Monocytes and Macrophages in Cancer: Unsuspected Roles.

The behavior of cancer is undoubtedly affected by stroma. Macrophages belong to this microenvironment and their presence correlates with reduced survival in most cancers. After a tumor-induced "immunoediting", these monocytes/macrophages, originally the first line of defense against tumor cells, undergo a phenotypic switch and become tumor-supportive and immunosuppressive.The influence of these tumor-associated macrophages (TAMs) on cancer is present in all traits of carcinogenesis. These cells participate in tumor initiation and growth, migration, vascularization, invasion and metastasis. Although metastasis is extremely clinically relevant, this step is always reliant on the angiogenic ability of tumors. Therefore, the formation of new blood vessels in tumors assumes particular importance as a limiting step for disease progression.Herein, the once unsuspected roles of macrophages in cancer will be discussed and their importance as a promising strategy to treat this group of diseases will be reminded.

Cysteine boosters the evolutionary adaptation to CoCl2 mimicked hypoxia conditions, favouring carboplatin resistance in ovarian cancer.

BACKGROUND: Ovarian cancer is the second most common gynaecologic malignancy and the most common cause of death from gynaecologic cancer, especially due to diagnosis at an advanced stage, when a cure is rare. As ovarian tumour grows, cancer cells are exposed to regions of hypoxia. Hypoxia is known to be partially responsible for tumour progression, metastasis and resistance to therapies. These suggest that hypoxia entails a selective pressure in which the adapted cells not only have a fitness increase under the selective environment, but also in non-selective adverse environments. In here, we used two different ovarian cancer cell lines - serous carcinoma (OVCAR3) and clear cell carcinoma (ES2) - in order to address the effect of cancer cells selection under normoxia and hypoxia mimicked by cobalt chloride on the evolutionary outcome of cancer cells. RESULTS: Our results showed that the adaptation to normoxia and CoCl2 mimicked hypoxia leads cells to display opposite strategies. Whereas cells adapted to CoCl2 mimicked hypoxia conditions tend to proliferate less but present increased survival in adverse environments, cells adapted to normoxia proliferate rapidly but at the cost of increased mortality in adverse environments. Moreover, results suggest that cysteine allows a quicker response and adaptation to hypoxic conditions that, in turn, are capable of driving chemoresistance. CONCLUSIONS: We showed that cysteine impacts the adaptation of cancer cells to a CoCl2 mimicked hypoxic environment thus contributing for hypoxia-drived platinum-based chemotherapeutic agents' resistance, allowing the selection of more aggressive phenotypes. These observations support a role of cysteine in cancer progression, recurrence and chemoresistance.

Metabolic cooperation between cancer and non-cancerous stromal cells is pivotal in cancer progression.

The way cancer cells adapt to microenvironment is crucial for the success of carcinogenesis, and metabolic fitness is essential for a cancer cell to survive and proliferate in a certain organ/tissue. The metabolic remodeling in a tumor niche is endured not only by cancer cells but also by non-cancerous cells that share the same microenvironment. For this reason, tumor cells and stromal cells constitute a complex network of signal and organic compound transfer that supports cellular viability and proliferation. The intensive dual-address cooperation of all components of a tumor sustains disease progression and metastasis. Herein, we will detail the role of cancer-associated fibroblasts, cancer-associated adipocytes, and inflammatory cells, mainly monocytes/macrophages (tumor-associated macrophages), in the remodeling and metabolic adaptation of tumors.

HNF1ß drives glutathione (GSH) synthesis underlying intrinsic carboplatin resistance of ovarian clear cell carcinoma (OCCC).

Chemoresistance to platinum-based antineoplastic agents is a consistent feature among ovarian carcinomas; however, whereas high-grade serous carcinoma (OSC) acquires resistance during chemotherapy, ovarian clear cell carcinoma (OCCC) is intrinsically resistant. The main objective of this study was to explore, in vitro and in vivo, if hepatocyte nuclear factor 1ß (HNF1ß) and glutaminolysis contribute for the resistance of OCCC to carboplatin through the intrinsically increased GSH bioavailability. To disclose the role of HNF1ß, experiments were also performed in an OSC cell line, which does not express HNF1ß. Metabolic profiles, GSH quantification, HNF1ß, and γ-glutamylcysteine ligase catalytic subunit (GCLC) and modifier subunit (GCLM) expression, cell cycle, and death were assessed in ES2 cell line (OCCC) and OVCAR3 cell line (OSC); HNF1ß knockdown was performed in ES2 and murine model of subcutaneous and peritoneal OCCC tumors was established to test buthionine sulphoxamine (BSO), as a sensitizer to carboplatin. Glutaminolysis is activated in ES2 and OVCAR3, though ES2 exclusively synthesizes amino acids and GSH. ES2 cells are more resistant to carboplatin than OVCAR3 and the abrogation of GSH production by BSO sensitizes ES2 to carboplatin. HNF1ß regulates the expression of GCLC, but not GCLM, and consequently GSH production in ES2. In vivo, BSO prior to carboplatin reduces dramatically subcutaneous tumor size and GSH levels, as well as peritoneal dissemination. Our study discloses HNF1ß as the mediator of intrinsic OCCC chemoresistance and sheds a light to re-explore a cancer adjuvant therapeutic approach using BSO to overcome the lack of efficient therapy in OCCC.

Glycidamide and cis-2-butene-1,4-dial (BDA) as potential carcinogens and promoters of liver cancer - An in vitro study.

Acrylamide and furan are environmental and food contaminants that are metabolized by cytochrome P450 2E1 (CYP2E1), giving rise to glycidamide and cis-2-butene-1,4-dial (BDA) metabolites, respectively. Both glycidamide and BDA are electrophilic species that react with nucleophilic groups, being able to introduce mutations in DNA and perform epigenetic remodeling. However, whereas these carcinogens are primarily metabolized in the liver, the carcinogenic potential of acrylamide and furan in this organ is still controversial, based on findings from experimental animal studies. With the ultimate goal of providing further insights into this issue, we explored in vitro, using a hepatocyte cell line and a hepatocellular carcinoma cell line, the putative effect of these metabolites as carcinogens and cancer promoters. Molecular alterations were investigated in cells that survive glycidamide and BDA toxicity. We observed that those cells express CD133 stemness marker, present a high proliferative capacity and display an adjusted expression profile of genes encoding enzymes involved in oxidative stress control, such as GCL-C, GSTP1, GSTA3 and CAT. These molecular changes seem to be underlined, at least in part, by epigenetic remodeling involving histone deacetylases (HDACs). Although more studies are needed, here we present more insights towards the carcinogenic capacity of glycidamide and BDA and also point out their effect in favoring hepatocellular carcinoma progression.

Monocytes as Endothelial Progenitor Cells (EPCs), Another Brick in the Wall to Disentangle Tumor Angiogenesis.

Bone marrow contains endothelial progenitor cells (EPCs) that, upon pro-angiogenic stimuli, migrate and differentiate into endothelial cells (ECs) and contribute to re-endothelialization and neo-vascularization. There are currently no reliable markers to characterize EPCs, leading to their inaccurate identification. In the past, we showed that, in a panel of tumors, some cells on the vessel wall co-expressed CD14 (monocytic marker) and CD31 (EC marker), indicating a putative differentiation route of monocytes into ECs. Herein, we disclosed monocytes as potential EPCs, using in vitro and in vivo models, and also addressed the cancer context. Monocytes acquired the capacity to express ECs markers and were able to be incorporated into blood vessels, contributing to cancer progression, by being incorporated in tumor neo-vasculature. Reactive oxygen species (ROS) push monocytes to EC differentiation, and this phenotype is reverted by cysteine (a scavenger and precursor of glutathione), which indicates that angiogenesis is controlled by the interplay between the oxidative stress and the scavenging capacity of the tumor microenvironment.

A Metabolomics-Inspired Strategy for the Identification of Protein Covalent Modifications.

Identification of protein covalent modifications (adducts) is a challenging task mainly due to the lack of data processing approaches for adductomics studies. Despite the huge technological advances in mass spectrometry (MS) instrumentation and bioinformatics tools for proteomics studies, these methodologies have very limited success on the identification of low abundant protein adducts. Herein we report a novel strategy inspired on the metabolomics workflows for the identification of covalently-modified peptides that consists on LC-MS data preprocessing followed by statistical analysis. The usefulness of this strategy was evaluated using experimental LC-MS data of histones isolated from HepG2 and THLE2 cells exposed to the chemical carcinogen glycidamide. LC-MS data was preprocessed using the open-source software MZmine and potential adducts were selected based on the m/z increments corresponding to glycidamide incorporation. Then, statistical analysis was applied to reveal the potential adducts as those ions are differently present in cells exposed and not exposed to glycidamide. The results were compared with the ones obtained upon the standard proteomics methodology, which relies on producing comprehensive MS/MS data by data dependent acquisition and analysis with proteomics data search engines. Our novel strategy was able to differentiate HepG2 and THLE2 and to identify adducts that were not detected by the standard methodology of adductomics. Thus, this metabolomics driven approach in adductomics will not only open new opportunities for the identification of protein epigenetic modifications, but also adducts formed by endogenous and exogenous exposure to chemical agents.

Cysteine allows ovarian cancer cells to adapt to hypoxia and to escape from carboplatin cytotoxicity.

Ovarian cancer is the second most common gynaecologic malignancy and the main cause of death from gynaecologic cancer, due to late diagnosis and chemoresistance. Studies have reported the role of cysteine in cancer, by contributing for hydrogen sulphide (H2S) generation and as a precursor of glutathione (GSH). However, the role of cysteine in the adaptation to hypoxia and therapy response remains unclear. We used several ovarian cancer cell lines, ES2, OVCAR3, OVCAR8, A2780 and A2780cisR, to clarify cysteine relevance in ovarian cancer cells survival upon hypoxia and carboplatin. Results show that ES2 and OVCAR8 cells presented a stronger dependence on cysteine availability upon hypoxia and carboplatin exposure than OVCAR3 cells. Interestingly, the A2780 cisR, but not A2780 parental cells, benefits from cysteine upon carboplatin exposure, showing that cysteine is crucial for chemoresistance. Moreover, GSH degradation and subsequent cysteine recycling pathway is associated with ovarian cancer as seen in peripheral blood serum from patients. Higher levels of total free cysteine (Cys) and homocysteine (HCys) were found in ovarian cancer patients in comparison with benign tumours and lower levels of GSH were found in ovarian neoplasms patients in comparison with healthy individuals. Importantly, the total and S-Homocysteinylated levels distinguished blood donors from patients with neoplasms as well as patients with benign from patients with malignant tumours. The levels of S-cysteinylated proteins distinguish blood donors from patients with neoplasms and the free levels of Cys in serum distinguish blood from patients with benign tumours from patients with malignant tumours. Herein we disclosed that cysteine contributes for a worse disease prognosis, allowing faster adaptation to hypoxia and protecting cells from carboplatin. The measurement of serum cysteine levels can be an effective tool for early diagnosis, for outcome prediction and follow up of disease progression.

Monocarboxylate transporter 1 (MCT1), a tool to stratify acute myeloid leukemia (AML) patients and a vehicle to kill cancer cells.

Dysregulation of glucose/lactate dynamics plays a role in cancer progression, and MCTs are key elements in metabolic remodeling. VEGF is a relevant growth factor in the maintenance of bone marrow microenvironment and it is also important in hematological diseases. Our aim was to investigate the role of VEGF in the metabolic adaptation of Acute myeloid leukemia (AML) cells by evaluating the metabolic profiles and cell features according to the AML lineage and testing lactate as a metabolic coin. Our in vitro results showed that AML promyelocytic (HL60) and monocytic (THP1) (but not erythroid- HEL) lineages are well adapted to VEGF and lactate rich environment. Their metabolic adaptation relies on high rates of glycolysis to generate intermediates for PPP to support cell proliferation, and on the consumption of glycolysis-generated lactate to supply biomass and energy production. VEGF orchestrates this metabolic network by regulating MCT1 expression. Bromopyruvic acid (BPA) was proven to be an effective cytotoxic in AML, possibly transported by MCT1. Our study reinforces that targeting metabolism can be a good strategy to fight cancer. MCT1 expression at the time of diagnosis can assist on the identification of AML patients that will benefit from BPA therapy. Additionally, MCT1 can be used in targeted delivery of conventional cytotoxic drugs.