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 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.

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.

Glucose deprivation entails oxidative stress and apoptosis in diverse cell types. Liver tissue shows high tolerance to nutritional stress, however regulation of survival in normal hepatocytes subjected to glucose restriction is unclear. We assessed the survival response of cultured hepatocytes subjected to glucose deprivation and analyzed the putative participation of protein kinase A (PKA) in this response. Six hours glucose deprivation induced a PKA dependent activation of apoptosis in cultured hepatocytes, without having an impact on non apoptotic death. Apoptotic activation associated to glucose restriction was secondary to an imbalance in cellular reactive oxygen species (ROS). In this condition, PKA inhibition led to an early prevention in mitochondrial ROS production and a late increase in scavenging enzymes transcript levels. These results supported the hypothesis that PKA could modulate glucose deprivation induced apoptotic activation by conditioning mitochondrial ROS production during glucose fasting. We presented additional evidence sustaining this model: First, glucose withdrawal led to a 95% increase in mitochondrial cAMP levels in cultured hepatocytes; second, activation of PKA significantly augmented hepatic mitochondrial ROS generation, whereas PKA inhibition elicited the opposite effect. Mitochondrial PKA signaling, previously proposed as an autonomic pathway adjusting respiration rate, emerges as a mechanism of controlling cell survival during glucose restriction.