13C Tracer Analysis and Metabolomics in Dormant Cancer Cells.

Over the last two decades, major advances in the field of tumor dormancy have been made. Yet, it is not completely understood how dormant disseminated tumor cells survive and transition to a proliferative state to generate a metastatic lesion. On the other hand, metabolic rewiring has been shown to influence metastasis development through the modulation of both intracellular signaling and the crosstalk between metastatic cells and their microenvironment. Thus, studying the metabolic features of dormant disseminated tumor cells has gained importance in understanding the dormancy process. Here, we describe a method to perform metabolomics and 13C tracer analysis in 3D cultures of dormant breast cancer cells.

Dysregulation of Mitochondrial Translation Caused by CBFB Deficiency Cooperates with Mutant PIK3CA and Is a Vulnerability in Breast Cancer.

Understanding functional interactions between cancer mutations is an attractive strategy for discovering unappreciated cancer pathways and developing new combination therapies to improve personalized treatment. However, distinguishing driver gene pairs from passenger pairs remains challenging. Here, we designed an integrated omics approach to identify driver gene pairs by leveraging genetic interaction analyses of top mutated breast cancer genes and the proteomics interactome data of their encoded proteins. This approach identified that PIK3CA oncogenic gain-of-function (GOF) and CBFB loss-of-function (LOF) mutations cooperate to promote breast tumor progression in both mice and humans. The transcription factor CBFB localized to mitochondria and moonlighted in translating the mitochondrial genome. Mechanistically, CBFB enhanced the binding of mitochondrial mRNAs to TUFM, a mitochondrial translation elongation factor. Independent of mutant PI3K, mitochondrial translation defects caused by CBFB LOF led to multiple metabolic reprogramming events, including defective oxidative phosphorylation, the Warburg effect, and autophagy/mitophagy addiction. Furthermore, autophagy and PI3K inhibitors synergistically killed breast cancer cells and impaired the growth of breast tumors, including patient-derived xenografts carrying CBFB LOF and PIK3CA GOF mutations. Thus, our study offers mechanistic insights into the functional interaction between mutant PI3K and mitochondrial translation dysregulation in breast cancer progression and provides a strong preclinical rationale for combining autophagy and PI3K inhibitors in precision medicine for breast cancer. SIGNIFICANCE: CBFB-regulated mitochondrial translation is a regulatory step in breast cancer metabolism and synergizes with mutant PI3K in breast cancer progression.

Involvement of redox signalling in tumour cell dormancy and metastasis.

Decades of research on oncogene-driven carcinogenesis and gene-expression regulatory networks only started to unveil the complexity of tumour cellular and molecular biology. This knowledge has been successfully implemented in the clinical practice to treat primary tumours. In contrast, much less progress has been made in the development of new therapies against metastasis, which are the main cause of cancer-related deaths. More recently, the role of epigenetic and microenviromental factors has been shown to play a key role in tumour progression. Free radicals are known to communicate the intracellular and extracellular compartments, acting as second messengers and exerting a decisive modulatory effect on tumour cell signalling. Depending on the cellular and molecular context, as well as the intracellular concentration of free radicals and the activation status of the antioxidant system of the cell, the signalling equilibrium can be tilted either towards tumour cell survival and progression or cell death. In this regard, recent advances in tumour cell biology and metastasis indicate that redox signalling is at the base of many cell-intrinsic and microenvironmental mechanisms that control disseminated tumour cell fate and metastasis. In this manuscript, we will review the current knowledge about redox signalling along the different phases of the metastatic cascade, including tumour cell dormancy, making emphasis on metabolism and the establishment of supportive microenvironmental connections, from a redox perspective.

An Olive-Derived Extract 20% Rich in Hydroxytyrosol Prevents ß-Amyloid Aggregation and Oxidative Stress, Two Features of Alzheimer Disease, via SKN-1/NRF2 and HSP-16.2 in Caenorhabditis elegans.

Olive milling produces olive oil and different by-products, all of them very rich in different bioactive compounds like the phenolic alcohol hydroxytyrosol. The aim of the present study was to investigate the effects of an olive fruit extract 20% rich in hydroxytyrosol on the molecular mechanisms associated with Alzheimer disease features like Aß- and tau- induced toxicity, as well as on oxidative stress in Caenorhabditis elegans. Moreover, characterization of the extracts, regarding the profile and content of phenolics, as well as total antioxidant ability, was investigated. The study of lethality, growth, pharyngeal pumping, and longevity in vivo demonstrated the lack of toxicity of the extract. One hundred µg/mL of extract treatment revealed prevention of oxidative stress and a delay in Aß-induced paralysis related with a lower presence of Aß aggregates. Indeed, the extract showed the ability to avoid a certain degree of proteotoxicity associated with aggregation of the tau protein. According to RNAi tests, SKN-1/NRF2 transcription factor and the overexpression of HSP-16.2 were mechanistically associated in the observed effects.

An oleuropein rich-olive (Olea europaea L.) leaf extract reduces ß-amyloid and tau proteotoxicity through regulation of oxidative- and heat shock-stress responses in Caenorhabditis elegans.

Olive tree-derived products have been associated with numerous benefits for health. The aim of the present study was to characterize an olive leaf extract enriched in oleuropein (OLE) concerning phenolic content and profile as well as antioxidant capacity. Short-term and long-term toxicity, including oxidative stress, was in vivo evaluated in the experimental model Caenorhabditis elegans. Moreover, the potential therapeutic effect of the extract against Aß induced- and tau protein induced-toxicity was also evaluated in C. elegans. OLE treatment did not exert toxicity. On the contrary, the extract was able to ameliorate oxidative stress and proteotoxicity related to Aß and tau aggregation. The potential molecular mechanisms present behind the observed results explored by RNAi technology revealed that DAF-16/FOXO and SKN-1/NRF2, elements of the insulin insulin-like signalling pathway, as well as HSP-16.2 overexpression were involved.

The central role of mitochondria in the relationship between dietary lipids and cancer progression.

Evidence demonstrates the importance of lipid metabolism and signaling in cancer cell biology. De novo lipogenesis is an important source of lipids for cancer cells, but exogenous lipid uptake remains essential for many cancer cells. Dietary lipids can modify lipids present in tumor microenvironment affecting cancer cell metabolism. Clinical trials have shown that diets rich in polyunsaturated fatty acids (PUFA) can negatively affect tumor growth. However, certain n-6 PUFAs can also contribute to cancer progression. Identifying the molecular mechanisms through which lipids affect cancer progression will provide an opportunity for focused dietary interventions that could translate into the development of personalized diets for cancer control. However, the effective mechanisms of action of PUFAs have not been fully clarified yet. Mitochondria controls ATP generation, redox homeostasis, metabolic signaling, apoptotic pathways and many aspects of autophagy, and it has been recognized to play a key role in cancer. The purpose of this review is to summarize the current evidence linking dietary lipids effects on mitochondrial aspects with consequences for cancer progression and the molecular mechanisms that underlie this association.

Reductive Stress, Bioactive Compounds, Redox-Active Metals, and Dormant Tumor Cell Biology to Develop Redox-Based Tools for the Treatment of Cancer.

Significance: Cancer is related to redox biology from many points of view, such as initiation and promotion, metabolism and growth, invasion and metastasis, vascularization, or through the interaction with the immune system. In addition, this extremely complex relationship depends on the redox homeostasis of each cellular compartment, which might be used to fight cancer. Recent Advances: New ways of modulating specific and little explored aspects of redox biology have been revealed, as well as new delivery methods or uses of previously known treatments against cancer. Here, we review the latest experimental evidence regarding redox biology in cancer treatment and analyze its potential impact in the development of improved and more effective antineoplastic therapies. Critical Issues: A critical issue that deserves particular attention is the understanding that both extremes of redox biology (i.e., oxidative stress [OS] and reductive stress) might be useful or harmful in relation to cancer prevention and treatment. Future Directions: Additional research is needed to understand how to selectively induce reductive or OS adequately to avoid cancer proliferation or to induce cancer cell death.

Cell-intrinsic survival signals. The role of autophagy in metastatic dissemination and tumor cell dormancy.

Metastasis is the main cause of cancer-related deaths. Disseminated tumor cells (DTCs), which seed metastasis, can remain undetected in a dormant state for decades after treatment of the primary tumor and their persistence is the main cause of late relapse and death in a substantial proportion of cancer patients. Understanding the mechanisms underlying the survival of dormant DTCs is of utmost importance to develop new therapies that effectively kill DTCs while in a quiescent state, therefore preventing metastatic disease and minimizing the chance of future relapses. Besides key interactions with the local microenvironment, dormant DTCs must integrate survival mechanisms to remain viable for long periods of time. Here, the pro-survival role of autophagy in tumor cell dissemination and dormant DTC maintenance are discussed, as well as the implications of the current knowledge for future research efforts.

Autophagy promotes the survival of dormant breast cancer cells and metastatic tumour recurrence.

Cancer recurrence after initial diagnosis and treatment is a major cause of breast cancer (BC) mortality, which results from the metastatic outbreak of dormant tumour cells. Alterations in the tumour microenvironment can trigger signalling pathways in dormant cells leading to their proliferation. However, processes involved in the initial and the long-term survival of disseminated dormant BC cells remain largely unknown. Here we show that autophagy is a critical mechanism for the survival of disseminated dormant BC cells. Pharmacologic or genetic inhibition of autophagy in dormant BC cells results in significantly decreased cell survival and metastatic burden in mouse and human 3D in vitro and in vivo preclinical models of dormancy. In vivo experiments identify autophagy gene autophagy-related 7 (ATG7) to be essential for autophagy activation. Mechanistically, inhibition of the autophagic flux in dormant BC cells leads to the accumulation of damaged mitochondria and reactive oxygen species (ROS), resulting in cell apoptosis.

Gene pathways associated with mitochondrial function, oxidative stress and telomere length are differentially expressed in the liver of rats fed lifelong on virgin olive, sunflower or fish oils.

This study investigates the effect of lifelong intake of different fat sources rich in monounsaturated (virgin olive oil), n6 polyunsaturated (sunflower oil) or n3 polyunsaturated (fish oil) fatty acids in the aged liver. Male Wistar rats fed lifelong on diets differing in the fat source were killed at 6 and at 24 months of age. Liver histopathology, mitochondrial ultrastructure, biogenesis, oxidative stress, mitochondrial electron transport chain, relative telomere length and gene expression profiles were studied. Aging led to lipid accumulation in the liver. Virgin olive oil led to the lowest oxidation and ultrastructural alterations. Sunflower oil induced fibrosis, ultrastructural alterations and high oxidation. Fish oil intensified oxidation associated with age, lowered electron transport chain activity and enhanced the relative telomere length. Gene expression changes associated with age in animals fed virgin olive oil and fish oil were related mostly to mitochondrial function and oxidative stress pathways, followed by cell cycle and telomere length control. Sunflower oil avoided gene expression changes related to age. According to the results, virgin olive oil might be considered the dietary fat source that best preserves the liver during the aging process.

Tumor cell dormancy as an adaptive cell stress response mechanism.

Metastases are responsible for most cancer-related deaths. The kinetics of tumor relapse is highly heterogeneous, ranging from recurrences shortly after diagnosis to years or even decades after the initial treatment. This subclinical period is known as tumor dormancy, in which residual disease remains in an undetectable state before finally appearing as an overtly proliferative metastasis. Despite recent advances in our understanding of the molecular mechanisms leading to tumor dormancy, it is still a poorly understood phase of cancer progression, which limits opportunities for the design of successful therapeutic interventions. The influence of the tumor microenvironment at the metastatic site and anti-metastatic immune responses have been shown to play a crucial role in the onset and maintenance of metastatic dormancy. However, there is still a significant gap in our understanding of how dormant cells remain viable in a quiescent state for long periods of time. Here, we review the latest experimental evidence shedding light on the biological processes that enable dormant tumor cells to endure the multiple stresses encountered at the metastatic site.

Exposure to bis(maltolato)oxovanadium(IV) increases levels of hepcidin mRNA and impairs the homeostasis of iron but not that of manganese.

The aim of this study was to examine whether alterations in iron homeostasis, caused by exposure to vanadium, are related to changes in the gene expression of hepatic hepcidin. Two groups of rats were examined: control and vanadium-exposed. Vanadium, as bis(maltolato)oxovanadium(IV) was supplied in the drinking water. The experiment had a duration of five weeks. Iron and manganese were measured in excreta, serum and tissues. Leptin, ferritin, IL-1ß, IL-6, TNF-α, red blood cells, haemoglobin and haematocrit were determined. Protein carbonyl group levels and hepcidin gene expression were determined in the liver. In the vanadium-exposed rats, iron absorption, serum iron and leptin and all haematological parameters decreased. Levels of IL-6, TNF-α and ferritin in serum and of iron in the liver, spleen and heart increased. In the liver, levels of protein carbonyl groups and hepcidin mRNA were also higher in the vanadium-exposed group. Exposure to vanadium did not modify manganese homeostasis. The results obtained from this study provide the first evidence that bis(maltolato)oxovanadium(IV) produces an increase in the gene expression of the hepcidin, possibly caused by an inflammatory process. Both factors could be the cause of alterations in Fe homeostasis and the appearance of anaemia. However, Mn homeostasis was not affected.

Hydroxytyrosol ameliorates oxidative stress and mitochondrial dysfunction in doxorubicin-induced cardiotoxicity in rats with breast cancer.

Oxidative stress is involved in several processes including cancer, aging and cardiovascular disease, and has been shown to potentiate the therapeutic effect of drugs such as doxorubicin. Doxorubicin causes significant cardiotoxicity characterized by marked increases in oxidative stress and mitochondrial dysfunction. Herein, we investigate whether doxorubicin-associated chronic cardiac toxicity can be ameliorated with the antioxidant hydroxytyrosol in rats with breast cancer. Thirty-six rats bearing breast tumors induced chemically were divided into 4 groups: control, hydroxytyrosol (0.5mg/kg, 5days/week), doxorubicin (1mg/kg/week), and doxorubicin plus hydroxytyrosol. Cardiac disturbances at the cellular and mitochondrial level, mitochondrial electron transport chain complexes I-IV and apoptosis-inducing factor, and oxidative stress markers have been analyzed. Hydroxytyrosol improved the cardiac disturbances enhanced by doxorubicin by significantly reducing the percentage of altered mitochondria and oxidative damage. These results suggest that hydroxytyrosol improve the mitochondrial electron transport chain. This study demonstrates that hydroxytyrosol protect rat heart damage provoked by doxorubicin decreasing oxidative damage and mitochondrial alterations.

Transcriptional shift identifies a set of genes driving breast cancer chemoresistance.

BACKGROUND: Distant recurrences after antineoplastic treatment remain a serious problem for breast cancer clinical management, which threats patients' life. Systemic therapy is administered to eradicate cancer cells from the organism, both at the site of the primary tumor and at any other potential location. Despite this intervention, a significant proportion of breast cancer patients relapse even many years after their primary tumor has been successfully treated according to current clinical standards, evidencing the existence of a chemoresistant cell subpopulation originating from the primary tumor. METHODS/FINDINGS: To identify key molecules and signaling pathways which drive breast cancer chemoresistance we performed gene expression analysis before and after anthracycline and taxane-based chemotherapy and compared the results between different histopathological response groups (good-, mid- and bad-response), established according to the Miller & Payne grading system. Two cohorts of 33 and 73 breast cancer patients receiving neoadjuvant chemotherapy were recruited for whole-genome expression analysis and validation assay, respectively. Identified genes were subjected to a bioinformatic analysis in order to ascertain the molecular function of the proteins they encode and the signaling in which they participate. High throughput technologies identified 65 gene sequences which were over-expressed in all groups (P ≤ 0·05 Bonferroni test). Notably we found that, after chemotherapy, a significant proportion of these genes were over-expressed in the good responders group, making their tumors indistinguishable from those of the bad responders in their expression profile (P ≤ 0.05 Benjamini-Hochgerg`s method). CONCLUSIONS: These data identify a set of key molecular pathways selectively up-regulated in post-chemotherapy cancer cells, which may become appropriate targets for the development of future directed therapies against breast cancer.

Curcumin and liver disease.

Liver diseases pose a major medical problem worldwide and a wide variety of herbs have been studied for the management of liver-related diseases. In this respect, curcumin has long been used in traditional medicine, and in recent years it has been the object of increasing research interest. In combating liver diseases, it seems clear that curcumin exerts a hypolipidic effect, which prevents the fatty acid accumulation in the hepatocytes that may result from metabolic imbalances, and which may cause nonalcoholic steatohepatitis. Another crucial protective activity of curcumin, not only in the context of chronic liver diseases but also regarding carcinogenesis and other age-related processes, is its potent antioxidant activity, which affects multiple processes and signaling pathways. The effects of curcumin on NF-κß are crucial to our understanding of the potent hepatoprotective role of this herb-derived micronutrient. Because curcumin is a micronutrient that is closely related to cellular redox balance, its properties and activity give rise to a series of molecular reactions that in every case and biological situation affect the mitochondria.

Impact of diet on breast cancer risk: a review of experimental and observational studies.

Breast cancer, which presents the highest global incidence of all female cancers, is caused by the interaction of genetic and environmental factors. Among the latter, diet has attracted considerable attention, as it is a modifiable risk factor and thus offers an opportunity to design preventive strategies. Nevertheless, only alcohol consumption has been unequivocally related to increased breast cancer risk. Despite the failure of observational studies in human populations to clearly define the nature of the relationship between specific nutrient exposures and breast cancer risk, in vivo and in vitro studies strongly suggest its existence. Moreover, studies at the molecular level have identified the putative action mechanism by which the nutritional constituents of specific foodstuffs may exert protective or enhancing effects with respect to breast cancer risk. The inadequate experimental design of some observational studies, or the occurrence of measurement errors and/or recall bias during data collection, or insufficient follow-up and subject characterization, may underlie these controversies. By improving the methods used to study the relationship between diet and breast cancer risk, and by applying new technologies linked to novel approaches such as "nutrigenomics," it might be possible to derive effective recommendations for breast cancer prevention and thus improve anti-cancer treatment.

Long-term effects of systemic cancer treatment on DNA oxidative damage: the potential for targeted therapies.

The main pathological consequence of free radical exposure is DNA damage, which is known to induce cell transformation and to facilitate important mutations in cancer progression. It is a matter of intense discussion whether the drug-induced production of free radicals limits the therapeutic efficacy of chemotherapeutics and enhances their toxicity or whether they may be enhanced to provoke cancer cell apoptosis. This paper reviews essential molecular processes to better understand the controversial role of free radicals in cancer development and progression, and discusses some novel therapeutic strategies based on oxidative stress induction and prevention.

Oxidative stress status in metastatic breast cancer patients receiving palliative chemotherapy and its impact on survival rates.

Antineoplastic agents are known to induce the production of free radicals leading to cell damage. These adverse effects may fuel the acquisition of new mutations and the development of treatment resistances. We selected 30 metastatic breast cancer patients receiving palliative chemotherapy, and paired blood samples, before and after chemotherapy, were extracted. We analysed DNA, lipid and protein oxidative damage markers and determined the extent of antioxidant and repair defences activation at the systemic level. We found that the DNA repair activity of the KU86 enzyme was significantly lower after chemotherapy and the antioxidant capacity of the plasma was significantly higher after treatment. Cox regression analysis revealed a significant effect of KU86 activity on the survival rates of those patients who received anthracyclines as part of their treatment. The high clinical heterogeneity of metastatic breast cancer patients warrants further studies to clarify the role of DNA repair and systemic antioxidant capacities during chemotherapy.

Does chemotherapy-induced oxidative stress improve the survival rates of breast cancer patients?

Antineoplastic agents induce oxidative stress leading to lipid, carbohydrate, protein, and DNA damage. We sought to explore the role of drug-induced oxidative stress on breast cancer patient's survival. We observed that neoadjuvant patients presented a marked raise in DNA damage and protein carbonyl levels after chemotherapy, whereas postchemotherapy DNA repair activity of the KU86 enzyme and total antioxidant capacity of the plasma were higher in the adjuvant group. With respect to patient's survival, we observed that increasing levels of KU86 and antioxidant capacity of the plasma during chemotherapy significantly influenced the survival rates of the patients, protecting from disease recurrence and death. Our results suggest that chemotherapy induces a certain level of systemic oxidative stress, which is maintained along successive clinical interventions and could influence the clinical outcome of the patients.

Free radicals in breast carcinogenesis, breast cancer progression and cancer stem cells. Biological bases to develop oxidative-based therapies.

Oxidative stress leads to lipid, carbohydrate, protein and DNA damage in biological systems and affects cell structure and function. Breast cancer cells are subjected to a high level of oxidative stress, both intracellular and extracellular. To survive, cancer cells must acquire adaptive mechanisms that counteract the toxic effects of free radicals exposure. These mechanisms may involve the activation of redox-sensitive transcription factors, increased expression of antioxidant enzymes and antiapoptotic proteins. Moreover, recent data maintain that different breast cancer cell types, show different intracellular antioxidant capacities that may determine their ability to resist radio and chemotherapy. The resistant cell type has been shown to correspond with tumor initiating cells, also known as cancer stem cells (CSCs), which are thought to be responsible for tumor initiation and metastasis. Abrogation of the above-mentioned adaptive mechanisms by redox regulation in cancer cells opens a promising research line that could have significant therapeutic applications.