The virus-induced protein APOBEC3G inhibits anoikis by activation of Akt kinase in pancreatic cancer cells.
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ABSTRACT:
SUBMITTER: Wu J
PROVIDER: S-EPMC4503957 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:HMGA1 proteins are architectural transcription factors that are overexpressed by pancreatic adenocarcinomas. Roles of HMGA1 in mediating the malignant phenotype of this cancer are poorly understood. We tested the hypothesis that overexpression of HMGA1 promotes resistance to anoikis (apoptosis induced by anchorage deprivation) in pancreatic cancer cells. HMGA1 cDNA was stably transfected into MiaPaCa2 human pancreatic adenocarcinoma cells (which have low baseline expression levels of HMGA1). Cells were grown in suspension on PolyHEMA-coated plates and their susceptibility to anoikis was assayed using flow cytometry. Overexpression of HMGA1 was associated with marked reductions in susceptibility to anoikis in concert with increases in Akt phosphorylation (Ser473) and in Akt kinase activity and with reductions in caspase 3 activation. Inhibition of phosphoinositidyl-3 (PI3-K)/Akt pathway with either the small molecule inhibitor LY294002 or dominant-negative Akt resulted in reversal of anoikis resistance induced by HMGA1 overexpression. Further, RNA interference-mediated HMGA1 silencing in MiaPaCa2 and BxPC3 (a human pancreatic adenocarcinoma cell line with high baseline levels of HMGA1 expression) cells resulted in significant increases in susceptibility to anoikis. Our findings suggest HMGA1 promotes anoikis resistance through a PI3-K/Akt-dependent mechanism. Given the putative associations between anoikis resistance and metastatic potential, HMGA1 represents a potential therapeutic target in pancreatic adenocarcinoma.
| S-EPMC2360112 | biostudies-literature
Project description:ObjectivesTobacco-derived carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) activates β-adrenergic receptor (β-AR) signaling through Src/focal adhesion kinases (FAKs)/mitogen-activated protein kinase to modulate proliferation, migration, and survival. Apigenin (4', 5, 7-trihydroxyflavone) is reported to attenuate proliferation and migration of cancer cells. This study was designed to determine the effects of apigenin on NNK-induced procarcinogenesis using human pancreatic cancer cells BxPC-3 and MIA PaCa-2, which express β-AR.MethodsProliferation and migration were assessed by standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and scratch assays. β-AR, FAK/mitogen-activated protein kinase and extracellular signal-regulated kinase (ERK) expression and activation were assessed by Western blotting and real-time polymerase chain reaction.Results4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone caused a dose- and time-dependent increase in BxPC-3 and MIA PaCa-2 cell proliferation that was inhibited by propranolol or apigenin. 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone also stimulated a time-dependent increase in FAK and ERK activation that was suppressed by propranolol or apigenin. 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone-enhanced gap closure at 24 hours was prevented by either propranolol or apigenin.ConclusionApigenin suppressed the effects of NNK on pancreatic cancer cell proliferation and migration that are mediated through the β-AR and its downstream signals FAK and ERK activation. These findings suggest a therapeutic role for this natural phytochemical in attenuating the procarcinogenic effects of NNK on pancreatic cancer proliferation and migration.
| S-EPMC3479318 | biostudies-literature
Project description:Ca2+/calmodulin-dependent protein kinase II (CaMKII), which is involved in the calcium signaling pathway, is an important regulator of cancer cell proliferation, motility, growth, and metastasis. The effects of CaMKII on hepatitis B virus (HBV) replication have never been evaluated. Here, we found that phosphorylated, active CaMKII is reduced during HBV replication. Similar to other members of the AMPK/AKT/mTOR signaling pathway associated with HBV replication, CaMKII, which is associated with this pathway, was found to be a novel regulator of HBV replication. Overexpression of CaMKII reduced the expression of covalently closed circular DNA (cccDNA), HBV RNAs, and replicative intermediate (RI) DNAs while activating AMPK and inhibiting the AKT/mTOR signaling pathway. Findings in HBx-deficient mutant-transfected HepG2 cells showed that the CaMKII-mediated AMPK/AKT/mTOR signaling pathway was independent of HBx. Moreover, AMPK overexpression reduced HBV cccDNA, RNAs, and RI DNAs through CaMKII activation. Although AMPK acts downstream of CaMKII, AMPK overexpression altered CaMKII phosphorylation, suggesting that CaMKII and AMPK form a positive feedback loop. These results demonstrate that HBV replication suppresses CaMKII activity, and that CaMKII upregulation suppresses HBV replication from cccDNA via AMPK and the AKT/mTOR signaling pathway. Thus, activation or overexpression of CaMKII may be a new therapeutic target against HBV infection.
| S-EPMC8950817 | biostudies-literature
Project description:G(q) protein-coupled receptors (G(q)PCRs) regulate various cellular processes, including mainly proliferation and differentiation. In a previous study we found that in prostate cancer cells, the G(q)PCR of gonadotropin-releasing hormone (GnRH) induces apoptosis by reducing the PKC-dependent AKT activity and elevating JNK phosphorylation. Because it was thought that G(q)PCRs mainly induce activation of AKT, we first undertook to examine how general this phenomenon is. In a screen of 21 cell lines we found that PKC activation results in the reduction of AKT activity, which correlates nicely with JNK activation and in some cases with apoptosis. To understand further the signaling pathways involved in this stimulation, we studied in detail SVOG-4O and αT3-1 cells. We found that prostaglandin F2α and GnRH agonist (GnRH-a) indeed induce significant Gα(q)- and PKC-dependent apoptosis in these cells. This is mediated by two signaling branches downstream of PKC, which converge at the level of MLK3 upstream of JNK. One branch consists of c-Src activation of the JNK cascade, and the second involves reduction of AKT activity that alleviates its inhibitory effect on MLK3 to allow the flow of the c-Src signal to JNK. At the MAPKK level, we found that the signal is transmitted by MKK7 and not MKK4. Our results present a general mechanism that mediates a G(q)PCR-induced, death receptor-independent, apoptosis in physiological, as well as cancer-related systems.
| S-EPMC3162461 | biostudies-literature
Project description:Cancer cells often rely on glycolysis to obtain energy and support anabolic growth. Several studies showed that glycolytic cells are susceptible to cell death when subjected to low glucose availability or to lack of glucose. However, some cancer cells, including glycolytic ones, can efficiently acquire higher tolerance to glucose depletion, leading to their survival and aggressiveness. Although increased resistance to glucose starvation has been shown to be a consequence of signaling pathways and compensatory metabolic routes activation, the full repertoire of the underlying molecular alterations remain elusive. Using omics and computational analyses, we found that cyclic adenosine monophosphate-Protein Kinase A (cAMP-PKA) axis activation is fundamental for cancer cell resistance to glucose starvation and anoikis. Notably, here we show that such a PKA-dependent survival is mediated by parallel activation of autophagy and glutamine utilization that in concert concur to attenuate the endoplasmic reticulum (ER) stress and to sustain cell anabolism. Indeed, the inhibition of PKA-mediated autophagy or glutamine metabolism increased the level of cell death, suggesting that the induction of autophagy and metabolic rewiring by PKA is important for cancer cellular survival under glucose starvation. Importantly, both processes actively participate to cancer cell survival mediated by suspension-activated PKA as well. In addition we identify also a PKA/Src mechanism capable to protect cancer cells from anoikis. Our results reveal for the first time the role of the versatile PKA in cancer cells survival under chronic glucose starvation and anoikis and may be a novel potential target for cancer treatment.
| S-EPMC4792400 | biostudies-literature
Project description:Elevating Akt activation is an obvious clinical strategy to prevent progressive neuronal death in neurological diseases. However, this endeavor has been hindered because of the lack of specific Akt activators. Here, from a cell-based high-throughput chemical genetic screening, we identified a small molecule SC79 that inhibits Akt membrane translocation, but paradoxically activates Akt in the cytosol. SC79 specifically binds to the PH domain of Akt. SC79-bound Akt adopts a conformation favorable for phosphorylation by upstream protein kinases. In a hippocampal neuronal culture system and a mouse model for ischemic stroke, the cytosolic activation of Akt by SC79 is sufficient to recapitulate the primary cellular function of Akt signaling, resulting in augmented neuronal survival. Thus, SC79 is a unique specific Akt activator that may be used to enhance Akt activity in various physiological and pathological conditions.
| S-EPMC3387065 | biostudies-literature
Project description:Akt is a critical protein kinase that drives cancer proliferation, modulates metabolism, and is activated by C-terminal phosphorylation. The current structural model for Akt activation by C-terminal phosphorylation has centered on intramolecular interactions between the C-terminal tail and the N lobe of the kinase domain. Here, we employ expressed protein ligation to produce site-specifically phosphorylated forms of purified Akt1 that are well suited for mechanistic analysis. Using biochemical, crystallographic, and cellular approaches, we determine that pSer473-Akt activation is driven by an intramolecular interaction between the C-tail and the pleckstrin homology (PH)-kinase domain linker that relieves PH domain-mediated Akt1 autoinhibition. Moreover, dual phosphorylation at Ser477/Thr479 activates Akt1 through a different allosteric mechanism via an apparent activation loop interaction that reduces autoinhibition by the PH domain and weakens PIP3 affinity. These results provide a new framework for understanding how Akt is controlled in cell signaling and suggest distinct functions for differentially modified Akt forms.
| S-EPMC6139374 | biostudies-literature
Project description:Inflammasome activation and subsequent IL-1? production is a driver of islet pathology in type 2 diabetes. Oligomers, but not mature amyloid fibrils, of human islet amyloid polypeptide (IAPP), which is co-secreted with insulin, trigger NOD-like receptor pyrin domain containing-3 (NLRP3) inflammasome activation. C4b-binding protein (C4BP), present in serum, binds to IAPP and affects transition of IAPP monomers and oligomers to amyloid fibrils. We therefore hypothesised that C4BP inhibits IAPP-mediated inflammasome activation and IL-1? production.Macrophages were exposed to IAPP in the presence or absence of plasma-purified human C4BP, and inflammasome activation was assessed by IL-1? secretion as detected by ELISA and reporter cell lines. IAPP fibrillation was assessed by thioflavin T assay. Uptake of IAPP-C4BP complexes and their effects on phagolysosomal stability were assessed by flow cytometry and confocal microscopy. The effect of C4BP regulation of IAPP-mediated inflammasome activation on beta cell function was assessed using a clonal rat beta cell line. Immunohistochemistry was used to examine the association of IAPP amyloid deposits and macrophage infiltration in isolated human and mouse pancreatic islets, and expression of C4BP from isolated human pancreatic islets was assessed by quantitative PCR, immunohistochemistry and western blot.C4BP significantly inhibited IAPP-mediated IL-1? secretion from primed macrophages at physiological concentrations in a dose-dependent manner. C4BP bound to and was internalised together with IAPP. C4BP did not affect IAPP uptake into phagolysosomal compartments, although it did inhibit its formation into amyloid fibrils. The loss of macrophage phagolysosomal integrity induced by IAPP incubation was inhibited by co-incubation with C4BP. Supernatant fractions from macrophages activated with IAPP inhibited both insulin secretion and viability of clonal beta cells in an IL-1?-dependent manner but the presence of C4BP during macrophage IAPP incubation rescued beta cell function and viability. In human and mouse islets, the presence of amyloid deposits correlated with higher numbers of infiltrating macrophages. Isolated human islets expressed and secreted C4BP, which increased with addition of IL-1?.IAPP deposition is associated with inflammatory cell infiltrates in pancreatic islets. C4BP blocks IAPP-induced inflammasome activation by preventing the loss of macrophage phagolysosomal integrity required for NLRP3 activation. The consequence of this is the preservation of beta cell function and viability. C4BP is secreted directly from human pancreatic islets and this increases in response to inflammatory cytokines. We therefore propose that C4BP acts as an extracellular chaperone protein that limits the proinflammatory effects of IAPP.
| S-EPMC5491568 | biostudies-other
Project description:Protein kinase B (Akt) is a key enzyme in the insulin signalling cascade, required for insulin-stimulated NO production in endothelial cells (ECs). Previous studies have suggested that AMP-activated protein kinase (AMPK) activation stimulates NO synthesis and enhances insulin-stimulated Akt activation, yet these studies have largely used indirect activators of AMPK. The effects of the allosteric AMPK activator A769662 on insulin signalling and endothelial function was therefore examined in cultured human macrovascular ECs. Surprisingly, A769662 inhibited insulin-stimulated NO synthesis and Akt phosphorylation in human ECs from umbilical veins (HUVECs) and aorta (HAECs). In contrast, the AMPK activators compound 991 and AICAR had no substantial inhibitory effect on insulin-stimulated Akt phosphorylation in ECs. Inhibition of AMPK with SBI-0206965 had no effect on the inhibition of insulin-stimulated Akt phosphorylation by A769662, suggesting the inhibitory action of A769662 is AMPK-independent. A769662 decreased IGF1-stimulated Akt phosphorylation yet had no effect on VEGF-stimulated Akt signalling in HUVECs, suggesting that A769662 attenuates early insulin/IGF1 signalling. The effects of A769662 on insulin-stimulated Akt phosphorylation were specific to human ECs, as no effect was observed in the human cancer cell lines HepG2 or HeLa, as well as in mouse embryonic fibroblasts (MEFs). A769662 inhibited insulin-stimulated Erk1/2 phosphorylation in HAECs and MEFs, an effect that was independent of AMPK in MEFs. Therefore, despite being a potent AMPK activator, A769662 has effects unlikely to be mediated by AMPK in human macrovascular ECs that reduce insulin sensitivity and eNOS activation.
| S-EPMC6321332 | biostudies-literature
Project description:Protein tyrosine kinase 6 (PTK6) is a non-receptor tyrosine kinase expressed in epithelial cancers. Disruption of Ptk6 decreases azoxymethane-induced colon tumorigenesis in mice by preventing signal transducer and activator of transcription 3 activation. Relocalization of PTK6 in prostate cancers contributes to increased growth. Although not expressed in normal breast or ovary, PTK6 promotes anchorage-independent survival of breast and ovarian tumor cells. We identified several potential PTK6 substrates in the human SW620 colon cancer cell line using mass spectrometry, including FAK (focal adhesion kinase). We show that FAK is a direct substrate of PTK6 in vitro and in vivo. Expression of membrane-targeted active PTK6 (Palm-PTK6-YF) induces constitutive activation of FAK and cell morphology changes, which are independent of SRC family kinases in Src-/-, Yes-/-, Fyn-/- (SYF) mouse embryonic fibroblasts (MEFs). Palm-PTK6-YF expressing SYF cells are transformed and overcome contact inhibition, form colonies in transformation assays, proliferate in suspension and form tumors in a xenograft model. Expression of FAK and Palm-PTK6-YF in Fak-/- MEFs synergistically activates AKT and protects cells against anoikis. However, expression of Palm-PTK6-YF in Akt1/2-/- MEFs fails to protect cells from anoikis, indicating AKT is critical in PTK6 and FAK-mediated survival signaling. In a conditional Pten knockout murine prostate cancer model, we identify prostate epithelial cells with enhanced activation of endogenous PTK6 and FAK at the plasma membrane. Knockdown of PTK6 in the PC3 human prostate cancer cell line disrupts FAK and AKT activation and promotes anoikis, which can be rescued by exogenous expression of FAK. Our data reveal important roles for a PTK6-FAK-AKT signaling axis in promoting anchorage-independent cell survival.
| S-EPMC3940264 | biostudies-other