Chronic Aroclor 1260 exposure alters the mouse liver proteome, selenoproteins, and metals in steatotic liver disease.

The prevalence of metabolic dysfunction-associated steatotic liver disease (MASLD) continues to increase due in part to the obesity epidemic and to environmental exposures to metabolism disrupting chemicals. A single gavage exposure of male mice to Aroclor 1260 (Ar1260), an environmentally relevant mixture of non-dioxin-like polychlorinated biphenyls (PCBs), resulted in steatohepatitis and altered RNA modifications in selenocysteine tRNA 34 weeks post-exposure. Unbiased approaches identified the liver proteome, selenoproteins, and levels of 25 metals. Ar1260 altered the abundance of 128 proteins. Enrichment analysis of the liver Ar1260 proteome included glutathione metabolism and translation of selenoproteins. Hepatic glutathione peroxidase 4 (GPX4) and Selenoprotein O (SELENOO) were increased and Selenoprotein F (SELENOF), Selenoprotein S (SELENOS), Selenium binding protein 2 (SELENBP2) were decreased with Ar1260 exposure. Increased copper, selenium (Se), and zinc and reduced iron levels were detected. These data demonstrate that Ar1260 exposure alters the (seleno)proteome, Se, and metals in MASLD-associated pathways.

Altered splicing factor and alternative splicing events in a mouse model of diet- and polychlorinated biphenyl-induced liver disease.

Non-alcoholic fatty liver disease (NAFLD) is associated with human environmental exposure to polychlorinated biphenyls (PCBs). Alternative splicing (AS) is dysregulated in steatotic liver disease and is regulated by splicing factors (SFs) and N-6 methyladenosine (m6A) modification. Here integrated analysis of hepatic mRNA-sequencing data was used to identify differentially expressed SFs and differential AS events (ASEs) in the livers of high fat diet-fed C57BL/6 J male mice exposed to Aroclor1260, PCB126, Aroclor1260 + PCB126, or vehicle control. Aroclor1260 + PCB126 co-exposure altered 100 SFs and replicate multivariate analysis of transcript splicing (rMATS) identified 449 ASEs in 366 genes associated with NAFLD pathways. These ASEs were similar to those resulting from experimental perturbations in m6A writers, readers, and erasers. These results demonstrate specific hepatic SF and AS regulatory mechanisms are disrupted by HFD and PCB exposures, contributing to the expression of altered isoforms that may play a role in NAFLD progression to NASH.

Changes in m6A in Steatotic Liver Disease.

Fatty liver disease is one of the major causes of morbidity and mortality worldwide. Fatty liver includes non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH), now replaced by a consensus group as metabolic dysfunction-associated steatotic liver disease (MASLD). While excess nutrition and obesity are major contributors to fatty liver, the underlying mechanisms remain largely unknown and therapeutic interventions are limited. Reversible chemical modifications in RNA are newly recognized critical regulators controlling post-transcriptional gene expression. Among these modifications, N6-methyladenosine (m6A) is the most abundant and regulates transcript abundance in fatty liver disease. Modulation of m6A by readers, writers, and erasers (RWE) impacts mRNA processing, translation, nuclear export, localization, and degradation. While many studies focus on m6A RWE expression in human liver pathologies, limitations of technology and bioinformatic methods to detect m6A present challenges in understanding the epitranscriptomic mechanisms driving fatty liver disease progression. In this review, we summarize the RWE of m6A and current methods of detecting m6A in specific genes associated with fatty liver disease.

MicroRNA regulation of the serine synthesis pathway in endocrine-resistant breast cancer cells.

Despite the successful combination of therapies improving survival of estrogen receptor α (ER+) breast cancer patients with metastatic disease, mechanisms for acquired endocrine resistance remain to be fully elucidated. The RNA binding protein HNRNPA2B1 (A2B1), a reader of N(6)-methyladenosine (m6A) in transcribed RNA, is upregulated in endocrine-resistant, ER+ LCC9 and LY2 cells compared to parental MCF-7 endocrine-sensitive luminal A breast cancer cells. The miRNA-seq transcriptome of MCF-7 cells overexpressing A2B1 identified the serine metabolic processes pathway. Increased expression of two key enzymes in the serine synthesis pathway (SSP), phosphoserine aminotransferase 1 (PSAT1) and phosphoglycerate dehydrogenase (PHGDH), correlates with poor outcomes in ER+ breast patients who received tamoxifen (TAM). We reported that PSAT1 and PHGDH were higher in LCC9 and LY2 cells compared to MCF-7 cells and their knockdown enhanced TAM sensitivity in these-resistant cells. Here we demonstrate that stable, modest overexpression of A2B1 in MCF-7 cells increased PSAT1 and PHGDH and endocrine resistance. We identified four miRNAs downregulated in MCF-7-A2B1 cells that directly target the PSAT1 3'UTR (miR-145-5p and miR-424-5p), and the PHGDH 3'UTR (miR-34b-5p and miR-876-5p) in dual luciferase assays. Lower expression of miR-145-5p and miR-424-5p in LCC9 and ZR-75-1-4-OHT cells correlated with increased PSAT1 and lower expression of miR-34b-5p and miR-876-5p in LCC9 and ZR-75-1-4-OHT cells correlated with increased PHGDH. Transient transfection of these miRNAs restored endocrine-therapy sensitivity in LCC9 and ZR-75-1-4-OHT cells. Overall, our data suggest a role for decreased A2B1-regulated miRNAs in endocrine resistance and upregulation of the SSP to promote tumor progression in ER+ breast cancer.

Disruption of the mouse liver epitranscriptome by long-term aroclor 1260 exposure.

Chronic environmental exposure to polychlorinated biphenyls (PCBs) is associated with non-alcoholic fatty liver disease (NAFLD) and exacerbated by a high fat diet (HFD). Here, chronic (34 wks.) exposure of low fat diet (LFD)-fed male mice to Aroclor 1260 (Ar1260), a non-dioxin-like (NDL) mixture of PCBs, resulted in steatohepatitis and NAFLD. Twelve hepatic RNA modifications were altered with Ar1260 exposure including reduced abundance of 2'-O-methyladenosine (Am) and N(6)-methyladenosine (m6A), in contrast to increased Am in the livers of HFD-fed, Ar1260-exposed mice reported previously. Differences in 13 RNA modifications between LFD- and HFD- fed mice, suggest that diet regulates the liver epitranscriptome. Integrated network analysis of epitranscriptomic modifications identified a NRF2 (Nfe2l2) pathway in the chronic, LFD, Ar1260-exposed livers and an NFATC4 (Nfatc4) pathway for LFD- vs. HFD-fed mice. Changes in protein abundance were validated. The results demonstrate that diet and Ar1260 exposure alter the liver epitranscriptome in pathways associated with NAFLD.

m6A readers, writers, erasers, and the m6A epitranscriptome in breast cancer.

Epitranscriptomic modification of RNA regulates human development, health, and disease. The true diversity of the transcriptome in breast cancer including chemical modification of transcribed RNA (epitranscriptomics) is not well understood due to limitations of technology and bioinformatic analysis. N-6-methyladenosine (m6A) is the most abundant epitranscriptomic modification of mRNA and regulates splicing, stability, translation, and intracellular localization of transcripts depending on m6A association with reader RNA-binding proteins. m6A methylation is catalyzed by the METTL3 complex and removed by specific m6A demethylase ALKBH5, with the role of FTO as an 'eraser' uncertain. In this review, we provide an overview of epitranscriptomics related to mRNA and focus on m6A in mRNA and its detection. We summarize current knowledge on altered levels of writers, readers, and erasers of m6A and their roles in breast cancer and their association with prognosis. We summarize studies identifying m6A peaks and sites in genes in breast cancer cells.

Polychlorinated biphenyls alter hepatic m6A mRNA methylation in a mouse model of environmental liver disease.

Exposure to polychlorinated biphenyls (PCBs) has been associated with liver injury in human cohorts and with nonalcoholic steatohepatitis (NASH) in mice fed a high fat diet (HFD). N (6)-methyladenosine (m6A) modification of mRNA regulates transcript fate, but the contribution of m6A modification on the regulation of transcripts in PCB-induced steatosis and fibrosis is unknown. This study tested the hypothesis that PCB and HFD exposure alters the levels of m6A modification in transcripts that play a role in NASH in vivo. Male C57Bl6/J mice were fed a HFD (12 wks) and administered a single oral dose of Aroclor1260, PCB126, or Aroclor1260 + PCB126. Genome-wide identification of m6A peaks was accomplished by m6A mRNA immunoprecipitation sequencing (m6A-RIP) and the mRNA transcriptome identified by RNA-seq. Exposure of HFD-fed mice to Aroclor1260 decreased the number of m6A peaks and m6A-containing genes relative to PCB vehicle control whereas PCB126 or the combination of Aroclor1260 + PCB126 increased m6A modification frequency. ∼41% of genes had one m6A peak and ∼49% had 2-4 m6A peaks. 117 m6A peaks were common in the four experimental groups. The Aroclor1260 + PCB126 exposure group showed the highest number (52) of m6A-peaks. qRT-PCR confirmed enrichment of m6A-containing fragments of the Apob transcript with PCB exposure. A1cf transcript abundance, m6A peak count, and protein abundance was increased with Aroclor1260 + PCB126 co-exposure. Irrespective of the PCB type, all PCB groups exhibited enriched pathways related to lipid/lipoprotein metabolism and inflammation through the m6A modification. Integrated analysis of m6A-RIP-seq and mRNA-seq identified 242 differentially expressed genes (DEGs) with increased or reduced number of m6A peaks. These data show that PCB exposure in HFD-fed mice alters the m6A landscape offering an additional layer of regulation of gene expression affecting a subset of gene responses in NASH.

Multiomics analysis of the impact of polychlorinated biphenyls on environmental liver disease in a mouse model.

Exposure to high fat diet (HFD) and persistent organic pollutants including polychlorinated biphenyls (PCBs) is associated with liver injury in human populations and non-alcoholic fatty liver disease (NAFLD) and steatohepatitis (NASH) in animal models. Previously, exposure of HFD-fed male mice to the non-dioxin-like (NDL) PCB mixture Aroclor1260, dioxin-like (DL) PCB126, or Aroclor1260 + PCB126 co-exposure caused toxicant-associated steatohepatitis (TASH) and differentially altered the liver proteome. Here unbiased mRNA and miRNA sequencing (mRNA- and miRNA- seq) was used to identify biological pathways altered in these liver samples. Fewer transcripts and miRs were up- or down- regulated by PCB126 or Aroclor1260 compared to the combination, suggesting that crosstalk between the receptors activated by these PCBs amplifies changes in the transcriptome. Pathway enrichment analysis identified "positive regulation of Wnt/ß-catenin signaling" and "role of miRNAs in cell migration, survival, and angiogenesis" for differentially expressed mRNAs and miRNAs, respectively. We evaluated the five miRNAs increased in human plasma with PCB exposure and suspected TASH and found that miR-192-5p was increased with PCB exposure in mouse liver. Although we observed little overlap between differentially expressed mRNA transcripts and proteins, biological pathway-relevant PCB-induced miRNA-mRNA and miRNA-protein inverse relationships were identified that may explain protein changes. These results provide novel insights into miRNA and mRNA transcriptome changes playing direct and indirect roles in the functional protein pathways in PCB-related hepatic lipid accumulation, inflammation, and fibrosis in a mouse model of TASH and its relevance to human liver disease in exposed populations.

Combined exposure to polychlorinated biphenyls and high-fat diet modifies the global epitranscriptomic landscape in mouse liver.

Exposure to a single dose of polychlorinated biphenyls (PCBs) and a 12-week high-fat diet (HFD) results in nonalcoholic steatohepatitis (NASH) in mice by altering intracellular signaling and inhibiting epidermal growth factor receptor signaling. Post-transcriptional chemical modification (PTM) of RNA regulates biological processes, but the contribution of epitranscriptomics to PCB-induced steatosis remains unknown. This study tested the hypothesis that PCB and HFD exposure alters the global RNA epitranscriptome in male mouse liver. C57BL/6J male mice were fed a HFD for 12 weeks and exposed to a single dose of Aroclor 1260 (20 mg/kg), PCB 126 (20 µg/kg), both Aroclor 1260 and PCB 126 or vehicle control after 2 weeks on HFD. Chemical RNA modifications were identified at the nucleoside level by liquid chromatography-mass spectrometry. From 22 PTM global RNA modifications, we identified 10 significant changes in RNA modifications in liver with HFD and PCB 126 exposure. Only two modifications were significantly different from HFD control liver in all three PCB exposure groups: 2'-O-methyladenosine (Am) and N(6)-methyladenosine (m6A). Exposure to HFD + PCB 126 + Aroclor 1260 increased the abundance of N(6), O(2)-dimethyladenosine (m6Am), which is associated with the largest number of transcript changes. Increased m6Am and pseudouridine were associated with increased protein expression of the writers of these modifications: Phosphorylated CTD Interacting Factor 1 (PCIF1) and Pseudouridine Synthase 10 (PUS10), respectively, in HFD + PCB 126- + Aroclor 1260-exposed mouse liver. Increased N1-methyladenosine (m1A) and m6A were associated with increased transcript levels of the readers of these modifications: YTH N6-Methyladenosine RNA Binding Protein 2 (YTHDF2), YTH Domain Containing 2 (YTHDC2), and reader FMRP Translational Regulator 1 (FMR1) transcript and protein abundance. The results demonstrate that PCB exposure alters the global epitranscriptome in a mouse model of NASH; however, the mechanism for these changes requires further investigation.

HNRNPA2B1 regulates tamoxifen- and fulvestrant-sensitivity and hallmarks of endocrine resistance in breast cancer cells.

Despite new combination therapies improving survival of breast cancer patients with estrogen receptor α (ER+) tumors, the molecular mechanisms for endocrine-resistant disease remain unresolved. Previously we demonstrated that expression of the RNA binding protein and N6-methyladenosine (m6A) reader HNRNPA2B1 (A2B1) is higher in LCC9 and LY2 tamoxifen (TAM)-resistant ERα breast cancer cells relative to parental TAM-sensitive MCF-7 cells. Here we report that A2B1 protein expression is higher in breast tumors than paired normal breast tissue. Modest stable overexpression of A2B1 in MCF-7 cells (MCF-7-A2B1 cells) resulted in TAM- and fulvestrant- resistance whereas knockdown of A2B1 in LCC9 and LY2 cells restored TAM and fulvestrant, endocrine-sensitivity. MCF-7-A2B1 cells gained hallmarks of TAM-resistant metastatic behavior: increased migration and invasion, clonogenicity, and soft agar colony size, which were attenuated by A2B1 knockdown in MCF-7-A2B1 and the TAM-resistant LCC9 and LY2 cells. MCF-7-A2B1, LCC9, and LY2 cells have a higher proportion of CD44+/CD24-/low cancer stem cells (CSC) compared to MCF-7 cells. MCF-7-A2B1 cells have increased ERα and reduced miR-222-3p that targets ERα. Like LCC9 cells, MCF-7-A2B1 have activated AKT and MAPK that depend on A2B1 expression and are growth inhibited by inhibitors of these pathways. These data support that targeting A2B1 could provide a complimentary therapeutic approach to reduce acquired endocrine resistance.

Serine synthesis influences tamoxifen response in ER+ human breast carcinoma.

Estrogen receptor-positive breast cancer (ER+ BC) is the most common form of breast carcinoma accounting for approximately 70% of all diagnoses. Although ER-targeted therapies have improved survival outcomes for this BC subtype, a significant proportion of patients will ultimately develop resistance to these clinical interventions, resulting in disease recurrence. Phosphoserine aminotransferase 1 (PSAT1), an enzyme within the serine synthetic pathway (SSP), has been previously implicated in endocrine resistance. Therefore, we determined whether expression of SSP enzymes, PSAT1 or phosphoglycerate dehydrogenase (PHGDH), affects the response of ER+ BC to 4-hydroxytamoxifen (4-OHT) treatment. To investigate a clinical correlation between PSAT1, PHGDH, and endocrine resistance, we examined microarray data from ER+ patients who received tamoxifen as the sole endocrine therapy. We confirmed that higher PSAT1 and PHGDH expression correlates negatively with poorer outcomes in tamoxifen-treated ER+ BC patients. Next, we found that SSP enzyme expression and serine synthesis were elevated in tamoxifen-resistant compared to tamoxifen-sensitive ER+ BC cells in vitro. To determine relevance to endocrine sensitivity, we modified the expression of either PSAT1 or PHGDH in each cell type. Overexpression of PSAT1 in tamoxifen-sensitive MCF-7 cells diminished 4-OHT inhibition on cell proliferation. Conversely, silencing of either PSAT1 or PHGDH resulted in greater sensitivity to 4-OHT treatment in LCC9 tamoxifen-resistant cells. Likewise, the combination of a PHGDH inhibitor with 4-OHT decreased LCC9 cell proliferation. Collectively, these results suggest that overexpression of serine synthetic pathway enzymes contribute to tamoxifen resistance in ER+ BC, which can be targeted as a novel combinatorial treatment option.

Regulation of breast cancer metastasis signaling by miRNAs.

Despite the decline in death rate from breast cancer and recent advances in targeted therapies and combinations for the treatment of metastatic disease, metastatic breast cancer remains the second leading cause of cancer-associated death in U.S. women. The invasion-metastasis cascade involves a number of steps and multitudes of proteins and signaling molecules. The pathways include invasion, intravasation, circulation, extravasation, infiltration into a distant site to form a metastatic niche, and micrometastasis formation in a new environment. Each of these processes is regulated by changes in gene expression. Noncoding RNAs including microRNAs (miRNAs) are involved in breast cancer tumorigenesis, progression, and metastasis by post-transcriptional regulation of target gene expression. miRNAs can stimulate oncogenesis (oncomiRs), inhibit tumor growth (tumor suppressors or miRsupps), and regulate gene targets in metastasis (metastamiRs). The goal of this review is to summarize some of the key miRNAs that regulate genes and pathways involved in metastatic breast cancer with an emphasis on estrogen receptor α (ERα+) breast cancer. We reviewed the identity, regulation, human breast tumor expression, and reported prognostic significance of miRNAs that have been documented to directly target key genes in pathways, including epithelial-to-mesenchymal transition (EMT) contributing to the metastatic cascade. We critically evaluated the evidence for metastamiRs and their targets and miRNA regulation of metastasis suppressor genes in breast cancer progression and metastasis. It is clear that our understanding of miRNA regulation of targets in metastasis is incomplete.