Sp1/Sp3 and DNA-methylation contribute to basal transcriptional activation of human podoplanin in MG63 versus Saos-2 osteoblastic cells

BACKGROUND: Podoplanin is a membrane mucin that, among a series of tissues, is expressed on late osteoblasts and osteocytes. Since recent findings have focussed on podoplanin's potential role as a tumour progression factor, we aimed at identifying regulatory elements conferring PDPN promoter activity. Here, we characterized the molecular mechanism controlling basal PDPN transcription in human osteoblast-like MG63 versus Saos-2 cells. RESULTS: We cloned and sequenced 2056 nucleotides from the 5'-flanking region of the PDPN gene and a computational search revealed that the TATA and CAAT box-lacking promoter possesses features of a growth-related gene, such as a GC-rich 5' region and the presence of multiple putative Sp1, AP-4 and NF-1 sites. Reporter gene assays demonstrated a functional promoter in MG63 cells exhibiting 30-fold more activity than in Saos-2 cells. In vitro DNase I footprinting revealed eight protected regions flanked by DNaseI hypersensitive sites within the region bp -728 to -39 present in MG63, but not in Saos-2 cells. Among these regions, mutation and supershift electrophoretic mobility shift assays (EMSA) identified four Sp1/Sp3 binding sites and two binding sites for yet unknown transcription factors. Deletion studies demonstrated the functional importance of two Sp1/Sp3 sites for PDPN promoter activity. Overexpression of Sp1 and Sp3 independently increased the stimulatory effect of the promoter and podoplanin mRNA levels in MG63 and Saos-2 cells. In SL2 cells, Sp3 functioned as a repressor, while Sp1 and Sp3 acted positively synergistic. Weak PDPN promoter activity of Saos-2 cells correlated with low Sp1/Sp3 nuclear levels, which was confirmed by Sp1/Sp3 chromatin immunoprecipitations in vivo. Moreover, methylation-sensitive Southern blot analyses and bisulfite sequencing detected strong methylation of CpG sites upstream of bp -464 in MG63 cells, but hypomethylation of these sites in Saos-2 cells. Concomitantly, treatment with the DNA methyltransferase inhibitor 5-azaCdR in combination with trichostatin A (TSA) downregulated podoplanin mRNA levels in MG63 cells, and region-specific in vitro methylation of the distal promoter suggested that DNA methylation rather enhanced than hindered PDPN transcription in both cell types. CONCLUSION: These data establish that in human osteoblast-like MG63 cells, Sp1 and Sp3 stimulate basal PDPN transcription in a concerted, yet independent manner, whereas Saos-2 cells lack sufficient nuclear Sp protein amounts for transcriptional activation. Moreover, a highly methylated chromatin conformation of the distal promoter region confers cell-type specific podoplanin upregulation versus Saos-2 cells

STAT3/LKB1 controls metastatic prostate cancer by regulating mTORC1/CREB pathway

Prostate cancer (PCa) is a common and fatal type of cancer in men. Metastatic PCa (mPCa) is a major factor contributing to its lethality, although the mechanisms remain poorly understood. PTEN is one of the most frequently deleted genes in mPCa. Here we show a frequent genomic co-deletion of PTEN and STAT3 in liquid biopsies of patients with mPCa. Loss of Stat3 in a Pten-null mouse prostate model leads to a reduction of LKB1/pAMPK with simultaneous activation of mTOR/CREB, resulting in metastatic disease. However, constitutive activation of Stat3 led to high LKB1/pAMPK levels and suppressed mTORC1/CREB pathway, preventing mPCa development. Metformin, one of the most widely prescribed therapeutics against type 2 diabetes, inhibits mTORC1 in liver and requires LKB1 to mediate glucose homeostasis. We find that metformin treatment of STAT3/AR-expressing PCa xenografts resulted in significantly reduced tumor growth accompanied by diminished mTORC1/CREB, AR and PSA levels. PCa xenografts with deletion of STAT3/AR nearly completely abrogated mTORC1/CREB inhibition mediated by metformin. Moreover, metformin treatment of PCa patients with high Gleason grade and type 2 diabetes resulted in undetectable mTORC1 levels and upregulated STAT3 expression. Furthermore, PCa patients with high CREB expression have worse clinical outcomes and a significantly increased risk of PCa relapse and metastatic recurrence. In summary, we have shown that STAT3 controls mPCa via LKB1/pAMPK/mTORC1/CREB signaling, which we have identified as a promising novel downstream target for the treatment of lethal mPCa

Spatial Proteomics for the Molecular Characterization of Breast Cancer

Breast cancer (BC) is a major global health issue, affecting a significant proportion of the female population and contributing to high rates of mortality. One of the primary challenges in the treatment of BC is the disease’s heterogeneity, which can lead to ineffective therapies and poor patient outcomes. Spatial proteomics, which involves the study of protein localization within cells, offers a promising approach for understanding the biological processes that contribute to cellular heterogeneity within BC tissue. To fully leverage the potential of spatial proteomics, it is critical to identify early diagnostic biomarkers and therapeutic targets, and to understand protein expression levels and modifications. The subcellular localization of proteins is a key factor in their physiological function, making the study of subcellular localization a major challenge in cell biology. Achieving high resolution at the cellular and subcellular level is essential for obtaining an accurate spatial distribution of proteins, which in turn can enable the application of proteomics in clinical research. In this review, we present a comparison of current methods of spatial proteomics in BC, including untargeted and targeted strategies. Untargeted strategies enable the detection and analysis of proteins and peptides without a predetermined molecular focus, whereas targeted strategies allow the investigation of a predefined set of proteins or peptides of interest, overcoming the limitations associated with the stochastic nature of untargeted proteomics. By directly comparing these methods, we aim to provide insights into their strengths and limitations and their potential applications in BC research

Production of scFv-Fc B6-11 fusion antibody and reconfirmation of binding to CD146.

<p>A: Western Blot analysis showing molecular size of scFv-Fc fusion antibodies B6-11, B6-112 and B6-117. The scFv-region of the phagemids was cloned into the pFUSE-mIgG2B-vector (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127169#pone.0127169.s005" target="_blank">S5 Fig</a>). Constructs were transfected into HEK293-cells. Cell culture supernatants were loaded on reducing (R) and non-reducing (NR) 10% SDS-PAGE, and nitrocellulose blots were probed with peroxidase-labeled anti-mouseFc antibodies. scFv-Fc fusion antibodies are secreted as approximate 140 kDa dimers, as seen under non-reducing condition (-DTT). Control: Fc only protein, produced from pFUSE vector without scFv insert. B: Immunoprecipitation with scFv-Fc B6-11 reconfirms CD146-binding. G1S1 lysates were incubated with scFv-Fc B6-11 and Fc only as control. Blots of immunoprecipitates were probed with anti-CD146 and anti-mouseFc antibodies. C: scFv-Fc B6-11 binds to recombinant CD146 in ELISA. scFv-Fc B6-11, commercial anti-CD146 antibody and Fc only were used on respective dilutions of recombinant human CD146 or BSA as control antigen coated on 96-well ELISA plates. Absorbance was measured at 450nm. D: scFv-Fc B6-11 fusion antibody binds to cells expressing CD146 in ELISA. Purified scFv-Fc B6-11 (light grey bars), commercial anti-CD146 antibody (dark grey bars) and Fc only (black bars) were added to monolayers of respective cell lines. Bound antibodies were detected using anti-Fc and HRP-conjugated anti-rabbit antibodies, and absorbance was measured at 450nm. Mean and standard deviation of triplicate experiments are given. E: On HDMECs, scFv-Fc B6-11 fusion antibody shows the same reactivity as a commercial anti-CD146 antibody. F: scFv-Fc fusion antibodies (red) reveal diverse membraneous labling patterns in co-immunofluorescent stainings with anti-CD31 antibody (green). Nuclei were counterstained with DAPI. Size bars: 50μm.</p

Frequency of diverse scFv antibody sequences among 557 intact clones.

<p>Out of 994 sequenced phage clones, 557 intact scFv sequences were derived, among which 166 diverse scFv sequences were identified (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127169#pone.0127169.s012" target="_blank">S2 Table</a>).</p><p>^a Frequency of unique scFv antibody sequences</p><p>^b Number of different scFv sequences</p><p>^c Number of scFv sequences with respective sequence diversity</p><p>^d Sequence occurrence was calculated as percentage of sequence count.</p><p>Frequency of diverse scFv antibody sequences among 557 intact clones.</p

Selection of scFv Antibody Fragments Binding to Human Blood versus Lymphatic Endothelial Surface Antigens by Direct Cell Phage Display

<div><p>The identification of marker molecules specific for blood and lymphatic endothelium may provide new diagnostic tools and identify new targets for therapy of immune, microvascular and cancerous diseases. Here, we used a phage display library expressing human randomized single-chain Fv (scFv) antibodies for direct panning against live cultures of blood (BECs) and lymphatic (LECs) endothelial cells in solution. After six panning rounds, out of 944 sequenced antibody clones, we retrieved 166 unique/diverse scFv fragments, as indicated by the V-region sequences. Specificities of these phage clone antibodies for respective compartments were individually tested by direct cell ELISA, indicating that mainly pan-endothelial cell (EC) binders had been selected, but also revealing a subset of BEC-specific scFv antibodies. The specific staining pattern was recapitulated by twelve phage-independently expressed scFv antibodies. Binding capacity to BECs and LECs and differential staining of BEC versus LEC by a subset of eight scFv antibodies was confirmed by immunofluorescence staining. As one antigen, CD146 was identified by immunoprecipitation with phage-independent scFv fragment. This antibody, B6-11, specifically bound to recombinant CD146, and to native CD146 expressed by BECs, melanoma cells and blood vessels. Further, binding capacity of B6-11 to CD146 was fully retained after fusion to a mouse Fc portion, which enabled eukaryotic cell expression. Beyond visualization and diagnosis, this antibody might be used as a functional tool. Overall, our approach provided a method to select antibodies specific for endothelial surface determinants in their native configuration. We successfully selected antibodies that bind to antigens expressed on the human endothelial cell surfaces <i>in situ</i>, showing that BECs and LECs share a majority of surface antigens, which is complemented by cell-type specific, unique markers.</p></div

LC-MS/MS identification of CD146 binding to scFv B6-11, and antigen confirmation by immunoprecipitation and ELISA.

<p>A: Alignment of LC-MS/MS identified peptides (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127169#pone.0127169.s004" target="_blank">S4 Fig</a>) with the sequence of MCAM/CD146MUC18. The eluates from scFv B6-11 immunoprecipitation were subjected to trypsin digestion (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127169#sec002" target="_blank">Materials and Methods</a> section) and subsequently analyzed by LC-MS/MS. B: Immunoprecipitation of CD146 by soluble scFv B6-11 from BEC lysates. Immune complexes were tested by Western blot in reducing conditions using commercial anti-CD146 antibody. Lane 1: input BEC lysate, lanes 2–5: immunoprecipitates with scFv B6-11, lanes 2 and 4: under addition of PNGase F. Treatment of BEC lysates with PNGase prior or after addition of scFv B6-11 had no influence on co-immunoprecipitation capacity of scFv B6-11, showing that scFv B6-11 binding to CD146 is glycosylation-independent. C: scFv B6-11 binds to immobilized extracellular domain of recombinant human CD146 in ELISA. An irrelevant antigen (BSA), a non-binding scFv and uncoated wells served as controls. scFv binding was detected with peroxidase-conjugated anti-His tag antibody (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127169#pone.0127169.s011" target="_blank">S1 Table</a>), and absorbance was measured at 450nm. Mean and standard deviation of triplicate experiments are given. D: CD146 expression of different cell lines as shown by immunoprobing with anti-CD146 antibody. CD146 is expressed in BECs, in A375, CRL1676, HTB71 melanoma cells, but not in primary LECs and HEK293 cells. The same blot was probed with anti-tubulin antibody for control of equal protein loads. E: scFv B6-11 stains cell lines expressing CD146 with similar intensity as commercial anti-CD146 antibody in ELISA. Negative controls were a non-binding scFv and 2^nd antibody only. F: Similar to commercial anti-CD146 antibody, scFv B6-11 stains BECs (upper lane, red) but not LECs (lower lane, green) in immunofluorescence. Size bars: 50μm. G: scFv B6-11 stains blood, but not lymphatic vessels in human skin. Double immunofluorescence staining of skin sample with Cy3-labeled scFv-B6-11 or anti-CD146 (red) and anti-PDPN (green) antibodies. Blood (BV) and lymphatic (LV) vessels are indicated by lines. Nuclei were counterstained with DAPI. Size bars: 50μm.</p

Fusion antibodies scFv-Fc B6-11, B6-112 and B6-117 stain blood vessels in the dermis.

<p>A: Representative images of double immunofluorescence stainings of frozen human skin sections with scFv-Fc fusion antibodies (red) in combination with anti-CD31 (green) show overlapping staining, but B: not with anti-PDPN antibody (green) as control. Nuclei were counter-stained with DAPI. Size bars: 50μm.</p

Enrichment of phage antibodies that bind to BECs and LECs with successive biopanning rounds.

<p>A: Numbers of recovered phage particles after each panning round on BECs and LECs. For each selection round, the phage output/input titer ratios are given on the y-axis, showing an increase on BECs (red line) and LECs (green line) when adding polyclonal ETH-2 phage. In contrast, control wild-type VCS-M13 (grey and black lines) phage did not shift significantly on either cell population. B: Enrichment of BEC- and LEC-binding phage antibodies as seen by FACS-analysis of phage antibody pools. Shown are representative histograms of BECs and LECs stained with phage antibodies after respective panning rounds (black line) and WT phage as negative control (grey-filled graphs). The number of cells (counts: Y-axis) is given as function of the fluorescence intensity of phage antibody staining of the cells (FL1-H: X-axis). Percentage of BECs and LECs shifted by phage binding is depicted in the graphics. In all experiments, cells were incubated with phage antibody pools, and cell-binding was detected by anti-M13 antibody and FITC-conjugated anti-rabbit antibody. Grey-filled graphs show WT phage binding as negative control. C: <i>BstN</i>I DNA fingerprint analysis of single scFv clones. Representative gel images of respective panning rounds (#1 - #6) performed on BECs and LECs are shown. Over the course of panning rounds, the number of intact clones is increasing. Phagemid DNA isolated from single clones was digested with <i>BstN</i>I and analyzed in agarose gel electrophoresis. M: 1 kbp molecular weight marker.</p

scFv-Fc B6-11 fusion antibody inhibits motility of BECs in a scratch wound assay.

<p>A: Migration analysis of BECs by scratch wound assay in the presence of scFv-Fc B6-11, Fc fragment or PBS as controls. Shown are representative images of wounds created in BEC monolayers (n = 3 in each group) at indicated time points. Size bars: 500μm. B: Reoccupation of the gap by BECs after t = 24 hrs and t = 48 hrs versus t = 0 hrs was measured using AxioVision 4.7 software. Areas repopulated by BECs and % of gap area newly covered by BECs were calculated at each timepoint. Assay was performed twice and values are means ± SD. Gap closure was analysed by one-way Anova (<i>P</i> < 0.05) followed by pairwise Student´s t-testing. Shown are significant differences between scFv-Fc versus Fc control, and scFv-Fc versus PBS control, respectively (<i>P</i>-values: * < 0.05, ** < 0.005, ***< 0.0005).</p