Evaluation of reverse phase protein array (RPPA)-based pathway-activation profiling in 84 non-small cell lung cancer (NSCLC) cell lines as platform for cancer proteomics and biomarker discovery

AbstractThe reverse phase protein array (RPPA) approach was employed for a quantitative analysis of 71 cancer-relevant proteins and phosphoproteins in 84 non-small cell lung cancer (NSCLC) cell lines and by monitoring the activation state of selected receptor tyrosine kinases, PI3K/AKT and MEK/ERK1/2 signaling, cell cycle control, apoptosis, and DNA damage. Additional information on NSCLC cell lines such as that of transcriptomic data, genomic aberrations, and drug sensitivity was analyzed in the context of proteomic data using supervised and non-supervised approaches for data analysis. First, the unsupervised analysis of proteomic data indicated that proteins clustering closely together reflect well-known signaling modules, e.g. PI3K/AKT- and RAS/RAF/ERK-signaling, cell cycle regulation, and apoptosis. However, mutations of EGFR, ERBB2, RAF, RAS, TP53, and PI3K were found dispersed across different signaling pathway clusters. Merely cell lines with an amplification of EGFR and/or ERBB2 clustered closely together on the proteomic, but not on the transcriptomic level. Secondly, supervised data analysis revealed that sensitivity towards anti-EGFR drugs generally correlated better with high level EGFR phosphorylation than with EGFR abundance itself. High level phosphorylation of RB and high abundance of AURKA were identified as candidates that can potentially predict sensitivity towards the aurora kinase inhibitor VX680. Examples shown demonstrate that the RPPA approach presents a useful platform for targeted proteomics with high potential for biomarker discovery. This article is part of a Special Issue entitled: Biomarkers: A Proteomic Challenge

Epigenetic suppression of human telomerase ( hTERT ) is mediated by the metastasis suppressor NME2 in a G-quadruplex–dependent fashion

Transcriptional activation of the human telomerase reverse transcriptase (hTERT) gene, which remains repressed in adult somatic cells, is critical during tumorigenesis. Several transcription factors and the epigenetic state of the hTERT promoter are known to be important for tight control of hTERT in normal tissues, but the molecular mechanisms leading to hTERT reactivation in cancer are not well-understood. Surprisingly, here we found occupancy of the metastasis suppressor non-metastatic 2 (NME2) within the hTERT core promoter in HT1080 fibrosarcoma cells and HCT116 colon cancer cells and NME2-mediated transcriptional repression of hTERT in these cells. We also report that loss of NME2 results in up-regulated hTERT expression. Mechanistically, additional results indicated that the RE1-silencing transcription factor (REST)–lysine-specific histone demethylase 1 (LSD1) co-repressor complex associates with the hTERT promoter in an NME2-dependent way and that this assembly is required for maintaining repressive chromatin at the hTERT promoter. Interestingly, a G-quadruplex motif at the hTERT promoter was essential for occupancy of NME2 and the REST repressor complex on the hTERT promoter. In light of this mechanistic insight, we studied the effects of G-quadruplex–binding ligands on hTERT expression and observed that several of these ligands repressed hTERT expression. Together, our results support a mechanism of hTERT epigenetic control involving a G-quadruplex promoter motif, which potentially can be targeted by tailored small molecules

Ubiquitin carboxyl-terminal hydrolase 1 (UCHL1) is a potential tumour suppressor in prostate cancer and is frequently silenced by promoter methylation

<p>Abstract</p> <p>Background</p> <p>We have previously reported significant downregulation of ubiquitin carboxyl-terminal hydrolase 1 (UCHL1) in prostate cancer (PCa) compared to the surrounding benign tissue. UCHL1 plays an important role in ubiquitin system and different cellular processes such as cell proliferation and differentiation. We now show that the underlying mechanism of UCHL1 downregulation in PCa is linked to its promoter hypermethylation. Furthermore, we present evidences that UCHL1 expression can affect the behavior of prostate cancer cells in different ways.</p> <p>Results</p> <p>Methylation specific PCR analysis results showed a highly methylated promoter region for UCHL1 in 90% (18/20) of tumor tissue compared to 15% (3/20) of normal tissues from PCa patients. Pyrosequencing results confirmed a mean methylation of 41.4% in PCa whereas only 8.6% in normal tissues. To conduct functional analysis of UCHL1 in PCa, UCHL1 is overexpressed in LNCaP cells whose UCHL1 expression is normally suppressed by promoter methylation and found that UCHL1 has the ability to decrease the rate of cell proliferation and suppresses anchorage-independent growth of these cells. In further analysis, we found evidence that exogenous expression of UCHL1 suppress LNCaP cells growth probably via p53-mediated inhibition of Akt/PKB phosphorylation and also via accumulation of p27kip1 a cyclin dependant kinase inhibitor of cell cycle regulating proteins. Notably, we also observed that exogenous expression of UCHL1 induced a senescent phenotype that was detected by using the SA-ß-gal assay and might be due to increased p14ARF, p53, p27kip1 and decreased MDM2.</p> <p>Conclusion</p> <p>From these results, we propose that UCHL1 downregulation via promoter hypermethylation plays an important role in various molecular aspects of PCa biology, such as morphological diversification and regulation of proliferation.</p

Identification of Clinically Relevant Protein Targets in Prostate Cancer with 2D-DIGE Coupled Mass Spectrometry and Systems Biology Network Platform

Prostate cancer (PCa) is the most common type of cancer found in men and among the leading causes of cancer death in the western world. In the present study, we compared the individual protein expression patterns from histologically characterized PCa and the surrounding benign tissue obtained by manual micro dissection using highly sensitive two-dimensional differential gel electrophoresis (2D-DIGE) coupled with mass spectrometry. Proteomic data revealed 118 protein spots to be differentially expressed in cancer (n = 24) compared to benign (n = 21) prostate tissue. These spots were analysed by MALDI-TOF-MS/MS and 79 different proteins were identified. Using principal component analysis we could clearly separate tumor and normal tissue and two distinct tumor groups based on the protein expression pattern. By using a systems biology approach, we could map many of these proteins both into major pathways involved in PCa progression as well as into a group of potential diagnostic and/or prognostic markers. Due to complexity of the highly interconnected shortest pathway network, the functional sub networks revealed some of the potential candidate biomarker proteins for further validation. By using a systems biology approach, our study revealed novel proteins and molecular networks with altered expression in PCa. Further functional validation of individual proteins is ongoing and might provide new insights in PCa progression potentially leading to the design of novel diagnostic and therapeutic strategies

Stereotypical Chronic Lymphocytic Leukemia B-Cell Receptors Recognize Survival Promoting Antigens on Stromal Cells

Chronic lymphocytic leukemia (CLL) is the most common leukemia in the Western world. Survival of CLL cells depends on their close contact with stromal cells in lymphatic tissues, bone marrow and blood. This microenvironmental regulation of CLL cell survival involves the stromal secretion of chemo- and cytokines as well as the expression of adhesion molecules. Since CLL survival may also be driven by antigenic stimulation through the B-cell antigen receptor (BCR), we explored the hypothesis that these processes may be linked to each other. We tested if stromal cells could serve as an antigen reservoir for CLL cells, thus promoting CLL cell survival by stimulation through the BCR. As a proof of principle, we found that two CLL BCRs with a common stereotyped heavy chain complementarity-determining region 3 (previously characterized as “subset 1”) recognize antigens highly expressed in stromal cells – vimentin and calreticulin. Both antigens are well-documented targets of autoantibodies in autoimmune disorders. We demonstrated that vimentin is displayed on the surface of viable stromal cells and that it is present and bound by the stereotyped CLL BCR in CLL-stroma co-culture supernatant. Blocking the vimentin antigen by recombinant soluble CLL BCR under CLL-stromal cell co-culture conditions reduces stroma-mediated anti-apoptotic effects by 20–45%. We therefore conclude that CLL BCR stimulation by stroma-derived antigens can contribute to the protective effect that the stroma exerts on CLL cells. This finding sheds a new light on the understanding of the pathobiology of this so far mostly incurable disease

Differentially expressed proteins in prostate cancer and functional characterization of proteins with altered expression

Summary Prostate cancer (PCa) is the most common type of cancer found in men from western countries and is the leading cancer death next to lung cancer and colorectal cancer. Proteomic studies on PCa identified a number of differentially expressed proteins and some of them were reported as potential markers, but clinical application of these markers is mostly missing. Most of the expression profiling studies have been carried out on radical prostatectomy specimens, formalin-fixed paraffin-embedded (FFPE) tissue sections, serum, urine and prostate fluids. To define the protein expression pattern of prostate biopsies, in the present study we investigated biopsy samples from benign prostate hyperplasia (BPH) and PCa patients by two-dimensional gel electrophoresis (BPH n=11 and PCa n=12) and mass spectrometry to identify potential biomarkers which might distinguish the two clinical situations. 2-DE results revealed 88 protein spots expressed differentially among hyperplasia and cancer groups with statistical significance. Interesting spots were analyzed by MALDI-TOF-MS-MS and 79 different proteins identified. The important proteins identified included, Prohibitin and NDRG1 tumor suppressor proteins, HSPs, cytoskeletal proteins, enzymes like DDAH1 and ALDH2. Prohibitin expression was investigated in detail at mRNA level and protein level using immunohistochemistry on prostatectomized specimens. We found that the level of mRNA for prohibitin correlates with the increased amount of protein indicating the involvement of changes at transcriptional level. Furthermore, immunohistochemistry revealed no staining in BPH, moderate staining in prostate intraepithelial neoplasia (PIN) and strong staining in PCa. From the list of differentially proteins compared to PCa, TPD52 is over expressed in prostate cancer and also mRNA estimation by real-time PCR confirmed over expression of TPD52 at transcriptional level in cancer. TPD52 is a protein over expressed in prostate and breast cancer due to gene amplification but its exact physiological function is not investigated in detail. In the present study, we explored the responsiveness of LNCaP cells after dysregulation of TPD52 expression. Transfection of LNCaP cells with specific shRNA giving efficient knockdown of TPD52 resulted in a significant cell death of the carcinoma LNCaP cells. As evidenced by the activation of caspases (caspase-3 and -9) and by the loss of mitochondrial membrane potential, cell death occurs due to apoptosis. The disruption of the mitochondrial membrane potential indicates that TPD52 acts upstream of the mitochondrial apoptotic reaction. To study the effect of TPD52 expression on cell proliferation, LNCaP cells were either transfected with EGFP-TPD52 or a specific shRNA. EGFP-TPD52 overexpressing cells showed an increased proliferation rate whereas TPD52-depleted cells showed a reverse effect. Additionally, we demonstrated that the exogenous expression of TPD52 promotes cell migration via ávâ3 integrin in prostate cancer cells through the activation of protein kinase B (PKB/Akt) pathway. In an attempt to identify new interacting proteins for TPD52, GST pulldown assays provided evidence for the physical interaction between TPD52 and Prx1 in LNCaP cells. Further, immunoprecipitation results confirmed this interaction. Our results demonstrates that protein profiling and mRNA studies can be performed on prostate biopsies. Moreover, our study revealed a significant up-regulation of prohibitin in prostate cancer compared to BPH which may be a potential marker to distinguish PCa and BPH. From the results for functional characterization of TPD52, we conclude that TPD52 plays an important role in various molecular events particularly in morphological diversification and dissemination of PCa. It may be a promising target to investigate further in detail to develop new therapeutic strategies to treat PCa patients. Caspases represent a family of cysteine proteases that are regarded as central executioners of apoptotic cell death. Activation of caspase cascade is an essential prerequisite in the induction of apoptosis in cellular systems. So far, in many tumors caspases were shown to be downregulated while anti-apoptotic Bcl-2 is up-regulated. To get insight in their putative role in PCa progression we determined the expression of caspase-1, uncleaved caspases 3 and 6, cleaved (activated) caspases 3 and 6, caspase-9 and antiapoptotic protein Bcl-2 in benign prostate epithelium (BPE) and prostate carcinoma. In the current study 20 prostates were obtained from patients undergoing radical prostatectomy due to PCa. Paraffin embedded prostate whole mounts were cut at (4 µm) and investigated immunohistochemically using anti-mouse monoclonal antibodies directed against caspases 1 and 9, uncleaved caspases 3 and 6, cleaved caspases 3 and 6, and Bcl-2. In BPE all caspases were localized in the cytoplasm of glandular cells. Comparing BPE to PCa, no differences were found for caspase-1, uncleaved caspases 3 and 6 as well as caspase-9. Immunostaining for cleaved caspases 3 and 6, however, revealed a statistically significant reduction in PCa compared to non-neoplastic tissue. Whereas in BPE Bcl-2 protein was detected in the basal compartment of epithelial gland cells no immunostaining was seen in PCa. As our results show a decreased amount of activated caspases may be due to the alterations of posttranslational cleavage rather than expression of caspases 3 and 6. This suggests that the modification in their activation pathway could play an important role during PCa progression.Summary Prostate cancer (PCa) is the most common type of cancer found in men from western countries and is the leading cancer death next to lung cancer and colorectal cancer. Proteomic studies on PCa identified a number of differentially expressed proteins and some of them were reported as potential markers, but clinical application of these markers is mostly missing. Most of the expression profiling studies have been carried out on radical prostatectomy specimens, formalin-fixed paraffin-embedded (FFPE) tissue sections, serum, urine and prostate fluids. To define the protein expression pattern of prostate biopsies, in the present study we investigated biopsy samples from benign prostate hyperplasia (BPH) and PCa patients by two-dimensional gel electrophoresis (BPH n=11 and PCa n=12) and mass spectrometry to identify potential biomarkers which might distinguish the two clinical situations. 2-DE results revealed 88 protein spots expressed differentially among hyperplasia and cancer groups with statistical significance. Interesting spots were analyzed by MALDI-TOF-MS-MS and 79 different proteins identified. The important proteins identified included, Prohibitin and NDRG1 tumor suppressor proteins, HSPs, cytoskeletal proteins, enzymes like DDAH1 and ALDH2. Prohibitin expression was investigated in detail at mRNA level and protein level using immunohistochemistry on prostatectomized specimens. We found that the level of mRNA for prohibitin correlates with the increased amount of protein indicating the involvement of changes at transcriptional level. Furthermore, immunohistochemistry revealed no staining in BPH, moderate staining in prostate intraepithelial neoplasia (PIN) and strong staining in PCa. From the list of differentially proteins compared to PCa, TPD52 is over expressed in prostate cancer and also mRNA estimation by real-time PCR confirmed over expression of TPD52 at transcriptional level in cancer. TPD52 is a protein over expressed in prostate and breast cancer due to gene amplification but its exact physiological function is not investigated in detail. In the present study, we explored the responsiveness of LNCaP cells after dysregulation of TPD52 expression. Transfection of LNCaP cells with specific shRNA giving efficient knockdown of TPD52 resulted in a significant cell death of the carcinoma LNCaP cells. As evidenced by the activation of caspases (caspase-3 and -9) and by the loss of mitochondrial membrane potential, cell death occurs due to apoptosis. The disruption of the mitochondrial membrane potential indicates that TPD52 acts upstream of the mitochondrial apoptotic reaction. To study the effect of TPD52 expression on cell proliferation, LNCaP cells were either transfected with EGFP-TPD52 or a specific shRNA. EGFP-TPD52 overexpressing cells showed an increased proliferation rate whereas TPD52-depleted cells showed a reverse effect. Additionally, we demonstrated that the exogenous expression of TPD52 promotes cell migration via ávâ3 integrin in prostate cancer cells through the activation of protein kinase B (PKB/Akt) pathway. In an attempt to identify new interacting proteins for TPD52, GST pulldown assays provided evidence for the physical interaction between TPD52 and Prx1 in LNCaP cells. Further, immunoprecipitation results confirmed this interaction. Our results demonstrates that protein profiling and mRNA studies can be performed on prostate biopsies. Moreover, our study revealed a significant up-regulation of prohibitin in prostate cancer compared to BPH which may be a potential marker to distinguish PCa and BPH. From the results for functional characterization of TPD52, we conclude that TPD52 plays an important role in various molecular events particularly in morphological diversification and dissemination of PCa. It may be a promising target to investigate further in detail to develop new therapeutic strategies to treat PCa patients. Caspases represent a family of cysteine proteases that are regarded as central executioners of apoptotic cell death. Activation of caspase cascade is an essential prerequisite in the induction of apoptosis in cellular systems. So far, in many tumors caspases were shown to be downregulated while anti-apoptotic Bcl-2 is up-regulated. To get insight in their putative role in PCa progression we determined the expression of caspase-1, uncleaved caspases 3 and 6, cleaved (activated) caspases 3 and 6, caspase-9 and antiapoptotic protein Bcl-2 in benign prostate epithelium (BPE) and prostate carcinoma. In the current study 20 prostates were obtained from patients undergoing radical prostatectomy due to PCa. Paraffin embedded prostate whole mounts were cut at (4 µm) and investigated immunohistochemically using anti-mouse monoclonal antibodies directed against caspases 1 and 9, uncleaved caspases 3 and 6, cleaved caspases 3 and 6, and Bcl-2. In BPE all caspases were localized in the cytoplasm of glandular cells. Comparing BPE to PCa, no differences were found for caspase-1, uncleaved caspases 3 and 6 as well as caspase-9. Immunostaining for cleaved caspases 3 and 6, however, revealed a statistically significant reduction in PCa compared to non-neoplastic tissue. Whereas in BPE Bcl-2 protein was detected in the basal compartment of epithelial gland cells no immunostaining was seen in PCa. As our results show a decreased amount of activated caspases may be due to the alterations of posttranslational cleavage rather than expression of caspases 3 and 6. This suggests that the modification in their activation pathway could play an important role during PCa progression

Innovation at OSDD – Unconventional vs Conventional Approaches

20-23OSDD is employing a plethora of innovative and unconventional approaches with a large network of scientists and drug-discovery platforms to provide a number of diverse and promising early-lead compounds that will feed into the drug discovery pipeline. </span

Fluvastatin mediated breast cancer cell death: a proteomic approach to identify differentially regulated proteins in MDA-MB-231 cells.

Statins are increasingly being recognized as anti-cancer agents against various cancers including breast cancer. To understand the molecular pathways targeted by fluvastatin and its differential sensitivity against metastatic breast cancer cells, we analyzed protein alterations in MDA-MB-231 cells treated with fluvastatin using 2-DE in combination with LC-MS/MS. Results revealed dys-regulation of 39 protein spots corresponding to 35 different proteins. To determine the relevance of altered protein profiles with breast cancer cell death, we mapped these proteins to major pathways involved in the regulation of cell-to-cell signaling and interaction, cell cycle, Rho GDI and proteasomal pathways using IPA analysis. Highly interconnected sub networks showed that vimentin and ERK1/2 proteins play a central role in controlling the expression of altered proteins. Fluvastatin treatment caused proteolysis of vimentin, a marker of epithelial to mesenchymal transition. This effect of fluvastatin was reversed in the presence of mevalonate, a downstream product of HMG-CoA and caspase-3 inhibitor. Interestingly, fluvastatin neither caused an appreciable cell death nor did modulate vimentin expression in normal mammary epithelial cells. In conclusion, fluvastatin alters levels of cytoskeletal proteins, primarily targeting vimentin through increased caspase-3- mediated proteolysis, thereby suggesting a role for vimentin in statin-induced breast cancer cell death

Solution Nuclear Magnetic Resonance Studies of Sterol Carrier Protein 2 Like 2 (SCP2L2) Reveal the Insecticide Specific Structural Characteristics of SCP2 Proteins in <i>Aedes aegypti</i> Mosquitoes

Sterol carrier protein 2 like 2 from <i>Aedes aegypti</i> (<i>Ae</i>SCP2L2) plays an important role in lipid transport in mosquitoes for its routine metabolic processes. Repeated unsuccessful attempts to crystallize ligand free SCP2L2 prompted us to undertake nuclear magnetic resonance (NMR) spectroscopy to determine its three-dimensional structure. We report here the three-dimensional structures and dynamics of apo-<i>Ae</i>SCP2L2 and its complex with palmitate. The ¹⁵N heteronuclear single-quantum coherence spectrum of apo-<i>Ae</i>SCP2L2 displayed multiple peaks for some of the amide resonances, implying the presence of multiple conformations in solution, which are transformed to a single conformation upon formation of the complex with plamitate. The three-dimensional structures of apo-<i>Ae</i>SCP2L2 and palmitated <i>Ae</i>SCP2L2 reveal an α/β mixed fold, with five β-strands and four α-helices, very similar to the other SCP2 protein structures. Unlike the crystal structure of palmitated <i>Ae</i>SCP2L2, both solution structures are monomeric. It is further confirmed by the rotational correlation times determined by NMR relaxation times (<i>T</i>₁ and <i>T</i>₂) of the amide protons. In addition, the palmitated <i>Ae</i>SCP2L2 structure contains two palmitate ligands, bound in the binding pocket, unlike the three palmitates bound in the dimeric form of <i>Ae</i>SCP2L2 in the crystals. The relaxation experiments revealed that complex formation significantly reduces the dynamics of the protein in solution

Mycobacterium tuberculosis virulence inhibitors discovered by Mycobacterium marinum high-throughput screening

High-throughput screening facilities do not generally support biosafety level 3 organisms such as Mycobacterium tuberculosis. To discover not only antibacterials, but also virulence inhibitors with either bacterial or host cell targets, an assay monitoring lung fibroblast survival upon infection was developed and optimized for 384-plate format and robotic liquid handling. By using Mycobacterium marinum as surrogate organism, 28,000 compounds were screened at biosafety level 2 classification, resulting in 49 primary hits. Exclusion of substances with unfavourable properties and known antimicrobials resulted in 11 validated hits of which 7 had virulence inhibiting properties and one had bactericidal effect also in wild type Mycobacterium tuberculosis. This strategy to discover virulence inhibitors using a model organism in high-throughput screening can be a valuable tool for other researchers working on drug discovery against tuberculosis and other biosafety level 3 infectious agents