Akt Regulates Drug-Induced Cell Death through Bcl-w Downregulation

Akt is a serine threonine kinase with a major role in transducing survival signals and regulating proteins involved in apoptosis. To find new interactors of Akt involved in cell survival, we performed a two-hybrid screening in yeast using human full-length Akt c-DNA as bait and a murine c-DNA library as prey. Among the 80 clones obtained, two were identified as Bcl-w. Bcl-w is a member of the Bcl-2 family that is essential for the regulation of cellular survival, and that is up-regulated in different human tumors, such as gastric and colorectal carcinomas. Direct interaction of Bcl-w with Akt was confirmed by immunoprecipitation assays. Subsequently, we addressed the function of this interaction: by interfering with the activity or amount of Akt, we have demonstrated that Akt modulates the amount of Bcl-w protein. We have found that inhibition of Akt activity may promote apoptosis through the downregulation of Bcl-w protein and the consequential reduction in interaction of Bcl-w with pro-apoptotic members of the Bcl-2 family. Our data provide evidence that Bcl-w is a new member of the Akt pathway and that Akt may induce anti-apoptotic signals at least in part through the regulation of the amount and activity of Bcl-w

The Identification of CELSR3 and Other Potential Cell Surface Targets in Neuroendocrine Prostate Cancer.

UNLABELLED Although recent efforts have led to the development of highly effective androgen receptor (AR)-directed therapies for the treatment of advanced prostate cancer, a significant subset of patients will progress with resistant disease including AR-negative tumors that display neuroendocrine features [neuroendocrine prostate cancer (NEPC)]. On the basis of RNA sequencing (RNA-seq) data from a clinical cohort of tissue from benign prostate, locally advanced prostate cancer, metastatic castration-resistant prostate cancer and NEPC, we developed a multi-step bioinformatics pipeline to identify NEPC-specific, overexpressed gene transcripts that encode cell surface proteins. This included the identification of known NEPC surface protein CEACAM5 as well as other potentially targetable proteins (e.g., HMMR and CESLR3). We further showed that cadherin EGF LAG seven-pass G-type receptor 3 (CELSR3) knockdown results in reduced NEPC tumor cell proliferation and migration in vitro. We provide in vivo data including laser capture microdissection followed by RNA-seq data supporting a causal role of CELSR3 in the development and/or maintenance of the phenotype associated with NEPC. Finally, we provide initial data that suggests CELSR3 is a target for T-cell redirection therapeutics. Further work is now needed to fully evaluate the utility of targeting CELSR3 with T-cell redirection or other similar therapeutics as a potential new strategy for patients with NEPC. SIGNIFICANCE The development of effective treatment for patients with NEPC remains an unmet clinical need. We have identified specific surface proteins, including CELSR3, that may serve as novel biomarkers or therapeutic targets for NEPC

Opposing transcriptional programs of KLF5 and AR emerge during therapy for advanced prostate cancer.

Endocrine therapies for prostate cancer inhibit the androgen receptor (AR) transcription factor. In most cases, AR activity resumes during therapy and drives progression to castration-resistant prostate cancer (CRPC). However, therapy can also promote lineage plasticity and select for AR-independent phenotypes that are uniformly lethal. Here, we demonstrate the stem cell transcription factor Krüppel-like factor 5 (KLF5) is low or absent in prostate cancers prior to endocrine therapy, but induced in a subset of CRPC, including CRPC displaying lineage plasticity. KLF5 and AR physically interact on chromatin and drive opposing transcriptional programs, with KLF5 promoting cellular migration, anchorage-independent growth, and basal epithelial cell phenotypes. We identify ERBB2 as a point of transcriptional convergence displaying activation by KLF5 and repression by AR. ERBB2 inhibitors preferentially block KLF5-driven oncogenic phenotypes. These findings implicate KLF5 as an oncogene that can be upregulated in CRPC to oppose AR activities and promote lineage plasticity

Rôle de la famille des Arrestines dans la voie de signalisation Notch chez les mammifères

Notch signaling is an evolutionary conserved pathway implicated in embryonic development and in adult tissue homeostasis. A number of post-translational modifications have been implicated in regulating the activity of Notch receptor and some of them affect the degradation of non-activated Notch receptor. The starting points of my PhD project are Drosophila findings showing that the adaptor protein Kurtz, the unique non-visual arrestin in Drosophila, is an essential regulator of Notch signaling. In mammals the arrestin family (excluding the visual arrestins) is composed of two sub-families: -arrestins and -arrestins. The main results that I have obtained show that both -arrestins and -arrestins are recruited to non-activated Notch receptor and allow Itch-mediated Notch ubiquitination in mammals. Biochemical evidence shows that an heterodimerization between -arrestins and the -arrestin ARRDC1 is required to promote Notch ubiquitination and its lysosomal degradation. To conclude, we show for the first time that the - -arrestin heterodimer is functionally involved in the degradation of Notch receptor, highlighting the existence of a cooperation between these adaptor proteins to regulate receptor traffickingLa voie de signalisation Notch est une voie conservée au cours de l évolution impliquée dans le développement embryonnaire et dans l'homéostasie tissuetaire chez les adultes. Un certain nombre de modifications post-traductionnelles ont été impliquées dans la régulation de l'activité du récepteur Notch et certaines d'entre elles ont une incidence sur la dégradation du récepteur Notch non activé. Le point de départ de mon projet de thèse est la découverte chez la Drosophile montrant que la protéine adaptateur Kurtz, l'unique arrestine non-visuelle de la Drosophile, est un régulateur essentiel de la voie de signalisation Notch. Chez les mammifères, la famille des arrestines (à l'exclusion des arrestines visuelles) est composée de deux sous-familles: b-arrestines et a-arrestines. Les principaux résultats que j'ai obtenus montrent que les deux arrestines a-b sont recrutées par le récepteur Notch non-activé et permettent l ubiquitination de Notch médiée par Itch chez les mammifères. Une hétérodimérisation entre une b-arrestine et l a-arrestine ARRDC1 est nécessaire pour promouvoir l'ubiquitination de Notch et la dégradation du récepteur Notch non activé dans le lysosome. Pour conclure, nous montrons pour la première fois que a et b-arrestines agissent comme des régulateurs négatifs de la signalisation Notch, mettant en évidence l'existence d'une coopération entre ces protéines adaptatrices pour réguler le trafic des récepteursPARIS-BIUSJ-Biologie recherche (751052107) / SudocSudocFranceF

(A) Akt activity regulates Bcl-w interaction with Bcl-2 family members.

<p>Flag-Bcl-w/HeLa cells were transfected with 2 µg of either HA-Akt D+ or HA-Akt D− cDNA, and 2 µg of EE-Bax cDNA for 48 hr. Cells were harvested and 1 mg of total lysate immunoprecipitated using an anti-Flag antibody. The immunoprecipitates were then blotted with an anti-EE antibody. Total protein was normalized using anti-EE, -HA or -β-actin antibodies, as indicated. (B) Flag Bcl-w/HeLa cells were transfected with 2 µg of HA-Akt D+ or HA-Akt D− cDNA, and 2 µg of either EE-Bad or EE-Bik cDNA, as indicated, for 48 hr. Cells were harvested and 1 mg of total lysate immunoprecipitated using anti-Flag antibody. The immunoprecipitates were then blotted with an anti-EE antibody. Total protein was normalized using anti-EE, -HA or -β-actin antibodies, as indicated. Inactivation of Akt induced a reduction of Bcl-w interaction with the pro-apoptotic Bcl-2 members.</p

Akt controls Bcl-w localization.

<p>(A) HeLa cells were subjected to fractionated separation of mitochondrial/cytosolic proteins using a mitochondria/cytosol fractionation kit (Biovision). Protein extracts were loaded onto 15% SDS polyacrilamide gel, and analyzed by western blot by anti-Bcl-w antibody. As a control of the mitochondrial fraction, an anti-cox4 antibody was used. (B) HeLa cells were transfected with 2 µg of HA-Akt WT, D+, or D− for 48 hrs. Cells were subjected to mitochondria/cytosol separation as above. Protein extracts were analyzed by western blot using anti-Bcl-w, anti-Akt, or anti-cox4 antibodies. (C) Cells were transfected with 100 nM of siAkt-RNA for 48 hrs. Cytosol and mitochondria were isolated as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004070#s2" target="_blank">methods</a> and analyzed by western blot using the indicated antibodies.</p

Akt activity regulates Bcl-w expression.

<p>(A) HeLa cells were transfected with 2 µg of HA-Akt wt, Akt D+, or HA-Akt D− cDNA and 2 µg Flag-Bcl-w for 48 hrs. Protein extracts were immunoprecipitated with an anti-HA monoclonal antibody. Immunoprecipitates were resolved on 12% SDS-PAGE and transferred to Hybond-C nitrocellulose. Membranes were incubated with anti-Flag antibody (0.2 µg/ml). 50 µg of total sample extracts were also analyzed by western blot using the indicated antibodies. Loading control was obtained using anti-β actin antibody. (B) HeLa cells were transfected with 4 µg of HA-Akt wt, HA-Akt D+, or HA-Akt D− cDNA for 48 hrs. Protein extracts were blotted with anti-Bcl-w antibody in order to detect endogenous levels of Bcl-w. Loading control was obtained with anti-β actin antibody. (C) Cells were transfected with 100 nM of siAkt-RNA for 48 hrs. Cellular proteins were solubilized and analyzed by western blot using the indicated antibodies. (D) HeLa cells were treated with 10, 20 or 40 µM of LY294002 for 24 hrs. Protein extracts were analyzed by western blot using the indicated antibodies. Loading control was obtained using anti-β actin antibody. (E) Bcl-w HeLa cells were treated with 10 µM of MG-132 for 8 hrs. 40 µg of protein extracts were analyzed by western blot with anti-Bcl-w antibodies. Loading control was obtained using anti-β actin antibody.</p

Effects of Bcl-w si RNA on cell death.

<p>(A) Cells were transfected with 150 nM of siBcl-w-RNAs for 72 hrs. Total lysates were analyzed by western blot using anti-Bcl-w antibodies. Loading control was obtained with an anti-β-actin antibody. (B, C) Cells were transfected with 150 nM of siBcl-w-RNAs for 48 hrs. Then, the cells were splitted into 96 wells and then treated with 30 µg/ml of cisplatin for 24 hr. Cell death was then analyzed with MTT (B) or propidium iodide staining and FACS analysis (C). Bcl-w down-regulation induces an increase of cell death.</p

Akt interacts with Bcl-w.

<p>(A) Co-immunoprecipitation of endogenous Akt with Bcl-w. Wt HeLa cells were lysed and 1 mg of protein extract immunoprecipitated using an anti-Bcl-w antibody. Immunoprecipitates and total lysates (50 µg) were separated on 12%SDS polyacrilamide gel and blotted with an anti-Akt antibody. As negative control, proteins were incubated with beads without antibody (B) Co-immunoprecipitation of transfected Akt with FLAG-Bcl-w or EE-BAD. HEK-293 cells were tansfected with 2 µg of HA-Akt and 2 µg of FLAG-Bcl-w or EE-BAD cDNAs, as indicated. After 48 hr, cells were lysed, and 1 mg of protein extract was immunoprecipitated using an anti-HA antibody. Immunoprecipitates were subsequently blotted with anti-HA, anti-Flag or anti-EE antibodies, as indicated. (C) HEK-293 cells were transfected with 2 µg of either wt-Bcl-w cDNA or the deletion mutants, Bcl-w/BH4 or Bcl-w/CT, as indicated. Protein extracts were immunoprecipitated using an anti-Akt antibody. Immunoprecipitates and total lysates were resolved on 12%SDS-PAGE and transferred to Hybond-C nitrocellulose. Membranes were incubated with an anti-FLAG antibody. Both deletion mutants, Bclw/BH4 and Bclw/CT, immunoprecipitated with Akt.</p