Chronic arsenic exposure suppresses proteasomal and autophagic protein degradation.

Ubiquitin Proteasomal System (UPS) and autophagy dysregulation initiate cancer. These pathways are regulated by zinc finger proteins. Trivalent inorganic arsenic (iAs) displaces zinc from zinc finger proteins disrupting functions of important cellular proteins. The effect of chronic environmental iAs exposure (100 nM) on UPS has not been studied. We tested the hypothesis that environmental iAs exposure suppresses UPS, activating autophagy as a compensatory mechanism. We exposed skin (HaCaT and Ker-CT; independent quadruplicates) and lung (BEAS-2B; independent triplicates) cell cultures to 0 or 100 nM iAs for 7 or 8 weeks. We quantified ER stress (XBP1 splicing employing Reverse Transcriptase -Polymerase Chain Reaction), proteasomal degradation (immunoblots), and initiation and completion of autophagy (immunoblots). We demonstrate that chronic iAs exposure suppresses UPS, initiates autophagy, but suppresses autophagic protein degradation in skin and lung cell lines. Our data suggest that chronic iAs exposure inhibits autophagy which subsequently suppresses UPS.

Clonal variability in chromosomal instability as a potential driver in the acquisition of tumorigenic phenotype in chronic arsenic-exposed and hsa-miR-186 overexpressing human keratinocytes.

Chronic arsenic exposure through drinking water is a global health issue, affecting >200 million people. Arsenic is a group I human carcinogen and causes chromosomal instability (CIN). Arsenic exposure is the second most common cause of skin cancer after UV radiation. hsa-miR-186 is overexpressed in arsenic-induced squamous cell carcinoma relative to premalignant hyperkeratosis. Among predicted targets of hsa-miR-186 are cell cycle regulators including regulators of mitotic progression. Disruption of mitotic progression can contribute to CIN. Thus, we hypothesized that hsa-miR-186 overexpression contributes to malignant transformation of arsenic exposed HaCaT cells by induction of CIN. Stable clones of HaCaT cells transfected with pEP-hsa-miR-186 expression vector or empty vector were maintained under puromycin selection and exposed to 0 or 100 nM NaAsO2 and cultured for 29 weeks. HaCaT clones overexpressing hsa-miR-186 and exposed to NaAsO2 showed increased CIN and anchorage independent growth at 29 weeks in a stochastic manner, in contrast to unexposed empty vector transfected clones. These results suggest that clonal variability mediates arsenic-induced carcinogenesis in hsa-miR-186 overexpressing human keratinocytes.

Iron metabolism in colorectal cancer: a balancing act.

BACKGROUND: Colorectal cancer (CRC) is the third most commonly diagnosed cancer and the second deadliest malignancy worldwide. Current dietary habits are associated with increased levels of iron and heme, both of which increase the risk of developing CRC. The harmful effects of iron overload are related to the induction of iron-mediated pro-tumorigenic pathways, including carcinogenesis and hyperproliferation. On the other hand, iron deficiency may also promote CRC development and progression by contributing to genome instability, therapy resistance, and diminished immune responses. In addition to the relevance of systemic iron levels, iron-regulatory mechanisms in the tumor microenvironment are also believed to play a significant role in CRC and to influence disease outcome. Furthermore, CRC cells are more prone to escape iron-dependent cell death (ferroptosis) than non-malignant cells due to the constitutive activation of antioxidant genes expression. There is wide evidence that inhibition of ferroptosis may contribute to the resistance of CRC to established chemotherapeutic regimens. As such, ferroptosis inducers represent promising therapeutic drugs for CRC. CONCLUSIONS AND PERSPECTIVES: This review addresses the complex role of iron in CRC, particularly in what concerns the consequences of iron excess or deprivation in tumor development and progression. We also dissect the regulation of cellular iron metabolism in the CRC microenvironment and emphasize the role of hypoxia and of oxidative stress (e.g. ferroptosis) in CRC. Finally, we underline some iron-related players as potential therapeutic targets against CRC malignancy.

miRNAs and arsenic-induced carcinogenesis.

Arsenic-induced carcinogenesis is a worldwide health problem. Identifying the molecular mechanisms responsible for the induction of arsenic-induced cancers is important for developing treatment strategies. MicroRNA (miRNA) dysregulation is known to affect development and progression of human cancer. Several studies have identified an association between altered miRNA expression in cancers from individuals chronically exposed to arsenic and in cell models for arsenic-induced carcinogenesis. This chapter provides a comprehensive review for miRNA dysregulation in arsenic-induced cancer.

miR-186 induces tetraploidy in arsenic exposed human keratinocytes.

Chronic inorganic arsenic (iAs) exposure in drinking water is a global issue affecting >225 million people. Skin is a major target organ for iAs. miRNA dysregulation and chromosomal instability (CIN) are proposed mechanisms of iAs-induced carcinogenesis. CIN is a cancer hallmark and tetraploid cells can better tolerate increase in chromosome number and aberration, contributing to the evolution of CIN. miR-186 is overexpressed in iAs-induced squamous cell carcinoma relative to iAs-induced hyperkeratosis. Bioinformatic analysis indicated that miR-186 targets mRNAs of important cell cycle regulators including mitotic checkpoint serine/threonine kinase B (BUB1) and cell division cycle 27 (CDC27). We hypothesized that miR-186 overexpression contributes to iAs-induced transformation of keratinocytes by targeting mitotic regulators leading to induction of CIN. Ker-CT cells, a near diploid human keratinocyte cell line, were transduced with miR-186 overexpressing or scrambled control lentivirus. Stable clones were isolated after puromycin selection. Clones transduced with lentivirus expressing either a scrambled control miRNA or miR-186 were maintained with 0 or 100 nM iAs for 4 weeks. Unexposed scrambled control clones were considered as passage matched controls. Chronic iAs exposure increased miR-186 expression in miR-186 clones. miR-186 overexpression significantly reduced CDC27 levels irrespective of iAs exposure. The percentage of tetraploid or aneuploid cells was increased in iAs exposed miR-186 clones. Aneuploidy can arise from a tetraploid intermediate. Suppression of CDC27 by miR-186 may lead to impairment of mitotic checkpoint complex formation and its ability to maintain cell cycle arrest leading to chromosome misalignment. As a result, cells overexpressing miR-186 and chronically exposed to iAs may have incorrect chromosome segregation and CIN. These data suggest that dysregulation of miRNA by iAs mediates tetraploidy, aneuploidy and chromosomal instability contributing to iAs-induced carcinogenesis.

Edible Insects: Perceptions of Marketing, Economic, and Social Aspects among Citizens of Different Countries.

Because edible insects (EI) have been, in recent years, recommended as a nutritious animal protein food with enormous environmental advantages over other sources of animal protein for human consumption, studies aimed at investigating the consumer perspective have become more prominent. Hence, this study intended to examine the perceptions of participants from different countries about the commercialization and economic and social impacts of edible insects. The study was made using a questionnaire survey, and data were collected in Brazil, Croatia, Greece, Latvia, Lebanon, Lithuania, Mexico, Poland, Portugal, Romania, Serbia, Slovenia, Spain, and Turkey. The final number of received answers was 7222 participants. For the treatment of the results, different statistical techniques were used: factor analysis, internal reliability by Cronbach's alpha, cluster analysis, ANOVA to test differences between groups, and Chi-square tests. The results obtained confirmed the validity of the scale, constituted by 12 out of the 14 items initially considered, distributed by 4 factors: the first related to the economic impact of EIs, the second related to the motivation for consumption of EIs, the third related to the places of purchase of EIs, and the fourth corresponding to a question presented to the participants as a false statement. A cluster analysis allowed identifying three clusters, with significant differences between them according to all the sociodemographic variables tested. Also, it was found that the participants expressed an exceptionally high level of agreement with aspects such as the difficulty in finding EIs on sale, knowledge acting as a strong motivator for EI consumption, and the role of personalities and influencers in increasing the will to consume EIs. Finally, practically all sociodemographic variables were found to be significantly associated with perceptions (country, sex, education, living environment, and income), but not age. In conclusion, the perceptions about EI commercialization were investigated and revealed differences among samples originating from different countries. Moreover, the sociodemographic characteristics of the participants were found to be strongly associated with their perceptions.

The Motivations for Consumption of Edible Insects: A Systematic Review.

The consumption of edible insects (EI) is traditional in many parts of the world, but not in others. In fact, despite globalization and the multiple advantages pointed out about the consumption of EI, there are still many countries where entomophagy is seen with disgust and aversion. This systematic review aimed to examine the motivations that influence the consumption of EI in diverse cultures and understand if there are differences between Western countries (WC) and insect-eating countries (IEC). It further evaluated whether the degree of acceptability was influenced by the form of consumption of the insects (eating whole insects or foods containing insects). This literature review was conducted in November 2021 within three databases, Web of Science, PubMed and Scopus, according to the Preferred Reporting of Items for Systematic Reviews and Meta-analysis and using PRISMA directives. From a total of 245 studies, 31 were selected to be included in this review, based on the inclusion criteria defined: only original research articles, from 2010 or beyond, and written in English. The results indicated that the main motivations that determine the consumption of EI are related to gender, age, sustainability, nutritional value, sensory attributes, tradition/culture, food neophobia, disgust and familiarity/past experiences. Moreover, whereas in IEC, there is a greater focus on factors related to sensory attributes, availability, affordability and preferences, in WC, there is a bigger emphasis on determinants such as nutritional value, sustainability, benefits, familiarity/past experience, tradition/culture, food neophobia and disgust. Finally, it was observed that people in WC are more willing to accept food products containing insects rather than the whole insect, which is one of the most promising points to be addressed in the future. Overall, this review highlights that there are numerous factors influencing the consumption of edible insects, and differences between WC and IEC are clear in what concerns the motivations of consumers. Hence, targeting market segments and consumers' characteristics has to be present when designing strategies to incentivize the consumption of EI in WC as a part of a global strategy for sustainability of food systems.

Human iNKT Cells Modulate Macrophage Survival and Phenotype.

CD1d-restricted invariant Natural Killer T (iNKT) cells are unconventional innate-like T cells whose functions highly depend on the interactions they establish with other immune cells. Although extensive studies have been reported on the communication between iNKT cells and macrophages in mice, less data is available regarding the relevance of this crosstalk in humans. Here, we dove into the human macrophage-iNKT cell axis by exploring how iNKT cells impact the survival and polarization of pro-inflammatory M1-like and anti-inflammatory M2-like monocyte-derived macrophages. By performing in vitro iNKT cell-macrophage co-cultures followed by flow cytometry analysis, we demonstrated that antigen-stimulated iNKT cells induce a generalized activated state on all macrophage subsets, leading to upregulation of CD40 and CD86 expression. CD40L blocking with a specific monoclonal antibody prior to co-cultures abrogated CD40 and CD86 upregulation, thus indicating that iNKT cells required CD40-CD40L co-stimulation to trigger macrophage activation. In addition, activated iNKT cells were cytotoxic towards macrophages in a CD1d-dependent manner, killing M1-like macrophages more efficiently than their naïve M0 or anti-inflammatory M2-like counterparts. Hence, this work highlighted the role of human iNKT cells as modulators of macrophage survival and phenotype, untangling key features of the human macrophage-iNKT cell axis and opening perspectives for future therapeutic modulation.

Testicular alterations in cryptorchid/orchiopexic rats chronically exposed to acrylamide or di-butyl-phthalate.

Exposure of Sprague-Dawley (SD) rats to acrylamide (AA) or di-butyl-phthalate (DBP) from the 12th gestational day to the 16th postnatal week (PNW) has been shown to reduce the effectiveness of orchiopexy in recovering the testicular alterations associated with experimental cryptorchidism established at weaning. Herein, we provide information about the long-term effects of AA or DBP on the testes of cryptorchid/orchiopexic rats. Male offspring exposed in utero to 10 mg/kg/day AA or 500 mg/kg/day DBP underwent bilateral surgical cryptorchidism at the 3rd PNW and orchiopexy at the 6th week, with continuous exposure to the chemicals through diet until the 58th week. Regardless of the test chemical, there were severe qualitative/quantitative alterations in the seminiferous tubules and increased numbers of Leydig cells. There was an increase and decrease in the number of tubules with c-Kit- and placental alkaline phosphatase-labeled germ cells, respectively, as compared to those in the control group, suggesting an imbalance between apoptosis and cell proliferation processes. The histological scores of the testicular lesions at the end of this one-year study were higher than those in the previous 16-week study, indicating that exposure of rats to the toxicants AA or DBP enhanced the testicular alterations induced by the chemicals beginning at the intra-uterine life, and impaired the effectiveness of orchiopexy in restoring the testes to normal morphology. Although the present experimental protocol does not completely replicate the natural human undescended testes, our findings may contribute to understanding the alterations occurring in cryptorchid/orchiopexic testes potentially exposed to exogenous chemicals for extended periods.

Chronic arsenic exposure suppresses ATM pathway activation in human keratinocytes.

An estimated 220 million people worldwide are chronically exposed to inorganic arsenic (iAs) primarily as a result of drinking iAs-contaminated water. Chronic iAs exposure is associated with a plethora of human diseases including skin lesions and multi-organ cancers. iAs is a known clastogen, inducing DNA double strand breaks (DSBs) in both exposed human populations and in vitro. However, iAs does not directly interact with DNA, suggesting that other mechanisms, such as inhibition of DNA repair and DNA Damage Response (DDR) signaling, may be responsible for iAs-induced clastogenesis. Recent RNA-sequencing data from human keratinocytes (HaCaT cells) indicate that mRNAs for phosphatases important for resolution of DDR signaling are induced as a result of chronic iAs exposure prior to epithelial to mesenchymal transition. Here, we report that phosphorylation of ataxia telengectasia mutated (ATM) protein at a critical site (pSer1981) important for DDR signaling, and downstream CHEK2 activation, are significantly reduced in two human keratinocyte lines as a result of chronic iAs exposure. Moreover, RAD50 expression is reduced in both of these lines, suggesting that suppression of the MRE11-RAD50-NBS1 (MRN) complex may be responsible for reduced ATM activation. Lastly, we demonstrate that DNA double strand break accumulation and DNA damage is significantly higher in human keratinocytes with low dose iAs exposure. Thus, inhibition of the MRN complex in iAs-exposed cells may be responsible for reduced ATM activation and reduced DSB repair by homologous recombination (HR). As a result, cells may favor error-prone DSB repair pathways to fix damaged DNA, predisposing them to chromosomal instability (CIN) and eventual carcinogenesis often seen resulting from chronic iAs exposure.

Temporal Modulation of Differential Alternative Splicing in HaCaT Human Keratinocyte Cell Line Chronically Exposed to Arsenic for up to 28 Wk.

BACKGROUND: Chronic arsenic exposure via drinking water is associated with an increased risk of developing cancer and noncancer chronic diseases. Pre-mRNAs are often subject to alternative splicing, generating mRNA isoforms encoding functionally distinct protein isoforms. The resulting imbalance in isoform species can result in pathogenic changes in critical signaling pathways. Alternative splicing as a mechanism of arsenic-induced toxicity and carcinogenicity is understudied. OBJECTIVE: This study aimed to accurately profile differential alternative splicing events in human keratinocytes induced by chronic arsenic exposure that might play a role in carcinogenesis. METHODS: Independent quadruplicate cultures of immortalized human keratinocytes (HaCaT) were maintained continuously for 28 wk with 0 or 100 nM sodium arsenite. RNA-sequencing (RNA-Seq) was performed with poly(A) RNA isolated from cells harvested at 7, 19, and 28 wk with subsequent replicate multivariate analysis of transcript splicing (rMATS) analysis to detect and quantify differential alternative splicing events. Reverse transcriptase-polymerase chain reaction (RT-PCR) for selected alternative splicing events was performed to validate RNA-Seq predictions. Functional enrichment was performed by gene ontology (GO) analysis of the differential alternative splicing event data set at each time point. RESULTS: At least 600 differential alternative splicing events were detected at each time point tested, comprising all the five main types of alternative splicing and occurring in both open reading frames (ORFs) and untranslated regions (UTRs). Based on functional relevance ELK4, SHC1, and XRRA1 were selected for validation of predicted alternative splicing events at 7 wk by RT-PCR. Densitometric analysis of RT-PCR data corroborated the rMATS predicted alternative splicing for all three events. Protein expression validation of the selected alternative splicing events was challenging given that very few isoform-specific antibodies are available. GO analysis demonstrated that the enriched terms in differential alternatively spliced mRNAs changed dynamically with the time of exposure. Notably, RNA metabolism and splicing regulation pathways were enriched at the 7-wk time point, when the greatest number of differentially alternatively spliced mRNAs are detected. Our preliminary proteomic analysis demonstrated that the expression of the canonical isoforms of the splice regulators DDX42, RMB25, and SRRM2 were induced upon chronic arsenic exposure, corroborating the splicing predictions. DISCUSSION: These results using cultures of HaCaT cells suggest that arsenic exposure disrupted an alternative splice factor network and induced time-dependent genome-wide differential alternative splicing that likely contributed to the changing proteomic landscape in arsenic-induced carcinogenesis. However, significant challenges remain in corroborating alternative splicing data at the proteomic level. https://doi.org/10.1289/EHP9676.

miRNA dysregulation is an emerging modulator of genomic instability.

Genomic instability consists of a range of genetic alterations within the genome that contributes to tumor heterogeneity and drug resistance. It is a well-established characteristic of most cancer cells. Genome instability induction results from defects in DNA damage surveillance mechanisms, mitotic checkpoints and DNA repair machinery. Accumulation of genetic alterations ultimately sets cells towards malignant transformation. Recent studies suggest that miRNAs are key players in mediating genome instability. miRNAs are a class of small RNAs expressed in most somatic tissues and are part of the epigenome. Importantly, in many cancers, miRNA expression is dysregulated. Consequently, this review examines the role of miRNA dysregulation as a causal step for induction of genome instability and subsequent carcinogenesis. We focus specifically on mechanistic studies assessing miRNA(s) and specific subtypes of genome instability or known modes of genome instability. In addition, we provide insight on the existing knowledge gaps within the field and possible ways to address them.

Time response of rat testicular alterations induced by cryptorchidism and orchiopexy.

Cryptorchidism is one of the main risk factors for infertility and testicular cancer. Orchiopexy surgery corrects cryptorchidism effects. Different models of cryptorchidism developed in the rat include surgery. We assessed testicular alterations in rats submitted to surgical cryptorchidism and examined their potential for reversibility at different time points in order to verify time dependency effect(s) on the recovery of the undescended testes. Cryptorchidism was induced in 3-week-old rats. Animals were euthanized 3, 6 or 11 weeks after surgery to evaluate the morphological progression of cryptorchidism-induced germinative epithelial alterations. Other groups underwent orchiopexy 3, 5 or 9 weeks after surgical cryptorchidism, before or after puberty. Animals were euthanized 3 or 8 weeks after orchiopexy. Controls underwent sham surgery at the same time points as the surgical groups. Cryptorchid testes showed decreased weight, germinative epithelial degeneration, apoptosis and vacuolation, corresponding to impairment of spermatogenesis and of Sertoli cells. Some tubules has a Sertoli cell-only pattern and atrophy. The intensity of damage was related to the duration of cryptorchidism. After orchiopexy, spermatogenesis completely recovered only when testicular relocation occurred before puberty and the interval for recovery was extended. These results indicate that age, sexual maturity and extension of germ cell damage were relevant for producing germ cell restoration and normal spermatogenesis. We provide original observations on the time dependency of testicular alterations induced by cryptorchidism and their restoration using morphologic, morphometric and immunohistochemical approaches. It may be useful to study germ cell impairment, progression and recovery in different experimental settings, including exposure to exogenous chemicals.

Development of a Questionnaire to Assess Knowledge and Perceptions about Edible Insects.

Edible insects (EI) have been consumed as traditional foods in many parts of the globe, but in other regions, they are not readily accepted, particularly in Western countries. However, because EI are suggested to constitute a more sustainable protein food as compared with other sources of animal protein, they can be considered a future food that could help mitigate hunger and malnutrition. Additionally, new gastronomic trends are already targeting this area for exploring new potentialities. The objective of this work was to develop and validate a questionnaire to assess consumers' perceptions and knowledge about EI in seven different domains: D1. Culture and Tradition, D2. Gastronomic Innovation and Gourmet Kitchen, D3. Environment and Sustainability, D4. Economic and Social Aspects, D5. Commercialization and Marketing, D6. Nutritional Aspects and D7. Health Effects. The 64 items were subjected to item analysis and reliability analysis for validation, and factor analysis was also conducted to identify a grouping structure. The results validated all the items of the seven subscales with high values of Cronbach's alpha (α = 0.732 for D1, α = 0.795 for D2, α = 0.882 for D3, α = 0.742 for D4, α = 0.675 for D5, α = 0.799 for D6 and α = 0.788 for D7). However, by eliminating 17 items, the final values of the alpha increased in all subscales. Factor analysis with extraction by principal component analysis with varimax rotation extracted 14 factors that explained, in total, 65% of the variance, although the first two factors were the most important (35.7% variance explained). In conclusion, the confirmed usefulness of the questionnaire has been hereby validated for assessing consumer perceptions of and knowledge about EI.

Arsenic-induced changes in miRNA expression in cancer and other diseases.

miRNAs (miRNA) are essential players regulating gene expression affecting cellular processes contributing to disease development. Dysregulated miRNA expression has been observed in numerous diseases including hepatitis, cardiovascular diseases and cancers. In cardiovascular diseases, several miRNAs function as mediators of pathogenic stress-related signaling pathways that may lead to an excessive extracellular matrix production and collagen deposition causing cardiac stress resulting in fibrosis. In cancers, many miRNAs function as oncogenes or tumor suppressors facilitating tumor growth, invasion and angiogenesis. Furthermore, the association between distinct miRNA profile and tumor development, progression and treatment response has identified miRNAs as potential biomarkers for disease diagnosis and prognosis. Growing evidence demonstrates changes in miRNA expression levels in experimental settings or observational studies associated with environmental chemical exposures such as arsenic. Arsenic is one of the most well-known human carcinogens. Long-term exposure through drinking water increases risk of developing skin, lung and urinary bladder cancers, as well as cardiovascular disease. The mechanism(s) by which arsenic causes disease remains elusive. Proposed mechanisms include miRNA dysregulation. Epidemiological studies identified differential miRNA expression between arsenic-exposed and non-exposed individuals from India, Bangladesh, China and Mexico. In vivo and in vitro studies have shown that miRNAs are critically involved in arsenic-induced malignant transformation. Few studies analyzed miRNAs in other diseases associated with arsenic exposure. Importantly, there is no consensus on a consistent miRNA profile for arsenic-induced cancers because most studies analyze only particular miRNAs. Identifying miRNA expression changes common among humans, rodents and cell lines might guide future miRNA investigations.

Arsenite Exposure Displaces Zinc from ZRANB2 Leading to Altered Splicing.

Exposure to arsenic, a class I carcinogen, affects 200 million people globally. Skin is the major target organ, but the molecular etiology of arsenic-induced skin carcinogenesis remains unclear. Arsenite (As3+)-induced disruption of alternative splicing could be involved, but the mechanism is unknown. Zinc finger proteins play key roles in alternative splicing. As3+ can displace zinc (Zn2+) from C3H1 and C4 zinc finger motifs (zfm's), affecting protein function. ZRANB2, an alternative splicing regulator with two C4 zfm's integral to its structure and splicing function, was chosen as a candidate for this study. We hypothesized that As3+ could displace Zn2+ from ZRANB2, altering its structure, expression, and splicing function. As3+/Zn2+ binding and mutual displacement experiments were performed with synthetic apo-peptides corresponding to each ZRANB2 zfm, employing a combination of intrinsic fluorescence, ultraviolet spectrophotometry, zinc colorimetric assay, and liquid chromatography-tandem mass spectrometry. ZRANB2 expression in HaCaT cells acutely exposed to As3+ (0 or 5 µM, 0-72 h; or 0-5 µM, 6 h) was examined by RT-qPCR and immunoblotting. ZRANB2-dependent splicing of TRA2B mRNA, a known ZRANB2 target, was monitored by reverse transcription-polymerase chain reaction. As3+ bound to, as well as displaced Zn2+ from, each zfm. Also, Zn2+ displaced As3+ from As3+-bound zfm's acutely, albeit transiently. As3+ exposure induced ZRANB2 protein expression between 3 and 24 h and at all exposures tested but not ZRANB2 mRNA expression. ZRANB2-directed TRA2B splicing was impaired between 3 and 24 h post-exposure. Furthermore, ZRANB2 splicing function was also compromised at all As3+ exposures, starting at 100 nm. We conclude that As3+ exposure displaces Zn2+ from ZRANB2 zfm's, changing its structure and compromising splicing of its targets, and increases ZRANB2 protein expression as a homeostatic response both at environmental/toxicological exposures and therapeutically relevant doses.

Overexpression of hsa-miR-186 induces chromosomal instability in arsenic-exposed human keratinocytes.

The mechanism of arsenic-induced skin carcinogenesis is not yet fully understood. Chromosomal instability contributes to aneuploidy and is a driving force in carcinogenesis. Arsenic causes mitotic arrest and induces aneuploidy. hsa-miR-186 overexpression is associated with metastatic cancers as well as arsenic-induced squamous cell carcinoma and is reported to target several mitotic regulators. Decreased levels of these proteins can dysregulate chromatid segregation contributing to aneuploidy. This work investigates the potential aneuploidogenic role of hsa-miR-186 in arsenic carcinogenesis. Clones of immortalized human keratinocytes (HaCaT) stably transfected with a hsa-miR-186 expression or empty vector were isolated. Three clones with high and low hsa-miR-186 expression determined by RT-qPCR were selected for further analysis and cultured with 0 or 100 nM NaAsO2 for 8 weeks. Analysis of mitoses revealed that chromosome number and structural abnormalities increased in cells overexpressing hsa-miR-186 and were further increased by arsenite exposure. Double minutes were the dominant structural aberrations. The peak number of chromosomes also increased. Cells with >220 to >270 chromosomes appeared after 2 months in hsa-miR-186 overexpressing cells, indicating multiple rounds of endomitosis had occurred. The fraction of cells with increased chromosome number or structural abnormalities did not increase in passage matched control cells. Levels of selected target proteins were determined by western blot. Expression of BUB1, a predicted hsa-miR-186 target was suppressed in hsa-miR-186 overexpressing clones, but increased with arsenite exposure. CDC27 remained constant under all conditions. These results suggest that overexpression of miR-186 in arsenic exposed tissues likely induces aneuploidy contributing to arsenic-induced carcinogenesis.

Arsenic-Induced Carcinogenesis: The Impact of miRNA Dysregulation.

Arsenic is a toxic metalloid widely present in the earth's crust, and is a proven human carcinogen. Chronic arsenic exposure mainly through drinking water causes skin, lung, and urinary bladder cancers, and is associated with liver, prostate, and kidney cancers, cardiovascular and neurological disorders, and diabetes. Several modes of action have been suggested in arsenic carcinogenesis. However, the molecular etiology of arsenic-induced cancer remains unclear. Recent evidence clearly indicates that gene expression modifications induced by arsenic may involve epigenetic alterations, including miRNA dysregulation. Many miRNAs have been implicated in different human cancers as a consequence of losses and or gains of miRNA function that contribute to cancer development. Progress in identifying miRNA dysregulation induced by arsenic has been made using different approaches and models. The present review discusses the recent data regarding dysregulated expression of miRNA in arsenic-induced malignant transformation in vitro, gaps in current understanding and deficiencies in current models for arsenic-induced carcinogenesis, and future directions of research that would improve our knowledge regarding the mechanisms involved in arsenic-induced carcinogenesis.

KRAS Oncogenic Signaling Extends beyond Cancer Cells to Orchestrate the Microenvironment.

KRAS is one of the most frequently mutated oncogenes in cancer, being a potent initiator of tumorigenesis, a strong inductor of malignancy, and a predictive biomarker of response to therapy. Despite the large investment to understand the effects of KRAS activation in cancer cells, pharmacologic targeting of KRAS or its downstream effectors has not yet been successful at the clinical level. Recent studies are now describing new mechanisms of KRAS-induced tumorigenesis by analyzing its effects on the components of the tumor microenvironment. These studies revealed that the activation of KRAS on cancer cells extends to the surrounding microenvironment, affecting the properties and functions of its constituents. Herein, we discuss the most emergent perspectives on the relationship between KRAS-mutant cancer cells and their microenvironment components. Cancer Res; 78(1); 7-14. ©2017 AACR.

Intricate Macrophage-Colorectal Cancer Cell Communication in Response to Radiation.

Both cancer and tumour-associated host cells are exposed to ionizing radiation when a tumour is subjected to radiotherapy. Macrophages frequently constitute the most abundant tumour-associated immune population, playing a role in tumour progression and response to therapy. The present work aimed to evaluate the importance of macrophage-cancer cell communication in the cellular response to radiation. To address this question, we established monocultures and indirect co-cultures of human monocyte-derived macrophages with RKO or SW1463 colorectal cancer cells, which exhibit higher and lower radiation sensitivity, respectively. Mono- and co-cultures were then irradiated with 5 cumulative doses, in a similar fractionated scheme to that used during cancer patients' treatment (2 Gy/fraction/day). Our results demonstrated that macrophages sensitize RKO to radiation-induced apoptosis, while protecting SW1463 cells. Additionally, the co-culture with macrophages increased the mRNA expression of metabolism- and survival-related genes more in SW1463 than in RKO. The presence of macrophages also upregulated glucose transporter 1 expression in irradiated SW1463, but not in RKO cells. In addition, the influence of cancer cells on the expression of pro- and anti-inflammatory macrophage markers, upon radiation exposure, was also evaluated. In the presence of RKO or SW1463, irradiated macrophages exhibit higher levels of pro-inflammatory TNF, IL6, CCL2 and CCR7, and of anti-inflammatory CCL18. However, RKO cells induce an increase of macrophage pro-inflammatory IL1B, while SW1463 cells promote higher pro-inflammatory CXCL8 and CD80, and also anti-inflammatory VCAN and IL10 levels. Thus, our data demonstrated that macrophages and cancer cells mutually influence their response to radiation. Notably, conditioned medium from irradiated co-cultures increased non-irradiated RKO cell migration and invasion and did not impact on angiogenesis in a chicken embryo chorioallantoic membrane assay. Overall, the establishment of primary human macrophage-cancer cell co-cultures revealed an intricate cell communication in response to ionizing radiation, which should be considered when developing therapies adjuvant to radiotherapy.