Combined deletion of Glut1 and Glut3 impairs lung adenocarcinoma growth

Glucose utilization increases in tumors, a metabolic process that is observed clinically by F-18-fluorodeoxyglucose positron emission tomography (F-18-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. F-18-FDG-PET analyses of tumors demonstrate that Glut1 and Glut3 loss decreases glucose uptake, which is mainly dependent on Glut1. Using C-13-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.

Metabolic plasticity and glucose transporter redundancy dictate lung adenocarcinoma growth

Caroline Anne-Lyse Contat

Lung cancers represent the leading cause of cancer-related deaths worldwide. These pulmonary cancers count several histological subgroups, whose non-small cell lung cancers, subject of our study. We were precisely interested in the most common subtype of non-small cell lung cancers, the adenocarcinomas, that are frequently mutated for the oncogene Kras and the tumor suppressors Tp53 and Lkb1. These tumors seeking to ensure their survival and their uncontrolled proliferation, especially modify their metabolism to sustain their increased and essential nutrient requirements. These cancer cells thus overexpress glucose transporters to augment their sugar uptake and maintain their functions. Although the high expression of certain transporters in pulmonary adenocarcinomas is associated with a poor vital prognostic, their implication in tumorigenesis remains poorly understood. Also, we focused on the two most expressed transporters in lung adenocarcinomas, GLUT1 and GLUT3, to determine and understand their putative role in the tumor cell-of-origin, but also in the initiation and growth of adenocarcinomas. To investigate these questions in vivo, we used genetically engineered mouse models, based on the frequent genetic alterations Kras, Tp53 and Lkb1. These models closely mimic human non-small cell lung cancer development. The intratracheal instillation of these mice with viral particles expressing Cre-recombinase allows the genetic recombinations and the subsequent initiation of pulmonary tumors. Being interesting in glucose transporters, we interbred the murine models KrasLSL-G12D/WT (K), KrasLSL-G12D/WT; Tp53lox/lox (KP), and KrasLSL-G12D/WT; Lkb1lox/lox (KL) with the models Slc2a1lox/lox (G1) and/or Slc2a3lox/lox (G3). Thus, we could specifically initiate the development of tumors expressing the oncogenic mutation KrasG12D, the deletion or not of Tp53 or Lkb1, and with or without glucose transporter Glut1 and/or Glut3. Our in vivo study then revealed that the only deletion of Glut1 in tumor cells was insufficient to impair their growth, and this, independently of the cell-of-origin or the stage of the lesions. Using electron microscopy and 13C-glucose tracing with correlated nanoscale secondary ion mass spectrometry (NanoSIMS), we demonstrated that the glucose-derived biomass accumulated into lamellar body-like organelles of the tumor cells. Moreover, this accumulation partially depends on Glut1. Ex vivo analyses showed besides that glycolysis in cancer cells was diminished in Glut1 absence, except when Glut3 was highly expressed. Similarly to Glut1, Glut3 expression suppression in cancer cells affects neither the initiation, nor the tumor expansion. In contrast, the combined deletion of Glut1 and Glut3 significantly decreases non-small cell lung cancer growth. Our results demonstrate the requirement to target both glucose transporters to impair pulmonary adenocarcinomas tumorigenesis.

EPFL2020

The role of Snail in non-small cell lung cancer

Svenja Johanna Groeneveld

The epithelial-to-mesenchymal transition (EMT) is a developmental program frequently reactivated in cancer. It plays an important role in several aspects of tumor progression, particularly in the acquisition of invasive capacities facilitating metastasis. Beyond invasion, EMT endows cancer cells with additional characteristics essential for metastatic dissemination and has been extensively studied in breast and colorectal cancer. Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related deaths worldwide and the majority of NSCLC patients is diagnosed with metastatic disease. The expression of the key EMT transcription factor Snail is correlated with a poor prognosis in NSCLC patients, while it is unclear whether Snail contributes to disease progression by actually facilitating metastasis formation. During EMT, the metabolism of a cancer cell changes, likely to meet the environmental challenges faced during the metastatic process. In this line, we have previously reported (Masin et al, 2014) that the glucose transporter GLUT3 is upregulated during EMT in human NSCLC cell lines. Here, we demonstrate that the rate-limiting enzyme of the hexosamine biosynthesis pathway (HBP) correlates with GLUT3 in NSCLC. This pathway produces a substrate for O-linked protein GlcNAcylations important during EMT, as they â for example â stabilize the Snail protein. Glutamine-fructose-6-phosphate amidotransferase 2 (GFPT2) was specifically upregulated during EMT, while its isoenzyme GFPT1 was unaffected. Furthermore, our results points towards a putative regulation of GLUT3 expression by GFPT2, possibly involving STAT3 signaling. We thus provide evidence for a possible reactivation of a developmental metabolic program during EMT in NSCLC. Chronic inflammation is an important disease-promoting factor during lung tumorigenesis, emphasizing the role of the immune system in this cancer type. Overexpressing or silencing Snail in the immunocompetent autochthonous KrasLSL-G12D/+;p53fl/fl mouse model of lung adenocarcinoma uncovered a substantial influence of Snail on the tumor microenvironment. An extensive analysis of tumor histology, gene and protein expression and immune infiltration revealed that while Snail did not contribute to metastasis formation, it accelerated growth and malignant progression of the lung tumors, reducing the survival time of the mice. Snail decreased B lymphocyte, while enhancing neutrophil infiltration of the tumors. Intriguingly, through the secretion of a soluble factor, Snail induced a feed-forward loop of neutrophil recruitment via Cxcl2, produced by neutrophils themselves. In our recently published collaborative work (Faget, Groeneveld et al, 2017), we identified neutrophils as main contributors to disease progression. We furthermore provided evidence for a vicious cycle formed between Snail expressing cancer cells and neutrophils in lung tumors, as neutrophils were found to impair angiogenesis, resulting in hypoxia and enhanced Snail expression. In addition, we found that Snail repressed the Dlk1-Dio3 locus, containing the genomeâs largest miRNA cluster and recently implicated in NSCLC, in tumor-infiltrating immune cells via a paracrine mechanism.

EPFL2018

Combinatorial targeting of angiogenesis with inducers of autophagy in the treatment of glioblastoma multiforme

Julie Laetitia Scotton

Malignant gliomas represent 80% of tumors developing in the central nervous system, with 50% being glioblastomas (GBM), the most aggressive form of the disease. The benefit from current therapies is very limited, the overall 5-year survival rate of patients with GBM being less than 5%. Thus, there is an urgent need for better therapies. It has been reported that the use of tricyclic antidepressants (TCAs) is linked with reduced incidence of gliomas. In line with these observations, our lab previously demonstrated that combining imipramine (IM), a TCA that activates adenylate cyclase and elevates production of cAMP, with ticlopidine (TIC), an inhibitor of the purinergic receptor P2Y12 that otherwise suppresses cAMP, results in a concerted increase in the levels of intracellular cAMP, which consequently stimulates increased autophagy in glioma cells, leading to autophagy-associated cell death (AACD) in culture and during orthotopic tumor growth. In several mouse models of glioma, the combination improved survival and reduced malignant grade. Seeking to assess the therapeutic translational potential of these repurposed drugs, particularly IM, we performed pre-clinical trials combining IM+/-TIC with anti-VEGF therapy, which is commonly used in patients with GBM; such anti-angiogenic therapy is associated with a transitory improvement in progression-free survival and quality of life, but does not translate into an overall survival benefit. Using genetic mouse models with enabling bioluminescent monitoring of the cancer cells, we treated established GBMs with imipramine, ticlopidine, and B20S, an anti-mouse VEGF monoclonal antibody. We observed transient stabilization of tumor growth, and the overall survival was significantly extended in the regimens combining B20S with IM +/- TIC compared to control and single treatments. Similar to clinical observations in GBM patients treated with anti-angiogenic therapy, mice treated with B20S alone had no survival benefit, implicating a synergetic effect for the combination of anti-VEGF treatment with imipramine and ticlopidine. We found that the combinatorial therapy elicited further increases in autophagy and displayed some indication of blood vessel normalization, concomitant with an elevation of CD8+T lymphocyte infiltration into the orthotopic tumors. Notably, we could partially reverse the beneficial effect of the combinations using a CD8+T cell-depleting antibody, revealing the involvement of the recruited CD8+T cells in the efficacy of the combinatorial treatment, in addition to the cell-intrinsic autophagy-associated death that is demonstrable in culture and in tumors. Together, these results suggest a strategy to improve anti-angiogenic therapy in patients with glioblastoma, by using repurposed FDA-approved drugs that markedly elevate autophagy in concert with anti-VEGF therapy. The recruitment of CD8+ T cells into the treated tumors and their evident contribution to therapeutic efficacy motivates ongoing experiments aimed to heighten and sustain the induced anti-tumor immune response.

EPFL2018

Metabolic plasticity and glucose transporter redundancy dictate lung adenocarcinoma growth

Caroline Anne-Lyse Contat

EPFL2020

Combinatorial targeting of angiogenesis with inducers of autophagy in the treatment of glioblastoma multiforme

Julie Laetitia Scotton

EPFL2018

The role of Snail in non-small cell lung cancer

Svenja Johanna Groeneveld

EPFL2018