Cyfip1 is a putative invasion suppressor in epithelial cancers.

Identification of bona fide tumor suppressors is often challenging because of the large number of genetic alterations present in most human cancers. To evaluate candidate genes present within chromosomal regions recurrently deleted in human cancers, we coupled high-resolution genomic analysis with a two-stage genetic study using RNA interference (RNAi). We found that Cyfip1, a subunit of the WAVE complex, which regulates cytoskeletal dynamics, is commonly deleted in human epithelial cancers. Reduced expression of CYFIP1 is commonly observed during invasion of epithelial tumors and is associated with poor prognosis in this setting. Silencing of Cyfip1 disturbed normal epithelial morphogenesis in vitro and cooperated with oncogenic Ras to produce invasive carcinomas in vivo. Mechanistically, we have linked alterations in WAVE-regulated actin dynamics with impaired cell-cell adhesion and cell-ECM interactions. Thus, we propose Cyfip1 as an invasion suppressor gene.

IPNA clinical practice recommendations for the diagnosis and management of children with steroid-sensitive nephrotic syndrome.

Idiopathic nephrotic syndrome is the most frequent pediatric glomerular disease, affecting from 1.15 to 16.9 per 100,000 children per year globally. It is characterized by massive proteinuria, hypoalbuminemia, and/or concomitant edema. Approximately 85-90% of patients attain complete remission of proteinuria within 4-6 weeks of treatment with glucocorticoids, and therefore, have steroid-sensitive nephrotic syndrome (SSNS). Among those patients who are steroid sensitive, 70-80% will have at least one relapse during follow-up, and up to 50% of these patients will experience frequent relapses or become dependent on glucocorticoids to maintain remission. The dose and duration of steroid treatment to prolong time between relapses remains a subject of much debate, and patients continue to experience a high prevalence of steroid-related morbidity. Various steroid-sparing immunosuppressive drugs have been used in clinical practice; however, there is marked practice variation in the selection of these drugs and timing of their introduction during the course of the disease. Therefore, international evidence-based clinical practice recommendations (CPRs) are needed to guide clinical practice and reduce practice variation. The International Pediatric Nephrology Association (IPNA) convened a team of experts including pediatric nephrologists, an adult nephrologist, and a patient representative to develop comprehensive CPRs on the diagnosis and management of SSNS in children. After performing a systematic literature review on 12 clinically relevant PICO (Patient or Population covered, Intervention, Comparator, Outcome) questions, recommendations were formulated and formally graded at several virtual consensus meetings. New definitions for treatment outcomes to help guide change of therapy and recommendations for important research questions are given.

Paediatric nephrology in under-resourced areas.

BACKGROUND: Nearly 50% of the world population and 60% of children aged 0 to 14 years live in low- or lower-middle-income countries. Paediatric nephrology (PN) in these countries is not a priority for allocation of limited health resources. This article explores advancements made and persisting limitations in providing optimal PN services to children in such under-resourced areas (URA). METHODS: Medline, PubMed and Google Scholar online databases were searched for articles pertaining to PN disease epidemiology, outcome, availability of services and infrastructure in URA. The ISN and IPNA offices were contacted for data, and two online questionnaire surveys of IPNA membership performed. Regional IPNA members were contacted for further detailed information. RESULTS: There is a scarcity of published data from URA; where available, prevalence of PN diseases, managements and outcomes are often reported to be different from high income regions. Deficiencies in human resources, fluoroscopy, nuclear imaging, immunofluorescence, electron microscopy and genetic studies were identified. Several drugs and maintenance kidney replacement therapy are inaccessible to the majority of patients. Despite these issues, regional efforts with support from international bodies have led to significant advances in PN services and infrastructure in many URA. CONCLUSIONS: Equitable distribution and affordability of PN services remain major challenges in URA. The drive towards acquisition of regional data, advocacy to local government and non-government agencies and partnership with international support bodies needs to be continued. The aim is to optimise and achieve global parity in PN training, investigations and treatments, initially focusing on preventable and reversible conditions.

Importance of clinical practice guidelines to practicing pediatric nephrologists and IPNA survey.

Clinical practice guidelines (CPGs) are systematically developed statements backed by scientific evidence to assist practitioners in management in clinical practice. An international cross-sectional survey was conducted by the IPNA to examine the perceptions of pediatric nephrologists on guidelines and their usage and to identify important diseases for future clinical practice guidelines (CPGs). The survey found that the majority of pediatric nephrologists find CPGs useful in clinical practice and admitted to using them most of the time. Developing CPGs is challenging and there are standards available to develop trustworthy guidelines. While evidence-based global guidelines are ideal, pediatric nephrologists expressed the desire that they address regional differences. Most respondents (89.2%) to the survey agreed that adult guidelines did not cover the pediatric perspective adequately and 71.4% opined that consensus-based pediatric guidelines can be developed when evidence for the pediatric population is lacking. The development of high-quality practice guidelines requires substantial resources and may not be feasible in resource-poor countries. Adaptation of an existing guideline has been suggested as an alternative and the ADAPTE collaboration provides a systematic approach to adapting guidelines. Several diseases where pediatric guidelines are needed as a priority including IgA and C3 glomerulopathy were identified in the survey. Implementation of guideline-based care is challenging and the survey found that lack of availability of guidelines (43%) and resources (22.8%) are important reasons for poor implementation in lower-middle and low-income countries. Perceived complexity of guidelines, physician attitudes, and lack of training also contribute to non-adherence to guidelines.

Loss of 5hmC identifies a new type of aberrant DNA hypermethylation in glioma.

Aberrant DNA hypermethylation is a hallmark of cancer although the underlying molecular mechanisms are still poorly understood. To study the possible role of 5-hydroxymethylcytosine (5hmC) in this process we analyzed the global and locus-specific genome-wide levels of 5hmC and 5-methylcytosine (5mC) in human primary samples from 12 non-tumoral brains and 53 gliomas. We found that the levels of 5hmC identified in non-tumoral samples were significantly reduced in gliomas. Strikingly, hypo-hydroxymethylation at 4627 (9.3%) CpG sites was associated with aberrant DNA hypermethylation and was strongly enriched in CpG island shores. The DNA regions containing these CpG sites were enriched in H3K4me2 and presented a different genuine chromatin signature to that characteristic of the genes classically aberrantly hypermethylated in cancer. As this 5mC gain is inversely correlated with loss of 5hmC and has not been identified with classical sodium bisulfite-based technologies, we conclude that our data identifies a novel 5hmC-dependent type of aberrant DNA hypermethylation in glioma.

Disaster Management in a Nephrology Service: Lessons Learned from Hurricane Maria.

BACKGROUND: Puerto Rico suffered a major humanitarian -crisis after Hurricane Maria. We describe our experience with patients with renal disease in an academic medical center. SUMMARY: A comprehensive emergency response plan should be developed, shared and discussed with the team and the patients prior to the hurricane. The needs of the staff should not be ignored to ensure their ability to participate as responders. Physical damage to facilities, lack of basic services, shortage of disposable products, and the inability to get to treatment centers are the most common threats. Preemptive dialysis can avoid serious complications. A contingency plan to move patients to another center should be prearranged in case the unit is rendered nonfunctional after the storm. Patients must receive preventive education about fluid and dietary restrictions and the possible use of potassium binding drugs if they cannot reach a dialysis unit. A list of alternative drugs that could be used if patients are not able to fill their medications is required. The Internet and social media proved to be an invaluable communication tool. A registry of patients with updated contact information, as well as contact information for relatives and a physical address where an emergency rescue team can be dispatched is essential. Water safety should be reinforced. Key Message: Our experience showed us that preparing for the worst is not enough. Advanced planning of a streamlined response is the best tactic to decrease harm.

Differential distribution and enrichment of non-coding RNAs in exosomes from normal and Cancer-associated fibroblasts in colorectal cancer.

Exosome production from cancer-associated fibroblasts seems to be an important driver of tumor progression. We report the first in-depth biotype characterization of ncRNAs, analyzed by Next Generation Sequencing and Bioinformatics, expressed in established primary human normal and cancer-associated fibroblasts (CAFs) from cancer and normal mucosa tissues from 9 colorectal cancer patients, and/or packaged in their derived exosomes. Differential representation and enrichment analyses based on these ncRNAs revealed a significant number of differences between the ncRNA content of exosomes and the expression patterns of the normal and cancer-associated fibroblast cells. ncRNA regulatory elements are specifically packaged in CAF-derived exosomes, supporting a specific cross-talk between CAFs and colon cancer cells and/or other stromal cells, mediated by exosomes. These sncRNAs are potential biomarkers present in cancer-associated fibroblast-derived exosomes, which should thereby contribute to developing new non-invasive diagnostic, prognostic and predictive methods for clinical applications in management of cancer patients.

Potassium regulation in the neonate.

Potassium, the major cation in intracelluar fluids, is essential for vital biological functions. Neonates maintain a net positive potassium balance, which is fundamental to ensure somatic growth but places these infants, especially those born prematurely, at risk for life-threatening disturbances in potassium concentration [K+] in the extracellular fluid compartment. Potassium conservation is achieved by maximizing gastrointestinal absorption and minimizing renal losses. A markedly low glomerular filtration rate, plus adaptations in tubular transport along the nephron, result in low potassium excretion in the urine of neonates. Careful evaluation of clinical data using reference values that are normal for the neonate's postmenstrual age is critical to avoid over-treating infants with laboratory results that represent physiologic values for their developmental stage. The treatment should be aimed at correcting the primary cause when possible. Alterations in the levels or sensitivity to aldosterone are common in neonates. In symptomatic patients, the disturbances in [K+] should be corrected promptly, with close electrocardiographic monitoring. Plasma [K+] should be monitored during the first 72 h of life in all premature infants born before 30 weeks of postmenstrual age as these infants are prone to develop non-oliguric hyperkalemia with potential serious complications.

A proteomic analysis reveals that Snail regulates the expression of the nuclear orphan receptor Nuclear Receptor Subfamily 2 Group F Member 6 (Nr2f6) and interleukin 17 (IL-17) to inhibit adipocyte differentiation.

Adipogenesis requires a differentiation program driven by multiple transcription factors, where PPARγ and C/EBPα play a central role. Recent findings indicate that Snail inhibits adipocyte differentiation in 3T3-L1 and murine mesenchymal stem cells (mMSC). An in-depth quantitative SILAC analysis of the nuclear fraction of Snail-induced alterations of 3T3-L1 cells was carried out. In total, 2251 overlapping proteins were simultaneously quantified in forward and reverse experiments. We observed 574 proteins deregulated by Snail1 using a fold-change ≥1.5, with 111 up- and 463 down-regulated proteins, respectively. Among other proteins, multiple transcription factors such as Trip4, OsmR, Nr2f6, Cbx6, and Prrx1 were down-regulated. Results were validated in 3T3-L1 cells and mMSC cells by Western blot and quantitative PCR. Knock-down experiments in 3T3-L1 cells demonstrated that only Nr2f6 (and Trip4 at minor extent) was required for adipocyte differentiation. Ectopic expression of Nr2f6 reversed the effects of Snail1 and promoted adipogenesis. Because Nr2f6 inhibits the expression of IL-17, we tested the effect of Snail on IL-17 expression. IL-17 and TNFα were among the most up-regulated pro-inflammatory cytokines in Snail-transfected 3T3-L1 and mMSC cells. Furthermore, the blocking of IL-17 activity in Snail-transfected cells promoted adipocyte differentiation, reverting Snail inhibition. In summary, Snail inhibits adipogenesis through a down-regulation of Nr2f6, which in turn facilitates the expression of IL-17, an anti-adipogenic cytokine. These results would support a novel and important role for Snail and Nr2f6 in obesity control.

Tumor-derived exosomes are enriched in ΔNp73, which promotes oncogenic potential in acceptor cells and correlates with patient survival.

Tumor-derived exosomes are emerging as local and systemic cell-to-cell mediators of oncogenic information through the horizontal transfer of mRNAs, microRNAs and proteins during tumorigenesis. The exosomal content has been described as biologically active when taken up by the recipient cell. Identifying the specific molecular cargo of exosomes will help to determine their function in specific steps of the tumorigenic process. Here we evaluate whether ΔNp73 is selectively packaged in tumor-derived exosomes, its function in the acceptor cells in vitro and in vivo and its prognosis potential in cancer. ΔNp73 messenger is enriched in tumor-derived exosomes, suggesting its active sorting in these microvesicles. We observed the transmission of this exosome cargo to different cell types and how it confers proliferation potential and chemoresistance to the acceptor cells in vitro and in animal models. Finally, our data support the potential prognostic value of exosomal ΔNp73 in colon cancer patients.

In-depth characterization of the secretome of colorectal cancer metastatic cells identifies key proteins in cell adhesion, migration, and invasion.

Liver metastasis in colorectal cancer is the major cause of cancer-related deaths. To identify and characterize proteins associated with colon cancer metastasis, we have compared the conditioned serum-free medium of highly metastatic KM12SM colorectal cancer cells with the parental, poorly metastatic KM12C cells using quantitative stable isotope labeling by amino acids in cell culture (SILAC) analyses on a linear ion trap-Orbitrap Velos mass spectrometer. In total, 1337 proteins were simultaneously identified in SILAC forward and reverse experiments. For quantification, 1098 proteins were selected in both experiments, with 155 proteins showing >1.5-fold change. About 52% of these proteins were secreted directly or using alternative secretion pathways. GDF15, S100A8/A9, and SERPINI1 showed capacity to discriminate cancer serum samples from healthy controls using ELISAs. In silico analyses of deregulated proteins in the secretome of metastatic cells showed a major abundance of proteins involved in cell adhesion, migration, and invasion. To characterize the tumorigenic and metastatic properties of some top up- and down-regulated proteins, we used siRNA silencing and antibody blocking. Knockdown expression of NEO1, SERPINI1, and PODXL showed a significant effect on cellular adhesion. Silencing or blocking experiments with SOSTDC1, CTSS, EFNA3, CD137L/TNFSF9, ZG16B, and Midkine caused a significant decrease in migration and invasion of highly metastatic cells. In addition, silencing of SOSTDC1, EFNA3, and CD137L/TNFSF9 reduced liver colonization capacity of KM12SM cells. Finally, the panel of six proteins involved in invasion showed association with poor prognosis and overall survival after dataset analysis of gene alterations. In summary, we have defined a collection of proteins that are relevant for understanding the mechanisms underlying adhesion, migration, invasion, and metastasis in colorectal cancer.

Different exosome cargo from plasma/bronchoalveolar lavage in non-small-cell lung cancer.

Tumor-derived exosomes mediate tumorigenesis by facilitating tumor growth, metastasis, development of drug resistance, and immunosuppression. However, little is known about the exosomes isolated from bronchoalveolar lavage (BAL) in patients with lung neoplasm. Exosomes isolated in plasma and BAL from 30 and 75 patients with tumor and nontumor pathology were quantified by acetylcholinesterase activity and characterized by Western Blot, Electron Microscopy, and Nanoparticle Tracking Analysis. Differences in exosome cargo were analyzed by miRNA quantitative PCR in pooled samples and validated in a second series of patients. More exosomes were detected in plasma than in BAL in both groups (P < 0.001). The most miRNAs evaluated by PCR array were detected in tumor plasma, tumor BAL, and nontumor BAL pools, but only 56% were detected in the nontumor plasma pool. Comparing the top miRNAs with the highest levels detected in each pool, we found close homology only between the BAL samples of the two pathologies. In tumor plasma, we found a higher percentage of miRNAs with increased levels than in tumor BAL or in nontumor plasma. The data reveal differences between BAL and plasma exosome amount and miRNA content.

Protumorigenic effects of Snail-expression fibroblasts on colon cancer cells.

Snail1 is a transcriptional factor that plays an important role in epithelial-mesenchymal transition and in the acquisition of invasive properties by epithelial cells. In colon tumors, Snail1 expression in the stroma correlates with lower specific survival of cancer patients. However, the role(s) of Snail1 expression in stroma and its association with patients' survival have not been determined. We used human primary carcinoma-associated fibroblasts (CAFs) or normal fibroblasts (NFs) and fibroblast cell lines to analyze the effects of Snail1 expression on the protumorigenic capabilities in colon cancer cells. Snail1 expression was higher in CAFs than in NFs and, as well as α-SMA, a classic marker of activated CAFs. Moreover, in tumor samples from 50 colon cancer patients, SNAI1 expression was associated with expression of other CAF markers, such as α-SMA and fibroblast activation protein. Interestingly, coculture of CAFs with colon cells induced a significant increase in epithelial cell migration and proliferation, which was associated with endogenous SNAI1 expression levels. Ectopic manipulation of Snail1 in fibroblasts demonstrated that Snail1 expression controlled migration as well as proliferation of cocultured colon cancer cells in a paracrine manner. Furthermore, expression of Snail1 in fibroblasts was required for the coadjuvant effect of these cells on colon cancer cell growth and invasion when coxenografted in nude mice. Finally, cytokine profile changes, particularly MCP-3 expression, in fibroblasts are put forward as mediators of Snail1-derived effects on colon tumor cell migration. In summary, these studies demonstrate that Snail1 is necessary for the protumorigenic effects of fibroblasts on colon cancer cells.

MicroRNA-22 is induced by vitamin D and contributes to its antiproliferative, antimigratory and gene regulatory effects in colon cancer cells.

Vitamin D deficiency is associated with the high risk of colon cancer and a variety of other diseases. The active vitamin D metabolite 1α,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) regulates gene transcription via its nuclear receptor (VDR), and posttranscriptional regulatory mechanisms of gene expression have also been proposed. We have identified microRNA-22 (miR-22) and several other miRNA species as 1,25(OH)(2)D(3) targets in human colon cancer cells. Remarkably, miR-22 is induced by 1,25(OH)(2)D(3) in a time-, dose- and VDR-dependent manner. In SW480-ADH and HCT116 cells, miR-22 loss-of-function by transfection of a miR-22 inhibitor suppresses the antiproliferative effect of 1,25(OH)(2)D(3). Additionally, miR-22 inhibition increases cell migration per se and decreases the antimigratory effect of 1,25(OH)(2)D(3) in both cell types. In silico analysis shows a significant overlap between genes suppressed by 1,25(OH)(2)D(3) and miR-22 putative target genes. Consistently, miR-22 inhibition abrogates the 1,25(OH)(2)D(3)-mediated suppression of NELL2, OGN, HNRPH1, RERE and NFAT5 genes. In 39 out of 50 (78%) human colon cancer patients, miR-22 expression was found lower in the tumour than in the matched normal tissue and correlated directly with that of VDR. Our results indicate that miR-22 is induced by 1,25(OH)(2)D(3) in human colon cancer cells and it may contribute to its antitumour action against this neoplasia.

The TP73 complex network: ready for clinical translation in cancer?

TP73 is a member of the TP53 family, whose deregulated expression has been reported in a wide variety of cancers and linked to patients' outcome. The fact that TP73 encodes a complex number of isoforms (TAp73 and ΔTAp73) with opposing functions and the cross-talk with other members of the family (TP53 and TP63) make it difficult to determine its clinical relevance. Here, we review the molecular mechanisms driving TAp73 and ΔTAp73 expression and how these variants inhibit or promote carcinogenesis. We also highlight the intricate interplay between TP53 family members. In addition, we comment on current pharmacological approaches targeting the TP73 pathway and those affecting the TAp73/ΔTAp73 ratio. Finally, we discuss the current data available in the literature that provide evidence on the role of TP73 variants in predicting prognosis. To date, most of the studies that evaluate the status levels of TP73 isoforms have been based on limited-size series. Despite this limitation, these publications highlight the correlation between high levels of the oncogenic forms and failure to respond to chemotherapy and/or shorter survival. Finally, we emphasize the need for studies to evaluate the significance of combining the deregulation of various members of the TP53 family in order to define patient outcome or their responsiveness to specific therapies.