Am J Cancer Res 2016;6(5):1066-1077 www.ajcr.us /ISSN:2156-6976/ajcr0027753 Original Article The aryl hydrocarbon receptor agonist benzo(a)pyrene reactivates LINE-1 in HepG2 cells through canonical TGF-β1 signaling: implications in hepatocellular carcinogenesis Elsa M Reyes-Reyes, Irma N Ramos, Marco A Tavera-Garcia, Kenneth S Ramos Division of Pulmonary, Allergy, Critical Care and Sleep Medicine, University of Arizona College of Medicine, Tucson, Arizona 85721, USA Received March 8, 2016; Accepted March 16, 2016; Epub May 1, 2016; Published May 15, 2016 Abstract: Long interspersed nuclear element-1 (L1) is a genetic element that mobilizes throughout the mammalian genome via retrotransposition and damages host DNA via mutational insertions, chromosomal rearrangements, and reprogramming of gene expression. The cellular mechanisms responsible for aberrant L1 expression during cancer pathogenesis are unclear. Previously, we have shown that L1 reactivation in several human cell lines is dependent upon the activation of aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor member of the PAS superfamily of proteins. We also showed that ectopic expression of L1 reprograms the HepG2 genome leading to epithelial-to-mesenchymal transition (EMT). Here we present evidence that reactivation of L1 and modu- lation of EMT in HepG2 cells by the AhR ligand benzo(a)pyrene (BaP) is effected through the canonical TGF-β1 signaling pathway. BaP increased TGF-β1 mRNA, SMAD2 phosphorylation and decreased expression of E-Cadherin. The functional relevance of these interactions and the involvement of TGFBR1/ALK5 and SMAD2/3 were confirmed by siRNA interference. Furthermore, expression of L1-encoded ORF1p was positively correlated with the activation of TGF-β1 signaling in human hepatocarcinoma samples at various stages of malignant progression. These results indicate that ligand-mediated AhR activation regulates L1 via canonical TGF-β1 signaling and raise important ques- tions about the molecular etiology of human hepatocarcinomas. Keywords: Long interspersed nuclear element-1, benzo(a)pyrene, AhR, TGF-β1, SMAD Introduction L1 retrotransposition is tightly regulated in somatic tissues via genetic and epigenetic Long interspersed nuclear element-1 (LINE-1 or mechanisms [5-7]. However, aberrant ORF1p L1) is a repetitive DNA sequence that mobilizes expression and new somatic L1 insertions have via a “copy and paste” retrotransposition mech- been detected in several epithelial cancers anism. A functional L1 element in humans is ~6 [8-11]. L1 retrotransposition is highly mutagen- kb in length and consists of a 5’ untranslated ic and may promote cancer through induction of region (UTR) with promoter activity, two open aberrant splicing, exon skipping, and genome reading frames, and a terminal 3’ UTR [1, 2]. L1 rearrangements that change gene expression encodes two proteins; ORF1p, a 40 kDa protein and promote genome instability [3, 4]. Our with nucleic acid binding activity and ORF2p, a group has shown that L1 reactivation exerts 150 kDa protein with endonuclease and retrotransposition-dependent and retrotrans- reverse transcriptase activities [3]. A complete position-independent functions that may be L1 retrotransposition cycle consists of tran- critical to neoplastic transformation and cancer scription of L1 RNA, export into the cytoplasm, progression [12, 13]. Ectopic expression of wild translation of ORF1 and ORF2, association of type or a mutant L1 protein incapable of ret- L1 RNA with ORF1 and ORF2 proteins to form rotransposition modulates the expression of a ribonucleoprotein (RNP) particles, import into large number of genetic targets involved in can- the nucleus, reverse transcription, and integra- cer through changes in cellular adhesion, tion into new genomic locations [4]. inflammation and metabolism [12, 13]. L1 also BaP-mediated AhR activation regulates L1 via canonical TGF-β1 signaling disrupts epithelial cell differentiation programs common cellular processes e.g. cell cycle pro- and induces epithelial-to-mesenchymal transi- gression, apoptosis, cell adhesion and interac- tion (EMT) [13], a process that facilitates tion with extracellular matrix [23]. Several stud- metastasis by promoting cell motility and con- ies have shown that AhR can regulate TGF-β1 ferring transformed cells the ability to invade signaling, through deregulation of TGF-β1 [14]. secretion, modulation of TGF-β1 expression or down-regulation of the latency-associated pro- A master promoter of EMT is transforming tein (LTBP-1) expression [24-26]. TGF-β1 also growth factor-β1 (TGF-β1) [15], a potent immu- regulates AhR expression and CYP1A1/1B1 nosuppressive and pro-inflammatory cytokine enzyme activity in a cell/tissue specific manner [16]. TGF-β1 plays a dual role in carcinogenesis, [27-29]. Thus, different mechanisms have been having the ability to inhibit proliferation of can- proposed to explain AhR and TGF-β1 crosstalk cer cells or to activate migration and invasive- in endothelium [30], regulatory T cells [31], ness through EMT [15] or neoangiogenesis Th17 cells [32] or dendritic cells [33]. The com- [17]. TGF-β1 signaling is mediated through a plexity of tissue-context specific mechanisms complex that includes type I and two type II in the regulation of L1 by AhR/TGF-β1 crosstalk transmembrane receptors with serine/threo- is the primary focus of the present nine kinase activity. Type II receptors phosphor- investigation. ylate type I components, while type 1 receptors transduce the signal from the cell membrane to Materials and methods the nucleus canonically through a complex of SMAD proteins or alternatively, non-canonical- Materials ly, through other signaling pathways e.g. Jun BaP was purchased from Ultra Scientific N-terminal kinase (JNK), p38 and extracellu- (Kingstown, RI). Recombinant human TGF-β1 lar signal-regulated kinases/mitogen-activated was purchased from R&D Systems (Minnea- protein kinases (ERK/MAPK) [18]. Interestingly, polis, MN). Monoclonal anti-GAPDH, and hor- in silico analysis of the L1 regulatory genetic seradish peroxidase (HRP) linked anti-mouse network [12], and biological validation in HepG2 IgG antibodies were from Santa Cruz Biotech cells [13], showed that some of genetic targets (Dallas, TX). Rabbit anti-AhR (13790), anti-E- of L1 are also targets of TGF-β1 (e.g. CCL2, cadherin (24E10), anti-vimentin (D21H3), anti- VCAM, CXCL1) [19-21]. These data suggested SMAD2 (5339), anti-phospho-SMAD2 (3108), that L1 activation and TGF-β1 signaling in hepa- anti-SMAD2/3 (8685), anti-TGFBR1 (3712), toma cells may be cooperative and important in and horseradish peroxidase (HRP) linked anti- hepatocarcinogenesis. rabbit IgG antibodies were from Cell Signaling Technology (Beverly, MA). Protein lysates from: The regulatory networks involved in L1 reactiva- normal limits liver tissue (male, case ID. tion during cell transformation and cancer pro- CU0000001489, Cat No. CP565754), staging I gression are not clear. We have previously hepatocellular liver carcinoma tissue (male, shown that L1 reactivation by the environmen- case ID. CU0000012132, Cat No. CP641361), tal carcinogen BaP is mediated through binding staging II hepatocellular liver carcinoma tissue to the aryl hydrocarbon receptor (AhR). AhR is a (male, case ID. CU0000005407, Cat No. ligand-activated transcription factor ubiqui- CP19427), staging IIIA hepatocellular liver car- tously distributed that translocates from the cinoma tissue (male, case ID. CU000001197, cytosol to the nucleus after ligation by polycy- Cat No. CP607175), and staging IV hepatocel- clic aromatic hydrocarbons. Ligand-bound AhR lular liver carcinoma tissue (male, case ID. forms a heterodimer with the AhR nuclear CU0000013002, Cat No. CP532216) were translocator (ARNT) and binds to a specific from OriGene (Rockville, MD). Pathological sequence to regulate gene expression. The staging of tissue samples followed established hallmark response of AhR activation is the tran- guidelines. DMSO was from American Type scriptional activation of cytochrome P4501A1 Culture Collection (ATCC). (CYP1A1) in hepatic parenchymal cells [22]. Polyclonal anti-human ORF1p antibody There exists a cell-specific and context-depen- dent crosstalk between AhR and TGF-β1. Both A custom made polyclonal antibody produced AhR and TGF-β1 participate in the regulation of by New England Peptide Inc. was diluted 1067 Am J Cancer Res 2016;6(5):1066-1077 BaP-mediated AhR activation regulates L1 via canonical TGF-β1 signaling Table 1. Oligonucleotides sequences used in this study 1% IGEPAL CA-630 (pH 7.5), supple- Sequence Description mented with protease and phospha- tase inhibitor cocktails (EMD Millipore) 5’-CCA AGTTGGAAAACACTCTGC-3’ ORF1-Forward-RT-PCR for 5 min at 4°C and then cleared by 5’-TGTGGCGTTCTCTGTATTTCC-3’ ORF1-Reverse-RT-PCR centrifugation at 16,000 × g for 10 5’-TCGACACATACACTCTCCCAAG-3’ ORF2-Forward-RT-PCR minutes at 4°C. All protein concentra- 5’-TGGTCCTGGACTCTTTTTGG-3’ ORF2-Reverse-RT-PCR tions were determined using the 5’-GGATACCAACTATTGCTTCAGCTCC-3’ TGF β1-Forward-RT-PCR bicinchoninic acid assay (Thermo 5’-AGGCTCCAAATATAGGGGCAGGGTC-3’ TGF β1-Reverse-RT-PCR Fisher Scientific). 5’-GATCATCAGCAATGCCTCCT-3’ GAPDH-Forward-RT-PCR Immunoblotting 5’-TGTGGTCATGAGTCCTTCCA-3’ GAPDH-Reverse-RT-PCR 5’-CGGAUGAAAUCCUGACGUAtt-3’ AHR-siRNA-Sense Total cell lysates were resolved by 5’-UACGUCAGGAUUUCAUCAGtt-3’ AHR-siRNA-Antisense SDS-Tris PAGE and transferred onto 5’-CAGUUACUGUGGAAGGAAUtt-3’ TGFBR1-siRNA-Sense polyvinylidine fluoride membranes