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.
- MeSH
- antigeny CD274 * metabolismus genetika MeSH
- jaderné proteiny metabolismus genetika MeSH
- kolorektální nádory * genetika metabolismus patologie MeSH
- lidé MeSH
- mukoproteiny * metabolismus genetika MeSH
- nádorové buněčné linie MeSH
- nukleofosmin * MeSH
- onkogenní proteiny * metabolismus genetika MeSH
- proteiny * metabolismus genetika MeSH
- regulace genové exprese u nádorů * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Persistent infection with high-risk types of human papillomaviruses (HPV) is a major cause of cervical cancer, and an important factor in other malignancies, for example, head and neck cancer. Despite recent progress in screening and vaccination, the incidence and mortality are still relatively high, especially in low-income countries. The mortality and financial burden associated with the treatment could be decreased if a simple, rapid, and inexpensive technology for HPV testing becomes available, targeting individuals for further monitoring with increased risk of developing cancer. Commercial HPV tests available in the market are often relatively expensive, time-consuming, and require sophisticated instrumentation, which limits their more widespread utilization. To address these challenges, novel technologies are being implemented also for HPV diagnostics that include for example, isothermal amplification techniques, lateral flow assays, CRISPR-Cas-based systems, as well as microfluidics, paperfluidics and lab-on-a-chip devices, ideal for point-of-care testing in decentralized settings. In this review, we first evaluate current commercial HPV tests, followed by a description of advanced technologies, explanation of their principles, critical evaluation of their strengths and weaknesses, and suggestions for their possible implementation into medical diagnostics.
- MeSH
- infekce papilomavirem * komplikace MeSH
- lidé MeSH
- lidské papilomaviry MeSH
- nádory děložního čípku * MeSH
- Papillomaviridae genetika MeSH
- technologie MeSH
- Check Tag
- lidé MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
Cancer is a genetic disease induced by mutations in DNA, in particular point mutations in important driver genes that lead to protein malfunctioning and ultimately to tumorigenesis. Screening for the most common DNA point mutations, especially in such genes as TP53, BRCA1 and BRCA2, EGFR, KRAS, or BRAF, is crucial to determine predisposition risk for cancer or to predict response to therapy. In this review, we briefly depict how these genes are involved in cancer, followed by a description of the most common techniques routinely applied for their analysis, including high-throughput next-generation sequencing technology and less expensive low-throughput options, such as real-time PCR, restriction fragment length polymorphism, or high resolution melting analysis. We then introduce benefits of electrochemical biosensors as interesting alternatives to the standard methods in terms of cost, speed, and simplicity. We describe most common strategies involved in electrochemical biosensing of point mutations, relying mostly on PCR or isothermal amplification techniques, and critically discuss major challenges and obstacles that, until now, prevented their more widespread application in clinical settings.
Estrogen receptor alpha (ER) is a key biomarker for breast cancer, and the presence or absence of ER in breast and other hormone-dependent cancers decides treatment regimens and patient prognosis. ER is activated after ligand binding - typically by steroid. 2682 steroid compounds were used in a molecular docking study to identify novel ligands for ER and to predict compounds that may show anticancer activity. The effect of the most promising compounds was determined by a novel luciferase reporter assay. Two compounds, 7 and 12, showing ER inhibitory activity comparable to clinical inhibitors such as tamoxifen or fulvestrant were selected. We propose that the inhibitory effect of compounds 7 and 12 on ER is related to the presence of a double bond in their D-ring, which may protect against ER activation by reducing the electron density of the keto group, or may undergo metabolism leading to an active compound. Western blotting revealed that compound 12 decreased the level of ER in the breast cancer cell line MCF7, which was associated with reduced expression of both isoforms of the progesterone receptor, a well-known downstream target of ER. However, compound 12 has a different mechanism of action from fulvestrant. Furthermore, we found that compound 12 interferes with mitochondrial functions, probably by disrupting the electron transport chain, leading to induction of the intrinsic apoptotic pathway even in ER-negative breast cancer cells. In conclusion, the combination of computational and experimental methods shown here represents a rapid approach to determine the activity of compounds towards ER. Our data will not only contribute to research focused on the regulation of ER activity but may also be useful for the further development of novel steroid receptor-targeted drugs applicable in clinical practice.
- MeSH
- alfa receptor estrogenů genetika metabolismus MeSH
- estradiol farmakologie terapeutické užití MeSH
- estron * farmakologie MeSH
- fulvestrant farmakologie terapeutické užití MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- nádory prsu * farmakoterapie metabolismus MeSH
- receptory pro estrogeny metabolismus MeSH
- simulace molekulového dockingu MeSH
- tamoxifen farmakologie MeSH
- Check Tag
- lidé MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Publikační typ
- abstrakt z konference MeSH
Breast cancers are a heterogeneous group of tumors classified according to their histological growth patterns and receptor expression characteristics. Intratumor heterogeneity also exists, with subpopulations of cells with different phenotypes found in individual cancers, including cells with stem or progenitor cell properties. At least two types of breast cancer stem cells (CSCs) exist, the epithelial and the basal/mesenchymal subtypes, although how these phenotypes are controlled is unknown. ΔNp63 is a basal cell marker and regulator of stem/progenitor cell activities in the normal mammary gland and is expressed in the basal-like CSC subpopulation in some estrogen receptor-positive (ER+) and/or human epidermal growth factor receptor 2-positive (HER2+) breast adenocarcinomas. Whilst p63 is known to directly impart CSC properties in luminal breast cancer cells, how p63 is regulated and induced in these cells is unknown. We initially confirmed the existence of a small subpopulation of ΔNp63+ cells in lymph node metastases of ER+ human ductal adenocarcinomas, indicating together with previous reports that ΔNp63+ tumor cells are present in approximately 40% of these metastases. Notably, ΔNp63+ cells show a preferential location at the edge of tumor areas, suggesting possible regulation of ΔNp63 by the tumor microenvironment. Subsequently, we showed that the high levels of ΔNp63 in basal non-transformed MCF-10A mammary epithelial cells rely on insulin in their culture medium, whilst ΔNp63 levels are increased in MCF-7 ER+ luminal-type breast cancer cells treated with insulin or insulin-like growth factor 1 (IGF-1). Mechanistically, small molecule inhibitors and siRNA gene knockdown demonstrated that induction of ΔNp63 by IGF-1 requires PI3K, ERK1/2, and p38 MAPK activation, and acts through FOXO transcriptional inactivation. We also show that metformin inhibits ΔNp63 induction. These data reveal an IGF-mediated mechanism to control basal-type breast CSCs, with therapeutic implications to modify intratumor breast cancer cell heterogeneity and plasticity.
The prevention and early diagnostics of precancerous stages are key aspects of contemporary oncology. In cervical cancer, well-organized screening and vaccination programs, especially in developed countries, are responsible for the dramatic decline of invasive cancer incidence and mortality. Cytological screening has a long and successful history, and the ongoing implementation of HPV triage with increased sensitivity can further decrease mortality. On the other hand, endometrial and ovarian cancers are characterized by a poor accessibility to specimen collection, which represents a major complication for early diagnostics. Therefore, despite relatively promising data from evaluating the combined effects of genetic variants, population screening does not exist, and the implementation of new biomarkers is, thus, necessary. The introduction of various circulating biomarkers is of potential interest due to the considerable heterogeneity of cancer, as highlighted in this review, which focuses exclusively on the most common tumors of the genital tract, namely, cervical, endometrial, and ovarian cancers. However, it is clearly shown that these malignancies represent different entities that evolve in different ways, and it is therefore necessary to use different methods for their diagnosis and treatment.
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
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.
- MeSH
- 5-methylcytosin metabolismus MeSH
- biosenzitivní techniky metody MeSH
- epigeneze genetická genetika MeSH
- lidé MeSH
- metylace DNA genetika fyziologie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
ΔNp63, also known as p40, regulates stemness of normal mammary gland epithelium and provides stem cell characteristics in basal and HER2-driven murine breast cancer models. Whilst ΔNp63/p40 is a characteristic feature of normal basal cells and basal-type triple-negative breast cancer, some receptor-positive breast cancers express ΔNp63/p40 and its overexpression imparts cancer stem cell-like properties in ER+ cell lines. However, the incidence of ER+ and HER2+ tumours that express ΔNp63/p40 is unclear and the phenotype of ΔNp63/p40+ cells in these tumours remains uncertain. Using immunohistochemistry with p63 isoform-specific antibodies, we identified a ΔNp63/p40+ tumour cell subpopulation in 100 of 173 (58%) non-triple negative breast cancers and the presence of this population associated with improved survival in patients with ER- /HER2+ tumours (p = 0.006). Furthermore, 41% of ER+ /PR+ and/or HER2+ locally metastatic breast cancers expressed ΔNp63/p40, and these cells commonly accounted for <1% of the metastatic tumour cell population that localised to the tumour/stroma interface, exhibited an undifferentiated phenotype and were CD44+ /ALDH- . In vitro studies revealed that MCF7 and T47D (ER+ ) and BT-474 (HER2+ ) breast cancer cell lines similarly contained a small subpopulation of ΔNp63/p40+ cells that increased in mammospheres. In vivo, MCF7 xenografts contained ΔNp63/p40+ cells with a similar phenotype to primary ER+ cancers. Consistent with tumour samples, these cells also showed a distinct location at the tumour/stroma interface, suggesting a role for paracrine factors in the induction or maintenance of ΔNp63/p40. Thus, ΔNp63/p40 is commonly present in a small population of tumour cells with a distinct phenotype and location in ER+ and/or HER2+ human breast cancers.
- MeSH
- fenotyp MeSH
- heterografty MeSH
- lidé MeSH
- myši MeSH
- nádorové biomarkery analýza metabolismus MeSH
- nádorové kmenové buňky metabolismus patologie MeSH
- nádorové supresorové proteiny metabolismus MeSH
- nádory prsu genetika metabolismus patologie MeSH
- receptor erbB-2 genetika metabolismus MeSH
- receptory pro estrogeny genetika metabolismus MeSH
- transkripční faktory metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Východiska: Výzkum posledního desetiletí potvrdil význam epigenetických procesů při vzniku, vývoji a léčbě nádorových onemocnění. Především sekvenování nové generace umožnilo zmapovat lidský epigenom a sledovat jeho změny během kancerogeneze. Tento přístup odhalil přímá napojení epigenetických abnormalit na mutace genů, které kontrolují metylaci DNA, sbalování a funkci DNA v chromatinu, nebo na metabolizmus buněk. Epigenetické změny DNA se vyskytují už v časných fázích vývoje nádorových onemocnění, a jsou tedy slibnými kandidáty na diagnostické a prognostické markery a současně epigenetické procesy představují vhodné cíle pro vývoj nových terapeutických látek. Získané poznatky o aberantní metylaci DNA umožňují dva různé pohledy na to, jak daná modifikace přispívá k vývoji nádorového onemocnění. První pohled předpokládá, že normální buňky podléhají transformaci vlivem řídicích mutací, kdy následné metylace de novo a demetylace DNA přispívají k řadě programových změn genové exprese. Alternativní přístup pohlíží na změny v metylaci DNA jako na důsledek např. stárnutí buněk. A právě tyto získané změny zvyšují citlivost DNA ke vzniku mutací a k následné onkogenní transformaci. Cíle: Cílem přehledového článku je shrnout dosud známé úlohy abnormální metylace DNA při vývoji nádorového onemocnění a představit již publikovanou alternativní teorii, která k dané problematice přistupuje méně obvyklým způsobem.
Background: Research in the last decade has confirmed the importance of epigenetic processes for the onset, development, and treatment of cancer. Next generation sequencing has allowed the inspection and mapping of the human epigenome and its monitoring for changes during carcinogenesis, which has revealed direct links between epigenetic abnormalities and mutations in genes that control DNA methylation and packing and those that function in chromatin dynamics and metabolism. Epigenetic changes that occur in the early stages of tumor progression thus represent promising candidates for diagnostic and prognostic markers, and epigenetic processes are suitable targets for the development of new therapeutic strategies. There are two contrasting views on how aberrant DNA methylation contributes to the development of cancer. The first view assumes that normal cells undergo transformation due to driver mutations and subsequent de novo methylation and DNA demethylation, resulting in global changes in gene expression. The second view considers changes in DNA methylation to be a consequence of cell aging, for example, and that the acquired changes increase the sensitivity of DNA to mutations and oncogenic transformation. Aims: The aim of the review article is to briefly summarize the role of abnormal DNA methylation in the development of cancer, and to present an alternative theory that considers the role of aberrant DNA methylation patterns in cancer from a new and unconventional perspective.
