Cancer stem cells and their unique role in metastatic spread.

Cancer stem cells (CSC) possess abilities generally associated with embryonic or adult stem cells, especially self-renewal and differentiation, but also dormancy and cellular plasticity that allow adaption to new environmental circumstances. These abilities are ideal prerequisites for the successful establishment of metastasis. This review highlights the role of CSCs in every step of the metastatic cascade from cancer cell invasion into blood vessels, survival in the blood stream, attachment and extravasation as well as colonization of the host organ and subsequent establishment of distant macrometastasis.

Therapy resistance mediated by cancer stem cells.

Cancer stem cells (CSC) possess abilities generally associated with embryonic or adult stem cells, especially self-renewal and differentiation. The CSC model assumes that this subpopulation of cells sustains malignant growth, which suggests a hierarchical organization of tumors in which CSCs are on top and responsible for the generation of intratumoral heterogeneity. Effective tumor therapy requires the eradication of CSC as they can support regrowth of the tumor resulting in recurrence. However, eradication of CSC is difficult because they frequently are therapy resistant. Therapy resistance is mediated by the acquisition of dormancy, increased DNA repair and drug efflux capacity, decreased apoptosis as well as the interaction between CSC and their supporting microenvironment, the CSC niche. This review highlights the role of CSC in chemo- and radiotherapy resistance as well as possible ways to overcome CSC mediated therapy resistance.

Therapy resistance mediated by exosomes.

Therapy resistance can arise within tumor cells because of genetic or phenotypic changes (intrinsic resistance), or it can be the result of an interaction with the tumor microenvironment (extrinsic resistance). Exosomes are membranous vesicles 40 to 100 nm in diameter constitutively released by almost all cell types, and mediate cell-to-cell communication by transferring mRNAs, miRNAs, DNAs and proteins causing extrinsic therapy resistance. They transfer therapy resistance by anti-apoptotic signalling, increased DNA-repair or delivering ABC transporters to drug sensitive cells. As functional mediators of tumor-stroma interaction and of epithelial to mesenchymal transition, exosomes also promote environment-mediated therapy resistance.Exosomes may be used in anticancer therapy exploiting their delivery function. They may effectively transfer anticancer drugs or RNAs in the context of gene therapy reducing immune stimulatory effects of these drugs and hydrophilic qualities facilitating crossing of cell membranes.

Concise Review: Prostate Cancer Stem Cells: Current Understanding.

Prostate cancer (PCa) is heterogeneous, harboring phenotypically diverse cancer cell types. PCa cell heterogeneity is caused by genomic instability that leads to the clonal competition and evolution of the cancer genome and by epigenetic mechanisms that result in subclonal cellular differentiation. The process of tumor cell differentiation is initiated from a population of prostate cancer stem cells (PCSCs) that possess many phenotypic and functional properties of normal stem cells. Since the initial reports on PCSCs in 2005, there has been much effort to elucidate their biological properties, including unique metabolic characteristics. In this Review, we discuss the current methods for PCSC enrichment and analysis, the hallmarks of PCSC metabolism, and the role of PCSCs in tumor progression. Stem Cells 2018;36:1457-1474.

Radiation resistance: Cancer stem cells (CSCs) and their enigmatic pro-survival signaling.

Despite the fact that radiation therapy is a highly effective therapeutic approach, a small intratumoral cell subpopulation known as "cancer stem cells" (CSCs) is radiation-resistant and possesses specific molecular properties protecting it against radiation-induced damage. The exact mechanisms of this radioresistance are still not fully elucidated, but they relate to these cells' enhanced DNA repair capacities and their low intracellular ROS concentrations, resulting from their up-regulation of ROS scavengers. The low ROS content is accompanied by disturbances in cell cycle regulation, so it can be assumed that either CSCs are quiescent or dormant themselves, or that this cell population consists of at least two cell subpopulations: the normally and the slowly proliferating cells (quiescent or dormant cells). Slowly dividing CSCs show concomitant dysregulation of the signaling molecules mediating both cell cycle progression and maintenance of cell stemness. Despite a massive accumulation of data concerning the mechanisms underlying DNA damage response in CSCs, it represents a challenge to researchers in the era of personalized medicine to elucidate the role of intracellular ROS and of signaling pathways associated with the radiation resistance of these cells; there is a clear need to understand the molecular mechanisms helping CSCs to survive radiation exposure.

Crosstalk between DNA repair and cancer stem cell (CSC) associated intracellular pathways.

DNA damaging agents (ionizing radiation and chemotherapeutics) are considered as most effective in cancer treatment. However, there is a subpopulation of carcinoma cells within the tumour demonstrating resistance to DNA damaging treatment approaches. It is suggested that limited tumour response to this kind of therapy can be associated with specific molecular properties of carcinoma stem cells (CSCs) representing the most refractory cell subpopulation. This review article presents novel data about molecular features of CSCs underlying DNA damage response and related intracellular signalling.

Putative biomarkers and therapeutic targets associated with radiation resistance.

Radiation therapy plays an important role in the management of malignant tumors, however, the problem of radiation resistance resulting in tumor recurrences after treatment is still unsolved. The emergence of novel biomarkers to predict cancer cell insensitivity to ionizing radiation could help to improve therapy results in cancer patients receiving radiation therapy. The proteomic approach could be effectively used to identify proteins associated with cancer radiation resistance. It is generally believed that radiation resistance could be associated with cancer stem cell persistence within the tumor. Therefore, determination of the molecular characteristics of cancer stem cells could provide additional possibilities to discover novel biomarkers to predict radiation resistance in cancer patients. This review addresses proteome-based findings that could be used for further biomarker identification and preclinical and clinical validation.

Chemoradiotherapy Combined with Brachytherapy for the Definitive Treatment of Esophageal Carcinoma.

This study aims to investigate the effect of dose escalation with brachytherapy (BT) as an addition to definitive chemoradiotherapy (CRT) on local control and survival in esophageal cancer. From 2001 to 2020, 183 patients with locally limited or locally advanced esophageal cancer received definitive CRT with or without brachytherapy in a two-center study. External-beam radiotherapy was delivered at 50.4 Gy in 1.8 Gy daily fractions, followed by a sequential boost to the primary tumor of 9 Gy in 1.8 Gy daily fractions if indicated. Intraluminal high dose rate (HDR) Ir-192 brachytherapy was performed on 71 patients at 10 Gy in two fractions, with one fraction per week. The combined systemic therapy schedules used included 5-fluorouracil/cisplatin or 5-fluorouracil alone. Cisplatin was not administered in patients receiving brachytherapy. The median local progression-free survival was significantly extended in the BT group (18.7 vs. 6.0 months; p < 0.0001), and the median local control was also significantly prolonged (30.5 vs. 11.3 months, p = 0.008). Overall survival (OS) significantly increased in the BT group (median OS 22.7 vs. 9.1 months, p < 0.0001). No significant difference in the overall rate of acute toxicities was observed; however, the rate of acute esophagitis was significantly higher in the BT group (94.4% vs. 81.2%). Likewise, the overall rate of late toxicities (43.7% vs. 18.8%) was significantly higher in the BT group, including the rate of esophageal stenosis (22.5% vs. 9.8%). There was no difference in the occurrence of life-threatening or lethal late toxicities (grades 4 and 5). Brachytherapy, after chemoradiation with single-agent 5-FU, represents a safe and effective alternative for dose escalation in the definitive treatment of esophageal cancer.

Erk1/2-Dependent HNSCC Cell Susceptibility to Erastin-Induced Ferroptosis.

Unfavorable clinical outcomes mean that cancer researchers must attempt to develop novel therapeutic strategies to overcome therapeutic resistance in patients with HNSCC. Recently, ferroptosis was shown to be a promising pathway possessing druggable targets, such as xCT (SLC7A11). Unfortunately, little is known about the molecular mechanisms underlying the susceptibility of HNSCC cells to ferroptosis. The goal of this study was to determine whether HNSCC cells with activated Erk1/2 are vulnerable to ferroptosis induction. Our results have shown that xCT (SLC7A11) was overexpressed in malignant tissues obtained from the patients with HNSCC, whereas normal mucosa demonstrated weak expression of the protein. In order to investigate the role of Erk1/2 in the decrease in cell viability caused by erastin, xCT-overexpressing FaDu and SCC25 HNSCC cells were used. The ravoxertinib-dependent inhibition of Erk1/2 signaling led to the decrease in erastin efficacy due to the effect on ROS production and the upregulation of ROS scavengers SOD1 and SOD2, resulting in repressed lipid peroxidation. Therefore, it was concluded that the erastin-dependent activation of ferroptosis seems to be a promising approach which can be further developed as an additional strategy for the treatment of HNSCC. As ferroptosis induction via erastin is strongly dependent on the expression of Erk1/2, this MAP kinase can be considered as a predictor for cancer cells' response to erastin.

Proteomics profiling of microdissected low- and high-grade prostate tumors identifies Lamin A as a discriminatory biomarker.

Proteomics screening methods for the identification of diagnostic and prognostic biomarkers in cancer are still lagging behind DNA- or RNA-based analysis. We used two-dimensional differential gel electrophoresis (2D-DIGE) in combination with laser capture microdissection (LCM) and MALDI-TOF/TOF mass spectrometry to determine differentially abundant proteins and candidate biomarkers in prostate cancer. Paired (benign and tumor) samples were isolated from 23 Gleason Score 6 (GS 6) and 23 Gleason Score 8 and higher (GS 8+) radical prostatectomy specimens and subjected to 2D-DIGE analysis. Minimal fluorescent dye labeling was applied and electrophoresis performed with triple samples (paired benign and tumor; internal control) for each case of tumor. Nineteen differently abundant proteins were identified by mass spectrometry and further validated. One half of them were associated with glycolysis and the Warburg effect; these were upregulated in tumors. The upregulation correlated with tumor dedifferentiation and might be relevant for selection of therapeutic strategies. Among the other proteins, heat shock protein 60 (HSP60) was significantly upregulated in tumor tissue compared to its benign counterpart. Furthermore, lamin A was statistically highly discriminatory between low and high Gleason score tumors and might serve as a new biomarker of tumor differentiation and prognosis.

Simvastatin is effective in killing the radioresistant breast carcinoma cells.

BACKGROUND: Statins, small molecular 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors, are widely used to lower cholesterol levels in lipid-metabolism disorders. Recent preclinical and clinical studies have shown that statins exert beneficial effects in the management of breast cancer by increasing recurrence free survival. Unfortunately, the underlying mechanisms remain elusive. MATERIALS AND METHODS: Simvastatin, one of the most widely prescribed lipophilic statins was utilized to investigate potential radiosensitizing effects and an impact on cell survival and migration in radioresistant breast cancer cell lines. RESULTS: Compared to parental cell counterparts, radioresistant MDA-MB-231-RR, T47D-RR andAu565-RR cells were characterized by upregulation of 3-hydroxy-3-methylglutharyl-coenzyme A reductase (HMGCR) expression accompanied by epithelial-to-mesenchymal transition (EMT) activation. Radioresistant breast cancer cells can be killed by simvastatin via mobilizing of a variety of pathways involved in apoptosis and autophagy. In the presence of simvastatin migratory abilities and vimentin expression is diminished while E-cadherin expression is increased. CONCLUSIONS: The present study suggests that simvastatin may effectively eradicate radioresistant breast carcinoma cells and diminish their mesenchymal phenotypes.

Ferritin H deficiency deteriorates cellular iron handling and worsens Salmonella typhimurium infection by triggering hyperinflammation.

Iron is an essential nutrient for mammals as well as for pathogens. Inflammation-driven changes in systemic and cellular iron homeostasis are central for host-mediated antimicrobial strategies. Here, we studied the role of the iron storage protein ferritin H (FTH) for the control of infections with the intracellular pathogen Salmonella enterica serovar Typhimurium by macrophages. Mice lacking FTH in the myeloid lineage (LysM-Cre+/+Fthfl/fl mice) displayed impaired iron storage capacities in the tissue leukocyte compartment, increased levels of labile iron in macrophages, and an accelerated macrophage-mediated iron turnover. While under steady-state conditions, LysM-Cre+/+Fth+/+ and LysM-Cre+/+Fthfl/fl animals showed comparable susceptibility to Salmonella infection, i.v. iron supplementation drastically shortened survival of LysM-Cre+/+Fthfl/fl mice. Mechanistically, these animals displayed increased bacterial burden, which contributed to uncontrolled triggering of NF-κB and inflammasome signaling and development of cytokine storm and death. Importantly, pharmacologic inhibition of the inflammasome and IL-1ß pathways reduced cytokine levels and mortality and partly restored infection control in iron-treated ferritin-deficient mice. These findings uncover incompletely characterized roles of ferritin and cellular iron turnover in myeloid cells in controlling bacterial spread and for modulating NF-κB and inflammasome-mediated cytokine activation, which may be of vital importance in iron-overloaded individuals suffering from severe infections and sepsis.

GLS-driven glutamine catabolism contributes to prostate cancer radiosensitivity by regulating the redox state, stemness and ATG5-mediated autophagy.

Radiotherapy is one of the curative treatment options for localized prostate cancer (PCa). The curative potential of radiotherapy is mediated by irradiation-induced oxidative stress and DNA damage in tumor cells. However, PCa radiocurability can be impeded by tumor resistance mechanisms and normal tissue toxicity. Metabolic reprogramming is one of the major hallmarks of tumor progression and therapy resistance. Specific metabolic features of PCa might serve as therapeutic targets for tumor radiosensitization and as biomarkers for identifying the patients most likely to respond to radiotherapy. The study aimed to characterize a potential role of glutaminase (GLS)-driven glutamine catabolism as a prognostic biomarker and a therapeutic target for PCa radiosensitization. Methods: We analyzed primary cell cultures and radioresistant (RR) derivatives of the conventional PCa cell lines by gene expression and metabolic assays to identify the molecular traits associated with radiation resistance. Relative radiosensitivity of the cell lines and primary cell cultures were analyzed by 2-D and 3-D clonogenic analyses. Targeting of glutamine (Gln) metabolism was achieved by Gln starvation, gene knockdown, and chemical inhibition. Activation of the DNA damage response (DDR) and autophagy was assessed by gene expression, western blotting, and fluorescence microscopy. Reactive oxygen species (ROS) and the ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG) were analyzed by fluorescence and luminescence probes, respectively. Cancer stem cell (CSC) properties were investigated by sphere-forming assay, CSC marker analysis, and in vivo limiting dilution assays. Single circulating tumor cells (CTCs) isolated from the blood of PCa patients were analyzed by array comparative genome hybridization. Expression levels of the GLS1 and MYC gene in tumor tissues and amino acid concentrations in blood plasma were correlated to a progression-free survival in PCa patients. Results: Here, we found that radioresistant PCa cells and prostate CSCs have a high glutamine demand. GLS-driven catabolism of glutamine serves not only for energy production but also for the maintenance of the redox state. Consequently, glutamine depletion or inhibition of critical regulators of glutamine utilization, such as GLS and the transcription factor MYC results in PCa radiosensitization. On the contrary, we found that a combination of glutamine metabolism inhibitors with irradiation does not cause toxic effects on nonmalignant prostate cells. Glutamine catabolism contributes to the maintenance of CSCs through regulation of the alpha-ketoglutarate (α-KG)-dependent chromatin-modifying dioxygenase. The lack of glutamine results in the inhibition of CSCs with a high aldehyde dehydrogenase (ALDH) activity, decreases the frequency of the CSC populations in vivo and reduces tumor formation in xenograft mouse models. Moreover, this study shows that activation of the ATG5-mediated autophagy in response to a lack of glutamine is a tumor survival strategy to withstand radiation-mediated cell damage. In combination with autophagy inhibition, the blockade of glutamine metabolism might be a promising strategy for PCa radiosensitization. High blood levels of glutamine in PCa patients significantly correlate with a shorter prostate-specific antigen (PSA) doubling time. Furthermore, high expression of critical regulators of glutamine metabolism, GLS1 and MYC, is significantly associated with a decreased progression-free survival in PCa patients treated with radiotherapy. Conclusions: Our findings demonstrate that GLS-driven glutaminolysis is a prognostic biomarker and therapeutic target for PCa radiosensitization.

Molecular heterogeneity in breast carcinoma cells with increased invasive capacities.

Background Metastatic progression of breast cancer is still a challenge in clinical oncology. Therefore, an elucidation how carcinoma cells belonging to different breast cancer subtypes realize their metastatic capacities is needed. The aim of this study was to elucidate a similarity of activated molecular pathways underlying an enhancement of invasiveness of carcinoma cells belonging to different breast carcinoma subtypes. Materials and methods In order to reach this aim, parental and invasive (INV) MDA-MB-231 (triple-negative), T47D (hormone receptor-positive), and Au565 (Her2-positive) breast carcinoma cells were used and their molecular phenotypes were compared using a proteomic approach. Results Independently from breast cancer subtypes, INV cells have demonstrated fibroblast-like morphology accompanied by enhancement of invasive and migratory capacities, increased expression of cancer stem cell markers, and delayed tumor growth in in vivo animal models. However, the global proteomic analysis has highlighted that INV cells were different in protein expressions from the parental cells, and Her2-positive Au565-INV cells showed the most pronounced molecular differences compared to the triple-negative MDA-MB-231-INV and hormone receptor-positive T47D-INV cells. Although Au565-INV breast carcinoma cells possessed the highest number of deregulated proteins, they had the lowest overlapping in proteins commonly expressed in MDA-MB-231-INV and T47D-INV cells. Conclusions We can conclude that hormone receptor-positive cells with increased invasiveness acquire the molecular characteristics of triple-negative breast cancer cells, whereas Her2-positive INV cells specifically changed their own molecular phenotype with very limited partaking in the involved pathways found in the MDA-MB-231-INV and T47D-INV cells. Since hormone receptor-positive invasive cells share their molecular properties with triple-negative breast cancer cells, we assume that these types of metastatic disease can be treated rather equally with an option to add anti-hormonal agents. In contrast, Her2-positive metastasis should be carefully evaluated for more effective therapeutic approaches which are distinct from the triple-negative and hormone-positive metastatic breast cancers.

MPC-6827: a small-molecule inhibitor of microtubule formation that is not a substrate for multidrug resistance pumps.

A novel series of 4-arylaminoquinazolines were identified from a cell-based screening assay as potent apoptosis inducers. Through structure-activity relationship studies, MPC-6827 and its close structural analogue, MPI-0441138, were discovered as proapoptotic molecules and mitotic inhibitors with potencies at low nanomolar concentrations in multiple tumor cell lines. Photoaffinity and radiolabeled analogues of MPC-6827 were found to bind a 55-kDa protein, and this binding was competed by MPC-6827, paclitaxel, and colchicine, but not vinblastine. MPC-6827 effectively inhibited the polymerization of tubulin in vitro, competed with colchicine binding, and disrupted the formation of microtubules in a variety of tumor cell lines, which together showed the molecular target as tubulin. Treatment of MCF-7 breast carcinoma or Jurkat leukemia cells with MPC-6827 led to pronounced G2-M cell cycle arrest followed by apoptosis. Apoptosis, as determined by terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling assay, was preceded by loss of mitochondrial membrane potential, cytochrome c translocation from mitochondria to nuclei, activation of caspase-3, and cleavage of poly(ADP-ribose) polymerase. MPC-6827 was equipotent in an in vitro growth inhibition assay in several cancer cell lines regardless of the expression levels of the multidrug resistance ABC transporters MDR-1 (Pgp-1), MRP-1, and BCRP-1. In B16-F1 allografts and in OVCAR-3, MIAPaCa-2, MCF-7, HT-29, MDA-MB-435, and MX-1 xenografts, statistically significant tumor growth inhibition was observed with MPC-6827. These studies show that MPC-6827 is a microtubule-disrupting agent with potent and broad-spectrum in vitro and in vivo cytotoxic activities and, therefore, MPC-6827 is a promising candidate for development as a novel therapeutic for multiple cancer types.

Quantitative proteomics and phosphoproteomics reveal novel insights into complexity and dynamics of the EGFR signaling network.

The epidermal growth factor receptor (EGFR/ErbB1/Her1) belongs to the ErbB family of receptor tyrosine kinases (RTKs) and is a key player in the regulation of cell proliferation, differentiation, survival, and migration. Overexpression and mutational changes of EGFR have been identified in a variety of human cancers and the regulation of EGFR signaling plays a critical role in tumor development and progression. Due to its biological significance the EGFR signaling network is a widely used model system for the development of analytical techniques. Novel quantitative proteomics and phosphoproteomics approaches play an important role in the characterization of signaling pathways in a time and stimulus dependent manner. Recent studies discussed in this review provide new insights into different aspects of EGFR signal transduction, such as regulation and dynamics of its phosphorylation sites, association with interaction partners and identification of regulated phosphoproteins. Correlation of data from functional proteomics studies with results from other fields of signal transduction research by systems biology will be necessary to integrate and translate these findings into successful clinical applications.

Intracellular signaling pathways regulating radioresistance of human prostate carcinoma cells.

Radiation therapy plays an important role in the management of prostate carcinoma. However, the problem of radioresistance and molecular mechanisms by which prostate carcinoma cells overcome cytotoxic effects of radiation therapy remains to be elucidated. In order to investigate possible intracellular mechanisms underlying the prostate carcinoma recurrences after radiotherapy, we have established three radiation-resistant prostate cancer cell lines, LNCaP-IRR, PC3-IRR, and Du145-IRR derived from the parental LNCaP, PC3, and Du145 prostate cancer cells by repetitive exposure to ionizing radiation. LNCaP-IRR, PC3-IRR, and Du145-IRR cells (prostate carcinoma cells recurred after radiation exposure (IRR cells)) showed higher radioresistance and cell motility than parental cell lines. IRR cells exhibited higher levels of androgen and epidermal growth factor (EGF) receptors and activation of their downstream pathways, such as Ras-mitogen-activated protein kinase (MAPK) and phosphatidyl inositol 3-kinase (PI3K)-Akt and Jak-STAT. In order to define additional mechanisms involved in the radioresistance development, we determined differences in the proteome profile of parental and IRR cells using 2-D DIGE followed by computational image analysis and MS. Twenty-seven proteins were found to be modulated in all three radioresistant cell lines compared to parental cells. Identified proteins revealed capacity to interact with EGF and androgen receptors related signal transduction pathways and were involved in the regulation of intracellular routs providing cell survival, increased motility, mutagenesis, and DNA repair. Our data suggest that radioresistance development is accompanied by multiple mechanisms, including activation of cell receptors and related downstream signal transduction pathways. Identified proteins regulated in the radioresistant prostate carcinoma cells can significantly intensify activation of intracellular signaling that govern cell survival, growth, proliferation, invasion, motility, and DNA repair. In addition, such analyses may be utilized in predicting cellular response to radiotherapy.

Increased numbers of endothelial progenitor cells in peripheral blood and tumor specimens in non-small cell lung cancer: a methodological challenge and an ongoing debate on the clinical relevance.

Bone marrow-derived endothelial progenitor cells (EPC) play an important role in neovascularisation and tumor growth. However, the clinical relevance of EPCs on blood vessel formation in non-small cell lung cancer (NSCLC) is unclear. EPC numbers in circulation are very low and therefore their detection is technically challenging. In the present study, 10 NSCLC patients and 5 healthy controls were included. Patients underwent blood analyses before and after surgery. EPCs were isolated from whole blood by magnetic cell sorting to CD34 (MACS). Afterwards, FACS analyses using antibodies against CD133, CD34, VEGFR2 and CD45 and and immunocytological staining to CD133 on cytospins (MCA) were performed. Cryostat sections of tumor samples were stained for CD133, CD31 and cytokeratin A7. Serum levels of the vascular endothelial growth factor (VEGF) were quantified by sandwich ELISA. Compared to the control group NSCLC patients showed significantly elevated EPC counts and VEGF levels in peripheral blood before and after surgery. From a methodological point of view, the tested procedure (MCA) was validated as compared to the standard FACS analyses (CD34+/VEGFR2+). MCA proved to have a very high sensitivity and even allowed the identification of singular positive EPCs.