Analysing Molecular Mechanism Related to Therapy- Resistance in In-vitro Models of Ovarian Cancer

Ovarian cancer is among the most common cause of cancer death and ranks first in the number of deaths each year in the field of gynaecological malignancies. This is due to its late diagnosis and the development of chemoresistance. Platinum derivates, including cisplatinum and carboplatin in combination with paclitaxel, are the first-line chemotherapeutic agents. Platinum derivates irreversibly intercalates into the DNA and creates inter- and intra-strand DNA cross-links. During cell division, platinum-DNA-adducts block the replication machinery, inducing DNA damage and apoptosis. Nearly all patients respond to first-line chemotherapy before it comes later to recurrence of the disease. At time of recurrence, tumours are usually more aggressive, form metastasis in secondary tissues and acquire resistance to conventional chemotherapeutics. Drug resistance is a common problem in tumour therapy not only restricted to ovarian cancer. It is characterized by gene mutations, increased DNA repair, reduced drug efficacy and enhanced drug clearance and detoxification. Up to now the complex molecular mechanism of chemoresistance is not well understood. Increasing evidence points towards AKT over-expression and alteration of the PI3K/AKT/mTOR cascade as a central mechanistic reason for this resistance

Non-Coding RNAs and Resistance to Anticancer Drugs in Gastrointestinal Tumors

Non-coding RNAs are important regulators of gene expression and transcription. It is well established that impaired non-coding RNA expression especially the one of long non-coding RNAs and microRNAs is involved in a number of pathological conditions including cancer. Non-coding RNAs are responsible for the development of resistance to anticancer treatments as they regulate drug resistance-related genes, affect intracellular drug concentrations, induce alternative signaling pathways, alter drug efficiency via blocking cell cycle regulation, and DNA damage response. Furthermore, they can prevent therapeutic-induced cell death and promote epithelial–mesenchymal transition (EMT) and elicit non-cell autonomous mechanisms of resistance. In this review, we summarize the role of non-coding RNAs for different mechanisms resulting in drug resistance (e.g., drug transport, drug metabolism, cell cycle regulation, regulation of apoptotic pathways, cancer stem cells, and EMT) in the context of gastrointestinal cancers

Characterisation of the immune-related transcriptome in resected biliary tract cancers

Although biliary tract cancers (BTCs) are known to have an inflammatory component, a detailed characterisation of immune-related transcripts has never been performed. In these studies, nCounter PanCancer Immune Profiling Panel was used to assess the expression of 770 immune-related transcripts in the tumour tissues (TTs) and matched adjacent tissues (ATs) of resected BTCs. Cox regression analysis and Kaplan-Meier methods were used to correlate findings with relapse-free survival (RFS). The first analysis in the TT and AT of an exploratory set (n = 22) showed deregulation of 39 transcripts associated with T-cell activation. Risk of recurrence was associated with a greater number of genes deregulated in AT in comparison to TT. Analysis in the whole set (n = 53) showed a correlation between AT cytotoxic T-lymphocyte antigen-4 (CTLA4) expression and RFS, which maintained statistical significance at multivariate analysis. CTLA4 expression correlated with forkhead box P3 (FOXP3) expression, suggesting enrichment in T regulatory cells. CTLA4 is known to act by binding to the cluster of differentiation 80 (CD80). No association was seen between AT CD80 expression and RFS. However, CD80 expression differentiated prognosis in patients who received adjuvant chemotherapy. We showed that the immunomodulatory transcriptome is deregulated in resected BTCs. Our study includes a small number of patients and does not enable to draw definitive conclusions; however, it provides useful insights into potential transcripts that may deserve further investigation in larger cohorts of patients. TRANSCRIPT PROFILING: Nanostring data have been submitted to GEO repository: GSE90698 and GSE906

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

MIR21 drives resistance to Heat Shock Protein 90 inhibition in cholangiocarcinoma

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

Modulation of biliary cancer chemo-resistance through microRNA-mediated rewiring of the expansion of CD133+ cells

Changes in single microRNA (MIR) expression have been associated with chemo-resistance in Biliary Tract Cancer (BTC). 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 of 997 LNA-MIR-inhibitors was performed in 6 CCA cell lines treated with Cisplatin-Gemcitabine (CG) seeking changes in cell viability. Validation experiments were performed with miRvana probes. MIR and gene expression was assessed by TaqMan-assay, RNA-sequencing and in-situ-hybridization in 4 indepedent cohorts of human BTC. Knock-out of microRNA was achieved by CRISPR-CAS9 in CCLP cells (MIR1249KO) and tested for effects on chemotherapy sensitivity in-vitro and in-vivo. High-throughput-screening revealed that MIR1249-inhibition enhanced chemotherapy sensitivity across all cell lines. MIR1249 expression was increased in 41% of cases in human BTC. In validation experiments, MIR1249-inhibition did not alter cell viability in untreated or DMSO-treated cells; however it did increase CG effect. MIR1249 expression was increased in CD133+ biliary cancer cells freshly isolated from the stem niche of human BTC, as well as in CD133+ chemo-resistant CCLP cells. MIR1249 modulated the chemotherapy-induced enrichment of CD133+ cells by controlling their clonal expansion via 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 tumour shrinkage after exposure to weekly CG, while WT models showed only stable disease over treatment

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

Maturation of the Language Network: From Inter- to Intrahemispheric Connectivities

Language development must go hand-in-hand with brain maturation. Little is known about how the brain develops to serve language processing, in particular, the processing of complex syntax, a capacity unique to humans. Behavioral reports indicate that the ability to process complex syntax is not yet adult-like by the age of seven years. Here, we apply a novel method to demonstrate that the basic neural basis of language, as revealed by low frequency fluctuation stemming from functional MRI data, differs between six-year-old children and adults in crucial aspects. Although the classical language regions are actively in place by the age of six, the functional connectivity between these regions clearly is not. In contrast to adults who show strong connectivities between frontal and temporal language regions within the left hemisphere, children's default language network is characterized by a strong functional interhemispheric connectivity, mainly between the superior temporal regions. These data indicate a functional reorganization of the neural network underlying language development towards a system that allows a close interplay between frontal and temporal regions within the left hemisphere

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

Funder: FP7 People: Marie-Curie Actions; FundRef: http://dx.doi.org/10.13039/100011264; Grant(s): CIG 334261OBJECTIVE: 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 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