Dynamic IL-6R/STAT3 signaling leads to heterogeneity of metabolic phenotype in pancreatic ductal adenocarcinoma cells

Malignancy is enabled by pro-growth mutations and adequate energy provision. However, global metabolic activation would be self-terminating if it depleted tumor resources. Cancer cells could avoid this by rationing resources, e.g., dynamically switching between “baseline” and “activated” metabolic states. Using single-cell metabolic phenotyping of pancreatic ductal adenocarcinoma cells, we identify MIA-PaCa-2 as having broad heterogeneity of fermentative metabolism. Sorting by a readout of lactic acid permeability separates cells by fermentative and respiratory rates. Contrasting phenotypes persist for 4 days and are unrelated to cell cycling or glycolytic/respiratory gene expression; however, transcriptomics links metabolically active cells with interleukin-6 receptor (IL-6R)-STAT3 signaling. We verify this by IL-6R/STAT3 knockdowns and sorting by IL-6R status. IL-6R/STAT3 activates fermentation and transcription of its inhibitor, SOCS3, resulting in delayed negative feedback that underpins transitions between metabolic states. Among cells manifesting wide metabolic heterogeneity, dynamic IL-6R/STAT3 signaling may allow cell cohorts to take turns in progressing energy-intense processes without depleting shared resources

Phenotypic screen of sixty-eight colorectal cancer cell lines identifies CEACAM6 and CEACAM5 as markers of acid-resistance

Elevated cancer metabolism releases lactic acid and CO2 into the under-perfused tumor microenvironment, resulting in extracellular acidosis. The surviving cancer cells must adapt to this selection pressure; thus, targeting tumor acidosis is a rational therapeutic strategy to manage tumor growth. However, none of the major approved treatments are based explicitly on disrupting acid handling, signaling, or adaptations, possibly because the distinction between acid-sensitive and acid-resistant phenotypes is not clear. Here, we report pH-related phenotypes of sixty-eight colorectal cancer (CRC) cell lines by measuring i) extracellular acidification as a readout of acid production by fermentative metabolism and ii) growth of cell biomass over a range of extracellular pH (pHe) levels as a measure of the acid sensitivity of proliferation. Based on these measurements, CRC cell lines were grouped along two dimensions as “acid-sensitive”/“acid-resistant” versus “low metabolic acid production”/“high metabolic acid production.” Strikingly, acid resistance was associated with the expression of CEACAM6 and CEACAM5 genes coding for two related cell-adhesion molecules, and among pH-regulating genes, of CA12. CEACAM5/6 protein levels were strongly induced by acidity, with a further induction under hypoxia in a subset of CRC lines. Lack of CEACAM6 (but not of CEACAM5) reduced cell growth and their ability to differentiate. Finally, CEACAM6 levels were strongly increased in human colorectal cancers from stage II and III patients, compared to matched samples from adjacent normal tissues. Thus, CEACAM6 is a marker of acid-resistant clones in colorectal cancer and a potential motif for targeting therapies to acidic regions within the tumors

Weight Loss and Sleep, Current Evidence in Animal Models and Humans

Sleep is a vital process essential for survival. The trend of reduction in the time dedicated to sleep has increased in industrialized countries, together with the dramatic increase in the prevalence of obesity and diabetes. Short sleep may increase the risk of obesity, diabetes and cardiovascular disease, and on the other hand, obesity is associated with sleep disorders, such as obstructive apnea disease, insomnia and excessive daytime sleepiness. Sleep and metabolic disorders are linked; therefore, identifying the physiological and molecular pathways involved in sleep regulation and metabolic homeostasis can play a major role in ameliorating the metabolic health of the individual. Approaches aimed at reducing body weight could provide benefits for both cardiometabolic risk and sleep quality, which indirectly, in turn, may determine an amelioration of the cardiometabolic phenotype of individuals. We revised the literature on weight loss and sleep, focusing on the mechanisms and the molecules that may subtend this relationship in humans as in animal models

Acid-adapted cancer cells alkalinize their cytoplasm by degrading the acid-loading membrane transporter Anion Exchanger 2 (SLC4A2)

Acidic environments reduce the intracellular pH (pHi) of most cells to levels that are sub-optimal for growth and cellular functions. Yet, cancers maintain an alkaline cytoplasm despite low extracellular pH (pHe). Raised pHi is thought to be beneficial for tumor progression and invasiveness. However, the transport mechanisms underpinning this adaptation have not been studied systematically. Here, we characterize the pHe-pHi relationships in 66 colorectal cancer cell lines and identify the acid-loading Anion Exchanger 2 (AE2, SLC4A2) as a regulator of resting pHi. Cells adapt to chronic extracellular acidosis by degrading AE2 protein, which raises pHi and reduces acid-sensitivity of growth. Acidity inhibits mTOR signaling, which stimulates lysosomal function and AE2 degradation, a process reversed by bafilomycin A1. We identify AE2 degradation as a mechanism for maintaining a conducive pHi in tumors. As an adaptive mechanism, inhibiting lysosomal degradation of AE2 is a potential therapeutic target

Correctors of mutant CFTR enhance subcortical cAMP-PKA signaling through modulating ezrin phosphorylation and cytoskeleton organization

The most common mutation of the cystic fibrosis transmembrane regulator (CFTR) gene, F508del, produces a misfolded protein resulting in its defective trafficking to the cell surface and an impaired chloride secretion. Pharmacological treatments partially rescue F508del CFTR activity either directly by interacting with the mutant protein and/or indirectly by altering the cellular protein homeostasis. Here, we show that the phosphorylation of ezrin together with its binding to phosphatidylinositol-4,5-bisphosphate (PIP2) tethers the F508del CFTR to the actin cytoskeleton, stabilizing it on the apical membrane and rescuing the sub-membrane compartmentalization of cAMP and activated PKA. Both the small molecules trimethylangelicin (TMA) and VX-809, which act as 'correctors' for F508del CFTR by rescuing F508del-CFTR-dependent chloride secretion, also restore the apical expression of phosphorylated ezrin and actin organization and increase cAMP and activated PKA submembrane compartmentalization in both primary and secondary cystic fibrosis airway cells. Latrunculin B treatment or expression of the inactive ezrin mutant T567A reverse the TMA and VX-809-induced effects highlighting the role of corrector-dependent ezrin activation and actin re-organization in creating the conditions to generate a sub-cortical cAMP pool of adequate amplitude to activate the F508del-CFTR-dependent chloride secretion

PKA and PDE4D3 anchoring to AKAP9 provides distinct regulation of cAMP signals at the centrosome

Previous work has shown that the protein kinase A (PKA)–regulated phosphodiesterase (PDE) 4D3 binds to A kinase–anchoring proteins (AKAPs). One such protein, AKAP9, localizes to the centrosome. In this paper, we investigate whether a PKA–PDE4D3–AKAP9 complex can generate spatial compartmentalization of cyclic adenosine monophosphate (cAMP) signaling at the centrosome. Real-time imaging of fluorescence resonance energy transfer reporters shows that centrosomal PDE4D3 modulated a dynamic microdomain within which cAMP concentration selectively changed over the cell cycle. AKAP9-anchored, centrosomal PKA showed a reduced activation threshold as a consequence of increased autophosphorylation of its regulatory subunit at S114. Finally, disruption of the centrosomal cAMP microdomain by local displacement of PDE4D3 impaired cell cycle progression as a result of accumulation of cells in prophase. Our findings describe a novel mechanism of PKA activity regulation that relies on binding to AKAPs and consequent modulation of the enzyme activation threshold rather than on overall changes in cAMP levels. Further, we provide for the first time direct evidence that control of cell cycle progression relies on unique regulation of centrosomal cAMP/PKA signals

HPV16 E7-Dependent Transformation Activates NHE1 through a PKA-RhoA-Iinduced Inhibition of p38alpha

Background: Neoplastic transformation originates from a large number of different genetic alterations. Despite this genetic variability, a common phenotype to transformed cells is cellular alkalinization. We have previously shown in human keratinocytes and a cell line in which transformation can be turned on and followed by the inducible expression of the E7 oncogene of human papillomavirus type 16 (HPV16), that intracellular alkalinization is an early and essential physiological event driven by the up-regulation of the Na/H-+(+) exchanger isoform 1 (NHE1) and is necessary for the development of other transformed phenotypes and the in vivo tumor formation in nude mice.Methodology: Here, we utilize these model systems to elucidate the dynamic sequence of alterations of the upstream signal transduction systems leading to the transformation-dependent activation of NHE1.Principal Findings: We observe that a down-regulation of p38 MAPK activity is a fundamental step in the ability of the oncogene to transform the cell. Further, using pharmacological agents and transient transfections with dominant interfering, constitutively active, phosphorylation negative mutants and siRNA strategy to modify specific upstream signal transduction components that link HPV16 E7 oncogenic signals to up-regulation of the NHE1, we demonstrate that the stimulation of NHE1 activity is driven by an early rise in cellular cAMP resulting in the down-stream inhibition of p38 MAPK via the PKA-dependent phosphorylation of the small G-protein, RhoA, and its subsequent inhibition.Conclusions: All together these data significantly improve our knowledge concerning the basic cellular alterations involved in oncogene-driven neoplastic transformation

A Barter Economy in Tumors: Exchanging Metabolites through Gap Junctions

To produce physiological functions, many tissues require their cells to be connected by gap junctions. Such diffusive coupling is important in establishing a cytoplasmic syncytium through which cells can exchange signals, substrates and metabolites. Often the benefits of connectivity become apparent solely at the multicellular level, leading to the notion that cells work for a common good rather than exclusively in their self-interest. In some tumors, gap junctional connectivity between cancer cells is reduced or absent, but there are notable cases where it persists or re-emerges in late-stage disease. Diffusive coupling will blur certain phenotypic differences between cells, which may seem to go against the establishment of population heterogeneity, a central pillar of cancer that stems from genetic instability. Here, building on our previous measurements of gap junctional coupling between cancer cells, we use a computational model to simulate the role of connexin-assembled channels in exchanging lactate and bicarbonate ions down their diffusion gradients. Based on the results of these simulations, we propose that an overriding benefit of gap junctional connectivity may relate to lactate/bicarbonate exchange, which would support an elevated metabolic rate in hypoxic tumors. In this example of barter, hypoxic cancer cells provide normoxic neighbors with lactate for mitochondrial oxidation; in exchange, bicarbonate ions, which are more plentiful in normoxic cells, are supplied to hypoxic neighbors to neutralize the H+ ions co-produced glycolytically. Both cells benefit, and so does the tumor

Dynamic IL-6R/STAT3 signaling leads to heterogeneity of metabolic phenotype in pancreatic ductal adenocarcinoma cells

Summary: Malignancy is enabled by pro-growth mutations and adequate energy provision. However, global metabolic activation would be self-terminating if it depleted tumor resources. Cancer cells could avoid this by rationing resources, e.g., dynamically switching between “baseline” and “activated” metabolic states. Using single-cell metabolic phenotyping of pancreatic ductal adenocarcinoma cells, we identify MIA-PaCa-2 as having broad heterogeneity of fermentative metabolism. Sorting by a readout of lactic acid permeability separates cells by fermentative and respiratory rates. Contrasting phenotypes persist for 4 days and are unrelated to cell cycling or glycolytic/respiratory gene expression; however, transcriptomics links metabolically active cells with interleukin-6 receptor (IL-6R)-STAT3 signaling. We verify this by IL-6R/STAT3 knockdowns and sorting by IL-6R status. IL-6R/STAT3 activates fermentation and transcription of its inhibitor, SOCS3, resulting in delayed negative feedback that underpins transitions between metabolic states. Among cells manifesting wide metabolic heterogeneity, dynamic IL-6R/STAT3 signaling may allow cell cohorts to take turns in progressing energy-intense processes without depleting shared resources