Fanny Dupuy

The Energy Sensor AMPK Regulates T Cell Metabolic Adaptation and Effector Responses In Vivo

Naive T cells undergo metabolic reprogramming to support the increased energetic and biosynthetic demands of effector T cell function. However, how nutrient availability influences T cell metabolism and function remains poorly understood. Here we report plasticity in effector T cell metabolism in response to changing nutrient availability. Activated T cells were found to possess a glucose-sensitive metabolic checkpoint controlled by the energy sensor AMP-activated protein kinase (AMPK) that regulated mRNA translation and glutamine-dependent mitochondrial metabolism to maintain T cell bioenergetics and viability. T cells lacking AMPKα1 displayed reduced mitochondrial bioenergetics and cellular ATP in response to glucose limitation in vitro or pathogenic challenge in vivo. Finally, we demonstrated that AMPKα1 is essential for T helper 1 (Th1) and Th17 cell development and primary T cell responses to viral and bacterial infections in vivo. Our data highlight AMPK-dependent regulation of metabolic homeostasis as a key regulator of T cell-mediated adaptive immunity.

PDK1-Dependent Metabolic Reprogramming Dictates Metastatic Potential in Breast Cancer

Metabolic reprogramming is a hallmark of cellular transformation, yet little is known about metabolic changes that accompany tumor metastasis. Here we show that primary breast cancer cells display extensive metabolic heterogeneity and engage distinct metabolic programs depending on their site of metastasis. Liver-metastatic breast cancer cells exhibit a unique metabolic program compared to bone- or lung-metastatic cells, characterized by increased conversion of glucose-derived pyruvate into lactate and a concomitant reduction in mitochondrial metabolism. Liver-metastatic cells displayed increased HIF-1α activity and expression of the HIF-1α target Pyruvate dehydrogenase kinase-1 (PDK1). Silencing HIF-1α reversed the glycolytic phenotype of liver-metastatic cells, while PDK1 was specifically required for metabolic adaptation to nutrient limitation and hypoxia. Finally, we demonstrate that PDK1 is required for efficient liver metastasis, and its expression is elevated in liver metastases from breast cancer patients. Our data implicate PDK1 as a key regulator of metabolism and metastatic potential in breast cancer.

The tumor suppressor folliculin regulates AMPK-dependent metabolic transformation

The Warburg effect is a tumorigenic metabolic adaptation process characterized by augmented aerobic glycolysis, which enhances cellular bioenergetics. In normal cells, energy homeostasis is controlled by AMPK; however, its role in cancer is not understood, as both AMPK-dependent tumor-promoting and -inhibiting functions were reported. Upon stress, energy levels are maintained by increased mitochondrial biogenesis and glycolysis, controlled by transcriptional coactivator PGC-1α and HIF, respectively. In normoxia, AMPK induces PGC-1α, but how HIF is activated is unclear. Germline mutations in the gene encoding the tumor suppressor folliculin (FLCN) lead to Birt-Hogg-Dubé (BHD) syndrome, which is associated with an increased cancer risk. FLCN was identified as an AMPK binding partner, and we evaluated its role with respect to AMPK-dependent energy functions. We revealed that loss of FLCN constitutively activates AMPK, resulting in PGC-1α-mediated mitochondrial biogenesis and increased ROS production. ROS induced HIF transcriptional activity and drove Warburg metabolic reprogramming, coupling AMPK-dependent mitochondrial biogenesis to HIF-dependent metabolic changes. This reprogramming stimulated cellular bioenergetics and conferred a HIF-dependent tumorigenic advantage in FLCN-negative cancer cells. Moreover, this pathway is conserved in a BHD-derived tumor. These results indicate that FLCN inhibits tumorigenesis by preventing AMPK-dependent HIF activation and the subsequent Warburg metabolic transformation.

Claudin-2 Promotes Breast Cancer Liver Metastasis by Facilitating Tumor Cell Interactions with Hepatocytes

ABSTRACT We previously identified claudin-2 as a functional mediator of breast cancer liver metastasis. We now confirm that claudin-2 levels are elevated in liver metastases, but not in skin metastases, compared to levels in their matched primary tumors in patients with breast cancer. Moreover, claudin-2 is specifically expressed in liver-metastatic breast cancer cells compared to populations derived from bone or lung metastases. The increased liver tropism exhibited by claudin-2-expressing breast cancer cells requires claudin-2-mediated interactions between breast cancer cells and primary hepatocytes. Furthermore, the reduction of the claudin-2 expression level, either in cancer cells or in primary hepatocytes, diminishes these heterotypic cell-cell interactions. Finally, we demonstrate that the first claudin-2 extracellular loop is essential for mediating tumor cell-hepatocyte interactions and the ability of breast cancer cells to form liver metastases in vivo. Thus, during breast cancer liver metastasis, claudin-2 shifts from acting within tight-junctional complexes to functioning as an adhesion molecule between breast cancer cells and hepatocytes.

Folliculin Regulates Ampk-Dependent Autophagy and Metabolic Stress Survival

Dysregulation of AMPK signaling has been implicated in many human diseases, which emphasizes the importance of characterizing AMPK regulators. The tumor suppressor FLCN, responsible for the Birt-Hogg Dubé renal neoplasia syndrome (BHD), is an AMPK-binding partner but the genetic and functional links between FLCN and AMPK have not been established. Strikingly, the majority of naturally occurring FLCN mutations predisposing to BHD are predicted to produce truncated proteins unable to bind AMPK, pointing to the critical role of this interaction in the tumor suppression mechanism. Here, we demonstrate that FLCN is an evolutionarily conserved negative regulator of AMPK. Using Caenorhabditis elegans and mammalian cells, we show that loss of FLCN results in constitutive activation of AMPK which induces autophagy, inhibits apoptosis, improves cellular bioenergetics, and confers resistance to energy-depleting stresses including oxidative stress, heat, anoxia, and serum deprivation. We further show that AMPK activation conferred by FLCN loss is independent of the cellular energy state suggesting that FLCN controls the AMPK energy sensing ability. Together, our data suggest that FLCN is an evolutionarily conserved regulator of AMPK signaling that may act as a tumor suppressor by negatively regulating AMPK function.

LKB1 is a central regulator of tumor initiation and pro-growth metabolism in ErbB2-mediated breast cancer

BackgroundGermline and somatic mutations in STK11, the gene encoding the serine/threonine kinase LKB1, are strongly associated with tumorigenesis. While loss of LKB1 expression has been linked to breast cancer, the mechanistic role of LKB1 in regulating breast cancer development, metastasis, and tumor metabolism has remained unclear.MethodsWe have generated and analyzed transgenic mice expressing ErbB2 in the mammary epithelium of LKB1 wild-type or LKB1-deficient mice. We have also utilized ErbB2-expressing breast cancer cells in which LKB1 levels have been reduced using shRNA approaches. These transgenic and xenograft models were characterized for the effects of LKB1 loss on tumor initiation, growth, metastasis and tumor cell metabolism.ResultsWe demonstrate that loss of LKB1 promotes tumor initiation and induces a characteristic shift to aerobic glycolysis (‘Warburg effect’) in a model of ErbB2-mediated breast cancer. LKB1-deficient breast cancer cells display enhanced early tumor growth coupled with increased cell migratory and invasive properties in vitro. We show that ErbB2-positive tumors deficient for LKB1 display a pro-growth molecular and phenotypic signature characterized by elevated Akt/mTOR signaling, increased glycolytic metabolism, as well as increased bioenergetic markers both in vitro and in vivo. We also demonstrate that mTOR contributes to the metabolic reprogramming of LKB1-deficient breast cancer, and is required to drive glycolytic metabolism in these tumors; however, LKB1-deficient breast cancer cells display reduced metabolic flexibility and increased apoptosis in response to metabolic perturbations.ConclusionsTogether, our data suggest that LKB1 functions as a tumor suppressor in breast cancer. Loss of LKB1 collaborates with activated ErbB2 signaling to drive breast tumorigenesis and pro-growth metabolism in the resulting tumors.

Metabolic Plasticity as a Determinant of Tumor Growth and Metastasis.

Cancer cells must adapt their metabolism to meet the energetic and biosynthetic demands that accompany rapid growth of the primary tumor and colonization of distinct metastatic sites. Different stages of the metastatic cascade can also present distinct metabolic challenges to disseminating cancer cells. However, little is known regarding how changes in cellular metabolism, both within the cancer cell and the metastatic microenvironment, alter the ability of tumor cells to colonize and grow in distinct secondary sites. This review examines the concept of metabolic heterogeneity within the primary tumor, and how cancer cells are metabolically coupled with other cancer cells that comprise the tumor and cells within the tumor stroma. We examine how metabolic strategies, which are engaged by cancer cells in the primary site, change during the metastatic process. Finally, we discuss the metabolic adaptations that occur as cancer cells colonize foreign metastatic microenvironments and how cancer cells influence the metabolism of stromal cells at sites of metastasis. Through a discussion of these topics, it is clear that plasticity in tumor metabolic programs, which allows cancer cells to adapt and grow in hostile microenvironments, is emerging as an important variable that may change clinical approaches to managing metastatic disease. Cancer Res; 76(18); 5201-8. ©2016 AACR.

Abstract 3367: Understanding the role of metabolic reprogramming in breast cancer progression and metastasis

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: The emergence of metastatic breast cancer is the most deadly aspect of the disease and once it has spread from the primary site, it is largely incurable. Important metabolic changes have been correlated with breast cancer progression and acquisition of the metastatic phenotype. One prominent example includes the well-established Warburg effect, which stipulates that cancer cells preferentially utilize rapid glycolytic metabolism over mitochondrial respiration to produce energy. Such a metabolic shift ensures that cancer cells can meet the bioenergetics and biosynthetic demands associated with increased proliferation. Objectives: The energetic requirements of individual cancer cells will greatly vary as the tumor grows and acquires malignant characteristics. The metabolic demands during tumor initiation will differ from those that occur during tumor growth at the primary site and dissemination to distant metastatic sites. Therefore, we hypothesize that distinct metabolic signatures are associated with each one of these steps and that a better characterization of these transitions, as well as the key regulators governing this process, will help identifying potential therapeutic targets. Results: Using a series of mouse mammary cancer cells derived from a spontaneous mammary tumor, which includes 67NR (tumorigenic/non-metastatic), 66cl4 (tumorigenic/lung metastatic) and 4T1 (tumorigenic/metastatic to multiple sites), we examined changes in several parameters of cellular metabolism that could be associated with different stages of tumor progression. Using mass spectrometry, we assessed the uptake and metabolic flux of labeled glucose through the cellular metabolic pathways. Our results suggest that there is a further shift towards a “Warburg-like” phenotype as tumor cells acquire aggressive characteristics. Using an in vivo selection approach on the 4T1 breast cancer cells, we have established subpopulations that aggressively form liver metastases. Glucose tracing experiments reveal an accumulation of lactate at the expense of reduced allocation of glucose towards citrate production suggesting that these cells have a further increased glycolytic phenotypes compared to the 4T1 parental cells. These results raise the possibility that unique metabolic processes and checkpoints are engaged in metastatic populations that contribute to their metastatic ability. PDK1 is an enzyme that prevents the uptake of pyruvate into the TCA cycle. We show that PDK1 is upregulated with the increase in metastatic potential. This has led us to hypothesize that the “Warburg-like” phenotype observed is mediated by a PDK1-induced switch favouring glycolysis. We demonstrate that PDK1 is required to form liver metastases in vivo following splenic injections. Ongoing experiments are being pursued to uncover the mechanism by which PDK1 is regulated and how it influences the metastatic process. Citation Format: Fanny Dupuy, Julianna Blagih, Sebastien Tabaries, Julie St-Pierre, Russell G. Jones, Peter M. Siegel. Understanding the role of metabolic reprogramming in breast cancer progression and metastasis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3367. doi:10.1158/1538-7445.AM2014-3367