They are mainly localised in the liver, participating in the phase I metabolism of a wide range of exogenous compounds and the biosynthesis and metabolism of endogenous hormones [ 2 ]. Apart from the liver, CYPs are also expressed in other tissues such as lung, kidney and hematopoietic tissue [ 3 ], and specific isoenzymes of the superfamily have been identified in tumours [ 4 ] where they are suggested to affect the response to anticancer therapy [ 4 , 5 ].
CYPs are highly conserved across species implying that, in addition to their function in the metabolism of xenobiotics, these enzymes possibly exert broader physiological functions [ 6 ]. Consistent with this view, the CYP2E1 isoenzyme has been implicated in a variety of pathological conditions such as diabetes, non-alcoholic steatohepatitis NASH and cancer, possibly as a result of its capacity to produce high levels of reactive oxygen species ROS [ 7 ].
CYP2E1 metabolizes several small molecules such as ethanol, acetaminophen and pro-carcinogens like nitrosamines and azo compounds [ 3 ].
High ROS levels, and hence oxidative stress due to increased CYP2E1 protein levels and induced enzymatic activity, are the main causes of various liver diseases associated with chronic alcohol consumption [ 8 ] and a variety of other pathophysiological conditions including diabetes type II and obesity [ 9 ].
Since CYP2E1 is a key determinant of the cellular redox state generating free radicals in a non-specific manner, even in the absence of a substrate the gene expression of this enzyme is tightly regulated [ 10 ]. Indeed, links between CYP2E1 protein levels and cytokines activity have been shown in recent reports [ 11 ] as well as variable CYP2E1 gene expression in numerous inflammatory diseases including cancer [ 12 , 13 ]. Autophagy is one of the pathways induced by elevated ROS levels which triggers the accumulation of various autophagy-regulated genes ATGs including beclin-1 and the light chain 3 LC3 [ 14 ], thereby stimulating the formation of the autophagosome in cancer [ 15 , 16 ].
Furthermore, oxidative stress and other cellular tensions, such as DNA damage and viral infection, impair the protein-folding process resulting in the accumulation of misfolded proteins within the endoplasmic reticulum ER lumen [ 17 ], stimulating the initiation of the unfolded protein response UPR [ 18 ]. UPR takes place in the ER lumen and is a major signal transduction pathway aiming to alleviate ER stress by removing accumulated unfolded proteins from this cellular compartment [ 18 ].
Taken together, the differential expression of CYP2E1 in different tumours and various stages of breast cancer, with its capacity to induce ROS production [ 7 ], raises the questions whether CYP2E1 cellular levels could be an indicator of breast cancer progression and which are the factors involved in its differential regulation of gene expression in the various stages of breast cancer.
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Here we present evidence to suggest that ectopically expressed CYP2E1-mediated oxidative stress regulates autophagy, ER stress and migratory potential and its gene expression is regulated by the p53 tumour suppressor in a cell-type-dependent manner in breast cancer cells. The process for chromatin immunoprecipitation ChIP has been described in the past [ 22 , 23 ]. Briefly chromatin was cross-linked with 1.
Upon activation, the endoribonuclease activity of IRE1 catalyses splicing and removal of an intron from the Xbox binding protein 1 XBP1 encoding mRNA thereby allowing its translation into a functional transcription factor [ 18 ]. The scratch wound assay was performed as described previously [ 25 ]. Briefly coverslips were placed in 6-well plates and an insert ibidi, Munich, Germany was placed onto the coverslip before cells were seeded.
It has been shown that CYP2E1 is one of the most active CYP isoforms in generating intracellular ROS [ 26 ] and oxygen radicals are associated with cancer development and metastasis [ 27 ]. Several observations link CYP2E1 with inflammatory reactions and carcinogenesis in different tissues [ 28 , 29 ] suggesting that CYP2E1 might be involved in the regulation of tumour growth [ 29 ].
Data represent fold induction of ROS levels. Error bars indicate standard deviation obtained from three independent experiments performed in duplicates. CYP2E1 is involved in orchestrating autophagy in breast cancer. One representative from three independent experiments is shown. Recently published observations have indicated that CYP2E1 is mainly localised within the endoplasmic reticulum and functions as metabolic enzyme by oxidising xenobiotics [ 32 ].
Due to their highly reactive properties, ROS have a short half-life and limited diffusion distance [ 33 ], therefore, they usually inflict cellular damage and impact molecular pathways only near the site of their production [ 34 ].
It is well known that generation of ROS, sensitisation of ER and initiation of the unfolded protein response [ 35 ] and autophagy are closely related processes [ 36 , 37 ]. Taken together all the above information triggered our interest to explore the potential involvement of CYP2E1 in the regulation of ER stress.
Cells were harvested and luciferase activity was measured. Data are representative of three independent experiments. Error bars represent standard deviation. The link between oxidative stress and metastasis has been demonstrated in several recent publications [ 38 , 39 ]. Taken together the role of CYP2E1 in inducing ROS generation and the fact that CYP2E1 is differentially expressed in early rather than later stages of breast cancer [ 19 ] implies that this cytochrome P isoenzyme might regulate migration of breast cancer cells.
Ectopic expression of CYP2E1 regulates migration of breast cancer cells. B Figure represents the calculations of migration distances of cells in A. Ethanol induces CYP2E1 enzymatic activity, and Bort is a proteasome inhibitor that has been shown to induce response to ER stress by stimulating the accumulation of misfolded proteins in the ER [ 42 ]. CYP2E1 regulates cell migration in breast cancer cell lines.
D Figure represents the calculations of migration distances of cells in C. Taking into account the fact that the high levels of ROS produced in cells due to the function of this enzyme could stimulate the transcriptional activity of the cancer-related transcription factor p53 [ 44 ] as well as that decreased CYP2E1 levels observed throughout cancer progression in different cancers [ 19 , 45 ] led us to explore the possibility that CYP2E1 was a possible p53 transcriptional target. Reasons explaining the lack of correlation between the CYP2E1 and p53 protein levels in all cell lines could be that p53 is not the only transcription factor that mediates CYP2E1 gene expression and that p53 might be stabilised and accumulated in MDA-MB and T47D cells but it is transcriptionally inactive.
CYP2E1 gene expression is under the transcriptional control of p Data presented as fold induction of luciferase activity in cells.
Results represent the average of three independent experiments. D Chromatin immuniprecipitated using specific p53 monoclonal antibody in non-treated or etoposide-treated MCF7 cells. To gain further insight in the transcriptional regulation of the CYP2E1 gene expression and investigate the potential role of p53 in the regulation of the cellular levels of this enzyme the luciferase activity of the CYP2E1-Luc reporter containing the putative p53 binding sites [ 44 ] identified in the regulatory region of the promoter of this gene was studied in MCF-7 breast cancer cells. Furthermore, to confirm whether the putative p53 binding sites were functional, the recruitment of p53 to these putative binding sites was followed in control or treated with etoposide MCF7 breast cancer cells using ChIP assay.
CYP2E1 is a drug-metabolising enzyme primarily expressed in hepatocytes, but has also been detected in other tissues including breast, brain, kidney and lungs [ 3 ]. CYP2E1 metabolises several pro-carcinogens including ethanol [ 7 ] and, for that reason, most of the research associated with CYP2E1 focuses on its role in liver diseases [ 8 ].
However, its functions in other tissues have not been extensively studied.
Clinical studies have indicated higher CYP2E1 gene expression in breast tumours than normal tissues [ 47 ] and decreasing CYP2E1 levels as carcinogenesis progresses [ 19 ] suggesting that this enzyme might serve important functions in breast carcinogenesis. In agreement with published observations [ 7 ], we detected increased intracellular ROS levels in breast cancer cells overexpressing CYP2E1 in a manner dependent on the status of p The p53 tumour suppressor plays an important role in the regulation of the cellular ROS generation and, in turn, ROS exert positive and negative effects on the p53 protein stability, transcriptional activity and gene target selectivity [ 46 ].
The pro-oxidant effects of p53 are mediated by another set of its target genes such as the pinduced gene 3 PIG3 [ 54 ] the p66Shc [ 55 ] and the Bcl2 family members bax and PUMA [ 50 , 56 ].
The mechanisms directing the p53 target selectivity to antioxidant or pro-oxidant transcriptional target genes, thereby determining the final outcome of the pmediated cellular redox state, include posttranslational modifications and recognition of specific DNA binding sites in the regulatory regions of the promoters of pro-oxidant subsets of genes by domains different than the p53 DNA binding domain proline-rich domain [ 50 , 54 , 57 ] and the affinity of p53 binding to the promoter of its diverse transcriptional targets follows a hierarchical order that is dependent on the type of stress [ 22 , 58 , 59 ].
In these terms, the amount of CYP2E1-mediated ROS generation could induce either antioxidant or pro-oxidant p53 outcomes in a tissue and cell-type-dependent manner. Since intracellular ROS levels are closely linked to the regulation of autophagy, we followed the autophagy biomarkers beclin-1, LC-3, Atg7 and Atg5 protein levels in breast cancer cells in which the CYP2E1 was either ectopically expressed or silenced. In line with potential tumour-suppressing function of autophagy, we identified that the protein levels of the autophagy markers [ 60 , 61 ], followed the same pattern as that of CYP2E1 in MDA-MB cells.
The role of p53 in linking cellular redox status energy metabolism and autophagy through transcription independent and transcription dependent mechanisms has been extensively investigated [ 62 — 64 ].
Taking into account the fact that CYP2E1 is predominantly localized in the endoplasmic reticulum [ 65 ] as well as the link between autophagy and ER stress [ 36 , 37 ] we investigated the possibility that CYP2E1 was part of the ER stress signalling transduction pathway. Integrated regulation of autophagy, ER stress and unfolded protein response determines the cell fate in breast cancer [ 61 ]. ER stress is able to both inhibit and activate the p53 pathway [ 66 , 67 ]. UPR is altered in many types of cancer [ 69 ], including breast cancer, and in some cases contributes to chemoresistance [ 24 , 35 , 70 , 71 ], highlighting the importance of our findings for cancer therapy.
The decreasing levels of CYP2E1 in advanced stages of breast cancer cells [ 19 ] together with our observations indicating that high cellular levels of CYP2E1 induce autophagy in MDA-MB cells and UPR in MCF7 cells, both of which are regulators of the tumour microenvironment [ 61 ], led us to investigate its role in regulating migration of breast cancer cells. To address this question, migration was followed in breast cancer cells expressing different levels of CYP2E1.
Our results provided evidence that CYP2E1 expression in breast cancer cells plays a role in the determination of migratory capacity. Another possible mechanism explaining the effects of the pCYP2E1-ROS generation axis on cell invasion and metastasis could be mediated by heparanase, which is a p53 transcriptional target [ 76 ] and is involved in the regulation of the ER stress-mediated breast cancer cell migration [ 77 ], but this hypothesis requires experimental validation.
To investigate the factors regulating CYP2E1 gene expression, its protein levels were followed in breast cancer cells with different p53 and estrogen receptor status treated with either ethanol, which induces CYP2E1 enzymatic activity, or the topoisomerase II inhibitor etoposide, which induces DNA damage and activates p53 response. Our results strengthened the hypothesis that p53 is a possible upstream regulator of CYP2E1 gene expression.
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Taken together, the results presented in this manuscript provide evidence to suggest that CYP2E1 plays an important role in breast carcinogenesis and the extent of the oxidative stress mediated by CYP2E1 determines distinct pmediated effects on autophagy, ER stress and metastasis in breast cancer cells. In addition, since this enzyme is involved in the metabolism of alcohol, observations described in this manuscript could be an additional link between chronic alcohol consumption and breast cancer [ 78 , 79 ].
Ectopic expression of CYP2E1 in breast cancer cells increase ROS generation, modulates autophagy and regulates ER stress and unfolded protein response in a cell-type-dependent manner. In addition, induction of CYP2E1 gene expression is under the transcriptional control of the p53 tumor suppressor and its activation inhibits migration of the highly invasive MDA-MB breast cancer cells. The results shown in this manuscript suggest that manipulation of CYP2E1 protein levels and enzymatic activity could be potentially exploited in breast cancer therapy.
Guengerich FP: Cytochrome Ps and other enzymes in drug metabolism and toxicity. AAPS J. Annu Rev Pharmacol Toxicol. Mutat Res. Mol Cancer Ther. Murray GI: The role of cytochrome P in tumour development and progression and its potential in therapy.
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J Pathol. Miksys S, Tyndale RF: Brain drug-metabolizing cytochrome P enzymes are active in vivo, demonstrated by mechanism-based enzyme inhibition. World J Gastroenterol.