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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.

Background During cancer progression, epithelial cancer cells can be reprogrammed into mesenchymal-like cells with increased migratory potential through the process of epithelial-mesenchymal transition (EMT), representing an essential step of tumor progression towards metastatic state. AGR2 protein was shown to regulate several cancer-associated processes including cellular proliferation, survival and drug resistance. Methods The expression of AGR2 was analyzed in cancer cell lines exposed to TGF-β alone or to combined treatment with TGF-β and the Erk1/2 inhibitor PD98059 or the TGF-β receptor specific inhibitor SB431542. The impact of AGR2 silencing by specific siRNAs or CRISPR/Cas9 technology on EMT was investigated by western blot analysis, quantitative PCR, immunofluorescence analysis, real-time invasion assay and adhesion assay. Results Induction of EMT was associated with decreased AGR2 along with changes in cellular morphology, actin reorganization, inhibition of E-cadherin and induction of the mesenchymal markers vimentin and N-cadherin in various cancer cell lines. Conversely, induction of AGR2 caused reversion of the mesenchymal phenotype back to the epithelial phenotype and re-acquisition of epithelial markers. Activated Smad and Erk signaling cascades were identified as mutually complementary pathways responsible for TGF-β-mediated inhibition of AGR2. Conclusion Taken together our results highlight a crucial role for AGR2 in maintaining the epithelial phenotype by preventing the activation of key factors involved in the process of EMT.

Despite extensive research, the molecular role of AGR2 in the progression and metastasis of colorectal cancer (CRC) has not been fully characterized. We used quantitative mass spectrometry (SWATH MS) to identify differentially expressed proteins in paired CRC cell models of the SW480 and SW620 cell lines in response to AGR2 protein level manipulation. Relying on the results from SWATH MS and subsequent immunochemical validation, we selected NMP3 as the top candidate protein associated with AGR2 in CRC tumour cells in our screen. RT‒qPCR and immunochemical analysis confirmed the involvement of AGR2-mediated regulation of NPM3 at the transcriptional and posttranscriptional levels. Since PD-L1 is a constituent of the NPM3 regulatory axis, we aimed to correlate the changes in PD-L1 to the differential expression of AGR2 in our cell models. We found that AGR2 positively regulates PD-L1 levels in both SW480 and SW620 cell lines; additionally, several different CRC patient transcriptome cohorts confirmed the association of AGR2 with PD-L1. Our work reveals a new AGR2-NPM3 regulatory axis and the involvement of AGR2 in the regulation of PD-L1, which paves the way for the association of AGR2 with immune evasion in CRC cells.

Catechol-O-methyl transferase (COMT) is involved in detoxification of catechol estrogens, playing cancer-protective role in cells producing or utilizing estrogen. Moreover, COMT suppressed migration potential of breast cancer (BC) cells. To delineate COMT role in metastasis of estrogen receptor (ER) dependent BC, we investigated the effect of COMT overexpression on invasion, transcriptome, proteome and interactome of MCF7 cells, a luminal A BC model, stably transduced with lentiviral vector carrying COMT gene (MCF7-COMT). 2D and 3D assays revealed that COMT overexpression associates with decreased cell invasion ( p  < 0.0001 for Transwell assay, p  < 0.05 for spheroid formation). RNA-Seq and LC-DIA-MS/MS proteomics identified genes associated with invasion ( FTO, PIR, TACSTD2, ANXA3, KRT80, S100P, PREX1, CLEC3A, LCP1 ) being downregulated in MCF7-COMT cells, while genes associated with less aggressive phenotype ( RBPMS, ROBO2, SELENBP, EPB41L2 ) were upregulated both at transcript (|log2FC|> 1, adj. p  < 0.05) and protein (|log2FC|> 0.58, q  < 0.05) levels. Importantly, proteins driving MET signaling were less abundant in COMT overexpressing cells, and pull-down confirmed interaction between COMT and Kunitz-type protease inhibitor 2 (SPINT2), a negative regulator of MET (log2FC = 5.10, q  = 1.04 −7 ). In conclusion, COMT may act as tumor suppressor in ER dependent BC not only by detoxification of catechol estrogens but also by suppressing cell invasion and interplay with MET pathway.

Brain metastases (BMs) are the most common intracranial tumors in adults and occur 3–10 times more frequently than primary brain tumors. Despite intensive multimodal therapies, including resection, radiotherapy, and chemotherapy, BMs are associated with poor prognosis and remain challenging to treat. BMs predominantly originate from primary lung (20–56%), breast (5–20%), and melanoma (7–16%) tumors, although they can arise from other cancer types less frequently. The metastatic cascade is a multistep process involving local invasion, intravasation into the bloodstream or lymphatic system, extravasation into normal tissue, and colonization of the distal site. After reaching the brain, circulating tumor cells (CTCs) breach the blood–brain barrier (BBB). The selective permeability of the BBB poses a significant challenge for therapeutic compounds, limiting the treatment efficacy of BMs. Understanding the mechanisms of tumor cell interactions with the BBB is crucial for the development of effective treatments. This review provides an in-depth analysis of the brain barriers, including the BBB, blood-spinal cord barrier, blood-meningeal barrier, blood-arachnoid barrier, and blood-cerebrospinal fluid barrier. It explores the molecular and cellular components of these barriers and their roles in brain metastasis, highlighting the importance of this knowledge for identifying druggable targets to prevent or limit BM formation.

Objectives Endometrial cancer (EC) is the most common malignancy of the female genital tract in developed countries, yet preventive screening remains unavailable, and diagnostic approaches are largely limited to symptomatic women. Despite advancements in precision oncology, the biology of precancerous lesions is less understood compared to advanced disease. To address this gap, we conducted a prospective case-control study analysing uterine lavage fluid from women undergoing diagnostic evaluation. The study included 257 participants: 80 diagnosed with endometrial intraepithelial neoplasia (EIN), 89 with early-stage EC, and 88 healthy controls. Using targeted next-generation sequencing, we examined genetic alterations in 22 selected genes associated with EC development. Results Our findings did not confirm a direct association between specific genetic mutations in uterine lavage fluid and the presence of EIN or early-stage EC ( p  = 0.501). Mutations were detected in both cases and controls, with a higher overall mutation burden observed in controls, suggesting potential background genomic alterations unrelated to EC development. In conclusion, while molecular profiling of uterine lavage fluid remains a promising concept for non-invasive diagnosis, our results highlight significant challenges in specificity. Further studies with larger cohorts and additional biomarkers are necessary to clarify its diagnostic relevance and clinical applicability.

The tumor suppressor p53 plays a key role in malignant transformation and tumor development. However, the frequency of p53 mutations within individual types of cancer is different, suggesting the existence of other mechanisms attenuating p53 tumor suppressor activity. Changes in upstream regulators of p53 such as MDM2 amplification and overexpression, expression of viral oncoproteins, estrogen receptor signaling, or changes in p53 transcriptional target genes were previously described in wild-type p53 tumors. We identified a novel pathway responsible for attenuation of p53 activity in human cancers. We demonstrate that AGR2, which is overexpressed in a variety of human cancers and provides a poor prognosis, up-regulates DUSP10 which subsequently inhibits p38 MAPK and prevents p53 activation by phosphorylation. Analysis of human breast cancers reveals that AGR2 specifically provides a poor prognosis in ER+ breast cancers with wild-type p53 but not ER- or mutant p53 breast cancers, and analysis of independent data sets show that DUSP10 levels also have prognostic significance in this specific sub-group of patients. These data not only reveal a novel pro-oncogenic signaling pathway mediating resistance to DNA damaging agents in human tumors, but also has implications for designing alternative strategies for modulation of wild-type p53 activity in cancer therapy. •The transcription factor p53 is the most intensively studied tumor suppressor.•AGR2-dependent signaling pathway blocks wt p53 transcriptional activity.•p53 activity is impaired via DUSP10 up-regulation causing inhibition of p38 MAPK.•AGR2 signaling towards p53 was demonstrated both in vitro and in vivo.

Immunochemical methods are used not only in clinical practice for the diagnosis of a wide range of diseases but also in basic and advanced research. Based on the unique reaction between the antibody and its respective antigens, it serves to specifically recognize target molecules in biological complex samples. Current methods of labelling antibodies with elemental labels followed by detection by inductively coupled plasma mass spectrometry (ICP-MS) allow detection of multiple antigens in parallel in a single analysis. Using the laser ablation (LA) modality (LA-ICP-MS), it is also possible to monitor the spatial distribution of biogenic elements. Moreover, the employment of metal nanoparticle-labeled antibodies expands the applicability also to molecular imaging by LA-ICP-MS. In this work, conjugates of model monoclonal antibody (DO-1, recognizing p53 protein) with various metal nanoparticles-based labels were created and utilized in dot-blot analysis in order to compare their benefits and disadvantages. Based on experiments with the p53 protein standard, commercial kits of gold nanoparticles proved to be the most suitable for the preparation of conjugates. The LA-ICP-MS demonstrated very good repeatability, wide linear dynamic range (0.1–14 ng), and limit of detection was calculated as a 1.3 pg of p53 protein.

High rates of mutation in the TP53 tumor suppressor gene have been found in many human cancers, including breast tumors, making p53 one of the most studied proteins in oncology. However, the prognostic and predictive value of alterations in this gene remains ambiguous. To analyze the clinical value of somatic TP53 mutations, we collected clinical and molecular data on 210 women with primary breast cancer. We found significant associations of p53 mutations with tumor grade, metastasis, molecular subtype, Her2 status and inverse correlations with estrogen and progesterone receptor status. Cox proportional hazard analysis confirmed a strong prognostic value of p53 mutation for overall survival rate and highlighted significant interactions with lymph node involvement and tumor size. In relation to treatment options, TP53 mutations were associated with poor response to anthracyclines and radiotherapy. Categorization of TP53 mutations according to their type and location revealed that patients with nonsense mutation have the poorest prognosis in comparison with wild-type cases and other types of mutations in this gene. Classification of TP53 mutations with respect to the degree of disturbance of protein structure showed association of disruptive mutations with poorer patients' outcome in contrast to wild-type and non-disruptive mutations. In conclusion, the present study confirms p53 as a potential predictive and prognostic factor in oncology practice and highlights the growing evidence that distinct types of mutations have different clinical impacts.

Cervical cancer is one of the most common types of cancer in women, with approximately 500,000 new cases and 250,000 deaths every year. Radiotherapy combined with chemotherapy represents the treatment of choice for advanced cervical carcinomas. The role of the chemotherapy is to increase the sensitivity of the cancer cells to irradiation. Cisplatin, the most commonly used drug for this purpose, has its limitations. Thus, we used a family of ferrocene derivatives (in addition, one new species was prepared using standard Schlenk techniques) and studied their effects on cervical cancer cells alone and in combination with irradiation. We applied colorimetric assay to determine the cytotoxicity of the compounds; flow cytometry to analyze the production of reactive oxygen species (ROS), cell cycle, and mitochondrial membrane potential (MMP); immunochemistry to study protein expression; and colony forming assay to evaluate changes in radiosensitivity. Treatment with ferrocenes exhibited significant cytotoxicity against cervical cancer cells, associated with increasing ROS production and MMP changes, suggesting the induction of apoptosis. The combined activity of ferrocenes and ionizing radiation highlighted ferrocenes as potential radiosensitizing drugs, while their higher single-agent toxicity in comparison with routinely used cisplatin could also be promising. Our results demonstrate antitumor activity of several tested ferrocenes both alone and in combination with radiotherapy.