Tumor immunoevasion via acidosis-dependent induction of regulatory tumor-associated macrophages.

Many tumors evolve sophisticated strategies to evade the immune system, and these represent major obstacles for efficient antitumor immune responses. Here we explored a molecular mechanism of metabolic communication deployed by highly glycolytic tumors for immunoevasion. In contrast to colon adenocarcinomas, melanomas showed comparatively high glycolytic activity, which resulted in high acidification of the tumor microenvironment. This tumor acidosis induced Gprotein-coupled receptor-dependent expression of the transcriptional repressor ICER in tumor-associated macrophages that led to their functional polarization toward a non-inflammatory phenotype and promoted tumor growth. Collectively, our findings identify a molecular mechanism of metabolic communication between non-lymphoid tissue and the immune system that was exploited by high-glycolytic-rate tumors for evasion of the immune system.

N-terminal alanine-rich (NTAR) sequences drive precise start codon selection resulting in elevated translation of multiple proteins including ERK1/2.

We report the discovery of N-terminal alanine-rich sequences, which we term NTARs, that act in concert with their native 5'-untranslated regions to promote selection of the proper start codon. NTARs also facilitate efficient translation initiation while limiting the production of non-functional polypeptides through leaky scanning. We first identified NTARs in the ERK1/2 kinases, which are among the most important signaling molecules in mammals. Analysis of the human proteome reveals that hundreds of proteins possess NTARs, with housekeeping proteins showing a particularly high prevalence. Our data indicate that several of these NTARs act in a manner similar to those found in the ERKs and suggest a mechanism involving some or all of the following features: alanine richness, codon rarity, a repeated amino acid stretch and a nearby second AUG. These features may help slow down the leading ribosome, causing trailing pre-initiation complexes (PICs) to pause near the native AUG, thereby facilitating accurate translation initiation. Amplification of erk genes is frequently observed in cancer, and we show that NTAR-dependent ERK protein levels are a rate-limiting step for signal output. Thus, NTAR-mediated control of translation may reflect a cellular need to precisely control translation of key transcripts such as potential oncogenes. By preventing translation in alternative reading frames, NTAR sequences may be useful in synthetic biology applications, e.g. translation from RNA vaccines.

Initiation of translation is essential for protein synthesis. A crucial step is the correct choice of the start AUG, which leads to the production of the fully functional polypeptide. To date, nucleotide composition next to the AUG has been considered the only determinant of start codon selection. Our work identifies a large family of proteins whose start codon choice is determined by an N-terminal alanine-rich sequence (NTAR) that enables efficient protein translation. Many of these proteins are encoded by housekeeping genes. Among them, the NTARs of the pivotal kinases ERK1 and ERK2 are highly optimized in humans, shaping ERK signal transduction by increasing the kinase quantity. Our findings could be useful for applied biology, especially for mRNA-based therapeutics.

Monitoring Mitochondrial Pyruvate Carrier Activity in Real Time Using a BRET-Based Biosensor: Investigation of the Warburg Effect.

The transport of pyruvate into mitochondria requires a specific carrier, the mitochondrial pyruvate carrier (MPC). The MPC represents a central node of carbon metabolism, and its activity is likely to play a key role in bioenergetics. Until now, investigation of the MPC activity has been limited. However, the recent molecular identification of the components of the carrier has allowed us to engineer a genetically encoded biosensor and to monitor the activity of the MPC in real time in a cell population or in a single cell. We report that the MPC activity is low in cancer cells, which mainly rely on glycolysis to generate ATP, a characteristic known as the Warburg effect. We show that this low activity can be reversed by increasing the concentration of cytosolic pyruvate, thus increasing oxidative phosphorylation. This biosensor represents a unique tool to investigate carbon metabolism and bioenergetics in various cell types.

Hypoxia and hypoxia-inducible factors promote the development of neointimal hyperplasia in arteriovenous fistula.

KEY POINTS: Patients with end-stage renal failure need arteriovenous fistulas (AVF) to undergo dialysis. However, AVFs present a high rate of failure as a result of excessive venous thickness. Excessive venous thickness may be a consequence of surgical dissection and change in oxygen concentration within the venous wall. We show that venous cells adapt their metabolism and growth depending on oxygen concentration, and drugs targeting the hypoxic response pathway modulate this response in vitro. We used the same drugs on a mouse model of AVF and show that direct or indirect inhibition of the hypoxia-inducible factors (HIFs) help decrease excessive venous thickness. Hypoxia and HIFs can be targets of therapeutic drugs to prevent excessive venous thickness in patients undergoing AVF surgical creation. ABSTRACT: Because the oxygen concentration changes in the venous wall, surrounding tissue and the blood during surgical creation of arteriovenous fistula (AVF), we hypothesized that hypoxia could contribute to AVF failure as a result of neointimal hyperplasia. We postulated that modulation of the hypoxia-inducible factors (HIF) with pharmacological compounds could promote AVF maturation. Fibroblasts [normal human fibroblasts (NHF)], smooth muscle cells [human umbilical vein smooth muscle cells (HUVSMC)] and endothelial cells [human umbilical vein endothelial cells (HUVEC)], representing the three layers of the venous wall, were tested in vitro for proliferation, cell death, metabolism, reactive oxygen species production and migration after silencing of HIF1/2-α or after treatment with deferioxamine (DFO), everolimus (Eve), metformin (Met), N-acetyl-l-cysteine (NAC) and topoisomerase I (TOPO), which modulate HIF-α stability or activity. Compounds that were considered to most probably modify intimal hyperplasia were applied locally to the vessels in a mouse model of aortocaval fistula. We showed, in vitro, that NHF and HUVSMC can adapt their metabolism and thus their growth depending on oxygen concentration, whereas HUVEC appears to be less flexible. siHIF1/2α, DFO, Eve, Met, NAC and TOPO can modulate metabolism and proliferation depending on the cell type and the oxygen concentration. In vivo, siHIF1/2α, Eve and TOPO decreased neointimal hyperplasia by 32%-50%, 7 days after treatment. Within the vascular wall, hypoxia and HIF-1/2 mediate early failure of AVF. Local delivery of drugs targeting HIF-1/2 could inhibit neointimal hyperplasia in a mouse model of AVF. Such compounds may be delivered during the surgical procedure for AVF creation to prevent early AVF failure.

Overcoming Therapeutic Challenges for Pancreatic Ductal Adenocarcinoma with xCT Inhibitors.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive and lethal cancers with a dismal 5-year survival rate of 5% and very limited efficacy of the current therapeutic regimens. The lethality of PDAC stems from asymptomatic early stage of the disease, its propensity to rapidly disseminate, as well as unusual, dense and highly active surrounding stroma. Fortunately, promising literature data suggests that exploiting newly contextualized type of cell death, termed "ferroptosis", has great potential for overcoming the major problems regarding PDAC treatment. A major player in this type of cell death is Glutamate/Cystine antiporter - xCT, which is responsible for the uptake of oxidized form of cysteine, and thus maintenance of intracellular amino acid and redox homeostasis. xCT seems to fulfill all requirements of the solid and specific molecular target for ferroptosis-based anti-cancer therapy. In this chapter we summarized mounting literature data supporting this hypothesis, but also, we pointed out some of the underexamined aspects of xCT-dependent (patho)physiology of the cancer cell, which have to be addressed in future studies. The abstract could be used as "informative abstract" for the online version.

Co-culture of human fibroblasts, smooth muscle and endothelial cells promotes osteopontin induction in hypoxia.

Arteriovenous fistulas (AVFs) are the preferred vascular access for haemodialysis of patients suffering from end-stage renal disease, a worldwide public health problem. However, they are prone to a high rate of failure due to neointimal hyperplasia and stenosis. This study aimed to determine if osteopontin (OPN) was induced in hypoxia and if OPN could be responsible for driving AVF failure. Identification of new factors that participate in remodelling of AVFs is a challenge. Three cell lines representing the cells of the three layers of the walls of arteries and veins, fibroblasts, smooth muscle cells and endothelial cells, were tested in mono- and co-culture in vitro for OPN expression and secretion in normoxia compared to hypoxia after silencing the hypoxia-inducible factors (HIF-1α, HIF-2α and HIF-1/2α) with siRNA or after treatment with an inhibitor of NF-kB. None of the cells in mono-culture showed OPN induction in hypoxia, whereas cells in co-culture secreted OPN in hypoxia. The changes in oxygenation that occur during AVF maturation up-regulate secretion of OPN through cell-cell interactions between the different cell layers that form AVF, and in turn, these promote endothelial cell proliferation and could participate in neointimal hyperplasia.

Dynamin inhibitors block activation of mTORC1 by amino acids independently of dynamin.

mTORC1 plays a crucial role in protein synthesis and cell proliferation and growth. It is activated by growth factors and amino acids, including essential amino acids (EAAs), such as leucine; Leu enters cells via the Leu transporter LAT1-4F2hc (also known as SLC7A5-SLC3A2) and potentially via endocytosis. Here, we investigated the contribution of the different routes of Leu entry into cells to mTORC1 activation using pharmacological inhibitors and cells that lack LAT1 or dynamin-1, -2 and -3. Our results show that LAT1 is the major route of Leu entry into cells and mTORC1 activation (∼70%), whereas dynamin-dependent endocytosis and macropinocytosis contribute minimally to both (5-15%). However, macropinocytosis contributes significantly (∼40%) to activation of mTORC1 by other EAAs. Surprisingly, the dynamin inhibitors dynasore and Dyngo 4A, which minimally inhibited Leu uptake, abolished mTORC1 activation independently of dynamin. Instead, dynasore inhibited RagA binding to Raptor, reduced mTORC1 recruitment to the lysosome, and inhibited Akt activation and TSC2-S939 phosphorylation; this resulted in inhibition of Rheb and mTORC1 activity. Our results suggest that these commonly used inhibitors of dynamin and endocytosis are potent suppressors of mTORC1 activation via off-target effects and not via dynamin inhibition.This article has an associated First Person interview with the first author of the paper.

From Malignant Progression to Therapeutic Targeting: Current Insights of Mesothelin in Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC), accounting for 90% of all pancreatic tumors, is a highly devastating disease with poor prognosis and rising incidence. The lack of available specific diagnostics tests and the limited treatment opportunities contribute to this pejorative issue. Over the last 10 years, a growing interest pointing towards mesothelin (MSLN) as a promising PDAC-associated antigen has emerged. The limited expression of MSLN in normal tissues (peritoneum, pleura and pericardium) and its overexpression in 80 to 90% of PDAC make it an attractive candidate for therapeutic management of PDAC patients. Moreover, its role in malignant progression related to its involvement in tumor cell proliferation and resistance to chemotherapy has highlighted the relevance of its targeting. Hence, several clinical trials are investigating anti-MSLN efficacy in PDAC. In this review, we provide a general overview of the different roles sustained by MSLN during PDAC progression. Finally, we also summarize the different MSLN-targeted therapies that are currently tested in the clinic.

Amino Acid Transporters Are a Vital Focal Point in the Control of mTORC1 Signaling and Cancer.

The mechanistic target of rapamycin complex 1 (mTORC1) integrates signals from growth factors and nutrients to control biosynthetic processes, including protein, lipid, and nucleic acid synthesis. Dysregulation in the mTORC1 network underlies a wide array of pathological states, including metabolic diseases, neurological disorders, and cancer. Tumor cells are characterized by uncontrolled growth and proliferation due to a reduced dependency on exogenous growth factors. The genetic events underlying this property, such as mutations in the PI3K-Akt and Ras-Erk signaling networks, lead to constitutive activation of mTORC1 in nearly all human cancer lineages. Aberrant activation of mTORC1 has been shown to play a key role for both anabolic tumor growth and resistance to targeted therapeutics. While displaying a growth factor-independent mTORC1 activity and proliferation, tumors cells remain dependent on exogenous nutrients such as amino acids (AAs). AAs are an essential class of nutrients that are obligatory for the survival of any cell. Known as the building blocks of proteins, AAs also act as essential metabolites for numerous biosynthetic processes such as fatty acids, membrane lipids and nucleotides synthesis, as well as for maintaining redox homeostasis. In most tumor types, mTORC1 activity is particularly sensitive to intracellular AA levels. This dependency, therefore, creates a targetable vulnerability point as cancer cells become dependent on AA transporters to sustain their homeostasis. The following review will discuss the role of AA transporters for mTORC1 signaling in cancer cells and their potential as therapeutic drug targets.

Inhibition of the amino-acid transporter LAT1 demonstrates anti-neoplastic activity in medulloblastoma.

Most cases of medulloblastoma (MB) occur in young children. While the overall survival rate can be relatively high, current treatments combining surgery, chemo- and radiotherapy are very destructive for patient development and quality of life. Moreover, aggressive forms and recurrences of MB cannot be controlled by classical therapies. Therefore, new therapeutic approaches yielding good efficacy and low toxicity for healthy tissues are required to improve patient outcome. Cancer cells sustain their proliferation by optimizing their nutrient uptake capacities. The L-type amino acid transporter 1 (LAT1) is an essential amino acid carrier overexpressed in aggressive human cancers that was described as a potential therapeutic target. In this study, we investigated the therapeutic potential of JPH203, a LAT1-specific pharmacological inhibitor, on two independent MB cell lines belonging to subgroups 3 (HD-MB03) and Shh (DAOY). We show that while displaying low toxicity towards normal cerebral cells, JPH203 disrupts AA homeostasis, mTORC1 activity, proliferation and survival in MB cells. Moreover, we demonstrate that a long-term treatment with JPH203 does not lead to resistance in MB cells. Therefore, this study suggests that targeting LAT1 with JPH203 is a promising therapeutic approach for MB treatment.

The glutamine transporter ASCT2 (SLC1A5) promotes tumor growth independently of the amino acid transporter LAT1 (SLC7A5).

The transporters for glutamine and essential amino acids, ASCT2 (solute carrier family 1 member 5, SLC1A5) and LAT1 (solute carrier family 7 member 5, SLC7A5), respectively, are overexpressed in aggressive cancers and have been identified as cancer-promoting targets. Moreover, previous work has suggested that glutamine influx via ASCT2 triggers essential amino acids entry via the LAT1 exchanger, thus activating mechanistic target of rapamycin complex 1 (mTORC1) and stimulating growth. Here, to further investigate whether these two transporters are functionally coupled, we compared the respective knockout (KO) of either LAT1 or ASCT2 in colon (LS174T) and lung (A549) adenocarcinoma cell lines. Although ASCT2KO significantly reduced glutamine import (>60% reduction), no impact on leucine uptake was observed in both cell lines. Although an in vitro growth-reduction phenotype was observed in A549-ASCT2KO cells only, we found that genetic disruption of ASCT2 strongly decreased tumor growth in both cell lines. However, in sharp contrast to LAT1KO cells, ASCT2KO cells displayed no amino acid (AA) stress response (GCN2/EIF2a/ATF4) or altered mTORC1 activity (S6K1/S6). We therefore conclude that ASCT2KO reduces tumor growth by limiting AA import, but that this effect is independent of LAT1 activity. These data were further supported by in vitro cell proliferation experiments performed in the absence of glutamine. Together these results confirm and extend ASCT2's pro-tumoral role and indicate that the proposed functional coupling model of ASCT2 and LAT1 is not universal across different cancer types.

Double genetic disruption of lactate dehydrogenases A and B is required to ablate the "Warburg effect" restricting tumor growth to oxidative metabolism.

Increased glucose consumption distinguishes cancer cells from normal cells and is known as the "Warburg effect" because of increased glycolysis. Lactate dehydrogenase A (LDHA) is a key glycolytic enzyme, a hallmark of aggressive cancers, and believed to be the major enzyme responsible for pyruvate-to-lactate conversion. To elucidate its role in tumor growth, we disrupted both the LDHA and LDHB genes in two cancer cell lines (human colon adenocarcinoma and murine melanoma cells). Surprisingly, neither LDHA nor LDHB knockout strongly reduced lactate secretion. In contrast, double knockout (LDHA/B-DKO) fully suppressed LDH activity and lactate secretion. Furthermore, under normoxia, LDHA/B-DKO cells survived the genetic block by shifting their metabolism to oxidative phosphorylation (OXPHOS), entailing a 2-fold reduction in proliferation rates in vitro and in vivo compared with their WT counterparts. Under hypoxia (1% oxygen), however, LDHA/B suppression completely abolished in vitro growth, consistent with the reliance on OXPHOS. Interestingly, activation of the respiratory capacity operated by the LDHA/B-DKO genetic block as well as the resilient growth were not consequences of long-term adaptation. They could be reproduced pharmacologically by treating WT cells with an LDHA/B-specific inhibitor (GNE-140). These findings demonstrate that the Warburg effect is not only based on high LDHA expression, as both LDHA and LDHB need to be deleted to suppress fermentative glycolysis. Finally, we demonstrate that the Warburg effect is dispensable even in aggressive tumors and that the metabolic shift to OXPHOS caused by LDHA/B genetic disruptions is responsible for the tumors' escape and growth.

Hypoxia protects against the cell death triggered by oxovanadium-galactomannan complexes in HepG2 cells.

BACKGROUND: Polysaccharides from various sources have been used in traditional medicine for centuries. The beneficial pharmacological effects of plant-derived polysaccharides include anti-tumor activity. METHODS: Here, we evaluated the anti-cancer effect of the MSAGM:VO complex under hypoxic conditions (1% oxygen). MSAGM:VO is a complex of the hydrolysate of galactomannan (MSAGM) from Schizolobium amazonicum with oxovanadium (IV/V). The hepatocellular carcinoma (HCC) cell line HepG2 was selected as HCC are one of the most hypoxic solid tumors. RESULTS: Our results showed that the strong apoptotic activity of MSAGM:VO observed in HepG2 cells under normoxic conditions was completely lost under hypoxic conditions. We found a dynamic balance between the pro- and anti-apoptotic members of the Bcl-2 protein family. The expressions of anti-apoptotic Mcl-1 and Bcl-XL increased in hypoxia, whereas the expression of pro-apoptotic Bax decreased. MSAGM:VO strongly induced autophagy, which was previously characterized as a pro-survival mechanism in hypoxia. These results demonstrate total elimination of the anti-cancer activity of MSAGM:VO with activation of autophagy under conditions of hypoxia. CONCLUSION: Although this study is a proof-of-concept of the impact of hypoxia on the potential of polysaccharides, further study is encouraged. The anti-tumor activity of polysaccharides could be achieved in normoxia or through raising the activity of the immune system. In addition, combination strategies for therapy with anti-autophagic drugs could be proposed.

Tumor Microenvironment: A Metabolic Player that Shapes the Immune Response.

Immune cells survey and patrol throughout the body and sometimes take residence in niche environments with distinct cellular subtypes and nutrients that may fluctuate from those in which they matured. Rooted in immune cell physiology are metabolic pathways and metabolites that not only deliver substrates and energy for growth and survival, but also instruct effector functions and cell differentiation. Unlike cancer cells, immune cells are not subject to a "Darwinian evolutionary pressure" that would allow them to adapt to developing tumors but are often irrevocably affected to local nutrient deprivation. Thus, immune cells must metabolically adapt to these changing conditions in order to perform their necessary functions. On the other hand, there is now a growing appreciation that metabolic changes occurring in cancer cells can impact on immune cell functionality and contribute to tumor immune evasion, and as such, there is a considerable and growing interest in developing techniques that target metabolism for immunotherapy. In this review, we discuss the metabolic plasticity displayed by innate and adaptive immune cells and highlight how tumor-derived lactate and tumor acidity restrict immunity. To our knowledge, this review outlines the most recent insights on how tumor microenvironment metabolically instructs immune responsiveness.

Targeting pH regulating proteins for cancer therapy-Progress and limitations.

Tumour acidity induced by metabolic alterations and incomplete vascularisation sets cancer cells apart from normal cellular physiology. This distinguishing tumour characteristic has been an area of intense study, as cellular pH (pHi) disturbances disrupt protein function and therefore multiple cellular processes. Tumour cells effectively utilise pHi regulating machinery present in normal cells with enhancements provided by additional oncogenic or hypoxia induced protein modifications. This overall improvement of pH regulation enables maintenance of an alkaline pHi in the continued presence of external acidification (pHe). Considerable experimentation has revealed targets that successfully disrupt tumour pHi regulation in efforts to develop novel means to weaken or kill tumour cells. However, redundancy in these pH-regulating proteins, which include Na+/H+ exchangers (NHEs), carbonic anhydrases (CAs), Na+/HCO3- co-transporters (NBCs) and monocarboxylate transporters (MCTs) has prevented effective disruption of tumour pHi when individual protein targeting is performed. Here we synthesise recent advances in understanding both normoxic and hypoxic pH regulating mechanisms in tumour cells with an ultimate focus on the disruption of tumour growth, survival and metastasis. Interactions between tumour acidity and other cell types are also proving to be important in understanding therapeutic applications such as immune therapy. Promising therapeutic developments regarding pH manipulation along with current limitations are highlighted to provide a framework for future research directives.

Dissecting the Process of Activation of Cancer-promoting Zinc-requiring Ectoenzymes by Zinc Metalation Mediated by ZNT Transporters.

Zinc-requiring ectoenzymes, including both secreted and membrane-bound enzymes, are considered to capture zinc in their active site for their activation in the early secretory pathway. This idea has been confirmed by our studies conducted using tissue-nonspecific alkaline phosphatase (TNAP), which is elaborately activated by means of a two-step mechanism by zinc transporter 5 (ZNT5)-ZNT6 heterodimers and ZNT7 homodimers, through protein stabilization followed by enzyme activation with zinc in the early secretory pathway. However, the molecular basis of the activation process in other zinc-requiring ectoenzymes remains largely unknown. In this study, we investigated this activation process by using three cancer-promoting zinc-requiring ectoenzymes, autotaxin (ATX), matrix metalloproteinase 9 (MMP9), and carbonic anhydrase IX (CAIX), and the chicken DT40 cell mutants that we generated; we specifically focused on clarifying whether the same or a similar activation mechanism operates in these ectoenzymes. ATX activation required ZNT5-ZNT6 heterodimers and ZNT7 homodimers in a manner similar to TNAP activation, although the protein stability of ATX was differently regulated from that of TNAP. MMP9 required ZNT5-ZNT6 heterodimers and ZNT7 homodimers for its activation as well as secretion; MMP9 was not secreted into the spent medium unless both zinc-transport complexes were present. Finally, CAIX activation by zinc was mediated not only by ZNT5-ZNT6 heterodimers and ZNT7 homodimers but also by ZNT4 homodimers; thus, these three zinc-transport complexes redundantly contribute to CAIX activation. Our results provide pivotal insights into the activation processes of zinc-requiring ectoenzymes, and furthermore, they offer novel insights for potential cancer therapy applications given the cancer-promoting potencies of ATX, MMP9, and CAIX.

Spontaneous chondroma formation in CD2-Cre-driven Erk-deficient mice.

Lineage-specific Cre Tg mice are widely used to delineate the functions of genes in a tissue-specific manner. Several T-cell-specific promoter cassettes have been developed; however, the activities of those promoters in non-T cells have not been investigated extensively. Here, we report that CD2-Cre-mediated deletion of Erk proteins by generating CD2-Cre × Erk1-/-Erk2flox/flox (Erk∆CD2-Cre) mice results in abnormal cartilage hyperplasia. Histological analysis revealed that this abnormality is caused by aberrant hyperplasia of chondrocytes. The presence of Erk-deficient T cells is not required for this chondroma formation, as it was similarly observed in the absence of T cells in a CD3ε-deficient background. In addition, adoptive transfer of bone marrow cells from Erk∆CD2-Cre mice to wild-type recipients did not cause chondroma formation, suggesting that Erk-deficient non-immune cells are responsible for this abnormality. By tracing Cre-expressed tissues using a ROSA26-STOP-RFP allele, we found that the chondroma emitted RFP fluorescence, indicating that functional Cre is expressed in hyperplastic chondrocytes in Erk∆CD2-Cre mice. Furthermore, RFP+ chondrocytes were also found in an Erk-sufficient background, albeit without aberrant growth. These results suggest that unexpected expression of CD2-driven Cre in chondrocytes generates Erk-deficient chondrocytes, resulting in hyperplastic cartilage formation. Recently, two independent reports showed that CD4-Cre-mediated Ras-Erk signaling ablation led to similar abnormal cartilage formation (Guittard, G., Gallardo, D. L., Li, W. et al. 2017. Unexpected cartilage phenotype in CD4-Cre-conditional SOS-deficient mice. Front. Immunol. 8:343; Wehenkel, M., Corr, M., Guy, C. S. et al. 2017. Extracellular signal-regulated kinase signaling in CD4-expressing cells inhibits osteochondromas. Front. Immunol. 8:482). Together with these reports, our study suggests that an unexpected link exists between T-like cell and chondrocyte lineages during ontogeny.

AKT1 restricts the invasive capacity of head and neck carcinoma cells harboring a constitutively active PI3 kinase activity.

BACKGROUND: In mammals, the AKT/PKB protein kinase family comprises three members (AKT1-3). PI3-Kinase (PI3K), a key oncogene involved in a wide variety of cancers, drives AKT activity. Constitutive activation of the PI3K/AKT pathway has been associated with tumorigenic properties including uncontrolled cell proliferation and survival, angiogenesis, promotion of cellular motility, invasiveness and metastasis. However, AKT1 activity has also been recently shown to repress the invasive properties of breast cancer cells in specific contexts. METHODS: This study used both pharmacological and shRNA approaches to inhibit AKT function, microscopy to characterize the cellular morphology, 3D spheroid models to assess migratory and invasive cellular capacities and a phenotypic screening approach based on electrical properties of the cells. RESULTS: Here we demonstrate that the alternative action of AKT1 on invasive properties of breast cancers can be extended to head and neck carcinomas, which exhibit constitutive activation of the PI3K/AKT pathway. Indeed, inhibition of AKT1 function by shRNA or a specific pharmacological inhibitor resulted in cellular spreading and an invasive phenotype. A phenotypic screening approach based on cellular electrical properties corroborated microscopic observations and provides a foundation for future high-throughput screening studies. This technique further showed that the inhibition of AKT1 signaling is phenocopied by blocking the mTORC1 pathway with rapamycin. CONCLUSION: Our study suggests that the repressive action of PI3K/AKT1 on cellular invasive properties may be a mechanism common to several cancers. Current and future studies involving AKT inhibitors must therefore consider this property to prevent metastases and consequently to improve survival.

ERK1 is dispensable for mouse pancreatic beta cell function but is necessary for glucose-induced full activation of MSK1 and CREB.

AIMS/HYPOTHESIS: Insufficient insulin secretion from pancreatic beta cells, which is associated with a decrease in beta cell mass, is a characteristic of type 2 diabetes. Extracellular signal-related kinase 1 and 2 (ERK1/2) inhibition in beta cells has been reported to affect insulin secretion, gene transcription and survival, although whether ERK1 and ERK2 play distinct roles is unknown. The aim of this study was to assess the individual roles of ERK1 and ERK2 in beta cells using ERK1 (also known as Mapk3)-knockout mice (Erk1 -/- mice) and pharmacological approaches. METHODS: NAD(P)H, free cytosolic Ca2+ concentration and insulin secretion were determined in islets. ERK1 and ERK2 subplasmalemmal translocation and activity was monitored using total internal reflection fluorescence microscopy. ERK1/2, mitogen and stress-activated kinase1 (MSK1) and cAMP-responsive element-binding protein (CREB) activation were evaluated by western blot and/or immunocytochemistry. The islet mass was determined from pancreatic sections. RESULTS: Glucose induced rapid subplasmalemmal recruitment of ERK1 and ERK2. When both ERK1 and ERK2 were inhibited simultaneously, the rapid transient peak of the first phase of glucose-induced insulin secretion was reduced by 40% (p < 0.01), although ERK1 did not appear to be involved in this process. By contrast, ERK1 was required for glucose-induced full activation of several targets involved in beta cell survival; MSK1 and CREB were less active in Erk1 -/- mouse beta cells (p < 0.01) compared with Erk1 +/+ mouse beta cells, and their phosphorylation could only be restored when ERK1 was re-expressed and not when ERK2 was overexpressed. Finally, the islet mass of Erk1 -/- mice was slightly increased in young animals (4-month-old mice) vs Erk1 +/+ mice (section occupied by islets [mean ± SEM]: 0.74% ± 0.03% vs 0.62% ± 0.04%; p < 0.05), while older mice (10 months old) were less prone to age-associated pancreatic peri-insulitis (infiltrated islets [mean ± SEM]: 7.51% ± 1.34% vs 2.03% ± 0.51%; p < 0.001). CONCLUSIONS/INTERPRETATION: ERK1 and ERK2 play specific roles in beta cells. ERK2 cannot always compensate for the lack of ERK1 but the absence of a clear-cut phenotype in Erk1 -/- mice shows that ERK1 is dispensable in normal conditions.

Hypoxia and cellular metabolism in tumour pathophysiology.

Cancer cells are optimised for growth and survival via an ability to outcompete normal cells in their microenvironment. Many of these advantageous cellular adaptations are promoted by the pathophysiological hypoxia that arises in solid tumours due to incomplete vascularisation. Tumour cells are thus faced with the challenge of an increased need for nutrients to support the drive for proliferation in the face of a diminished extracellular supply. Among the many modifications occurring in tumour cells, hypoxia inducible factors (HIFs) act as essential drivers of key pro-survival pathways via the promotion of numerous membrane and cytosolic proteins. Here we focus our attention on two areas: the role of amino acid uptake and the handling of metabolic acid (CO2 /H+ ) production. We provide evidence for a number of hypoxia-induced proteins that promote cellular anabolism and regulation of metabolic acid-base levels in tumour cells including amino-acid transporters (LAT1), monocarboxylate transporters, and acid-base regulating carbonic anhydrases (CAs) and bicarbonate transporters (NBCs). Emphasis is placed on current work manipulating multiple CA isoforms and NBCs, which is at an interesting crossroads of gas physiology as they are regulated by hypoxia to contribute to the cellular handling of CO2 and pHi regulation. Our research combined with others indicates that targeting of HIF-regulated membrane proteins in tumour cells will provide promising future anti-cancer therapeutic approaches and we suggest strategies that could be potentially used to enhance these tactics.