A multi-omics approach reveals mechanisms of nanomaterial toxicity and structure-activity relationships in alveolar macrophages.

In respect to the high number of released nanomaterials and their highly variable properties, novel grouping approaches are required based on the effects of nanomaterials. Proper grouping calls for a combination of an experimental setup with a higher number of structurally similar nanomaterials and for employing integrated omics approaches to identify the mode of action. Here, we analyzed the effects of seven well-characterized NMs comprising different chemical compositions, sizes and chemical surface modifications on the rat alveolar macrophage cell line NR8383. The NMs were investigated at three doses ranging from 2.5 to 10 µg/cm2 after 24 h incubation using an integrated multi-omics approach involving untargeted proteomics, targeted metabolomics, and src homology 2 (SH2) profiling. By using Weighted Gene Correlation Network Analysis (WGCNA) for the integrative data, we identified correlations of molecular pathways with physico-chemical properties and toxicological endpoints. The three investigated SiO2 variants induced strong alterations in all three omics approaches and were, therefore, be classified as "active." Two organic phthalocyanines showed minor responses and Mn2O3 induced a different molecular response pattern than the other NMs. WGCNA revealed that agglomerate size and surface area as well as LDH release are among the most important parameters correlating with nanotoxicology. Moreover, we identified key drivers that can serve as representative biomarker candidates, supporting the value of multi-omics approaches to establish integrated approaches to testing and assessment (IATAs).

An in-depth multi-omics analysis in RLE-6TN rat alveolar epithelial cells allows for nanomaterial categorization.

BACKGROUND: Nanomaterials (NMs) can be fine-tuned in their properties resulting in a high number of variants, each requiring a thorough safety assessment. Grouping and categorization approaches that would reduce the amount of testing are in principle existing for NMs but are still mostly conceptual. One drawback is the limited mechanistic understanding of NM toxicity. Thus, we conducted a multi-omics in vitro study in RLE-6TN rat alveolar epithelial cells involving 12 NMs covering different materials and including a systematic variation of particle size, surface charge and hydrophobicity for SiO2 NMs. Cellular responses were analyzed by global proteomics, targeted metabolomics and SH2 profiling. Results were integrated using Weighted Gene Correlation Network Analysis (WGCNA). RESULTS: Cluster analyses involving all data sets separated Graphene Oxide, TiO2_NM105, SiO2_40 and Phthalocyanine Blue from the other NMs as their cellular responses showed a high degree of similarities, although apical in vivo results may differ. SiO2_7 behaved differently but still induced significant changes. In contrast, the remaining NMs were more similar to untreated controls. WGCNA revealed correlations of specific physico-chemical properties such as agglomerate size and redox potential to cellular responses. A key driver analysis could identify biomolecules being highly correlated to the observed effects, which might be representative biomarker candidates. Key drivers in our study were mainly related to oxidative stress responses and apoptosis. CONCLUSIONS: Our multi-omics approach involving proteomics, metabolomics and SH2 profiling proved useful to obtain insights into NMs Mode of Actions. Integrating results allowed for a more robust NM categorization. Moreover, key physico-chemical properties strongly correlating with NM toxicity were identified. Finally, we suggest several key drivers of toxicity that bear the potential to improve future testing and assessment approaches.

Analyzing expression and phosphorylation of the EGF receptor in HNSCC.

Overexpression of the epidermal growth factor receptor (EGFR) in head and neck squamous cell carcinomas (HNSCC) is considered to cause increased EGFR activity, which adds to tumorigenicity and therapy resistance. Since it is still unclear, whether EGFR expression is indeed associated with increased activity in HNSCC, we analyzed the relationship between EGFR expression and auto-phosphorylation as a surrogate marker for activity. We used a tissue micro array, fresh frozen HNSCC tumor and corresponding normal tissue samples and a large panel of HNSCC cell lines. While we observed substantial overexpression only in approximately 20% of HNSCC, we also observed strong discrepancies between EGFR protein expression and auto-phosphorylation in HNSCC cell lines as well as in tumor specimens using Western blot and SH2-profiling; for the majority of HNSCC EGFR expression therefore seems not to be correlated with EGFR auto-phosphorylation. Blocking of EGFR activity by cetuximab and erlotinib points to increased EGFR activity in samples with increased basal auto-phosphorylation. However, we could also identify cells with low basal phosphorylation but relevant EGFR activity. In summary, our data demonstrate that EGFR expression and activity are not well correlated. Therefore EGFR positivity is no reliable surrogate marker for EGFR activity, arguing the need for alternative biomarkers or functional predictive tests.

Immature O-glycans recognized by the macrophage glycoreceptor CLEC10A (MGL) are induced by 4-hydroxy-tamoxifen, oxidative stress and DNA-damage in breast cancer cells.

BACKGROUND: Ligands of the C-type lectin CLEC10A such as Tn and sialyl-Tn representing early intermediates of O-glycosylation are hallmarks of many human malignancies. A variety of regulatory mechanisms underlying their expression are being discussed. METHODS: CLEC10A ligands were detected in various tissues and cells using the recombinant glycan-binding domain of CLEC10A. In normal breast and endometrium, presence of ligands was correlated to the female cycle. Estrogen- and stress dependent induction of CLEC10A ligands was analyzed in MCF7 and T47D cells exposed to 4-hydroxy-tamoxifen (Tam), zeocin and hydrogen peroxide. The expression and localization of CLEC10A ligands was analyzed by Western blot and immunofluorescence. In breast cancer patients CLEC10A ligand expression and survival was correlated by Kaplan-Meyer analysis. RESULT: We observed binding of CLEC10A in normal endometrial and breast tissues during the late phase of the female hormonal cycle suggesting a suppressive effect of female sex hormones on CLEC10A ligand expression. Accordingly, CLEC10A ligands were induced in MCF7- and T47D breast cancer cells after Tam treatment and accumulated on the cell surface and in the endosomal/lysosomal compartment. Phagocytosis experiments indicate that macrophages preferentially internalize CLEC10A ligands coated beads and Tam treated MCF7 cells. CLEC10A ligands were also expressed after the addition of zeocin and hydrogen-peroxide. Each substance induced the production of ROS indicating reactive oxygen species as a unifying mechanism of CLEC10A ligand induction. Mechanistically, increased expression of GalNAc-transferase 6 (GalNT6) and translocation of GalNT2 and GalNT6 from cis- towards trans-Golgi compartment was observed, while protein levels of COSMC and T-synthase remained unaffected. In breast cancer patients, positivity for CLEC10A staining in tumor tissues was associated with improved outcome and survival. CONCLUSION: CLEC10A ligands are inducible by hormone depletion, 4-hydroxy-tamoxifen and agents inducing DNA damage and oxidative stress. Our results indicate that CLEC10A acts as a receptor for damaged and dead cells and may play an important role in the uptake of cell debris by macrophages and dendritic cells.

Lineage-specific control of TFIIH by MITF determines transcriptional homeostasis and DNA repair.

The melanocytic lineage, which is prominently exposed to ultraviolet radiation (UVR) and radiation-independent oxidative damage, requires specific DNA-damage response mechanisms to maintain genomic and transcriptional homeostasis. The coordinate lineage-specific regulation of intricately intertwined DNA repair and transcription is incompletely understood. Here we demonstrate that the Microphthalmia-associated transcription factor (MITF) directly controls general transcription and UVR-induced nucleotide excision repair by transactivation of GTF2H1 as a core element of TFIIH. Thus, MITF ensures the rapid resumption of transcription after completion of strand repair and maintains transcriptional output, which is indispensable for survival of the melanocytic lineage including melanoma in vitro and in vivo. Moreover, MITF controls c-MYC implicated in general transcription by transactivation of far upstream binding protein 2 (FUBP2/KSHRP), which induces c-MYC pulse regulation through TFIIH, and experimental depletion of MITF results in consecutive loss of CDK7 in the TFIIH-CAK subcomplex. Targeted for proteasomal degradation, CDK7 is dependent on transactivation by MITF or c-MYC to maintain a steady state. The dependence of TFIIH-CAK on sequence-specific MITF and c-MYC constitutes a previously unrecognized mechanism feeding into super-enhancer-driven or other oncogenic transcriptional circuitries, which supports the concept of a transcription-directed therapeutic intervention in melanoma.

Combined inhibition of receptor tyrosine and p21-activated kinases as a therapeutic strategy in childhood ALL.

Receptor tyrosine kinase (RTK)-dependent signaling has been implicated in the pathogenesis of acute lymphoblastic leukemia (ALL) of childhood. However, the RTK-dependent signaling state and its interpretation with regard to biological behavior are often elusive. To decipher signaling circuits that link RTK activity with biological output in vivo, we established patient-derived xenograft ALL (PDX-ALL) models with dependencies on fms-like tyrosine kinase 3 (FLT3) and platelet-derived growth factor receptor ß (PDGFRB), which were interrogated by phosphoproteomics using iTRAQ mass spectrometry. Signaling circuits were determined by receptor type and cellular context with few generic features, among which we identified group I p21-activated kinases (PAKs) as potential therapeutic targets. Growth factor stimulation markedly increased catalytic activities of PAK1 and PAK2. RNA interference (RNAi)-mediated or pharmacological inhibition of PAKs using allosteric or adenosine triphosphate (ATP)-competitive compounds attenuated cell growth and increased apoptosis in vitro. Notably, PAK1- or PAK2-directed RNAi enhanced the antiproliferative effects of the type III RTK and protein kinase C inhibitor midostaurin. Treatment of FLT3- or PDGFRB-dependent ALLs with ATP-competitive PAK inhibitors markedly decreased catalytic activities of both PAK isoforms. In FLT3-driven ALL, this effect was augmented by coadministration of midostaurin resulting in synergistic effects on growth inhibition and apoptosis. Finally, combined treatment of FLT3 D835H PDX-ALL with the ATP-competitive group I PAK inhibitor FRAX486 and midostaurin in vivo significantly prolonged leukemia progression-free survival compared with midostaurin monotherapy or control. Our study establishes PAKs as potential downstream targets in RTK-dependent ALL of childhood, the inhibition of which might help prevent the selection or acquisition of resistance mutations toward tyrosine kinase inhibitors.

Identification of Tyrosine Phosphorylated Proteins by SH2 Domain Affinity Purification and Mass Spectrometry.

Phosphotyrosine signaling plays a major role in the control of many important biological functions such as cell proliferation and apoptosis. Deciphering of phosphotyrosine-dependent signaling is therefore of great interest paving the way for the understanding of physiological and pathological processes of signal transduction. On the basis of the specific binding of SH2 domains to phosphotyrosine residues, we here present an experimental workflow for affinity purification and subsequent identification of tyrosine phosphorylated proteins by mass spectrometry. In combination with SH2 profiling, a broadly applicable platform for the characterization of phosphotyrosine profiles in cell extracts, our pull down strategy enables researchers by now to identify proteins in signaling cascades which are differentially phosphorylated and selectively recognized by distinct SH2 domains.

SH2 Domain Histochemistry.

Among posttranslational modifications, the phosphorylation of tyrosine residues is a key modification in cell signaling. Because of its biological importance, characterization of the cellular state of tyrosine phosphorylation is of great interest. Based on the unique properties of endogenously expressed SH2 domains recognizing tyrosine phosphorylated signaling proteins with high specificity we have developed an alternative approach, coined SH2 profiling, enabling us to decipher complex patterns of tyrosine phosphorylation in various normal and cancerous tissues. So far, SH2 profiling has largely been applied for the analysis of protein extracts with the limitation that information on spatial distribution and intensity of tyrosine phosphorylation within a tissue is lost. Here, we describe a novel SH2 domain based strategy for differential characterization of the state of tyrosine phosphorylation in formaldehyde-fixed and paraffin-embedded tissues. This approach demonstrates that SH2 domains may serve as very valuable tools for the analysis of the differential state of tyrosine phosphorylation in primary tissues fixed and processed under conditions frequently applied by routine pathology laboratories.

The Protein Tyrosine Phosphatase Rptpζ Suppresses Osteosarcoma Development in Trp53-Heterozygous Mice.

Osteosarcoma (OS), a highly aggressive primary bone tumor, belongs to the most common solid tumors in growing children. Since specific molecular targets for OS treatment remain to be identified, surgical resection combined with multimodal (neo-)adjuvant chemotherapy is still the only way to help respective individuals. We have previously identified the protein tyrosine phosphatase Rptpζ as a marker of terminally differentiated osteoblasts, which negatively regulates their proliferation in vitro. Here we have addressed the question if Rptpζ can function as a tumor suppressor protein inhibiting OS development in vivo. We therefore analyzed the skeletal phenotype of mice lacking Ptprz1, the gene encoding Rptpζ on a tumor-prone genetic background, i.e. Trp53-heterozygosity. By screening a large number of 52 week old Trp53-heterozygous mice by contact radiography we found that Ptprz1-deficiency significantly enhanced OS development with 19% of the mice being affected. The tumors in Ptprz1-deficient Trp53-heterozygous mice were present in different locations (spine, long bones, ribs), and their OS nature was confirmed by undecalcified histology. Likewise, cell lines derived from the tumors were able to undergo osteogenic differentiation ex vivo. A comparison between Ptprz1-heterozygous and Ptprz1-deficient cultures further revealed that the latter ones displayed increased proliferation, a higher abundance of tyrosine-phosphorylated proteins and resistance towards the influence of the growth factor Midkine. Our findings underscore the relevance of Rptpζ as an attenuator of proliferation in differentiated osteoblasts and raise the possibility that activating Rptpζ-dependent signaling could specifically target osteoblastic tumor cells.

Deciphering of ADP-induced, phosphotyrosine-dependent signaling networks in human platelets by Src-homology 2 region (SH2)-profiling.

Tyrosine phosphorylation plays a central role in signal transduction controlling many important biological processes. In platelets, the activity of several signaling proteins is controlled by tyrosine phosphorylation ensuring proper platelet activation and aggregation essential for regulation of the delicate balance between bleeding and hemostasis. Here, we applied Src-homology 2 region (SH2)-profiling for deciphering of the phosphotyrosine state of human platelets activated by adenosine diphosphate (ADP). Applying a panel of 31 SH2-domains, rapid and complex regulation of the phosphotyrosine state of platelets was observed after ADP stimulation. Specific inhibition of platelet P2Y receptors by synthetic drugs revealed a major role for the P2Y1 receptor in tyrosine phosphorylation. Concomitant activation of protein kinase A (PKA) abolished ADP-induced tyrosine phosphorylation in a time and concentration-dependent manner. Given the fact that PKA activity is negatively regulated by the P2Y12 receptor, our data provide evidence for a novel link of synergistic control of the state of tyrosine phosphorylation by both P2Y receptors. By SH2 domain pull down and MS/MS analysis, we identified distinct tyrosine phosphorylation sites in cell adhesion molecules, intracellular adapter proteins and phosphatases suggesting a major, functional role of tyrosine phosphorylation of theses candidate proteins in ADP-dependent signaling in human platelets.