DNA methylation, i.e., addition of methyl group to 5′-carbon of cytosine residues in CpG dinucleotides, is an important epigenetic modification regulating gene expression, and thus implied in many cellular processes. Deregulation of DNA methylation is strongly associated with onset of various diseases, including cancer. Here, we review how DNA methylation affects carcinogenesis process and give examples of solid tumors where aberrant DNA methylation is often present. We explain principles of methods developed for DNA methylation analysis at both single gene and whole genome level, based on (i) sodium bisulfite conversion, (ii) methylation-sensitive restriction enzymes, and (iii) interactions of 5-methylcytosine (5mC) with methyl-binding proteins or antibodies against 5mC. In addition to standard methods, we describe recent advances in next generation sequencing technologies applied to DNA methylation analysis, as well as in development of biosensors that represent their cheaper and faster alternatives. Most importantly, we highlight not only advantages, but also disadvantages and challenges of each method.

Anterior gradient 2 (AGR2) is an endoplasmic reticulum (ER)-resident protein disulfide isomerase (PDI) known to be overexpressed in many human epithelial cancers and is involved in cell migration, cellular transformation, angiogenesis, and metastasis. This protein inhibits the activity of the tumor suppressor p53, and its expression levels can be used to predict cancer patient outcome. However, the precise network of AGR2-interacting partners and clients remains to be fully characterized. Herein, we used label-free quantification and also stable isotope labeling with amino acids in cell culture–based LC–MS/MS analyses to identify proteins interacting with AGR2. Functional annotation confirmed that AGR2 and its interaction partners are associated with processes in the ER that maintain intracellular metabolic homeostasis and participate in the unfolded protein response, including those associated with changes in cellular metabolism, energy, and redox states in response to ER stress. As a proof of concept, the interaction between AGR2 and PDIA3, another ER-resident PDI, was studied in more detail. Pathway analysis revealed that AGR2 and PDIA3 play roles in protein folding in ER, including post-translational modification and in cellular response to stress. We confirmed the AGR2–PDIA3 complex formation in cancer cells, which was enhanced in response to ER stress. Accordingly, molecular docking characterized potential quaternary structure of this complex; however, it remains to be elucidated whether AGR2 rather contributes to PDIA3 maturation in ER, the complex directly acts in cellular signaling, or mediates AGR2 secretion. Our study provides a comprehensive insight into the protein–protein interaction network of AGR2 by identifying functionally relevant proteins and related cellular and biochemical pathways associated with the role of AGR2 in cancer cells.

Epithelial-mesenchymal transition (EMT) is a process involved not only in morphogenesis and embryonic development, but also in cancer progression, whereby tumor cells obtain a more aggressive metastatic phenotype. Anterior gradient protein 2 (AGR2) maintains the epithelial phenotype and blocks the induction of EMT, thus playing an undeniable role in tumor progression. However, the mechanism through which AGR2 expression is regulated, not only during EMT, but also in the early stages of cancer development, remains to be elucidated. In the present study, we show an inverse correlation of AGR2 with ZEB1 (zinc finger enhancer binding protein, δEF1) that was verified by analysis of several independent clinical data sets of lung adenocarcinomas. We also identified the ZEB1 binding site within the AGR2 promoter region and confirmed AGR2 as a novel molecular target of ZEB1. The overexpression of ZEB1 decreased the promoter activity of the AGR2 gene, which resulted in reduced AGR2 protein level and the acquisition of a more invasive phenotype of these lung cancer cells. Conversely, silencing of ZEB1 led not only to increased levels of AGR2 protein, but also attenuated the invasiveness of tumor cells. The AGR2 knockout, vice versa, increased ZEB1 expression, indicating that the ZEB1/AGR2 regulatory axis may function in a double negative feedback loop. In conclusion, we revealed for the first time that ZEB1 regulates AGR2 at the transcriptional level, while AGR2 presence contributes to ZEB1 mRNA degradation. Thus, our data identify a new regulatory mechanism between AGR2 and ZEB1, two rivals in the EMT process, tightly associated with the development of metastasis.

The TGF-β signaling pathway is involved in numerous cellular processes, and its deregulation may result in cancer development. One of the key processes in tumor progression and metastasis is epithelial to mesenchymal transition (EMT), in which TGF-β signaling plays important roles. Recently, AGR2 was identified as a crucial component of the cellular machinery responsible for maintaining the epithelial phenotype, thereby interfering with the induction of mesenchymal phenotype cells by TGF-β effects in cancer. Here, we performed transcriptomic profiling of A549 lung cancer cells with CRISPR-Cas9 mediated AGR2 knockout with and without TGF-β treatment. We identified significant changes in transcripts associated with focal adhesion and eicosanoid production, in particular arachidonic acid metabolism. Changes in transcripts associated with the focal adhesion pathway were validated by RT-qPCR of COL4A1, COL4A2, FLNA, VAV3, VEGFA, and VINC mRNAs. In addition, immunofluorescence showed the formation of stress fibers and vinculin foci in cells without AGR2 and in response to TGF-β treatment, with synergistic effects observed. These findings imply that both AGR2 downregulation and TGF-β have a role in focal adhesion formation and cancer cell migration and invasion. Transcripts associated with arachidonic acid metabolism were downregulated after both AGR2 knockout and TGF-β treatment and were validated by RT-qPCR of GPX2, PTGS2, and PLA2G4A. Since PGE₂ is a product of arachidonic acid metabolism, its lowered concentration in media from AGR2-knockout cells was confirmed by ELISA. Together, our results demonstrate that AGR2 downregulation and TGF-β have an essential role in focal adhesion formation; moreover, we have identified AGR2 as an important component of the arachidonic acid metabolic pathway.

The unfolded protein response (UPR) is a critical adaptive program triggered upon cellular stresses that profoundly reshapes the transcriptome and translatome. In the very first minutes of cellular stress, translation blockage, RNA decay and RNA granules formation prompt the synthesis of proteins essential to the stress response. Due to the dynamic nature of these processes, investigating translation upon stress has proven to be challenging; therefore, our understanding of these mechanisms and translatome rewiring upon stress remains limited. Here, we exploit O-Propargyl-puromycin (OPP) labelling of de novo peptides followed by LC-MS/MS to identify de novo proteins translated upon endoplasmic reticulum (ER) stress. Combined with transcriptomic analyses, our approach reveals that ER stress profoundly impacts the synthesis of core splicing factor proteins leading to a significant reshaping of the splicing landscape. We identify a signature of seven splicing events consistently occurring in mammalian cells exposed to ER stress. Using pharmacological, genetic, phosphoproteomic and sequencing approaches, we demonstrate that this specific signature is driven by PERK activation and is dependent on the axis CLK1/SRSF1. Our findings identify PERK/CLK1/SRSF1 -mediated splicing regulation as a new facet of ER stress, defining an ER ^i-splice signature spanning healthy and malignant tissues.