Arsenic-Induced Carcinogenesis: The Impact of miRNA Dysregulation

Abstract

Carcinogenesis is a complicated process that, despite much progress in delineating the role of mutation, is still incompletely understood. Recent studies have provided evidence that microRNAs (miRNAs or miRs) dysregulation plays a role in carcinogenesis of many tissue types. Focused studies in arsenic-induced cancers suggest that mutations may play a lesser role in arsenic-induced carcinogenesis (States, 2015). Instead, the findings from many studies have shown that miRNAs are critically involved in arsenic-induced malignant transformation. Here, we explore the recent data on dysregulation of miRNA expression in arsenic-exposed cells in vitro and discuss gaps in current understanding and deficiencies in current models for arsenic-induced carcinogenesis.

ARSENIC

Arsenic is a toxic metalloid present in the earth’s crust, and is one of the most well-known human carcinogens (IARC Working Group on the Evaluation of Carcinogenic Risks to Humans., 2012). Chronic arsenic exposure in drinking water is a global issue affecting at least 70 countries and >140 million people (Mukherjee et al., 2006). The U.S. Environmental Protection Agency (USEPA) and the World Health Organization (WHO) have set the Maximum Contaminant Level (MCL) for arsenic in water at 10 ppb (10 μg/l). However, a recent study showed that 2.1 million people in the U.S. use water from domestic wells with predicted arsenic concentration >10 μg/l (Ayotte et al., 2017). Epidemiological evidence has shown that long-term arsenic exposure is associated with human diseases such as skin lesions, cardiovascular disease, neurological disorders, and diabetes, and increased risk of developing skin, lung, and urinary bladder cancers (Chen et al., 2017; States, 2015). Several modes of action have been suggested in arsenic carcinogenesis including DNA repair inhibition, comutagenicity, oxidative stress, cell proliferation, aneuploidy, interaction with zinc finger proteins and epigenetic alterations including changes in DNA methylation, histone modification, and miRNA expression (Bailey and Fry, 2014; Reichard and Puga, 2010; States, 2015; Zhou et al., 2014). Recent in vitro studies support an association of exposure to arsenic with altered miRNA expression and alternative mRNA splicing (Al-Eryani et al., 2018; Riedmann et al., 2015).

miRNAs

Biogenesis and Biological Function

miRNAs are part of the epigenome and compose a dominating class of small RNAs in most somatic tissues. They are 21–22 nucleotide long RNAs that mediate posttranscriptional gene silencing by guiding Argonaute (AGO) proteins to mRNA targets (Ha and Kim, 2014; Hausser and Zavolan, 2014). miRNA biogenesis occurs in both the nucleus and the cytoplasm. The formation of mature miRNA is a 2-step process (Figure 1). In the first step, the primary transcript (pri-miRNA) is cleaved in the nucleus by the microprocessor complex into pre-miRNA (∼85-nucleotide pre-miRNA hairpin) (Peng and Croce, 2016). Then, the trimmed pre-miRNA is transported from the nucleus to the cytoplasm where the pre-miRNA is processed to a ∼22-nucleotide single-stranded mature miRNA (Peng and Croce, 2016). Mature miRNA is incorporated into an RNA-induced silencing complex (RISC) and guides the RISC to the target mRNA to regulate translation (Peng and Croce, 2016).

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Figure 1.

miRNA biogenesis. The multiple steps of miRNAs biogenesis in both the nucleus and cytoplasm and their role in regulating their target mRNAs.

miRNAs target cytosolic mRNA by hybridization to complementary sequences, typically in 3′-untranslated regions (UTRs) called the “seed region” leading to decreased translation, deadenylation, or degradation of the mRNA (Bushati and Cohen, 2007; Hayes et al., 2014). Extensive pairing of miRNAs with their target mRNAs, eg, the seed region of mRNA matches perfectly with the targeting miRNA, leads to the degradation of the mRNA. However, imperfect base pairing leads to repression of mRNA translation and the possibility of restoring translation once the repressor miRNA is degraded (Macfarlane and Murphy, 2010).

miRNAs play a crucial role during developmental processes, apoptosis, and cell proliferation, and in regulating translation of most mammalian protein-coding genes (Friedman et al., 2009). Some miRNAs can also regulate a large number of zinc finger (ZNF) genes by directly targeting their coding regions (Huang et al., 2010). Dysregulation of miRNA is often associated with development, progression, and response to therapy of viral, immune-related and neurodegenerative diseases, and cancer (Li and Kowdley, 2012). The involved mechanisms of miRNA dysregulation in cancer include transcriptional dysregulation, epigenetic alterations, defects in the miRNA biogenesis pathway, and chromosomal instability (Peng and Croce, 2016).

Role in Carcinogenesis

miRNAs regulate molecular pathways by targeting various oncogenes and tumor suppressors having a function in cancer and stem cell biology, angiogenesis, the epithelial-mesenchymal transition, metastasis, and drug resistance (Humphries et al., 2016). Dysregulated miRNAs have a fundamental activity in cancer development and progression and may act as a novel class of oncogenes (by suppressing tumor suppressor genes) or of tumor suppressor genes (by suppressing oncogenes) (Figure 2) (Lotterman et al., 2008; Zhou et al., 2017). For example, miRNA let-7a down regulated MYC, a proto oncogene, in Burkitt lymphoma cells inhibiting cell growth induced by MYC (Sampson et al., 2007). On the other hand, overexpression of miR-504 negatively regulated p53, decreasing the p53-mediated apoptosis, and cell cycle arrest in response to stress (Hu et al., 2010). These 2 examples highlight the importance of microRNAs in tumorigenesis. Aberrant miRNA expression has been found in different cancers, including breast, colon, gastric, lung, prostate, and thyroid (Reddy, 2015). Thus, identification of miRNAs differentially expressed between normal and tumor tissues may help to define the contribution of miRNA in carcinogenesis initiation and progression and additionally may function as biomarkers for cancer diagnosis and prognosis (Humphries et al., 2016). A further role of miRNA may be implicated with chemical carcinogenesis since many studies have linked dysregulated miRNA expression and function after chemical exposure. Moreover, recent data have shown that arsenic can alter miRNA expression patterns in in vitro and in vivo models of arsenic-induced carcinogenesis (Table 1) (Humphries et al., 2016; Ren et al., 2015).

Table 1.

miRNA Dysregulation in Models of Arsenic-Induced Carcinogenesis

Type of Study	Target Organ	Cell Type or Animal Strain	Arsenic Exposure	miRNA	miRNA Change	References
In vitro	Skin	HaCaT	500 nM for 4 weeks	21, 200a, 141	Upregulation	(32)
		HaCaT	0.05 ppm for 8 weeks	21	Upregulation	(33)
		HaCaT	1 μM for 15 weeks	let-7a, let-7b, let7-c	Downregulation	(34)
	Lung	16-HBE	2.5 μM for 13 weeks	155	Upregulation	(5)
		HBE	1 μM for 30 passages	21	Upregulation	(36)
		BEAS-2B	0.5 μM for 24 weeks	21	Upregulation	(38)
		BEAS-2B	1 μM for 26 weeks	199-a	Downregulation	(40)
	Urinary bladder	HUC1	1 μM for 48 and 60 weeks	200a, 200b, 200c	Downregulation	(41)
	Liver	L-02	1, 2, 4, or 8 μM for 24 h	21	Upregulation	(42)
		L-02	2 μM for 15 weeks	191	Upregulation	(43)
In vivo	Liver	Sprague Dawley rats	0.1, 1, 10, and 100 mg/l for 60 days	151, 183	Upregulation	(24)
				423, 26a, 148b	Downregulation

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Figure 2.

miRNAs as tumor suppressors and oncogenes. miRNA may act as transducer/mediator among oncogene and tumor suppressor genes leading to tumor formation (adapted from Paranjape et al., 2009).

miRNA DYSREGULATION IN ARSENIC-INDUCED CARCINOGENESIS

After the identification of miRNAs, several studies were performed exploring environmental carcinogens such as arsenic, altered miRNA expression profile, and carcinogenesis. The first work evaluating the impact of arsenic exposure on miRNA expression was published in 2006 (Marsit et al., 2006). Until 2010, relatively few studies in this field had been published. Some of these studies have focused on the therapeutic potential of arsenic in leukemia by modulating miRNAs, resulting in apoptosis inhibition (Gao et al., 2010; Li et al., 2010). Since then, a variety of studies have assessed the involvement of miRNAs mediating arsenic-induced carcinogenesis. As for example, miR-200 (Sahu, 2012), miR-190, (Ren et al., 2010), and miR-21 (Gonzalez et al., 2015; Sherr et al., 2016; Ye et al., 2017). However, as indicated by some authors, the majority of these studies have been performed in vitro using different cell lines, but the functional consequences of miRNA altered profile still need to be addressed (Bailey and Fry, 2014; Ren et al., 2010; Sahu, 2012). In the past 5 years, progress in identifying miRNA changes after arsenic exposure has been made using different approaches and models.

CURRENT DATA GAPS IN ARSENIC-INDUCED CARCINOGENESIS

Oral administration of arsenic to rodent models has provided significant information on human arsenic carcinogenesis although there is still a general lack of animal models, particularly an in vivo model for arsenic-induced skin cancer. The available studies evaluating arsenic and skin in rodents did not provide evidence of arsenic carcinogenicity by itself, rather, they demonstrated that arsenic is not a complete carcinogen for rodent skin (Burns et al., 2004; Motiwale et al., 2005; Rossman et al., 2001; Uddin et al., 2005), indicating that the biological response of humans to arsenic is different from that of rodents. Thus, the in vitro studies are the major source of information regarding arsenic-induced skin carcinogenesis. However, the in vitro models used to evaluate arsenic toxicity often use immortalized cell lines instead of primary cells. Although results provided by immortalized cell systems are generally consistent, not only may they not adequately represent primary cells but also may provide different results due to differences between immortalized and primary cells. Immortalized cell lines provide unlimited supply of material and should maintain functional features of the primary cells, although serial passages of cells could lead to genotypic and phenotypic variation over time and genetic drift could also cause heterogeneity in cultures. For these reasons, immortalized cells may not adequately mimic primary cells and may provide different results (Kaur and Dufour, 2012). A better understanding of how arsenic changes primary cells is essential to integrate epidemiological evidence and experimental data on arsenic and cancer risk.

Another issue concerning in vitro data consists of the arsenic concentration used in most of the peer-reviewed literature, which do not represent internal arsenic exposures at physiological levels. Epidemiological studies had measured arsenic physiological levels as ∼100 nM (Pi, 2000), a lower level than those that have been used in many arsenic-induced transformation studies. The high concentrations of arsenic might induce molecular responses and biological effects in vitro that might not reflect the chronic health outcomes experienced by individuals exposed to low to moderate arsenic concentrations. A large number of pathways and proteins have been analyzed in order to investigate arsenic effects. The levels of tumor protein P53 (TP53) expression remain conflicting, having been reported to be enhanced or decreased after arsenic treatment, depending on the cell line, dosing, and timing of exposure (Huang et al., 2008). Because TP53 plays an important role in the induction of cell cycle arrest, DNA repair signaling and/or apoptosis, further investigation is needed to explain the differences found in TP53 levels after arsenic exposure (Fischer, 2017). These results could clarify early signaling events caused by arsenic, delineating some of the details involved in cancer induction.

FUTURE DIRECTIONS

The mechanisms of arsenic-induced carcinogenesis are not yet clear. Several mechanisms, including epigenetic alterations are proposed. miRNAs are part of the epigenome and play an important role in gene regulation and cellular development (Leichter et al., 2017). The studies discussed above strongly suggest that dysregulated miRNA expression plays a critical role in arsenic-induced tumorigenesis and carcinogenesis although there are still some important aspects to consider in further investigations. First, the use of primary cells and physiologically relevant arsenic exposure could provide more relevant details about mechanisms involved in arsenic-induced cancer in humans. Second, findings from epidemiological and in vivo arsenic-related studies must be considered for choosing potential miRNAs for future research. Current studies analyzed selected miRNAs based on previous data showing differential expression in human tumors in general but not related to arsenic exposure. An unpublished pioneering work identified differential miRNA expression between human arsenic-induced premalignant and malignant skin lesions (Al-Eryani et al., manuscript submitted). Thus, further investigation could consider the identified miRNAs as targets for understanding their role in carcinogenesis induced by chronic exposure to arsenic. Furthermore, longitudinal studies following the miRNA differential expression throughout the transformation process are necessary to evaluate the critical changes during arsenic-associated transformation and carcinogenesis. Such information would improve our knowledge regarding the mechanisms involved in arsenic-induced carcinogenesis and direct the next steps of research.

FUNDING

The authors were supported in part by NIH-NIEHS grants 5R21ES023627-02 and 1R01ES027778-01A1.

REFERENCES