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

A schematic representation of the complex interplay of TGF-β-signaling pathways regulating TIMP-3 expression in human fibroblasts.

<p>Stimulation of human gingival fibroblasts with TGF-β results in activation of Smad3, ERK1/2 and p38 MAPK pathways. Activation of all three pathways is required for induction of TIMP-3 expression by TGF-β Smad3 associates with Smad4 and mediates induction of TIMP-3 expression by TGF-β. ERK1/2 and p38 MAPK pathways both co-operate with Smad3 in mediating the induction of TIMP-3 expression by TGF-β.</p

Expression of Smad4 rescues the TGF-β response of TIMP-3 and PAI-1 in Smad4 null fibroblasts.

<p>(<b>A</b>) EF7WT (wild-type) and EF7KO (Smad4 deficient) fibroblasts were transduced with recombinant adenovirus for HA-tagged Smad4 (RAdSmad4), or with empty control virus RAd66 at MOI 100 (EF7WT) or 300 (EF7KO). After 36 h incubation cell lysates were harvested and analyzed by Western blotting to detect the levels of endogenous and exogenous Smad4. Anti-HA antibody was used to detect adenovirally delivered Smad4 (upper panel) and anti-Smad4 to detect endogenous Smad4 (lower panel). (<b>B</b>) EF7KO fibroblasts were infected with adenoviruses RAdSmad4 or RAd66. After 36 h incubation the cells were stimulated with TGF-β1 (5 ng/ml) for different periods of time, as indicated. Total RNA was extracted and analyzed by qRT-PCR to determine TIMP-3 and PAI-1 mRNA levels. mRNA expression (mean ± SEM from two separate experiments, both run with duplicates) is shown relative to 18S ribosomal RNA. *p<0.05, t-test).</p

TGF-β induces TIMP-3 gene expression in a Smad-dependent manner in fibroblasts.

<p>(<b>A</b>) Human gingival fibroblasts were treated with TGF-β1 (5 ng/ml) for 24 h. Thereafter, total cellular RNAs were harvested and analyzed for the expression of TIMP-3, TIMP-1, PAI-1, and GAPDH mRNAs by Northern blotting. (<b>B</b>) EF7WT and EF7Smad4KO (Smad4 deficient) cells were treated with TGF-β1 (5 ng/ml) for 3 h and 12 h or left untreated (control). Total RNA was extracted and TIMP-3 and PAI-1 gene expression was determined by qRT-PCR. mRNA expression (mean+SD) is shown relative to 18S ribosomal RNA (n = 4). *p<0.05, **p<0.005 (t-test) for TGF-β vs. control cultures.</p

Smad3, p38α and ERK1/2 cooperate in the induction of TIMP-3 gene expression in human fibroblasts.

<p>(<b>A</b>) Human gingival fibroblasts were serum starved for 18 h, and treated for 1 h with PD98059 (30 µM), or SB203580 (10 µM), specific chemical inhibitors for MEK1 or p38, respectively. Subsequently, TGF-β1 (5 ng/ml) was added, and the cultures incubated for 16 h. Total cellular RNAs were harvested and analyzed for the levels of TIMP-3, TIMP-1, PAI-1 and GAPDH mRNAs by Northern blot hybridizations. (<b>B</b>) Human gingival fibroblasts were transduced with recombinant adenoviruses for wild-type p38α (RAdp38α), constitutively active MKK3b (RAdMKK3bE), Smad3 (RAdSmad3), Smad4 (RAdSmad4), or with empty control virus (RAd66) at MOI 500, and incubated for 24 h. Total cellular RNA was analyzed with Northern blot hybridizations for the expression of TIMP-3, TIMP-1, PAI-1, and GAPDH mRNAs. (<b>C</b>) Human gingival fibroblasts were transduced with recombinant adenoviruses for constitutively active MEK1 (RAdMEK1CA), constitutively active MKK3b (RAdMKK3bE), Smad3 (RAdSmad3) and control virus RAd66 as in (<b>B</b>). Total cellular RNA was analyzed with Northern blot hybridizations for the expression of TIMP-3, TIMP-1, PAI-1, and GAPDH mRNAs.</p

Smad3 mediates TGF-β-elicited induction of TIMP-3 expression in human fibroblasts.

<p>(<b>A</b>) Normal human gingival fibroblasts were transduced with recombinant adenoviruses for Smad2 (RAdSmad2), Smad3 (RAdSmad3), dominant negative Smad3 (RAdSmad3DN), or with empty control virus (RAd66) at MOI 500, and incubated for 18 h. Thereafter, the cells were treated with TGF-β1 for 24 h. The cell layers were harvested for RNA extraction and analyzed for the expression of TIMP-3 or GAPDH by Northern blot hybridizations. (<b>B</b>) Normal human gingival fibroblasts were infected with RAdSmad3, RAdSmad3DN, adenovirus for Smad7 (RAdSmad7), or with empty control virus (RAdpCA3) as in (<b>A</b>). Cells were treated with TGF-β1 for 24 h, the cell layers harvested and analyzed for the expression of TIMP-3 by Western blotting. Equal loading was confirmed by stripping and reprobing the same filter for β-actin.</p

Association of plasma MMP8 levels with the extent of lymph node metastasis and risk of distant metastasis

note: controls + pN0 comprises healthy women and breast cancer patients without detectable lymph node or distant metastasis; pN1+pN2 comprises patients with moderate lymph node metastasis but without distant metastasis; pN3 comprises patients with extensive lymph node metastasis and a high risk of distant metastasis (box-whisker diagrams with median, 1quartile, 3quartile and non-outlier range).<p><b>Copyright information:</b></p><p>Taken from "Plasma MMP1 and MMP8 expression in breast cancer: Protective role of MMP8 against lymph node metastasis"</p><p>http://www.biomedcentral.com/1471-2407/8/77</p><p>BMC Cancer 2008;8():77-77.</p><p>Published online 20 Mar 2008</p><p>PMCID:PMC2278147.</p><p></p

Differences in plasma levels of MMP1 and MMP8 between patients with and without inflammatory breast cancer (box-whisker diagrams with median, 1quartile, 3quartile and non-outlier range)

<p><b>Copyright information:</b></p><p>Taken from "Plasma MMP1 and MMP8 expression in breast cancer: Protective role of MMP8 against lymph node metastasis"</p><p>http://www.biomedcentral.com/1471-2407/8/77</p><p>BMC Cancer 2008;8():77-77.</p><p>Published online 20 Mar 2008</p><p>PMCID:PMC2278147.</p><p></p

Additional file 1: Table S1. of Loss of MMP-8 in ductal carcinoma in situ (DCIS)-associated myoepithelial cells contributes to tumour promotion through altered adhesive and proteolytic function

Summary of breast tissue examined for MMP8. (DOC 27 kb