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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 mediatedAGR2knockout 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 ofCOL4A1,COL4A2,FLNA,VAV3,VEGFA, andVINCmRNAs. 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 bothAGR2knockout and TGF-β treatment and were validated by RT-qPCR ofGPX2,PTGS2, andPLA2G4A. Since PGE2is a product of arachidonic acid metabolism, its lowered concentration in media fromAGR2-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.

International audience; 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.

Summary Chromatin is a barrier to efficient DNA repair, as it hinders access and processing of certain DNA lesions. ALC1/CHD1L is a nucleosome-remodeling enzyme that responds to DNA damage, but its precise function in DNA repair remains unknown. Here we report that loss of ALC1 confers sensitivity to PARP inhibitors, methyl-methanesulfonate, and uracil misincorporation, which reflects the need to remodel nucleosomes following base excision by DNA glycosylases but prior to handover to APEX1. Using CRISPR screens, we establish that ALC1 loss is synthetic lethal with homologous recombination deficiency (HRD), which we attribute to chromosome instability caused by unrepaired DNA gaps at replication forks. In the absence of ALC1 or APEX1, incomplete processing of BER intermediates results in post-replicative DNA gaps and a critical dependence on HR for repair. Hence, targeting ALC1 alone or as a PARP inhibitor sensitizer could be employed to augment existing therapeutic strategies for HRD cancers., Graphical Abstract, Highlights • Loss of ALC1 nucleosome remodeling confers PARPi, MMS, and formyl-dU sensitivity • ALC1 is required after lesion excision by DNA glycosylases prior to APEX1 • Loss of ALC1 is synthetic lethal with homologous recombination deficiency (HRD) • Endogenous alkylated base damage is a source of synthetic lethality with HRD, Hewitt et al. report that loss of the chromatin-remodeling enzyme ALC1 leads to persistent BER intermediates and a critical dependency on HR for repair. Combined loss of ALC1 and HR is synthetic lethal, which can be partially rescued by blocking excision of endogenous alkylation damage by the glycosylase MPG.

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, &delta, EF1) 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.

International audience; Human anterior gradient proteins AGR2 and AGR3 are overexpressed in a variety of adenocarcinomas and are often secreted in cancer patients' specimens, which suggests a role for AGR proteins in intra and extracellular compartments. Although these proteins exhibit high sequence homology, AGR2 is predominantly described as a pro-oncogene and a potential prognostic biomarker. However, little is known about the function of AGR3. Therefore, the aim of the present study was to investigate the role of AGR3 in breast cancer. The results demonstrated that breast cancer cells secrete AGR3. Furthermore, it was revealed that extracellular AGR3 (eAGR3) regulates tumor cell adhesion and migration. The current study indicated that the pharmacological and genetic perturbation of Src kinase signaling, through treatment with Dasatinib (protein kinase inhibitor) or investigating cells that express a dominant-negative form of Src, significantly abrogated eAGR3-mediated breast cancer cell migration. Therefore, the results indicated that eAGR3 may control tumor cell migration via activation of Src kinases. The results of the present study indicated that eAGR3 may serve as a microenvironmental signaling molecule in tumor-associated processes.

There is growing epidemiological evidence indicating an association between diabetes mellitus and the increased incidence of colorectal cancer (CRC). The preferred initial and most widely used pharmacological agent for the treatment of type 2 diabetes is metformin, which in parallel reduces the risk of CRC and improves patient prognosis. AMP-activated protein kinase (AMPK) appears to be tightly associated with the beneficial metabolic effects of metformin, serving as a cellular energy sensor activated in response to a variety of conditions that deplete cellular energy levels. Such conditions include nutrient starvation (particularly glucose), hypoxia and exposure to toxins that inhibit the mitochondrial respiratory chain complex. The aim of the present study was to determine the effect of metformin on CRC cell lines, with different levels of anterior gradient 2 (AGR2) expression, exposed to 5-fluorouracil (5-FU) and oxaliplatin, alone or in combination with metformin. AGR2 has recently emerged as a factor involved in colon carcinogenesis. In AGR2-knockout cells, markedly higher levels of phosphorylated-AMPK were observed in comparison with control cells transfected with GFP-scrambled guide RNA, which indicated that the presence of AGR2 may interfere with the metformin-dependent activation of AMPK. In addition, metformin in combination with 5-FU and oxaliplatin induced ROS production and attenuated autophagy. This effect was enhanced in AGR2-knockout cells.

Chemotherapy is an essential treatment that still plays a vital role in cancer treatment worldwide. The ferrocene derivatives of the general formula [Fe{(η5‑C5H4CH2(p‑C6H4)CH2(N‑het)}2] bearing modified six and five membered N-heterocycles were tested in vitro for their cytotoxic properties against ovarian cancer cell lines A2780 and SK-OV-3. These ferrocene complexes displayed cytotoxicity in low micromolar concentrations against both cell lines. To study cellular uptake of particular ferrocenes into tumor cells, we used differential pulse voltammetry and ICP-MS. We confirmed the crucial role of transferrin receptors in the process of intracellular accumulation of these ferrocenes. Interestingly, the rate of intracellular accumulation of particular ferrocenes clearly mirrored the cytotoxicity of these organometallic compounds. Deeper investigation of the mechanism by which ferrocenes kill tumor cells revealed induction of apoptosis associated with significant increase of reactive oxygen species. In conclusion, our screening identified several ferrocene derivatives exerting promising cytostatic activity in vitro. Further investigation led to the identification of the mechanism of action of these potential anticancer agents, which represents an important milestone in preclinical anticancer drug discovery programs. This work was supported by the project MEYS-NPS I-LO1413, MH CZ-DRO (MMCI, 00209805) and Czech Science Foundation project 17-05838S.

Background Cervical cancer is the fourth most common cancer in women and is usually associated with human papillomavirus infection. Viral infections are usually characterized by morphological changes of epithelial cells; however, it is difficult to determine using recently available screening methods whether the changes are caused by productive infection or by malignant disease. Thus, new efforts are required to find novel diagnostic biomarkers of cervical cancer, such as miRNAs, which are small non-coding RNAs involved in the regulation of gene expression. Materials and methods miR-34c levels in cervical cancer cell lines were determined by the droplet-digital polymerase chain reaction. Changes in miR-34c expression in vitro were achieved by transient transfection with a specific miRNA mimic and inhibitor oligonucleotides. Cell proliferation was analyzed by crystal violet staining followed by spectrophotometric measurements. The effect on migratory properties was studied using a „scratch“ assay. Western blotting analysis was used to determine the expression of selected proteins. Results The downregulation of miR-34c expression was associated with a slight increase in cellular proliferation and a significant increase in cell migration. The analysis of miR-34c expression performed on a set of 39 dysplastic tissues and 35 samples of healthy controls subsequently revealed a significant difference (p l 0.01) in the level of this miRNA. Conclusion Comparative expression analysis revealed lower expression of miR-34c in cervical precanceroses than in normal untransformed epithelium. in vitro modulation of miR-34c expression revealed its tumor suppressor role in cervical malignancies. Key words: cervical cancer - HPV - miRNA - HSIL - hsa-miR-34c - precancerosis This work was supported by the projects MEYS - NPS I - LO1413, P206/12/G151 and MH CZ - DRO (MMCI, 00209805). The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers. Accepted: 16. 7. 2018.

Background Due to the irreplaceable role of chemotherapy in cancer treatment, research has focused on improving the efficacies of individual drugs and minimizing, or completely suppressing, their negative side effects. Based on long-term experience and the results of clinical trials, the selection of appropriate treatment is currently based on classical clinical diagnostic criteria, such as tumor size, grade, and the presence or absence of standard markers. However, complications arise due to variability between patients and tumor heterogeneity. Characterization of intratumoral heterogeneity and acquisition of more reliable drug performance indicators should improve personalized therapy. Development and selection of suitable models are therefore important issues in cancer research focused on predicting sensitivity to therapy. Aim This work provides an overview of various chemosensitivity tests that have been previously employed and those that are currently used. Great emphasis is placed on comparing 2D and 3D cell culture models, since their importance and popularity are increasing. Particular attention is paid to in vivo systems, which have significantly improved recently and are tested in clinical trials to predict responses to therapy. Conclusion This work provides a brief overview of chemosensitivity tests, focusing on the importance of individual tests and their application in decision-making and patient stratification to improve the clinical responses of patients and the development of targeted personalized therapy.Key words: cell culture techniques - personalized medicine - drug screening - biological models - tumor cell lines - carcinoma - cytotoxicity assays This work was supported by the project MEYSNPS I-LO1413 and GACR 17-05838S. The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers.Submitted: 21. 9. 2017Accepted: 20. 12. 2017.

Dynamic, punctual and perfectly coordinated cellular response to internal and external stimuli is a crucial prerequisite for adaptation of mammalian cells to all changes that occur during cellular development under physiological conditions. Hijacking this ability is characteristic for tumor cells that are capable to adapt to unfavorable conditions which contribute to the formation and development of cancer during the process of tumor formation and progression. By changing key mechanisms, malignant cells can avoid cell death and thus allow development and spread of the tumor. The changes at the genetic level are manifested by various phenotypic characteristics, through which tumor cells are able to escape defense mechanisms, to acquire resistance to treatment, to invade and to create secondary tumors. In recent years, one of the most studied properties include changes in energy metabolism, when tumor cells specifically control reprogramming of the main metabolic pathways for their own benefit and to satisfy their increased needs not only for energy, but also for building materials required for increased proliferation. To adapt to extracellular conditions, it is necessary that cells undergo morphological changes, where modifications in the cell shape through reorganization of cytoskeletal filaments allow tumor cells to increase their invasiveness and other aggressive features. Clarifying these changes together with understanding of the switch in the genetic program within cancer cells, which allows them to overcome different stages of differentiation from cancer stem cells to fully differentiated cells, would be an important prerequisite for identification of the cancer cell "weaknesses" and may lead to improved cancer treatment. The ability of tumor cells to alter the rules of their own organism thus represents an important challenge for oncological research.Key words: cellular reprogramming - cancer cell plasticity - cancer metabolism - tumor heterogeneity - cytoskeleton remodeling - metastasis - oncogenesisThis work was supported by the project MEYS - NPS I - LO1413.The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study.The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers.Submitted: 3. 7. 2016Accepted: 11. 8. 2016.

Background Dissemination of cancer cells from the primary tumor and establishment of therapy-resistant distant metastases is the most common cause of human cancer deaths. The primary tumor consists of a heterogeneous population of cancer cells that have to overcome activity of the immune system, insufficient delivery of nutrients and oxygen, chemotherapy, radiotherapy etc. that lead to the selection of resistant and plastic cancer cells. Another selection pressure during metastatic spread gives rise to resistant subpopulations of cells, capable of surviving and proliferating in the hostile microenvironment of distant tissues. Aim In this article, individual steps of the metastatic cascade are described as well as the mechanisms and signaling pathways that cancer cells use to deal with them. Metastatic process is generally inefficient and only very few cells released from the primary tumor develop into metastases. This success is enabled by pro-metastatic mutations, accumulated due to the selection pressure and also by cooperation of non-transformed cells that secrete supporting factors. Conclusion Recent advances in research provide deeper insights into the complex processes that lead to formation and dissemination of cancer cells. Deciphering the key points of metastatic cascade and principles of its regulation will perhaps lead to development of efficient therapeutics targeting metastatic cells.Key words: metastasis - carcinoma - vascular endothelial growth factor A - epithelial-mesenchymal transitioThis work was supported by the project MEYS - NPS I - LO1413.The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study.The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers.Submitted: 6. 5. 2016Accepted: 19. 5. 2016.