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Cancer cells consume large amounts of nutrients, largely glucose, in excess of metabolic needs to maintain aberrant growth. Converting all of glucose to CO2 via oxidative phosphorylation to generate ATP runs counter to the needs of the cell as macromolecules precursors are needed in order to create a new cell, such as glycolytic intermediates for non-essential amino acids synthesis, ribose for nucleic acids, and acetyl-CoA and NADPH for fatty acid synthesis.1 These metabolic features support cancer cells to survive under adverse conditions and serve to promote tumor progression, invasiveness, and metastasis. Therefore, most cancers have developed mechanisms to exploit glucose uptake and flux through the glycolytic pathway. Glucose uptake is largely regulated by the expression of glucose transporters (GLUT), among which GLUT1 (SLC2A1) regulates basal glucose uptake in all cells and is consistently over-expressed in breast cancer cells. In contrast GLUT4 (SLC2A4) is translocated from the cytosol to the plasma membrane upon stimulation by insulin, while GLUT2 (SLC2A2) expression changes in response to glucose concentrations, such as post-fasting conditions.2 Regulation of GLUT expression in cancer cells is largely influences by increasing expression of oncogenes, including PI3K/Akt/GSK3, mTOR, c-Myc, and hypoxia inducible factors-1 (HIF1). HIF1α levels have also been reported to be increased by an mTOR-dependent mechanism. Importantly, c-Myc and HIF1α encode the same genes of glycolysis leading to increased glucose uptake and metabolic reprogramming.3 On the other hand, loss of p53 promotes glucose uptake by regulating GLUT1 and GLUT4 expression. While hypoxia results in a shift toward glycolysis through HIF1α stabilization, it also has other important consequences, including stimulating the production of angiogenic growth factors and cytokines to increase angiogenesis, and hence gain nutrients from the blood supply. Furthermore, hypoxia has been shown to increase exosome secretion in breast cancer cells and additionally modulates the extracellular matrix (ECM) remodeling through the secretion of proteins associated with cell migration and ECM degradation.4 Exosome release by breast cancer cells has also demonstrated a pro-metastatic effect through the release of miR-105, which destroys endothelial gap junctions allowing enhanced intravasation and extravasation of cancer cells.5 Furthermore, circulating miR-105 and miR-122 were elevated in the serum of breast cancer patients with metastatic reoccurrence, which urged us to explore the function of extracellular miR-122.6 With these key concepts in mind, we sought to determine if breast cancer could enhance its access to glucose by minimizing the competition by niche cells. Furthermore we asked if this effect could be induced prior to the arrival of circulating tumor cells in order to create an environment to support survival and hence promote metastatic colonization.7 We demonstrated that miR-122 targets the glycolytic enzyme pyruvate kinase (PKM), resulting in down-regulation of GLUT1 (SLC2A1) and consequently glucose uptake. Furthermore, the secretion of miR-122 by cancer cells has 2 important functions: 1) by eliminating miR-122 from the intracellular compartment, breast cancer cells release the brake on PKM and GLUT1 suppression, gaining them the ability to increase their glucose consumption; and 2) by suppressing PKM and GLUT1 in niche cells, cancer reduces the competition for nutrients. The net result of these dual actions results in the increased ability for circulating tumor cells to colonize a new tissue during a critical time when increased nutrients are needed for increased biomass and proliferation (Fig. 1). To demonstrate these concepts, we used 3 animal models: 1) Extracellular vesicles (EVs) injection followed by circulating tumor cell injection, 2) poorly metastatic xenograft mammary tumors with miR-122 over-expression using MCFDCIS.com cells, and 3) anti-miR-122 intervention using highly metastatic xenograft tumors derived from MDA-MB-231 cells. The first and second models demonstrated that high miR-122 levels resulted in enhanced metastasis to the brain and lungs while concurrently decreasing glucose uptake due to modulations in PKM and GLUT1 expression. Importantly, anti-miR-122 treatment in a metastatic breast cancer model demonstrated a therapeutic effect by reducing metastasis to the brain and lungs. We also established that the suppression of glucose uptake in niche tissues is induced in a pre-metastatic stage, providing evidence that this effect is not induced by the metastases but in preparation for colonization of the pre-metastatic niche. MiR-122 intervention was capable of restoring GLUT1 expression and glucose uptake in the brain and lungs. Hence, we have identified an important function of breast cancer secreted miR-122 in promoting metastasis through systemic reprogramming of glucose metabolism, which contributes to the emerging concept that reprogrammed stromal metabolism to manipulate energy availability for cancer cells can fuel metastatic colonization and therefore represents an important aspect in the treatment of cancer metastasis. Figure 1. Schematic representation of the secretion of miR-122 in exosomes by cancer cells. The export of miR-122 allows cancer cells to increase their glucose uptake by enhancing GLUT1 expression to support cancer cell growth, while simultaneously suppressing ...

The human organic cation transporter, OCT1 (SLC22A1), a member of the SLC22 family, was first cloned in 1997.1 Since then OCT1 has been characterized as a major drug transporter in the liver, transporting important pharmacologic agents including the anti-diabetic drug, metformin and the anti-cancer drug, oxaliplatin. Genetic polymorphisms of OCT1 have been associated with variation in the disposition and response to drugs such as codeine2 and metformin,3 and with variation in serum levels of various metabolites (http://www.genome.gov/gwastudies/). Initial studies in Oct1−/− mice showed that deletion of Oct1 resulted in no observable physiologic changes. Our recent report contradicts these initial findings, and suggests that OCT1 has an important physiologic role beyond xenobiotic disposition, in mammals. Notably, we found that OCT1 is a high capacity thiamine transporter, and as such plays an important role in carbohydrate and lipid metabolism in hepatocytes.3 Our findings were based on integrated experimental approaches using cells, tissues, mouse models, metabolomic methods, and various molecular approaches to characterize the endogenous role of OCT1 including its principal substrate.3 A key observation, that Oct1 deficiency alleviated hepatic steatosis in leptin deficient mice, inspired our further investigations. Our discovery (using metabolomic and isotopic uptake methods) that thiamine is the principal endogenous substrate of OCT1 provided the rationale for the reduced hepatic steatosis in the Oct1−/− mice. In particular, the thiamine derivative, thiamine pyrophosphate (TPP), is crucial for converting pyruvate to acetyl-CoA for glucose oxidative phosphorylation (ATP production) and fatty acid synthesis. The “short fall” in glycolytic energy production caused by OCT1 disruption in the knockout mice triggered increases in fatty acid oxidation to compensate for the energy requirement of the cell (Fig. 1). The dual effects caused by OCT1 deficiency (reduced glucose oxidation and increased β-oxidation) modulated the energy flow in the hepatocytes. Figure 1. The physiological role of OCT1 in the liver. Thiamine enters hepatocytes through OCT1. Deletion or inhibition of OCT1 reduces thiamine and TPP levels [1] and hence reduces glucose oxidation and de novo lipogenesis. The reduced ATP from glucose [2] activates ... Supporting the decreased glycolytic energy production, reductions in glucose oxidation rates, ATP levels and levels of key gluconeogenic enzymes, such as glucose-6-phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase (PEPCK) (5) were observed in the livers of Oct1 deficient mice. Further, OCT1 expression was predominantly restricted to the peri-venous zone responsible for glycolysis and lipogenesis.3,4 Finally, our re-discovery that thiamine deficiency in rodents results in reduced hepatic steatosis implicates thiamine deficiency in the liver as the potential cause of the reduced hepatic steatosis in the Oct1 deficient mice. Beyond an understanding of basic physiology, our study has 3 critical implications. First, the findings suggest that there is a dose-response relationship for thiamine in terms of fat accumulation in the liver. The epidemic of beriberi that occurred in the early part of the 20th century was abated by thiamine supplementation in various products such as rice and bread. However our study raises an important point. Can thiamine supplementation be taken too far? Is too much thiamine a culprit for fatty liver disease? Clearly, the role of thiamine needs to be examined in light of the current epidemic in metabolic syndrome and diabetes. Second, our findings suggest that OCT1 represents an excellent target for the treatment of fatty liver disease and nonalcoholic steatohepatitis (NASH), an increasing health problem with limited therapeutic options.5 Developing small molecules or monoclonal antibodies against OCT1 would open a new window for NASH treatment. Finally, our study has important implications for the mechanisms of action of metformin, which is widely prescribed for the treatment of type 2 diabetes mellitus. How metformin acts has been the subject of numerous studies.6,7 Our study suggests that in addition to transporting metformin, OCT1 may serve as a target for metformin. In particular, parallel effects of metformin and Oct1 deficiency in mice were noted. For example, Oct1 deficiency and administration of metformin both result in reduced hepatic steatosis, and increased phosphorylation of AMPK and ACC. Moreover, we observed that metformin competitively inhibited OCT1-mediated thiamine uptake in transfected cells. In vivo, Oct1 deficiency resulted in substantially reduced intestinal and liver levels of thiamine; similarly, metformin treatment resulted in reduced intestinal and systemic plasma levels of thiamine and a trend toward reduced liver levels of thiamine. Modulation of thiamine levels by metformin (via OCT1 and perhaps other thiamine transporters) may be critically important in its beneficial effects on diabetes, hepatic steatosis, obesity and cancer. Collectively, the findings of our study present a compelling role for OCT1 in thiamine disposition, hepatic steatosis and energy production, and potentially in the mechanisms of metformin action (Fig. 1). However, in closing, several other points are worth mentioning. Firstly, exploration of the role of other endogenous substrates of OCT1, for example, serotonin, is needed. Secondly, studies of interactions between metformin and thiamine at transporters beyond OCT1 are needed to understand the role of metformin in thiamine disposition in other tissues. Finally, studies are clearly needed that differentiate the role of OCT1 in the cellular uptake of metformin and as a target for the drug.

Resistance to chemotherapy is a major factor responsible for cancer recurrence and failure of therapy. Recent studies have disclosed that the nature of the extracellular matrix and the crosstalk between the stroma and the malignant cells have substantial effects on the sensitivity to standard chemotherapeutic agents. The αV-containing integrins play a central role in the interaction between the matrix and tumor cells, and together with its β integrin partners participates in the regulation of numerous signal transduction pathways through the activation of FAK, Src and related kinases. However, little is known about how αV integrin modulates cellular sensitivity to chemotherapeutic agents, particularly the platinum-containing drugs. Reduced drug accumulation due to impaired influx is a common feature of cells that have acquired resistance to cisplatin (cDDP) and carboplatin. Both are quite polar and do not cross lipid membranes readily. There is now a substantial body of evidence, derived from multiple cell models, that the copper (Cu) influx transporter CTR1 regulates the cellular pharmacology of these drugs.1 The discovery that CTR1 accounts for a significant fraction of the cDDP uptake introduced the concept that cDDP exploits its mimicry of the chemical characteristics of Cu+1 to get into the cell where it is now known to form complexes with several Cu-binding proteins containing a CXXC motif.2 We recently discovered that the loss of the cell-ECM interaction mediated by αV is associated with an increase in CTR1 expression, enhanced cDDP accumulation and DNA adduct formation, and greater sensitivity to the cytotoxic effect of cDDP.3 This suggests that the signals generated by the αV-containing integrins normally serve to repress CTR1 levels. The linkage between αV and CTR1 expression is of potential clinical significance because elevated hCTR1 expression, as determined by immunohistochemical and mRNA expression profiling analysis, is associated with favorable treatment outcome in several diseases including ovarian, non-small cell lung and endometrial cancers in which cDDP is used as part of primary therapy.

Kinase activation and substrate phosphorylation commonly form the backbone of signaling cascades. Bone morphogenetic proteins (BMPs), a subclass of TGF-β family ligands, induce activation of their signaling effectors, the Smads, through C-terminal phosphorylation by transmembrane receptor kinases. However, the slow kinetics of Smad activation in response to BMP suggests a preceding step in the initiation of BMP signaling. We now show that arginine methylation, which is known to regulate gene expression, yet also modifies some signaling mediators, initiates BMP-induced Smad signaling. BMP-induced receptor complex formation promotes interaction of the methyltransferase PRMT1 with the inhibitory Smad6, resulting in Smad6 methylation and relocalization at the receptor, leading to activation of effector Smads through phosphorylation. PRMT1 is required for BMP-induced biological responses across species, as evidenced by the role of its ortholog Dart1 in BMP signaling during Drosophila wing development. Activation of signaling by arginine methylation may also apply to other signaling pathways.

SummaryAerobic glycolysis (the Warburg effect) is a core hallmark of cancer, but the molecular mechanisms underlying it remain unclear. Here, we identify an unexpected central role for mTORC2 in cancer metabolic reprogramming where it controls glycolytic metabolism by ultimately regulating the cellular level of c-Myc. We show that mTORC2 promotes inactivating phosphorylation of class IIa histone deacetylases, which leads to the acetylation of FoxO1 and FoxO3, and this in turn releases c-Myc from a suppressive miR-34c-dependent network. These central features of activated mTORC2 signaling, acetylated FoxO, and c-Myc levels are highly intercorrelated in clinical samples and with shorter survival of GBM patients. These results identify a specific, Akt-independent role for mTORC2 in regulating glycolytic metabolism in cancer.

Enhanced signaling by the small guanosine triphosphatase Ras is common in T cell acute lymphoblastic leukemia/lymphoma (T-ALL), but the underlying mechanisms are unclear. We identified the guanine nucleotide exchange factor RasGRP1 (Rasgrp1 in mice) as a Ras activator that contributes to leukemogenesis. We found increased RasGRP1 expression in many pediatric T-ALL patients, which is not observed in rare early T cell precursor T-ALL patients with KRAS and NRAS mutations, such as K-Ras[superscript G12D]. Leukemia screens in wild-type mice, but not in mice expressing the mutant K-Ras[superscript G12D] that encodes a constitutively active Ras, yielded frequent retroviral insertions that led to increased Rasgrp1 expression. Rasgrp1 and oncogenic K-Ras[superscript G12D] promoted T-ALL through distinct mechanisms. In K-Ras[superscript G12D] T-ALLs, enhanced Ras activation had to be uncoupled from cell cycle arrest to promote cell proliferation. In mouse T-ALL cells with increased Rasgrp1 expression, we found that Rasgrp1 contributed to a previously uncharacterized cytokine receptor–activated Ras pathway that stimulated the proliferation of T-ALL cells in vivo, which was accompanied by dynamic patterns of activation of effector kinases downstream of Ras in individual T-ALLs. Reduction of Rasgrp1 abundance reduced cytokine-stimulated Ras signaling and decreased the proliferation of T-ALL in vivo. The position of RasGRP1 downstream of cytokine receptors as well as the different clinical outcomes that we observed as a function of RasGRP1 abundance make RasGRP1 an attractive future stratification marker for T-ALL., National Institutes of Health (U.S.). Pioneer Award, National Cancer Institute (U.S.). Physical Sciences-Oncology Center (U54CA143874), National Institutes of Health (U.S.). (P01 AI091580)