CADM1 is essential for KSHV-encoded vGPCR-and vFLIP-mediated chronic NF-κB activation

<div><p>Approximately 12% of all human cancers worldwide are caused by infections with oncogenic viruses. Kaposi's sarcoma herpesvirus/human herpesvirus 8 (KSHV/HHV8) is one of the oncogenic viruses responsible for human cancers, including Kaposi’s sarcoma (KS), Primary Effusion Lymphoma (PEL), and the lymphoproliferative disorder multicentric Castleman’s disease (MCD). Chronic inflammation mediated by KSHV infection plays a decisive role in the development and survival of these cancers. NF-κB, a family of transcription factors regulating inflammation, cell survival, and proliferation, is persistently activated in KSHV-infected cells. The KSHV latent and lytic expressing oncogenes involved in NF-κB activation are vFLIP/K13 and vGPCR, respectively. However, the mechanisms by which NF-κB is activated by vFLIP and vGPCR are poorly understood. In this study, we have found that a host molecule, Cell Adhesion Molecule 1 (CADM1), is robustly upregulated in KSHV-infected PBMCs and KSHV-associated PEL cells. Further investigation determined that both vFLIP and vGPCR interacted with CADM1. The PDZ binding motif localized at the carboxyl terminus of CADM1 is essential for both vGPCR and vFLIP to maintain chronic NF-κB activation. Membrane lipid raft associated CADM1 interaction with vFLIP is critical for the initiation of IKK kinase complex and NF-κB activation in the PEL cells. In addition, CADM1 played essential roles in the survival of KSHV-associated PEL cells. These data indicate that CADM1 plays key roles in the activation of NF-κB pathways during latent and lytic phases of the KSHV life cycle and the survival of KSHV-infected cells.</p></div

CADM1 expression is upregulated in KSHV-infected cells.

<p>(A-B) Quantitative real-time PCR (qRT-PCR) analysis of CADM1 from HeLa and primary human B cells with and without KSHV infection. (C-D) qRT-PCR analysis of CADM1 from KSHV-associated PEL cell lines (BC-1, BC-3, BCBL-1, and UM-PEL-3) (error bars, s.e.m. of triplicate samples). (E-G) Western blot analysis of CADM1 and LANA in KSHV-infected HeLa, human primary B cells, and PEL cell lines (BC-1, BC-3, BCBL-1, and UM-PEL-3).</p

Membrane-associated CADM1 mediates vFLIP and NEMO interactions and IKK complex activation in lipid rafts.

<p>(A) BC-3 cells were stained with DAPI, anti-vFLIP, anti-CADM1, and cholera toxin B conjugated with red fluorescence to detect GM-1 and subjected to confocal microscopy. (B) Lipid raft fractionations from BC-3 cells stably expressing control scrambled shRNA or CADM1 shRNA were subjected to immunoprecipitation with anti-vFLIP. Samples immunoprecipitated with anti-vFLIP were immunoblotted with anti-vFLIP, anti-NEMO, and anti-CADM1. Lysates from lipid rafts fractions were examined for vFLIP, phospho-IKKα/β, total IKKα, IKKβ, NEMO, CADM1, ERK1 (marker for soluble fractions), and Lyn (lipid raft protein marker).</p

CADM1 is required for KSHV vFLIP to activate NF-κB.

<p>(A) NF-κB luciferase assay using lysates of HeLa cells expressing increasing amounts of CADM1 or vFLIP. HeLa cells were transfected with increasing amounts of either CADM1 or vFLIP with κB‐TATA Luc and pRL‐tk plasmids. After 36 hours, lysates were subjected to dual luciferase assays. The lysates were also subjected to immunoblotting to examine CADM1 and vFLIP expression using anti-Flag antibody. (B) NF-κB luciferase assay using lysates of HeLa cells stably expressing control scrambled shRNA or three different CADM1 shRNAs and transfected with pRL-tk, κB-TATA Luc and vFLIP as indicated. Immunoblot analyses of CADM1 protein expression in HeLa cells after transduction with lentiviruses expressing different shRNAs targeting distinct sequences of the CADM1 transcript. (C) NF-κB luciferase assay using lysates of <i>Cadm1</i>^<i>+/+</i> and <i>Cadm1</i>^{<i>−/−</i>} MEFs transfected with pRL-tk internal control Renilla luciferase plasmid, κB-TATA Luc and vFLIP as indicated. The lysates were also subjected to immunoblotting to examine vFLIP expression. (D) A schematic overview of the FLAG-CADM1 deletion mutants ΔSP, ΔCP, ΔEC, ΔPDZ-BM and ΔFERM. (E) NF-κB luciferase assay of lysates of <i>Cadm1</i>^{<i>−/−</i>} MEFs transfected with an NF-κB firefly luciferase reporter and a renilla luciferase vector reporter together with empty vector or an expression vector for Flag-tagged wild-type CADM1, CADM1 ΔSP, CADM1 ΔCP, CADM1 ΔEC, CADM1 ΔPDZ-BM and CADM1 ΔFERM-BM with vFLIP. The lysates were also subjected to immunoblotting to examine expression of vFLIP and Flag for wild-type and deletion mutants of CADM1. Error bars represent s.e.m. of triplicates.</p

CADM1 is required for PEL cell survival.

<p>(A) Requirement of CADM1 for the viability of PEL cell lines. Cell viability assay was performed 96 hours after BC-1, BC-3, and BCBL-1 cells were transduced with lentiviruses expressing the indicated shRNAs. Relative cell viability (%) was expressed as a percentage relative to the control cells. (B) CADM1 protein was knocked down in BC-1, BC-3, and BCBL-1 cells after lentiviral transduction expressing the indicated shRNAs. Immunoblotting was performed with whole cell lysates. (C) Flow cytometric analysis of PEL cell lines transduced with shRNAs as described in (A). Cells were stained with both annexin-V-Alexa Fluor 488 and propidium iodide (PI). The distribution of cells is indicated as a percentage in each quadrant.</p

Lentiviral‐mediated transfer of wild-type CADM1 in CADM1-depleted PEL cells restores cell viability.

<p>(A) Suppression of CADM1 expression after 3’UTR CADM1 siRNA transfection in HeLa cells. Lysates were immunoblotted with anti-CADM1 and anti-β‐actin antibodies. (B-D) BC-1, BC-3, and BCBL-1 cells were transfected with 3’UTR CADM1 siRNA followed by transduction of lentiviruses containing wild-type Flag-CADM1 as indicated. Relative cell viability (%) was expressed as a percentage relative to the control cells. Whole cell lysates were immunoblotted with anti-CADM1, anti-β‐actin, and anti-Flag antibodies.</p

vFLIP interacts with CADM1.

<p>(A) HeLa cells were transfected with Flag-vFLIP. After 48 hours, cells were lysed and immunoprecipitated with either anti-Flag or control anti-IgG, followed by immunoblotting with anti-CADM1 and anti-Flag antibodies (Flag-vFLIP co-migrated with the IgG light chain). Lysates were examined for Flag-vFLIP and CADM1 expression. (B) Primary <i>Cadm1</i>^{<i>−/−</i>} MEFs were transfected with Flag-vFLIP expression vector, with or without Flag-CADM1. After 48 hours post-transfection, lysates were immunoprecipitated with anti-vFLIP and detected by immunoblotting with anti-CADM1 and vFLIP antibodies. Lysates were immunoblotted with anti-vFLIP and anti-CADM1 antibodies. (C) Lysates from PEL cell lines (BC-1, BC-3, and BCBL-1) were immunoprecipitated with either anti-CADM1 or control anti-IgG, followed by immunoblotting with anti-vFLIP and anti-CADM1. Lysates were examined for vFLIP and CADM1 expression. (D) Lysates from PEL cell lines (BC-1, BC-3, and BCBL-1) were immunoprecipitated with either anti-vFLIP or control anti-IgG, followed by immunoblotting with anti-CADM1 and anti-vFLIP. Lysates were examined for vFLIP and CADM1 expression.</p

CADM1 expression is required for vFLIP-induced NF-κB activation.

<p>(A) Lysates from PEL cell lines (BC-1, BC-3, and BCBL-1) stably expressing control scrambled shRNA or CADM1 shRNA were subjected to immunoblotting with total anti-IκBα, anti-phospho-IκBα, anti-CADM1, and anti-β-actin antibodies. (B) Nuclear extracts from PEL cell lines (BC-1, BC-3, and BCBL-1) stably expressing control scrambled shRNA or CADM1 shRNA were used for NF-κB and Oct-1 EMSA, and cytoplasmic extracts were subjected to immunoblotting with anti-CADM1 and anti-β-actin antibodies. (C) Primary <i>Cadm1</i>^<i>+/+</i> and <i>Cadm1</i>^{<i>−/−</i>} MEFs were transfected with vFLIP plasmid. After 48 h, lysates were subjected to immunoblotting with anti-phospho-IκBα, anti-CADM1, and anti-Flag antibodies. (D) Nuclear extracts from primary <i>Cadm1</i>^<i>+/</i>+ and <i>Cadm1</i>^{<i>−/−</i>} MEFs transfected with vFLIP were used for NF-κB and Oct-1 EMSA, and cytoplasmic extracts were subjected to immunoblotting with anti-Flag antibody. (E) Quantitative real-time PCR (qRT-PCR) analysis of <i>Tnf and Il-6</i> from <i>Cadm1</i>^<i>+/</i>+ and <i>Cadm1</i>^{<i>−/−</i>} MEFs expressing vFLIP for 48 h. Lysates were subjected to immunoblotting with anti-Flag for vFLIP protein expression.</p