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Cell senescence deters the activation of various oncogenes. Induction of senescence is, therefore, a potentially effective strategy to interfere with vital processes in tumor cells. Sphingosine-1-phosphate receptor 1 (S1PR1) has been implicated in various cancer types, including ovarian cancer. The mechanism by which S1PR1 regulates ovarian cancer cell senescence is currently elusive. In this study, we demonstrate that S1PR1 was highly expressed in human ovarian cancer tissues and cell lines. S1PR1 deletion inhibited the proliferation and migration of ovarian cancer cells. S1PR1 deletion promoted ovarian cancer cell senescence and sensitized ovarian cancer cells to cisplatin chemotherapy. Exposure of ovarian cancer cells to sphingosine-1-phosphate (S1P) increased the expression of 3-phosphatidylinositol-dependent protein kinase 1 (PDK1), decreased the expression of large tumor suppressor 1/2 (LATS1/2), and induced phosphorylation of Yes-associated protein (p-YAP). Opposite results were obtained in S1PR1 knockout cells following pharmacological inhibition. After silencing LATS1/2 in S1PR1-deficient ovarian cancer cells, senescence was suppressed and S1PR1 expression was increased concomitantly with YAP expression. Transcriptional regulation of S1PR1 by YAP was confirmed by chromatin immunoprecipitation. Accordingly, the S1PR1-PDK1-LATS1/2-YAP pathway regulates ovarian cancer cell senescence and does so through a YAP-mediated feedback loop. S1PR1 constitutes a druggable target for the induction of senescence in ovarian cancer cells. Pharmacological intervention in the S1PR1-PDK1-LATS1/2-YAP signaling axis may augment the efficacy of standard chemotherapy.

PPM1D phosphatase, also called wild-type p53-induced phosphatase 1, promotes tumor development by inactivating the p53 tumor suppressor pathway. RBM38 RNA-binding protein, also called RNPC1 and a target of p53, inhibits p53 messenger RNA (mRNA) translation, which can be reversed by GSK3 protein kinase via phosphorylation of RBM38 at serine 195. Here we showed that ectopic expression of RBM38 increases, whereas knockdown of RBM38 inhibits, PPM1D mRNA translation. Consistent with this, we found that RBM38 directly binds to PPM1D 3′-untranslated region (3′-UTR) and promotes expression of a heterologous reporter gene that carries PPM1D 3′-UTR in a dose-dependent manner. Interestingly, we showed that PPM1D directly interacts with and dephosphorylates RBM38 at serine 195. Furthermore, we showed that PPM1D modulates p53 mRNA translation and p53-dependent growth suppression through dephosphorylation of RBM38. These findings provide evidence that the crosstalk between PPM1D and RBM38, both of which are targets and modulators of p53, has a critical role in p53 expression and activity. [ABSTRACT FROM AUTHOR]

A growing body of evidences indicate that deregulation of translation contributes to tumourigenesis. In tumours, alterations of translational control of specific mRNAs encoding oncogenes or tumour suppressors have been extensively reported. Moreover, restricting the rate of protein synthesis has been shown to delays tumourigenesis in C-Myc overexpressing or PTEN deleted mice models. Finally, the specific inhibition of RNA polymerase I (RNA pol I) has been shown to kill cancer cells without affecting normal cells. It thus emerges that a tight coordination between the rate of global protein synthesis and a defined translational program is required to prevent tumour development. In this review, we expose the evidences supporting that p53 acts as a translational regulator. In addition, this review discusses the notion that the ability to maintain both a selective translational program and a low level of protein synthesis could directly contribute to the p53 tumour-suppressor activity. [ABSTRACT FROM AUTHOR]

Angiogenesis is required for tumour growth and is induced principally by vascular endothelial growth factor A (VEGF-A). VEGF-A pre-mRNA is alternatively spliced at the terminal exon to produce two families of isoforms, pro- and anti-angiogenic, only the former of which is upregulated in prostate cancer (PCa). In renal epithelial cells and colon cancer cells, the choice of VEGF splice isoforms is controlled by the splicing factor SRSF1, phosphorylated by serine-arginine protein kinase 1 (SRPK1). Immunohistochemistry staining of human samples revealed a significant increase in SRPK1 expression both in prostate intra-epithelial neoplasia lesions as well as malignant adenocarcinoma compared with benign prostate tissue. We therefore tested the hypothesis that the selective upregulation of pro-angiogenic VEGF in PCa may be under the control of SRPK1 activity. A switch in the expression of VEGF165 towards the anti-angiogenic splice isoform, VEGF165b, was seen in PC-3 cells with SRPK1 knockdown (KD). PC-3 SRPK1-KD cells resulted in tumours that grew more slowly in xenografts, with decreased microvessel density. No effect was seen as a result of SRPK1-KD on growth, proliferation, migration and invasion capabilities of PC-3 cells in vitro. Small-molecule inhibitors of SRPK1 switched splicing towards the anti-angiogenic isoform VEGF165b in PC-3 cells and decreased tumour growth when administered intraperitoneally in an orthotopic mouse model of PCa. Our study suggests that modulation of SRPK1 and subsequent inhibition of tumour angiogenesis by regulation of VEGF splicing can alter prostate tumour growth and supports further studies for the use of SRPK1 inhibition as a potential anti-angiogenic therapy in PCa. [ABSTRACT FROM AUTHOR]

As a sensor of cellular energy status, the AMP-activated protein kinase (AMPK) is believed to act in opposition to the metabolic phenotypes favored by proliferating tumor cells. Consequently, compounds known to activate AMPK have been proposed as cancer therapeutics. However, the extent to which the anti-neoplastic properties of these agonists are mediated by AMPK is unclear. Here we examined the AMPK dependence of six commonly used AMPK agonists (metformin, phenformin, 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), 2-deoxy-D-glucose (2DG), salicylate and A-769662) and their influence on cellular processes often deregulated in tumor cells. We demonstrate that the majority of these agonists display AMPK-independent effects on cell proliferation and metabolism with only the synthetic activator, A-769662, exerting AMPK-dependent effects on these processes. We find that A-769662 promotes an AMPK-dependent increase in mitochondrial spare respiratory capacity. Finally, contrary to the view of AMPK activity being tumor suppressive, we find that A-769662 confers a selective proliferative advantage to tumor cells growing under nutrient deprivation. Our results indicate that many of the antigrowth properties of these agonists cannot be attributed to AMPK activity in cells, and thus any observed effects using these agonists should be confirmed using AMPK-deficient cells. Ultimately, our data urge caution not only regarding the type of AMPK agonist proposed for cancer treatment but also the context in which they are used. [ABSTRACT FROM AUTHOR]

The Pim protein kinases contribute to transformation by enhancing the activity of oncogenic Myc and Ras, which drives significant metabolic changes during tumorigenesis. In this report, we demonstrate that mouse embryo fibroblasts (MEFs) lacking all three isoforms of Pim protein kinases, triple knockout (TKO), cannot tolerate the expression of activated K-Ras (K-RasG12V) and undergo cell death. Transduction of K-RasG12V into these cells markedly increased the level of cellular reactive oxygen species (ROS). The addition of N-acetyl cysteine attenuated ROS production and reversed the cytotoxic effects of K-RasG12V in the TKO MEFs. The altered cellular redox state caused by the loss of Pim occurred as a result of lower levels of metabolic intermediates in the glycolytic and pentose phosphate pathways as well as abnormal mitochondrial oxidative phosphorylation. TKO MEFs exhibit reduced levels of superoxide dismutase (Sod), glutathione peroxidase 4 (Gpx4) and peroxiredoxin 3 (Prdx3) that render them susceptible to killing by K-RasG12V-mediated ROS production. In contrast, the transduction of c-Myc into TKO cells can overcome the lack of Pim protein kinases by regulating cellular metabolism and Sod2. In the absence of the Pim kinases, c-Myc transduction permitted K-RasG12V-induced cell growth by decreasing Ras-induced cellular ROS levels. These results demonstrate that the Pim protein kinases have an important role in regulating cellular redox, metabolism and K-Ras-stimulated cell growth. [ABSTRACT FROM AUTHOR]

Homeodomain-interacting protein kinase 2 (HIPK2) is a potential tumor suppressor that has a crucial role in the DNA damage response (DDR) by regulating cell-cycle checkpoint activation and apoptosis. However, it is unclear whether HIPK2 exerts distinct roles in DNA damage repair. The aim of this study was to identify novel target molecule(s) of HIPK2, which mediates HIPK2-dependent DNA damage repair. HIPK2-knockdown human colon cancer cells (HCT116) or hipk1/hipk2 double-deficient mouse embryonic fibroblasts could not remove histone H2A.X phosphorylated at Ser139 (γH2A.X) after irradiation with a sublethal dose (10 J/m2) of ultraviolet (UV)-C, resulting in apoptosis. Knockdown of HIPK2 in p53-null HCT116 cells similarly promoted the UV-C-induced γH2A.X accumulation and apoptosis. Proteomic analysis of HIPK2-associated proteins using liquid chromatography-tandem mass spectrometry identified heterochromatin protein 1γ (HP1γ) as a novel target for HIPK2. Immunoprecipitation experiments with HCT116 cells expressing FLAG-tagged HIPK2 and one of the HA-tagged HP1 family members demonstrated that HIPK2 specifically associated with HP1γ, but not with HP1α or HP1β, through its chromo-shadow domain. Mutation of the HP1box motif (883-PTVSV-887) within HIPK2 abolished the association. HP1γ knockdown also enhanced accumulation of γH2A.X and apoptosis after sublethal UV-C irradiation. In vitro kinase assay demonstrated an HP1γ-phosphorylating activity of HIPK2. Sublethal UV-C irradiation phosphorylated HP1γ. This phosphorylation was absent in endogenous HIPK2-silenced cells with HIPK2 3'UTR siRNA. Overexpression of FLAG-HIPK2, but not the HP1box-mutated or kinase-dead HIPK2 mutant, in the HIPK2-silenced cells increased HP1γ binding to trimethylated (Lys9) histone H3 (H3K9me3), rescued the UV-C-induced phosphorylation of HP1γ, triggered release of HP1γ from histone H3K9me3 and suppressed γH2A.X accumulation. Our results suggest that HIPK2-dependent phosphorylation of HP1γ may participate in the regulation of dynamic interaction between HP1γ and histone H3K9me3 to promote DNA damage repair. This HIPK2/HP1γ pathway may uncover a new functional aspect of HIPK2 as a tumor suppressor. [ABSTRACT FROM AUTHOR]

Cancer cells mainly metabolize by glycolysis (the Warburg effect) and are better adapted to resist metabolic stress. Metastasis-associated in colon cancer-1 (MACC1) is an oncogene promoting gastric cancer (GC) growth and metastasis, and its expression positively correlates with GC progression. However, it is unknown why MACC1 elevates with GC progression and what is its role in cancer metabolism. In this study, we discovered that MACC1 expression was significantly upregulated via adenosine monophosphate-activated protein kinase signaling in response to glucose deprivation-induced metabolic stress. Clinical observation demonstrates that MACC1 expression was higher in advanced stage GC. MACC1 expression was proved to be positively correlated with the maximum standardized uptake value of 18F-deoxyglucose in the patients, and MACC1 enhanced 18F-deoxyglucose uptake in GC cells and the xenografts. The underlying mechanism was that MACC1 promoted the Warburg effect by upregulating the activities and expressions of a series of glycolytic enzymes, including hexokinase, pyruvate dehydrogenase kinase and lactate dehydrogenase, in GC cells. This metabolic shift enhanced cell viability and resistance to apoptosis by facilitating ATP generation, reducing the reactive oxygen species production and stabilizing the mitochondrial membrane potential. In contrast, MACC1-silenced or the Warburg effect-blocked GC cells were more vulnerable to metabolic stress. In conclusion, metabolic stress is one of the mechanisms that elevate MACC1 expression in GC, and MACC1 upregulation compensatively ensures GC growth against metabolic stress by facilitating the Warburg effect. [ABSTRACT FROM AUTHOR]

The LKB1 tumor suppressor gene encodes a master kinase that coordinates the regulation of energetic metabolism and cell polarity. We now report the identification of a novel isoform of LKB1 (named ΔN-LKB1) that is generated through alternative transcription and internal initiation of translation of the LKB1 mRNA. The ΔN-LKB1 protein lacks the N-terminal region and a portion of the kinase domain. Although ΔN-LKB1 is catalytically inactive, it potentiates the stimulating effect of LKB1 on the AMP-activated protein kinase (AMPK) metabolic sensor through a direct interaction with the regulatory autoinhibitory domain of AMPK. In contrast, ΔN-LKB1 negatively interferes with the LKB1 polarizing activity. Finally, combining in vitro and in vivo approaches, we showed that ΔN-LKB1 has an intrinsic oncogenic property. ΔN-LKB1 is expressed solely in the lung cancer cell line, NCI-H460. Silencing of ΔN-LKB1 decreased the survival of NCI-H460 cells and inhibited their tumorigenicity when engrafted in nude mice. In conclusion, we have identified a novel LKB1 isoform that enhances the LKB1-controlled AMPK metabolic activity but inhibits LKB1-induced polarizing activity. Both the LKB1 tumor suppressor gene and the oncogene ΔN-LKB1 are expressed from the same locus and this may account for some of the paradoxical effects of LKB1 during tumorigenesis. [ABSTRACT FROM AUTHOR]

Hec1 (highly expressed in cancer 1) or Nek2 (NIMA-related kinase 2) is often overexpressed in cancers with poor prognosis. Both are critical mitotic regulators, and phosphorylation of Hec1 S165 by Nek2 is required for proper chromosome segregation. Therefore, inactivation of Hec1 and Nek2 by targeting their interaction with small molecules represents an ideal strategy for tackling these types of cancers. Here we showed that new derivatives of INH (inhibitor for Nek2 and Hec1 binding) bind to Hec1 at amino acids 394-408 on W395, L399 and K400 residues, effectively blocking Hec1 phosphorylation on S165 by Nek2, and killing cancer cells at the nanomolar range. Mechanistically, the D-box (destruction-box) region of Nek2 specifically binds to Hec1 at amino acids 408-422, immediately adjacent to the INH binding motif. Subsequent binding of Nek2 to INH-bound Hec1 triggered proteasome-mediated Nek2 degradation, whereas the Hec1 binding defective Nek2 mutant, Nek2 R361L, resisted INH-induced Nek2 degradation. This finding unveils a novel drug-action mechanism where the binding of INHs to Hec1 forms a virtual death-trap to trigger Nek2 degradation and eventually cell death. Furthermore, analysis of the gene expression profiles of breast cancer patient samples revealed that co-elevated expressions of Hec1 and Nek2 correlated with the shortest survival. Treatment of mice with this kind of tumor with INHs significantly suppressed tumor growth without obvious toxicity. Taken together, the new INH derivatives are suitable for translation into clinical application. [ABSTRACT FROM AUTHOR]

The HSP90 molecular chaperone plays a key role in the maturation, stability and activation of its clients, including many oncogenic proteins. Kinases are a substantial and important subset of clients requiring the key cochaperone CDC37. We sought an improved understanding of protein kinase chaperoning by CDC37 in cancer cells. CDC37 overexpression in human colon cancer cells increased CDK4 protein levels, which was negated upon CDC37 knockdown. Overexpressing CDC37 increased CDK4 protein half-life and enhanced binding of HSP90 to CDK4, consistent with CDC37 promoting kinase loading onto chaperone complexes. Against expectation, expression of C-terminus-truncated CDC37 (ΔC-CDC37) that lacks HSP90 binding capacity did not affect kinase client expression or activity; moreover, as with wild-type CDC37 overexpression, it augmented CDK4-HSP90 complex formation. However, although truncation blocked binding to HSP90 in cells, ΔC-CDC37 also showed diminished client protein binding and was relatively unstable. CDC37 mutants with single and double point mutations at residues M164 and L205 showed greatly reduced binding to HSP90, but retained association with client kinases. Surprisingly, these mutants phenocopied wild-type CDC37 overexpression by increasing CDK4-HSP90 association and CDK4 protein levels in cells. Furthermore, expression of the mutants was sufficient to protect kinase clients CDK4, CDK6, CRAF and ERBB2 from depletion induced by silencing endogenous CDC37, indicating that CDC37's client stabilising function cannot be inactivated by substantially reducing its direct interaction with HSP90. However, CDC37 could not compensate for loss of HSP90 function, showing that CDC37 and HSP90 have their own distinct and non-redundant roles in maintaining kinase clients. Our data substantiate the important function of CDC37 in chaperoning protein kinases. Furthermore, we demonstrate that CDC37 can stabilise kinase clients by a mechanism that is not dependent on a substantial direct interaction between CDC37 and HSP90, but nevertheless requires HSP90 activity. These results have significant implications for therapeutic targeting of CDC37. [ABSTRACT FROM AUTHOR]

Prostate cancer is the most commonly diagnosed malignancy among men in industrialized countries, accounting for the second leading cause of cancer-related deaths. Although we now know that the androgen receptor (AR) is important for progression to the deadly advanced stages of the disease, it is poorly understood what AR-regulated processes drive this pathology. Here we demonstrate that AR regulates prostate cancer cell growth via the metabolic sensor 5′-AMP-activated protein kinase (AMPK), a kinase that classically regulates cellular energy homeostasis. In patients, activation of AMPK correlated with prostate cancer progression. Using a combination of radiolabeled assays and emerging metabolomic approaches, we also show that prostate cancer cells respond to androgen treatment by increasing not only rates of glycolysis, as is commonly seen in many cancers, but also glucose and fatty acid oxidation. Importantly, this effect was dependent on androgen-mediated AMPK activity. Our results further indicate that the AMPK-mediated metabolic changes increased intracellular ATP levels and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α)-mediated mitochondrial biogenesis, affording distinct growth advantages to the prostate cancer cells. Correspondingly, we used outlier analysis to determine that PGC-1α is overexpressed in a subpopulation of clinical cancer samples. This was in contrast to what was observed in immortalized benign human prostate cells and a testosterone-induced rat model of benign prostatic hyperplasia. Taken together, our findings converge to demonstrate that androgens can co-opt the AMPK-PGC-1α signaling cascade, a known homeostatic mechanism, to increase prostate cancer cell growth. The current study points to the potential utility of developing metabolic-targeted therapies directed toward the AMPK-PGC-1α signaling axis for the treatment of prostate cancer. [ABSTRACT FROM AUTHOR]

ATM, the protein kinase mutated in the rare human disease ataxia telangiectasia (A-T), has been the focus of intense scrutiny over the past two decades. Initially this was because of the unusual radiosensitive phenotype of cells from A-T patients, and latterly because investigating ATM signalling has yielded valuable insights into the DNA damage response, redox signalling and cancer. With the recent explosion in genomic data, ATM alterations have been revealed both in the germline as a predisposing factor for cancer and as somatic changes in tumours themselves. Here we review these findings, as well as advances in the understanding of ATM signalling mechanisms in cancer and ATM inhibition as a strategy for cancer treatment. [ABSTRACT FROM AUTHOR]

Superior cervical ganglia 10 (SCG10), as a microtubule (MT) destabilizer, maintains MT homeostasis and has a critical role in neuronal development, but Its function in tumorigenesis has not been characterized. In the present study, we demonstrated that p21-activated kinase 4 (PAK4)-mediated SCG10 phosphorylation regulates MT homeostasis in metastatic gastric cancer. Our results indicate that SCG10 is a physiological substrate of PAK4, which is phosphorylated on serine 50 (Ser50) in a PAK4-dependent manner. Phosphorylated SCG10 regulated MT dynamics to promote gastric cancer cell migration and invasion in vitro and metastasis in a xenograft mouse models. Inhibiting PAK4, either by LCH-7749944 or RNA interference, resulted in the inhibition of Ser50 phosphorylation and a blockade to cell invasion, suggesting that PAK4-SCG10 signaling occurs in gastric cancer cell invasion. Moreover, we demonstrated a strong positive correlation between PAK4 and phospho-Ser50 SCG10 expression in gastric cancer samples. We also showed that high expression of SCG10 phospho-Ser50 is highly correlated to an aggressive phenotype of clinical gastric cancer. These findings revealed a novel function of SCG10 in promoting invasive potential of gastric cancer cells, suggesting that blocking PAK4-mediated SCG10 phosphorylation might be a potential therapeutic strategy for metastasis of gastric cancer. [ABSTRACT FROM AUTHOR]

Glioblastomas (GBMs), the most common and malignant brain tumors, are highly resistant to current therapies. The failure of targeted therapies against aberrantly activated oncogenic signaling, such as that of the EGFR-PI3K/Akt pathway, underscores the urgent need to understand alternative downstream pathways and to identify new molecular targets for the development of more effective treatments for gliomas. Here, we report that EGFRvIII (ΔEGFR/de2-7EGFR), a constitutively active EGFR mutant that is frequently co-overexpressed with EGFR in clinical GBM tumors, promotes glioma growth and invasion through protein kinase A (PKA)-dependent phosphorylation of Dock180, a bipartite guanine nucleotide exchange factor (GEF) for Rac1. We demonstrate that EGFRvIII induces serine phosphorylation of Dock180, stimulates Rac1 activation and glioma cell migration. Treatments of glioma cells using the PKA inhibitors H-89 and KT5720, overexpression of a PKA inhibitor (PKI), and in vitro PKA kinase assays show that EGFRvIII induction of serine phosphorylation of Dock180 is PKA-dependent. Significantly, PKA induces phosphorylation of Dock180 at amino acid residue S1250 that resides within its Rac1-activating DHR-2 domain. Expression of the Dock180S1250L mutant, but not wild type Dock180WT, protein in EGFRvIII-expressing glioma cells inhibited receptor-stimulated cell proliferation, survival, migration in vitro and glioma tumor growth and invasion in vivo. Together, our findings describe a novel mechanism by which EGFRvIII drives glioma tumorigenesis and invasion through PKA-dependent phosphorylation of Dock180, thereby suggesting that targeting EGFRvIII-PKA-Dock180-Rac1 signaling axis could provide a novel pathway to develop potential therapeutic strategies for malignant gliomas. [ABSTRACT FROM AUTHOR]

Protein kinase D2 (PKD2) is a member of the PKD family of serine/threonine kinases, a subfamily of the CAMK super-family. PKDs have a critical role in cell motility, migration and invasion of cancer cells. Expression of PKD isoforms is deregulated in various tumours and PKDs, in particular PKD2, have been implicated in the regulation of tumour angiogenesis. In order to further elucidate the role of PKD2 in tumours, we investigated the signalling context of this kinase by performing an extensive substrate screen by in vitro expression cloning (IVEC). We identified a novel splice variant of calcium and integrin-binding protein 1, termed CIB1a, as a potential substrate of PKD2. CIB1 is a widely expressed protein that has been implicated in angiogenesis, cell migration and proliferation, all important hallmarks of cancer, and CIB1a was found to be highly expressed in various cancer cell lines. We identify Ser118 as the major PKD2 phosphorylation site in CIB1a and show that PKD2 interacts with CIB1a via its alanine and proline-rich domain. Furthermore, we confirm that CIB1a is indeed a substrate of PKD2 also in intact cells using a phosphorylation-specific antibody against CIB1a-Ser118. Functional analysis of PKD2-mediated CIB1a phosphorylation revealed that on phosphorylation, CIB1a mediates tumour cell invasion, tumour growth and angiogenesis by mediating PKD-induced vascular endothelial growth factor secretion by the tumour cells. Thus, CIB1a is a novel mediator of PKD2-driven carcinogenesis and a potentially interesting therapeutic target. [ABSTRACT FROM AUTHOR]

Ataxia Telangiectasia Mutated (ATM) kinase, a central regulator of the DNA damage response, regulates the activity of several E3-ubiquitin ligases, and the ubiquitination-proteasome system is a consistent target of ATM. ITCH is an E3-ubiquitin ligase that modulates the ubiquitination of several targets, therefore participating to the regulation of several cellular responses, such as the DNA damage response, tumor necrosis factorα (TNFα), Notch and Hedgehog signaling, and the differentiation of 'naive' lymphocytes into T helper type 2 cells. Here we uncover ATM as a novel positive modulator of ITCH E3-ubiquitin ligase activity. A single residue on ITCH protein, S161, which is part of an ATM SQ consensus motif, is required for ATM-dependent activation of ITCH. ATM activity enhances ITCH enzymatic activity, which in turn drives the ubiquitination and degradation of c-FLIP-L and c-Jun, previously identified as ITCH substrates. Importantly, ATM-deficient mice show resistance to hepatocyte cell death, similarly to Itch-deficient animals, providing in vivo genetic evidence for this circuit. Our data identify ITCH as a novel component of the ATM-dependent signaling pathway and suggest that the impairment of the correct functionality of ITCH caused by Atm deficiency may contribute to the complex clinical features linked to Ataxia Telangiectasia. [ABSTRACT FROM AUTHOR]

Integrin-linked kinase (ILK) and p38MAPK are protein kinases that transduce extracellular signals regulating cell migration and actin cytoskeletal organization. ILK-dependent regulation of p38MAPK is critical for mammalian kidney development and in smooth muscle cell migration, however, specific p38 isoforms has not been previously examined in ILK-regulated responses. Signaling by ILK and p38MAPK is often dysregulated in bladder cancer, and here we report a strong positive correlation between protein levels of ILK and p38β, which is the predominant isoform found in bladder cancer cells, as well as in patient-matched normal bladder and tumor samples. Knockdown by RNA interference of either p38β or ILK disrupts serum-induced, Rac1-dependent migration and actin cytoskeletal organization in bladder cancer cells. Surprisingly, ILK knockdown causes the selective reduction in p38β cellular protein level, without inhibiting p38β messenger RNA (mRNA) expression. The loss of p38β protein in ILK-depleted cells is partially rescued by the 26S proteasomal inhibitor MG132. Using co-precipitation and bimolecular fluorescent complementation assays, we find that ILK selectively forms cytoplasmic complexes with p38β. In situ proximity ligation assays further demonstrate that serum-stimulated assembly of endogenous ILK-p38β complexes is sensitive to QLT-0267, a small molecule ILK kinase inhibitor. Finally, inhibition of ILK reduces the amplitude and period of serum-induced activation of heat shock protein 27 (Hsp27), a target of p38β implicated in actin cytoskeletal reorganization. Our work identifies Hsp27 as a novel target of ILK-p38β signaling complexes, playing a key role in bladder cancer cell migration. [ABSTRACT FROM AUTHOR]

S6 kinases (S6Ks) are mechanistic target of rapamycin substrates that participate in cell growth control. S6Ks phosphorylate ribosomal protein S6 (rpS6) and additional proteins involved in the translational machinery, although the functional roles of these modifications remain elusive. Here we analyze the S6K-dependent transcriptional and translational regulation of gene expression by comparing whole-genome microarray of total and polysomal mouse liver RNA after feeding. We show that tissue lacking S6Ks 1 and 2 (S6K1 and S6K2), displays a defect in the ribosome biogenesis (RiBi) transcriptional program after feeding. Over 75% of RiBi factors are controlled by S6K, including Nop56, Nop14, Gar1, Rrp9, Rrp15, Rrp12 and Pwp2 nucleolar proteins. Importantly, the reduced activity of RiBi transcriptional promoters in S6K1;S6K2−/− cells is also observed in rpS6 knock-in mutants that cannot be phosphorylated. As ribosomal protein synthesis is not affected by these mutations, our data reveal a distinct and specific aspect of RiBi under the control of rpS6 kinase activity, that is, the RiBi transcriptional program. [ABSTRACT FROM AUTHOR]

The transcription factor GLI2 has an important role in the transduction of Hedgehog signaling and thereby regulates tumorigenesis in a wide variety of human tumors. However, the mechanisms controlling GLI2 protein expression and stabilization are incompletely understood. In this study, we show that the mitogen-activated protein kinase MEK1 modulates GLI2 both at the mRNA and protein level. Constitutively activated MEK1 prolonged the half-life of GLI2 and increased its nuclear translocation, accompanied by attenuated ubiquitination of GLI2 protein. RSK2, a protein kinase lying downstream of MEK-ERK cascade, mimicked the effect of MEK on GLI2 stabilization. MEK1 and RSK2 failed to augment the half-life of GLI2 lacking GSK-3β phosphorylation sites, indicating that MEK-RSK stabilizes GLI2 by controlling targeting GSK-3β-mediated phosphorylation and ubiquitination of GLI2. The significance of MEK-RSK stabilization was demonstrated in experiments showing that activation of MEK-RSK paralleled higher protein level of GLI2 in several multiple myelomas (MM) cells relative to normal B cells. Moreover, combined treatment with RSK and GLI inhibitors led to an enhanced apoptosis of MM cells. Thus, our results indicate that MEK-RSK cascade positively regulates GLI2 stabilization and represses its degradation via inhibiting GSK-3β-dependent phosphorylation and ubiquitination of GLI2. [ABSTRACT FROM AUTHOR]

The phosphatidylinositol 3′ kinase (PI3K) pathway is involved in many cellular processes including cell proliferation, survival and glucose transport, and is implicated in various disease states, such as cancer and diabetes. Although there have been numerous studies dissecting the role of PI3K signaling in different cell types and disease models, the mechanism by which PI3K signaling regulates embryonic stem (ES) cell fate remains unclear. It is believed that in addition to proliferation and tumorigenesis, PI3K activity may also be important for ES cell self-renewal. Paling et al. reported that the inhibition of PI3K led to a reduction in the ability of leukemia inhibitory factor to maintain self-renewal, causing cells to differentiate. Studies in our lab have revealed that ES cells completely lacking glycogen synthase kinase-3 (GSK-3) remain undifferentiated compared with wild-type ES cells. GSK-3 is negatively regulated by PI3K, suggesting that PI3K may have a vital role in maintaining pluripotency in ES cells through GSK-3. By using a modified Flp recombinase system, we expressed activated alleles of 3-phosphoinositide-dependent protein kinase-1 and protein kinase B to create stable, isogenic ES cell lines to further study the role of the PI3K signaling pathway in stem cell fate determination. In vitro characterization of the transgenic cell lines revealed a strong tendency toward the maintenance of pluripotency, and this phenotype was found to be independent of canonical Wnt signal transduction. In summary, PI3K signaling is sufficient to maintain the self-renewal and survival of stem cells. As this pathway is frequently mutationally activated in cancers, its effect on suppressing differentiation may contribute to its oncogenicity. [ABSTRACT FROM AUTHOR]

We have recently identified apoptosis-antagonizing transcription factor (AATF), tumor-susceptibility gene 101 (TSG101) and zipper-interacting protein kinase (ZIPK) as novel coactivators of the androgen receptor (AR). The mechanisms of coactivation remained obscure, however. Here we investigated the interplay and interdependence between these coactivators and the AR using the endogenous prostate specific antigen (PSA) gene as model for AR-target genes. Chromatin immunoprecipitation in combination with siRNA-mediated knockdown revealed that recruitment of AATF and ZIPK to the PSA enhancer was dependent on AR, whereas recruitment of TSG101 was dependent on AATF. Association of AR and its coactivators with the PSA enhancer or promoter occurred in cycles. Dissociation of AR-transcription complexes was due to degradation because inhibition of the proteasome system by MG132 caused accumulation of AR at enhancer/promoter elements. Moreover, inhibition of degradation strongly reduced transcription, indicating that continued and efficient transcription is based on initiation, degradation and reinitiation cycles. Interestingly, knockdown of ZIPK by siRNA had a similar effect as MG132, leading to reduced transcription but enhanced accumulation of AR at androgen-response elements. In addition, knockdown of ZIPK, as well as overexpression of a dominant-negative ZIPK mutant, diminished polyubiquitination of AR. Furthermore, ZIPK cooperated with the E3 ligase Mdm2 in AR-dependent transactivation, assembled into a single complex on chromatin and phosphorylated Mdm2 in vitro. These results suggest that ZIPK has a crucial role in regulation of ubiquitination and degradation of the AR, and hence promoter clearance and efficient transcription. [ABSTRACT FROM AUTHOR]

Adhesion to the extracellular matrix (ECM) is critical for epithelial tissue homeostasis and function. ECM detachment induces metabolic stress and programmed cell death via anoikis. ECM-detached mammary epithelial cells are able to rapidly activate autophagy allowing for survival and an opportunity for re-attachment. However, the mechanisms controlling detachment-induced autophagy remain unclear. Here we uncover that the kinase PERK rapidly promotes autophagy in ECM-detached cells by activating AMP-activated protein kinase (AMPK), resulting in downstream inhibition of mTORC1-p70S6K signaling. LKB1 and TSC2, but not TSC1, are required for PERK-mediated inhibition of mammalian target of rapamycinin MCF10A cells and mouse embryo fibroblast cells. Importantly, this pathway shows fast kinetics, is transcription-independent and is exclusively activated during ECM detachment, but not by canonical endoplasmic reticulum stressors. Moreover, enforced PERK or AMPK activation upregulates autophagy and causes luminal filling during acinar morphogenesis by perpetuating a population of surviving autophagic luminal cells that resist anoikis. Hence, we identify a novel pathway in which suspension-activated PERK promotes the activation of LKB1, AMPK and TSC2, leading to the rapid induction of detachment-induced autophagy. We propose that increased autophagy, secondary to persistent PERK and LKB1-AMPK signaling, can robustly protect cells from anoikis and promote luminal filling during early carcinoma progression. [ABSTRACT FROM AUTHOR]

Focal adhesion turnover is a complex process required for cell migration. We have previously shown that the Ste20-like kinase (SLK) is required for cell migration and efficient focal adhesion (FA) turnover in a FA kinase (FAK)-dependent manner. However, the role of SLK in this process remains unclear. Using a candidate substrate approach, we show that SLK phosphorylates the adhesion adapter protein paxillin on serine 250. Serine 250 phosphorylation is required for paxillin redistribution and cell motility. Mutation of paxillin serine 250 prevents its phosphorylation by SLK in vitro and results in impaired migration in vivo as evidenced by an accumulation of phospho-FAK-Tyr397 and altered FA turnover rates. Together, our data suggest that SLK phosphorylation of paxillin on serine 250 is required for FAK-dependent FA dynamics. [ABSTRACT FROM AUTHOR]

Viral and pharmacological inducers of protein kinase RNA-activated (PKR)-like ER kinase (PERK) were shown to accelerate the phosphorylation-dependent degradation of the IFNAR1 chain of the Type 1 interferon (IFN) receptor and to limit cell sensitivity to IFN. Here we report that hypoxia can elicit these effects in a PERK-dependent manner. The altered fate of IFNAR1 affected by signaling downstream of PERK depends on phosphorylation of eIF2α (eukaryotic translational initiation factor 2-α) and ensuing activation of p38α kinase. Activators of other eIF2α kinases such as PKR or GCN2 (general control nonrepressed-2) are also capable of eliminating IFNAR1 and blunting IFN responses. Modulation of constitutive PKR activity in human breast cancer cells stabilizes IFNAR1 and sensitizes these cells to IFNAR1-dependent anti-tumorigenic effects. Although downregulation of IFNAR1 and impaired IFNAR1 signaling can be elicited in response to amino-acid deficit, the knockdown of GCN2 in melanoma cells reverses these phenotypes. We propose that, in cancer cells and the tumor microenvironment, activation of diverse eIF2α kinases followed by IFNAR1 downregulation enables multiple cellular components of tumor tissue to evade the direct and indirect anti-tumorigenic effects of Type 1 IFN. [ABSTRACT FROM AUTHOR]

We have recently identified nc886 (pre-miR-886 or vtRNA2-1) as a novel type of non-coding RNA that inhibits activation of protein kinase R (PKR). PKR's pro-apoptotic role through eukaryotic initiation factor 2 α (eIF2α) phosphorylation is well established in the host defense against viral infection. Paradoxically, some cancer patients have elevated PKR activity; however, its cause and consequence are not understood. Initially, we evaluated the expression of nc886, PKR and eIF2α in non-malignant cholangiocyte and cholangiocarcinoma (CCA) cells. nc886 is repressed in CCA cells and this repression is the cause of PKR's activation therein. nc886 alone is necessary and sufficient for suppression of PKR via direct physical interaction. Consistently, artificial suppression of nc886 in cholangiocyte cells activates the canonical PKR/eIF2α cell death pathway, suggesting a potential significance of the nc886 suppression and the consequent PKR activation in eliminating pre-malignant cells during tumorigenesis. In comparison, active PKR in CCA cells does not induce phospho-eIF2α nor apoptosis, but promotes the pro-survival nuclear factor-κB pathway. Thus, PKR has a dual life or death role during tumorigenesis. Similarly to the CCA cell lines, nc886 tends to be decreased but PKR tends to be activated in our clinical samples from CCA patients. Collectively from our data, we propose a tumor surveillance model for nc886's role in the PKR pathway during tumorigenesis. [ABSTRACT FROM AUTHOR]

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive neoplastic disease. Gemcitabine, the currently used chemotherapeutic drug for PDAC, elicits only minor benefits, because of the development of escape pathways leading to chemoresistance. Herein, we aimed at investigating the involvement of the mitogen activating protein kinase interacting kinase (MNK)/eIF4E pathway in the acquired drug resistance of PDAC cells. Screening of a cohort of PDAC patients by immunohistochemistry showed that eIF4E phosphorylation correlated with disease grade, early onset of disease and worse prognosis. In PDAC cell lines, chemotherapeutic drugs induced MNK-dependent phosphorylation of eIF4E. Importantly, pharmacological inhibition of MNK activity synergistically enhanced the cytostatic effect of gemcitabine, by promoting apoptosis. RNA interference (RNAi) experiments indicated that MNK2 is mainly responsible for eIF4E phosphorylation and gemcitabine resistance in PDAC cells. Furthermore, we found that gemcitabine induced the expression of the oncogenic splicing factor SRSF1 and splicing of MNK2b, a splice variant that overrides upstream regulatory pathways and confers increased resistance to the drug. Silencing of SRSF1 by RNAi abolished this splicing event and recapitulated the effects of MNK pharmacological or genetic inhibition on eIF4E phosphorylation and apoptosis in gemcitabine-treated cells. Our results highlight a novel pro-survival pathway triggered by gemcitabine in PDAC cells, which leads to MNK2-dependent phosphorylation of eIF4E, suggesting that the MNK/eIF4E pathway represents an escape route utilized by PDAC cells to withstand chemotherapeutic treatments. [ABSTRACT FROM AUTHOR]

Rictor and its binding partner Sin1 are indispensable components of mTORC2 (mammalian target of rapamycin complex 2). The mTORC2 signaling complex functions as the regulatory kinase of the distinct members of AGC kinase family known to regulate cell proliferation and survival. In the early chemotaxis studies in Dictyostelium, the rictor's ortholog has been identified as a regulator of cell migration. How rictor regulates cell migration is poorly characterized. Here we show that rictor regulates cell migration by controlling a potent inhibitor of Rho proteins known as the Rho-GDP dissociation inhibitor 2 (RhoGDI2). On the basis of on our proteomics study we identified that the rictor-dependent deficiency in cell migration is caused by upregulation of RhoGDI2 leading to a low activity of Rac and Cdc42. We found that a suppression of RhoGDI2 by rictor is not related to the Sin1 or raptor function that excludes a role of mTORC2 or mTORC1 in regulation of RhoGDI2. Our study reveals that rictor by suppressing RhoGDI2 promotes activity of the Rho proteins and cell migration. [ABSTRACT FROM AUTHOR]

Prostate cancer is initially androgen-dependent but, over time, usually develops hormone- and chemo-resistance. The present study investigated a role for p21-activated kinase 4 (PAK4) in prostate cancer progression. PAK4 activation was markedly inhibited by H89, a specific protein kinase A (PKA) inhibitor, and PAK4 was activated by the elevation of cAMP. The catalytic subunit of PKA interacted with the regulatory domain of PAK4, and directly phosphorylated PAK4 at serine 474 (S474). Catalytically active PAK4 enhanced the transcriptional activity of CREB independent of S133 phosphorylation. Stable knockdown of PAK4 in PC-3 and DU145 prostate cancer cells inhibited tumor formation in nude mice. Decreased tumorigenicity correlated with decreased expression of CREB and its targets, including Bcl-2 and cyclin A1. Additionally, in androgen-dependent LNCap-FGC cells, PAK4 regulated cAMP-induced neuroendocrine differentiation, which is known to promote tumor progression. Finally, PAK4 enhanced survival and decreased apoptosis following chemotherapy. These results suggested that PAK4 regulates progression toward hormone- and chemo-resistance in prostate cancer, and this study identified both a novel activation mechanism and potential downstream effector pathways. Therefore, PAK4 may be a promising therapeutic target in prostate cancer. [ABSTRACT FROM AUTHOR]

Homologous recombination (HR) and nonhomologous end joining (NHEJ) are two distinct DNA double-stranded break (DSB) repair pathways. Here, we report that DNA-dependent protein kinase (DNA-PK), the core component of NHEJ, partnering with DNA-damage checkpoint kinases ataxia telangiectasia mutated (ATM) and ATM- and Rad3-related (ATR), regulates HR repair of DSBs. The regulation was accomplished through modulation of the p53 and replication protein A (RPA) interaction. We show that upon DNA damage, p53 and RPA were freed from a p53-RPA complex by simultaneous phosphorylations of RPA at the N-terminus of RPA32 subunit by DNA-PK and of p53 at Ser37 and Ser46 in a Chk1/Chk2-independent manner by ATR and ATM, respectively. Neither the phosphorylation of RPA nor of p53 alone could dissociate p53 and RPA. Furthermore, disruption of the release significantly compromised HR repair of DSBs. Our results reveal a mechanism for the crosstalk between HR repair and NHEJ through the co-regulation of p53-RPA interaction by DNA-PK, ATM and ATR. [ABSTRACT FROM AUTHOR]

Loss of PTEN tumor suppressor enhances metastatic risk in breast cancer, although the underlying mechanisms are poorly defined. We report that homozygous deletion of PTEN in mammary epithelial cells induces tubulin-based microtentacles (McTNs) that facilitate cell reattachment and homotypic aggregation. Treatment with contractility-modulating drugs showed that McTNs in PTEN−/− cells are suppressible by controlling the actin cytoskeleton. Because outward microtubule extension is counteracted by actin cortical contraction, increased activity of actin-severing proteins could release constraints on McTN formation in PTEN−/− cells. One such actin-severing protein, cofilin, is activated in detached PTEN−/− cells that could weaken the actin cortex to promote McTNs. Expression of wild-type cofilin, an activated mutant (S3A), and an inactive mutant (S3E) demonstrated that altering cofilin phosphorylation directly affects McTNs formation. Chemical inhibition of PI3K did not reduce McTNs or inactivate cofilin in PTEN−/− cells. Additionally, knock-in expression of the two most common PI3K-activating mutations observed in human cancer patients did not increase McTNs or activate cofilin. PTEN loss and PI3K activation also caused differential activation of the cofilin regulators, LIM-kinase1 (LIMK) and Slingshot-1L (SSH). Furthermore, McTNs were suppressed and cofilin was inactivated by restoration of PTEN in the PTEN−/− cells, indicating that both the elevation of McTNs and the activation of cofilin are specific results arising from PTEN loss. These data identify a novel mechanism by which PTEN loss could remodel the cortical actin network to facilitate McTNs that promote tumor cell reattachment and aggregation. Using isogenic MCF-10A PTEN−/− and PIK3CA mutants, we have further demonstrated that there are clear differences in activation of cofilin, LIMK and SSH between PTEN loss and PI3K activation, providing a new evidence that these mutations yield distinct cytoskeletal phenotypes, which could have an impact on tumor biology. [ABSTRACT FROM AUTHOR]

Brca1 deficiency leads to the development of breast cancer. We previously found that Brca1 deficiency activates the Akt oncogenic pathway. Reduced expression of Brca1 was highly correlated with increased activated Akt in human breast cancer samples. Furthermore, activation of Akt1 was involved in Brca1-deficiency-mediated tumorigenesis in mice. Defective homologous recombination (HR) is thought to be a major contributor to tumorigenesis in Brca1 deficiency. Here, we show that Akt1 promotes chromosome instability in Brca1-deficent cells. DNA breaks in Brca1-deficent cells are aberrantly joined into complex chromosome rearrangements by a process dependent on Akt1. Depletion of Akt1 increases HR in Brca1-mutant cells, which is rescued by expression of wild-type, but not mutant Akt1 with deletion of Brca1-binding domain. Mechanistically, activated Akt1 in Brca1-deficient cells impairs Chk1 nuclear localization and subsequently disrupts interaction of Chk1 and Rad51 leading to HR defects. Our results indicate that Brca1 deficiency might activate Akt1 contributing to tumorigenesis through regulation of the Chk1-Rad51 signaling. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) have been involved in the pathogenesis of different types of cancer; however, their function in pituitary tumorigenesis remains poorly understood. Cyclic-AMP-dependent protein kinase-defective pituitaries occasionally form aggressive growth-hormone (GH)-producing pituitary tumors in the background of hyperplasia caused by haploinsufficiency of the protein kinase's main regulatory subunit, PRKAR1A. The molecular basis for this development remains unknown. We have identified a 17-miRNA signature of pituitary tumors formed in the background of hyperplasia (caused in half of the cases by PRKAR1A-mutations). We selected two miRNAs on the basis of their functional screen analysis: inhibition of miR-26b expression and upregulation of miR-128 suppressed the colony formation ability and invasiveness of pituitary tumor cells. Furthermore, we identified that miR-26b and miR-128 affected pituitary tumor cell behavior through regulation of their direct targets, PTEN and BMI1, respectively. In addition, we found that miR-128 through BMI1 direct binding on the PTEN promoter affected PTEN expression levels and AKT activity in the pituitary tumor cells. Our in vivo data revealed that inhibition of miR-26b and overexpression of miR-128 could suppress pituitary GH3 tumor growth in xenografts. Taken together, we have identified a miRNA signature for GH-producing pituitary tumors and found that miR-26b and miR-128 regulate the activity of the PTEN-AKT pathway in these tumors. This is the first suggestion of the possible involvement of miRNAs regulating the PTEN-AKT pathway in GH-producing pituitary tumor formation in the context of hyperplasia or due to germline PRKAR1A defects. MiR-26b suppression and miR-128 upregulation could have therapeutic potential in GH-producing pituitary tumor patients. [ABSTRACT FROM AUTHOR]

The ERK1/2 (extracellular signal-regulated kinase 1 and 2) pathway, comprising the protein kinases RAF (v-raf-1 murine leukemia viral oncogene homolog 1), MEK1/2 (mitogen-activated protein kinase or ERK kinase 1 and 2) and ERK1/2 is frequently de-regulated in human cancers, due to mutations in RAS or BRAF (v-raf-1 murine leukemia viral oncogene homolog B1). New, highly selective inhibitors of BRAF and MEK1/2 have shown promise in clinical trials, including in previously intractable diseases such as melanoma. However, drug-resistant tumour cells invariably emerge leading to disease progression. It is important to understand the mechanisms underlying such acquired resistance since this may lead to the development of rational strategies either to delay its onset or to overcome it once established. It also offers unique insights into the plasticity of signalling pathways, which may in turn inform our understanding of the basic biology of these pathways and lead to the validation of new drug targets. Several recent reports have identified diverse mechanisms of acquired resistance to MEK1/2 or BRAF inhibitors. In this article, we review these studies, discuss the different mechanisms, identify common themes and consider their therapeutic implications. [ABSTRACT FROM AUTHOR]

The oncogenic kinase PAK4 was recently found to be involved in the regulation of the G1 phase and the G2/M transition of the cell cycle. We have also identified that PAK4 regulates Ran GTPase activity during mitosis. Here, we show that after entering mitosis, PAK4-depleted cells maintain a prolonged metaphase-like state. In these cells, chromosome congression to the metaphase plate occurs with normal kinetics but is followed by an extended period during which membrane blebbing and spindle rotation are observed. These bipolar PAK4-depleted metaphase-like spindles have a defective astral microtubule (MT) network and are not centered in the cell but are in close contact with the cell cortex. As the metaphase-like state persists, centrosome fragmentation occurs, chromosomes scatter from the metaphase plate and move toward the spindle poles with an active spindle assembly checkpoint, a phenotype that is reminiscent of cohesion fatigue. PAK4 also regulates the acto-myosin cytoskeleton and we report that PAK4 depletion results in the induction of cortical membrane blebbing during prometaphase arrest. However, we show that membrane blebs, which are strongly enriched in phospho-cofilin, are not responsible for the poor anchoring of the spindle. As PAK4 depletion interferes with the localization of components of the dynein/dynactin complexes at the kinetochores and on the astral MTs, we propose that loss of PAK4 could induce a change in the activities of motor proteins. [ABSTRACT FROM AUTHOR]

In chronic myelogenous leukemia, the constitutive activation of the BCR-ABL kinase transforms cells to an addicted state that requires glucose metabolism for survival. We investigated S6K1, a protein kinase that drives glycolysis in leukemia cells, as a target for counteracting glucose-dependent survival induced by BCR-ABL. BCR-ABL potently activated S6K1-dependent signaling and glycolysis. Although S6K1 knockdown or rapamycin treatment suppressed glycolysis in BCR-ABL-transformed cells, these treatments did not induce cell death. Instead, loss of S6K1 triggered compensatory activation of fatty-acid oxidation, a metabolic program that can support glucose-independent cell survival. Fatty-acid oxidation in response to S6K1 inactivation required the expression of the fatty-acid transporter carnitine palmitoyl transferase 1c, which was recently linked to rapamycin resistance in cancer. Finally, addition of an inhibitor of fatty-acid oxidation significantly enhanced cytotoxicity in response to S6K1 inactivation. These data indicate that S6K1 dictates the metabolic requirements mediating BCR-ABL survival and provide a rationale for combining targeted inhibitors of signal transduction, with strategies to interrupt oncogene-induced metabolism. [ABSTRACT FROM AUTHOR]

The Warburg effect in cancer cells has been proposed to involve several mechanisms, including adaptation to hypoxia, oncogenes activation or loss of oncosuppressors and impaired mitochondrial function. In previous papers, it has been shown that K-ras transformed mouse cells are much more sensitive as compared with normal cells to glucose withdrawal (undergoing apoptosis) and present a high glycolytic rate and a strong reduction of mitochondrial complex I. Recent observations suggest that transformed cells have a derangement in the cyclic adenosine monophosphate/cAMP-dependent protein kinase (cAMP/PKA) pathway, which is known to regulate several mitochondrial functions. Herein, the derangement of the cAMP/PKA pathway and its impact on transformation-linked changes of mitochondrial functions is investigated. Exogenous stimulation of PKA activity, achieved by forskolin treatment, protected K-ras-transformed cells from apoptosis induced by glucose deprivation, enhanced complex I activity, intracellular adenosine triphosphate (ATP) levels, mitochondrial fusion and decreased intracellular reactive oxygen species (ROS) levels. Several of these effects were almost completely prevented by inhibiting the PKA activity. Short-time treatment with compounds favoring mitochondrial fusion strongly decreased the cellular ROS levels especially in transformed cells. These findings support the notion that glucose shortage-induced apoptosis, specific of K-ras-transformed cells, is associated to a derangement of PKA signaling that leads to mitochondrial complex I decrease, reduction of ATP formation, prevalence of mitochondrial fission over fusion, and thereby opening new approaches for development of anticancer drugs. [ABSTRACT FROM AUTHOR]

BAG3 is a co-chaperone of the heat shock protein (Hsp) 70, is expressed in many cell types upon cell stress, however, its expression is constitutive in many tumours. We and others have previously shown that in neoplastic cells BAG3 exerts an anti-apoptotic function thus favoring tumour progression. As a consequence we have proposed BAG3 as a target of antineoplastic therapies. Here we identify a novel role for BAG3 in regulation of neo-angiogenesis and show that its downregulation results in reduced angiogenesis therefore expanding the role of BAG3 as a therapeutical target. In brief we show that BAG3 is expressed in endothelial cells and is essential for the interaction between ERK and its phosphatase DUSP6, as a consequence its removal results in reduced binding of DUSP6 to ERK and sustained ERK phosphorylation that in turn determines increased levels of p21 and p15 and cell-cycle arrest in the G1 phase. [ABSTRACT FROM AUTHOR]

Insulin-like growth factor II (IGF-II) mRNA-binding protein 3 (IMP3) is emerging as a useful indicator of the progression and outcome of several cancers. IMP3 expression is associated with triple-negative breast carcinomas (TNBCs), which are aggressive tumors associated with poor outcome. In this study, we addressed the hypothesis that signaling pathways, which are characteristic of TNBCs, impact the expression of IMP3 and that IMP3 contributes to the function of TNBCs. The data obtained reveal that IMP3 expression is repressed specifically by estrogen receptor β (ERβ) and its ligand 3βA-diol but not by ERα. Epidermal growth factor receptor (EGFR) signaling and consequent activation of the mitogen-activated protein kinase pathway induce IMP3 transcription and expression. Interestingly, we discovered that the EGFR promoter contains an imperfect estrogen response element and that ERβ represses EGFR transcription. These data support a mechanism in which ERβ inhibits IMP3 expression indirectly by repressing the EGFR. This mechanism relates to the biology of TNBC, which is characterized by diminished ERβ and increased EGFR expression. We also demonstrate that IMP3 contributes to the migration and invasion of breast carcinoma cells. Given that IMP3 is an mRNA-binding protein, we determined that it binds several key mRNAs that could contribute to migration and invasion, including CD164 (endolyn) and MMP9. Moreover, expression of these mRNAs is repressed by ERβ and enhanced by EGFR signaling, consistent with our proposed mechanism for the regulation of IMP3 expression in breast cancer cells. Our findings show that IMP3 is an effector of EGFR-mediated migration and invasion and they provide the first indication of how this important mRNA-binding protein is regulated in cancer. [ABSTRACT FROM AUTHOR]

Chk1 both arrests replication forks and enhances repair of DNA damage by phosphorylating downstream effectors. Although there has been a concerted effort to identify effectors of Chk1 activity, underlying mechanisms of effector action are still being identified. Metnase (also called SETMAR) is a SET and transposase domain protein that promotes both DNA double-strand break (DSB) repair and restart of stalled replication forks. In this study, we show that Metnase is phosphorylated only on Ser495 (S495) in vivo in response to DNA damage by ionizing radiation. Chk1 is the major mediator of this phosphorylation event. We had previously shown that wild-type (wt) Metnase associates with chromatin near DSBs and methylates histone H3 Lys36. Here we show that a Ser495Ala (S495A) Metnase mutant, which is not phosphorylated by Chk1, is defective in DSB-induced chromatin association. The S495A mutant also fails to enhance repair of an induced DSB when compared with wt Metnase. Interestingly, the S495A mutant demonstrated increased restart of stalled replication forks compared with wt Metnase. Thus, phosphorylation of Metnase S495 differentiates between these two functions, enhancing DSB repair and repressing replication fork restart. In summary, these data lend insight into the mechanism by which Chk1 enhances repair of DNA damage while at the same time repressing stalled replication fork restart. [ABSTRACT FROM AUTHOR]

Snail1 is a transcriptional factor essential for triggering epithelial-to-mesenchymal transition. Moreover, Snail1 promotes resistance to apoptosis, an effect associated to PTEN gene repression and Akt stimulation. In this article we demonstrate that Snail1 activates Akt at an additional level, as it directly binds to and activates this protein kinase. The interaction is observed in the nucleus and increases the intrinsic Akt activity. We determined that Akt2 is the isoform interacting with Snail1, an association that requires the pleckstrin homology domain in Akt2 and the C-terminal half in Snail1. Snail1 enhances the binding of Akt2 to the E-cadherin (CDH1) promoter and Akt2 interference prevents Snail1 repression of CDH1 gene. We also show that Snail1 binding increases Akt2 intrinsic activity on histone H3 and have identified Thr45 as a residue modified on this protein. Phosphorylation of Thr45 in histone H3 is sensitive to Snail1 and Akt2 cellular levels; moreover, Snail1 upregulates the binding of phosphoThr45 histone H3 to the CDH1 promoter. These results uncover an unexpected role of Akt2 in transcriptional control and point out to phosphorylation of Thr45 in histone H3 as a new epigenetic mark related to Snail1 and Akt2 action. [ABSTRACT FROM AUTHOR]

We integrated four gene expression profile data sets, namely two different pair-matched stage I lung adenocarcinoma data sets, secondary metastatic tumors vs benign tumors and lung tumor metastasizes to the brain, and we identified one kinase, T-LAK Cell-Originated Protein Kinase (TOPK), as a putative gene that promotes metastasis. To delineate the role of TOPK in lung cancer, we showed that overexpression of TOPK, but not a catalytically inactive form of TOPK, can enhance the migration and invasion of lung fibroblasts or cells with low TOPK expression. In addition, TOPK-induced cell migration was shown to be a PI3K/AKT-dependent event. TOPK concurrently promoted AKT phosphorylation at Ser473 and decreased the phosphatase and tensin homolog (PTEN) levels, whereas TOPK knockdown had the reverse effects. LY294002, a PI3K inhibitor, did not inhibit the TOPK-induced decrease in PTEN, and co-expression of PTEN significantly reduced TOPK-induced AKT phosphorylation in a dose-dependent manner; these results indicate that the TOPK-mediated PTEN decrease has an upstream role in regulating PI3K/AKT-stimulated migration. Using immunohistochemical analysis of lung cancer tissue samples, we showed that a high TOPK expression level correlates strongly with reduced overall and disease-free survivals. Moreover, an inverse correlation between TOPK and PTEN expression was present and is consistent with the biochemical findings. Finally, a combination of high TOPK and low PTEN expression was inversely correlated with overall and disease-free survivals, independent of other pathologic staging factors. Our results suggest that TOPK is a potential therapeutic target in lung cancer that promotes cell migration by modulating a PI3K/PTEN/AKT-dependent signaling pathway; they also suggest that high TOPK expression, either alone or in combination with a low level of PTEN, may serve as a prognostic marker for lung cancer. [ABSTRACT FROM AUTHOR]

Although the hyper-glycosylated transmembrane protein Mucin 1 (MUC1) is aberrantly overexpressed in human breast carcinoma, the biological significance of MUC1 overexpression is unclear. This study showed that MUC1 expression promoted the synthesis and secretion of vascular endothelial growth factor (VEGF) through the AKT signaling pathway. Increase VEGF production through MUC1 expression had a number of effect. First, MUC1 transfection increased expression of VEGF in breast cancer cells. Second, MUC1-mediated VEGF induction was attenuated by a chemical inhibitor of AKT or MUC1 knock-down by MUC1 siRNA. Third, MUC1 expression led to the activation of insulin-like growth factor-1 receptor, which correlated with VEGF expression. In addition, when MDA-MB-231 human breast cancer cells were directly injected into NOD/SCID mice, MUC1 expression accelerated xenograft tumor growth in vivo. Finally, MUC1 expression enhanced tumor growth and angiogenesis in a PyMT-MMTV/hMUC1 transgenic mouse model. Concurrent with these results, analysis of a human tissue microarray identified a high correlation between MUC1 and VEGF expression in human breast carcinoma. The current report is the first to demonstrate that MUC1 expression promotes angiogenesis in human breast cancer in vivo and in vitro. [ABSTRACT FROM AUTHOR]

Cisplatin (CDDP: cis-diamminedichloroplatinum) resistance is a major hurdle in the treatment of human ovarian cancer (OVCA). A better understanding of the mechanisms of CDDP resistance can greatly improve therapeutic outcome for patients. A determinant of CDDP sensitivity in OVCA, p53, is activated by checkpoint kinase 1 (Chk1) in response to DNA damage. Although the oncogenic phosphatase protein phosphatase magnesium-dependent 1 (PPM1D) can deactivate both p53 and Chk1 through site-specific dephosphorylation, whether PPM1D has a role in CDDP resistance is unknown. Here, using pair-matched wild-type p53 CDDP-sensitive (OV2008) and -resistant (C13*) cells, and p53-compromised CDDP-resistant cells (A2780cp, OCC-1, OVCAR-3 and SKOV3), we have demonstrated (i) the existence of site-specific differences in phospho-Ser-Chk1 content between sensitive and resistant cells in response to CDDP; (ii) PPM1D, but not phosphoinositide-3-kinase-related kinase Ataxia Telangiectasia and Rad3 related protein (ATR), is important in the regulation of CDDP-induced Chk1 activation and OVCA cell chemosensitivity; (iii) PPM1D downregulation sensitizes resistant cells to CDDP primarily by activating Chk1 and p53. Our findings establish for the first time that PPM1D confers CDDP resistance in OVCA cells through attenuating CDDP-induced, Chk1-mediated, p53-dependent apoptosis. These findings extend the current knowledge on the molecular and cellular basis of cisplatin resistance and offer the rationale for PPMID as a potential target for treatment of chemoresistant OVCA. [ABSTRACT FROM AUTHOR]

The cyclin-dependent kinase (CDK) inhibitor p27 has an important role in cell cycle regulation. Reduced expression of p27 is commonly associated with poor prognosis in many malignancies, including gastric cancer. Cytoplasmic p27 mislocalization may be an additional indicator of high-grade tumors and poor prognosis in cancer. As chronic infection by Helicobacter pylori is the most important risk factor for gastric cancer development, we evaluated the effects of H. pylori on p27 expression and localization in gastric cancer cells. Co-culture of gastric cells with H. pylori induced cytoplasmic p27 expression and reduced nuclear p27 expression in vitro. Cytoplasmic p27 expression was associated with and dependent upon phosphorylation of p27 at T157 and T198: wild-type p27 accumulated in the cytoplasm, but non-phosphorylatable mutants affecting T157 or T198 were nuclear in H. pylori-infected cells. These post-translational p27 changes were secondary to activation of cellular phosphoinositide-3 kinase (PI3K) and AKT signaling pathways, and dependent upon a functional H. pylori cag pathogenicity island. We investigated the clinical significance of cytoplasmic p27 mislocalization in 164 cases of resected gastric cancer in tissue microarrays. In 97 cases (59%), cytoplasmic p27 mislocalization was observed, and this was associated with increased mortality in multivariate analysis. These results show that H. pylori infection induces AKT/PI3K-mediated phosphorylation of p27 at T157 and T198 to cause cytoplasmic p27 mislocalization in gastric cancer, and that p27 mislocalization is an adverse prognostic feature in gastric cancer. This is the first demonstration of the translocation of a specific bacterial virulence factor that post-translationally regulates a host cell CDK inhibitor. This is of particular significance, because p27 has both tumor-suppressive and oncogenic activities, depending upon its subcellular localization. Cytoplasmic mislocalization of p27 induced by H. pylori may be an important mechanistic link between H. pylori infection and gastric carcinogenesis. [ABSTRACT FROM AUTHOR]

Estrogen receptor-α (ERα, ESR1) is a pivotal transcriptional regulator of breast cancer physiology and is targeted by endocrine therapies. Loss of ERα activity or expression is an indication of endocrine resistance and is associated with increased risk of tumor recurrence and worse prognosis. In this study, we sought to investigate whether elements of the tumor microenvironment, namely macrophages, would impact on ERα and we found that macrophage-derived factors caused loss of ERα expression in breast cancer cells. Conditioned media from macrophages caused activation of several intracellular pathways in breast cancer cells of which c-Src, protein kinase c and mitogen-activated protein kinase (MAPK) were essential for loss of ERα expression. Moreover, a prolonged hyperactivation of MAPK was observed. The activation of this kinase cascade resulted in recruitment of extracellular signal regulated kinase 2 (ERK2) directly to chromatin at the ESR1 gene locus in a process that was dependent upon activation and recruitment of the c-Jun transcription factor. Thus, we identify a novel mechanism for loss of ERα expression in breast cancer cells via macrophage activation of kinase cascades in the cancer cells causing transcriptional repression of the ESR1 gene by a direct chromatin action of a c-Jun/ERK2 complex. The findings in this study support an alternative mechanism, not intrinsic to the tumor cell but derived from the cross-talk with the tumor microenvironment, that could lead to endocrine resistance and might be targeted therapeutically to prevent loss of ERα expression in breast tumors. [ABSTRACT FROM AUTHOR]

The c-Jun transcription factor is a highly unstable oncoprotein. Several ubiquitin ligases mediate c-Jun degradation. However, c-Jun can be stabilized once it is phosphorylated at the N-terminus by c-Jun N-terminal kinases (JNKs) or other protein kinases. This phosphorylation decreases c-Jun ubiquitination and degradation. The underlying mechanism for this phenomenon is still unknown. Here, we show that receptor for activated C-kinase 1 (Rack1) can bind with c-Jun and ubiquitin ligase Fbw7 to form a complex. When c-Jun is phosphorylated at the N-terminus, c-Jun is released from the complex and cannot be ubiquitinated by Fbw7, which leads to increased stabilization and accumulation of c-Jun. These results reveal that Rack1 has a very important role in tumorigenesis by maintaining the stability of c-Jun that has been phosphorylated at its N-terminus by JNKs or other kinases. [ABSTRACT FROM AUTHOR]