables, we used the droplet digital PCR (ddPCR) technique., Results: We investigated a panel of five miRNAs in the sera of two independent cohorts of breast cancer patients and disease-free controls. The study showed that miR-148b-3p and miR-652-3p levels were significantly lower in the serum of breast cancer patients than that in controls in both cohorts. For these two miRNAs, the stratification of breast cancer patients versus controls was confirmed by receiver operating characteristic curve analyses. In addition, we showed that higher levels of serum miR-10b-5p were associated with clinicobiological markers of poor prognosis., Conclusions: The study revealed the usefulness of the ddPCR approach for the quantification of circulating miRNAs. The use of the ddPCR quantitative approach revealed very good agreement between two independent cohorts in terms of comparable absolute miRNA concentrations and consistent trends of dysregulation in breast cancer patients versus controls. Overall, this study supports the use of the quantitative ddPCR approach for monitoring the absolute levels of diagnostic and prognostic tumor-specific circulating miRNAs.

Background: The miR-9 family microRNAs have been identified as a tumor suppressor miRNA in cancers. We postulated that miR-9-1, miR-9-2 and miR-9-3 might be inactivated by DNA hypermethylation in chronic lymphocytic leukemia (CLL)., Methods: Methylation of miR-9-1, miR-9-2 and miR-9-3 was studied in eight normal controls including normal bone marrow, buffy coat, and CD19-sorted peripheral blood B-cells from healthy individuals, seven CLL cell lines, and seventy-eight diagnostic CLL samples by methylation-specific polymerase chain reaction., Results: The promoters of miR-9-3 and miR-9-1 were both unmethylated in normal controls, but methylated in five (71.4%) and one of seven CLL cell lines respectively. However, miR-9-2 promoter was methylated in normal controls including CD19 + ve B-cells, hence suggestive of a tissue-specific but not tumor-specific methylation, and thus not further studied. Different MSP statuses of miR-9-3, including complete methylation, partial methylation, and complete unmethylation, were verified by quantitative bisulfite methylation analysis. 5-Aza-2'-deoxycytidine treatment resulted in miR-9-3 promoter demethylation and re-expression of pri-miR-9-3 in I83-E95 and WAC3CD5+ cells, which were homozygously methylated for miR-9-3. Moreover, overexpression of miR-9 led to suppressed cell proliferation and enhanced apoptosis together with downregulation of NFκB1 in I83-E95 cells, supporting a tumor suppressor role of miR-9-3 in CLL. In primary CLL samples, miR-9-3 was detected in 17% and miR-9-1 methylation in none of the patients at diagnosis. Moreover, miR-9-3 methylation was associated with advanced Rai stage (≥ stage 2) (P = 0.04)., Conclusions: Of the miR-9 family, miR-9-3 is a tumor suppressor miRNA relatively frequently methylated, and hence silenced in CLL; whereas miR-9-1 methylation is rare in CLL. The role of miR-9-3 methylation in the constitutive activation of NFκB signaling pathway in CLL warrants further study.

Background: The microRNA 125b is a double-faced gene expression regulator described both as a tumor suppressor gene (in solid tumors) and an oncogene (in hematologic malignancies). In human breast cancer, it is one of the most down-regulated miRNAs and is able to modulate ERBB2/3 expression. Here, we investigated its targets in breast cancer cell lines after miRNA-mimic transfection. We examined the interactions of the validated targets with ERBB2 oncogene and the correlation of miR-125b expression with clinical variables., Methods: MiR-125b possible targets were identified after transfecting a miRNA-mimic in MCF7 cell line and analyzing gene expression modifications with Agilent microarrays and Sylamer bioinformatic tool. Erythropoietin (EPO) and its receptor (EPOR) were validated as targets of miR-125b by luciferase assay and their expression was assessed by RT-qPCR in 42 breast cancers and 13 normal samples. The molecular talk between EPOR and ERBB2 transcripts, through miR-125b, was explored transfecting MDA-MD-453 and MDA-MB-157 with ERBB2 RNA and using RT-qPCR., Results: We identified a panel of genes down-regulated after miR-125b transfection and putative targets of miR-125b. Among them, we validated erythropoietin (EPO) and its receptor (EPOR) - frequently overexpressed in breast cancer--as true targets of miR-125b. Moreover, we explored possible correlations with clinical variables and we found a down-regulation of miR-125b in metastatic breast cancers and a significant positive correlation between EPOR and ERBB2/HER2 levels, that are both targets of miR-125b and function as competing endogenous RNAs (ceRNAs)., Conclusions: Taken together our results show a mechanism for EPO/EPOR and ERBB2 co-regulation in breast cancer and confirm the importance of miR-125b in controlling clinically-relevant cancer features.

In vivo use of monoclonal antibodies (mAbs) has become a mainstay of routine clinical practice in the treatment of various human diseases. A number of molecules can serve as targets, according to the condition being treated. Now entering human clinical trials, CD38 molecule is a particularly attractive target because of its peculiar pattern of expression and its twin role as receptor and ectoenzyme. This review provides a range of analytical perspectives on the current progress in and challenges to anti-CD38 mAb therapy. We present a synopsis of the evidence available on CD38, particularly in myeloma and chronic lymphocytic leukemia (CLL). Our aim is to make the data from basic science helpful and accessible to a diverse clinical audience and, at the same time, to improve its potential for in vivo use. The topics covered include tissue distribution and signal implementation by mAb ligation and the possibility of increasing cell density on target cells by exploiting information about the molecule's regulation in combination with drugs approved for in vivo use. Also analyzed is the behavior of CD38 as an enzyme: CD38 is a component of a pathway leading to the production of adenosine in the tumor microenvironment, thus inducing local anergy. Consequently, not only might CD38 be a prime target for mAb-mediated therapy, but its functional block may contribute to general improvement in cancer immunotherapy and outcomes.

Background: Fludarabine, is one of the most active single agents in the treatment of chronic lymphocytic leukemia (CLL). Over time, however, virtually all CLL patients become fludarabine-refractory. To elucidate whether microRNAs are involved in the development of fludarabine resistance, we analyzed the expression of 723 human miRNAs before and 5-days after fludarabine mono-therapy in 17 CLL patients which were classified as responder or refractory to fludarabine treatment based on NCI criteria., Results: By comparing the expression profiles of these two groups of patients, we identified a microRNA signature able to distinguish refractory from sensitive CLLs. The expression of some microRNAs was also able to predict fludarabine resistance of 12 independent CLL patients. Among the identified microRNAs, miR-148a, miR-222 and miR-21 exhibited a significantly higher expression in non-responder patients either before and after fludarabine treatment. After performing messenger RNA expression profile of the same patients, the activation of p53-responsive genes was detected in fludarabine responsive cases only, therefore suggesting a possible mechanism linked to microRNA deregulation in non-responder patients. Importantly, inhibition of miR-21 and miR-222 by anti-miRNA oligonucleotides induced a significant increase in caspase activity in fludarabine-treated p53-mutant MEG-01 cells, suggesting that miR-21 and miR-222 up-regulation may be involved in the establishment of fludarabine resistance., Conclusions: This is the first report that reveals the existence of a microRNA profile that differentiate refractory and sensitive CLLs, either before and after fludarabine mono-therapy. A p53 dysfunctional pathway emerged in refractory CLLs and could contribute in explaining the observed miRNA profile. Moreover, this work indicates that specific microRNAs can be used to predict fludarabine resistance and may potentially be used as therapeutic targets, therefore establishing an important starting point for future studies.

Background: The importance of non-coding RNAs (ncRNAs) as fine regulators of eukaryotic gene expression has emerged by several studies focusing on microRNAs (miRNAs). miRNAs represent a newly discovered family of non coding-RNAs. They are thought to be crucial players of human hematopoiesis and related tumorigenesis and to represent a potential tool to detect the early stages of cancer. More recently, the expression regulation of numerous long ncRNAs has been linked to cell growth, differentiation and cancer although the molecular mechanism of their function is still unknown.NB4 cells are promyelocytic cells that can be induced to differentiation upon retinoic acid (ATRA) treatment and represent a feasible model to study changes of non coding RNAs expression between cancer cells and their terminally differentiated counterpart., Findings: we screened, by microarray analysis, the expression of 243 miRNAs and 492 human genes transcribing for putative long ncRNAs different from miRNAs in NB4 cells before and after ATRA induced differentiation. Our data show that 8 miRNAs, and 58 long ncRNAs were deregulated by ATRA induced NB4 differentiation., Conclusion: our data suggest that ATRA-induced differentiation lead to deregulation of a large number of the ncRNAs that can play regulatory roles in both tumorigenesis and differentiation.

Background: Colorectal cancer develops through two main genetic instability pathways characterized by distinct pathologic features and clinical outcome., Results: We investigated colon cancer samples (23 characterized by microsatellite stability, MSS, and 16 by high microsatellite instability, MSI-H) for genome-wide expression of microRNA (miRNA) and mRNA. Based on combined miRNA and mRNA gene expression, a molecular signature consisting of twenty seven differentially expressed genes, inclusive of 8 miRNAs, could correctly distinguish MSI-H versus MSS colon cancer samples. Among the differentially expressed miRNAs, various members of the oncogenic miR-17-92 family were significantly up-regulated in MSS cancers. The majority of protein coding genes were also up-regulated in MSS cancers. Their functional classification revealed that they were most frequently associated with cell cycle, DNA replication, recombination, repair, gastrointestinal disease and immune response., Conclusion: This is the first report that indicates the existence of differences in miRNA expression between MSS versus MSI-H colorectal cancers. In addition, the work suggests that the combination of mRNA/miRNA expression signatures may represent a general approach for improving bio-molecular classification of human cancer.