Identification of two HLA-A*0201 immunogenic epitopes of lactate dehydrogenase C (LDHC): potential novel targets for cancer immunotherapy

Lactate dehydrogenase C (LDHC) is an archetypical cancer testis antigen with limited expression in adult tissues and re-expression in tumors. This restricted expression pattern together with the important role of LDHC in cancer metabolism renders LDHC a potential target for immunotherapy. This study is the first to investigate the immunogenicity of LDHC using T cells from healthy individuals. LDHC-specific T cell responses were induced by in vitro stimulation with synthetic peptides, or by priming with autologous peptide-pulsed dendritic cells. We evaluated T cell activation by IFN-γ ELISpot and determined cytolytic activity of HLA-A*0201-restricted T cells in breast cancer cell co-cultures. In vitro T cell stimulation induced IFN-γ secretion in response to numerous LDHC-derived peptides. Analysis of HLA-A*0201 responses revealed a significant T cell activation after stimulation with peptide pools 2 (PP2) and 8 (PP8). The PP2- and PP8-specific T cells displayed cytolytic activity against breast cancer cells with endogenous LDHC expression within a HLA-A*0201 context. We identified peptides LDHC41−55 and LDHC288−303 from PP2 and PP8 to elicit a functional cellular immune response. More specifically, we found an increase in IFN-γ secretion by CD8 + T cells and cancer-cell-killing of HLA-A*0201/LDHC positive breast cancer cells by LDHC41−55- and LDHC288−303-induced T cells, albeit with a possible antigen recognition threshold. The majority of induced T cells displayed an effector memory phenotype. To conclude, our findings support the rationale to assess LDHC as a targetable cancer testis antigen for immunotherapy, and in particular the HLA-A*0201 restricted LDHC41–55 and LDHC288–303 peptides within LDHC.Other Information Published in: Cancer Immunology, Immunotherapy License: https://creativecommons.org/licenses/by/4.0See article on publisher's website: http://dx.doi.org/10.1007/s00262-020-02480-4</p

PRAME promotes epithelial-to-mesenchymal transition in triple negative breast cancer

The triple negative breast cancer (TNBC) paradox marks a major challenge in the treatment-decision making process. TNBC patients generally respond better to neoadjuvant chemotherapy compared to other breast cancer patients; however, they have a substantial higher risk of disease recurrence. We evaluated the expression of the tumor-associated antigen PReferentially Antigen expressed in MElanoma (PRAME) as a prognostic biomarker in breast cancer and explored its role in cell migration and invasion, key hallmarks of progressive and metastatic disease. TCGA and GTeX datasets were interrogated to assess the expression of PRAME in relation to overall and disease-free survival. The role of PRAME in cell migration and invasion was investigated using gain- and loss-of-function TNBC cell line models. We show that PRAME promotes migration and invasion of TNBC cells through changes in expression of E-cadherin, N-cadherin, vimentin and ZEB1, core markers of an epithelial-to-mesenchymal transition. Mechanistic analysis of PRAME-overexpressing cells showed an upregulation of 11 genes (SNAI1, TCF4, TWIST1, FOXC2, IL1RN, MMP2, SOX10, WNT11, MMP3, PDGFRB, and JAG1) and downregulation of 2 genes (BMP7 and TSPAN13). Gene ontology analyses revealed enrichment of genes that are dysregulated in ovarian and esophageal cancer and are involved in transcription and apoptosis. In line with this, interrogation of TCGA and GTEx data demonstrated an increased PRAME expression in ovarian and esophageal tumor tissues in addition to breast tumors where it is associated with worse survival. Our findings indicate that PRAME plays a tumor-promoting role in triple negative breast cancer by increasing cancer cell motility through EMT-gene reprogramming. Therefore, PRAME could serve as a prognostic biomarker and/or therapeutic target in TNBC.Other Information Published in: Journal of Translational Medicine License: http://creativecommons.org/licenses/by/4.0/See article on publisher's website: http://dx.doi.org/10.1186/s12967-018-1757-3</p

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

Tax requires CADM1 for NF-κB activation.

<p>(A) Lentiviral Tax was transduced in Jurkat T-cells stably expressing control scrambled shRNA or CADM1 shRNA. After 48 h, lysates were subjected to immunoblotting with anti-IκBα, anti-phospho-IκBα, anti-CADM1, anti-Tax, and anti-β-actin antibodies. (B) Primary <i>Cadm1</i>^<i>+/+</i> and <i>Cadm1</i>^{<i>−/−</i>} MEFs were transduced with Tax-expressing lentiviruses. After 48 h, lysates were subjected to immunoblotting with anti-IκBα, anti-phospho-IκBα, anti-CADM1, anti-Tax, and anti-β-actin antibodies. (C) Nuclear extracts from lentiviral expressing Tax in primary <i>Cadm1</i>^<i>+/+</i> and <i>Cadm1</i>^{<i>−/−</i>} MEFs were used for NF-κB and Oct-1 EMSA, and cytoplasmic extract were subjected to immunoblotting with anti-Tax, anti-CADM1, and anti-β-actin antibodies. (D) Lysates from HTLV-1 transformed C8166, MT-2, and MT-4 cells stably expressing control scrambled shRNA or CADM1 shRNA were subjected to immunoblotting with anti-IκBα, anti-phospho-IκBα, anti-CADM1, anti-Tax, and anti-β-actin antibodies. (E) Nuclear extracts from HTLV-1 transformed C8166, MT-2, and MT-4 cells 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. (F) Primary <i>Cadm1</i>^<i>+/+</i> and <i>Cadm1</i>^{<i>−/−</i>} MEFs were transduced with Tax-expressing lentiviruses as described for panel B. After 48 hours, RNA was prepared and subjected to RT-PCR for A20, IL-6, Bfl-1, Tax, and GAPDH expression.</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

Tax induces CADM1 expression.

<p>(A) CADM1 expression in foot, tail, spleen and bone marrow (BM) tissues derived from spontaneous tumors of 14 month old control Tax-negative and Tax-positive transgenic mice. Immunoblotting was performed with anti-CADM1, Tax, and β-actin antibodies. CADM1 expression in lentiviral-transduced empty vector wildtype Tax or Tax mutants (M22 or M47) in primary MEFs (B) and Jurkat T-cells (C) was analyzed with anti-CADM1, SOCS1, Tax, and β-actin antibodies.</p

Membrane associated CADM1 mediates K63-linked polyubiquitination of Tax and links Tax adaptor proteins in the lipid rafts.

<p>(A) MT-2 cells were stained with DAPI, anti-Tax, anti-CADM1, and anti-GM-130, and subjected to confocal microscopy. (B) MT-2 cells were stained with DAPI, anti-Tax, anti-CADM1, and cholera toxin B conjugated with red fluorescence to detect GM-1, and subjected to confocal microscopy. (C) Lipid raft fractionations from MT-2 cells stably expressing control scrambled shRNA or CADM1 shRNA were split into half and subjected to immunoprecipitation with either anti-Tax or anti-CADM1. Samples immunoprecipitated with anti-Tax were immunoblotted with anti-K63-ubi and anti-Tax. Samples immunoprecipitated with anti-CADM1 were immunoblotted with anti-CADM1, anti-TAX₁BP₁, anti-Tax, anti-NEMO, anti-Ubc13, and anti-NRP antibodies. Lysates from lipid rafts fractions were examined for Tax, phospho-IKKα/β, total IKKα, IKKβ, NEMO, CADM1, Ubc13, TAX₁BP₁, NRP, ERK1 (marker for soluble fractions), LAT (lipid raft protein marker) and GM1 (lipid raft marker).</p