Inhibition of homologous recombination repair by Mirin in ovarian cancer ameliorates carboplatin therapy response in vitro.

Chemoresistance poses one of the most significant challenges of cancer therapy. Carboplatin (CbPt) is one of the most used chemotherapeutics in ovarian cancer (OVC) treatment. MRE11 constitutes a part of homologous recombination (HR), which is responsible for the repair of CbPt-induced DNA damage, particularly DNA crosslinks. The study's main aim was to address the role of HR in CbPt chemoresistance in OVC and to evaluate the possibility of overcoming CbPt chemoresistance by Mirin-mediated MRE11 inhibition in an OVC cell line. Lower expression of MRE11 was associated with better overall survival in a cohort of OVC patients treated with platinum drugs (TCGA dataset, p < 0.05). Using in vitro analyses, we showed that the high expression of HR genes drives the CbPt chemoresistance in our CbPt-resistant cell line model. Moreover, the HR inhibition by Mirin not only increased sensitivity to carboplatin (p < 0.05) but also rescued the sensitivity in the CbPt-resistant model (p < 0.05). Our results suggest that MRE11 inhibition with Mirin may represent a promising way to overcome OVC resistance. More therapy options will ultimately lead to better personalized cancer therapy and improvement of patients' survival.

Differences in genome, transcriptome, miRNAome, and methylome in synchronous and metachronous liver metastasis of colorectal cancer.

Despite distant metastases being the critical factor affecting patients' survival, they remain poorly understood. Our study thus aimed to molecularly characterize colorectal cancer liver metastases (CRCLMs) and explore whether molecular profiles differ between Synchronous (SmCRC) and Metachronous (MmCRC) colorectal cancer. This characterization was performed by whole exome sequencing, whole transcriptome, whole methylome, and miRNAome. The most frequent somatic mutations were in APC, SYNE1, TP53, and TTN genes. Among the differently methylated and expressed genes were those involved in cell adhesion, extracellular matrix organization and degradation, neuroactive ligand-receptor interaction. The top up-regulated microRNAs were hsa-miR-135b-3p and -5p, and the hsa-miR-200-family while the hsa-miR-548-family belonged to the top down-regulated. MmCRC patients evinced higher tumor mutational burden, a wider median of duplications and deletions, and a heterogeneous mutational signature than SmCRC. Regarding chronicity, a significant down-regulation of SMOC2 and PPP1R9A genes in SmCRC compared to MmCRC was observed. Two miRNAs were deregulated between SmCRC and MmCRC, hsa-miR-625-3p and has-miR-1269-3p. The combined data identified the IPO5 gene. Regardless of miRNA expression levels, the combined analysis resulted in 107 deregulated genes related to relaxin, estrogen, PI3K-Akt, WNT signaling pathways, and intracellular second messenger signaling. The intersection between our and validation sets confirmed the validity of our results. We have identified genes and pathways that may be considered as actionable targets in CRCLMs. Our data also provide a valuable resource for understanding molecular distinctions between SmCRC and MmCRC. They have the potential to enhance the diagnosis, prognostication, and management of CRCLMs by a molecularly targeted approach.

MUC13-miRNA-4647 axis in colorectal cancer: Prospects to identifications of risk factors and clinical outcomes.

MUC13, a transmembrane mucin glycoprotein, is overexpressed in colorectal cancer (CRC), however, its regulation and functions are not fully understood. It has been shown that MUC13 protects colonic epithelial cells from apoptosis. Therefore, studying MUC13 and MUC13-regulated pathways may reveal promising therapeutic approaches for CRC treatment. Growing evidence suggests that microRNAs (miRs) are involved in the development and progression of CRC. In the present study, the MUC13-miR-4647 axis was addressed in association with survival of patients. miR-4647 is predicted in silico to bind to the MUC13 gene and was analyzed by RT-qPCR in 187 tumors and their adjacent non-malignant mucosa of patients with CRC. The impact of previously mentioned genes on survival and migration abilities of cancer cells was validated in vitro. Significantly upregulated MUC13 (P=0.02) in was observed tumor tissues compared with non-malignant adjacent mucosa, while miR-4647 (P=0.05) showed an opposite trend. Higher expression levels of MUC13 (log-rank P=0.05) were associated with worse patient's survival. The ectopic overexpression of studied miR resulted in decreased migratory abilities and worse survival of cells. Attenuated MUC13 expression levels confirmed the suppression of colony forming of CRC cells. In summary, the present data suggested the essential role of MUC13-miR-4647 in patients' survival, and this axis may serve as a novel therapeutic target. It is anticipated MUC13 may hold significant potential in the screening, diagnosis and treatment of CRC.

An investigation of DNA damage and DNA repair in chemical carcinogenesis triggered by small-molecule xenobiotics and in cancer: Thirty years with the comet assay.

In the present review we addressed the determination of DNA damage induced by small-molecule carcinogens, considered their persistence in DNA and mutagenicity in in vitro and in vivo systems over a period of 30 years. The review spans from the investigation of the role of DNA damage in the cascade of chemical carcinogenesis. In the nineties, this concept evolved into the biomonitoring studies comprising multiple biomarkers that not only reflected DNA/chromosomal damage, but also the potential of the organism for biotransformation/elimination of various xenobiotics. Since first years of the new millennium, dynamic system of DNA repair and host susceptibility factors started to appear in studies and a considerable knowledge has been accumulated on carcinogens and their role in carcinogenesis. It was understood that the final biological links bridging the arising DNA damage and cancer onset remain to be elucidated. In further years the community of scientists learnt that cancer is a multifactorial disease evolving over several decades of individual´s life. Moreover, DNA damage and DNA repair are inseparable players also in treatment of malignant diseases, but affect substantially other processes, such as degeneration. Functional monitoring of DNA repair pathways and DNA damage response may cast some light on above aspects. Very little is currently known about the relationship between telomere homeostasis and DNA damage formation and repair. DNA damage/repair in genomic and mitochondrial DNA and crosstalk between these two entities emerge as a new interesting topic.

Scalable Production of Monodisperse Bioactive Spider Silk Nanowires.

Elongated protein-based micro- and nanostructures are of great interest for a wide range of biomedical applications, where they can serve as a backbone for surface functionalization and as vehicles for drug delivery. Current production methods for protein constructs lack precise control of either shape and dimensions or render structures fixed to substrates. This work demonstrates production of recombinant spider silk nanowires suspended in solution, starting with liquid bridge induced assembly (LBIA) on a substrate, followed by release using ultrasonication, and concentration by centrifugation. The significance of this method lies in that it provides i) reproducability (standard deviation of length <13% and of diameter <38%), ii) scalability of fabrication, iii) compatibility with autoclavation with retained shape and function, iv) retention of bioactivity, and v) easy functionalization both pre- and post-formation. This work demonstrates how altering the function and nanotopography of a surface by nanowire coating supports the attachment and growth of human mesenchymal stem cells (hMSCs). Cell compatibility is further studied through integration of nanowires during aggregate formation of hMSCs and the breast cancer cell line MCF7. The herein-presented industrial-compatible process enables silk nanowires for use as functionalizing agents in a variety of cell culture applications and medical research.

MiR-140 leads to MRE11 downregulation and ameliorates oxaliplatin treatment and therapy response in colorectal cancer patients.

Cancer therapy failure is a fundamental challenge in cancer treatment. One of the most common reasons for therapy failure is the development of acquired resistance of cancer cells. DNA-damaging agents are frequently used in first-line chemotherapy regimens and DNA damage response, and DNA repair pathways are significantly involved in the mechanisms of chemoresistance. MRE11, a part of the MRN complex involved in double-strand break (DSB) repair, is connected to colorectal cancer (CRC) patients' prognosis. Our previous results showed that single-nucleotide polymorphisms (SNPs) in the 3' untranslated region (3'UTR) microRNA (miRNA) binding sites of MRE11 gene are associated with decreased cancer risk but with shorter survival of CRC patients, which implies the role of miRNA regulation in CRC. The therapy of colorectal cancer utilizes oxaliplatin (oxalato(trans-l-1,2-diaminocyclohexane)platinum), which is often compromised by chemoresistance development. There is, therefore, a crucial clinical need to understand the cellular processes associated with drug resistance and improve treatment responses by applying efficient combination therapies. The main aim of this study was to investigate the effect of miRNAs on the oxaliplatin therapy response of CRC patients. By the in silico analysis, miR-140 was predicted to target MRE11 and modulate CRC prognosis. The lower expression of miR-140 was associated with the metastatic phenotype (p < 0.05) and poor progression-free survival (odds ratio (OR) = 0.4, p < 0.05). In the in vitro analysis, we used miRNA mimics to increase the level of miR-140 in the CRC cell line. This resulted in decreased proliferation of CRC cells (p < 0.05). Increased levels of miR-140 also led to increased sensitivity of cancer cells to oxaliplatin (p < 0.05) and to the accumulation of DNA damage. Our results, both in vitro and in vivo, suggest that miR-140 may act as a tumor suppressor and plays an important role in DSB DNA repair and, consequently, CRC therapy response.

Genetic Susceptibility in Understanding of Pancreatic Ductal Adenocarcinoma Risk: A Decade-Long Effort of the PANDORA Consortium.

Pancreatic cancer, a complex disease, emerges as a severe health problem worldwide and it exhibits a poor prognosis and high mortality. Risk factors associated with sporadic pancreatic cancer remain poorly understood, even less is known about disease prognosis due to its rapid progression. The PANcreatic Disease ReseArch (PANDoRA) consortium, of which the authors are members, was established to coordinate the efforts of different research groups to uncover new genetic factors for pancreatic cancer risk, response to treatment, and patient survival. PANDoRA consortium has contributed to the identification of several low-penetrance risk loci for the disease both by candidate variants approach and genome-wide association studies, including those in cell-cycle and DNA damage response, telomere homeostasis, SCL and ABC transporters, ABO locus variability, mitochondrial metabolism and it participated on collaborative genome-wide association study approach and implementation of a search for functional-based pancreatic cancer risk loci and long noncoding RNAs. Complex studies covering genetic, environmental and microenvironmental factors in the pancreatic cancer onset, progression and its prognosis are warranted.

Analysis of MicroRNA Expression Changes During the Course of Therapy In Rectal Cancer Patients.

MicroRNAs (miRNAs) regulate gene expression in a tissue-specific manner. However, little is known about the miRNA expression changes induced by the therapy in rectal cancer (RC) patients. We evaluated miRNA expression levels before and after therapy and identified specific miRNA signatures reflecting disease course and treatment responses of RC patients. First, miRNA expression levels were assessed by next-generation sequencing in two plasma samplings (at the time of diagnosis and a year after) from 20 RC patients. MiR-122-5p and miR-142-5p were classified for subsequent validation in plasma and plasma extracellular vesicles (EVs) on an independent group of RC patients (n=107). Due to the intrinsic high differences in miRNA expression levels between samplings, cancer-free individuals (n=51) were included in the validation phase to determine the baseline expression levels of the selected miRNAs. Expression levels of these miRNAs were significantly different between RC patients and controls (for all p <0.001). A year after diagnosis, miRNA expression profiles were significantly modified in patients responding to treatment and were no longer different from those measured in cancer-free individuals. On the other hand, patients not responding to therapy maintained low expression levels in their second sampling (miR-122-5p: plasma: p=0.05, EVs: p=0.007; miR-142-5p: plasma: p=0.008). Besides, overexpression of miR-122-5p and miR-142-5p in RC cell lines inhibited cell growth and survival. This study provides novel evidence that circulating miR-122-5p and miR-142-5p have a high potential for RC screening and early detection as well as for the assessment of patients' outcomes and the effectiveness of treatment schedule.

DNA Repair and Ovarian Carcinogenesis: Impact on Risk, Prognosis and Therapy Outcome.

There is ample evidence for the essential involvement of DNA repair and DNA damage response in the onset of solid malignancies, including ovarian cancer. Indeed, highpenetrance germline mutations in DNA repair genes are important players in familial cancers: BRCA1, BRCA2 mutations or mismatch repair, and polymerase deficiency in colorectal, breast, and ovarian cancers. Recently, some molecular hallmarks (e.g., TP53, KRAS, BRAF, RAD51C/D or PTEN mutations) of ovarian carcinomas were identified. The manuscript overviews the role of DNA repair machinery in ovarian cancer, its risk, prognosis, and therapy outcome. We have attempted to expose molecular hallmarks of ovarian cancer with a focus on DNA repair system and scrutinized genetic, epigenetic, functional, and protein alterations in individual DNA repair pathways (homologous recombination, non-homologous end-joining, DNA mismatch repair, base- and nucleotide-excision repair, and direct repair). We suggest that lack of knowledge particularly in non-homologous end joining repair pathway and the interplay between DNA repair pathways needs to be confronted. The most important genes of the DNA repair system are emphasized and their targeting in ovarian cancer will deserve further attention. The function of those genes, as well as the functional status of the entire DNA repair pathways, should be investigated in detail in the near future.

Ganoderma Lucidum induces oxidative DNA damage and enhances the effect of 5-Fluorouracil in colorectal cancer in vitro and in vivo.

The first-line chemotherapy of colorectal cancer (CRC), besides surgery, comprises administration of 5-Fluorouracil (5FU). Apart from cytotoxic effect on cancer cells, 5FU may also cause adverse side effects. Ganoderma Lucidum (GLC) is a mushroom used in Traditional Eastern Medicine. We propose that natural compounds, particularly GLC extracts, may sensitize cancer cells to conventional chemotherapeutics. This combination therapy could lead to more selective cancer cell death and may improve the response to the therapy and diminish the adverse effects of anticancer drugs. Here we demonstrate that GLC induced oxidative DNA damage selectively in colorectal cancer cell lines, whereas it protected non-malignant cells from the accumulation of reactive oxygen species. Accumulation of DNA damage caused sensitization of cancer cells to 5FU resulting in improved anticancer effect of 5FU. The results obtained in colorectal cell lines were confirmed in in vivo study: GLC co-treatment with 5FU increased the survival of treated mice and reduced the tumor volume in comparison with group treated with 5FU alone. Combination of conventional chemotherapeutics and natural compounds is a promising approach, which may reduce the effective curative dose of anticancer drugs, suppress their adverse effects and ultimately lead to better quality of life of CRC patients.

Label-Free Surface Protein Profiling of Extracellular Vesicles by an Electrokinetic Sensor.

Small extracellular vesicles (sEVs) generated from the endolysosomal system, often referred to as exosomes, have attracted interest as a suitable biomarker for cancer diagnostics, as they carry valuable biological information and reflect their cells of origin. Herein, we propose a simple and inexpensive electrical method for label-free detection and profiling of sEVs in the size range of exosomes. The detection method is based on the electrokinetic principle, where the change in the streaming current is monitored as the surface markers of the sEVs interact with the affinity reagents immobilized on the inner surface of a silica microcapillary. As a proof-of-concept, we detected sEVs derived from the non-small-cell lung cancer (NSCLC) cell line H1975 for a set of representative surface markers, such as epidermal growth factor receptor (EGFR), CD9, and CD63. The detection sensitivity was estimated to be ∼175000 sEVs, which represents a sensor surface coverage of only 0.04%. We further validated the ability of the sensor to measure the expression level of a membrane protein by using sEVs displaying artificially altered expressions of EGFR and CD63, which were derived from NSCLC and human embryonic kidney (HEK) 293T cells, respectively. The analysis revealed that the changes in EGFR and CD63 expressions in sEVs can be detected with a sensitivity in the order of 10% and 3%, respectively, of their parental cell expressions. The method can be easily parallelized and combined with existing microfluidic-based EV isolation technologies, allowing for rapid detection and monitoring of sEVs for cancer diagnosis.

lncRNAs in Non-Malignant Tissue Have Prognostic Value in Colorectal Cancer.

Although colorectal cancer (CRC) is the third most frequent cause of cancer related death in Europe, clinically relevant biomarkers for therapy guidance and prognosis are insufficiently reliable. Long non-coding RNAs (lncRNAs) are RNAs over 200 nucleotides long that are not translated into proteins but can influence biological processes. There is emerging evidence for their involvement in solid cancer as oncogenes, tumour suppressors or regulators of cell proliferation and metastasis development. The goal of this study was to evaluate the prognostic effect of selected lncRNAs in a retrospective study on CRC patients from the Czech Republic. We used a quantitative PCR approach to measure the expression in paired non-malignant and tumour tissue samples of CRC patients of nine lncRNAs previously shown to be involved in cancer progression-ANRIL, CCAT1, GAS5, linc-ROR, MALAT1, MIR155HG, PCAT1, SPRY4-IT1 and TUG1. Associations between expression and expression ratios and clinical characteristics and survival were assessed by using univariable Cox proportional hazards models, Kaplan-Meier estimations with the Gehan-Wilcoxon test, the Mann-Whitney U test, the Kruskal-Wallis test and Spearman's correlations. A comparison of expression in tumour tissue (TT) and non-malignant mucosa tissue (MT) showed significant upregulation of CCAT1 and linc-ROR in TT (p < 0.001 and p = 0.001, respectively) and downregulation of ANRIL, MIR155HG and MALAT1 (p = 0.001, p = 0.010, p = 0.001, respectively). Linc-ROR was significantly associated with the presence of synchronous metastases (p = 0.033). For individual tissue types, lower MIR155HG expression in TT was correlated with both shorter overall survival (p = 0.008) and shorter disease-free survival (p = 0.040). In MT, expression ratios of CCAT1/ANRIL and CCAT1/MIR155HG were associated with overall survival (p = 0.005 and p = 0.006, respectively). Our results revealed that changes in expression of lncRNAs between MT and TT hold potential to be used as prognostic biomarkers in CRC patients. Moreover, the ratios of CCAT1 to ANRIL and MIR155HG in MT also exhibit potential for prognosis assessment without tumour sampling. Our results also indicate that cancer progression is associated with detrimental system-wide changes in patient tissue, which might govern patient survival even after successful elimination of tumour or cancerous cells.

Electrokinetic effect for molecular recognition: A label-free approach for real-time biosensing.

We present a simple and inexpensive method for label-free detection of biomolecules. The method monitors the changes in streaming current in a fused silica capillary as target biomolecules bind to immobilized receptors on the inner surface of the capillary. To validate the concept, we show detection and time response of different protein-ligand and protein-protein systems: biotin-avidin and biotin-streptavidin, barstar-dibarnase and Z domain-immunoglobulin G (IgG). We show that specific binding of these biomolecules can be reliably monitored using a very simple setup. Using sequential injections of various proteins at a diverse concentration range and as well as diluted human serum we further investigate the capacity of the proposed technique to perform specific target detection from a complex sample. We also investigate the time for the signal to reach equilibrium and its dependence on analyte concentration and demonstrate that the current setup can be used to detect biomolecules at a concentration as low as 100pM without requiring any advanced device fabrication procedures. Finally, an analytical model based on diffusion theory has been presented to explain the dependence of the saturation time on the analyte concentration and capillary dimensions and how reducing length and inner diameter of the capillary is predicted to give faster detection and in practice also lower limit of detection.

Sensitive, rapid and quantitative detection of substance P in serum samples using an integrated microfluidic immunochip.

Miniaturized diagnostic devices hold the promise of accelerate the specific and sensitive detection of various biomarkers, which can translate into many areas of medicine - from cheaper clinical trials, to early diagnosis and treatment of complex diseases. Therefore, we report on a disposable integrated chip-based capillary immunoassay featuring a microfluidic ELISA format combining electrochemical detection and low-cost fabrication employing a dry film photoresist, Vacrel(®) 8100. The readily accessible carboxylate groups on the material surface allow fast and high yield immobilization of biomolecules using amine-specific coupling via reactive esters requiring no laborious surface pretreatment. The integrated microfluidic system provides a convenient platform for a flow-through immunoassay. Capillary force is used for easy reagent delivery and loading the chip channel. We performed rapid quantification of serum level of substance P, a potential biomarker of acute neuroinflammation, using the developed microfluidic immunochip. Our miniaturized assay demonstrated a sensitive electrochemical detection of the antigen at 15.4pgml(-1) (11.5pM) using only 5µl of the biological fluid while cutting the total assay preparation time in half and the read-out time to 10min. Combining microfluidics and fabrication suitable for mass production with the capability of testing clinically relevant samples creates conditions for the construction of low-cost and portable point of care diagnostic devices with minimal auxiliary electronics.

A micro-cantilever sensor chip based on contact angle analysis for a label-free troponin I immunoassay.

Cantilever sensors have been extensively explored as a promising technique for real-time and label-free analyses in biological systems. A major sensing principle utilized by state-of-the-art cantilever sensors is based on analyte-induced surface stress changes, which result in static bending of a cantilever. The sensor performance, however, suffers from the intrinsically small change in surface stress induced by analytes, especially for molecular recognition such as antigen-antibody binding. Through the contact angle change on a tailored solid surface, it is possible to convert a tiny surface stress into a capillary force-a much larger physical quantity needed for a practical sensor application. In this work, a micro-cantilever sensor based on contact angle analysis (CAMCS) was proposed to effectively enhance the sensitivity of a sensor in proportion to the square of the length to thickness ratio of the cantilever structure. CAMCS chips were fabricated using a standard complementary-metal-oxide-semiconductor (CMOS) process to demonstrate a 1250-fold enhancement in the sensitivity of surface stress to bioanalyte adsorption using a piezoresistive sensing method. A real-time and label-free troponin I (cTnI) immunoassay, which is now widely used in clinics and considered a gold standard for the early diagnosis and prognosis of cardiovascular disease, was performed to demonstrate cTnI detection levels as low as 1 pg mL(-1). The short detection time of this assay was within several minutes, which matches the detection time of commercially available instruments that are based on fluorescence-labeling techniques.