Gemcitabine alters the proteasome composition and immunopeptidome of tumour cells

Abstract

The antigenic makeup of tumour cells can have a profound effect on the progression of cancer and success of immunotherapies. Therefore, one strategy to improve the efficacy of cancer treatments is to augment the antigens displayed by tumours. The present study explores how the recognition of tumour cells may be altered by non-cytotoxic concentrations of gemcitabine (GEM). Testing a panel of chemotherapeutics in human cancer cell lines in vitro, it was found that GEM increased surface expression of HLA-A,B,C and that underlying this were specific increases in β-2-microglobulin and immunoproteasome subunit proteins. Furthermore, the peptide antigen repertoire displayed on HLA class I was altered, revealing a number of novel antigens, many of which that were derived from proteins involved in the DNA-damage response. Changes in the nature of the peptide antigens eluted from HLA-A,B,C after GEM treatment consisted of amino acid anchor-residue modifications and changes in peptide length which rendered peptides likely to favour alternative HLA-alleles and increased their predicted immunogenicity. Signalling through the MAPK/ERK and NFκB/RelB pathways was associated with these changes. These data may explain observations made in previous in vivo studies, advise as to which antigens should be used in future vaccination protocols and reinforce the idea that chemotherapy and immunotherapy could be used in combination.

Figure 1.

GEM increased HLA class I expression on tumour cells in a β2 m-associated manner. a) Representative histograms showing change in HLA-A,B,C MFI in response to culture with drugs at IC₂₅. b) The effect of chemotherapy drugs on surface expression of HLA-A,B,C on tumour cells as measured by flow cytometry. Data are expressed relative to untreated controls. Mean and standard deviation is plotted and values are significantly different (**** = p<0.0001, ** = p<0.01) to controls by one-way ANOVA with Dunnett's multiple comparisons test. n = 3. c) Blots representative of three experiments showing expression of HLA α-heavy chains and β2 m proteins in untreated (Un), IFNγ-treated (1000 IU/ml) or GEM-treated (100 nM) tumour cells. d) Transcription of the β2 m gene was increased in HCT116 cells in response to GEM, as assessed by qPCR. n = 1. e) Mean fold-change in mRNA for HLA class I genes in response to GEM. n = 3. f) Tumour cells transfected with human β2 m expressing plasmid had increased surface expression of HLA class I 48 hours after transfection, as measured by flow cytometry. Means and standard deviations are plotted and mock-transfected and β2 m-transfected are significantly different by student's paired t-test. For A549 and MCF-7, n = 3, for HCT116 n = 6.

Figure 2.

GEM induced immunoproteasome subunits in tumour cells. a) Immunoproteasome expression was assessed by Western blot after 24 hour culture with 100 nM GEM. IFNγ (1000 IU/ml) was used as a positive control. Blots are representative of three separate experiments. b) Proteasomes were isolated from HCT116 cells either untreated (Un) or treated with 100 nM GEM (G) for 24 hours. Expression of proteasome and immunoproteasome subunits in crude cell lysate, unbound and proteasome pull-out fractions was then measured. Representative blots from three separate experiments are shown.

Figure 3.

Peptide ligands eluted from HLA-A,B,C on HCT116 cells treated with 100 nM GEM were different from those found on untreated control cells. All panels represent mean and standard deviation values of three separate peptide elution experiments and show differences in the proportion of peptides with various characteristics between those found exclusively on treated or exclusively on control cells in terms of: a) Peptide length, b) C-terminal amino acid, c) Predicted HLA-allele binding, d) Predicted immunogenicity. Values significant different from controls by student's t-test are indicated (** = p<0.01). Due to the anomalous appearance of a number of 11 and 12mer peptides in one of the three peptide elution experiments, data from this particular experiment was removed from the analysis of a) and replaced by peptide length data from a small pilot study.

Figure 4.

GEM alters the immunopeptidome of tumour cells. a) Relative abundance of peptides from MDM2, RPA70 and TOPO1 proteins was assessed in control and GEM-treated HCT116 cells. n = 2. b) The relative abundance of all peptides present in both control and GEM treated cells in all three experiments is shown. Values significantly different from controls by student's paired t-test are indicated (* = p<0.05). c) Western blots showing the expression of proteins that have an altered peptide representation in GEM-treated cells are shown. Blots are representative of three separate experiments.

Figure 5.

MEK and NFκB are associated with GEM-mediated HLA-A,B,C upregulation. For all panels n = 3 and involve HCT116 cells unless otherwise stated. a) Phosphorylation of ERK1/2 was increased in response to GEM in HCT116, A549 and MCF-7 cells. Representative blots from MCF-7 cells are shown. b) GEM-mediated HLA-A,B,C upregulation was reduced by inhibiting ERK signalling. HCT116 cells were untreated (Un) or cultured with 100 nM GEM, +/− ERK inhibitor U0126 (-I ERK) at 1 μM or 10 μM and HLA-A,B,C measured by flow cytometry. n = 4. c) As b) but measuring intracellular levels of LMP2 expression by Western blot. d) Representative blot showing the effect of U0126 on expression of LMP2 and pERK. e) Expression of p52 and RelB were measured in the cytoplasmic and nuclear fractions of HCT116 cells treated with GEM for various durations. Representative blots are shown. Anti-β-tubulin and lamin A/C antibodies used as loading and purity controls for cytoplasmic and nuclear fractions, respectively. f) HCT116 tumour cells were treated with 100 nM GEM +/−IKK-specific NFκB inhibitor. The effect on upregulation of HLA-A,B,C at the surface of the tumour cells was then assessed. b), c) and f) Values significantly different from GEM-treated by one-way ANOVA with Dunnett's test for multiple comparisons are shown. * = p<0.05, ** = p<0.01.