Metabolic plasticity and glucose transporter redundancy dictate lung adenocarcinoma growth

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.

Combined deletion of Glut1 and Glut3 impairs lung adenocarcinoma growth

Pierre-Benoit Bernard Ancey, Caroline Anne-Lyse Contat, Stéphane Laurent Escrig, Rolf Gruetter, Louise Helene Soegaard Jensen, Graham Knott, Bernard Lanz, Mario Gaetano Lepore, Anders Meibom, Etienne Meylan, Justine Pascual, Anouk Rossier, Silvia Sabatino, Nadine Zangger

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.

2020

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

GLUT1 and GLUT3 in non-small cell lung cancer

Mark Jonathan Masin

While altered metabolism is a well-established tumor cell trait, the specifics of metabolic rewiring during cancer development and progression are yet to be fully understood. The Warburg effect, or aerobic glycolysis, has been described in a wide array of malignancies and has long been considered as an inefficient mean to fill energetic requirements. Emerging evidence however supports a role that extends beyond that, with glycolytic intermediates diverting to anabolic pathways to allow growth and proliferation. Importantly, the first and most critical rate-limiting step in glycolysis is glucose uptake. Fittingly, high affinity, high capacity glucose transporters such as GLUT1 and to a lesser extent GLUT3, are upregulated in cancer. Given the centrality of glycolysis in tumor biology and reported clinical observations relating to glucose transporter expression patterns, the present work focuses on characterizing the role of GLUT1 and GLUT3 in the context of non-small cell lung cancer (NSCLC), the most prevalent histological subtype of lung cancer. Linking glucose metabolism and a program involved in early steps of metastasis, part of the findings uncovers an aberrant expression of GLUT3 as integral to the epithelial-mesenchymal transition (EMT) in NSCLC. Indeed, a unique association between GLUT3 and a mesenchymal status is observed in a panel of human NSCLC cell lines. Furthermore, GLUT3 expression is increased during EMT by a mechanism that involves direct binding of EMT mediating transcription factor ZEB1 to the GLUT3 (SLC2A3) gene. Data also supports a functional role of GLUT3 in proliferation, as inhibiting GLUT3 expression reduces glucose import and proliferation of mesenchymal lung tumor cells, whereas ectopic expression in epithelial cells sustains proliferation in low glucose. In an analysis of a large microarray data collection of human NSCLC samples, GLUT3 expression is found to correlate with EMT markers, and is prognostic of poor overall survival. Taken collectively, the data reveal an important role for GLUT3 in lung cancer, when tumor cells loose their epithelial characteristics to become more invasive. Owing to the restricted expression of this transporter in healthy individuals, its presence in lung tumor cells may represent a noteworthy prospect for targeted therapy. The contrastingly ubiquitous nature of GLUT1 in normal and transformed tissue warrants in vivo investigation to determine expression patterns and contribution tumor development. To that end, generation of autochthonous lung cancer mouse models allows assessment of the effect of tumor exclusive GLUT1 loss. Ensuing preliminary results allude to a possible role of GLUT1 in specific histological lesions. The significance of this specificity is currently under investigation.