Regulatory chromatin rewiring promotes metabolic switching during adaptation to oncogenic receptor tyrosine kinase inhibition.

Oesophageal adenocarcinoma (OAC) patients show poor survival rates and there are few targeted molecular therapies available. However, components of the receptor tyrosine kinase (RTK) driven pathways are commonly mutated in OAC, typified by high frequency amplifications of the RTK ERBB2. ERBB2 can be therapeutically targeted, but this has limited clinical benefit due to the acquisition of drug resistance. Here we examined how OAC cells adapt to ERBB2 inhibition as they transition to a drug resistant state. ERBB2 inhibition triggers widespread remodelling of the accessible chromatin landscape and the underlying gene regulatory networks. The transcriptional regulators HNF4A and PPARGC1A play a key role in this network rewiring. Initially, inhibition of cell cycle associated gene expression programmes is observed, with compensatory increases in the programmes driving changes in metabolic activity. Both PPARGC1A and HNF4A are required for the acquisition of resistance to ERBB2 inhibition and PPARGC1A is instrumental in promoting a switch to dependency on oxidative phosphorylation. Our work therefore reveals the molecular pathways that support the acquisition of a resistant state and points to potential new therapeutic strategies to combat cellular adaptation and ensuing drug resistance.

Insulin protects acinar cells during pancreatitis by preserving glycolytic ATP supply to calcium pumps.

Acute pancreatitis (AP) is serious inflammatory disease of the pancreas. Accumulating evidence links diabetes with severity of AP, suggesting that endogenous insulin may be protective. We investigated this putative protective effect of insulin during cellular and in vivo models of AP in diabetic mice (Ins2Akita) and Pancreatic Acinar cell-specific Conditional Insulin Receptor Knock Out mice (PACIRKO). Caerulein and palmitoleic acid (POA)/ethanol-induced pancreatitis was more severe in both Ins2Akita and PACIRKO vs control mice, suggesting that endogenous insulin directly protects acinar cells in vivo. In isolated pancreatic acinar cells, insulin induced Akt-mediated phosphorylation of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 2 (PFKFB2) which upregulated glycolysis thereby preventing POA-induced ATP depletion, inhibition of the ATP-dependent plasma membrane Ca2+ ATPase (PMCA) and cytotoxic Ca2+ overload. These data provide the first mechanistic link between diabetes and severity of AP and suggest that phosphorylation of PFKFB2 may represent a potential therapeutic strategy for treatment of AP.

Targeting the Calcium Signalling Machinery in Cancer.

Cancer is caused by excessive cell proliferation and a propensity to avoid cell death, while the spread of cancer is facilitated by enhanced cellular migration, invasion, and vascularization. Cytosolic Ca2+ is central to each of these important processes, yet to date, there are no cancer drugs currently being used clinically, and very few undergoing clinical trials, that target the Ca2+ signalling machinery. The aim of this review is to highlight some of the emerging evidence that targeting key components of the Ca2+ signalling machinery represents a novel and relatively untapped therapeutic strategy for the treatment of cancer.

Plasma Membrane Ca2+ ATPase Isoform 4 (PMCA4) Has an Important Role in Numerous Hallmarks of Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is largely resistant to standard treatments leading to poor patient survival. The expression of plasma membrane calcium ATPase-4 (PMCA4) is reported to modulate key cancer hallmarks including cell migration, growth, and apoptotic resistance. Data-mining revealed that PMCA4 was over-expressed in pancreatic ductal adenocarcinoma (PDAC) tumors which correlated with poor patient survival. Western blot and RT-qPCR revealed that MIA PaCa-2 cells almost exclusively express PMCA4 making these a suitable cellular model of PDAC with poor patient survival. Knockdown of PMCA4 in MIA PaCa-2 cells (using siRNA) reduced cytosolic Ca2+ ([Ca2+]i) clearance, cell migration, and sensitized cells to apoptosis, without affecting cell growth. Knocking down PMCA4 had minimal effects on numerous metabolic parameters (as assessed using the Seahorse XF analyzer). In summary, this study provides the first evidence that PMCA4 is over-expressed in PDAC and plays a role in cell migration and apoptotic resistance in MIA PaCa-2 cells. This suggests that PMCA4 may offer an attractive novel therapeutic target in PDAC.

Cutting off the fuel supply to calcium pumps in pancreatic cancer cells: role of pyruvate kinase-M2 (PKM2).

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) has poor survival and treatment options. PDAC cells shift their metabolism towards glycolysis, which fuels the plasma membrane calcium pump (PMCA), thereby preventing Ca2+-dependent cell death. The ATP-generating pyruvate kinase-M2 (PKM2) is oncogenic and overexpressed in PDAC. This study investigated the PKM2-derived ATP supply to the PMCA as a potential therapeutic locus. METHODS: PDAC cell growth, migration and death were assessed by using sulforhodamine-B/tetrazolium-based assays, gap closure assay and poly-ADP ribose polymerase (PARP1) cleavage, respectively. Cellular ATP and metabolism were assessed using luciferase/fluorescent-based assays and the Seahorse XFe96 analyzer, respectively. Cell surface biotinylation identified membrane-associated proteins. Fura-2 imaging was used to assess cytosolic Ca2+ overload and in situ Ca2+ clearance. PKM2 knockdown was achieved using siRNA. RESULTS: The PKM2 inhibitor (shikonin) reduced PDAC cell proliferation, cell migration and induced cell death. This was due to inhibition of glycolysis, ATP depletion, inhibition of PMCA and cytotoxic Ca2+ overload. PKM2 associates with plasma membrane proteins providing a privileged ATP supply to the PMCA. PKM2 knockdown reduced PMCA activity and reduced the sensitivity of shikonin-induced cell death. CONCLUSIONS: Cutting off the PKM2-derived ATP supply to the PMCA represents a novel therapeutic strategy for the treatment of PDAC.

Metabolic regulation of the PMCA: Role in cell death and survival.

The plasma membrane Ca2+-ATPase (PMCA) is a ubiquitously expressed, ATP-driven Ca2+ pump that is critical for maintaining low resting cytosolic Ca2+ ([Ca2+]i) in all eukaryotic cells. Since cytotoxic Ca2+ overload has such a central role in cell death, the PMCA represents an essential "linchpin" for the delicate balance between cell survival and cell death. In general, impaired PMCA activity and reduced PMCA expression leads to cytotoxic Ca2+ overload and Ca2+ dependent cell death, both apoptosis and necrosis, whereas maintenance of PMCA activity or PMCA overexpression is generally accepted as being cytoprotective. However, the PMCA has a paradoxical role in cell death depending on the cell type and cellular context. The PMCA can be differentially regulated by Ca2+-dependent proteolysis, can be maintained by a localised glycolytic ATP supply, even in the face of global ATP depletion, and can be profoundly affected by the specific phospholipid environment that it sits within the membrane. The major focus of this review is to highlight some of the controversies surrounding the paradoxical role of the PMCA in cell death and survival, challenging the conventional view of ATP-dependent regulation of the PMCA and how this might influence cell fate.

Chronic Pancreatitis in the 21st Century - Research Challenges and Opportunities: Summary of a National Institute of Diabetes and Digestive and Kidney Diseases Workshop.

A workshop was sponsored by the National Institute of Diabetes and Digestive and Kidney Diseases to focus on research gaps and opportunities in chronic pancreatitis (CP) and its sequelae. This conference marked the 20th year anniversary of the discovery of the cationic trypsinogen (PRSS1) gene mutation for hereditary pancreatitis. The event was held on July 27, 2016, and structured into 4 sessions: (1) pathophysiology, (2) exocrine complications, (3) endocrine complications, and (4) pain. The current state of knowledge was reviewed; many knowledge gaps and research needs were identified that require further investigation. Common themes included the need to design better tools to diagnose CP and its sequelae early and reliably, identify predisposing risk factors for disease progression, develop standardized protocols to distinguish type 3c diabetes mellitus from other types of diabetes, and design effective therapeutic strategies through novel cell culture technologies, animal models mimicking human disease, and pain management tools. Gene therapy and cystic fibrosis conductance regulator potentiators as possible treatments of CP were discussed. Importantly, the need for CP end points and intermediate targets for future drug trials was emphasized.

Glucose metabolic phenotype of pancreatic cancer.

AIM: To construct a global "metabolic phenotype" of pancreatic ductal adenocarcinoma (PDAC) reflecting tumour-related metabolic enzyme expression. METHODS: A systematic review of the literature was performed using OvidSP and PubMed databases using keywords "pancreatic cancer" and individual glycolytic and mitochondrial oxidative phosphorylation (MOP) enzymes. Both human and animal studies investigating the oncological effect of enzyme expression changes and inhibitors in both an in vitro and in vivo setting were included in the review. Data reporting changes in enzyme expression and the effects on PDAC cells, such as survival and metastatic potential, were extracted to construct a metabolic phenotype. RESULTS: Seven hundred and ten papers were initially retrieved, and were screened to meet the review inclusion criteria. 107 unique articles were identified as reporting data involving glycolytic enzymes, and 28 articles involving MOP enzymes in PDAC. Data extraction followed a pre-defined protocol. There is consistent over-expression of glycolytic enzymes and lactate dehydrogenase in keeping with the Warburg effect to facilitate rapid adenosine-triphosphate production from glycolysis. Certain isoforms of these enzymes were over-expressed specifically in PDAC. Altering expression levels of HK, PGI, FBA, enolase, PK-M2 and LDA-A with metabolic inhibitors have shown a favourable effect on PDAC, thus identifying these as potential therapeutic targets. However, the Warburg effect on MOP enzymes is less clear, with different expression levels at different points in the Krebs cycle resulting in a fundamental change of metabolite levels, suggesting that other essential anabolic pathways are being stimulated. CONCLUSION: Further characterisation of the PDAC metabolic phenotype is necessary as currently there are few clinical studies and no successful clinical trials targeting metabolic enzymes.

The Plasma Membrane Calcium Pump in Pancreatic Cancer Cells Exhibiting the Warburg Effect Relies on Glycolytic ATP.

Evidence suggests that the plasma membrane Ca(2+)-ATPase (PMCA), which is critical for maintaining a low intracellular Ca(2+) concentration ([Ca(2+)]i), utilizes glycolytically derived ATP in pancreatic ductal adenocarcinoma (PDAC) and that inhibition of glycolysis in PDAC cell lines results in ATP depletion, PMCA inhibition, and an irreversible [Ca(2+)]i overload. We explored whether this is a specific weakness of highly glycolytic PDAC by shifting PDAC cell (MIA PaCa-2 and PANC-1) metabolism from a highly glycolytic phenotype toward mitochondrial metabolism and assessing the effects of mitochondrial versus glycolytic inhibitors on ATP depletion, PMCA inhibition, and [Ca(2+)]i overload. The highly glycolytic phenotype of these cells was first reversed by depriving MIA PaCa-2 and PANC-1 cells of glucose and supplementing with α-ketoisocaproate or galactose. These culture conditions resulted in a significant decrease in both glycolytic flux and proliferation rate, and conferred resistance to ATP depletion by glycolytic inhibition while sensitizing cells to mitochondrial inhibition. Moreover, in direct contrast to cells exhibiting a high glycolytic rate, glycolytic inhibition had no effect on PMCA activity and resting [Ca(2+)]i in α-ketoisocaproate- and galactose-cultured cells, suggesting that the glycolytic dependence of the PMCA is a specific vulnerability of PDAC cells exhibiting the Warburg phenotype.

Insulin protects pancreatic acinar cells from palmitoleic acid-induced cellular injury.

Acute pancreatitis is a serious and sometimes fatal inflammatory disease where the pancreas digests itself. The non-oxidative ethanol metabolites palmitoleic acid (POA) and POA-ethylester (POAEE) are reported to induce pancreatitis caused by impaired mitochondrial metabolism, cytosolic Ca(2+) ([Ca(2+)]i) overload and necrosis of pancreatic acinar cells. Metabolism and [Ca(2+)]i are linked critically by the ATP-driven plasma membrane Ca(2+)-ATPase (PMCA) important for maintaining low resting [Ca(2+)]i. The aim of the current study was to test the protective effects of insulin on cellular injury induced by the pancreatitis-inducing agents, ethanol, POA, and POAEE. Rat pancreatic acinar cells were isolated by collagenase digestion and [Ca(2+)]i was measured by fura-2 imaging. An in situ [Ca(2+)]i clearance assay was used to assess PMCA activity. Magnesium green (MgGreen) and a luciferase-based ATP kit were used to assess cellular ATP depletion. Ethanol (100 mM) and POAEE (100 µM) induced a small but irreversible Ca(2+) overload response but had no significant effect on PMCA activity. POA (50-100 µM) induced a robust Ca(2+) overload, ATP depletion, inhibited PMCA activity, and consequently induced necrosis. Insulin pretreatment (100 nm for 30 min) prevented the POA-induced Ca(2+) overload, ATP depletion, inhibition of the PMCA, and necrosis. Moreover, the insulin-mediated protection of the POA-induced Ca(2+) overload was partially prevented by the phosphoinositide-3-kinase (PI3K) inhibitor, LY294002. These data provide the first evidence that insulin directly protects pancreatic acinar cell injury induced by bona fide pancreatitis-inducing agents, such as POA. This may have important therapeutic implications for the treatment of pancreatitis.

The inhibitory effect of simvastatin and aspirin on histamine responsiveness in human vascular endothelial cells.

Statins and aspirin deliver well-established cardiovascular benefits resulting in their increased use as combined polypills to decrease risk of stroke and heart disease. However, the direct endothelial effect of combined statin/aspirin cotreatment remains unclear. Histamine is an inflammatory mediator that increases vascular permeability, and so we examined the effect of treating human umbilical vein endothelial cells (HUVECs) for 24 h with 1 µM simvastatin and 100 µM aspirin on histamine responsiveness. Subsequent histamine (1 µM) challenge increased intracellular calcium (Ca(2+)i) concentration, an effect that was significantly inhibited by combined simvastatin/aspirin pretreatment but not when then the compounds were given separately, even at 10-fold higher concentrations. In contrast, the Ca(2+)i mobilization response to ATP challenge (10 µM) was not inhibited by combined simvastatin/aspirin pretreatment. The H1 receptor antagonist pyrilamine significantly inhibited both histamine-induced Ca(2+)i mobilization and extracellular signal-regulated kinase (ERK) activation, whereas ranitidine (H2 receptor antagonist) was without effect. However, combined simvastatin/aspirin pretreatment failed to decrease H1 receptor protein expression ruling out receptor downregulation as the mechanism of action. Histamine-induced ERK activation was also inhibited by atorvastatin pretreatment, while simvastatin further inhibited histamine-induced vascular endothelial cadherin phosphorylation as well as altered HUVEC morphology and inhibited actin polymerization. Therefore, in addition to the known therapeutic benefits of statins and aspirin, here we provide initial cellular evidence that combined statin/aspirin treatment inhibits histamine responsiveness in HUVECs.

Glycolytic ATP fuels the plasma membrane calcium pump critical for pancreatic cancer cell survival.

Pancreatic cancer is an aggressive cancer with poor prognosis and limited treatment options. Cancer cells rapidly proliferate and are resistant to cell death due, in part, to a shift from mitochondrial metabolism to glycolysis. We hypothesized that this shift is important in regulating cytosolic Ca(2+) ([Ca(2+)]i), as the ATP-dependent plasma membrane Ca(2+) ATPase (PMCA) is critical for maintaining low [Ca(2+)]i and thus cell survival. The present study aimed to determine the relative contribution of mitochondrial versus glycolytic ATP in fuelling the PMCA in human pancreatic cancer cells. We report that glycolytic inhibition induced profound ATP depletion, PMCA inhibition, [Ca(2+)]i overload, and cell death in PANC1 and MIA PaCa-2 cells. Conversely, inhibition of mitochondrial metabolism had no effect, suggesting that glycolytic ATP is critical for [Ca(2+)]i homeostasis and thus survival. Targeting the glycolytic regulation of the PMCA may, therefore, be an effective strategy for selectively killing pancreatic cancer while sparing healthy cells.

Insulin protects pancreatic acinar cells from cytosolic calcium overload and inhibition of plasma membrane calcium pump.

Acute pancreatitis is a serious and sometimes fatal inflammatory disease of the pancreas without any reliable treatment or imminent cure. In recent years, impaired metabolism and cytosolic Ca(2+) ([Ca(2+)](i)) overload in pancreatic acinar cells have been implicated as the cardinal pathological events common to most forms of pancreatitis, regardless of the precise causative factor. Therefore, restoration of metabolism and protection against cytosolic Ca(2+) overload likely represent key therapeutic untapped strategies for the treatment of this disease. The plasma membrane Ca(2+)-ATPase (PMCA) provides a final common path for cells to "defend" [Ca(2+)](i) during cellular injury. In this paper, we use fluorescence imaging to show for the first time that insulin treatment, which is protective in animal models and clinical studies of human pancreatitis, directly protects pancreatic acinar cells from oxidant-induced cytosolic Ca(2+) overload and inhibition of the PMCA. This protection was independent of oxidative stress or mitochondrial membrane potential but appeared to involve the activation of Akt and an acute metabolic switch from mitochondrial to predominantly glycolytic metabolism. This switch to glycolysis appeared to be sufficient to maintain cellular ATP and thus PMCA activity, thereby preventing Ca(2+) overload, even in the face of impaired mitochondrial function.

Calcium-activated K+ channels increase cell proliferation independent of K+ conductance.

The intermediate-conductance calcium-activated potassium channel (IK1) promotes cell proliferation of numerous cell types including endothelial cells, T lymphocytes, and several cancer cell lines. The mechanism underlying IK1-mediated cell proliferation was examined in human embryonic kidney 293 (HEK293) cells expressing recombinant human IK1 (hIK1) channels. Inhibition of hIK1 with TRAM-34 reduced cell proliferation, while expression of hIK1 in HEK293 cells increased proliferation. When HEK293 cells were transfected with a mutant (GYG/AAA) hIK1 channel, which neither conducts K(+) ions nor promotes Ca(2+) entry, proliferation was increased relative to mock-transfected cells. Furthermore, when HEK293 cells were transfected with a trafficking mutant (L18A/L25A) hIK1 channel, proliferation was also increased relative to control cells. The lack of functional activity of hIK1 mutants at the cell membrane was confirmed by a combination of whole cell patch-clamp electrophysiology and fura-2 imaging to assess store-operated Ca(2+) entry and cell surface immunoprecipitation assays. Moreover, in cells expressing hIK1, inhibition of ERK1/2 and JNK kinases, but not of p38 MAP kinase, reduced cell proliferation. We conclude that functional K(+) efflux at the plasma membrane and the consequent hyperpolarization and enhanced Ca(2+) entry are not necessary for hIK1-induced HEK293 cell proliferation. Rather, our data suggest that hIK1-induced proliferation occurs by a direct interaction with ERK1/2 and JNK signaling pathways.

Differential regulation of calcium-activated potassium channels by dynamic intracellular calcium signals.

Calcium (Ca(2+))-activated K(+) (K(Ca)) channels regulate membrane excitability and are activated by an increase in cytosolic Ca(2+) concentration ([Ca(2+)](i)), leading to membrane hyperpolarization. Most patch clamp experiments that measure K(Ca) currents use steady-state [Ca(2+)] buffered within the patch pipette. However, when cells are stimulated physiologically, [Ca(2+)](i) changes dynamically, for example during [Ca(2+)](i) oscillations. Therefore, the aim of the present study was to examine the effect of dynamic changes in [Ca(2+)](i) on small (SK3), intermediate (hIK1), and large conductance (BK) channels. HEK293 cells stably expressing each K(Ca) subtype in isolation were used to simultaneously measure agonist-evoked [Ca(2+)](i) signals, using indo-1 fluorescence, and current/voltage, using perforated patch clamp. Agonist-evoked [Ca(2+)](i) oscillations induced a corresponding K(Ca) current that faithfully followed the [Ca(2+)](i) in 13-50% of cells, suggesting a good synchronization. However, [Ca(2+)](i) and K(Ca) current was much less synchronized in 50-76% of cells that exhibited Ca(2+)-independent current events (55% of SK3-, 50% of hIK1-, and 53% of BK-expressing cells) and current-independent [Ca(2+)](i) events (18% SK3- and 33% of BK-expressing cells). Moreover, in BK-expressing cells, where [Ca(2+)](i) and K(Ca) current was least synchronized, 36% of total [Ca(2+)](i) spikes occurred without activating a corresponding K(Ca) current spike, suggesting that BK(Ca) channels were either inhibited or had become desensitized. This desynchronization between dynamic [Ca(2+)](i) and K(Ca) current suggests that this relationship is more complex than could be predicted from steady-state [Ca(2+)](i) and K(Ca) current. These phenomena may be important for encoding stimulus-response coupling in various cell types.

Plasma membrane calcium pump regulation by metabolic stress.

The plasma membrane Ca(2+)-ATPase (PMCA) is an ATP-driven pump that is critical for the maintenance of low resting [Ca(2+)](i) in all eukaryotic cells. Metabolic stress, either due to inhibition of mitochondrial or glycolytic metabolism, has the capacity to cause ATP depletion and thus inhibit PMCA activity. This has potentially fatal consequences, particularly for non-excitable cells in which the PMCA is the major Ca(2+) efflux pathway. This is because inhibition of the PMCA inevitably leads to cytosolic Ca(2+) overload and the consequent cell death. However, the relationship between metabolic stress, ATP depletion and inhibition of the PMCA is not as simple as one would have originally predicted. There is increasing evidence that metabolic stress can lead to the inhibition of PMCA activity independent of ATP or prior to substantial ATP depletion. In particular, there is evidence that the PMCA has its own glycolytic ATP supply that can fuel the PMCA in the face of impaired mitochondrial function. Moreover, membrane phospholipids, mitochondrial membrane potential, caspase/calpain cleavage and oxidative stress have all been implicated in metabolic stress-induced inhibition of the PMCA. The major focus of this review is to challenge the conventional view of ATP-dependent regulation of the PMCA and bring together some of the alternative or additional mechanisms by which metabolic stress impairs PMCA activity resulting in cytosolic Ca(2+) overload and cytotoxicity.

Oxidant-induced inhibition of the plasma membrane Ca2+-ATPase in pancreatic acinar cells: role of the mitochondria.

Impairment of the normal spatiotemporal pattern of intracellular Ca(2+) ([Ca(2+)](i)) signaling, and in particular, the transition to an irreversible "Ca(2+) overload" response, has been implicated in various pathophysiological states. In some diseases, including pancreatitis, oxidative stress has been suggested to mediate this Ca(2+) overload and the associated cell injury. We have previously demonstrated that oxidative stress with hydrogen peroxide (H(2)O(2)) evokes a Ca(2+) overload response and inhibition of plasma membrane Ca(2+)-ATPase (PMCA) in rat pancreatic acinar cells (Bruce JI and Elliott AC. Am J Physiol Cell Physiol 293: C938-C950, 2007). The aim of the present study was to further examine this oxidant-impaired inhibition of the PMCA, focusing on the role of the mitochondria. Using a [Ca(2+)](i) clearance assay in which mitochondrial Ca(2+) uptake was blocked with Ru-360, H(2)O(2) (50 microM-1 mM) markedly inhibited the PMCA activity. This H(2)O(2)-induced inhibition of the PMCA correlated with mitochondrial depolarization (assessed using tetramethylrhodamine methylester fluorescence) but could occur without significant ATP depletion (assessed using Magnesium Green fluorescence). The H(2)O(2)-induced PMCA inhibition was sensitive to the mitochondrial permeability transition pore (mPTP) inhibitors, cyclosporin-A and bongkrekic acid. These data suggest that oxidant-induced opening of the mPTP and mitochondrial depolarization may lead to an inhibition of the PMCA that is independent of mitochondrial Ca(2+) handling and ATP depletion, and we speculate that this may involve the release of a mitochondrial factor. Such a phenomenon may be responsible for the Ca(2+) overload response, and for the transition between apoptotic and necrotic cell death thought to be important in many disease states.