Targeting the Lactate Transporter MCT1 in Endothelial Cells Inhibits Lactate-Induced HIF-1 Activation and Tumor Angiogenesis

Switching to a glycolytic metabolism is a rapid adaptation of tumor cells to hypoxia. Although this metabolic conversion may primarily represent a rescue pathway to meet the bioenergetic and biosynthetic demands of proliferating tumor cells, it also creates a gradient of lactate that mirrors the gradient of oxygen in tumors. More than a metabolic waste, the lactate anion is known to participate to cancer aggressiveness, in part through activation of the hypoxia-inducible factor-1 (HIF-1) pathway in tumor cells. Whether lactate may also directly favor HIF-1 activation in endothelial cells (ECs) thereby offering a new druggable option to block angiogenesis is however an unanswered question. In this study, we therefore focused on the role in ECs of monocarboxylate transporter 1 (MCT1) that we previously identified to be the main facilitator of lactate uptake in cancer cells. We found that blockade of lactate influx into ECs led to inhibition of HIF-1-dependent angiogenesis. Our demonstration is based on the unprecedented characterization of lactate-induced HIF-1 activation in normoxic ECs and the consecutive increase in vascular endothelial growth factor receptor 2 (VEGFR2) and basic fibroblast growth factor (bFGF) expression. Furthermore, using a variety of functional assays including endothelial cell migration and tubulogenesis together with in vivo imaging of tumor angiogenesis through intravital microscopy and immunohistochemistry, we documented that MCT1 blockers could act as bona fide HIF-1 inhibitors leading to anti-angiogenic effects. Together with the previous demonstration of MCT1 being a key regulator of lactate exchange between tumor cells, the current study identifies MCT1 inhibition as a therapeutic modality combining antimetabolic and anti-angiogenic activities

Modulation of CD4 T Cell Response According to Tumor Cytokine Microenvironment

The advancement of knowledge on tumor biology over the past decades has demonstrated a close link between tumor cells and cells of the immune system. In this context, cytokines have a major role because they act as intermediaries in the communication into the tumor bed. Cytokines play an important role in the homeostasis of innate and adaptive immunity. In particular, they participate in the differentiation of CD4 T lymphocytes. These cells play essential functions in the anti-tumor immune response but can also be corrupted by tumors. The differentiation of naïve CD4 T cells depends on the cytokine environment in which they are activated. Additionally, at the tumor site, their activity can also be modulated according to the cytokines of the tumor microenvironment. Thus, polarized CD4 T lymphocytes can see their phenotype evolve, demonstrating functional plasticity. Knowledge of the impact of these cytokines on the functions of CD4 T cells is currently a source of innovation, for therapeutic purposes. In this review, we discuss the impact of the major cytokines present in tumors on CD4 T cells. In addition, we summarize the main therapeutic strategies that can modulate the CD4 response through their impact on cytokine production

Transcription Factor Binding Studies in CD4+ T Cells: siRNA Transfection, Chromatin Immunoprecipitation, and Liquid Luminescent DNA Precipitation Assay.

ProtocolTh9 CellsVolume 1585 of the series Methods in Molecular Biology pp 167-177International audienceCD4+ T cells are key components of the immune system that shape the anticancer immune response in animal models and in humans among other responses. The biology of CD4+ T cells is complex because naïve CD4+ T cells can differentiate into various subpopulations with various functions that depend on the milieu. Recently, a new population of IL-9-secreting T cells called Th9 cells has been described. These cells are characterized by their ability to produce IL-9. These cells were described to be involved in parasite infections and allergic inflammation. However, some reports described their presence in the tumor bed in mice and humans. Molecular events that account for Th9 effector properties and differentiation are still elusive. To study these mechanisms, downregulation of gene expression and protein-DNA interaction detection allow to identify and study putative transcription factors regulating gene expression during their differentiation. Here, we describe three methods allowing the study of transcription factors in Th9 cells: siRNA transfection for downregulation of gene expression, and chromatin immunoprecipitation and liquid luminescent DNA precipitation assay to detect protein-DNA interactions

A Short Caspase-3 Isoform Inhibits Chemotherapy- Induced Apoptosis by Blocking Apoptosome Assembly

Alternative splicing of caspase-3 produces a short isoform caspase-3s that antagonizes caspase-3 apoptotic activity. However, the mechanism of apoptosis inhibition by caspase-3s remains unknown. Here we show that exogenous caspase-3 sensitizes MCF-7 and HBL100 breast cancers cells to chemotherapeutic treatments such as etoposide and methotrexate whereas co-transfection with caspase-3s strongly inhibits etoposide and methotrexate-induced apoptosis underlying thus the anti-apoptotic role of caspase-3s. In caspase-3 transfected cells, lamin-A and a-fodrin were cleaved when caspase-3 was activated by etoposide or methotrexate. When caspase-3s was co-transfected, this cleavage was strongly reduced. Depletion of caspase-3 by RNA interference in HBL100 containing endogenous caspase-3s caused reduction in etoposide and methotrexate-induced apoptosis, whereas the depletion of caspase-3s sensitized cells to chemotherapy. In the presence of caspase-3s, a lack of interaction between caspase-3 and caspase-9 was observed. Immunoprecipitation assays showed that caspase-3s binds the pro-forms of caspase-3. This result suggested that the absence of interaction with caspase-9 when both variants of caspase-3 are present contribute to block the apoptosome assembly and inhibit apoptosis. These data support that caspases-3s negatively interferes with caspase-3 activation and apoptosis in breast cancer, and tha

Impact of Lipid Metabolism on Antitumor Immune Response

Over the past decade, metabolic reprogramming has been defined as a hallmark of cancer. More recently, a large number of studies have demonstrated that metabolic reprogramming can modulate the differentiation and functions of immune cells, and thus modify the antitumor response. Increasing evidence suggests that modified energy metabolism could be responsible for the failure of antitumor immunity. Indeed, tumor-infiltrating immune cells play a key role in cancer, and metabolic switching in these cells has been shown to help determine their phenotype: tumor suppressive or immune suppressive. Recent studies in the field of immunometabolism focus on metabolic reprogramming in the tumor microenvironment (TME) by targeting innate and adaptive immune cells and their associated anti- or protumor phenotypes. In this review, we discuss the lipid metabolism of immune cells in the TME as well as the effects of lipids; finally, we expose the link between therapies and lipid metabolism

Impact of Lipid Metabolism on Antitumor Immune Response

Over the past decade, metabolic reprogramming has been defined as a hallmark of cancer. More recently, a large number of studies have demonstrated that metabolic reprogramming can modulate the differentiation and functions of immune cells, and thus modify the antitumor response. Increasing evidence suggests that modified energy metabolism could be responsible for the failure of antitumor immunity. Indeed, tumor-infiltrating immune cells play a key role in cancer, and metabolic switching in these cells has been shown to help determine their phenotype: tumor suppressive or immune suppressive. Recent studies in the field of immunometabolism focus on metabolic reprogramming in the tumor microenvironment (TME) by targeting innate and adaptive immune cells and their associated anti- or protumor phenotypes. In this review, we discuss the lipid metabolism of immune cells in the TME as well as the effects of lipids; finally, we expose the link between therapies and lipid metabolism