Search

Additional file 6: Supplementary Table S1. EDI3 (GPCPD1) expression in human breast cancer tissue was evaluated in publicly available Affymetrix HG U133 Plus 2.0 gene expression microarray dataset that were downloaded from the Gene Expression Omnibus webportal (GEO) together with available clinicopathological data.

Additional file 8: Supplementary Table S3. Association of EDI3 expression for the three probesets on the Affymetrix HG U133 Plus 2.0 array and available clinicopathological parameters shown for A, the six datasets combined, as well as separately for B, GSE16446, C, GSE19615, D, GSE28844, E, GSE32646, F, GSE6532, and G, GSE9195. P value from the Mann Whitney or Kruskal-Wallis U test.

Additional file 1: Supplementary Figure S1. EDI3 is highly expressed in ER-HER2+ cells and is regulated by HER2. A (left panel), HER2 mRNA expression measured using qRT-PCR. Quantification of A (right panel), HER2 and B, ER protein expression from immunoblotting as represented in Figure 1D. C, Immunofluorescence staining, and D, metabolite levels by NMR in a panel of breast cancer cell lines representing the different breast cancer subtypes. E, HER2 and EDI3 mRNA, and F, protein expression in HCC1954 cells upon HER2 silencing. G, EDI3 and HER2 mRNA expression and H, EDI3, pHER2, and HER2 protein expression in SKBR3 cells after silencing EDI3 with siRNA. I, EDI3 and HER2 mRNA expression and J, EDI3, pHER2, and HER2 protein expression in HCC1954 cells after silencing EDI3 with siRNA. K, Representative Western blots showing EDI3, pHER2 and HER2 protein expression in HCC1954 cells treated with 0.1 µM or 1.0 µM lapatinib for 24, 48, 72 and 96h, with corresponding quantification of protein levels. Data are mean ± SD or mean ± SE for HER2 protein expression in panel A of at least three independent experiments (*, P

Additional file 3: Supplementary Figure S3. Effect of targeting EDI3 on viability, alone or in combination with lapatinib, in ER-HER2+ and ER+HER2+ breast cancer cell lines. A-B, EDI3 mRNA (left panels) and protein expression (right panels) after silencing EDI3 using siRNA in A, BT474 and B, EFM192A cells. C, Effect of inhibiting HER2 using increasing concentrations of lapatinib on viability in SKBR3, HCC1954, BT474 and EFM192A cells. D-G Influence of silencing EDI3 with siRNA (oligo #2) and inhibiting HER2 with lapatinib (0.01 µM, 0.1 µM and 1 µM), as well as the combined inhibition of both EDI3 and HER2 on viability in D, SKBR3, E, HCC1954, F, BT474 and G, EFM192A cells. H, Effect of inhibiting HER2 using different concentrations of trastuzumab on viability in SKBR3, HCC1954, BT474 and EFM192A cells. I-L Influence of inhibiting EDI3 with siRNA (oligos #1 and #2) and HER2 with trastuzumab (1 µg/ml and 10 µg/ml), as well as the combined inhibition of both EDI3 and HER2 on viability in I, SKBR3, J, HCC1954, K, BT474 and L, EFM192A. Data are mean ± SD (A-C and H) or mean ± SE (D-G and I-L) of at least three independent experiments (*, P

Additional file 2: Supplementary Figure S2. GSK3β and mTOR are key proteins in HER2-mediated upregulation of EDI3. A, Quantification of HER2, EDI3 and p-Akt protein expression after inhibiting PI3K with 5 and 10 µM LY294002 for 3 and 7 days in MCF7-NeuT cells. B, Representative Western blot and HER2, EDI3 and p-PKCα/βII protein expression quantification after inhibiting PLCγ with 1 and 3 µM U73122 for 3 and 7 days in MCF7-NeuT cells. C, Representative Western blot and quantification of HER2, EDI3 and p-ERK1/2 protein expression after inhibiting MEK with 5 and 25 µM PD98059 for 3 and 7 days in MCF7-NeuT cells. D, HER2, EDI3 and p-mTOR protein expression after inhibiting mTORC1 with 1 and 3 µM everolimus for 3 and 7 days in MCF7-NeuT cells. E, HER2, EDI3 and p-mTOR protein expression after inhibiting mTORC1 with 1 and 3 µM everolimus for 1, 2 and 3 days in HCC1954 cells. F, HER2, EDI3 and p-mTOR protein expression after inhibiting mTORC1 with 1 and 3 µM everolimus for 1, 2 and 3 days in SKBR3 cells. G, HER2, EDI3 and β-catenin protein expression after inhibiting GSKβ with 1 and 2.5 µM CHIR-99021 for 3 and 7 days in MCF7/NeuT cells. H, HER2, EDI3 and β-catenin protein expression after inhibiting GSKβ with 1 and 2.5 µM CHIR-99021 for 1, 2 and 3 days in HCC1954 cells. I, HER2, EDI3 and β-catenin protein expression after inhibiting GSKβ with 1 and 2.5 µM CHIR-99021 for 1, 2 and 3 days in SKBR3 cells. Graphs D-I are quantification of Western blots exemplified in Fig. 3C-H, respectively. J, EDI3 mRNA expression after inhibiting c-Myc with 5 and 10 µM 10074-G5, K, NFκB with 5 and 10 µM SC75741, or L, KLF5 with 5 and 10 µM SR18662 for 1, 2 and 3 days in SKBR3 cells. Data are mean ± SD of at least three independent experiments (*, P

Additional file 7: Supplementary Table S2. List of used reagents including A, QuantiTect primer assays, B, siRNA and shRNA oligos, and C, antibodies.

Additional file 5: Supplemental Figure S5. Inducibly silencing EDI3 inhibits colony formation and cell viability. A, Representative images (top) and corresponding quantification (bottom) of colony number (left) and size (right) formed by HCC1954 shNEG and HCC1954 shEDI3 (oligos shEDI3 #1, #2, #3) cells treated with 0 or 0.1 µg/ml doxycycline. B, Viability (RFU) after treating HCC1954 shNEG and HCC1954 shEDI3 (oligos shEDI3 #1, #2) with 0.1 or 1 µg/ml doxycycline. All in vitro data are mean ± SD of three independent experiments. (*, P < 0.05; **, P < 0.01; ***, P < 0.001). RFU, relative fluorescence units.

Additional file 4: Supplementary Figure S4. Inhibiting EDI3 with dipyridamole, alone or in combination with HER2-targeting therapy, in ER-HER2+ tumors in mice. A-D EDI3 activity in percent after treatment with dipyridamole at different concentrations in A, HCC1954 B, SKBR3, C, BT474 and D, EFM192 cells. E, Cell viability in percent of vehicle control after treatment with different concentrations of dipyridamole in SKBR3, HCC1954, BT474 and EFM192A cells (upper panel) and EC50-values with corresponding 95% confidence intervals (CI) (lower panel). F, Concentration of dipyridamole in plasma after a single oral dose of 120 mg/kg dipyridamole in mice over time. G, Mouse weight (grams) after treatment with dipyridamole, lapatinib and the combination for up to 4 weeks. H, Images of CD1 nude mice with tumors encircled, after 4-week treatment of vehicle, dipyridamole, lapatinib and the combination of both. I, Plasma and tumor concentrations (µM) of dipyridamole (upper panel) and lapatinib (lower panel) after treatment with each compound alone or in combination (D+L). Data (A-E) are mean ± SD of three independent experiments; mean ± SD of three CD1 mice (F); mean ± SD 5 to 6 mice (G); and representative images of 4 mice per condition H. D+L (combined dipyridamole and lapatinib treatment); BQL (below quantification limit); EC50 (half maximal effective concentration).

Diffuse large B-cell lymphoma (DLBCL) is commonly classified by gene expression profiling according to its cell of origin (COO) into activated B-cell (ABC)-like and germinal center B-cell (GCB)-like subgroups. Here we report the application of label-free nano-liquid chromatography - Sequential Window Acquisition of all THeoretical fragment-ion spectra – mass spectrometry (nanoLC-SWATH-MS) to the COO classification of DLBCL in formalin-fixed paraffin-embedded (FFPE) tissue. To generate a protein signature capable of predicting Affymetrix-based GCB scores, the summed log2-transformed fragment ion intensities of 780 proteins quantified in a training set of 42 DLBCL cases were used as independent variables in a penalized zero-sum elastic net regression model with variable selection. The eight-protein signature obtained showed an excellent correlation (r = 0.873) between predicted and true GCB scores and yielded only 9 (21.4%) minor discrepancies between the three classifications: ABC, GCB, and unclassified. The robustness of the model was validated successfully in two independent cohorts of 42 and 31 DLBCL cases, the latter cohort comprising only patients aged >75 years, with Pearson correlation coefficients of 0.846 and 0.815, respectively, between predicted and NanoString nCounter based GCB scores. We further show that the 8-protein signature is directly transferable to both a triple quadrupole and a Q Exactive quadrupole-Orbitrap mass spectrometer, thus obviating the need for proprietary instrumentation and reagents. This method may therefore be used for robust and competitive classification of DLBCLs on the protein level.

Figure S4. ADGRE5 IHC staining of cell line and patient tissue samples. (A) Burkitt Lymphoma (BL): Cell lines (1–5: Blue-1 Bl-41, BL-2, DG-75, CA-46) and primary tumor tissues (7–14) are manly ADGRE5 positive. (B) Diffuse large B cell lymphoma without MYC break (DLBCLneg): Cell lines (1–4: Karpass-422, U2932-R1, HT, WSU-DLCL2) and primary tumor tissues (5–19). (C) DLBCLpos: Cell lines (1–3: Carnaval, U2932-R2, SU-DHL-10) and primary tumor tissues (4–6). DLBCLpos and DLBCLneg are manly negative for ADGRE5. Strong positive staining in some tissue sections results from macrophages or T-cells. (PDF 4769 kb)

Deficiency of methylthioadenosine phosphorylase (MTAP) supports melanoma development and progression through accumulation of its substrate 5'-methylthioadenosine (MTA), which leads amongst others to a constitutive inhibition of protein arginine methyltransferases (PRMTs) and activation of the transcription factor AP-1 via the receptor ADORA2B. Genetic association studies have also suggested that genetic polymorphism in MTAP may modulate the risk of melanoma. Here, we investigated the only globally common non-synonymous single nucleotide polymorphism (SNP) reported to date for MTAP. The SNP rs7023954 is located in exon 3 (c. 166G>A), and leads to the conservative substitution of one branched-chain amino acid residue (valine) for another (isoleucine) at position 56 (p. Val56lle). Whereas genotype frequencies in normal and primary melanoma tissues or cell lines were in Hardy-Weinberg equilibrium based on cDNA amplicon sequencing, a marked (P = 0.00019) deviation was observed in metastatic melanoma tissues and cell lines due to a deficit of heterozygotes. Enzyme assays conducted on the co-dominantly expressed alleles revealed no difference in the conversion rate of MTA to adenine and 5-methylthioribose-1-phosphate, indicating that this known enzymatic activity does not modulate the tumor suppressive function of MTAP.

cAMP and cGMP are well established second messengers that are essential for numerous (patho)physiological processes. These purine cyclic nucleotides activate cAK and cGK, respectively. Recently, the existence of cCMP was described, and a possible function for this cyclic nucleotide was investigated. It was postulated that cCMP plays a role as a second messenger. However, the functions regulated by cCMP are mostly unknown. To elucidate probable functions, cCMP-binding and -activated proteins were identified using different methods. We investigated the effect of cCMP on purified cyclic nucleotide-dependent protein kinases and lung and jejunum tissues of wild type (WT), cGKI-knockout (cGKI KO) and cGKII-knockout (cGKII KO) mice. The catalytic activity of protein kinases was measured by a (γ-32P) ATP kinase assay. Cyclic nucleotide-dependent protein kinases (cAK, cGKI and cGKII) in WT tissue lysates were stimulated by cCMP. In contrast, there was no stimulation of phosphorylation in KO tissue lysates. Competitive binding assays identified cAK, cGKI, and cGKII as cCMP-binding proteins. An interaction between cCMP/MAPK and a protein-protein complex of MAPK/cGK were detected via cCMP affinity chromatography and co-immunoprecipitation, respectively. These complexes were abolished or reduced in jejunum tissues from cGKI KO or cGKII KO mice. In contrast, these complexes were observed in the lung tissues from WT, cGKI KO and cGKII KO mice. Moreover, cCMP was also able to stimulate the phosphorylation of MAPK. These results suggest that MAPK signaling is regulated by cGMP-dependent protein kinases upon activation by cCMP. Based on these results, we propose that additional cCMP-dependent protein kinases that are capable of modulating MAPK signaling could exist. Hence, cCMP could potentially act as a second messenger in the cAK/cGK and MAPK signaling pathways and play an important role in physiological processes of the jejunum and lung.

The complement system is an essential part of the innate immune system by acting as a first line of defense which is stabilized by properdin, the sole known positive regulator of the alternative complement pathway. Dysregulation of complement can promote a diversity of human inflammatory diseases which are treated by complement inhibitors. Here, we generated a novel blocking monoclonal antibody (mAb) against properdin and devised a new diagnostic assay for this important complement regulator. Mouse mAb 1340 specifically detected native properdin from human samples with high avidity. MAb 1340 inhibited specifically the alternative complement mediated cell lysis within a concentration range of 1–10 µg/mL. Thus, in vitro anti-properdin mAb 1340 was up to fifteen times more efficient in blocking the complement system as compared to anti-C5 or anti-Ba antibodies. Computer-assisted modelling suggested a three-dimensional binding epitope in a properdin-C3(H2O)-clusterin complex to be responsible for the inhibition. Recovery of properdin in a newly established sandwich ELISA using mAb 1340 was determined at 80–125% for blood sample dilutions above 1:50. Reproducibility assays showed a variation below 25% at dilutions less than 1:1,000. Systemic properdin concentrations of healthy controls and patients with age-related macular degeneration or rheumatic diseases were all in the range of 13–30 µg/mL and did not reveal significant differences. These initial results encourage further investigation into the functional role of properdin in the development, progression and treatment of diseases related to the alternative complement pathway. Thus, mAb 1340 represents a potent properdin inhibitor suitable for further research to understand the exact mechanisms how properdin activates the complement C3-convertase and to determine quantitative levels of properdin in biological samples.