Cell Culture Mass Spectrometry Research Articles

Combinatorial treatment with statins and niclosamide prevents CRC dissemination by unhinging the MACC1-β-catenin-S100A4 axis of metastasis

Colorectal cancer (CRC) is the second-most common malignant disease worldwide, and metastasis is the main culprit of CRC-related death. Metachronous metastases remain to be an unpredictable, unpreventable, and fatal complication, and tracing the molecular chain of events that lead to metastasis would provide mechanistically linked biomarkers for the maintenance of remission in CRC patients after curative treatment. We hypothesized, that Metastasis-associated in colorectal cancer-1 (MACC1) induces a secretory phenotype to enforce metastasis in a paracrine manner, and found, that the cell-free culture medium of MACC1-expressing CRC cells induces migration. Stable isotope labeling by amino acids in cell culture mass spectrometry (SILAC-MS) of the medium revealed, that S100A4 is significantly enriched in the MACC1-specific secretome. Remarkably, both biomarkers correlate in expression data of independent cohorts as well as within CRC tumor sections. Furthermore, combined elevated transcript levels of the metastasis genes MACC1 and S100A4 in primary tumors and in blood plasma robustly identifies CRC patients at high risk for poor metastasis-free (MFS) and overall survival (OS). Mechanistically, MACC1 strengthens the interaction of β-catenin with TCF4, thus inducing S100A4 synthesis transcriptionally, resulting in elevated secretion to enforce cell motility and metastasis. In cell motility assays, S100A4 was indispensable for MACC1-induced migration, as shown via knock-out and pharmacological inhibition of S100A4. The direct transcriptional and functional relationship of MACC1 and S100A4 was probed by combined targeting with repositioned drugs. In fact, the MACC1-β-catenin-S100A4 axis by statins (MACC1) and niclosamide (S100A4) synergized in inhibiting cancer cell motility in vitro and metastasis in vivo. The MACC1-β-catenin-S100A4 signaling axis is causal for CRC metastasis. Selectively repositioned drugs synergize in restricting MACC1/S100A4-driven metastasis with cross-entity potential.

Proteomics reveal cap-dependent translation inhibitors remodel the translation machinery and translatome.

SUMMARYTactical disruption of protein synthesis is an attractive therapeutic strategy, with the first-in-class eIF4A-targeting compound zotatifin in clinical evaluation for cancer and COVID-19. The full cellular impact and mechanisms of these potent molecules are undefined at a proteomic level. Here, we report mass spectrometry analysis of translational reprogramming by rocaglates, cap-dependent initiation disruptors that include zotatifin. We find effects to be far more complex than simple “translational inhibition” as currently defined. Translatome analysis by TMT-pSILAC (tandem mass tag-pulse stable isotope labeling with amino acids in cell culture mass spectrometry) reveals myriad upregulated proteins that drive hitherto unrecognized cytotoxic mechanisms, including GEF-H1-mediated anti-survival RHOA/JNK activation. Surprisingly, these responses are not replicated by eIF4A silencing, indicating a broader translational adaptation than currently understood. Translation machinery analysis by MATRIX (mass spectrometry analysis of active translation factors using ribosome density fractionation and isotopic labeling experiments) identifies rocaglate-specific dependence on specific translation factors including eEF1ε1 that drive translatome remodeling. Our proteome-level interrogation reveals that the complete cellular response to these historical “translation inhibitors” is mediated by comprehensive translational landscape remodeling.

Integrated Cell Culture-Mass Spectrometry Method for Infectious Human Virus Monitoring

Tools for detecting infectious human viruses in environmental samples are essential for protecting human health. Here, we developed an Integrated Cell Culture-Mass Spectrometry (ICC-MS) method that detects infectious viruses by identifying viral proteins that replicate in cell cultures. The ICC-MS method was first validated and optimized with murine hepatitis viruses (MHV) spiked in culture media and wastewater concentrates and then applied to detect human viruses in untreated and treated wastewater samples. Reoviruses were detected in wastewater samples when the method was applied with Vero, BGMK, and BSC-1 cell lines. Strain-specific viral peptides from reovirus type 1, type 2, and type 3 were detected in primary influent, secondary effluent (pre-UV treatment), and UV-treated effluent. We propose that the ICC-MS method is a promising technique for directly detecting infectious human viruses in environmental samples without the need for primer design. This method can be expanded in future work to detect ...

Identification and validation of DOCK4 as a potential biomarker for risk of bone metastasis development in patients with early breast cancer.

Skeletal metastasis occurs in around 75% of advanced breast cancers, with the disease incurable once cancer cells disseminate to bone, but there remains an unmet need for biomarkers to identify patients at high risk of bone recurrence. This study aimed to identify such a biomarker and to assess its utility in predicting response to adjuvant zoledronic acid (zoledronate). We used quantitative proteomics (stable isotope labelling by amino acids in cell culture‐mass spectrometry; SILAC‐MS) to compare protein expression in a bone‐homing variant (BM1) of the human breast cancer cell line MDA‐MB‐231 with parental non‐bone‐homing cells to identify novel biomarkers for risk of subsequent bone metastasis in early breast cancer. SILAC‐MS showed that dedicator of cytokinesis protein 4 (DOCK4) was upregulated in bone‐homing BM1 cells, confirmed by western blotting. BM1 cells also had enhanced invasive ability compared with parental cells, which could be reduced by DOCK4‐shRNA. In a training tissue microarray (TMA) comprising 345 patients with early breast cancer, immunohistochemistry followed by Cox regression revealed that high DOCK4 expression correlated with histological grade (p = 0.004) but not oestrogen receptor status (p = 0.19) or lymph node involvement (p = 0.15). A clinical validation TMA used tissue samples and the clinical database from the large AZURE adjuvant study (n = 689). Adjusted Cox regression analyses showed that high DOCK4 expression in the control arm (no zoledronate) was significantly prognostic for first recurrence in bone (HR 2.13, 95%CI 1.06–4.30, p = 0.034). No corresponding association was found in patients who received zoledronate (HR 0.812, 95%CI 0.176–3.76, p = 0.790), suggesting that treatment with zoledronate may counteract the higher risk for bone relapse from high DOCK4‐expressing tumours. High DOCK4 expression was not associated with metastasis to non‐skeletal sites when these were assessed collectively. In conclusion, high DOCK4 in early breast cancer is significantly associated with aggressive disease and with future bone metastasis and is a potentially useful biomarker for subsequent bone metastasis risk. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

English

Isorhamnetin (Iso) exhibits antitumor effects in promyelotic leukemia and lung cancers. However, its effect in liver cancer is not clear. This study explored the effects of Iso in human liver cancer cells and profiled the differential proteins upon Iso treatment, which contributes to a better understanding of its anticancer mechanisms. Stable isotope labeling with amino acids in cell culture-mass spectrometry: ESI-Iontrap (SILAC-MS) was used to study the differentially proteomic profile of Iso-treated HepG2 cells. Iso inhibits HepG2 cell proliferation in a dose-dependent manner.  A total of 84 proteins are identified, among which 7 proteins were up-regulated while 77 proteins were down-regulated. Iso inhibits HepG2 cell proliferation by the induction of apoptosis and the inhibition of protein synthesis of human liver cancer cell. Key words:  Isorhamnetin, Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC), differential proteomic profile, HepG2 cells.

In-Depth Proteomics Identifies a Role for Autophagy in Controlling Reactive Oxygen Species Mediated Endothelial Permeability

Endothelial cells (ECs) form the inner layer of blood vessels and physically separate the blood from the surrounding tissue. To support tissues with nutrients and oxygen, the endothelial monolayer is semipermeable. When EC permeability is altered, blood vessels are not functional, and this is associated with disease. A comprehensive knowledge of the mechanisms regulating EC permeability is key in developing strategies to target this mechanism in pathologies. Here we have used an in vitro model of human umbilical vein endothelial cells mimicking the formation of a physiologically permeable vessel and performed time-resolved in-depth molecular profiling using stable isotope labeling by amino acids in cell culture mass spectrometry (MS)-proteomics. Autophagy is induced when ECs are assembled into a physiologically permeable monolayer. By using siRNA and drug treatment to block autophagy in combination with functional assays and MS proteomics, we show that ECs require autophagy flux to maintain intracellular reactive oxygen species levels, and this is required to maintain the physiological permeability of the cells.

Differential ICAM3 Gene Expression Correlates with Susceptibility to Natural Killer Cell-Mediated Lysis in Multiple Myeloma

Introduction We previously reported on the generation of highly activated/expanded natural killer cells (ENKs) after coculture with K562 cells modified to express membrane bound IL15 and 41BB-ligand. These cells have potent antimyeloma properties in vitro , in a NGS mouse model, and are safe when given to advanced multiple myeloma (MM) patients. (Szmania et al , J Immunother 2015) A potential obstacle to the effectiveness of ENK-based immunotherapy of MM is the evasion of immune recognition. We have generated 4 MM cell lines (OPM2, JJN3, ANBL6, and INA-6) which are resistant to ENK-mediated lysis to study mechanisms of resistance. These lines were derived from parental lines by repeated challenge with ENKs and maintained resistance long term when cultured without further exposure to ENKs.(Garg et al , Blood 2012, 120:4020) We have shown by stable isotope labeling with amino acids in cell culture-mass spectrometry, gene expression profiling (GEP), and flow cytometry that ICAM3 is downregulated in the ENK-resistant version of OPM2 (OPM2-R) compared to the parental OPM2. (OPM2-P; Garg et al , Blood 2013, 122:3105) We investigated OPM2-P and OPM2-R by whole exome sequencing (WES) and RNA sequencing (RNAseq) with a focus on ICAM3, evaluated ICAM3 cell surface expression on patient myeloma cells, and studied the importance of ICAM3 expression on ENK functionality. Methods DNA and RNA were extracted from OPM2-P and OPM2-R cells using the Qiagen AllPrep kit. WES libraries were prepared with the Agilent qXT and Agilent SureSelect Clinical Research Exome kits with additional baits covering the Ig and MYC loci. RNAseq libraries were prepared using the Illumina TruSeq stranded mRNA kit. Samples were sequenced 100bp PE on an Illumina HiSeq2500. Samples for WES were sequenced to a mean coverage of >120x and RNAseq to a target of >100M reads. WES data were aligned to the Ensembl GRCh37/hg19 human reference using BWA mem. Somatic variants were called MuTect. RNAseq data were analyzed using Tuxedo Suite. Data were aligned to the Ensembl GRCh37/hg19 human reference using TopHat with Bowtie2. Transcriptome reconstruction, quantification and differential analysis was performed using CuffLinks. ENK-mediated lysis of myeloma cells was measured by 4 hour chromium release assay in the presence of isotype or ICAM3 blocking antibody. Bone marrow aspirates were obtained from MM patients after informed consent in accordance with the Declaration of Helsinki. Primary myeloma cells were selected with CD138-coated immunomagnetic beads and ICAM3 expression was assessed by flow cytometry gated on viable CD138 positive cells. Results There was no mutation in ICAM3 in OPM2-R by WES, but RNAseq found a significant reduction in ICAM3 RNA in OPM2-R compared to OPM2-P ( p Conclusion Our findings demonstrate that MM patients harbor ICAM3-negative myeloma populations in varying frequencies, and we hypothesize that these cells may be similarly resistant to ENK-mediated lysis. Functional assays exploring this question are in progress. By understanding the mechanisms of ENK resistance and immune escape in MM, we hope to elucidate a surrogate biomarker which will allow us to select subjects who are most likely to benefit from cellular immunotherapeutic strategies for enrollment in future ENK-based clinical trials. Additionally, the ICAM3/LFA-1 interaction is also important for adhesion of T cells to their targets; therefore, down-regulation of ICAM3 may also have functional implications in the efficacy of T cell-based therapies for MM. Disclosures Garg: University of Arkansas for Medical Sciences: Employment. Chavan: University of Arkansas for Medical Sciences: Employment. Stone: University of Arkansas for Medical Sciences: Employment. Stivers: University of Arkansas for Medical Sciences: Employment. Warden: University of Arkansas for Medical Sciences: Employment. Skinner: University of Arkansas for Medical Sciences: Employment. Lingo: University of Arkansas for Medical Sciences: Employment. Greenway: University of Arkansas for Medical Sciences: Employment. Khan: University of Arkansas for Medical Sciences: Employment. Johann: University of Arkansas for Medical Sciences: Employment. Heuck: Millenium: Other: Advisory Board; Celgene: Consultancy; Janssen: Other: Advisory Board; University of Arkansas for Medical Sciences: Employment; Foundation Medicine: Honoraria. Barlogie: University of Arkansas for Medical Sciences: Employment. Morgan: University of Arkansas for Medical Sciences: Employment; Weismann Institute: Honoraria; MMRF: Honoraria; Bristol Myers Squibb: Honoraria, Membership on an entity9s Board of Directors or advisory committees; CancerNet: Honoraria; Takeda: Honoraria, Membership on an entity9s Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity9s Board of Directors or advisory committees. Epstein: University of Arkansas for Medical Sciences: Employment. van Rhee: University of Arkansa for Medical Sciences: Employment.

Discovery of Histone Modification Crosstalk Networks by Stable Isotope Labeling of Amino Acids in Cell Culture Mass Spectrometry (SILAC MS)

In this paper we describe an approach that combines stable isotope labeling of amino acids in cells culture, high mass accuracy liquid chromatography tandem mass spectrometry and a novel data analysis approach to accurately determine relative peptide post-translational modification levels. This paper describes the application of this approach to the discovery of novel histone modification crosstalk networks in Saccharomyces cerevisiae. Yeast histone mutants were generated to mimic the presence/absence of 44 well-known modifications on core histones H2A, H2B, H3, and H4. In each mutant strain the relative change in H3 K79 methylation and H3 K56 acetylation were determined using stable isotope labeling of amino acids in cells culture. This approach showed relative changes in H3 K79 methylation and H3 K56 acetylation that are consistent with known histone crosstalk networks. More importantly, this study revealed additional histone modification sites that affect H3 K79 methylation and H3 K56 acetylation.

Identification of Core Components and Transient Interactors of the Peroxisomal Importomer by Dual-Track Stable Isotope Labeling with Amino Acids in Cell Culture Analysis

The importomer complex plays an essential role in the biogenesis of peroxisomes by mediating the translocation of matrix proteins across the organellar membrane. A central part of this highly dynamic import machinery is the docking complex consisting of Pex14p, Pex13p, and Pex17p that is linked to the RING finger complex (Pex2p, Pex10p, Pex12p) via Pex8p. To gain detailed knowledge on the molecular players governing peroxisomal matrix protein import and, thus, the integrity and functionality of peroxisomes, we aimed at a most comprehensive investigation of stable and transient interaction partners of Pex14p, the central component of the importomer. To this end, we performed a thorough quantitative proteomics study based on epitope tagging of Pex14p combined with dual-track stable isotope labeling with amino acids in cell culture-mass spectrometry (SILAC-MS) analysis of affinity-purified Pex14p complexes and statistics. The results led to the establishment of the so far most extensive Pex14p interactome, comprising 9 core and further 12 transient components. We confirmed virtually all known Pex14p interaction partners including the core constituents of the importomer as well as Pex5p, Pex11p, Pex15p, and Dyn2p. More importantly, we identified new transient interaction partners (Pex25p, Hrr25p, Esl2p, prohibitin) that provide a valuable resource for future investigations on the functionality, dynamics, and regulation of the peroxisomal importomer.

Quantitative Proteomics Analysis Reveals BAG3 as a Potential Target To Suppress Severe Acute Respiratory Syndrome Coronavirus Replication

The discovery of a novel coronavirus (CoV) as the causative agent of severe acute respiratory syndrome (SARS) has highlighted the need for a better understanding of CoV replication. The replication of SARS-CoV is highly dependent on host cell factors. However, relatively little is known about the cellular proteome changes that occur during SARS-CoV replication. Recently, we developed a cell line expressing a SARS-CoV subgenomic replicon and used it to screen inhibitors of SARS-CoV replication. To identify host proteins important for SARS-CoV RNA replication, the protein profiles of the SARS-CoV replicon cells and parental BHK21 cells were compared using a quantitative proteomic strategy termed "stable-isotope labeling by amino acids in cell culture-mass spectrometry" (SILAC-MS). Our results revealed that, among the 1,081 host proteins quantified in both forward and reverse SILAC measurements, 74 had significantly altered levels of expression. Of these, significantly upregulated BCL2-associated athanogene 3 (BAG3) was selected for further functional studies. BAG3 is involved in a wide variety of cellular processes, including cell survival, cellular stress response, proliferation, migration, and apoptosis. Our results show that inhibition of BAG3 expression by RNA interference led to significant suppression of SARS-CoV replication, suggesting the possibility that upregulation of BAG3 may be part of the machinery that SARS-CoV relies on for replication. By correlating the proteomic data with these functional studies, the findings of this study provide important information for understanding SARS-CoV replication.

Combined Integrin Phosphoproteomic Analyses and Small Interfering RNA–Based Functional Screening Identify Key Regulators for Cancer Cell Adhesion and Migration

Integrins interact with extracellular matrix (ECM) and deliver intracellular signaling for cell proliferation, survival, and motility. During tumor metastasis, integrin-mediated cell adhesion to and migration on the ECM proteins are required for cancer cell survival and adaptation to the new microenvironment. Using stable isotope labeling by amino acids in cell culture-mass spectrometry, we profiled the phosphoproteomic changes induced by the interactions of cell integrins with type I collagen, the most common ECM substratum. Integrin-ECM interactions modulate phosphorylation of 517 serine, threonine, or tyrosine residues in 513 peptides, corresponding to 357 proteins. Among these proteins, 33 key signaling mediators with kinase or phosphatase activity were subjected to small interfering RNA-based functional screening. Three integrin-regulated kinases, DBF4, PAK2, and GRK6, were identified for their critical role in cell adhesion and migration possibly through their regulation of actin cytoskeleton arrangement. Altogether, we not only depict an integrin-modulated phosphorylation network during cell-ECM protein interactions but also reveal novel regulators for cell adhesion and migration.