UBE2D3 facilitates NHEJ by orchestrating ATM signalling through multi-level control of RNF168.

Maintenance of genome integrity requires tight control of DNA damage response (DDR) signalling and repair, with phosphorylation and ubiquitination representing key elements. How these events are coordinated to achieve productive DNA repair remains elusive. Here we identify the ubiquitin-conjugating enzyme UBE2D3 as a regulator of ATM kinase-induced DDR that promotes non-homologous end-joining (NHEJ) at telomeres. UBE2D3 contributes to DDR-induced chromatin ubiquitination and recruitment of the NHEJ-promoting factor 53BP1, both mediated by RNF168 upon ATM activation. Additionally, UBE2D3 promotes NHEJ by limiting RNF168 accumulation and facilitating ATM-mediated phosphorylation of KAP1-S824. Mechanistically, defective KAP1-S824 phosphorylation and telomeric NHEJ upon UBE2D3-deficiency are linked to RNF168 hyperaccumulation and aberrant PP2A phosphatase activity. Together, our results identify UBE2D3 as a multi-level regulator of NHEJ that orchestrates ATM and RNF168 activities. Moreover, they reveal a negative regulatory circuit in the DDR that is constrained by UBE2D3 and consists of RNF168- and phosphatase-mediated restriction of KAP1 phosphorylation.

Large-scale identification of rodenticide resistance in Rattus norvegicus and Mus musculus in the Netherlands based on Vkorc1 codon 139 mutations.

BACKGROUND: Resistance to rodenticides has been reported globally and poses a considerable problem for efficacy in pest control. The most-documented resistance to rodenticides in commensal rodents is associated with mutations in the Vkorc1 gene, in particular in codon 139. Resistance to anticoagulant rodenticides has been reported in the Netherlands since 1989. A study from 2013 showed that 25% of 169 Norway rats (Rattus norvegicus) had a mutation at codon 139 of the Vkorc1 gene. To gain insight in the current status of rodenticide resistance amongst R. norvegicus and house mice Mus musculus in the Netherlands, we tested these rodents for mutations in codon 139 of the Vkorc1 gene. In addition, we collected data from pest controllers on their use of rodenticides and experience with rodenticide resistance. RESULTS: A total of 1801 rodent samples were collected throughout the country consisting of 1404 R. norvegicus and 397 M. musculus. In total, 15% of R. norvegicus [95% confidence interval (CI): 13-17%] and 38% of M. musculus (95% CI: 33-43%) carried a genetic mutation at codon 139 of the Vkorc1 gene. CONCLUSION: This study demonstrates genetic mutations at codon 139 of the Vkorc1 gene in M. musculus in the Netherlands. Resistance to anticoagulant rodenticides is present in R. norvegicus and M. musculus in multiple regions in the Netherlands. The results of this comprehensive study provide a baseline and facilitate trend analyses of Vkorc1 codon 139 mutations and evaluation of integrated pest management (IPM) strategies as these are enrolled in the Netherlands. © 2022 The Dutch Pest and Wildlife. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Seasonal dynamics of tick burden and associated Borrelia burgdorferi s.l. and Borrelia miyamotoi infections in rodents in a Dutch forest ecosystem.

Ixodes ricinus ticks transmit Borrelia burgdorferi sensu lato (s.l.) as well as Borrelia miyamotoi. Larvae become infected when feeding on infected rodents, with horizontal transmission of B. burgdorferi and horizontal and vertical transmission of B. miyamotoi. We studied seasonal dynamics of infection rates of I. ricinus and their rodent hosts, and hence transmission risk of these two distinctly different Borrelia species. Rodents were live-trapped and inspected for ticks from May to November in 2013 and 2014 in a forest in The Netherlands. Trapped rodents were temporarily housed in the laboratory and detached ticks were collected. Borrelia infections were determined from the trapped rodents and collected ticks. Borrelia burgdorferi s.l. and B. miyamotoi were found in ticks as well as in rodents. Rodent density was higher in 2014, whereas tick burden as well as the Borrelia infection rates in rodents were higher in 2013. The density of B. miyamotoi-infected nymphs did not differ between the years. Tick burdens were higher on Apodemus sylvaticus than on Myodes glareolus, and higher on males than on females. Borrelia-infection rate of rodents varied strongly seasonally, peaking in summer. As the larval tick burden also peaked in summer, the generation of infected nymphs was highest in summer. We conclude that the heterogeneity of environmental and host-specific factors affects the seasonal transmission of Borrelia spp., and that these effects act more strongly on horizontally transmitted B. burgdorferi spp. than on the vertically transmitted B. miyamotoi.

MAD2L2 dimerization and TRIP13 control shieldin activity in DNA repair.

MAD2L2 (REV7) plays an important role in DNA double-strand break repair. As a member of the shieldin complex, consisting of MAD2L2, SHLD1, SHLD2 and SHLD3, it controls DNA repair pathway choice by counteracting DNA end-resection. Here we investigated the requirements for shieldin complex assembly and activity. Besides a dimerization-surface, HORMA-domain protein MAD2L2 has the extraordinary ability to wrap its C-terminus around SHLD3, likely creating a very stable complex. We show that appropriate function of MAD2L2 within shieldin requires its dimerization, mediated by SHLD2 and accelerating MAD2L2-SHLD3 interaction. Dimerization-defective MAD2L2 impairs shieldin assembly and fails to promote NHEJ. Moreover, MAD2L2 dimerization, along with the presence of SHLD3, allows shieldin to interact with the TRIP13 ATPase, known to drive topological switches in HORMA-domain proteins. We find that appropriate levels of TRIP13 are important for proper shieldin (dis)assembly and activity in DNA repair. Together our data provide important insights in the dependencies for shieldin activity.

Effectiveness and costs of interventions to reduce the within-farm Toxoplasma gondii seroprevalence on pig farms in the Netherlands.

BACKGROUND: The parasite Toxoplasma gondii (T. gondii) is recognized as one of the major foodborne pathogens with a high human disease burden. To control T. gondii infections in pigs, European Food Safety Agency (EFSA) advises serological testing of pigs and audits of pig farms to identify risk factors for T. gondii infection. In line with this approach, the aim of the current study was to assess the effectiveness and costs of intervention measures implemented to reduce the T. gondii seroprevalence on finishing pig farms in the Netherlands. A crossover clinical trial was conducted at five case farms were their own control and the cross-over moment was the implementation of interventions to reduce risk factors. Each of the case farms had a farm-specific intervention strategy with one principal intervention measure (neutering of cats, professional rodent control or covering food storage). RESULTS: All finishing pig farms (n = 5) showed a reduction in T. gondii seroprevalence within one year of implementing the intervention strategy. Cat neutering (n = 3) and feed coverage (n = 1) showed statistically significant reductions in seroprevalence. Rodent control (n = 1) did not show a statistically significant reduction. The estimated reduction in seroprevalence in response to the neutering of cats and feed coverage were 67 and 96 %, respectively. CONCLUSIONS: Our work demonstrates that it is possible to reduce the within-farm T. gondii seroprevalence within one year after interventions were implemented to reduce T. gondii risk factors. This information is essential and encouraging for policy makers, food business operators, and farmers to implement in their risk assessment and to apply to food safety control systems.

REV7: Jack of many trades.

The HORMA domain protein REV7, also known as MAD2L2, interacts with a variety of proteins and thereby contributes to the establishment of different complexes. With doing so, REV7 impacts a diverse range of cellular processes and gained increasing interest as more of its activities became uncovered. REV7 has important roles in translesion synthesis and mitotic progression, and acts as a central component in the recently discovered shieldin complex that operates in DNA double-strand break repair. Here we discuss the roles of REV7 in its various complexes, focusing on its activity in genome integrity maintenance. Moreover, we will describe current insights on REV7 structural features that allow it to be such a versatile protein.

Efficacy of Management and Monitoring Methods to Prevent Post-Harvest Losses Caused by Rodents.

The presence of pest rodents around food production and storage sites is one of many underlying problems contributing to food contamination and loss, particularly influencing food and nutrition security in low-income countries. By reducing both pre- and post-harvest losses by rodents, millions of food-insecure people would benefit. As there are limited quantitative data on post-harvest rice losses due to rodents, our objectives were to assess stored rice losses in local households from eight rural communities and two rice milling factories in Bangladesh and to monitor the effect of different rodent control strategies to limit potential losses. Four treatments were applied in 2016 and 2017, (i) untreated control, (ii) use of domestic cats, (iii) use of rodenticides, (iv) use of snap-traps. In total, over a two-year period, 210 rodents were captured from inside people's homes, with Rattus rattus trapped most often (n = 91), followed by Mus musculus (n = 75) and Bandicota bengalensis (n = 26). In the milling stations, 68 rodents were trapped, of which 21 were M. musculus, 19 R. rattus, 17 B. bengalensis, 8 Rattus exulans, and 3 Mus terricolor. In 2016, losses from standardised baskets of rice within households were between 13.6% and 16.7%. In 2017, the losses were lower, ranging from 0.6% to 2.2%. Daily rodent removal by trapping proved to be most effective to diminish stored produce loss. The effectiveness of domestic cats was limited.

H3K36 dimethylation by MMSET promotes classical non-homologous end-joining at unprotected telomeres.

The epigenetic environment plays an important role in DNA damage recognition and repair, both at DNA double-strand breaks and at deprotected telomeres. To increase understanding on how DNA damage responses (DDR) at deprotected telomeres are regulated by modification and remodeling of telomeric chromatin we screened 38 methyltransferases for their ability to promote telomere dysfunction-induced genomic instability. As top hit we identified MMSET, a histone methyltransferase (HMT) causally linked to multiple myeloma and Wolf-Hirschhorn syndrome. We show that MMSET promotes non-homologous end-joining (NHEJ) at deprotected telomeres through Ligase4-dependent classical NHEJ, and does not contribute to Ligase3-dependent alternative NHEJ. Moreover, we show that this is dependent on the catalytic activity of MMSET, enabled by its SET-domain. Indeed, in absence of MMSET H3K36-dimethylation (H3K36me2) decreases, both globally and at subtelomeric regions. Interestingly, the level of MMSET-dependent H3K36me2 directly correlates with NHEJ-efficiency. We show that MMSET depletion does not impact on recognition of deprotected telomeres by the DDR-machinery or on subsequent recruitment of DDR-factors acting upstream or at the level of DNA repair pathway choice. Our data are most consistent with an important role for H3K36me2 in more downstream steps of the DNA repair process. Moreover, we find additional H3K36me2-specific HMTs to contribute to NHEJ at deprotected telomeres, further emphasizing the importance of H3K36me2 in DNA repair.

Wild rodents and insectivores as carriers of pathogenic Leptospira and Toxoplasma gondii in The Netherlands.

Small mammals such as rodents can to carry zoonotic pathogens. Currently, there is impaired knowledge on zoonotic pathogens in rodents and insectivores in the Netherlands. This limits opportunities for preventive measures and complicates risk-assessments for zoonotic transmission to humans. Leptospira spp. and Toxoplasma gondii are present on a list of prioritized emerging pathogens in the Netherlands and were therefore the focus of this study. Both pathogens have the ability to survive under moist environmental conditions. In total, a group of 379 small mammals (rodents & insectivores) were tested on pathogenic Leptospira spp., and 312 on T. gondii. Rodents and insectivores were trapped at various sites, but mostly on pig and dairy farms throughout the country. Over five percent of the animals (5.3%, n = 379) tested positive for Leptospira DNA, and five of the animals (1.6%, n = 312) tested were positive for T. gondii DNA. The animals positive for T.gondii were all brown rats and the ones for Leptospira spp. were various species. Our results show that insectivores and rodents might be used as an indicator for the environmental contamination and/or the contamination in wildlife for Leptospira spp.

Evidence of Toxoplasma gondii in Rodents from Bangladesh.

Rodents contribute to the life cycle of the protozoan parasite Toxoplasma gondii as an intermediate host and key prey animal of cats, the definitive host. As there is limited scientific knowledge available about the incidence and prevalence of T. gondii in commensal rodents in many Asian countries, we tested rodents from a commercial rice mill and eight local villages in Bangladesh for the presence of T. gondii DNA using rodent brain material preserved in ethanol. Overall, 10 of 296 (3.4%) rodent samples tested positive for Toxoplasma DNA. Our results indicate that rodents present in food production and food storage facilities may carry T. gondii.

Prevalence of Leptospira Infection in Rodents from Bangladesh.

Worldwide, Leptospira infection poses an increasing public health problem. In 2008, leptospirosis was recognised as a re-emerging zoonosis of global importance with South-East Asia being one of the most significant centres of the disease. Rodents are thought to be the most important host for a variety of Leptospira serovars. Because Bangladesh offers a suitable humid climate for the survival of these pathogenic bacteria, the presence of rodents could be a serious risk for human infection, especially in peri-urban areas or locations where food is stored. In order to gain more understanding of the multi-host epidemiology, a prevalence study was conducted in Comilla, Bangladesh to determine the presence of pathogenic Leptospira species in rodents. Real-time Polymerase Chain Reaction (qPCR) and sequencing showed that 13.1% (61/465) of the trapped rodents were infected with pathogenic Leptospira. Sequencing of the qPCR products identified the presence of three species: Leptospira interrogans, Leptospira borgpetersenii, and Leptospira kirschneri. Rodents of the genus, Bandicota, were significantly more likely to be positive than those of the genus, Rattus and Mus. Our results confirm the importance of rodents as hosts of pathogenic Leptospira and indicate that human exposure to pathogenic Leptospira may be considerable, also in places where food (rice) is stored for longer times. This study emphasizes the need to improve rodent management at such locations and to further quantify the public health impacts of this neglected emerging zoonosis in Bangladesh.

Shieldin complex promotes DNA end-joining and counters homologous recombination in BRCA1-null cells.

BRCA1 deficiencies cause breast, ovarian, prostate and other cancers, and render tumours hypersensitive to poly(ADP-ribose) polymerase (PARP) inhibitors. To understand the resistance mechanisms, we conducted whole-genome CRISPR-Cas9 synthetic-viability/resistance screens in BRCA1-deficient breast cancer cells treated with PARP inhibitors. We identified two previously uncharacterized proteins, C20orf196 and FAM35A, whose inactivation confers strong PARP-inhibitor resistance. Mechanistically, we show that C20orf196 and FAM35A form a complex, 'Shieldin' (SHLD1/2), with FAM35A interacting with single-stranded DNA through its C-terminal oligonucleotide/oligosaccharide-binding fold region. We establish that Shieldin acts as the downstream effector of 53BP1/RIF1/MAD2L2 to promote DNA double-strand break (DSB) end-joining by restricting DSB resection and to counteract homologous recombination by antagonizing BRCA2/RAD51 loading in BRCA1-deficient cells. Notably, Shieldin inactivation further sensitizes BRCA1-deficient cells to cisplatin, suggesting how defining the SHLD1/2 status of BRCA1-deficient tumours might aid patient stratification and yield new treatment opportunities. Highlighting this potential, we document reduced SHLD1/2 expression in human breast cancers displaying intrinsic or acquired PARP-inhibitor resistance.

The CST Complex Mediates End Protection at Double-Strand Breaks and Promotes PARP Inhibitor Sensitivity in BRCA1-Deficient Cells.

Selective elimination of BRCA1-deficient cells by inhibitors of poly(ADP-ribose) polymerase (PARP) is a prime example of the concept of synthetic lethality in cancer therapy. This interaction is counteracted by the restoration of BRCA1-independent homologous recombination through loss of factors such as 53BP1, RIF1, and REV7/MAD2L2, which inhibit end resection of DNA double-strand breaks (DSBs). To identify additional factors involved in this process, we performed CRISPR/SpCas9-based loss-of-function screens and selected for factors that confer PARP inhibitor (PARPi) resistance in BRCA1-deficient cells. Loss of members of the CTC1-STN1-TEN1 (CST) complex were found to cause PARPi resistance in BRCA1-deficient cells in vitro and in vivo. We show that CTC1 depletion results in the restoration of end resection and that the CST complex may act downstream of 53BP1/RIF1. These data suggest that, in addition to its role in protecting telomeres, the CST complex also contributes to protecting DSBs from end resection.

H4K20me2 distinguishes pre-replicative from post-replicative chromatin to appropriately direct DNA repair pathway choice by 53BP1-RIF1-MAD2L2.

The main pathways for the repair of DNA double strand breaks (DSBs) are non-homologous end-joining (NHEJ) and homologous recombination directed repair (HDR). These operate mutually exclusive and are activated by 53BP1 and BRCA1, respectively. As HDR can only succeed in the presence of an intact copy of replicated DNA, cells employ several mechanisms to inactivate HDR in the G1 phase of cell cycle. As cells enter S-phase, these inhibitory mechanisms are released and HDR becomes active. However, during DNA replication, NHEJ and HDR pathways are both functional and non-replicated and replicated DNA regions co-exist, with the risk of aberrant HDR activity at DSBs in non-replicated DNA. It has become clear that DNA repair pathway choice depends on inhibition of DNA end-resection by 53BP1 and its downstream factors RIF1 and MAD2L2. However, it is unknown how MAD2L2 accumulates at DSBs to participate in DNA repair pathway control and how the NHEJ and HDR repair pathways are appropriately activated at DSBs with respect to the replication status of the DNA, such that NHEJ acts at DSBs in pre-replicative DNA and HDR acts on DSBs in post-replicative DNA. Here we show that MAD2L2 is recruited to DSBs in H4K20 dimethylated chromatin by forming a protein complex with 53BP1 and RIF1 and that MAD2L2, similar to 53BP1 and RIF1, suppresses DSB accumulation of BRCA1. Furthermore, we show that the replication status of the DNA locally ensures the engagement of the correct DNA repair pathway, through epigenetics. In non-replicated DNA, saturating levels of the 53BP1 binding site, di-methylated lysine 20 of histone 4 (H4K20me2), lead to robust 53BP1-RIF1-MAD2L2 recruitment at DSBs, with consequent exclusion of BRCA1. Conversely, replication-associated 2-fold dilution of H4K20me2 promotes the release of the 53BP1-RIF1-MAD2L2 complex and favours the access of BRCA1. Thus, the differential H4K20 methylation status between pre-replicative and post-replicative DNA represents an intrinsic mechanism that locally ensures appropriate recruitment of the 53BP1-RIF1-MAD2L2 complex at DNA DSBs, to engage the correct DNA repair pathway.

The need to implement the landscape of fear within rodent pest management strategies.

Current reactive pest management methods have serious drawbacks such as the heavy reliance on chemicals, emerging genetic rodenticide resistance and high secondary exposure risks. Rodent control needs to be based on pest species ecology and ethology to facilitate the development of ecologically based rodent management (EBRM). An important aspect of EBRM is a strong understanding of rodent pest species ecology, behaviour and spatiotemporal factors. Gaining insight into the behaviour of pest species is a key aspect of EBRM. The landscape of fear (LOF) is a mapping of the spatial variation in the foraging cost arising from the risk of predation, and reflects the levels of fear a prey species perceives at different locations within its home range. In practice, the LOF maps habitat use as a result of perceived fear, which shows where bait or traps are most likely to be encountered and used by rodents. Several studies have linked perceived predation risk of foraging animals with quitting-harvest rates or giving-up densities (GUDs). GUDs have been used to reflect foraging behaviour strategies of predator avoidance, but to our knowledge very few papers have directly used GUDs in relation to pest management strategies. An opportunity for rodent control strategies lies in the integration of the LOF of rodents in EBRM methodologies. Rodent management could be more efficient and effective by concentrating on those areas where rodents perceive the least levels of predation risk. © 2017 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

PARP1 Links CHD2-Mediated Chromatin Expansion and H3.3 Deposition to DNA Repair by Non-homologous End-Joining.

The response to DNA double-strand breaks (DSBs) requires alterations in chromatin structure to promote the assembly of repair complexes on broken chromosomes. Non-homologous end-joining (NHEJ) is the dominant DSB repair pathway in human cells, but our understanding of how it operates in chromatin is limited. Here, we define a mechanism that plays a crucial role in regulating NHEJ in chromatin. This mechanism is initiated by DNA damage-associated poly(ADP-ribose) polymerase 1 (PARP1), which recruits the chromatin remodeler CHD2 through a poly(ADP-ribose)-binding domain. CHD2 in turn triggers rapid chromatin expansion and the deposition of histone variant H3.3 at sites of DNA damage. Importantly, we find that PARP1, CHD2, and H3.3 regulate the assembly of NHEJ complexes at broken chromosomes to promote efficient DNA repair. Together, these findings reveal a PARP1-dependent process that couples ATP-dependent chromatin remodeling with histone variant deposition at DSBs to facilitate NHEJ and safeguard genomic stability.

Prevalence of Toxoplasma gondii in common moles (Talpa europaea).

BACKGROUND: The prevalence of Toxoplasma gondii in common moles, Talpa europaea, was investigated in order to determine whether moles can serve as an indicator species for T. gondii infections in livestock. FINDINGS: In total, 86 moles were caught from 25 different sites in the Netherlands. Five different trapping habitats were distinguished: pasture, garden, forest, roadside, and recreation area. No positive samples (brain cysts) were found during microscopic detection (n = 70). Using the Latex Agglutination Test (LAT), sera of 70 moles were examined, whereby no sample reacted with T. gondii antigen. Real Time-PCR tests on brain tissue showed 2 positive samples (2.3%). CONCLUSIONS: Because of the low number of positives in our study, the use of the common mole as an indicator species for livestock infections is currently not recommended.

RAS and RHO families of GTPases directly regulate distinct phosphoinositide 3-kinase isoforms.

RAS proteins are important direct activators of p110α, p110γ, and p110δ type I phosphoinositide 3-kinases (PI3Ks), interacting via an amino-terminal RAS-binding domain (RBD). Here, we investigate the regulation of the ubiquitous p110ß isoform of PI3K, implicated in G-protein-coupled receptor (GPCR) signaling, PTEN-loss-driven cancers, and thrombocyte function. Unexpectedly, RAS is unable to interact with p110ß, but instead RAC1 and CDC42 from the RHO subfamily of small GTPases bind and activate p110ß via its RBD. In fibroblasts, GPCRs couple to PI3K through Dock180/Elmo1-mediated RAC activation and subsequent interaction with p110ß. Cells from mice carrying mutations in the p110ß RBD show reduced PI3K activity and defective chemotaxis, and these mice are resistant to experimental lung fibrosis. These findings revise our understanding of the regulation of type I PI3K by showing that both RAS and RHO family GTPases directly regulate distinct ubiquitous PI3K isoforms and that RAC activates p110ß downstream of GPCRs.

Beyond KRAS mutation status: influence of KRAS copy number status and microRNAs on clinical outcome to cetuximab in metastatic colorectal cancer patients.

BACKGROUND: KRAS mutation is a negative predictive factor for treatment with anti-epidermal growth factor receptor (EGFR) antibodies in metastatic colorectal cancer (mCRC). Novel predictive markers are required to further improve the selection of patients for this treatment. We assessed the influence of modification of KRAS by gene copy number aberration (CNA) and microRNAs (miRNAs) in correlation to clinical outcome in mCRC patients treated with cetuximab in combination with chemotherapy and bevacizumab. METHODS: Formalin-fixed paraffin-embedded primary tumour tissue was used from 34 mCRC patients in a phase III trial, who were selected based upon their good (n = 17) or poor (n = 17) progression-free survival (PFS) upon treatment with cetuximab in combination with capecitabine, oxaliplatin, and bevacizumab. Gene copy number at the KRAS locus was assessed using high resolution genome-wide array CGH and the expression levels of 17 miRNAs targeting KRAS were determined by real-time PCR. RESULTS: Copy number loss of the KRAS locus was observed in the tumour of 5 patients who were all good responders including patients with a KRAS mutation. Copy number gains in two wild-type KRAS tumours were associated with a poor PFS. In KRAS mutated tumours increased miR-200b and decreased miR-143 expression were associated with a good PFS. In wild-type KRAS patients, miRNA expression did not correlate with PFS in a multivariate model. CONCLUSIONS: Our results indicate that the assessment of KRAS CNA and miRNAs targeting KRAS might further optimize the selection of mCRC eligible for anti-EGFR therapy.

MicroRNAs in colorectal cancer metastasis.

Metastatic disease is the major cause of death in colorectal cancer (CRC) patients. The metastatic process is highly inefficient and comprises multiple sequential steps. While many genetic factors relevant in this process have already been identified, the epigenetic factors underlying each step still remain obscure. MicroRNAs (miRNAs) are key regulators in tumourigenesis, but their role in the development of cancer metastasis is poorly investigated. The majority of miRNAs involved in the metastatic process have been identified in breast cancer cell lines, and in CRC less data are available. We review the role of miRNAs in the metastatic pathway of CRC, including escape of apoptosis, epithelial-mesenchymal transition (EMT), angiogenesis, and invasion. Better understanding of the complex role of miRNAs in the development of CRC metastases may provide new insights that could be of therapeutic consequence.