Differentially expressed proteins in Korean GC patients
To identify DEPs in GC tissues, we performed label-free proteomics using 9 pairs of cancer and matched normal stomach tissues. We found that 72 and 29 proteins were commonly up- or down-regulated, respectively, in at least 5 GC tissues compared with normal control tissues (Fig. 1, Additional file 2)
To investigate the Gene Ontology categories of the DEPs, up- or down-regulated proteins were loaded into the Panther database (www.pantherdb.org) for categorization according to biological processes. Among the 72 up-regulated proteins identified in GCs, 42, 34, 23, and 21 proteins were allocated to the metabolic process, cellular process, localization, and cellular component organization or biogenesis categories, respectively. In contrast, among the 29 down-regulated proteins, 13, 11, 10, and 9 proteins were allocated to the multicellular organismal process, metabolic process, developmental process, and cellular process categories, respectively (Fig. 1).
To further investigate the molecular and cellular etiology underlying the pathogenesis of Korean GC, the interactions between DEPs in Korean GC tissues were investigated using the Search Tool for Recurring Instances of Neighboring Genes (STRING) database (www.STRING-db.org). Fig. 2 presents the results of STRING analysis. Red nodes indicate up-regulated proteins, and blue represents down-regulated proteins in cancer tissues. The colored lines between nodes represent the various types of interaction evidence [14]. The DEPs were further divided into 6 clusters that contained most of the highly interacting DEPs and 5 groups that did not interact within groups but had similar known or predicted functions (Additional file 2).
Altered expression of proteins regulating the actin cytoskeleton and motor activity
Cluster I contained components of two major pathways: regulation of the actin cytoskeleton and motor activities. Interestingly, Actin-binding proteins and cross-linkers, such as Actinin a-4 (ACTN4), Actinin a-1, Actinin a-1 skeletal muscle, Moesin (MSN), Vinculin, and Transgelin, were consistently down-regulated in GCs. The expression levels of ACTN4 in pancreatic, ovarian, lung, and salivary gland cancer [15] and MSN in breast cancer [16] were altered. In contrast, the levels of proteins regulating actin polymerization, such as Actin-related protein 3 Homolog, IQ motif-containing GTPase activating protein 1 (IQGAP1), Adenylate cyclase-associated protein 1, GDP dissociation inhibitor 2, and Rho GTPase activating protein 1, were significantly increased in GCs. Among these proteins, up-regulation of IQGAP1 is known to be involved in tumorigenesis in lung, ovarian, stomach, and colon cancers [17].
Motor activity regulatory components, such as Myosin light chain 9, regulatory (MYL9), Myosin light chain 6, alkali, smooth muscle and non-muscle, Tropomyosin 1-a (TPM1), Tropomyosin 2-b, and Tropomyosin 3 (TPM3), were down-regulated in GCs. However, Myosin Heavy Chain 9, non-muscle (MYH9) was up-regulated. Among these proteins, MYL9 [18], TPM1 [19] and MYH9 [20] are known to be involved in tumorigenesis in various cancers. Other down-regulated components included intermediate filament components such as Vimentin and Desmin (Fig. 2I), which are highly associated with hepatocellular carcinoma [21] and colorectal cancer [22], respectively.
Significantly up-regulated major components of microtubules
Among the diverse Tubulins present in humans, 6 a-Tubulins [Tubulin a (TUBA)-1A, TUBA-1B, TUBA-1C, TUBA-3E, TUBA-4A, and TUBA-8], 7 b-Tubulins [Tubulin b (TUBB), TUBB-2A, TUBB-2B, TUBB-3, TUBB-4A, TUBB-4B, TUBB-6, and TUBB-8], and two regulatory components [Filamin A (FLNA), Chaperonin containing TCP1 subunit 6A] were significantly up-regulated in GCs (Fig. 2 II). b-Tubulins are established targets for anti-cancer drugs [23], and FLNA was reported to be up-regulated in breast cancer tissues [24].
Altered ion-binding proteins in GCs
Among the DEPs, iron- and oxygen-binding proteins, such as Hemoglobin (HB) subunit-b, HB subunit-g2, HB subunit-d, HB subunit-e1, and HB subunit-g1 were significantly down-regulated in GCs. In addition, the binding of Albumin to Ca2+, Na+, and K+ was down-regulated in GCs. In contrast, another iron-binding protein, Transferrin, and the zinc-containing Carbonic anhydrase enzymes were significantly up-regulated in GCs (Fig. 2 III).
Up-regulated glycolysis metabolism-associated proteins in GCs
Proteins in Cluster IV are involved in various metabolic processes. Among these proteins, Glyceraldehyde-3-phosphate dehydrogenase, Enolase 1 (ENO1), ENO2, Glucose-6-phosphate isomerase (GPI), Pyruvate kinase muscle (PKM), and Phosphoglycerate Kinase 1 (PGK1) are highly associated with glycolysis. These genes were up-regulated and closely associated each other. This cluster also included the down-regulated Malate Dehydrogenase 1, ATP synthase H+ transporting mitochondrial F1 complex a-subunit and cardiac muscle and the up-regulated Citrate Synthase found in mitochondria (Fig. 2 IV).
Most molecular chaperone-related proteins were up-regulated
Cluster V (Fig. 2V) included two hub molecular chaperones, Heat Shock Protein (HSP) 90 kDa cytosolic-a class A member 1 and HSP90 a-family class b member 1, which strongly interacted with HSP family A member 1 like, HSP family A member 2, HSP family A member 5, HSP family A member 6, HSP family A member 8, HSP family A member 9, HSP family D member 1, Hypoxia up-regulated 1, and HSP B member 1 (HSPB1). With the exception of HSPB1, all HSPs were up-regulated in GCs.
Up-regulated proteins with protein folding and trafficking activities
Cluster VI (Fig. 2VI) included up-regulated proteins that localized to the Golgi apparatus (GA) or endoplasmic reticulum (ER) and regulated protein folding and trafficking. Valosin-containing protein, Major histocompatibility complex class 1 (HLA)-B, HLA-C, Ribophorin II (RPN2), Protein disulfide isomerase family A (PDIA) member-3, PDIA member-6, Prolyl 4-hydroxylase subunit-b, Calnexin (CANX), Calreticulin (CALR), Thioredoxin domain containing 5, and Glucosidase II a-subunit were significantly up-regulated proteins.
Up-regulated proteins involved in protein synthesis
Three proteins involved in protein synthesis: Eukaryotic translation elongation factor (EEF)-2, EEF-1A1, and EEF-1A2, were up-regulated and interacted with components in other clusters. Among these proteins, EEF1A2 is involved in tumorigenesis in ovarian cancer [25].
Down-regulated proto-oncogenes and up-regulated peptidase inhibitors in GCs
Proto-oncogenes, such as Anterior gradient 2 [26, 27], Anterior gradient 3 [27], Ras suppressor-1 [28], SET nuclear proto-oncogene [29], Tryptophanyl-tRNA synthetase [30], and Ubiquitin-like modifier activating enzyme 1 [31], were significantly down-regulated in GCs (Fig. 2B). In addition, Serpin peptidase inhibitor clade (SERPIN)-A member 1 (SPERPINA1), which is involved in tumor progression in GCs [32] and colorectal cancer [33], and SERPIN-H member 1, which has been identified as a potential biomarker for early-stage hepatocellular carcinoma [34], were found to be significantly increased in GCs in the present study.
Altered proteins involved in energy metabolism and cellular structural components
Creatine kinase B, which regulates energy homeostasis in tissues and is decreased in cervical cancers [35], was down-regulated in GCs. In contrast, ATPase Na+/K+-transporting subunit a-1, which maintains energy homeostasis; mitochondrial Aldehyde dehydrogenase 2 family, which oxidizes aldehydes to generate carboxylic acids; and Gastric type Lipase F, an enzyme involved in the digestion of dietary triglycerides, were significantly up-regulated in GCs.
Other proteins with multiple domains that are important for organizing cellular structures also exhibited alterations. For example, POTE Ankyrin domain family member (POTE)-J and POTEI were down- and up-regulated in GCs, respectively. Lumican, a member of the small leucine-rich proteoglycan family that regulates collagen fibril organization and is involved in prostate cancers [36], was significantly down-regulated in GCs. Major vault protein is highly overexpressed in drug-resistant cancers [37]. Karyopherin subunit b-1, Junction Plakoglobin, Lamin A/C, Multiple PDZ domain crumbs cell polarity complex component, Clathrin heavy chain, and Leucine-rich pentatricopeptide repeat-containing were up-regulated.
Label-free proteomic analysis identified 2 fusion proteins in GC
We searched MS data obtained from 9 pairs of GC and normal tissues using the COSMIC fusion gene database. Twenty-six candidate fusion proteins were obtained (Additional file 2). We manually examined whether peptides spanning the junction of two proteins were identified by mass spectrometry. Only 2 fusion proteins were confirmed to have a corresponding peptide spanning two proteins: TPM4-ALK and hnRNPA2B1-FAM96A (Additional file 2).
To gain further insight into the possible roles of the 2 fusion proteins in tumorigenesis, the 3-D structures of the two fusion proteins were predicted by performing homology modeling. The 3-D structure of the TPM4-ALK fusion protein showed that it contains an ALK kinase domain and a TPM that primarily consists of an alpha helix and may assemble into parallel dimeric coiled-coils with normal TPM4 or other TPM4-ALK fusion proteins (Fig. 3a). The 3-D modeling of the hnRNPA2B1-FAM96A fusion protein revealed that the fusion protein presented as a dimer, given that FAM96A is known to form dimers [38]. The ability of the fusion proteins to dimerize suggested that the localization of the fusion proteins could be different from that of the normal proteins and might alter cellular and molecular processes.
Increased size of bands recognized by monoclonal TPM4 antibodies in GC tissues
To confirm the presence of TPM4-ALK fusion proteins, we performed Western blot analysis using monoclonal antibodies specific to TPM3 or TPM4. Anti-TPM4 antibodies recognized bands of increased sizes that were not detected in normal gastric tissues. However, anti-TPM3 antibodies did not detect any additional bands. Only one band from GC tissues showed the same migration pattern observed in normal gastric tissues (Fig. 4).