Additional file 4 of Mutant p53-ENTPD5 control of the calnexin/calreticulin cycle: a druggable target for inhibiting integrin-α5-driven metastasis

Additional file 4: Supplemental Figure S4. ENTPD5 co-localizes with PDIA3 in the endoplasmic reticulum. Shown are representative confocal immunofluorescence microscopy images of MIA PaCa-2 cells stained with antibodies against the endoplasmic reticulum marker PDIA3 (protein disulfide isomerase family A member 3, also known as ERp57) and ENTPD5. Shown is the Pearson correlation coefficient for co-localization calculated with the Coloc 2 plug-in in ImageJ

Additional file 6 of Mutant p53-ENTPD5 control of the calnexin/calreticulin cycle: a druggable target for inhibiting integrin-α5-driven metastasis

Additional file 6: Supplemental Figure S6. α-glucosidase inhibitors block mutant p53-dependent ITGA5 expression and function. a RT-qPCR analysis of ITGA5 mRNA expression in MIA PaCa-2 cells treated with Acarbose and UV-4 as indicated. Shown is the GAPDH-normalized expression relative to vehicle-treated control cells as 100% (n=3 replicates). b Adhesion kinetics on FN of Acarbose-treated MIA PaCa-2 cells with tet-inducible ENTPD5 expression. c-d Migration and invasion of Acarbose-treated MIA PaCa-2 cells in the absence and presence of FN. e-f MIA PaCa-2 cells were treated with Acarbose or UV-4 and analyzed by real-time live cell imaging. e Confluence curves. Shown is the mean confluence of n=3 replicates. Shading for vehicle-treated control cells indicates the SD. b Proliferation was quantified as the area under the confluence curve and normalized to vehicle-treated control cells as 100%. All results are shown as mean ± SD (n=3 replicates)

Additional file 2 of Mutant p53-ENTPD5 control of the calnexin/calreticulin cycle: a druggable target for inhibiting integrin-α5-driven metastasis

Additional file 2: Supplemental Figure S2. Kaplan-Meier plots showing disease-free survival for patients with the indicated cancer types (PDAC, pancreatic ductal adenocarcinoma; LUAD, lung adenocarcinoma; BRCA, breast cancer; OVCA, ovarian carcinoma) stratified into high vs. low ITGA5 or ITGB1 mRNA expressing groups. Plots were generated and data statistically analyzed with GEPIA2 [46]

LBH589 modulated the expression of surface markers in cancer and immune effector cells.

<p>(<b>A</b>) Flow cytometry for the cell surface marker MICA/B on HL cells after 4 and 16 hours incubation time with 0, 10 or 20 nM LBH589. One representative experiment of three is shown. Shaded grey = IgG control; dashed line = 0 nM, solid grey line = 10 nM and black line = 20 nM. The isotype control experiment was performed in the presence of 20 nM LBH589. (<b>B</b>) The expression of CD30 on the surface of L428 and L540 cells was detected via flow cytometry. CD30 was significantly decreased after treatment with 20 nM LBH589 (48 h; n = 3; two-sided t-test). A representative histogram is shown in Fig. S1A in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079502#pone.0079502.s001" target="_blank">File S1</a>. (<b>C</b>) Shed CD30 (sCD30) was measured via ELISA in cell culture supernatants. Upon treatment with 10 nM LBH589, sCD30 levels were significantly lower in L428 supernatants but not for L540 supernatants (t = 48 h; n = 6 per cell line; two-sided, paired t-test). (<b>D/E</b>) Flow cytometry to detect activating NK cell receptors (<b>D</b>) and T cell receptors (<b>E</b>). NK and T cells were treated for 24 hours with 20 nM LBH589. n = 4, two-sided, paired t-test. T cells: n = 3 for CD3 and n = 5 for CD28; two-sided, paired t-test. Representative histograms are shown in Fig. S1 D and E in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079502#pone.0079502.s001" target="_blank">File S1</a>, respectively. MFI = mean fluorescent intensity. (<b>F/G</b>) The activation marker CD69 was upregulated on both, NK cells (<b>F</b>) and T cells (<b>G</b>) but not at statistically significant levels (p>0.05). Representative histograms are shown in Fig. S1 F in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079502#pone.0079502.s001" target="_blank">File S1</a>.</p

Additional file 7 of Mutant p53-ENTPD5 control of the calnexin/calreticulin cycle: a druggable target for inhibiting integrin-α5-driven metastasis

Additional file 7: Supplemental Figure S7. Degradation of mutant p53 blocks ITGA5-mediated cancer cell motility. a RT-qPCR analysis of ITGA5 mRNA expression in MIA PaCa-2 cells treated with Ganet (0.5 or 1 μM) or SAHA+AAG (10 or 15 μM). Shown is the GAPDH-normalized expression relative to vehicle-treated control cells as 100% (n=3 replicates). b Western blot of p53-null H1299 cells for ITGA5 and ENTPD5 expression following treatment as in (a). c-d MIA PaCa-2 cells were treated as in (a) and analyzed by real-time live cell imaging. c Confluence curves. Shown is the mean confluence of n=3 replicates. Shading for vehicle-treated control cells indicates the SD. d Proliferation was quantified as the area under the confluence curve and normalized to vehicle-treated control cells as 100%. e Adhesion on FN of p53-null H1299 cells treated as in (a). f-h MIA PaCa-2 pIND-ENTPD5 cells (with tet-inducible expression of ENTPD5) were treated with doxycycline (+tet), Ganet (0.5 µM) and ITGA5-blocking antibody (α5) as indicated. f Adhesion on FN. g-h Invasion in the presence of FN. Statistical significance was tested using one-way ANOVA followed by Dunnett’s multiple comparisons test: ****, p<0.0001; ns, not significant. All results are shown as mean ± SD (n=3 replicates)

Additional file 5 of Mutant p53-ENTPD5 control of the calnexin/calreticulin cycle: a druggable target for inhibiting integrin-α5-driven metastasis

Additional file 5: Supplemental Figure S5. CANX/CALR chaperones are essential for mutant p53-dependent ITGA5 activity. a FN-mediated adhesion of MIA PaCa-2 cells with tet-inducible expression of ENTPD5. Cells were treated with doxycycline 24 hours before transfection of indicated siRNAs and analyzed as in Fig. 2. b Migration and invasion of MIA PaCa-2 cells in the absence and presence of FN following depletion of CANX, CALR, and UGGT. All results are shown as mean ± SD (n=3 independent experiments). Statistical significance was tested using two-way ANOVA followed by Dunnett’s multiple comparisons test: ***, p<0.001; ****, p<0.0001; ns, not significant. Mock: non-transfected cells; nsi: non-targeting siRNA control

Additional file 3 of Mutant p53-ENTPD5 control of the calnexin/calreticulin cycle: a druggable target for inhibiting integrin-α5-driven metastasis

Additional file 3: Supplemental Figure S3. Mutant p53 signaling via ENTPD5 is required for FN-mediated cell adhesion, migration, and invasion. a-d Adhesion kinetics of (a) H1975, (b) MDA-MB-231, (c) OC58 and (d) OC121 cells to fibronectin (FN) following siRNA-mediated depletion of p53, ENTPD5, ITGA5 or ITGB1. Adhesion is expressed as the percentage of the seeded cell number. e-f Proliferation effect of mutant p53 and ENTPD5. MIA PaCa-2 cells were transfected with siRNAs targeting p53 and ENTPD5 and analyzed by real-time live cell imaging. Non-targeting siRNA (nsi) is shown as control. e Confluence curves. Shown is the mean confluence of n=3 replicates. Shading for nsi-transfected cells indicates the SD. f Proliferation was quantified as the area under the confluence curve and normalized to nsi control cells as 100%. Shown is the mean ± SD (n=3 replicates

Additional file 1 of Mutant p53-ENTPD5 control of the calnexin/calreticulin cycle: a druggable target for inhibiting integrin-α5-driven metastasis

Additional file 1: Supplemental Figure S1. Mutp53 regulation of ENTPD5, ITGA5, and ITGB1 in different cancer cell lines. a-e Western blot for p53, ITGA5, and ENTPD5, in p53-mutated breast (MDA-MB-231), lung (PC-9 and H1975) and ovarian (OC58 and OC121) cancer cells, transfected with siRNAs targeting p53 or ENTPD5 as indicated. nsi, non-targeting siRNA. g RT-qPCR analysis of ITGA5 mRNA expression in MIA PaCa-2 cells transfected with siRNAs targeting p53, ENTPD5, CANX, CALR, UGGT or ITGA5. Shown is the log2-fold mean normalized expression (MNE) ± SD (n=3 replicates). Statistical significance was tested using one-way ANOVA followed by Dunnett’s multiple comparisons test versus control: ****, p<0.0001; ns, not significant. g-j Western blot for p53, ITGB1, and ENTPD5, in p53-mutated pancreatic (MIA PaCa-2), breast (MDA-MB-231), lung (H1975) and ovarian (OC58) cancer cells, transfected with siRNAs as indicated. nsi, non-targeting siRNA

LBH589 modulated lymphocyte secretion of cytokines.

<p>(<b>A</b>) ELISA for IFNgamma from supernatants of cultured cells. Left panel: PBMCs and L428 cells were cultured separately. L428 cells were co-cultured with PBMCs (middle panel) and CD3+ cells (right panel; t = 36 hours). In indirect co-cultures, cells were separated by the membrane. The ratios of effector to tumour cells were 1∶1 with PBMCs and 1∶2 with CD3+ cells. PBMC/L428 co-culture did not show significant results due to high intra-group variance. For CD3+ cells, a significant IFNgamma increase in direct co-culture compared with indirect co-culture (p = 0.0198) as well as a significant downregulation upon LBH589-treatment (p = 0.0192) was observed (two-way ANOVA analysis; solid lines indicate the comparison of indirect versus direct co-culture; dashed lines the effect of LBH589 [also in B) and C]). (<b>B</b>) ELISA for TNFalpha and settings like in A). For both, PBMC/L428 and CD3+/L428 co-cultures, the effect of indirect vs. direct co-culture was not significant (p = 0.6637 and p = 0.3187, respectively), however the LBH589 treatment increased the TNFalpha secretion significantly (middle and right subpanels; p = 0.0059 and 0.0006, respectively; two-way ANOVA analysis). (<b>C</b>) ELISA for TNFalpha release in lymphocyte/L428 co-cultures. The ratio of effector to tumour cells was 1∶4 with CD4+ and CD8+ cells each and 1∶10 in NK cell co-cultures. The ratio of effector to tumour cells was 1∶4 with CD4+ and CD8+ cells each; and 1∶10 in NK cell co-cultures. LBH589 increased significantly the TNFalpha secretion in co-cultures of L428 with CD4+, CD8+ and NK cells (p = 0.0001, p = 0.0017 and p = 0.0253, respectively). The direct versus indirect co-culture method showed a significant impact on CD4+ (p = 0.0006) and CD8+ lymphocytes (p = 0.0328; two-way ANOVA analysis). The ratio of effector to tumour cells was 1∶4 with CD4+ and CD8+ cells each and 1∶10 in NK cell co-cultures.</p

LBH589 enhanced cytotoxicity and worked synergistically with gemcitabine.

<p>(<b>A/B</b>) Killing assays in which target cells and PBMCs were pre-treated for 4 h with (<b>A</b>) 10 nM LBH or (<b>B</b>) 200 ng/ml TNFalpha. The panels show one representative experiment of three. The ratios of effector to target cells are indicated below the axis. In addition, the summary bar graph for the effector to target cell ratio 50∶1 of three independent experiments is given for both experimental settings (paired, two-sided t-test). (<b>C/D</b>) Combination regimens were tested with sublethal doses adjusted to each compound and cell line. L428 cells and L540 cells were treated with 10 nM LBH589 (LBH) and (<b>C</b>) Gemcitabine (GMZ; 500 ng/ml for L428 and 0.5 ng/ml for L540; n = 3) or (<b>D</b>) Everolismus (RAD; 5 µM for L428; 0.5 µM for L540; n = 3 each; paired, two-sided t-test).</p