Par-4 inhibits Akt and suppresses Ras-induced lung tumorigenesis
2008; Springer Nature; Volume: 27; Issue: 16 Linguagem: Inglês
10.1038/emboj.2008.149
ISSN1460-2075
AutoresJayashree Joshi, Pablo J. Fernández-Marcos, Anita Gálvez, Ramars Amanchy, Juan F. Linares, Angeles Durán, Peterson Pathrose, Michael Leitges, Marta Cañamero, Manuel Collado, Clara Salas, Manuel Serrano, Jorge Moscat, Marı́a T. Diaz-Meco,
Tópico(s)Ubiquitin and proteasome pathways
ResumoArticle24 July 2008free access Par-4 inhibits Akt and suppresses Ras-induced lung tumorigenesis Jayashree Joshi Jayashree Joshi Department of Cancer and Cell Biology, University of Cincinnati College of Medicine, Cincinnati, OH, USA Search for more papers by this author Pablo J Fernandez-Marcos Pablo J Fernandez-Marcos Spanish National Cancer Research Center (CNIO), Melchor Fernandez Almagro 3, Madrid, Spain Search for more papers by this author Anita Galvez Anita Galvez Department of Cancer and Cell Biology, University of Cincinnati College of Medicine, Cincinnati, OH, USA Search for more papers by this author Ramars Amanchy Ramars Amanchy Department of Cancer and Cell Biology, University of Cincinnati College of Medicine, Cincinnati, OH, USA Search for more papers by this author Juan F Linares Juan F Linares Department of Cancer and Cell Biology, University of Cincinnati College of Medicine, Cincinnati, OH, USA Search for more papers by this author Angeles Duran Angeles Duran Department of Cancer and Cell Biology, University of Cincinnati College of Medicine, Cincinnati, OH, USA Search for more papers by this author Peterson Pathrose Peterson Pathrose Department of Cancer and Cell Biology, University of Cincinnati College of Medicine, Cincinnati, OH, USA Search for more papers by this author Michael Leitges Michael Leitges The Biotechnology Centre of Oslo, University of Oslo, Oslo, Norway Search for more papers by this author Marta Cañamero Marta Cañamero Spanish National Cancer Research Center (CNIO), Melchor Fernandez Almagro 3, Madrid, Spain Search for more papers by this author Manuel Collado Manuel Collado Spanish National Cancer Research Center (CNIO), Melchor Fernandez Almagro 3, Madrid, Spain Search for more papers by this author Clara Salas Clara Salas Department of Pathology, Hospital Universitario Puerta de Hierro, Madrid, Spain Search for more papers by this author Manuel Serrano Manuel Serrano Spanish National Cancer Research Center (CNIO), Melchor Fernandez Almagro 3, Madrid, Spain Search for more papers by this author Jorge Moscat Corresponding Author Jorge Moscat Department of Cancer and Cell Biology, University of Cincinnati College of Medicine, Cincinnati, OH, USA Search for more papers by this author Maria T Diaz-Meco Corresponding Author Maria T Diaz-Meco Department of Cancer and Cell Biology, University of Cincinnati College of Medicine, Cincinnati, OH, USA Search for more papers by this author Jayashree Joshi Jayashree Joshi Department of Cancer and Cell Biology, University of Cincinnati College of Medicine, Cincinnati, OH, USA Search for more papers by this author Pablo J Fernandez-Marcos Pablo J Fernandez-Marcos Spanish National Cancer Research Center (CNIO), Melchor Fernandez Almagro 3, Madrid, Spain Search for more papers by this author Anita Galvez Anita Galvez Department of Cancer and Cell Biology, University of Cincinnati College of Medicine, Cincinnati, OH, USA Search for more papers by this author Ramars Amanchy Ramars Amanchy Department of Cancer and Cell Biology, University of Cincinnati College of Medicine, Cincinnati, OH, USA Search for more papers by this author Juan F Linares Juan F Linares Department of Cancer and Cell Biology, University of Cincinnati College of Medicine, Cincinnati, OH, USA Search for more papers by this author Angeles Duran Angeles Duran Department of Cancer and Cell Biology, University of Cincinnati College of Medicine, Cincinnati, OH, USA Search for more papers by this author Peterson Pathrose Peterson Pathrose Department of Cancer and Cell Biology, University of Cincinnati College of Medicine, Cincinnati, OH, USA Search for more papers by this author Michael Leitges Michael Leitges The Biotechnology Centre of Oslo, University of Oslo, Oslo, Norway Search for more papers by this author Marta Cañamero Marta Cañamero Spanish National Cancer Research Center (CNIO), Melchor Fernandez Almagro 3, Madrid, Spain Search for more papers by this author Manuel Collado Manuel Collado Spanish National Cancer Research Center (CNIO), Melchor Fernandez Almagro 3, Madrid, Spain Search for more papers by this author Clara Salas Clara Salas Department of Pathology, Hospital Universitario Puerta de Hierro, Madrid, Spain Search for more papers by this author Manuel Serrano Manuel Serrano Spanish National Cancer Research Center (CNIO), Melchor Fernandez Almagro 3, Madrid, Spain Search for more papers by this author Jorge Moscat Corresponding Author Jorge Moscat Department of Cancer and Cell Biology, University of Cincinnati College of Medicine, Cincinnati, OH, USA Search for more papers by this author Maria T Diaz-Meco Corresponding Author Maria T Diaz-Meco Department of Cancer and Cell Biology, University of Cincinnati College of Medicine, Cincinnati, OH, USA Search for more papers by this author Author Information Jayashree Joshi1,‡, Pablo J Fernandez-Marcos2,‡, Anita Galvez1, Ramars Amanchy1, Juan F Linares1, Angeles Duran1, Peterson Pathrose1, Michael Leitges3, Marta Cañamero2, Manuel Collado2, Clara Salas4, Manuel Serrano2, Jorge Moscat 1 and Maria T Diaz-Meco 1 1Department of Cancer and Cell Biology, University of Cincinnati College of Medicine, Cincinnati, OH, USA 2Spanish National Cancer Research Center (CNIO), Melchor Fernandez Almagro 3, Madrid, Spain 3The Biotechnology Centre of Oslo, University of Oslo, Oslo, Norway 4Department of Pathology, Hospital Universitario Puerta de Hierro, Madrid, Spain ‡These authors contributed equally to this work *Corresponding authors: Department of Cancer and Cell Biology, University of Cincinnati College of Medicine, 3125 Eden Ave., Cincinnati, OH 45267, USA. Tel.: +1 513 558 8419; Fax: +1 513 558 5061; E-mail: [email protected] or E-mail: [email protected] The EMBO Journal (2008)27:2181-2193https://doi.org/10.1038/emboj.2008.149 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The atypical PKC-interacting protein, Par-4, inhibits cell survival and tumorigenesis in vitro, and its genetic inactivation in mice leads to reduced lifespan, enhanced benign tumour development and low-frequency carcinogenesis. Here, we demonstrate that Par-4 is highly expressed in normal lung but reduced in human lung cancer samples. We show, in a mouse model of lung tumours, that the lack of Par-4 dramatically enhances Ras-induced lung carcinoma formation in vivo, acting as a negative regulator of Akt activation. We also demonstrate in cell culture, in vivo, and in biochemical experiments that Akt regulation by Par-4 is mediated by PKCζ, establishing a new paradigm for Akt regulation and, likely, for Ras-induced lung carcinogenesis, wherein Par-4 is a novel tumour suppressor. Introduction Inactivating mutations and deletions of tumour suppressor genes along with gain-of-function mutations in proto-oncogenes give rise to cells that can grow independent of external proliferation and survival cues and result in tumour transformation and cancer (Hanahan and Weinberg, 2000). Among the proto-oncogenes most frequently altered in human tumours are the small GTPases of the Ras family, which have been found to be mutated in at least 25% of human lung adenocarcinomas (Bos, 1989). This is of particular interest because lung neoplasia is the leading cancer-related cause of death in the United States, with an estimated incidence of 213 380 new cases and 160 390 deaths in 2007 (http://www.cancer.gov/cancertopics/wyntk/lung). Mouse lung cancer models are available that reproduce the human disease relatively faithfully, thus allowing the study of the cellular and molecular basis of this neoplasia at an organismal level (Fisher et al, 2001; Meuwissen and Berns, 2005). The two main types of lung cancer are small-cell lung cancer and non-small-cell lung cancer (NSCLC). Among the latter, the most prevalent type is the adenocarcinoma, which accounts for more than 40% of all lung cancers (http://www.cancer.gov/cancertopics/wyntk/lung). Current treatments do not lead to a cure for most patients with lung cancer. Targeted anti-tumour therapies are likely to prove more effective, but their development will require a better understanding of the signalling cascades involved in the initiation and progression of this type of tumour. In this regard, Ras oncogenes trigger a myriad of signalling pathways, of which only a few have been characterized in detail, such as the Raf-MEK and PI3 Kinase-Akt signalling cascades (Downward, 2003; Malumbres and Barbacid, 2003). With regard to tumour suppressors, we have reported the reduction of Par-4 (also known as PAWR) levels in Ras-transformed cells (Barradas et al, 1999). In the case of cultured fibroblasts, this Par-4 reduction is a required event for Ras to manifest its full transforming potential (Barradas et al, 1999). However, its role in more physiological cancer models is still unclear. Par-4 was initially identified in an in vitro differential screen for pro-apoptotic genes in human carcinoma cell lines (Sells et al, 1994). The Par-4 gene maps to chromosome 12q21, a region frequently deleted in certain malignancies (Johnstone et al, 1998), and encodes a protein (38 kDa) containing a leucine zipper domain in the COOH-terminal region, which interacts with a variety of proteins (Moscat and Diaz-Meco, 2003), including the atypical protein kinases (aPKCs), PKCζ and PKCλ/ι (Diaz-Meco et al, 1996). Among the mechanisms by which Par-4 triggers apoptosis, the best established one, which is supported by studies in knockout (KO) mice, is through inhibition of the aPKCs and the ensuing downmodulation of NF-κB and its prosurvival transcriptional targets, such as X-linked inhibitor of apoptosis (XIAP) (Garcia-Cao et al, 2003, 2005; Lafuente et al, 2003). These previous studies all point to a link between Par-4 downregulation and cancer. Indeed, we have recently shown that Par-4-null mice develop spontaneous benign prostate neoplasias and endometrial carcinomas, thus implicating Par-4 deficiency in the development of tumours (Garcia-Cao et al, 2005). In addition, our recent data also demonstrate, using cDNA arrays, quantitative reverse-transcription PCR, and by immunohistochemistry (IHC), that Par-4 is downregulated in approximately 40% of human endometrial carcinomas (Moreno-Bueno et al, 2007). Lungs, along with prostate (see below) and endometrium (Moreno-Bueno et al, 2007), are the tissues that exhibit the highest levels of Par-4. We reasoned that if Par-4 is a tumour suppressor, its loss in the lung would lead to increased tumorigenicity. In this study we provide compelling evidence that Par-4 deficiency in the lung leads not only to enhanced NF-κB but also to increased Akt activity, thus making Par-4-deficient lungs more sensitive to Ras-induced oncogenesis. We demonstrate that the negative actions of Par-4 on Akt are cell autonomous and mediated by the ability of PKCζ to phosphorylate Akt at Ser124, which impacts the phosphorylation status of Ser473 and Thr308, two critical residues for its enzymatic activity (Cantley, 2002). Akt's Thr308 is phosphorylated by PDK1 in response to activation of PI 3-kinase, which generates PIP3, which by binding the pleckstrin homology domain of Akt and PDK1, makes that residue accessible to the action of PDK1 (Manning and Cantley, 2007). Ser473 has been shown to be phosphorylated by an mTOR–Rictor complex, termed as TORC2, which in contrast to the TORC1 complex, composed of mTOR and raptor that phosphorylate the Ser396 of S6K1, is insensitive to rapamycin (Sarbassov et al, 2005; Bhaskar and Hay, 2007). Our observations reported here unveil a novel role for Par-4 and PKCζ and reveal a novel mechanism of action for the regulation of Akt involving PKCζ phosphorylation of Ser124, which is negatively regulated by Par-4 and which is likely important in lung cancer in vivo. Results Tissue distribution of Par-4 and loss in human tumours Our previous observations have demonstrated that Par-4 KO mice have decreased lifespan and display higher tumour incidence with ageing. These tumours are mainly prostate intraepithelial neoplasias (PIN) and endometrial carcinomas, and low-frequency lung adenocarcinomas (Garcia-Cao et al, 2005). This, together with the fact that Par-4 is absent in about 58% of human prostate cancers (our unpublished observations) and in about 40% of human endometrial carcinomas (Moreno-Bueno et al, 2007), indicates that Par-4 can be considered as a tumour suppressor. Thus, it is of interest to determine whether Par-4 also has tumour-suppressive activity in other organs. We reasoned that Par-4 will most likely have an important function as a tumour suppressor in those organs that exhibit the highest levels of Par-4 expression. Therefore, we analysed, by immunoblotting with a specific anti-Par-4 antibody, the levels of this protein in different normal mouse tissues. Interestingly, the Par-4 protein is highly expressed in the lung, with levels comparable with those of prostate (Figure 1A). Prostate displays the highest Par-4 mRNA levels, whereas lung, liver and kidney also expressed high levels as compared with other tissues such as brain and intestine (Figure 1B). On the basis of the high Par-4 protein levels in the lung in mouse samples, we determined the distribution of Par-4 in human and mouse lung sections by IHC. The data of Figure 1C show that Par-4 is expressed mostly in the epithelial cells of the airways both in human and mouse lung, as well as in the alveoli. Figure 1.Par-4 is highly expressed in the lung and is downregulated in human lung tumours. (A) Par-4 protein expression in normal mouse tissues. (B) Par-4 mRNA levels in normal mouse tissues. (C) Par-4 distribution in human and mouse tissues, determined by IHC. (D) Commercial tissue microarrays of NSCLC human samples (n=133) were stained with anti-Par-4 antibody. Par-4 expression was analysed in adenocarcinoma and squamous cell carcinoma tumours compared with normal lung tissue. A representative example of positive and negative tumour samples and normal control for Par-4 staining is shown at two magnifications ( × 2.5 and × 20). Scale bar=50 μm. Download figure Download PowerPoint We hypothesized, then, that the loss of Par-4 in the lung would be instrumental for lung cancer initiation and progression. To begin addressing this possibility, we next determined Par-4 expression levels in tissue microarrays of human NSCLC. Interestingly, 47% (n=133) of NSCLC were negative for Par-4 as determined by IHC (Figure 1D). Importantly, there is a clear correlation between the loss of Par-4 and the type of tumour. That is, 41% of the adenocarcinomas were negative for Par-4 expression, whereas only 6% of squamous cell carcinomas show negative staining for Par-4. Also, when the adenocarcinomas are stratified by grade, it is clear that 74% of grade III tumours have lost Par-4 expression, whereas 59% of grade I-II are negative for Par-4. In summary, Par-4 levels are reduced in a significant number of human lung malignancies, strongly suggesting that this protein may have an important function in lung cancer prevention. Par-4 deficiency enhances Ras-induced lung cancer in vivo Par-4 deficiency leads to only benign tumorigenicity in prostates, and to low-frequency lung carcinomas (Garcia-Cao et al, 2005). As Par-4 is reduced most significantly in adenocarcinomas, which are the type of lung tumours that best correlate with the expression of mutant oncogenic Ras (Bos, 1989), we next wanted to test the hypothesis that loss of Par-4 favours the tumorigenic actions of this oncogene. To determine whether Ras-induced lung cancer may be influenced by Par-4, we asked whether in vivo genetic ablation of Par-4 would affect tumour development in lungs. To do this, we used a mouse model of pulmonary adenocarcinoma in which oncogenic Ras was introduced by a knock-in strategy and is inducibly expressed in an endogenous manner (Guerra et al, 2003). This model leads to lung adenomas and adenocarcinomas in which the likely target cell is the type II pneumocyte, evidenced by the observation that the resulting tumour cells express surfactant protein C, a marker of type II pneumocytes (Guerra et al, 2003; Tuveson et al, 2004). This is a physiologically relevant model for human cancer, as it has been reported that, in addition to Clara cells, type II pneumocytes are the most likely precursors of human lung adenocarcinomas (Fisher et al, 2001; Tuveson et al, 2004; Meuwissen and Berns, 2005). To address whether the role of Par-4 is to physiologically restrain Ras-induced lung tumorigenesis, we tested the hypothesis that expression of oncogenic Ras in a Par-4−/− background would increase tumour burden as compared with that in Ras-expressing WT mice. Therefore, Par-4 KO mice were bred to the mice expressing oncogenic Ras in the lung. In this model, a limited percentage of oncogenic Ras-expressing lung broncho-alveolar cells undergo malignant transformation leading to adenomas and adenocarcinomas (Guerra et al, 2003). Therefore, this is an ideal in vivo model to determine whether the loss of Par-4 under these conditions would lead to increased tumorigenicity in the lung. Results in Figure 2A demonstrate that the tumour burden in oncogenic-Ras-expressing WT lungs is approximately 18% of the total lung tissue, whereas that parameter is significantly higher in Par-4 KO mice (about 75%). Tumour incidence in the Ras-expressing Par-4 KO lungs was significantly higher as compared with the Ras-expressing WT controls (Figure 2B). The lifespan was dramatically reduced in Ras-expressing Par-4 KO mice as compared with the other genotypes (Figure 2C). The loss of Par-4 also accelerated the progression of Ras-initiated lung adenocarcinomas, as the percentage of high-grade tumours was significantly higher in the Par-4 KO mice as compared with their corresponding WT controls (Figure 2D). Consistent with increased tumorigenesis in the Par-4 KO lungs, proliferation was also enhanced, as determined by increased Ki67 staining compared with WT controls, both in the normal alveolar tissue (Figure 2E, upper panels) and in the tumours (Figure 2E, lower panels) of Ras-expressing lungs. We have reported previously in vitro downregulation of Par-4 in cultured, oncogenic Ras-expressing fibroblasts (Barradas et al, 1999). Surprisingly, the levels of Par-4 were similar in normal lung of WT mice and in cancer tissues of the oncogenic Ras-expressing mice (Figure 2F). Therefore, our observation that the loss of Par-4 dramatically enhances Ras tumorigenic potential in lung (Figure 2A and B) is consistent with: (1) oncogenic Ras expression not being sufficient to downregulate Par-4 in vivo; and (2) the ability of Par-4 inactivation in tumours to facilitate the development of Ras full-transforming potential. Globally, these data strongly suggest that Par-4 is a physiologically relevant negative regulator of lung tumorigenesis. Figure 2.Par-4 cooperates with Ras-induced tumorigenesis in the lung. (A) H&E staining of lungs from WT and Par-4 KO mice crossed with K-ras+/V12, RERT2T/T mice and analysed 5 months after activation of the K-rasV12 allele by injection of 4-hydroxytamoxifen (n=5 per genotype). Overall tumour burden was determined by quantification of the tumour area as a percentage of total area of H&E-stained tissue (right panel). (B) Number of total tumours per mouse in WT and Par-4 KO mice. (C) Survival of mice of different genotypes represented as percentage of total; the cause of death was asphyxiation. (D) Loss of Par-4 leads to increased Ras-induced high-grade lung adenocarcinomas. (E) Representative sections and quantification of proliferation index measured as percentage of positive nuclear staining for Ki67 in lung sections from normal alveolar or tumour tissue from WT and Par-4 KO Ras-expressing mice. (F) Par-4 expression levels in the lung from different mouse genotypes. **, P<0.01; ****, P<0.0001. Download figure Download PowerPoint Signalling alterations in Par-4-deficient lungs To gain understanding of the molecular and cellular mechanism(s) whereby Par-4 restrains lung tumorigenesis, we next analysed the state of different signalling pathways in lungs from WT and Par-4-deficient mice under basal conditions. We have recently demonstrated in cell cultures of Par-4-deficient EFs, and in Par-4 KO prostates and uteri, that the loss of Par-4 produces enhanced NF-κB activation, which leads to the increased expression of XIAP, a well-established anti-apoptotic downstream target of NF-κB (Garcia-Cao et al, 2003, 2005). Consistent with this, our analysis of lung extracts from Par-4 KO mice revealed increased levels of XIAP as compared with lung extracts from WT controls (Figure 3A). Par-4 constitutively inhibits aPKCs, as demonstrated in vivo by increased phospho-PKCζ staining in lung sections from Par-4 KO mice as compared with WT controls, both in the alveoli and in the airways (Supplementary Figure 1A). Consistently, PKCζ activity in lung extracts is also increased in the Par-4 KO mice, as determined in an in vitro enzymatic assay in PKCζ immunoprecipitates (Supplementary Figure 1B). Figure 3.Increased levels of XIAP and activation of RelA and Akt in lungs of Par-4 KO mice. (A) The levels of XIAP, phospho-Akt-S473, Akt, Par-4 and actin were determined in lung extracts from WT and Par-4 KO mice. These are representative experiments where there were at least two others with similar results. (B, C) Sections of alveolar lung tissue from WT and Par-4 KO mice were stained by IHC with anti-RelA (B) or anti-phospho-Akt-S473 (C) antibody and scored for the number of cells with nuclear staining. Results are the mean±s.d. of 10 different fields per mouse, with a total of five mice for each condition. Scale bar=50 μm. **, P<0.01; ****, P<0.003. Download figure Download PowerPoint As PKCζ is required for the nuclear translocation of NF-κB in lung (Leitges et al, 2001), we next determined by IHC analysis whether the loss of Par-4 in the lung leads to enhanced nuclear levels of RelA in this tissue. The percentage of cells staining positive for nuclear RelA (also known as p65) was significantly increased in Par-4 KO lungs as compared with WT controls (Figure 3B). Therefore, the loss of Par-4 enhances basal NF-κB activity in lungs, which most likely accounts for the increased XIAP levels (Figure 3A). These results are consistent with the increased proliferation observed in Par-4 KO lungs, as enhanced NF-κB would result in higher cell survival during the oncogenic transformation process (Karin, 2006a, 2006b). This observation provides in vivo confirmation of our previous observations on the effect of Par-4 deficiency in EFs, prostate and uterus (Garcia-Cao et al, 2003, 2005), and extends their relevance to the lung. When the activation of other signalling molecules related to cell survival and proliferation was determined in lung extracts, we observed that the lack of Par-4 correlated with increased Akt phosphorylation of Ser473 in lung extracts (Figure 3A). Ser473 phosphorylation is a bona fide marker of Akt activation (Cantley, 2002), indicating that the Par-4 KO lungs have increased levels of Akt activity under basal conditions. These results were confirmed by IHC analysis of lung sections, which demonstrated increased phospho-Akt-Ser473 staining in vivo in Par-4−/− lung alveolar (Figure 3C) and bronchiolar (not shown) cells, as compared with WT controls. Importantly, levels of phospho-Akt-Ser473 were also significantly augmented in Ras-expressing Par-4 KO tumours as compared with WT tumours (Figure 4A and B). Surprisingly, nuclear RelA levels were not detectable in Ras-expressing WT or Par-4 KO tumours (Figure 4C), although the Par-4 KO cells in those tumours showed enhanced cytosolic staining for RelA (Figure 4C). However, increased RelA nuclear translocation was observed in the Par-4 KO, oncogenic Ras-expressing alveolar normal and hyperplastic tissues as compared with WT controls (Supplementary Figure 2A). Phospho-Akt-Ser473 levels in the Par-4 KO mice were increased as compared with WT controls in all oncogenic Ras-expressing lung tissues analysed (Supplementary Figure 2B). These results suggest that increased levels of Akt activity, but probably not of NF-κB, correlate with Par-4 KO-enhanced Ras tumorigenesis. These are important observations because they establish Par-4 as a novel critical negative regulator, in the lung, of two essential prosurvival cascades, NF-κB and Akt. The mechanism whereby Par-4 regulates NF-κB is well established and most likely involves PKCζ (Leitges et al, 2001; Garcia-Cao et al, 2003). However, the results of this article unveil an unexpected regulatory role of Par-4 as a negative modulator of Akt that deserves further analysis. Figure 4.Increased activation of Akt but not RelA in Ras-expressing Par-4 KO lung tumours. Sections of lung tumours from WT and Par-4 KO mice expressing Ras were stained by IHC with anti-phospho-Akt-S473 (A) or anti-RelA (C) antibody. (B) Quantitation of cells showing positive nuclear staining for phospho-Akt-S473. Results are the mean±s.d. of 10 different fields per mouse, with a total of five mice for each condition. Scale bar=50 μm. ***, P<0.001. Download figure Download PowerPoint Cell-autonomous inhibition of Akt by Par-4 To investigate how Par-4 influences Akt Ser473 phosphorylation, we first determined whether the upregulation of Akt activity by Par-4 deficiency is cell autonomous, and if it can be reverted in KO cells by ectopically reintroducing Par-4. To address these two important issues, we used anti-Par-4 and anti-XIAP antibodies to perform immunofluorescence analysis of EFs from WT and Par-4-deficient mice. Both types of fibroblasts were co-cultured on the same coverslips so that the staining conditions for the different types of cells (WT and KO) would be identical for the different antibodies. Results in Figure 5A (upper panel) show that KO cells (which are easily identified because they stain negatively for Par-4) give a much stronger signal for XIAP, consistent with Par-4 being a negative regulator of XIAP expression (Garcia-Cao et al, 2003, 2005). As the antibodies to detect phospho-Akt-Ser473 are not compatible with those that detect Par-4, but are compatible with those for XIAP, we assessed phospho-Akt levels in cells with elevated XIAP levels (thus identified as Par-4−/−). The lower panel of Figure 5A shows a representative experiment. Interestingly, cells that were Par-4−/− displayed higher phospho-Akt-Ser473 levels, demonstrating a clear inverse correlation between Par-4 and Akt activity. Furthermore, when EFs were made quiescent by serum starvation and subsequently activated by serum, the levels of phospho-Akt were more potently stimulated in the Par-4 KO cells than in the WT controls, in both time- and dose-dependent manners, and in both the cytoplasm and the nucleus (Figure 5B and C). Importantly, when Par-4 was re-expressed in KO EFs, the activation of Akt was severely compromised (Figure 5D). These results support the notion that Par-4 is a cell-autonomous negative regulator of Akt in the lung and EFs. To test whether this is also true in human cells, we used a Par-4 siRNA to deplete endogenous Par-4 levels in human 293 cells and in the A549 human lung adenocarcinoma cell line. Cells were treated with control or Par-4-specific siRNAs, after which they were kept for 24 h in serum-free medium conditions and then stimulated with serum. Data in Figure 5E and F clearly demonstrate that the knockdown of Par-4 provokes enhanced serum-activated phospho-Akt-Ser473 levels in A549 and 293 human cells, respectively. These data strongly indicate that the extent of phosho-Akt-Ser473 activation is linked to Par-4 levels in several cell systems, including human lung cancer cells. Figure 5.Par-4 deficiency induces increased nuclear phospho-Akt in vivo. (A) Confocal immunofluorescence on WT and Par-4 KO EFs seeded on the same coverslip, double-stained for Par-4 and XIAP (upper panels), or XIAP and phospho-Akt-S473 (lower panels). WT and Par-4 KO EFs stimulated with serum (FCS) for different durations or a dose–response for 15 min (C) in total extracts (C, D) or in nuclear and cytosol extracts (B). Reconstitution of Par-4 KO EFs with Par-4 restored phospho-Akt-S473 levels to basal levels (D). (E, F) A549 or 293 cells treated with a control siRNA or with Par-4-specific siRNA were stimulated with serum for different times and the levels of phospho-Akt-S473 were determined. Knockdown of Par-4 was confirmed by immunoblot. (G) WT and Par-4 KO EFs stimulated with serum were analysed by immunoblot for phospho-Akt-S473 and phospho-Akt-T308 levels as well as the Akt substrates, Gsk3β-S9 and Foxo3-T32. (H) Knockdown of Akt blocks the increased cell proliferation induced by knockdown of Par-4 in A549 cells. Cell number was determined by trypan blue exclusion. Knockdown of Par-4 and Akt was analysed by immunoblot. These are representative experiments where there were at least two others with similar results. Download figure Download PowerPoint Thr308 is also critical for Akt activity and function and is phosphorylated by PDK1 (Manning et al, 2002). To test whether the loss of Par-4 impacts not only Akt's Ser473 but also Thr308, we determined by immunoblotting with a specific anti-phospho-Akt-Thr308 antibody the levels of this phosphorylation in EFs from Par-4 KO and WT controls that have been either serum-starved or restimulated for 10 and 30 min with serum. We also tested in these extracts the phosphorylation levels of GSK3β and FOXO3, two direct substrates of Akt. From the data of Figure 5G, it
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