Journal: Cell Communication and Signaling : CCS

Article Title: MAPK ERK5 is a novel regulator of MHC-I in cancer cells

doi: 10.1186/s12964-026-02780-9

Figure Lengend Snippet: ERK5 inhibitor and PROTAC degrader enhance MHC-I membrane levels in 3D cultures of cancer cells and tumor xenografts. A , B 3D spheroid cultures of EC Ishikawa cells were treated for 24 h with either vehicle, 5 μM JWG-071 or 5 μM GW284543 ( A ), or with either vehicle, 300 nM PROTAC negative control INY-06–089 (Cont.), or 300 nM ERK5 PROTAC INY-06–061 ( B ). MHC-I surface expression was determined by FC analysis. A A representative image of untreated 3D cultures on day five is shown. Right histograms show the percentage of MHC-I expressing cells and MFI values. B Upper histograms show the percentage of MHC-I expressing cells and MFI values. Immunoblot shows ERK5 protein levels. A-B Data are the mean ± SD of a representative experiment from three independent experiments, each performed in triplicate. ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001, as determined by two-tailed Student’s t -test ( A ) or one-way ANOVA followed by Bonferroni multiple comparisons test ( B ). C Immunohistochemical analysis of the expression of surface MHC-I in tumors from nude mice engrafted with Ishikawa cells and treated either with vehicle or 50 mg/kg JWG-071 for 7 days. Scatter plot shows the percentage of cells expressing membrane MHC-I ( N = 5 mice per group, 5 fields per tumor; mean ± SD). Significance as determined by Mann–Whitney test

Article Snippet: ERK5 inhibitor JWG-071 (MedChemExpress), MEK5 inhibitor GW284543 (CliniSciences), ERK5 proteolysis-targeting chimera (PROTAC) INY-06–061 and its negative control INY-06–089 (a gift from Prof. N. Gray, [ ]) and proteasome inhibitor MG-132 (MedChemExpress) were resuspended in dimethyl sulfoxide (DMSO, Sigma).

Techniques: Membrane, Negative Control, Expressing, Western Blot, Two Tailed Test, Immunohistochemical staining, MANN-WHITNEY

Journal: Cell Communication and Signaling : CCS

Article Title: MAPK ERK5 is a novel regulator of MHC-I in cancer cells

doi: 10.1186/s12964-026-02780-9

Figure Lengend Snippet: ERK5 does not regulate transcription of classical HLA-I genes or antigen-processing pathway. A , B ERK5/MEK5 inhibition does not alter mRNA levels of HLA-A , HLA-B , HLA-C , B2M , TAP1 and TAP2 genes. Ishikawa EC cells ( A ) or IMR-32 NBL cells ( B ) were treated for 24 h with either vehicle, or 5 μM JWG-071, or 5 μM GW284543 . Relative gene expression, normalized to TBP mRNA was assessed by RT-qPCR. KLF2 relative expression was used as a control for ERK5 or MEK5 inhibition. C , D Effect of ERK5 inhibition on the transcriptional programs of Ishikawa EC cells. Cells were treated for 24 h with 5 μM JWG-071, followed by RNA-seq analysis. Panels show gene set enrichment analysis (GSEA) for the Hallmark “Angiogenesis” and the GoBP “Cell migration” ( C ), and for the Reactome “Class I MHC mediated antigen processing presentation” pathway, the KEGG “antigen processing and presentation by MHC Class I” pathway, and the Biocarta “MHC Pathway” ( D ). E ERK5 inhibition does not enhance Golgi-to-plasma membrane transport of MHC-I. Ishikawa cells were treated for 24 h with either vehicle, or 5 μM JWG-071, or 250 nM brefeldin A (Bref.A), or the combination of both, and MHC-I surface expression was determined by FC. Histograms show MHC-I MFI values and the percentages of cells expressing MHC-I. F Effect of ERK5 inhibition and of the lysosome inhibitors chloroquine (CQ) and bafilomycin A (Baf.), on MHC-I levels. Ishikawa cells were treated for 24 h with either vehicle, or 5 μM JWG-071, or 10 µM chloroquine/150 nM bafilomycin (A), or the combination of both. Total MHC-I protein levels were determined by immunoblot analysis. Histograms show the quantification of MHC-I levels, referred to Hsp90. Autophagy marker LC3-I/II was used as a control for lysosome inhibitors, and Hsp90 as loading control. Data represent mean ± SD from three ( A , B , E ) or two ( F ) independent experiments. ns, not significant; ∗ p < 0.05; ∗∗ , $$ p < 0.005; ∗∗∗ p < 0.001; ∗∗∗∗ , $$$$ p < 0.0001, one-way ANOVA followed by Bonferroni multiple comparisons test

Article Snippet: ERK5 inhibitor JWG-071 (MedChemExpress), MEK5 inhibitor GW284543 (CliniSciences), ERK5 proteolysis-targeting chimera (PROTAC) INY-06–061 and its negative control INY-06–089 (a gift from Prof. N. Gray, [ ]) and proteasome inhibitor MG-132 (MedChemExpress) were resuspended in dimethyl sulfoxide (DMSO, Sigma).

Techniques: Inhibition, Gene Expression, Quantitative RT-PCR, Expressing, Control, RNA Sequencing, Migration, Clinical Proteomics, Membrane, Western Blot, Marker

Journal: Cell Communication and Signaling : CCS

Article Title: MAPK ERK5 is a novel regulator of MHC-I in cancer cells

doi: 10.1186/s12964-026-02780-9

Figure Lengend Snippet: ERK5 inhibition enhances CD8 + T cell–mediated apoptosis of cancer cells. A Schematic representation of the protocol used to generate tumor-specific CD8 + T cells and their co-culture with human cancer cells. Tumor-specific antigens derived from IMR-32 or Ishikawa cancer cells were used to stimulate dendritic cells (DCs) derived from PBMCs of three healthy donors. Subsequently, T cells were activated and expanded. Finally, CD8 + T cells were isolated and co-cultured for 18 h with cancer cells pre-treated with either vehicle or JWG-071. The cytotoxic activity of CD8 + T cells in cancer cells was monitored by flow cytometry analysis of apoptosis (Annexin V/PI). B Representative dot plots of flow cytometry apoptosis assays for IMR-32 cells. C Bar graphs show the percentages of early (Annexin V +/PI-) and late (Annexin V +/PI +) apoptotic IMR-32 cells ( n = 3). Effector CD8 + T cells: target IMR-32 cells (E:T) ratios were 8:1 for donors 1 and 2, and 2:1 for donor 3. D Percentage of MHC-I + IMR-32 cells treated with either vehicle or JWG-071 and co-cultured with tumor-specific CD8 + T cells from three different donors. E Percentages of early and late apoptotic cells ( n = 3) for flow cytometry apoptosis assays performed with Ishikawa cells. Effector CD8 + T cells: target Ishikawa cells (E:T) ratios were 16:1, 8:1 and 12:1, respectively. F Percentage of MHC-I + Ishikawa cells treated with either vehicle or JWG-071 and co-cultured with tumor-specific CD8 + T cells from three different donors. Data represents mean ± SD from three independent experiments. ∗∗∗∗ p < 0.0001, ∗∗∗ p < 0.001, ∗∗ p < 0.005, ∗ p < 0.05, determined by two-way ANOVA followed by Tukey multiple comparisons test

Article Snippet: ERK5 inhibitor JWG-071 (MedChemExpress), MEK5 inhibitor GW284543 (CliniSciences), ERK5 proteolysis-targeting chimera (PROTAC) INY-06–061 and its negative control INY-06–089 (a gift from Prof. N. Gray, [ ]) and proteasome inhibitor MG-132 (MedChemExpress) were resuspended in dimethyl sulfoxide (DMSO, Sigma).

Techniques: Inhibition, Co-Culture Assay, Derivative Assay, Isolation, Cell Culture, Activity Assay, Flow Cytometry

Journal: Cell Communication and Signaling : CCS

Article Title: MAPK ERK5 is a novel regulator of MHC-I in cancer cells

doi: 10.1186/s12964-026-02780-9

Figure Lengend Snippet: ERK5 and MEK5 inhibitors enhance MHC-I surface expression in cancer cells without affecting PD-L1. A Effect of ERK5/MEK5 inhibitors in cancer cells with low basal MHC-I expression. IMR-32, Ishikawa and LNCaP cells were treated for 24 h with vehicle, 5 μM JWG-071 (ERK5i) or 5 μM GW284543 (MEK5i), and MHC-I surface expression was assessed by flow cytometry (FC). Left panels show representative FC histograms, and right panels show percentages of cells expressing MHC-I and the mean fluorescence intensity (MFI) values. B Effect of ERK5/MEK5 inhibitors in cancer cells with high basal MHC-I expression. ARK1, A549 and SW620 cells were treated as in ( A ), and MHC-I membrane expression was determined by FC. Representative FC histograms and MFI values are presented. C Effect of ERK5/MEK5 inhibitors in membrane PD-L1 expression in cancer cells with low basal MHC-I levels. IMR-32, Ishikawa and LNCaP cells were treated as in ( A ), or with 20 ng/mL IFN-γ or 8 µM MG-132 or 10 ng/mL TNF-α as positive controls. PD-L1 surface expression was measured by FC. Left panels show representative FC histograms, and right panels show percentages of cells expressing PD-L1 and MFI values. D Effect of ERK5/MEK5 inhibitors on total MHC-I protein levels. The indicated cells were treated with vehicle, 5 μM JWG-071 or 5 μM GW284543 for 24 h, and MHC-I protein levels were determined by immunoblot. The numbers indicate relative MHC-I protein levels referred to vehicle (mean ± SD from three independent experiments). Immunoblot ( D ) or qPCR analysis of p21 mRNA levels, as well as relative KLF2 gene expression ( E ) were used as controls for ERK5 inhibition. mRNA levels were normalized to TBP mRNA levels. F Effect of 5 μM ERK5i on MHC-I expression in Ishikawa ERK5 wild type (ERK5 +/+ ) and ERK5 CRISPR-KO (ERK5 −/− ) cells. Cells were treated 24 h and MHC-I membrane levels were assessed by FC (left panels show percentages of cells expressing MHC-I and MFI values), and total MHC-I protein levels by immunoblot (right panels). p21 levels were used as a control for ERK5 inhibition ( G ) Total MHC-I and ERK5 protein levels inversely correlate in cancer cells. Immunoblot analysis show MHC-I and ERK5 expression in the indicated cells. Right plot shows Pearson’s pairwise correlation analysis of relative levels of ERK5 and MHC-I protein expression in the cell lines tested. Data represents mean ± SD from three independent experiments. ns, not significant; ∗ p < 0.05; ∗∗ p < 0.005; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001, as determined by one-way ANOVA followed by Bonferroni multiple comparisons test

Article Snippet: ERK5 inhibitor JWG-071 (MedChemExpress), MEK5 inhibitor GW284543 (CliniSciences), ERK5 proteolysis-targeting chimera (PROTAC) INY-06–061 and its negative control INY-06–089 (a gift from Prof. N. Gray, [ ]) and proteasome inhibitor MG-132 (MedChemExpress) were resuspended in dimethyl sulfoxide (DMSO, Sigma).

Techniques: Expressing, Flow Cytometry, Fluorescence, Membrane, Western Blot, Gene Expression, Inhibition, CRISPR, Control

Journal: Cell Communication and Signaling : CCS

Article Title: MAPK ERK5 is a novel regulator of MHC-I in cancer cells

doi: 10.1186/s12964-026-02780-9

Figure Lengend Snippet: ERK5 targeted degradation upregulates MHC-I membrane levels in cancer cells, whereas ERK5 overexpression reduces MHC-I expression. A - C ERK5 PROTAC degrader enhances MHC-I membrane levels in cancer cells with low basal MHC-I surface expression. IMR-32 ( A ), Ishikawa ( B ) and LNCaP ( C ) cells were treated for 24 h with vehicle, 300 nM PROTAC negative control INY-06–089, or 300 nM ERK5 PROTAC INY-06–061, and surface MHC-I (upper panels) and total MHC-I were determined by FC and immunoblot, respectively. GAPDH levels are shown as loading control. D ERK5 PROTAC degrader does not alter MHC-I in cancer cells with high basal MHC-I membrane levels. ARK1, A549 and SW620 cells were treated and analyzed as in ( A ). E ERK5 overexpression impairs MHC-I membrane expression in endometrial cancer cells. Ishikawa cells were co-transfected with vectors encoding for GST-tagged ERK5 and GFP, and MHC-I membrane expression was analyzed by FC within the GFP + cell population. Left bar graphs show the percentage of MHC-I expressing cells and the MFI values. Immunoblots show levels of ERK5 and MHC-I. p21 protein expression was used as control for ERK5 activity, and Hsp90 as loading control. The numbers indicate relative MHC-I protein levels (mean ± SD from three independent experiments). Results represent mean ± SD from three independent experiments. ns, not significant; ∗∗ p < 0.005; ∗∗∗∗ p < 0.0001 as determined by one-way ANOVA followed by Bonferroni multiple comparisons test ( A-D ) or two-tailed Student’s t -test ( E )

Article Snippet: ERK5 inhibitor JWG-071 (MedChemExpress), MEK5 inhibitor GW284543 (CliniSciences), ERK5 proteolysis-targeting chimera (PROTAC) INY-06–061 and its negative control INY-06–089 (a gift from Prof. N. Gray, [ ]) and proteasome inhibitor MG-132 (MedChemExpress) were resuspended in dimethyl sulfoxide (DMSO, Sigma).

Techniques: Membrane, Over Expression, Expressing, Negative Control, Western Blot, Control, Transfection, Activity Assay, Two Tailed Test