Introduction: treatments targeting the Human Epidermal Growth Factor Receptor 2 (HER2/ERBB2) have... more Introduction: treatments targeting the Human Epidermal Growth Factor Receptor 2 (HER2/ERBB2) have improved the natural history of HER2-positive breast cancer. However, except HER2 protein expression and gene amplification, there is no predictive biomarker to guide the HER2-targeted therapies. We developed Parallel reaction monitoring (PRM) a powerful approach, to quantify and evaluate key proteins involved in the HER2 pathway and/or anti-HER2 treatment sensitivity. Results: in BCLs, PRM measurements correlated with western blot immunocytochemistry and transcriptomic data. At baseline, higher expression of HER2, EGFR, PTEN and HER3 but lower expression of phospho-HER2 correlated with trastuzumab sensitivity. Under trastuzumab, PRM demonstrated a decrease in HER2 and an increase in phospho-HER2, which correlated with drug sensitivity. The opposite was observed under lapatinib. HER2 quantification was also correlated with immunohistochemistry in PDXs and clinical breast cancer samples. Discussion: in conclusion, PRM-based assay, developed to quantify proteins of the HER2 pathway in breast cancer samples revealed a large magnitude of expression, which may have relevance in terms of treatment sensitivity. Materials and Methods: we first evaluated PRM in term of sensitivity, linearity and reproducibility. PRM was then applied to breast cancer cell lines (BCLs) including BCLs exposed to anti-HER2 agents, patient-derived xenografts (PDXs) and frozen breast cancer samples.
Despite the increased interest in secretomes associated with paracrine/autocrine mechanisms, the ... more Despite the increased interest in secretomes associated with paracrine/autocrine mechanisms, the majority of mass spectrometric cell secretome studies have been performed using serum-free medium (SFM). On the other hand, serum-containing medium (SCM) is not recommended very much because the secretome obtained with SCM is easily contaminated with fetal bovine serum (FBS) proteins. In this study, through the combination of bioorthogonal non-canonical amino acid tagging (BONCAT) and pulsed-SILAC (pSILAC), we analyzed differentially secreted proteins between SFM and SCM in a cancer-derived human cell, U87MG, and a mesenchymal stem cell derived from human Wharton's jelly (hWJ-MSCs). In most cases, the bioinformatic tools predicted a protein to be truly secretory when the secretion level of the protein was more in SCM than in SFM. In the case of hWJ-MSCs, the amount of proteins secreted in SCM for 24 hours was larger than that of SFM (log 2 fold change = 0.96), even considering different cell proliferation rates. hWJ-MSCs proteins secreted more in SCM included several positive markers of MSC paracrine factors implicated in angiogenesis, neurogenesis and osteogenesis, and upstream regulators of cell proliferation. Our study suggests the analysis of the secretome should be processed in SCM that promotes cell proliferation and secretion. Cytokines, growth factors, and enzymes are secreted or released into culture medium or body fluids. The secretome that encompasses them all changes over time depending on the changes of environmental factors or disease state and can act as a reporter for the health state of a patient 1. Therefore, it is important to understand the composition and dynamic changes of secretome during cell proliferation, development, and a certain pathological or environmental stimuli. They might also be a source of drug monitoring and disease diagnostic/prognostic biomarkers 2. The number of cell secretome studies has been increased for the past decade. However, many researchers have utilized serum-free media (SFM) to identify secreted proteins 3. Cells growing under serum condition, usually 10% fetal bovine serum (FBS), are transferred to SFM and incubated for several hours before collection of the media for mass spectrometric (MS) analysis. Because the secreted proteins are mostly low abundant (as low as ng/mL) when compared to high abundant contaminating proteins derived from serum-containing culture media (~5 mg/mL), the FBS proteins often mask the low abundant secreted proteins, which makes it difficult to detect
Schwann cells (SCs), the primary glia in the peripheral nervous system (PNS), display remarkable ... more Schwann cells (SCs), the primary glia in the peripheral nervous system (PNS), display remarkable plasticity in that fully mature SCs undergo dedifferentiation and convert to repair SCs upon nerve injury. Dedifferentiated SCs provide essential support for PNS regeneration by producing signals that enhance the survival and axon regrowth of damaged neurons, but the identities of neurotrophic factors remain incompletely understood. Here we show that SCs express and secrete progranulin (PGRN), depending on the differentiation status of SCs. PGRN expression and secretion markedly increased as primary SCs underwent dedifferentiation, while PGRN secretion was prevented by administration of cAMP, which induced SC differentiation. We also found that sciatic nerve injury, a physiological trigger of SC dedifferentiation, induced PGRN expression in SCs in vivo. These results suggest that dedifferentiated SCs express and secrete PGRN that functions as a paracrine factor to support the survival and axon growth of neighboring neurons after injury.
To discover serological colorectal cancer (CRC) markers, we analyzed cell line secretome to gathe... more To discover serological colorectal cancer (CRC) markers, we analyzed cell line secretome to gather proteins of higher potential to be secreted from tissues into circulation. A total of 898 human proteins were identified, of which 62.2% were predicted to be released or shed from cells. The identified proteins were compared with tissue proteomes to find candidate proteins whose expressions were elevated in tumor tissues compared with normal tissues as revealed by (i) quantitative proteomic analysis based on cICAT and mTRAQ or (ii) data mining of immunohistochemical images piled in Human Protein Atlas database. By applying various stringent criteria, 11 candidate proteins were selected. Among these, we validated an significant increase (p = 0.0018) of melanotransferrin (TRFM) at the plasma level of CRC patients through Western blotting, using 130 plasma samples containing 30 healthy controls, 80 CRC patients, and 20 patients of other diseases. Finally, we measured the expression level of TRFM in 325 plasma samples containing 77 healthy controls and 228 CRC patients (34.6 ± 4.2 ng/mL and 67.0 ± 6.4 ng/mL, p < 0.0001) through ELISA and demonstrated the area under the receiver operating characteristic curve of 0.723 (p < 0.0001) with a 92.5% specificity, 48.2% sensitivity, and 95.7% positive predictive value. Furthermore, unlike CEA and PAI-1, up-regulation of TRFM in pathological stages I & II groups compared with stages III & IV groups lead us to expect the use TRFM for early-stage diagnosis of CRC. In this study, we suggest TRFM as a potential serological marker for CRC and expect our discovery strategy to help identify highly cancer-specific and body-fluid-accessible biomarkers.
We propose to use cRFP (common Repository of FBS Proteins) in the MS (mass spectrometry) raw data... more We propose to use cRFP (common Repository of FBS Proteins) in the MS (mass spectrometry) raw data search of cell secretome. cRFP is a small supplementary sequence list of highly abundant fetal bovine serum proteins added to the reference database in use. The aim behind using cRFP is to prevent the contaminant FBS proteins from being misidentified as other proteins in the reference database, just as we would use cRAP (common Repository of Adventitious Proteins) to prevent contaminant proteins present either by accident or through unavoidable contacts from being misidentified as other proteins. We expect it to be widely used in experiments where the proteins are obtained from serum-free media after thorough washing of the cells, or from a complex media such as SILAC, or from extracellular vesicles directly.
An amendment to this paper has been published and can be accessed via a link at the top of the pa... more An amendment to this paper has been published and can be accessed via a link at the top of the paper.
Despite the increased interest in secretomes associated with paracrine/autocrine mechanisms, the ... more Despite the increased interest in secretomes associated with paracrine/autocrine mechanisms, the majority of mass spectrometric cell secretome studies have been performed using serum-free medium (SFM). On the other hand, serum-containing medium (SCM) is not recommended very much because the secretome obtained with SCM is easily contaminated with fetal bovine serum (FBS) proteins. In this study, through the combination of bioorthogonal non-canonical amino acid tagging (BONCAT) and pulsed-SILAC (pSILAC), we analyzed differentially secreted proteins between SFM and SCM in a cancer-derived human cell, U87MG, and a mesenchymal stem cell derived from human Wharton’s jelly (hWJ-MSCs). In most cases, the bioinformatic tools predicted a protein to be truly secretory when the secretion level of the protein was more in SCM than in SFM. In the case of hWJ-MSCs, the amount of proteins secreted in SCM for 24 hours was larger than that of SFM (log2 fold change = 0.96), even considering different c...
Schwann cells (SCs), the primary glia in the peripheral nervous system (PNS), display remarkable ... more Schwann cells (SCs), the primary glia in the peripheral nervous system (PNS), display remarkable plasticity in that fully mature SCs undergo dedifferentiation and convert to repair SCs upon nerve injury. Dedifferentiated SCs provide essential support for PNS regeneration by producing signals that enhance the survival and axon regrowth of damaged neurons, but the identities of neurotrophic factors remain incompletely understood. Here we show that SCs express and secrete progranulin (PGRN), depending on the differentiation status of SCs. PGRN expression and secretion markedly increased as primary SCs underwent dedifferentiation, while PGRN secretion was prevented by administration of cAMP, which induced SC differentiation. We also found that sciatic nerve injury, a physiological trigger of SC dedifferentiation, induced PGRN expression in SCs in vivo. These results suggest that dedifferentiated SCs express and secrete PGRN that functions as a paracrine factor to support the survival and axon growth of neighboring neurons after injury.
Despite the wide use of mesenchymal stromal cells (MSCs) for paracrine support in clinical trials... more Despite the wide use of mesenchymal stromal cells (MSCs) for paracrine support in clinical trials, their variable and heterogeneous supporting activity pose major challenges. While three-dimensional (3D) MSC cultures are emerging as alternative approaches, key changes in cellular characteristics during 3D-spheroid formation remain unclear. Here, we show that MSCs in 3D spheroids undergo further progression towards the epithelial-mesenchymal transition (EMT), driven by upregulation of EMT-promoting microRNAs and suppression of EMT-inhibitory miRNAs. The shift of EMT in MSCs is associated with widespread histone modifications mimicking the epigenetic reprogramming towards enhanced chromatin dynamics and stem cell-like properties, but without changes in their surface phenotype. Notably, these molecular shifts towards EMT in 3D MSCs caused enhanced stem cell niche activity, resulting in higher stimulation of hematopoietic progenitor self-renewal and cancer stem cell metastasis. Moreover, miRNA-mediated induction of EMT in 2D MSCs were sufficient to mimic the enhanced niche activity of 3D spheroid MSCs. Thus, the molecular hierarchy in the EMT gradient among phenotypically indistinguishable MSCs revealed the previously unrecognized functional parameters in MSCs, and the EMT-enhanced "naïve" mesenchymal state represents an 'activated mesenchymal niche' in 3D spheroid MSCs. Mesenchymal stromal cells (MSCs) are nonhematopoietic adherent cell populations derived from various organs, including the bone marrow (BM), adipose tissue, and placental tissue. Studies have shown that the primary mode of action of MSCs is establishment of a regenerative microenvironment in response to tissue injury, thereby stimulating the regeneration of endogenous stem cells, such as hematopoietic stem cells (HSCs), neuronal stem cells, or other tissue-specific stem cells 1, 2. During development, MSCs are derived from the neural crest through the epithelial-mesenchymal transition (EMT) to form the hematopoietic niche in the BM, or from mesenchymal sclerotome that contribute to the osteochondral differentiation 3. After birth, MSCs derived from pericytes in various organs 4 can give rise to self-renewing mesenchymal progenitors, which contribute to stem cell niche in a heterotrophic BM model 5. These MSCs are frequently expanded by in-vitro culture and these ex vivo cultured MSCs have been widely used for cell therapy in a variety of clinical trials. To date, over 320 clinical trials have been registered (www.clinicaltrials.org) for cell therapies aimed at supporting the regeneration of various types of tissue-specific stem cells 1, 2. However, the outcomes of clinical trials utilizing ex vivo expanded MSCs have been characterized by significant variations in outcomes, as exemplified in trials performed to enhance hematopoietic engraftment or immune suppression 6, 7. Accordingly, the cellular identities of ex-vivo expanded MSCs become controversial. Indeed, it is unclear whether these cultured MSCs can represent the in vivo nature of MSCs because they are selectively outgrown from subsets of MSCs during expansion culture 8. Similarly, studies have shown that MSCs expanded under conventional in vitro culture by plastic adherence undergo functional and phenotypic changes during the passage
Non-small-cell lung cancer (NSCLC) constitutes approximately 80% of all diagnosed lung cancers, a... more Non-small-cell lung cancer (NSCLC) constitutes approximately 80% of all diagnosed lung cancers, and diagnostic markers detectable in the plasma/serum of NSCLC patients are greatly needed. In this study, we established a pipeline for the discovery of markers using 9 transcriptome datasets from publicly available databases and profiling of six lung cancer cell secretomes. Thirty-one out of 312 proteins that overlapped between twofold differentially expressed genes and identified cell secretome proteins were detected in the pooled plasma of lung cancer patients. To quantify the candidates in the serum of NSCLC patients, multiple-reaction-monitoring mass spectrometry (MRM-MS) was performed for five candidate biomarkers. Finally, two potential biomarkers (BCHE and GPx3; AUC = 0.713 and 0.673, respectively) and one two-marker panel generated by logistic regression (BCHE/GPx3; AUC = 0.773) were identified. A validation test was performed by ELISA to evaluate the reproducibility of GPx3 and BCHE expression in an independent set of samples (BCHE and GPx3; AUC = 0.630 and 0.759, respectively, BCHE/GPx3 panel; AUC = 0.788). Collectively, these results demonstrate the feasibility of using our pipeline for marker discovery and our MRM-MS platform for verifying potential biomarkers of human diseases.
Fibroblast growth factor 11 (FGF11) is an intracellular FGF. Although induction of FGF11 by hypox... more Fibroblast growth factor 11 (FGF11) is an intracellular FGF. Although induction of FGF11 by hypoxia has been observed in several cell types, the molecular function of FGF11 is not clearly understood yet. Here, we investigated the role of FGF11 under hypoxia. We identified hypoxia-inducible factor-1α (HIF-1α) as an interacting protein of FGF11 using immunoprecipitation and mass spectrometry. FGF11 knockdown decreased HIF-1α protein, while FGF11 overexpression increased it, without affecting HIF-1α mRNA. Protein stability test and ubiquitination assay showed that FGF11 increased HIF-1α stability by acting upstream of proteasomal degradation. Altogether, these results suggest a cross-regulation between HIF-1α and FGF11, through which hypoxia-induced FGF11 reinforces hypoxia responses by enhancing the stability of HIF-1α.
To discover serological colorectal cancer (CRC) markers, we analyzed cell line secretome to gathe... more To discover serological colorectal cancer (CRC) markers, we analyzed cell line secretome to gather proteins of higher potential to be secreted from tissues into circulation. A total of 898 human proteins were identified, of which 62.2% were predicted to be released or shed from cells. The identified proteins were compared with tissue proteomes to find candidate proteins whose expressions were elevated in tumor tissues compared with normal tissues as revealed by (i) quantitative proteomic analysis based on cICAT and mTRAQ or (ii) data mining of immunohistochemical images piled in Human Protein Atlas database. By applying various stringent criteria, 11 candidate proteins were selected. Among these, we validated an significant increase (p = 0.0018) of melanotransferrin (TRFM) at the plasma level of CRC patients through Western blotting, using 130 plasma samples containing 30 healthy controls, 80 CRC patients, and 20 patients of other diseases. Finally, we measured the expression level of TRFM in 325 plasma samples containing 77 healthy controls and 228 CRC patients (34.6 ± 4.2 ng/mL and 67.0 ± 6.4 ng/mL, p < 0.0001) through ELISA and demonstrated the area under the receiver operating characteristic curve of 0.723 (p < 0.0001) with a 92.5% specificity, 48.2% sensitivity, and 95.7% positive predictive value. Furthermore, unlike CEA and PAI-1, up-regulation of TRFM in pathological stages I & II groups compared with stages III & IV groups lead us to expect the use TRFM for early-stage diagnosis of CRC. In this study, we suggest TRFM as a potential serological marker for CRC and expect our discovery strategy to help identify highly cancer-specific and body-fluid-accessible biomarkers.
Lymph node metastasis (LNM) is recognized as an important factor in the progression of tumor mali... more Lymph node metastasis (LNM) is recognized as an important factor in the progression of tumor malignancy. It is required to discover molecular markers for the prediction of LNM in gastric cancers (GCs). An isotope coded affinity tag (ICAT) method and mass spectrometry were used for the quantitative profiling of LNM-related proteins. Western blot analysis of the identified proteins and immunohistochemistry on a tissue microarray comprising 120 GC cases were performed for validation. We identified 151 differentially expressed proteins (DEPs) with an abundance ratio greater than 1.5-fold. The proteins upregulated in LNM-negative GCs were largely populated with proteins related to cell death. Among the DEPs, galectin-2 was further tested because its expression level was significantly higher in LNM-negative GCs (~12-fold, p &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.0001) and its expression is known to be not ubiquitous but confined to the gastrointestinal tract. Immunohistochemical analysis revealed that low expression of galectin-2 was significantly associated with LNM (p = 0.031) and advanced clinical stage (p = 0.024). The association of low galectin-2 with LNM was found even in early GCs (p = 0.020). Our results show that proteomic analysis coupled with immunohistochemistry using tissue microarray is a useful tool for identifying LNM-associated proteins in GC. Also, loss of galectin-2 might play an important role in the aggressiveness of GC.
Cancer cell secretome is considered a potential source for the discovery of cancer markers. In th... more Cancer cell secretome is considered a potential source for the discovery of cancer markers. In this study, the secretomes of four breast cancer (BC) cell lines (Hs578T, MCF-7, MDA-MB-231 and SK-BR-3) were profiled with LC-MS/MS analysis. A total of 1,410 proteins were identified with less than 1% FDR, of which about 55% (796 proteins) were predicted to be secreted from cells. To find BC-specific proteins among the secreted proteins, data of immunohistochemical staining piled in Human Protein Atlas were investigated by comparing the data of BC tissues with those of normal tissues. By applying various criteria, including higher expression level in BC tissues, higher predicted potential of secretion and enough number of tandem mass spectra, 12 biomarker candidate proteins including Ganglioside GM2 activator (GM2A) were selected for confirmation. Western blot and ELISA for plasma samples of healthy controls and BC patients revealed elevation of GM2A in BC patients, especially the ones w...
Mass spectrometric (MS) data of human cell secretomes are usually run through the conventional hu... more Mass spectrometric (MS) data of human cell secretomes are usually run through the conventional human database for identification. However, the search may result in false identifications due to contamination of the secretome with fetal bovine serum (FBS) proteins. To overcome this challenge, here we provide a composite protein database including human as well as 199 FBS protein sequences for MS data search of human cell secretomes. Searching against the human-FBS database returned more reliable results with fewer false-positive and false-negative identifications compared to using either a human only database or a human-bovine database. Furthermore, the improved results validated our strategy without complex experiments like SILAC. We expect our strategy to improve the accuracy of human secreted protein identification and to also add value for general use.
Lung cancer-related transcription factors (TFs) were identified by integrating previously reporte... more Lung cancer-related transcription factors (TFs) were identified by integrating previously reported genomic, transcriptomic, and proteomic data and were quantified by multiple reaction monitoring (MRM) in various cell lines. All experiments were performed without affinity depletion or subfractionation of cell lysates. Since the target proteins were expected to be present in low abundance, we experimentally optimized MRM transition parameters with chemically synthesized peptides. Quantitation was based on stable isotope-labeled standard peptides (SIS peptides). Out of 288 MRM measurements (36 peptides representing 28 TFs × 8 cell lines), 241 were successfully obtained within a quantitation limit of 15 amol, 221 measurements (91.7%) showed coefficients of variation (CVs) of ≤20%, and 149 (61.8%) showed CVs of ≤10%, quantifying as low as 19.4 amol/μg protein for STAT2 with a CV of 6.3% in an A549 cell. Comparisons between MRM measurements and levels of the corresponding mRNAs revealed linear, nonlinear, or no relationship between protein and mRNA levels, indicating the need for an MRM assay. An integrative analysis of MRM and gene expression profiles from doxorubicinresistant H69AR and sensitive H69 cells further showed that 14 differentially expressed TFs, such as STAT1 and SMAD4, regulated genes associated with drug resistance and cell differentiation-related processes. Thus, the analytical performance of MRM for the quantitation of low abundance TFs suggests its usefulness for biological application.
Introduction: treatments targeting the Human Epidermal Growth Factor Receptor 2 (HER2/ERBB2) have... more Introduction: treatments targeting the Human Epidermal Growth Factor Receptor 2 (HER2/ERBB2) have improved the natural history of HER2-positive breast cancer. However, except HER2 protein expression and gene amplification, there is no predictive biomarker to guide the HER2-targeted therapies. We developed Parallel reaction monitoring (PRM) a powerful approach, to quantify and evaluate key proteins involved in the HER2 pathway and/or anti-HER2 treatment sensitivity. Results: in BCLs, PRM measurements correlated with western blot immunocytochemistry and transcriptomic data. At baseline, higher expression of HER2, EGFR, PTEN and HER3 but lower expression of phospho-HER2 correlated with trastuzumab sensitivity. Under trastuzumab, PRM demonstrated a decrease in HER2 and an increase in phospho-HER2, which correlated with drug sensitivity. The opposite was observed under lapatinib. HER2 quantification was also correlated with immunohistochemistry in PDXs and clinical breast cancer samples. Discussion: in conclusion, PRM-based assay, developed to quantify proteins of the HER2 pathway in breast cancer samples revealed a large magnitude of expression, which may have relevance in terms of treatment sensitivity. Materials and Methods: we first evaluated PRM in term of sensitivity, linearity and reproducibility. PRM was then applied to breast cancer cell lines (BCLs) including BCLs exposed to anti-HER2 agents, patient-derived xenografts (PDXs) and frozen breast cancer samples.
Despite the increased interest in secretomes associated with paracrine/autocrine mechanisms, the ... more Despite the increased interest in secretomes associated with paracrine/autocrine mechanisms, the majority of mass spectrometric cell secretome studies have been performed using serum-free medium (SFM). On the other hand, serum-containing medium (SCM) is not recommended very much because the secretome obtained with SCM is easily contaminated with fetal bovine serum (FBS) proteins. In this study, through the combination of bioorthogonal non-canonical amino acid tagging (BONCAT) and pulsed-SILAC (pSILAC), we analyzed differentially secreted proteins between SFM and SCM in a cancer-derived human cell, U87MG, and a mesenchymal stem cell derived from human Wharton's jelly (hWJ-MSCs). In most cases, the bioinformatic tools predicted a protein to be truly secretory when the secretion level of the protein was more in SCM than in SFM. In the case of hWJ-MSCs, the amount of proteins secreted in SCM for 24 hours was larger than that of SFM (log 2 fold change = 0.96), even considering different cell proliferation rates. hWJ-MSCs proteins secreted more in SCM included several positive markers of MSC paracrine factors implicated in angiogenesis, neurogenesis and osteogenesis, and upstream regulators of cell proliferation. Our study suggests the analysis of the secretome should be processed in SCM that promotes cell proliferation and secretion. Cytokines, growth factors, and enzymes are secreted or released into culture medium or body fluids. The secretome that encompasses them all changes over time depending on the changes of environmental factors or disease state and can act as a reporter for the health state of a patient 1. Therefore, it is important to understand the composition and dynamic changes of secretome during cell proliferation, development, and a certain pathological or environmental stimuli. They might also be a source of drug monitoring and disease diagnostic/prognostic biomarkers 2. The number of cell secretome studies has been increased for the past decade. However, many researchers have utilized serum-free media (SFM) to identify secreted proteins 3. Cells growing under serum condition, usually 10% fetal bovine serum (FBS), are transferred to SFM and incubated for several hours before collection of the media for mass spectrometric (MS) analysis. Because the secreted proteins are mostly low abundant (as low as ng/mL) when compared to high abundant contaminating proteins derived from serum-containing culture media (~5 mg/mL), the FBS proteins often mask the low abundant secreted proteins, which makes it difficult to detect
Schwann cells (SCs), the primary glia in the peripheral nervous system (PNS), display remarkable ... more Schwann cells (SCs), the primary glia in the peripheral nervous system (PNS), display remarkable plasticity in that fully mature SCs undergo dedifferentiation and convert to repair SCs upon nerve injury. Dedifferentiated SCs provide essential support for PNS regeneration by producing signals that enhance the survival and axon regrowth of damaged neurons, but the identities of neurotrophic factors remain incompletely understood. Here we show that SCs express and secrete progranulin (PGRN), depending on the differentiation status of SCs. PGRN expression and secretion markedly increased as primary SCs underwent dedifferentiation, while PGRN secretion was prevented by administration of cAMP, which induced SC differentiation. We also found that sciatic nerve injury, a physiological trigger of SC dedifferentiation, induced PGRN expression in SCs in vivo. These results suggest that dedifferentiated SCs express and secrete PGRN that functions as a paracrine factor to support the survival and axon growth of neighboring neurons after injury.
To discover serological colorectal cancer (CRC) markers, we analyzed cell line secretome to gathe... more To discover serological colorectal cancer (CRC) markers, we analyzed cell line secretome to gather proteins of higher potential to be secreted from tissues into circulation. A total of 898 human proteins were identified, of which 62.2% were predicted to be released or shed from cells. The identified proteins were compared with tissue proteomes to find candidate proteins whose expressions were elevated in tumor tissues compared with normal tissues as revealed by (i) quantitative proteomic analysis based on cICAT and mTRAQ or (ii) data mining of immunohistochemical images piled in Human Protein Atlas database. By applying various stringent criteria, 11 candidate proteins were selected. Among these, we validated an significant increase (p = 0.0018) of melanotransferrin (TRFM) at the plasma level of CRC patients through Western blotting, using 130 plasma samples containing 30 healthy controls, 80 CRC patients, and 20 patients of other diseases. Finally, we measured the expression level of TRFM in 325 plasma samples containing 77 healthy controls and 228 CRC patients (34.6 ± 4.2 ng/mL and 67.0 ± 6.4 ng/mL, p < 0.0001) through ELISA and demonstrated the area under the receiver operating characteristic curve of 0.723 (p < 0.0001) with a 92.5% specificity, 48.2% sensitivity, and 95.7% positive predictive value. Furthermore, unlike CEA and PAI-1, up-regulation of TRFM in pathological stages I & II groups compared with stages III & IV groups lead us to expect the use TRFM for early-stage diagnosis of CRC. In this study, we suggest TRFM as a potential serological marker for CRC and expect our discovery strategy to help identify highly cancer-specific and body-fluid-accessible biomarkers.
We propose to use cRFP (common Repository of FBS Proteins) in the MS (mass spectrometry) raw data... more We propose to use cRFP (common Repository of FBS Proteins) in the MS (mass spectrometry) raw data search of cell secretome. cRFP is a small supplementary sequence list of highly abundant fetal bovine serum proteins added to the reference database in use. The aim behind using cRFP is to prevent the contaminant FBS proteins from being misidentified as other proteins in the reference database, just as we would use cRAP (common Repository of Adventitious Proteins) to prevent contaminant proteins present either by accident or through unavoidable contacts from being misidentified as other proteins. We expect it to be widely used in experiments where the proteins are obtained from serum-free media after thorough washing of the cells, or from a complex media such as SILAC, or from extracellular vesicles directly.
An amendment to this paper has been published and can be accessed via a link at the top of the pa... more An amendment to this paper has been published and can be accessed via a link at the top of the paper.
Despite the increased interest in secretomes associated with paracrine/autocrine mechanisms, the ... more Despite the increased interest in secretomes associated with paracrine/autocrine mechanisms, the majority of mass spectrometric cell secretome studies have been performed using serum-free medium (SFM). On the other hand, serum-containing medium (SCM) is not recommended very much because the secretome obtained with SCM is easily contaminated with fetal bovine serum (FBS) proteins. In this study, through the combination of bioorthogonal non-canonical amino acid tagging (BONCAT) and pulsed-SILAC (pSILAC), we analyzed differentially secreted proteins between SFM and SCM in a cancer-derived human cell, U87MG, and a mesenchymal stem cell derived from human Wharton’s jelly (hWJ-MSCs). In most cases, the bioinformatic tools predicted a protein to be truly secretory when the secretion level of the protein was more in SCM than in SFM. In the case of hWJ-MSCs, the amount of proteins secreted in SCM for 24 hours was larger than that of SFM (log2 fold change = 0.96), even considering different c...
Schwann cells (SCs), the primary glia in the peripheral nervous system (PNS), display remarkable ... more Schwann cells (SCs), the primary glia in the peripheral nervous system (PNS), display remarkable plasticity in that fully mature SCs undergo dedifferentiation and convert to repair SCs upon nerve injury. Dedifferentiated SCs provide essential support for PNS regeneration by producing signals that enhance the survival and axon regrowth of damaged neurons, but the identities of neurotrophic factors remain incompletely understood. Here we show that SCs express and secrete progranulin (PGRN), depending on the differentiation status of SCs. PGRN expression and secretion markedly increased as primary SCs underwent dedifferentiation, while PGRN secretion was prevented by administration of cAMP, which induced SC differentiation. We also found that sciatic nerve injury, a physiological trigger of SC dedifferentiation, induced PGRN expression in SCs in vivo. These results suggest that dedifferentiated SCs express and secrete PGRN that functions as a paracrine factor to support the survival and axon growth of neighboring neurons after injury.
Despite the wide use of mesenchymal stromal cells (MSCs) for paracrine support in clinical trials... more Despite the wide use of mesenchymal stromal cells (MSCs) for paracrine support in clinical trials, their variable and heterogeneous supporting activity pose major challenges. While three-dimensional (3D) MSC cultures are emerging as alternative approaches, key changes in cellular characteristics during 3D-spheroid formation remain unclear. Here, we show that MSCs in 3D spheroids undergo further progression towards the epithelial-mesenchymal transition (EMT), driven by upregulation of EMT-promoting microRNAs and suppression of EMT-inhibitory miRNAs. The shift of EMT in MSCs is associated with widespread histone modifications mimicking the epigenetic reprogramming towards enhanced chromatin dynamics and stem cell-like properties, but without changes in their surface phenotype. Notably, these molecular shifts towards EMT in 3D MSCs caused enhanced stem cell niche activity, resulting in higher stimulation of hematopoietic progenitor self-renewal and cancer stem cell metastasis. Moreover, miRNA-mediated induction of EMT in 2D MSCs were sufficient to mimic the enhanced niche activity of 3D spheroid MSCs. Thus, the molecular hierarchy in the EMT gradient among phenotypically indistinguishable MSCs revealed the previously unrecognized functional parameters in MSCs, and the EMT-enhanced "naïve" mesenchymal state represents an 'activated mesenchymal niche' in 3D spheroid MSCs. Mesenchymal stromal cells (MSCs) are nonhematopoietic adherent cell populations derived from various organs, including the bone marrow (BM), adipose tissue, and placental tissue. Studies have shown that the primary mode of action of MSCs is establishment of a regenerative microenvironment in response to tissue injury, thereby stimulating the regeneration of endogenous stem cells, such as hematopoietic stem cells (HSCs), neuronal stem cells, or other tissue-specific stem cells 1, 2. During development, MSCs are derived from the neural crest through the epithelial-mesenchymal transition (EMT) to form the hematopoietic niche in the BM, or from mesenchymal sclerotome that contribute to the osteochondral differentiation 3. After birth, MSCs derived from pericytes in various organs 4 can give rise to self-renewing mesenchymal progenitors, which contribute to stem cell niche in a heterotrophic BM model 5. These MSCs are frequently expanded by in-vitro culture and these ex vivo cultured MSCs have been widely used for cell therapy in a variety of clinical trials. To date, over 320 clinical trials have been registered (www.clinicaltrials.org) for cell therapies aimed at supporting the regeneration of various types of tissue-specific stem cells 1, 2. However, the outcomes of clinical trials utilizing ex vivo expanded MSCs have been characterized by significant variations in outcomes, as exemplified in trials performed to enhance hematopoietic engraftment or immune suppression 6, 7. Accordingly, the cellular identities of ex-vivo expanded MSCs become controversial. Indeed, it is unclear whether these cultured MSCs can represent the in vivo nature of MSCs because they are selectively outgrown from subsets of MSCs during expansion culture 8. Similarly, studies have shown that MSCs expanded under conventional in vitro culture by plastic adherence undergo functional and phenotypic changes during the passage
Non-small-cell lung cancer (NSCLC) constitutes approximately 80% of all diagnosed lung cancers, a... more Non-small-cell lung cancer (NSCLC) constitutes approximately 80% of all diagnosed lung cancers, and diagnostic markers detectable in the plasma/serum of NSCLC patients are greatly needed. In this study, we established a pipeline for the discovery of markers using 9 transcriptome datasets from publicly available databases and profiling of six lung cancer cell secretomes. Thirty-one out of 312 proteins that overlapped between twofold differentially expressed genes and identified cell secretome proteins were detected in the pooled plasma of lung cancer patients. To quantify the candidates in the serum of NSCLC patients, multiple-reaction-monitoring mass spectrometry (MRM-MS) was performed for five candidate biomarkers. Finally, two potential biomarkers (BCHE and GPx3; AUC = 0.713 and 0.673, respectively) and one two-marker panel generated by logistic regression (BCHE/GPx3; AUC = 0.773) were identified. A validation test was performed by ELISA to evaluate the reproducibility of GPx3 and BCHE expression in an independent set of samples (BCHE and GPx3; AUC = 0.630 and 0.759, respectively, BCHE/GPx3 panel; AUC = 0.788). Collectively, these results demonstrate the feasibility of using our pipeline for marker discovery and our MRM-MS platform for verifying potential biomarkers of human diseases.
Fibroblast growth factor 11 (FGF11) is an intracellular FGF. Although induction of FGF11 by hypox... more Fibroblast growth factor 11 (FGF11) is an intracellular FGF. Although induction of FGF11 by hypoxia has been observed in several cell types, the molecular function of FGF11 is not clearly understood yet. Here, we investigated the role of FGF11 under hypoxia. We identified hypoxia-inducible factor-1α (HIF-1α) as an interacting protein of FGF11 using immunoprecipitation and mass spectrometry. FGF11 knockdown decreased HIF-1α protein, while FGF11 overexpression increased it, without affecting HIF-1α mRNA. Protein stability test and ubiquitination assay showed that FGF11 increased HIF-1α stability by acting upstream of proteasomal degradation. Altogether, these results suggest a cross-regulation between HIF-1α and FGF11, through which hypoxia-induced FGF11 reinforces hypoxia responses by enhancing the stability of HIF-1α.
To discover serological colorectal cancer (CRC) markers, we analyzed cell line secretome to gathe... more To discover serological colorectal cancer (CRC) markers, we analyzed cell line secretome to gather proteins of higher potential to be secreted from tissues into circulation. A total of 898 human proteins were identified, of which 62.2% were predicted to be released or shed from cells. The identified proteins were compared with tissue proteomes to find candidate proteins whose expressions were elevated in tumor tissues compared with normal tissues as revealed by (i) quantitative proteomic analysis based on cICAT and mTRAQ or (ii) data mining of immunohistochemical images piled in Human Protein Atlas database. By applying various stringent criteria, 11 candidate proteins were selected. Among these, we validated an significant increase (p = 0.0018) of melanotransferrin (TRFM) at the plasma level of CRC patients through Western blotting, using 130 plasma samples containing 30 healthy controls, 80 CRC patients, and 20 patients of other diseases. Finally, we measured the expression level of TRFM in 325 plasma samples containing 77 healthy controls and 228 CRC patients (34.6 ± 4.2 ng/mL and 67.0 ± 6.4 ng/mL, p < 0.0001) through ELISA and demonstrated the area under the receiver operating characteristic curve of 0.723 (p < 0.0001) with a 92.5% specificity, 48.2% sensitivity, and 95.7% positive predictive value. Furthermore, unlike CEA and PAI-1, up-regulation of TRFM in pathological stages I & II groups compared with stages III & IV groups lead us to expect the use TRFM for early-stage diagnosis of CRC. In this study, we suggest TRFM as a potential serological marker for CRC and expect our discovery strategy to help identify highly cancer-specific and body-fluid-accessible biomarkers.
Lymph node metastasis (LNM) is recognized as an important factor in the progression of tumor mali... more Lymph node metastasis (LNM) is recognized as an important factor in the progression of tumor malignancy. It is required to discover molecular markers for the prediction of LNM in gastric cancers (GCs). An isotope coded affinity tag (ICAT) method and mass spectrometry were used for the quantitative profiling of LNM-related proteins. Western blot analysis of the identified proteins and immunohistochemistry on a tissue microarray comprising 120 GC cases were performed for validation. We identified 151 differentially expressed proteins (DEPs) with an abundance ratio greater than 1.5-fold. The proteins upregulated in LNM-negative GCs were largely populated with proteins related to cell death. Among the DEPs, galectin-2 was further tested because its expression level was significantly higher in LNM-negative GCs (~12-fold, p &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.0001) and its expression is known to be not ubiquitous but confined to the gastrointestinal tract. Immunohistochemical analysis revealed that low expression of galectin-2 was significantly associated with LNM (p = 0.031) and advanced clinical stage (p = 0.024). The association of low galectin-2 with LNM was found even in early GCs (p = 0.020). Our results show that proteomic analysis coupled with immunohistochemistry using tissue microarray is a useful tool for identifying LNM-associated proteins in GC. Also, loss of galectin-2 might play an important role in the aggressiveness of GC.
Cancer cell secretome is considered a potential source for the discovery of cancer markers. In th... more Cancer cell secretome is considered a potential source for the discovery of cancer markers. In this study, the secretomes of four breast cancer (BC) cell lines (Hs578T, MCF-7, MDA-MB-231 and SK-BR-3) were profiled with LC-MS/MS analysis. A total of 1,410 proteins were identified with less than 1% FDR, of which about 55% (796 proteins) were predicted to be secreted from cells. To find BC-specific proteins among the secreted proteins, data of immunohistochemical staining piled in Human Protein Atlas were investigated by comparing the data of BC tissues with those of normal tissues. By applying various criteria, including higher expression level in BC tissues, higher predicted potential of secretion and enough number of tandem mass spectra, 12 biomarker candidate proteins including Ganglioside GM2 activator (GM2A) were selected for confirmation. Western blot and ELISA for plasma samples of healthy controls and BC patients revealed elevation of GM2A in BC patients, especially the ones w...
Mass spectrometric (MS) data of human cell secretomes are usually run through the conventional hu... more Mass spectrometric (MS) data of human cell secretomes are usually run through the conventional human database for identification. However, the search may result in false identifications due to contamination of the secretome with fetal bovine serum (FBS) proteins. To overcome this challenge, here we provide a composite protein database including human as well as 199 FBS protein sequences for MS data search of human cell secretomes. Searching against the human-FBS database returned more reliable results with fewer false-positive and false-negative identifications compared to using either a human only database or a human-bovine database. Furthermore, the improved results validated our strategy without complex experiments like SILAC. We expect our strategy to improve the accuracy of human secreted protein identification and to also add value for general use.
Lung cancer-related transcription factors (TFs) were identified by integrating previously reporte... more Lung cancer-related transcription factors (TFs) were identified by integrating previously reported genomic, transcriptomic, and proteomic data and were quantified by multiple reaction monitoring (MRM) in various cell lines. All experiments were performed without affinity depletion or subfractionation of cell lysates. Since the target proteins were expected to be present in low abundance, we experimentally optimized MRM transition parameters with chemically synthesized peptides. Quantitation was based on stable isotope-labeled standard peptides (SIS peptides). Out of 288 MRM measurements (36 peptides representing 28 TFs × 8 cell lines), 241 were successfully obtained within a quantitation limit of 15 amol, 221 measurements (91.7%) showed coefficients of variation (CVs) of ≤20%, and 149 (61.8%) showed CVs of ≤10%, quantifying as low as 19.4 amol/μg protein for STAT2 with a CV of 6.3% in an A549 cell. Comparisons between MRM measurements and levels of the corresponding mRNAs revealed linear, nonlinear, or no relationship between protein and mRNA levels, indicating the need for an MRM assay. An integrative analysis of MRM and gene expression profiles from doxorubicinresistant H69AR and sensitive H69 cells further showed that 14 differentially expressed TFs, such as STAT1 and SMAD4, regulated genes associated with drug resistance and cell differentiation-related processes. Thus, the analytical performance of MRM for the quantitation of low abundance TFs suggests its usefulness for biological application.
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Papers by Jihye Shin