FRA1 is essential for the maintenance of the oncogenic phenotype induced by in vitro long-term arsenic exposure.

Arsenic induces oncogenic effects activating stress-related signalling pathways. This can result in the over-activation of the AP-1 protein, specifically its FRA1 component. FRA1 is a transcription factor frequently overexpressed in epithelial tumors, where it can regulate the expression of different target genes. Accordingly, FRA1 could play an essential role in the in vitro cell transformation induced by arsenic. FRA1 levels were monitored in MEF cells throughout their transformation stages during 40 weeks of long-term 2 µM arsenic exposure. Interestingly, the results show a progressive FRA1 overexpression with time (60-fold and 11-fold for mRNA and pFRA/non-pFRA1, respectively, at week 40), which may be responsible for the observed altered expression in the FRA1 downstream target genes Pten, Pdcd4, Tpm1, Tgfb1, Tgfb2, Zeb1, Zeb2, and Twist. The levels of MAPKs (ERK, p38, and JNK) and other known players upstream from FRA1 were assessed at equivalent time-points, and ERK, p38 and RAS were pinpointed as potential candidates involved in arsenic-induced FRA1 activation. Furthermore, FRA1 stable knockdown under chronic arsenic exposure settings elicits a remarkable impact on the features relative to the cells' oncogenic phenotype. Notably, FRA1 knockdown cells present a 30% diminished proliferation rate, a 50% lowered migration and invasion potential, a 50% reduction in senescence, and a 30-60% reduced tumorsphere-forming ability. This work is the first to demonstrate the important role of FRA1 in the development and aggressiveness of the in vitro transformed phenotype induced by long-term arsenic exposure.

Role of As3mt and Mth1 in the genotoxic and carcinogenic effects induced by long-term exposures to arsenic in MEF cells.

DNA damage plays a crucial role in the transforming potential of the human carcinogen arsenic. The arsenic biotransformation enzyme AS3MT is known to participate in the generation of ROS after arsenic exposure, whereas MTH1 sanitizes oxidized dNTP pools to prevent the incorporation of damaged bases into DNA. In this work, we sought to assess the role of these two enzymes in the genotoxic and carcinogenic effects of arsenic exposure. Thus, mouse embryonic fibroblasts (MEF), transformed by chronic arsenite exposure, were monitored for DNA damage by the comet and the micronucleus assays at different time-of-exposure intervals lasting for 50 weeks. Results indicate that the oxidative and DNA damage of chronically exposed MEF cells increased time-dependently up to the point of transformation. As3mt expression followed a pattern like that of DNA damage, and its forced inhibition by shRNA technology before transformation resulted in a DNA damage decrease. On the other hand, Mth1 mRNA levels increased after the transformation point, and its forced knock-down increased significantly the levels of DNA damage and decreased the aggressiveness of the oncogenic phenotype. Thus, As3mt and Mth1 have important differential roles in the accumulation of DNA damage linked to the transformation process: while As3mt contributes to the genotoxic effects before the transformation, Mth1 prevents the DNA damage fixation after the acquisition of the oncogenic phenotype. This study demonstrates the influence of As3mt and Mth1 in arsenic DNA damage induction and it is the first to present Mth1 as a candidate modulator biomarker of the tumoral phenotype.

Differentially Expressed mRNA Targets of Differentially Expressed miRNAs Predict Changes in the TP53 Axis and Carcinogenesis-Related Pathways in Human Keratinocytes Chronically Exposed to Arsenic.

Arsenic is a widely distributed toxic natural element. Chronic arsenic ingestion causes several cancers, especially skin cancer. Arsenic-induced cancer mechanisms are not well defined, but several studies indicate that mutation is not the driving force and that microRNA expression changes play a role. Chronic low arsenite exposure malignantly transforms immortalized human keratinocytes (HaCaT), serving as a model for arsenic-induced skin carcinogenesis. Early changes in miRNA expression in HaCaT cells chronically exposed to arsenite will reveal early steps in transformation. HaCaT cells were maintained with 0/100 nM NaAsO2 for 3 and 7 weeks. Total RNA was purified. miRNA and mRNA expression was assayed using Affymetrix microarrays. Targets of differentially expressed miRNAs were collected from TargetScan 6.2, intersected with differentially expressed mRNAs using Partek Genomic Suite software, and mapped to their pathways using MetaCore software. MDM2, HMGB1 and TP53 mRNA, and protein levels were assayed by RT-qPCR and Western blot. Numerous miRNAs and mRNAs involved in carcinogenesis pathways in other systems were differentially expressed at 3 and 7 weeks. A TP53 regulatory network including MDM2 and HMGB1 was predicted by the miRNA and mRNA networks. Total TP53 and TP53-S15-phosphorylation were induced. However, TP53-K382-hypoacetylation suggested that the induced TP53 is inactive in arsenic exposed cells. Our data provide strong evidence that early changes in miRNAs and target mRNAs may contribute to arsenic-induced carcinogenesis.

Oxidative DNA damage enhances the carcinogenic potential of in vitro chronic arsenic exposures.

Chronic exposure to arsenic is known to increase the incidence of cancer in humans. Our previous work demonstrated that environmentally relevant arsenic exposures generate an accelerated accumulation of pre-carcinogen 8-OH-dG DNA lesions under Ogg1-deficient backgrounds, but it remains unproved whether this observed arsenic-induced oxidative DNA damage (ODD) is certainly important in terms of cancer. Here, isogenic MEF Ogg1 (+/+) cells and MEF Ogg1 (-/-) cells-unable to properly eliminate 8-OH-dG from DNA-were exposed to 0.5, 1 and 2 µM of sodium arsenite for 40 weeks. The acquisition of an in vitro cancer-like phenotype was assessed throughout the exposure; matrix metalloproteinase (MMP) activities were measured by zymography, colony formation and promotion were evaluated by soft agar assay, and cellular invasiveness was measured by the transwell assay. Alterations in cellular morphology, growth and differentiation status were also included as complementary measures of transformation. MEF Ogg1 (-/-) cells showed a cancer-associated phenotype after 30 weeks of exposure, as indicated by morphological changes, increased proliferation, deregulated differentiation status, increased MMPs secretion, anchorage-independent cell growth and enhancement of tumor growth and invasiveness. Conversely, MEF Ogg1 (+/+) cells did not present changes in morphology or proliferation, exhibited a milder degree of gene deregulation and needed 10 weeks of additional exposure to the highest arsenite doses to show tumor enhancing effects. Thus, Ogg1 genetic background and arsenic-induced 8-OH-dG proved relevant for arsenic-mediated carcinogenic effects. To our knowledge, this is the first study directly linking ODD with arsenic carcinogenesis.

Reduced cellular DNA repair capacity after environmentally relevant arsenic exposure. Influence of Ogg1 deficiency.

Inorganic arsenic (i-As) is a genotoxic and carcinogenic environmental contaminant known to affect millions of people worldwide. Our previous work demonstrated that chronic sub-toxic i-As concentrations were able to induce biologically significant levels of genotoxic and oxidative DNA damage that were strongly influenced by the Ogg1 genotype. In order to study the nature of the observed levels of damage and the observed differences between MEF Ogg1(+/+) and Ogg1(-/-) genetic backgrounds, the genotoxic and oxidative DNA repair kinetics of 18-weeks exposed MEF cells were evaluated by the comet assay. Results indicate that MEF Ogg1(+/+) and Ogg1(-/-) cells chronically exposed to i-As repair the DNA damage induced by arsenite, potassium bromide and UVC radiation less efficiently than control cells, being that observation clearly more pronounced in MEF Ogg1(-/-) cells. Consequently, exposed cells accumulate a higher percentage of unrepaired DNA damage at the end of the repair period. As an attempt to eliminate i-As associated toxicity, chronically exposed MEF Ogg1(-/-) cells overexpress the arsenic metabolizing enzyme As3mt. This adaptive response confers cells a significant resistance to i-As-induced cell death, but at expenses of accumulating high levels of DNA damage due to their repair impairment. Overall, the work presented here evidences that i-As chronic exposure disrupts the normal cellular repair function, and that oxidative DNA damage-and Ogg1 deficiency-exacerbates this phenomenon. The observed cell death resistance under a chronic scenario of genotoxic and oxidative stress may in turn contribute to the carcinogenic effects of i-As.

Arsenic exposure disrupts the normal function of the FA/BRCA repair pathway.

Chronic arsenic exposure is known to enhance the genotoxicity/carcinogenicity of other DNA-damaging agents by inhibiting DNA repair activities. Interference with nucleotide excision repair and base excision repair are well documented, but interactions with other DNA repair pathways are poorly explored so far. The Fanconi anemia FA/BRCA pathway is a DNA repair mechanism required for maintaining genomic stability and preventing cancer. Here, interactions between arsenic compounds and the FA/BRCA pathway were explored by using isogenic FANCD2(-/-) (FA/BRCA-deficient) and FANCD2(+/+) (FA/BRCA-corrected) human fibroblasts. To study whether arsenic disrupts the normal FA/BRCA function, FANCD2(+/+) cells were preexposed to subtoxic concentrations of the trivalent arsenic compounds methylarsonous acid (MMA(III)) and arsenic trioxide (ATO) for 2 weeks. The cellular response to mitomicin-C, hydroxyurea, or diepoxybutane, typical inducers of the studied pathway, was then evaluated and compared to that of FANCD2(-/-) cells. Our results show that preexposure to the trivalent arsenicals MMA(III) and ATO induces in corrected cells, a cellular FA/BRCA-deficient phenotype characterized by hypersensitivity, enhanced accumulation in the G2/M compartment and increased genomic instability--measured as micronuclei. Overall, our data demonstrate that environmentally relevant arsenic exposures disrupt the normal function of the FA/BRCA activity, supporting a novel source of arsenic co- and carcinogenic effects. This is the first study linking arsenic exposure with the FA/BRCA DNA repair pathway.