Bioinformatics analysis of the data.
Bioinformatics analysis was performed to classify proteins based on subcellular localization and biological function. We performed classification based on annotations in the Human Protein Reference Database (HPRD; www.hprd.org
) in compliance with Gene Ontology (GO) standards. The summary includes fold-changes for differentially expressed proteins in ESCC. It also includes biological domains and motifs obtained from HPRD. The distribution of proteins identified in our study based on subcellular localization is shown in and according to biological processes is shown in . Among the 687 identified proteins, 206 contained signal peptides (SP), 26 contained a transmembrane (TM) domain and 13 contained both a TM domain and a SP motif. The MS and MS/MS spectra of representative known and novel proteins are represented in .
Figure 2 Classification of proteins by gene ontology based on their cellular localization and biological process. (A) Distribution of proteins based on their cellular component using gene ontology classifier. (B) Distribution of proteins based on their biological (more ...)
Figure 3 MS and MS/MS spectra of known and novel upregulated proteins in ESCC tissues as compared with the adjacent normal epithelia. MS and MS/MS spectra of peptide from representative differentially expressed proteins identified in this study. (A) Periostin (more ...) Known overexpressed proteins identified in this study.
Among the overexpressed proteins in ESCC tissues, we found number of proteins that had been previously described in ESCC, confirming the validity of the quantitative proteomic approach undertaken by us in this study. A partial list of known upregulated proteins is shown in . Among the proteins previously shown to be upregulated in ESCC were periostin (POSTN
thrombospondin 1 (THBS1
) and fascin 1 (FSCN1
). In one of the earlier studies on mRNA profiling of ESCC tissues, periostin was 11-fold upregulated in ESCC.24
In the current quantitative tissue proteomic study periostin was 7-fold upregulated at the protein level in cancer tissue as compared with adjacent normal tissue.
Partial list of overexpressed proteins that were previously reported in esophageal squamous cell carcinoma
Thrombospondin 1 is an extracellular adhesive glycoprotein that mediates cell-cell contact and is widely expressed. In the current study, thrombospondin 1 was 8-fold upregulated in cancer. Thrombospondin 1 has been previously shown to be overexpressed in ESCC and its overexpression correlated with regional lymph node invasion and poor survival of patients.24
We have identified fascin 1 (FSCN1
), an actin bundling protein, which plays diverse roles in cell-cell interaction, cell migration and is involved in increasing cell motility in transformed cells which was 2.5-fold upregulated in ESCC. In one of the previous studies, fascin 1 was shown to be upregulated in ESCC as compared with normal esophageal epithelia.25
Stomatin-like protein 2 (SLP2
) is a mitochondrial membrane protein involved in maintaining the mitochondrial membrane potential. Stomatin-like protein 2 affects cell motility and proliferation potentially by effecting the energy cycle within the cell, since it has a control on mitochondrial membrane potential. Stomatin-like protein 2 overexpression has been correlated with tumorigenesis and metastasis. In our study, Stomatin-like protein 2 was 3-fold upregulated in ESCC. Wang et al. showed the correlation of Stomatin-like protein 2 downregulation with inhibition of proliferation and cell motility using wound healing assay and transwell assay in KYSE150 cell line. They also showed by IHC labeling that Stomatin-like protein 2 was overexpressed in ESCC tissues.26
Fas associated via death domain (FADD
) is a member of the TNF superfamily which mediates apoptotic signaling via its death domain. It is an adaptor protein that binds to Fas and TNF receptors upon ligand binding, thus mediating downstream apoptotic signaling. In a study by Xue et al. FADD
was shown to be overexpressed in ESCC.27
In our study, FADD
was found to be 2-fold upregulated in ESCC tissue. Transgelin (TAGLN
), also known as SM22
, is an actin bundling protein which contains an actin binding region which is made of positively charged amino acids and CLIK domain (C-terminal calponin-like module). It is involved in binding actin filaments and in the process form podosomes. It is known that transgelin dysregulation results in cells becoming more malignant and invasive.28
A proteomic study performed by Qi et al. showed that transgelin was upregulated in ESCC based on a comparative 2DE analysis of ESCC and normal esophageal tissue.29
Our study is in concordance with study performed by Qi et al. as we also observed 2-fold upregulation of transgelin in ESCC.
Novel overexpressed proteins identified in this study.
There was a subset of proteins that were observed to be upregulated, which have not been previously described in the context of ESCC. A partial list of these novel and upregulated proteins is shown in . Among these upregulated proteins, PDIA4, PLEC1
, have not been described in the context of ESCC. Cofilin 1 (CFL1
) was found to be 3-fold upregulated in ESCC. Among other upregulated proteins in ESCC, ribophorin II (RPN2
) was found to be 2-fold upregulated in our study. Ribophorin II is an ER membranous protein involved in N-glycosylation of newly synthesized proteins in ER. Overexpression of RPN2
with respect to lymphovascular invasion is associated with poor survival in gastric cancer.30
Carbonyl reductase 1 (CBR1
) and carbonyl reductase 3 (CBR3
) are cytoplasmic NADPH-dependent reductase which has been shown to be involved in reduction of a broad range of molecules including quinones, prostaglandins and antitumor agents. Carbonyl reductase 1 was shown to be overexpressed in hepatocellular carcinoma under oxidative stress conditions like hypoxia.31
In the current study, carbonyl reductase 1 and carbonyl reductase 3 were 2-fold upregulated in ESCC. Cofilin 1 is an actin binding protein which is involved in polymerization and depolymerization of actin. It has been shown to be overexpressed in non-small cell lung cancer (NSCLC) and in this study overexpression of cofilin 1 was positively correlated with increased cellular invasiveness and resistance. Hence it was shown to be an effective prognostic and drug resistance marker.32
Partial list of novel proteins identified as overexpressed in esophageal squamous cell carcinoma
Downregulated proteins in ESCC.
There was a subset of proteins that were downregulated in the context of ESCC. 97 proteins were downregulated ≥2-fold in ESCC tissue as compared with adjacent normal epithelia. Among the downregulated and known proteins, junction plakoglobin (JUP
) was found to be 3-fold downregulated in ESCC as compared with normal. We have also identified Cystatin A (CSTA
) and was found to be 3-fold downregulated in cancer. Osteoglycin (OGN
) is a small protein belonging to the family of proteoglycans, which contains leucine rich repeats (LRR). It is involved in cellular growth, differentiation, cell growth and migration. It has been shown in many studies that downregulation of osteoglycin is related to increased metastasis.33–35
In the current study, osteoglycin was 3-fold downregulated in ESCC tissue as compared with adjacent normal epithelia.
Annexin 1 (ANXA1
) is a calcium dependent phosholipid binding protein, phospholipids are precursor for arachidonic acid which are in turn used in biosynthesis of prostaglandins and leukotrienes known proinflammatory molecules. Annexin 1 is localized to the cytoplasmic face of the plasma membrane, where they bind to phospholipids upon stimulation and prevent access to phospholipase A2 (PLA2G4A
). Annexin 1 is known to be downregulated in esophageal adenocarcinoma and squamous cell carcinomas,27,36–41
many studies have showed that ANXA1
downregulation correlates with poorly differentiated status of oral squamous cell carcinoma cells. We also found that ANXA1
was 3-fold downregulated in ESCC tissue.
Another molecule found to be downregulated in ESCC was cytokeratin 4 (KRT4
), which is a basic cytokeratin usually present as a heterodimer with the acidic cytokeratin 13 (KRT13
). It is expressed in differentiated layers of the mucosal and esophageal epithelia and has been shown to be downregulated during progression of ESCC. Xue et al. analyzed cytokeratin 4 in a panel of proteins using tissue microarrays of different stages of tumors varying from precursor lesions to metastasis.27
Downregulation of cytokeratin 4 was observed during progression from dysplasia and carcinoma in situ to metastasis. In our study, cytokeratin 4 was 9-fold downregulated in ESCC. Lastly, our quantitative proteomics study provides validation for several molecules at the protein level that were reported earlier at the mRNA level and in concordance with current study, including, cystatin A (CSTA
) and small proline-rich protein 3 (SPRR3
Validation of novel candidate biomarkers by immunohistochemical staining.
Some of the candidate proteins identified in our study were further tested by IHC labeling to assess whether they could serve as potential biomarkers for ESCC. The selection of proteins was based on the extent of overexpression, biological importance, involvement in other tumors and availability of commercial antibodies suitable for IHC labeling. We selected plectin 1 (PLEC1
), prosaposin (PSAP
) and protein disulfide isomerase 4 (PDIA4
) for further validation in formalin fixed paraffin embedded tissue sections. All of these proteins were overexpressed at the protein level with ≥2-fold change in ESCC as compared with the adjacent normal epithelium (see ), and were validated using IHC labeling. A statistical analysis was performed to determine the significance of differential expression of PDIA4, PSAP
between ESCC and normal tissues using a Chi-Square test. There was a statistically significant (p < 0.05) difference in the expression of PDIA4, PLEC1
between tumor and normal controls. The IHC staining pattern of these molecules in tumor and normal tissues is summarized in . The IHC scores for all the ESCC patients for plectin 1, prosaposin and protein disulfide isomerase A 4 are provided in Table S5
Summary of IHC labeling for the validated molecules—PLEC1, PSAP and PDIA4 in tumor and normal tissue
Plectin 1 (PLEC1).
Plectin 1 (PLEC1
) is the member of the plakin family. It is a multi-domain protein involved in crosslinking different cytoskeletal proteins and plays an important role in maintaining cell architecture and shape.42
Plectin was originally isolated as an intermediate filament binding protein. It is localized in the basal layer of epidermal cells where it appears to be a component of both hemidesmosomes and desmosomes.43,44
Plectin is also involved in connecting cytokeratins to α6β4 integrin at hemidesmosomes. It has been shown that PLEC1
is involved in microfilament network reorganization during apoptosis since caspase-8 cleaves PLEC1
upon activation during early stages of apoptosis. However this cytoskeletal remodeling is disturbed in cancer cells since most apoptotic pathways are dysregulated in cancer cells leading to accumulation of PLEC1
. In one study on pancreatic intraductal papillary mucinous neoplasms (IPMN), it was shown that PLEC1
was upregulated in malignant IPMNs.45
In our study, PLEC1 was 2-fold upregulated in ESCC tissue. Immunohistochemical labeling for PLEC1
showed overexpression of PLEC1
in 84/100 ESCC cases and expression in the majority of cases was cytoplasmic and membranous. The staining pattern of PLEC1
in representative ESCC and normal esophageal tissue is shown in .
Figure 4 Validation of Plectin 1 using immunohistochemical labeling. Representative sections from tissue microarrays stained with anti-Plectin is shown (A) expression of Plectin 1 in representative normal esophageal squamous mucosa and (B) expression of plectin (more ...)
Prosaposin is a lysosomal compartmental protein involved in catabolism of sphingolipids with small sugar chains. It is 526 amino acids long with a molecular weight of 58 kDa and is present as both membrane and secreted forms in the lysosome. It facilitates degradation of sphingolipids by making the lipid substrate more accessible to the soluble degradative enzymes in the lumen of lysosome.46
Prosaposin is a precursor protein which is partially cleaved into structurally related activator proteins saposin A, B, C and D. These saposins are known to play different biological roles. Prosaposin is a known neurotrophic factor47
and prosaposin deficiency or deletion is lethal in human and mice. It has been shown that prosaposin is amplified and overexpressed in number of androgen independent human prostate cancer cell lines (metastatic cell lines-PC-3, DU-145, MDA-PCa 2b, M-12, NCI-H660).48,49
In some of the early studies PSAP
has been shown to prevent apoptosis and promote survival in prostate cancer cells50
is shown to upregulate androgen receptor (AR), prostate specific antigen (PSA) in prostate cancer cells (LNCaP cells).51
In the process, it acts as androgen-agonist and its growth promoting effect may give a selective growth advantage to these prostate cancer cells, hence in the process it acts as an androgen regulated gene. In our study, PSAP
was 4-fold upregulated in ESCC tissue. Immunohistochemical labeling for PSAP
showed overexpression of PSAP
in 94/100 ESCC cases and expression in the majority of cases was cytoplasmic. The staining pattern of PSAP
in representative ESCC and normal esophageal tissue is shown in .
Figure 5 Validation of Prosaposin using immunohistochemical labeling. Representative sections from tissue microarrays stained with anti-prosaposin is shown (A) expression of prosaposin in representative normal esophageal squamous mucosa and (B) expression of prosaposin (more ...)
Protein disulfide isomerase 4 (PDIA4).
Protein disulfide isomerase 4 is an enzyme resident in the endoplasmic reticulum which is a thiol isomerase involved in facilitating the formation, reduction and rearrangement of disulfide bonds in nascent proteins. The extent of sequence identity between different members of PDI family is relatively low; however they share active-site motifs and domain structure. PDIA4
is 645 amino acids long and has a molecular weight of 72 kDa; thus it is also known as ERP72
. Most protein disulfide isomerases have three conserved domains—A, B and C.52,53
Domain A is a catalytically active thioredoxin like domain, domain B is a catalytically inactive thioredoxin like domain and domain C is a highly acidic region which is frequently used to bind to peptides.54 PDIA4
differs from other PDIA family members other in having 3 active thioredoxin domains rather than two with the domain distribution being C-A-A-B-B-A. Like other PDIA family members, PDIA4
is a stress induced protein and hence it is seen in many different cancers especially in tumor induced hypoxic states. In one of our earlier studies where we analyzed the secretome from ESCC and normal esophageal cell lines, PDIA4
along with PDIA3
was shown to be upregulated in the secretome of ESCC cell lines as compared with normal esophageal cell line.55
In this study, PDIA4
was again observed to be 2-fold upregulated in ESCC tissue. Immunohistochemical labeling for PDIA4
showed overexpression of PDIA4
in 92/100 ESCC cases and expression in the majority of cases was cytoplasmic. The staining pattern of PDIA4
in representative ESCC and normal esophageal tissue is shown in .
Figure 6 Validation of protein disulfide isomerase A4 using immunohistochemical labeling. Representative sections from tissue microarrays stained with anti-protein disulfide isomerase A4 as shown (A) expression of protein disulfide isomerase A4 in representative (more ...)