Reductive carboxylation supports growth in tumour cells with defective mitochondria.

Mitochondrial metabolism provides precursors to build macromolecules in growing cancer cells. In normally functioning tumour cell mitochondria, oxidative metabolism of glucose- and glutamine-derived carbon produces citrate and acetyl-coenzyme A for lipid synthesis, which is required for tumorigenesis. Yet some tumours harbour mutations in the citric acid cycle (CAC) or electron transport chain (ETC) that disable normal oxidative mitochondrial function, and it is unknown how cells from such tumours generate precursors for macromolecular synthesis. Here we show that tumour cells with defective mitochondria use glutamine-dependent reductive carboxylation rather than oxidative metabolism as the major pathway of citrate formation. This pathway uses mitochondrial and cytosolic isoforms of NADP(+)/NADPH-dependent isocitrate dehydrogenase, and subsequent metabolism of glutamine-derived citrate provides both the acetyl-coenzyme A for lipid synthesis and the four-carbon intermediates needed to produce the remaining CAC metabolites and related macromolecular precursors. This reductive, glutamine-dependent pathway is the dominant mode of metabolism in rapidly growing malignant cells containing mutations in complex I or complex III of the ETC, in patient-derived renal carcinoma cells with mutations in fumarate hydratase, and in cells with normal mitochondria subjected to acute pharmacological ETC inhibition. Our findings reveal the novel induction of a versatile glutamine-dependent pathway that reverses many of the reactions of the canonical CAC, supports tumour cell growth, and explains how cells generate pools of CAC intermediates in the face of impaired mitochondrial metabolism.

Embryonic and tumorigenic pathways converge via Nodal signaling: role in melanoma aggressiveness.

Bidirectional cellular communication is integral to both cancer progression and embryological development. In addition, aggressive tumor cells are phenotypically plastic, sharing many properties with embryonic cells. Owing to the similarities between these two types of cells, the developing zebrafish can be used as a biosensor for tumor-derived signals. Using this system, we show that aggressive melanoma cells secrete Nodal (a potent embryonic morphogen) and consequently can induce ectopic formation of the embryonic axis. We further show that Nodal is present in human metastatic tumors, but not in normal skin, and thus may be involved in melanoma pathogenesis. Inhibition of Nodal signaling reduces melanoma cell invasiveness, colony formation and tumorigenicity. Nodal inhibition also promotes the reversion of melanoma cells toward a melanocytic phenotype. These data suggest that Nodal signaling has a key role in melanoma cell plasticity and tumorigenicity, thereby providing a previously unknown molecular target for regulating tumor progression.

Mitochondrial metabolism and ROS generation are essential for Kras-mediated tumorigenicity.

Otto Warburg's theory on the origins of cancer postulates that tumor cells have defects in mitochondrial oxidative phosphorylation and therefore rely on high levels of aerobic glycolysis as the major source of ATP to fuel cellular proliferation (the Warburg effect). This is in contrast to normal cells, which primarily utilize oxidative phosphorylation for growth and survival. Here we report that the major function of glucose metabolism for Kras-induced anchorage-independent growth, a hallmark of transformed cells, is to support the pentose phosphate pathway. The major function of glycolytic ATP is to support growth under hypoxic conditions. Glutamine conversion into the tricarboxylic acid cycle intermediate alpha-ketoglutarate through glutaminase and alanine aminotransferase is essential for Kras-induced anchorage-independent growth. Mitochondrial metabolism allows for the generation of reactive oxygen species (ROS) which are required for Kras-induced anchorage-independent growth through regulation of the ERK MAPK signaling pathway. We show that the major source of ROS generation required for anchorage-independent growth is the Q(o) site of mitochondrial complex III. Furthermore, disruption of mitochondrial function by loss of the mitochondrial transcription factor A (TFAM) gene reduced tumorigenesis in an oncogenic Kras-driven mouse model of lung cancer. These results demonstrate that mitochondrial metabolism and mitochondrial ROS generation are essential for Kras-induced cell proliferation and tumorigenesis.

Hypoxia. 2. Hypoxia regulates cellular metabolism.

Adaptation to lowering oxygen levels (hypoxia) requires coordinated downregulation of metabolic demand and supply to prevent a mismatch in ATP utilization and production that might culminate in a bioenergetic collapse. Hypoxia diminishes ATP utilization by downregulating protein translation and the activity of the Na-K-ATPase. Hypoxia diminishes ATP production in part by lowering the activity of the electron transport chain through activation of the transcription factor hypoxia-inducible factor-1. The decrease in electron transport limits the overproduction of reactove oxygen species during hypoxia and slows the rate of oxygen depletion to prevent anoxia. In this review, we discuss these mechanisms that diminish metabolic supply and demand for adaptation to hypoxia.

Human embryonic stem cell microenvironment suppresses the tumorigenic phenotype of aggressive cancer cells.

Embryonic stem cells sustain a microenvironment that facilitates a balance of self-renewal and differentiation. Aggressive cancer cells, expressing a multipotent, embryonic cell-like phenotype, engage in a dynamic reciprocity with a microenvironment that promotes plasticity and tumorigenicity. However, the cancer-associated milieu lacks the appropriate regulatory mechanisms to maintain a normal cellular phenotype. Previous work from our laboratory reported that aggressive melanoma and breast carcinoma express the embryonic morphogen Nodal, which is essential for human embryonic stem cell (hESC) pluripotency. Based on the aberrant expression of this embryonic plasticity gene by tumor cells, this current study tested whether these cells could respond to regulatory cues controlling the Nodal signaling pathway, which might be sequestered within the microenvironment of hESCs, resulting in the suppression of the tumorigenic phenotype. Specifically, we discovered that metastatic tumor cells do not express the inhibitor to Nodal, Lefty, allowing them to overexpress this embryonic morphogen in an unregulated manner. However, exposure of the tumor cells to a hESC microenvironment (containing Lefty) leads to a dramatic down-regulation in their Nodal expression concomitant with a reduction in clonogenicity and tumorigenesis accompanied by an increase in apoptosis. Furthermore, this ability to suppress the tumorigenic phenotype is directly associated with the secretion of Lefty, exclusive to hESCs, because it is not detected in other stem cell types, normal cell types, or trophoblasts. The tumor-suppressive effects of the hESC microenvironment, by neutralizing the expression of Nodal in aggressive tumor cells, provide previously unexplored therapeutic modalities for cancer treatment.

Hypoxia/reoxygenation: a dynamic regulator of lysyl oxidase-facilitated breast cancer migration.

Fluctuating oxygen levels characterize the microenvironment of many cancers and tumor hypoxia is associated with increased invasion and metastatic potential concomitant with a poor prognosis. Similarly, the expression of lysyl oxidase (LOX) in breast cancer facilitates tumor cell migration and is associated with estrogen receptor negative status and reduced patient survival. Here we demonstrate that hypoxia/reoxygenation drives poorly invasive breast cancer cells toward a more aggressive phenotype by up-regulating LOX expression and catalytic activity. Specifically, hypoxia markedly increased LOX protein expression; however, catalytic activity (beta-aminopropionitrile inhibitable hydrogen peroxide production) was significantly reduced under hypoxic conditions. Moreover, poorly invasive breast cancer cells displayed a marked increase in LOX-dependent FAK/Src activation and cell migration following hypoxia/reoxygenation, but not in response to hypoxia alone. Furthermore, LOX expression is only partially dependent on hypoxia inducible factor-1 (HIF-1alpha) in poorly invasive breast cancer cells, as hypoxia mimetics and overexpression of HIF-1alpha could not up-regulate LOX expression to the levels observed under hypoxia. Clinically, LOX expression positively correlates with tumor progression and co-localization with hypoxic regions (defined by HIF-1alpha expression) in ductal carcinoma in situ and invasive ductal carcinoma primary tumors. However, positive correlation is lost in metastatic tumors, suggesting that LOX expression is independent of a hypoxic environment at later stages of tumor progression. This work demonstrates that both hypoxia and reoxygenation are necessary for LOX catalytic activity which facilitates breast cancer cell migration through a hydrogen peroxide-mediated mechanism; thereby illuminating a potentially novel mechanism by which poorly invasive cancer cells can obtain metastatic competency.

Epigenetic transdifferentiation of normal melanocytes by a metastatic melanoma microenvironment.

The clinical management of cutaneous melanoma would benefit significantly from a better understanding of the molecular changes that occur during melanocytic progression to a melanoma phenotype. To gain unique insights into this process, we developed a three-dimensional in vitro model that allows observations of normal human melanocytes interacting with a metastatic melanoma matrix to determine whether these normal cells could be reprogrammed by inductive cues in the tumor cell microenvironment. The results show the epigenetic transdifferentiation of the normal melanocytic phenotype to that of an aggressive melanoma-like cell with commensurate increased migratory and invasive ability with no detectable genomic alterations. Removal of the transdifferentiated melanocytes from the inductive metastatic melanoma microenvironment results in a reversion to their normal phenotype. However, a normal melanocyte microenvironment had no epigenetic influence on the phenotype of metastatic melanoma cells. This novel approach identifies specific genes involved in the transdifferentiation of melanocytes to a more aggressive phenotype, which may offer significant therapeutic value.

Metformin inhibits mitochondrial complex I of cancer cells to reduce tumorigenesis.

Recent epidemiological and laboratory-based studies suggest that the anti-diabetic drug metformin prevents cancer progression. How metformin diminishes tumor growth is not fully understood. In this study, we report that in human cancer cells, metformin inhibits mitochondrial complex I (NADH dehydrogenase) activity and cellular respiration. Metformin inhibited cellular proliferation in the presence of glucose, but induced cell death upon glucose deprivation, indicating that cancer cells rely exclusively on glycolysis for survival in the presence of metformin. Metformin also reduced hypoxic activation of hypoxia-inducible factor 1 (HIF-1). All of these effects of metformin were reversed when the metformin-resistant Saccharomyces cerevisiae NADH dehydrogenase NDI1 was overexpressed. In vivo, the administration of metformin to mice inhibited the growth of control human cancer cells but not those expressing NDI1. Thus, we have demonstrated that metformin's inhibitory effects on cancer progression are cancer cell autonomous and depend on its ability to inhibit mitochondrial complex I.DOI: http://dx.doi.org/10.7554/eLife.02242.001.

Mammary serine protease inhibitor (Maspin) binds directly to interferon regulatory factor 6: identification of a novel serpin partnership.

Since its reported discovery in 1994, maspin (mammary serine protease inhibitor) has been characterized as a class II tumor suppressor by its ability to promote apoptosis and inhibit cell invasion. Maspin is highly expressed in normal mammary epithelial cells but reduced or absent in aggressive breast carcinomas. However, despite efforts to characterize the mechanism(s) by which maspin functions as a tumor suppressor, its molecular characterization has remained somewhat elusive. Therefore, in an attempt to identify maspin-interacting proteins and thereby gain insight into the functional pathways of maspin, we employed a maspin-baited yeast two-hybrid system and subsequently identified Interferon Regulatory Factor 6 (IRF6) as a maspin-binding protein. IRF6 belongs to the IRF family of transcription factors, which is best known for its regulation of interferon and interferon-inducible genes following a pathogenic stimulus. Although many of the IRF family members have been well characterized, IRF6 remains poorly understood. We report that IRF6 is expressed in normal mammary epithelial cells and that it directly associates with maspin in a yeast two-hybrid system and in vitro. The interaction occurs via the conserved IRF protein association domain and is regulated by phosphorylation of IRF6. We have shown that, similar to maspin, IRF6 expression is inversely correlated with breast cancer invasiveness. We further demonstrated that the transient re-expression of IRF6 in breast cancer cells results in an increase of N-cadherin and a redistribution of vimentin commensurate with changes in cell morphology, suggestive of an epithelial-to-mesenchymal transition event. Concomitantly, we showed that maspin acts as a negative regulator of this process. These findings help to elucidate the molecular mechanisms of maspin and suggest an interactive role between maspin and IRF6 in regulating cellular phenotype, the loss of which can lead to neoplastic transformation.