Vault RNAs: hidden gems in RNA and protein regulation.

Non-coding RNAs are important regulators of differentiation during embryogenesis as well as key players in the fine-tuning of transcription and furthermore, they control the post-transcriptional regulation of mRNAs under physiological conditions. Deregulated expression of non-coding RNAs is often identified as one major contribution in a number of pathological conditions. Non-coding RNAs are a heterogenous group of RNAs and they represent the majority of nuclear transcripts in eukaryotes. An evolutionary highly conserved sub-group of non-coding RNAs is represented by vault RNAs, named since firstly discovered as component of the largest known ribonucleoprotein complexes called "vault". Although they have been initially described 30 years ago, vault RNAs are largely unknown and their molecular role is still under investigation. In this review we will summarize the known functions of vault RNAs and their involvement in cellular mechanisms.

EGFR amplification and outcome in a randomised phase III trial of chemotherapy alone or chemotherapy plus panitumumab for advanced gastro-oesophageal cancers.

OBJECTIVE: Epidermal growth factor receptor (EGFR) inhibition may be effective in biomarker-selected populations of advanced gastro-oesophageal adenocarcinoma (aGEA) patients. Here, we tested the association between outcome and EGFR copy number (CN) in pretreatment tissue and plasma cell-free DNA (cfDNA) of patients enrolled in a randomised first-line phase III clinical trial of chemotherapy or chemotherapy plus the anti-EGFR monoclonal antibody panitumumab in aGEA (NCT00824785). DESIGN: EGFR CN by either fluorescence in situ hybridisation (n=114) or digital-droplet PCR in tissues (n=250) and plasma cfDNAs (n=354) was available for 474 (86%) patients in the intention-to-treat (ITT) population. Tissue and plasma low-pass whole-genome sequencing was used to screen for coamplifications in receptor tyrosine kinases. Interaction between chemotherapy and EGFR inhibitors was modelled in patient-derived organoids (PDOs) from aGEA patients. RESULTS: EGFR amplification in cfDNA correlated with poor survival in the ITT population and similar trends were observed when the analysis was conducted in tissue and plasma by treatment arm. EGFR inhibition in combination with chemotherapy did not correlate with improved survival, even in patients with significant EGFR CN gains. Addition of anti-EGFR inhibitors to the chemotherapy agent epirubicin in PDOs, resulted in a paradoxical increase in viability and accelerated progression through the cell cycle, associated with p21 and cyclin B1 downregulation and cyclin E1 upregulation, selectively in organoids from EGFR-amplified aGEA. CONCLUSION: EGFR CN can be accurately measured in tissue and liquid biopsies and may be used for the selection of aGEA patients. EGFR inhibitors may antagonise the antitumour effect of anthracyclines with important implications for the design of future combinatorial trials.

Modulation of Biliary Cancer Chemo-Resistance Through MicroRNA-Mediated Rewiring of the Expansion of CD133+ Cells.

BACKGROUND AND AIMS: Changes in single microRNA (miRNA) expression have been associated with chemo-resistance in biliary tract cancers (BTCs). However, a global assessment of the dynamic role of the microRNome has never been performed to identify potential therapeutic targets that are functionally relevant in the BTC cell response to chemotherapy. APPROACH AND RESULTS: High-throughput screening (HTS) of 997 locked nucleic acid miRNA inhibitors was performed in six cholangiocarcinoma cell lines treated with cisplatin and gemcitabine (CG) seeking changes in cell viability. Validation experiments were performed with mirVana probes. MicroRNA and gene expression was assessed by TaqMan assay, RNA-sequencing, and in situ hybridization in four independent cohorts of human BTCs. Knockout of microRNA was achieved by CRISPR-CAS9 in CCLP cells (MIR1249KO) and tested for effects on chemotherapy sensitivity in vitro and in vivo. HTS revealed that MIR1249 inhibition enhanced chemotherapy sensitivity across all cell lines. MIR1249 expression was increased in 41% of cases in human BTCs. In validation experiments, MIR1249 inhibition did not alter cell viability in untreated or dimethyl sulfoxide-treated cells; however, it did increase the CG effect. MIR1249 expression was increased in CD133+ biliary cancer cells freshly isolated from the stem cell niche of human BTCs as well as in CD133+ chemo-resistant CCLP cells. MIR1249 modulated the chemotherapy-induced enrichment of CD133+ cells by controlling their clonal expansion through the Wnt-regulator FZD8. MIR1249KO cells had impaired expansion of the CD133+ subclone and its enrichment after chemotherapy, reduced expression of cancer stem cell markers, and increased chemosensitivity. MIR1249KO xenograft BTC models showed tumor shrinkage after exposure to weekly CG, whereas wild-type models showed only stable disease over treatment. CONCLUSIONS: MIR1249 mediates resistance to CG in BTCs and may be tested as a target for therapeutics.

MIR21 Drives Resistance to Heat Shock Protein 90 Inhibition in Cholangiocarcinoma.

BACKGROUND & AIMS: Cholangiocarcinomas (CCA) are resistant to chemotherapy, so new therapeutic agents are needed. We performed a screen to identify small-molecule compounds that are active against CCAs. Levels of microRNA 21 (MIR21 or miRNA21) are increased in CCAs. We investigated whether miRNA21 mediates resistance of CCA cells and organoids to HSP90 inhibitors. METHODS: We performed a high-throughput screen of 484 small-molecule compounds to identify those that reduced viability of 6 human CCA cell lines. We tested the effects of HSP90 inhibitors on cells with disruption of the MIR21 gene, cells incubated with MIR21 inhibitors, and stable cell lines with inducible expression of MIR21. We obtained CCA biopsies from patients, cultured them as organoids (patient-derived organoids). We assessed their architecture, mutation and gene expression patterns, response to compounds in culture, and when grown as subcutaneous xenograft tumors in mice. RESULTS: Cells with IDH1 and PBRM1 mutations had the highest level of sensitivity to histone deacetylase inhibitors. HSP90 inhibitors were effective in all cell lines, irrespective of mutations. Sensitivity of cells to HSP90 inhibitors correlated inversely with baseline level of MIR21. Disruption of MIR21 increased cell sensitivity to HSP90 inhibitors. CCA cells that expressed transgenic MIR21 were more resistant to HSP90 inhibitors than cells transfected with control vectors; inactivation of MIR21 in these cells restored sensitivity to these agents. MIR21 was shown to target the DnaJ heat shock protein family (Hsp40) member B5 (DNAJB5). Transgenic expression of DNAJB5 in CCA cells that overexpressed MIR21 re-sensitized them to HSP90 inhibitors. Sensitivity of patient-derived organoids to HSP90 inhibitors, in culture and when grown as xenograft tumors in mice, depended on expression of miRNA21. CONCLUSIONS: miRNA21 appears to mediate resistance of CCA cells to HSP90 inhibitors by reducing levels of DNAJB5. HSP90 inhibitors might be developed for the treatment of CCA and miRNA21 might be a marker of sensitivity to these agents.

Microsatellite instability in gastric cancer: molecular bases, clinical perspectives, and new treatment approaches.

Gastric cancer is one of the most aggressive malignancies, with limited treatment options in both locally advanced and metastatic setting, resulting in poor prognosis. Based on genomic characterization, stomach tumour has recently been described as a heterogeneous disease composed by different subtypes, each of them with peculiar molecular aspects and specific clinical behaviour. With an incidence of 22% among all western gastric tumour cases, stomach cancer with microsatellite instability was identified as one of these subgroups. Retrospective studies and limited prospective trials reported differences between gastric cancers with microsatellite stability and those with instability, mainly concerning clinical and pathological features, but also in regard to immunological microenvironment, correlation with prognostic value, and responses to treatment. In particular, gastric cancer with microsatellite instability constitutes a small but relevant subgroup associated with older age, female sex, distal stomach location, and lower number of lymph-node metastases. Emerging data attribute to microsatellite instability status a favourable prognostic meaning, whereas the poor outcomes reported after perioperative chemotherapy administration suggest a detrimental role of cytotoxic drugs in this gastric cancer subgroup. The strong immunogenicity and the widespread expression of immune-checkpoint ligands make microsatellite instability subtype more vulnerable to immunotherapeutic approach, e.g., with anti-PD-L1 and anti-CTLA4 antibodies. Since gastric cancer with microsatellite instability shows specific features and clinical behaviour not overlapping with microsatellite stable disease, microsatellite instability test might be suitable for inclusion in a diagnostic setting for all tumour stages to guarantee the most targeted and effective treatment to every patient.

Functional imaging and circulating biomarkers of response to regorafenib in treatment-refractory metastatic colorectal cancer patients in a prospective phase II study.

OBJECTIVE: Regorafenib demonstrated efficacy in patients with metastatic colorectal cancer (mCRC). Lack of predictive biomarkers, potential toxicities and cost-effectiveness concerns highlight the unmet need for better patient selection. DESIGN: Patients with RAS mutant mCRC with biopsiable metastases were enrolled in this phase II trial. Dynamic contrast-enhanced (DCE) MRI was acquired pretreatment and at day 15 post-treatment. Median values of volume transfer constant (Ktrans), enhancing fraction (EF) and their product KEF (summarised median values of Ktrans× EF) were generated. Circulating tumour (ct) DNA was collected monthly until progressive disease and tested for clonal RAS mutations by digital-droplet PCR. Tumour vasculature (CD-31) was scored by immunohistochemistry on 70 sequential tissue biopsies. RESULTS: Twenty-seven patients with paired DCE-MRI scans were analysed. Median KEF decrease was 58.2%. Of the 23 patients with outcome data, >70% drop in KEF (6/23) was associated with higher disease control rate (p=0.048) measured by RECIST V. 1.1 at 2 months, improved progression-free survival (PFS) (HR 0.16 (95% CI 0.04 to 0.72), p=0.02), 4-month PFS (66.7% vs 23.5%) and overall survival (OS) (HR 0.08 (95% CI 0.01 to 0.63), p=0.02). KEF drop correlated with CD-31 reduction in sequential tissue biopsies (p=0.04). RAS mutant clones decay in ctDNA after 8 weeks of treatment was associated with better PFS (HR 0.21 (95% CI 0.06 to 0.71), p=0.01) and OS (HR 0.28 (95% CI 0.07-1.04), p=0.06). CONCLUSIONS: Combining DCE-MRI and ctDNA predicts duration of anti-angiogenic response to regorafenib and may improve patient management with potential health/economic implications.

Bromodomain and extra-terminal domain inhibition modulates the expression of pathologically relevant microRNAs in diffuse large B-cell lymphoma.

Aberrant changes in microRNA expression contribute to lymphomagenesis. Bromodomain and extra-terminal domain inhibitors such as OTX015 (MK-8628, birabresib) have demonstrated preclinical and clinical activity in hematologic tumors. MicroRNA profiling of diffuse large B-cell lymphoma cells treated with OTX015 revealed changes in the expression levels of a limited number of microRNAs, including miR-92a-1-5p, miR-21-3p, miR-155-5p and miR-96-5p. Analysis of publicly available chromatin immunoprecipitation sequencing data of diffuse large B-cell lymphoma cells treated with bromodomain and extra-terminal domain (BET) inhibitors showed that the BET family member BRD4 bound to the upstream regulatory regions of multiple microRNA genes and that this binding decreased following BET inhibition. Alignment of our microRNA profiling data with the BRD4 chromatin immunoprecipitation sequencing data revealed that microRNAs downregulated by OTX015 also exhibited reduced BRD4 binding in their promoter regions following treatment with another bromodomain and extra-terminal domain inhibitor, JQ1, indicating that BRD4 contributes directly to microRNA expression in lymphoma. Treatment with bromodomain and extra-terminal domain inhibitors also decreased the expression of the arginine methyltransferase PRMT5, which plays a crucial role in B-cell transformation and negatively modulates the transcription of miR-96-5p. The data presented here indicate that in addition to previously observed effects on the expression of coding genes, bromodomain and extra-terminal domain inhibitors also modulate the expression of microRNAs involved in lymphomagenesis.

Wnt signalling modulates transcribed-ultraconserved regions in hepatobiliary cancers.

OBJECTIVE: Transcribed-ultraconserved regions (T-UCR) are long non-coding RNAs which are conserved across species and are involved in carcinogenesis. We studied T-UCRs downstream of the Wnt/ß-catenin pathway in liver cancer. DESIGN: Hypomorphic Apc mice (Apcfl/fl) and thiocetamide (TAA)-treated rats developed Wnt/ß-catenin dependent hepatocarcinoma (HCC) and cholangiocarcinoma (CCA), respectively. T-UCR expression was assessed by microarray, real-time PCR and in situ hybridisation. RESULTS: Overexpression of the T-UCR uc.158- could differentiate Wnt/ß-catenin dependent HCC from normal liver and from ß-catenin negative diethylnitrosamine (DEN)-induced HCC. uc.158- was overexpressed in human HepG2 versus Huh7 cells in line with activation of the Wnt pathway. In vitro modulation of ß-catenin altered uc.158- expression in human malignant hepatocytes. uc.158- expression was increased in CTNNB1-mutated human HCCs compared with non-mutated human HCCs, and in human HCC with nuclear localisation of ß-catenin. uc.158- was increased in TAA rat CCA and reduced after treatment with Wnt/ß-catenin inhibitors. uc.158- expression was negative in human normal liver and biliary epithelia, while it was increased in human CCA in two different cohorts. Locked nucleic acid-mediated inhibition of uc.158- reduced anchorage cell growth, 3D-spheroid formation and spheroid-based cell migration, and increased apoptosis in HepG2 and SW1 cells. miR-193b was predicted to have binding sites within the uc.158- sequence. Modulation of uc.158- changed miR-193b expression in human malignant hepatocytes. Co-transfection of uc.158- inhibitor and anti-miR-193b rescued the effect of uc.158- inhibition on cell viability. CONCLUSIONS: We showed that uc.158- is activated by the Wnt pathway in liver cancers and drives their growth. Thus, it may represent a promising target for the development of novel therapeutics.

Sequence variation in mature microRNA-608 and benefit from neo-adjuvant treatment in locally advanced rectal cancer patients.

Single nucleotide polymorphisms (SNPs) in microRNA genes have been associated with colorectal cancer (CRC) risk, survival and response to treatment. Conflicting results are available on the association between rs4919510, a SNP in mature miR-608 and clinical outcome in CRC. Here, we analyzed the association between rs4919510 and benefit from perioperative treatment in a randomised phase II trial of neoadjuvant Capecitabine and Oxaliplatin (CAPOX) followed by chemo-radiotherapy, surgery and adjuvant CAPOX ± Cetuximab in high-risk locally advanced rectal cancer (LARC). A total of 155/164 (94.5%) patients were assessable. 95 (61.3%) were homozygous for CC, 55 (35.5%) heterozygous (CG) and 5 (3.2%) homozygous for GG. Median follow-up was 64.9 months. In the CAPOX arm the 5-year progression-free survival (PFS) and overall survival (OS) rates were 54.6% and 60.7% for CC and 82.0% and 82.1% for CG/GG, respectively (HR PFS 0.13, 95% CI: 0.12-0.83, P = 0.02; HR OS 0.38, 95% CI: 0.14-1.01, P = 0.05). In the CAPOX-C arm PFS and OS were 73.2 and 82.2%, respectively for CC carriers and 64.6 and 73.1% for CG/GG carriers (HR PFS 1.38, 95% CI: 0.61-3.13, P = 0.44; HR OS 1.34, 95% CI: 0.52-3.48, P = 0.55). An interaction was found between study treatment and rs4919510 genotype for both PFS (P = 0.02) and OS (P = 0.07). This is the first study investigating rs4919510 in LARC. The CC genotype appeared to be associated with worse prognosis compared to the CG/GG genotype in patients treated with chemotherapy and chemo-radiotherapy alone. Addition of Cetuximab to chemotherapy and chemo-radiotherapy in CC carriers appeared to improve clinical outcome.

MicroRNA 193b-3p as a predictive biomarker of chronic kidney disease in patients undergoing radical nephrectomy for renal cell carcinoma.

BACKGROUND: A significant proportion of patients undergoing radical nephrectomy (RN) for clear-cell renal cell carcinoma (RCC) develop chronic kidney disease (CKD) within a few years following surgery. Chronic kidney disease has important health, social and economic impact and no predictive biomarkers are currently available. MicroRNAs (miRs) are small non-coding RNAs implicated in several pathological processes. METHODS: Primary objective of our study was to define miRs whose deregulation is predictive of CKD in patients treated with RN. Ribonucleic acid from formalin-fixed paraffin embedded renal parenchyma (cortex and medulla isolated separately) situated >3 cm from the matching RCC was tested for miR expression using nCounter NanoString technology in 71 consecutive patients treated with RN for RCC. Validation was performed by RT-PCR and in situ hybridisation. End point was post-RN CKD measured 12 months post-operatively. Multivariable logistic regression and decision curve analysis were used to test the statistical and clinical impact of predictors of CKD. RESULTS: The overexpression of miR-193b-3p was associated with high risk of developing CKD in patients undergoing RN for RCC and emerged as an independent predictor of CKD. The addition of miR-193b-3p to a predictive model based on clinical variables (including sex and estimated glomerular filtration rate) increased the sensitivity of the predictive model from 81 to 88%. In situ hybridisation showed that miR-193b-3p overexpression was associated with tubule-interstitial inflammation and fibrosis in patients with no clinical or biochemical evidence of pre-RN nephropathy. CONCLUSIONS: miR-193b-3p might represent a useful biomarker to tailor and implement surveillance strategies for patients at high risk of developing CKD following RN.

Circulating microRNA Analysis in a Prospective Co-clinical Trial Identifies MIR652-3p as a Response Biomarker and Driver of Regorafenib Resistance Mechanisms in Colorectal Cancer.

PURPOSE: The multi-kinase inhibitor (mKi) regorafenib has demonstrated efficacy in chemorefractory patients with metastatic colorectal cancer (mCRC). However, lack of predictive biomarkers and concerns over significant toxicities hamper the use of regorafenib in clinical practice. EXPERIMENTAL DESIGN: Serial liquid biopsies were obtained at baseline and monthly until disease progression in chemorefractory patients with mCRC treated with regorafenib in a phase II clinical trial (PROSPECT-R n = 40; NCT03010722) and in a multicentric validation cohort (n = 241). Tissue biopsies collected at baseline, after 2 months and at progression in the PROSPECT-R trial were used to establish patient-derived organoids (PDO) and for molecular analyses. MicroRNA profiling was performed on baseline bloods using the NanoString nCounter platform and results were validated by digital-droplet PCR and/or ISH in paired liquid and tissue biopsies. PDOs co-cultures and PDO-xenotransplants were generated for functional analyses. RESULTS: Large-scale microRNA expression analysis in longitudinal matched liquid and tissue biopsies from the PROSPECT-R trial identified MIR652-3p as a biomarker of clinical benefit to regorafenib. These findings were confirmed in an independent validation cohort and in a "control" group of 100 patients treated with lonsurf. Using ex vivo co-culture assays paired with single-cell RNA-sequencing of PDO established pre- and post-treatment, we modeled regorafenib response observed in vivo and in patients, and showed that MIR652-3p controls resistance to regorafenib by impairing regorafenib-induced lethal autophagy and by orchestrating the switch from neo-angiogenesis to vessel co-option. CONCLUSIONS: Our results identify MIR652-3p as a potential biomarker and as a driver of cell and non-cell-autonomous mechanisms of resistance to regorafenib.

MicroRNAs as biomarkers for trastuzumab-based therapy in HER2-positive advanced oesophago-gastric cancer patients.

Background: This study aimed to identify microRNAs (miRs) as circulating biomarkers of resistance to first-line trastuzumab-based therapy in advanced HER2-positive oesophago-gastric cancer patients. Methods: A high-throughput 1015 Exiqon miRCURY LNA™ microRNA inhibitor library screen was performed in trastuzumab-treated HER2-positive NCI-N87 and HER2-negative FLO-1 oesophago-gastric cancer cell lines. NanoString nCounter® miR analysis was performed in NCI-N87, FLO-1, and MAGIC trial (ISRCTN93793971) formalin-fixed paraffin-embedded (FFPE) oesophago-gastric cancer patient samples. MiR-148a-3p copies in plasma samples were quantified using digital droplet polymerase chain reaction (ddPCR) from HER2-positive oesophago-gastric cancer patients treated with standard-of-care trastuzumab-based therapy within the FOrMAT (NCT02112357) and PLATFORM (NCT02678182) clinical trials. The primary endpoints were overall survival (OS) for plasma miR-148a-3p HIGH (>median) versus LOW (≤median). The secondary endpoints were progression-free survival (PFS) and 3-month progression-free rates (PFRs) miR-148a-3p HIGH versus LOW. PLATFORM sensitivity analysis normalised miR-148a-3p (NmiR-148a-3p). Results: The inhibition of miR-148a-3p reduced NCI-N87 relative cell viability (<0.6) and expression was high (>242) in NCI-N87 and HER2-positive MAGIC trial patients (n=5). Normalised-miR-148a-3p (NmiR-148a-3p) LOW versus HIGH demonstrated a statistically significant difference in 3-month PFRs (n=23; OR, 0.11 [0.02-0.78]; p=0.027; aOR, 0.03 [0.001-0.71], p=0.029) but no difference in OS or PFS. There was no statistically significant relationship between miR-148-3p LOW versus HIGH for OS (PLATFORM, n=62; hazard ratio [HR], 0.98 [0.57-1.66]; p=0.933; FOrMAT, n=8; HR, 0.54 [0.13-2.31]; p=0.322), PFS (n=62; HR, 1.08 [0.65-1.81]; p=0.759; FOrMAT, n=8; HR, 1.26 [0.31-5.07]; p=0.714), or PFRs (PLATFORM, n=31; odds ratio [OR], 0.67 [0.2-2.8]; p=0.577). Conclusion: Normalised miR-148a-3p may be a relevant biomarker for trastuzumab-based therapy in advanced HER2-positive oesophago-gastric cancer patients.

NTRK fusions in colorectal cancer: clinical meaning and future perspective.

INTRODUCTION: Despite the efforts of the scientific community, the prognosis of metastatic colorectal cancer (mCRC) remains poor. Actionable gene fusions such as Neurotrophic Tropomyosin Receptor Kinases (NTRK) rearrangements are rare but might represent a new target to improve outcomes in this setting. The first-generation TRK inhibitors, larotrectinib and entrectinib, have demonstrated efficacy and safety in mCRC cancer patients exhibiting NTRK pathogenic fusions. Moreover, second-generation molecules are emerging, able to overcome the acquired resistance to NTRK blocking. AREAS COVERED: This review aims to report the current knowledge and the available evidence on NTRK fusion in mCRC, with a focus on molecular bases, clinical characteristics, prognostic meaning, and new therapeutic approaches, from the perspective of the clinical oncologist. EXPERT OPINION: Considering the limited options associated with the treatment of mCRC patients, the possibility of identifying new molecular biomarkers is an urgent clinical need. The availability of new molecular targets and the combinations of different agents might represent the true breakthrough point, allowing for change in the clinical course of colorectal cancer patients.

Challenges and perspectives for immunotherapy in oesophageal cancer: A look to the future (Review).

Oesophageal cancer is one of the most aggressive malignancies with limited treatment options, thus resulting in a high morbidity and mortality. With 5­year survival rates of only 5­10%, oesophageal cancer holds a dismal prognosis for patients. In order to improve overall survival, the early diagnosis and tools for patient stratification for personalized treatment are urgent needs. A minority of oesophageal cancers belong to the spectrum of Lynch syndrome­associated cancers and are characterized by microsatellite instability (MSI). Microsatellite instability is a consequence of defective mismatch repair protein functions and it has been well characterized in other gastrointestinal tumours, such as colorectal and gastric cancer. In the latter, high levels of MSI are associated with a better prognosis and with an increased benefit to immune­based therapies. Therefore, similar therapeutic approaches could offer an opportunity of treatment for oesophageal cancer patients with MSI. Apart from immune checkpoint inhibitors, other immunotherapies such as adoptive T­cell transfer, peptide vaccine and oncolytic viruses are under investigation in oesophageal cancer patients. In the present review, the rationale and current knowledge about immunotherapies in oesophageal cancer are summarised.

MIR21-induced loss of junctional adhesion molecule A promotes activation of oncogenic pathways, progression and metastasis in colorectal cancer.

Junctional adhesion molecules (JAMs) play a critical role in cell permeability, polarity and migration. JAM-A, a key protein of the JAM family, is altered in a number of conditions including cancer; however, consequences of JAM-A dysregulation on carcinogenesis appear to be tissue dependent and organ dependent with significant implications for the use of JAM-A as a biomarker or therapeutic target. Here, we test the expression and prognostic role of JAM-A downregulation in primary and metastatic colorectal cancer (CRC) (n = 947). We show that JAM-A downregulation is observed in ~60% of CRC and correlates with poor outcome in four cohorts of stages II and III CRC (n = 1098). Using JAM-A knockdown, re-expression and rescue experiments in cell line monolayers, 3D spheroids, patient-derived organoids and xenotransplants, we demonstrate that JAM-A silencing promotes proliferation and migration in 2D and 3D cell models and increases tumour volume and metastases in vivo. Using gene-expression and proteomic analyses, we show that JAM-A downregulation results in the activation of ERK, AKT and ROCK pathways and leads to decreased bone morphogenetic protein 7 expression. We identify MIR21 upregulation as the cause of JAM-A downregulation and show that JAM-A rescue mitigates the effects of MIR21 overexpression on cancer phenotype. Our results identify a novel molecular loop involving MIR21 dysregulation, JAM-A silencing and activation of multiple oncogenic pathways in promoting invasiveness and metastasis in CRC.

Modulation of pancreatic cancer cell sensitivity to FOLFIRINOX through microRNA-mediated regulation of DNA damage.

FOLFIRINOX, a combination of chemotherapy drugs (Fluorouracil, Oxaliplatin, Irinotecan -FOI), provides the best clinical benefit in pancreatic ductal adenocarcinoma (PDAC) patients. In this study we explore the role of miRNAs (MIR) as modulators of chemosensitivity to identify potential biomarkers of response. We find that 41 and 84 microRNA inhibitors enhance the sensitivity of Capan1 and MiaPaCa2 PDAC cells respectively. These include a MIR1307-inhibitor that we validate in further PDAC cell lines. Chemotherapy-induced apoptosis and DNA damage accumulation are higher in MIR1307 knock-out (MIR1307KO) versus control PDAC cells, while re-expression of MIR1307 in MIR1307KO cells rescues these effects. We identify binding of MIR1307 to CLIC5 mRNA through covalent ligation of endogenous Argonaute-bound RNAs cross-linking immunoprecipitation assay. We validate these findings in an in vivo model with MIR1307 disruption. In a pilot cohort of PDAC patients undergoing FOLFIRONX chemotherapy, circulating MIR1307 correlates with clinical outcome.

Personalized molecular targeted therapy in advanced, recurrent hepatocellular carcinoma after liver transplantation: a proof of principle.

BACKGROUND & AIMS: The advent of molecular medicine that targets specific pathways is changing the therapeutic approach to hepatocellular carcinoma. For several aberrantly activated pathways in hepatocarcinoma, surrogate markers of activation can be assessed by immunohistochemistry, although associations with in vivo response to targeted therapies are still lacking. METHODS: A patient, who presented with hepatic and extra-hepatic hepatocarcinoma recurrence 11 years after liver transplantation, was assessed for beta-catenin, pERK, and pS6 in primary and secondary tumor specimens, in order to define a possible activation of the Wnt, Ras/MAPK and Akt/mTOR pathways and design a personalized targeted therapy in absence of alternative treatment options. Moreover, mutation analysis of the beta-catenin gene (CTNNB1) and DNA microsatellite analyses were performed. RESULTS: The identification of the same mutation in the beta-catenin gene, as well as the same microsatellite pattern in tumor tissues taken 11 years apart, proved that the observed hepatocarcinoma was a true recurrence. Nuclear beta-catenin and pS6 in tumor cells were positive, whereas pERK was positive only in the peritumoral endothelium. This pattern of immunohistochemistry, after failure of sorafenib alone, lead to the choice to add the mTOR inhibitor, everolimus, to sorafenib. Three months later a 50% tumor reduction was observed, and after 6 months a further reduction of tumor vital components was confirmed, while a grade II gastrointestinal bleeding episode occurred. CONCLUSIONS: A personalized approach aimed to treat recurrent hepatocarcinoma is possible through analysis of tumoral molecular pathways. Partial success of the selected combination of sorafenib and everolimus supports the pivotal role of mTOR signalling and highlights the importance of reliable biomarkers to route the best molecular-based therapeutic options in HCC.

Integrative approach for prioritizing cancer genes in sporadic colon cancer.

The current multistep carcinogenesis models of colon cancer do not fully capture the genetic heterogeneity of the disease, which is additionally complicated by the presence of passenger and driver genetic alterations. The aim of this study was to select in the context of this significant heterogeneity additional genes functionally related to colon cancer development. High-throughput copy number and gene expression data of 36 microsatellite stable sporadic colon cancers resected from patients of a single institution characterized for mutations in APC, KRAS, TP53 and loss of 18q were analyzed. Genes whose expression correlated with the underlying copy number pattern were selected, and their association with the above listed mutations and overall survival was evaluated. Gain of 20q was strongly associated with TP53 mutation, and overall survival with alterations on 7p, 8p, 13q, 18q, and 20q. An association with 18q loss and gain of 8q24 was also observed. New candidate genes with a potential role in colon cancer are PLCG1 on 20q, DBC1 on 8q21, and NDGR1 on 8p24. In addition, an unexpected pattern of loss and mutability was found in the region upstream of the KRAS gene. By integrating copy number alterations with gene expression and mutations in colon cancer associated genes, we have developed a strategy that identifies previously known molecular features and additional players in the molecular landscape of colon cancer.

MicroRNAs as mediators of drug resistance mechanisms.

MicroRNAs are small RNA transcripts involved in fine-tuning of several cellular mechanisms and pathways crucial for maintaining cells' homeostasis like apoptosis, differentiation, inflammation and cell-cycle regulation. They act by regulation of gene expression at post-transcriptional level through fine-tuning of target proteins expression. Expression of microRNAs is cell-type specific and since their discovery they have been proven to be deregulated in various disorders including cancer. Several lines of evidence are emerging that link microRNAs to drug resistance mechanisms in tumours given their important role in modulating oncogenic and tumour suppressive mechanisms. This review will focus on latest knowledge of the roles and mechanisms of microRNAs as mediators to drug resistance and the implications for future therapies.

Immune-Based Therapies and the Role of Microsatellite Instability in Pancreatic Cancer.

Pancreatic cancer is one of the most aggressive malignancies with limited treatment options thus resulting in high morbidity and mortality. Among all cancers, with a five-year survival rates of only 2-9%, pancreatic cancer holds the worst prognostic outcome for patients. To improve the overall survival, an earlier diagnosis and stratification of cancer patients for personalized treatment options are urgent needs. A minority of pancreatic cancers belong to the spectrum of Lynch syndrome-associated cancers and are characterized by microsatellite instability (MSI). MSI is a consequence of defective mismatch repair protein functions and it has been well characterized in other gastrointestinal tumors such as colorectal and gastric cancer. In the latter, high levels of MSI are linked to a better prognosis and to an increased benefit to immune-based therapies. Therefore, the same therapies could offer an opportunity of treatment for pancreatic cancer patients with MSI. In this review, we summarize the current knowledge about immune-based therapies and MSI in pancreatic cancer.