The signaling pathways that govern survival response in hepatic cancer cells subjected to nutritional restriction have not been clarified yet. In this study we showed that liver cancer cells undergoing glucose deprivation both arrested in G0/G1 and died mainly by apoptosis. Treatment with the AMPK activator AICAR phenocopied the effect of glucose deprivation on cell survival, whereas AMPK silencing in HepG2/C3A, HuH-7 or SK-Hep-1 cells blocked the cell cycle arrest and the increase in apoptotic death induced by glucose starvation. Both AMPK and PKA were promptly activated after glucose withdrawal. PKA signaling had a dual role during glucose starvation: whereas it elicited an early decreased in cell viability, it later improved this parameter. We detected AMPK phosphorylation (AMPKα(Ser173)) by PKA, which was increased in glucose starved cells and was associated with diminution of AMPK activation. To better explore this inhibitory effect, we constructed a hepatocarcinoma derived cell line which stably expressed an AMPK mutant lacking that PKA phosphorylation site: AMPKα1(S173C). Expression of this mutant significantly decreased viability in cells undergoing glucose starvation. Furthermore, after 36 h of glucose deprivation, the index of AMPKα1(S173C) apoptotic cells doubled the apoptotic index observed in control cells. Two main remarks arise: 1. AMPK is the central signaling kinase in the scenario of cell cycle arrest and death induced by glucose starvation in hepatic cancer cells; 2. PKA phosphorylation of Ser173 comes out as a strong control point that limits the antitumor effects of AMPK in this situation.

Hepatocellular carcinoma (HCC) is a highly metastatic cancer with very poor prognosis. AMP activated kinase (AMPK) constitutes a candidate to inhibit HCC progression. First, AMPK is downregulated in HCC. Second, glucose starvation induces apoptosis in HCC cells via AMPK. Correspondingly, metformin activates AMPK and inhibits HCC cell proliferation. Nevertheless, the effect of AMPK activation on HCC cell invasiveness remains elusive. Here, migration/invasion was studied in HCC cells exposed to metformin and glucose starvation. Cell viability, proliferation and differentiation, as well as AMPK and PKA activation were analyzed. In addition, invasiveness in mutants of the AMPKα activation loop was assessed. Metformin decreased cell migration, invasion and epithelial-mesenchymal transition, and interference with AMPKα expression avoided metformin actions. Those antitumor effects were potentiated by glucose deprivation. Metformin activated AMPK at the same time that inhibited PKA, and both effects were enhanced by glucose starvation. Given that AMPKα(S173) phosphorylation by PKA decreases AMPK activation, we hypothesized that the reduction of PKA inhibitory effect by metformin could explain the increased antitumor effects observed. Supporting this, in AMPK activating conditions, cell migration/invasion was further impaired in AMPKα(S173C) mutant cells. Metformin emerges as a strong inhibitor of migration/invasion in HCC cells, and glucose restriction potentiates this effect.

The acquisition of a migratory phenotype is central in processes as diverse as embryo differentiation and tumor metastasis. An early event in this phenomenon is the generation of a nucleuscentrosome-Golgi back-to-front axis. AKAP350 (also known as AKAP9) is a Golgi and centrosome scaffold protein that is involved in microtubule nucleation. AKAP350 interacts with CIP4 (also known as TRIP10), a cdc42 effector that regulates actin dynamics. The present study aimed to characterize the participation of centrosomal AKAP350 in the acquisition of migratory polarity, and the involvement of CIP4 in the pathway. The decrease in total or in centrosomal AKAP350 led to decreased formation of the nucleus-centrosomeGolgi axis and defective cell migration. CIP4 localized at the centrosome, which was enhanced in migratory cells, but inhibited in cells with decreased centrosomal AKAP350. A decrease in the CIP4 expression or inhibition of the CIP4-AKAP350 interaction also led to defective cell polarization. Centrosome positioning, but not nuclear movement, was affected by loss of CIP4 or AKAP350 function. Our results support a model in which AKAP350 recruits CIP4 to the centrosome, providing a centrosomal scaffold to integrate microtubule and actin dynamics, thus enabling centrosome polarization and ensuring cell migration directionality.

Most epithelial cells contain apical membrane structures associated to bundles of actin filaments, which constitute the brush border. Whereas microtubule participation in the maintenance of the brush border identity has been characterized, their contribution to de novo microvilli organization remained elusive. Hereby, using a cell model of individual enterocyte polarization, we found that nocodazole induced microtubule depolymerization prevented the de novo brush border formation. Microtubule participation in brush border actin organization was confirmed in polarized kidney tubule MDCK cells. We also found that centrosome, but not Golgi derived microtubules, were essential for the initial stages of brush border development. During this process, microtubule plus ends acquired an early asymmetric orientation toward the apical membrane, which clearly differs from their predominant basal orientation in mature epithelia. In addition, overexpression of the microtubule plus ends associated protein CLIP170, which regulate actin nucleation in different cell contexts, facilitated brush border formation. In combination, the present results support the participation of centrosomal microtubule plus ends in the activation of the polarized actin organization associated to brush border formation, unveiling a novel mechanism of microtubule regulation of epithelial polarity.

Identification of a CIP4 PKA phosphorylation site involved in the regulation of cancer cell invasiveness and metastasis, Cancer Letters,

The organization of epithelial cells to form hollow organs with a single lumen requires the accurate three-dimensional arrangement of cell divisions. Mitotic spindle orientation is defined by signaling pathways that provide molecular links between specific spots at the cell cortex and astral microtubules, which have not been fully elucidated. AKAP350 is a centrosomal/Golgi scaffold protein, implicated in the regulation of microtubule dynamics. Using 3D epithelial cell cultures, we found that cells with decreased AKAP350 expression (AKAP350KD) formed polarized cysts with abnormal lumen morphology. Analysis of mitotic cells in AKAP350KD cysts indicated defective spindle alignment. We established that AKAP350 interacts with EB1, a microtubule associated protein that regulates spindle orientation, at the spindle poles. Decrease of AKAP350 expression lead to a significant reduction of EB1 levels at spindle poles and astral microtubules. Conversely, overexpression of EB1 rescued the defective spindle orientation induced by deficient AKAP350 expression. The specific delocalization of the AKAP350/EB1complex from the centrosome decreased EB1 levels at astral microtubules and lead to the formation of 3D-organotypic structures which resembled AKAP350KD cysts. We conclude that AKAP350 recruits EB1 to the spindle poles, ensuring EB1 presence at astral microtubules and proper spindle orientation during epithelial morphogenesis.

Cardiac function was measured in 16 prepubertal Ecuadorean patients with growth hormone receptor deficiency given insulin‐like growth factor I (IGF‐I) during part of a clinical trial. The IGF‐I was given subcutaneously twice daily at a dose of 40 μg/kg on days 1 and 2, 80 μg/kg on days 3 and 4, and 120 μg/kg thereafter. Heart rate was determined at baseline (pretreatment) and on days 1–7 by repeated palpation of the radial artery and at baseline and on days 2, 4 and 7 by continuous portable Holter monitoring. Heart rate measured by both methods rose progressively with increasing doses of IGF‐I. The mean palpated pulse exceeded baseline on each treatment day and was significantly higher on day 5 than day 4 and significantly higher on day 3 than day 2. The mean Holter heart rate was significantly higher on day 4 than on day 2 and significantly higher on day 2 than at baseline. Non‐significant glucose and electrolyte changes did not appear to be associated with the cardiac events.

Background and aims Non‐alcoholic fatty liver (NAFLD) and its more serious form non‐alcoholic steatohepatitis increase risk of hepatocellular carcinoma (HCC). Lipid metabolic alterations and its role in HCC development remain unclear. SPARC (Secreted Protein, Acidic and Rich in Cysteine) is involved in lipid metabolism, NAFLD and diabetes, but the effects on hepatic lipid metabolism and HCC development is unknown. The aim of this study was to evaluate the role of SPARC in HCC development in the context of NAFLD. Methods Primary hepatocyte cultures from knockout (SPARC−/−) or wild‐type (SPARC+/+) mice, and HepG2 cells were used to assess the effects of free fatty acids on lipid accumulation, expression of lipogenic genes and de novo triglyceride (TG) synthesis. A NAFLD‐HCC model was stabilized on SPARC−/− or SPARC+/+ mice. Correlations among SPARC, lipid metabolism‐related gene expression patterns and clinical prognosis were studied using HCC gene expression dataset. Results SPARC−/− mice increases hepatic lipid deposits over time. Hepatocytes from SPARC−/− mice or inhibition of SPARC by an antisense adenovirus in HepG2 cells resulted in increased TG deposit, expression of lipid‐related genes and nuclear translocation of SREBP1c. Human HCC database analysis revealed that SPARC negatively correlated with genes involved in lipid metabolism, and with poor survival. In NAFLD‐HCC murine model, the absence of SPARC accelerates HCC development. RNA‐seq study revealed that pathways related to lipid metabolism, cellular detoxification and proliferation were upregulated in SPARC−/− tumour‐bearing mice. Conclusions The absence of SPARC is associated with an altered hepatic lipid metabolism, and an accelerated NAFLD‐related HCC development.