Targeting Tumor Angiogenesis with the Selective VEGFR-3 Inhibitor EVT801 in Combination with Cancer Immunotherapy.

The receptor tyrosine kinase VEGFR-3 plays a crucial role in cancer-induced angiogenesis and lymphangiogenesis, promoting tumor development and metastasis. Here, we report the novel VEGFR-3 inhibitor EVT801 that presents a more selective and less toxic profile than two major inhibitors of VEGFRs (i.e., sorafenib and pazopanib). As monotherapy, EVT801 showed a potent antitumor effect in VEGFR-3-positive tumors, and in tumors with VEGFR-3-positive microenvironments. EVT801 suppressed VEGF-C-induced human endothelial cell proliferation in vitro and tumor (lymph)angiogenesis in different tumor mouse models. In addition to reduced tumor growth, EVT801 decreased tumor hypoxia, favored sustained tumor blood vessel homogenization (i.e., leaving fewer and overall larger vessels), and reduced important immunosuppressive cytokines (CCL4, CCL5) and myeloid-derived suppressor cells (MDSC) in circulation. Furthermore, in carcinoma mouse models, the combination of EVT801 with immune checkpoint therapy (ICT) yielded superior outcomes to either single treatment. Moreover, tumor growth inhibition was inversely correlated with levels of CCL4, CCL5, and MDSCs after treatment with EVT801, either alone or combined with ICT. Taken together, EVT801 represents a promising anti(lymph)angiogenic drug for improving ICT response rates in patients with VEGFR-3 positive tumors. Significance: The VEGFR-3 inhibitor EVT801 demonstrates superior selectivity and toxicity profile than other VEGFR-3 tyrosine kinase inhibitors. EVT801 showed potent antitumor effects in VEGFR-3-positive tumors, and tumors with VEGFR-3-positive microenvironments through blood vessel homogenization, and reduction of tumor hypoxia and limited immunosuppression. EVT801 increases immune checkpoint inhibitors' antitumor effects.

GLUT1 Expression in Tumor-Associated Neutrophils Promotes Lung Cancer Growth and Resistance to Radiotherapy.

Neutrophils are the most abundant circulating leucocytes and are essential for innate immunity. In cancer, pro- or antitumor properties have been attributed to tumor-associated neutrophils (TAN). Here, focusing on TAN accumulation within lung tumors, we identify GLUT1 as an essential glucose transporter for their tumor supportive behavior. Compared with normal neutrophils, GLUT1 and glucose metabolism increased in TANs from a mouse model of lung adenocarcinoma. To elucidate the impact of glucose uptake on TANs, we used a strategy with two recombinases, dissociating tumor initiation from neutrophil-specific Glut1 deletion. Loss of GLUT1 accelerated neutrophil turnover in tumors and reduced a subset of TANs expressing SiglecF. In the absence of GLUT1 expression by TANs, tumor growth was diminished and the efficacy of radiotherapy was augmented. Our results demonstrate the importance of GLUT1 in TANs, which may affect their pro- versus antitumor behavior. These results also suggest targeting metabolic vulnerabilities to favor antitumor neutrophils. SIGNIFICANCE: Lung tumor support and radiotherapy resistance depend on GLUT1-mediated glucose uptake in tumor-associated neutrophils, indicating that metabolic vulnerabilities should be considered to target both tumor cells as well as innate immune cells. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/9/2345/F1.large.jpg.

Anti-Ly6G binding and trafficking mediate positive neutrophil selection to unleash the anti-tumor efficacy of radiation therapy.

The anti-Ly6G antibody is used to deplete Ly6Gpos neutrophils and study their role in diverse pathologies. However, depletion is never absolute, as Ly6Glow neutrophils resistant to depletion rapidly emerge. Studying the functionality of these residual neutrophils is necessary to interpret anti-Ly6G-based experimental designs. In vitro, we found anti-Ly6G binding induced Ly6G internalization, surface Ly6G paucity, and primed the oxidative burst of neutrophils upon TNF α co-stimulation. In vivo, we found neutrophils resistant to anti-Ly6G depletion exhibited anti-neutrophil-cytoplasmic-antibodies. In the pre-clinical KrasLox-STOP-Lox-G12D/WT; Trp53Flox/Flox mouse lung tumor model, abnormal neutrophil accumulation and aging was accompanied with an N2-like SiglecFpos polarization and ly6g downregulation. Consequently, SiglecFpos neutrophils exposed to anti-Ly6G reverted to Ly6Glow and were resistant to depletion. Noting that anti-Ly6G mediated neutrophil depletion alone had no anti-tumor effect, we found a long-lasting rate of tumor regression (50%) by combining anti-Ly6G with radiation-therapy, in this model reputed to be refractory to standard anticancer therapies. Mechanistically, anti-Ly6G regulated neutrophil aging while radiation-therapy enhanced the homing of anti-Ly6G-boundSiglecFneg neutrophils to tumors. This anti-tumor effect was recapitulated by G-CSF administration prior to RT and abrogated with an anti-TNFα antibody co-administration. In summary, we report that incomplete depletion of neutrophils using targeted antibodies can intrinsically promote their oxidative activity. This effect depends on antigen/antibody trafficking and can be harnessed locally using select delivery of radiation-therapy to impair tumor progression. This underutilized aspect of immune physiology may be adapted to expand the scope of neutrophil-related research.

Combined deletion of Glut1 and Glut3 impairs lung adenocarcinoma growth.

Glucose utilization increases in tumors, a metabolic process that is observed clinically by 18F-fluorodeoxyglucose positron emission tomography (18F-FDG-PET). However, is increased glucose uptake important for tumor cells, and which transporters are implicated in vivo? In a genetically-engineered mouse model of lung adenocarcinoma, we show that the deletion of only one highly expressed glucose transporter, Glut1 or Glut3, in cancer cells does not impair tumor growth, whereas their combined loss diminishes tumor development. 18F-FDG-PET analyses of tumors demonstrate that Glut1 and Glut3 loss decreases glucose uptake, which is mainly dependent on Glut1. Using 13C-glucose tracing with correlated nanoscale secondary ion mass spectrometry (NanoSIMS) and electron microscopy, we also report the presence of lamellar body-like organelles in tumor cells accumulating glucose-derived biomass, depending partially on Glut1. Our results demonstrate the requirement for two glucose transporters in lung adenocarcinoma, the dual blockade of which could reach therapeutic responses not achieved by individual targeting.

Durable and controlled depletion of neutrophils in mice.

Neutrophils are an essential part of the innate immune system. To study their importance, experimental studies often aim to deplete these cells, generally by injecting anti-Ly6G or anti-Gr1 antibodies. However, these approaches are only partially effective, transient or lack specificity. Here we report that neutrophils remaining after anti-Ly6G treatment are newly derived from the bone marrow, instead of depletion escapees. Mechanistically, newly generated, circulating neutrophils have lower Ly6G membrane expression, and consequently reduced targets for anti-Ly6G-mediated depletion. To overcome this limitation, we develop a double antibody-based depletion strategy that enhances neutrophil elimination by anti-Ly6G treatment. This approach achieves specific, durable and controlled reduction of neutrophils in vivo, and may be suitable for studying neutrophil function in experimental models.

Glucose transporters in cancer - from tumor cells to the tumor microenvironment.

Solute carriers of the glucose transporter (GLUT) family mediate the first step for cellular glucose usage. The upregulation of GLUTs has been reported in numerous cancer types as a result of perturbation of gene expression or protein relocalization or stabilization. Because they enable to sustain the energy demand required by tumor cells for various biochemical programs, they are promising targets for the development of anticancer strategies. Recently, important biological insights have come from the fine crystal structure determination of several GLUTs; these advances will likely catalyze the development of new selective inhibitory compounds. Furthermore, deregulated glucose metabolism of nontumor cells in the tumor mass is beginning to be appreciated and could have major implications for our understanding of how glucose uptake by specific cell types influences the behavior of neighboring cells in the same microenvironment. In this review, we discuss some of the deregulation mechanisms of glucose transporters, their genetic and pharmacological targeting in cancer, and new functions they may have in nontumor cells of the tumor environment or beyond glucose uptake for glycolysis.

Lowly methylated region analysis identifies EBF1 as a potential epigenetic modifier in breast cancer.

Breast cancer (BC) encompasses heterogeneous pathologies with different subtypes exhibiting distinct molecular changes, including those related to DNA methylation. However, the role of these changes in mediating BC heterogeneity is poorly understood. Lowly methylated regions (LMRs), non-CpG island loci that usually contain transcription factor (TF) binding sites, have been suggested to act as regulatory elements that define cellular identity. In this study, we aimed to identify the key subtype-specific TFs that may lead to LMR generation and shape the BC methylome and transcription program. We initially used whole-genome bisulfite sequencing (WGBS) data available at The Cancer Genome Atlas (TCGA) portal to identify subtype-specific LMRs. Differentially methylated regions (DMRs) within the BC PAM50 subtype-specific LMRs were selected by comparing tumors and normal tissues in a larger TCGA cohort assessed by HumanMethylation450 BeadChip (450K) arrays and TF enrichment analyses were performed. To assess the impact of LMRs on gene expression, TCGA RNA sequencing data were downloaded and Pearson correlations between methylation levels of loci presenting subtype-specific TF motifs and expression of the nearest genes were calculated. WGBS methylome data revealed a large number of LMRs for each of the BC subtypes. Analysis of these LMRs in the 450K datasets available for a larger sample set identified 7,765, 5,657, and 19 differentially methylated positions (DMPs) between normal adjacent tissues and tumor tissues from basal, luminal, and HER2-enriched subtypes, respectively. Unsupervised clustering showed that the discriminatory power of the top DMPs was remarkably strong for basal BC. Interestingly, in this particular subtype, we found 4,409 differentially hypomethylated positions grouped into 1,185 DMRs with a strong enrichment for the early B-cell factor 1 (EBF1) motifs. The methylation levels of the DMRs containing EBF1 motifs showed a strong negative correlation with the expression of 719 nearby genes, including BTS2 and CD74, two oncogenes known to be specific for basal BC subtype and for poor outcome. This study identifies LMRs specific to the three main BC subtypes and reveals EBF1 as a potentially important regulator of BC subtype-specific methylation and gene expression program.

Mutant CTNNB1 and histological heterogeneity define metabolic subtypes of hepatoblastoma.

Hepatoblastoma is the most common malignant pediatric liver cancer. Histological evaluation of tumor biopsies is used to distinguish among the different subtypes of hepatoblastoma, with fetal and embryonal representing the two main epithelial components. With frequent CTNNB1 mutations, hepatoblastoma is a Wnt/ß-catenin-driven malignancy. Considering that Wnt activation has been associated with tumor metabolic reprogramming, we characterized the metabolic profile of cells from hepatoblastoma and compared it to cells from hepatocellular carcinoma. First, we demonstrated that glucose transporter GLUT3 is a direct TCF4/ß-catenin target gene. RNA sequencing enabled to identify molecular and metabolic features specific to hepatoblastoma and revealed that several glycolytic enzymes are overexpressed in embryonal-like compared to fetal-like tumor cells. This led us to implement successfully three biomarkers to distinguish embryonal from fetal components by immunohistochemistry from a large panel of human hepatoblastoma samples. Functional analyses demonstrated that embryonal-like hepatoblastoma cells are highly glycolytic and sensitive to hexokinase-1 silencing. Altogether, our findings reveal a new, metabolic classification of human hepatoblastoma, with potential future implications for patients' diagnosis and treatment.

TET-Catalyzed 5-Hydroxymethylation Precedes HNF4A Promoter Choice during Differentiation of Bipotent Liver Progenitors.

Understanding the processes that govern liver progenitor cell differentiation has important implications for the design of strategies targeting chronic liver diseases, whereby regeneration of liver tissue is critical. Although DNA methylation (5mC) and hydroxymethylation (5hmC) are highly dynamic during early embryonic development, less is known about their roles at later stages of differentiation. Using an in vitro model of hepatocyte differentiation, we show here that 5hmC precedes the expression of promoter 1 (P1)-dependent isoforms of HNF4A, a master transcription factor of hepatocyte identity. 5hmC and HNF4A expression from P1 are dependent on ten-eleven translocation (TET) dioxygenases. In turn, the liver pioneer factor FOXA2 is necessary for TET1 binding to the P1 locus. Both FOXA2 and TETs are required for the 5hmC-related switch in HNF4A expression. The epigenetic event identified here may be a key step for the establishment of the hepatocyte program by HNF4A.

Genomic responses to hepatitis B virus (HBV) infection in primary human hepatocytes.

Viral infections are able to modify the host's cellular programs, with DNA methylation being a biological intermediate in this process. The extent to which viral infections deregulate gene expression and DNA methylation is not fully understood. In the case of Hepatitis B virus (HBV), there is evidence for an interaction between viral proteins and the host DNA methylation machinery. We studied the ability of HBV to modify the host transcriptome and methylome, using naturally infected primary human hepatocytes to better mimic the clinical setting.Gene expression was especially sensitive to culture conditions, independently of HBV infection. However, we identified non-random changes in gene expression and DNA methylation occurring specifically upon HBV infection. There was little correlation between expression and methylation changes, with transcriptome being a more sensitive marker of time-dependent changes induced by HBV. In contrast, a set of differentially methylated sites appeared early and were stable across the time course experiment. Finally, HBV-induced DNA methylation changes were defined by a specific chromatin context characterized by CpG-poor regions outside of gene promoters.These data support the ability of HBV to modulate host cell expression and methylation programs. In addition, it may serve as a reference for studies addressing the genome-wide consequences of HBV infection in human hepatocytes.

Aberrant DNA methylation of imprinted loci in hepatocellular carcinoma and after in vitro exposure to common risk factors.

BACKGROUND: Hepatocellular carcinoma (HCC) is among the most frequent human malignancies and a major cause of cancer-related death worldwide. It is characterized by late detection and fast progression, and it is believed that epigenetic disruption may be one of the molecular mechanisms leading to hepatocarcinogenesis. Previous studies from our group revealed that HCC tumors exhibit specific DNA methylation signatures associated with major risk factors and tumor progression. Imprinted genes are mono-allelically expressed in a parent-of-origin-dependent manner and have been suggested to be more susceptible to deregulation in cancer. To test this notion, we performed a targeted analysis of DNA methylation in known imprinted genes, using HCC samples and in vitro models of carcinogenic exposure. RESULTS: Analysis of HCC DNA methylation in two independent datasets showed that differentially methylated loci are significantly enriched in imprinted genes. Most of the promoters of imprinted genes were found hypomethylated in HCC tumors compared to surrounding tissues, contrasting with the frequent promoter hypermethylation observed in tumors. We next investigated the status of methylation of the imprinting control region (ICR) of different imprinted clusters and found that the 15q11-13 ICR was significantly hypomethylated in tumors relative to their surrounding tissues. In addition, expression of imprinted genes within this cluster was frequently deregulated in a gene-specific manner, suggesting distinct mechanisms of regulation in this region. Finally, primary human hepatocytes and hepatocyte-like HepaRG cells displayed higher methylation variability in certain imprinted loci after natural hepatitis B virus (HBV) infection and after lipid accumulation, respectively. CONCLUSION: The methylation status of a large panel of imprinted genes was found deregulated in HCC, suggesting a major role of this mechanism during hepatocarcinogenesis. In vitro models support the hypothesis of imprinted gene methylation as a potential marker of environmental exposures.

Dynamic imbalance between cancer cell subpopulations induced by transforming growth factor beta (TGF-ß) is associated with a DNA methylome switch.

BACKGROUND: Distinct subpopulations of neoplastic cells within tumors, including hepatocellular carcinoma (HCC), display pronounced ability to initiate new tumors and induce metastasis. Recent evidence suggests that signals from transforming growth factor beta (TGF-ß) may increase the survival of these so called tumor initiating cells leading to poor HCC prognosis. However, how TGF-ß establishes and modifies the key features of these cell subpopulations is not fully understood. RESULTS: In the present report we describe the differential DNA methylome of CD133-negative and CD133-expressing liver cancer cells. Next, we show that TGF-ß is able to increase the proportion of CD133+ cells in liver cancer cell lines in a way that is stable and persistent across cell division. This process is associated with stable genome-wide changes in DNA methylation that persist through cell division. Differential methylation in response to TGF-ß is under-represented at promoter CpG islands and enriched at gene bodies, including a locus in the body of the de novo DNA methyl-transferase DNMT3B gene. Moreover, phenotypic changes induced by TGF-ß, including the induction of CD133, are impaired by siRNA silencing of de novo DNA methyl-transferases. CONCLUSIONS: Our study reveals a self-perpetuating crosstalk between TGF-ß signaling and the DNA methylation machinery, which can be relevant in the establishment of cellular phenotypes. This is the first indication of the ability of TGF-ß to induce genome-wide changes in DNA methylation, resulting in a stable change in the proportion of liver cancer cell subpopulations.

MicroRNA miR-30 family regulates non-attachment growth of breast cancer cells.

BACKGROUND: A subset of breast cancer cells displays increased ability to self-renew and reproduce breast cancer heterogeneity. The characterization of these so-called putative breast tumor-initiating cells (BT-ICs) may open the road for novel therapeutic strategies. As microRNAs (miRNAs) control developmental programs in stem cells, BT-ICs may also rely on specific miRNA profiles for their sustained activity. To explore the notion that miRNAs may have a role in sustaining BT-ICs, we performed a comprehensive profiling of miRNA expression in a model of putative BT-ICs enriched by non-attachment growth conditions. RESULTS: We found breast cancer cells grown under non-attachment conditions display a unique pattern of miRNA expression, highlighted by a marked low expression of miR-30 family members relative to parental cells. We further show that miR-30a regulates non-attachment growth. A target screening revealed that miR-30 family redundantly modulates the expression of apoptosis and proliferation-related genes. At least one of these targets, the anti-apoptotic protein AVEN, was able to partially revert the effect of miR-30a overexpression. Finally, overexpression of miR-30a in vivo was associated with reduced breast tumor progression. CONCLUSIONS: miR30-family regulates the growth of breast cancer cells in non-attachment conditions. This is the first analysis of target prediction in a whole family of microRNAs potentially involved in survival of putative BT-ICs.