Proteomics, Morphoproteomics, and Targeted Therapy of Gastric Carcinoma



Proteomics, Morphoproteomics, and Targeted Therapy of Gastric Carcinoma


Wei Feng

Dongfeng Tan



INTRODUCTION

Gastric carcinoma has a dismal outcome due to the lack of practical screening methods and to the high percentage of cases presenting with advanced disease. Surgical resection remains the mainstay of gastric carcinoma treatment. However, the rate of local tumor recurrence and lymph node metastasis after curative surgical treatment is high (ranging from 45% to 88%, depending on the type of resection and lymph node dissection).1 Furthermore, present standard chemotherapy regimens of cisplatin, infusional 5 fluorouracil, and epirubicin produce low and frequently shortlived response rates (20%-40%).2

The lack of effective treatment for gastric carcinoma has led to intensive research to identify markers for early detection, tumor resistance to drugs, and tumor aggressiveness, as well as to identify new molecular targets of therapy. These efforts have consisted of approaches from the deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and protein levels. In this chapter, we highlight the therapeutic implications of studies in gastric carcinoma via proteomics (the systematic large-scale study of proteins) and morphoproteomics (the combined study of histopathology molecular biology, and protein chemistry).


PROTEOMICS

The term “proteome” was first coined by Marc Wilkins and Keith Williams as the whole spectrum of proteins encoded in a single genome expressed under distinct conditions.3 Following the success of the systemic human genome project, the classical study of protein chemistry has also evolved into a high-throughput and holistic science of the proteome known as proteomics. Proteomics has become an exciting field in biomedical research, as it provides an improved understanding of actual cellular functions. However, proteomics is complicated by the dynamic nature of the proteome, which is subject to posttranscriptional alterations such as alternative splicing and posttranslational modifications such as phosphorylation, glycosylation, and degradation.


Proteomic Techniques

In cancer proteomics, the protein expression profiles between cancerous tissues and corresponding noncancerous tissues are characterized, quantified, and compared. After total protein extraction from tissues, a proteomic study typically involves the concentration and separation of
proteins utilizing two-dimensional gel electrophoresis (2-DE), which resolves various proteins by isoelectric point (pI) and relative molecular mass (Mr). Proteins are then subsequently identified by mass spectrometry (MS). However, these techniques are limited in the detection of lowabundance proteins by the requirement of large protein samples. At present, advancements in isolation and analysis techniques such as liquid chromatography (LC), laser capture microdissection, isotope-coded affinity tagging (ICAT), matrix-assisted laser desorption/ioinization (MALDI), surface-enhanced laser desorption/ionization (SELDI), and time-of-flight MS have allowed for a quantitatively accurate nonselective approach to analyze proteomic changes in tumor cells.

Because cancer tissue is heterogeneous and comprises neoplastic cells as well as variable numbers of fibroblasts, adipocytes, endothelial cells, lymphocytes, and histiocytes, microdissection techniques such as laser capture microdissection (LCM) have been used to sample only the targeted neoplastic cells for further proteomic analysis. LCM is, however, labor intensive and limited by the large quantity of samples required for a complete proteomic study, since up to 15 hours of LCM are needed to isolate the required number of cells per 2-DE gel analysis (approximately 250,000 cells).4 Separation of proteins is most commonly achieved by one-dimensional (1-D) or two-dimensional gel electrophoresis (2-DE). By combining the concentrating technique of isoelectric focusing with the separation technique based on differences in molecular mass, 2-DE can resolve > 10,000 different compounds. LC is another alternative method for protein separation, but in typical LC techniques the advantage of keeping the protein in solution is compromised by considerably lower separation resolution than is achieved with 2-DE.3 The development of 2-D LC coupled with ICAT and MS has allowed for the identification of a large number of proteins, including proteins of low abundance.

After concentrating and separation steps, MS is then utilized to identify the protein/peptide composition by determining its molecular weight, chemical structure, and posttranslational modifications. The principle of MS consists of first ionizing protein/peptide compounds to generate charged molecules and then characterizing these compounds by measuring their mass-to-charge ratios (m/z). Various techniques are combined with the MS principle in proteomic applications in biomedical research. The large biomolecules, such as proteins and peptides, studied in proteomics are very fragile and tend to fragment when ionized by conventional ionization methods. This problem is overcome by MALDI, which is a soft ionization technique used in MS that combines the analyte with a solid matrix material to absorb most of the pulsed laser beam energy during the ionizing process and allows very large molecules to be preserved for further analysis. SELDI is a modified MALDI technique, with an added selective step wherein a chemically functional surface with biochemical affinity for a certain subset of proteins or peptides is used before combining the analyte with the solid matrix material.8 MALDI or SELDI can then be combined with time of flight MS (TOF-MS), where mass spectra are determined by measuring flight times of ionized compounds accelerated by an electric field over a fixed distance.3,5 MS data gathered are then analyzed via various online databases, such as ExPASy from the Swiss Institute of Bioinformatics or BLAST from the National Center for Biotechnology Information, to identify individual proteins.

Beyond merely identifying protein composition, a need exists to quantitatively and differentially characterize the proteome in neoplastic and normal tissues in cancer proteomics. This characterization can be achieved by difference gel electrophoresis (DIGE) or the more common ICAT procedure. In both techniques, markers are used to identify two or more samples (e.g., neoplastic and normal tissues) to allow for direct quantitative measurements between samples coresolved on the same gel electrophoresis or LC and subsequent MS analysis. DIGE utilizes fluorescent dyes (CyDyes, Cy2, Cy3, Cy5) covalently bound to protein samples, whereas ICAT utilizes isotope tags (12C or13C) to label protein samples. Whereas DIGE requires separate gel electrophoresis to be performed to separate labeled samples before MS analysis, ICAT allows for direct MS analysis of the separated LC sample.4


Proteomic Applications in Gastric Carcinoma

Cancer proteomics have been applied to gastric carcinoma in various studies to identify particular proteins that show differential expression in cancer tissues, serum, gastric juices, and tumor
cell lines. Various types of proteins, from structural cytoskeletal proteins, stress-related and chaperoning proteins, acute-phase proteins, glycolytic enzymes, enzymes involved in metabolism and cellular proliferation, tumor suppressor proteins to stomach-specific proteins, are found to be either upregulated or downregulated. Some of these proteins are potential markers for diagnosis of carcinoma or preneoplastic lesions, some are potential prognostic markers, and some are potential markers to predict therapy response.

Both small- and large-scale proteomic studies examining paired patient gastric carcinoma tumor tissues and nonneoplastic tissues using 2-DE MALDI-TOF/SELDI-TOF, LC MS, or LC tandem MS (MS/MS) have been conducted with varying results. Table 16-1 summarizes some of these studies. As with many proteomic studies, resulting data among different studies show little overlap, a finding thought to result from several issues. First, the heterogeneous nature of tissues and the underutilization of microdissection techniques cause suboptimal differential comparison between tumor cells and nonneoplastic epithelium, since other cells and connective tissues were included in the analysis. Second, the sample sizes of these studies were usually small. Even with these limitations, these studies identified many potential markers for further study. Nishigaki et al.6 identified differential expression of proteins in gastric carcinoma, which are involved in mitotic checkpoint regulation (MAD1L1 and EB1), apoptosis (HSP27), as well as mitochondrial reduction-oxidative balance. A proteomic analysis by Zhang et al.7 showed that there is decreased expression in MAWBP, a binding protein for MAWD, which is in turn an inhibitor of transcriptional activation mediated by transforming growth factor beta (TGF-β).

Additional proteomic studies were also performed on gastric juice and serum in attempts to identify serum markers for practical applications in early detection of gastric carcinoma. A study of patient gastric fluids from Singapore at Humphrey Oei Institute of Cancer Research showed that 106 significantly different proteomic features could be used to distinguish benign from gastric carcinoma cases with 88% sensitivity and 93% specificity.8 Similar studies have also been performed on serum. Ren et al.9 compared serum proteomic spectra in patients with gastric carcinoma before and after operation and showed that the serum protein profile consisting of four proteins (heat-shock protein 27, glucose-regulated protein, prohibitin, protein disulfide isomerase A3) could be used to differentiate normal from gastric carcinoma cases with 95.7% sensitivity and 92.5% specificity. These studies show that proteomics results have potential applications in early diagnosis of gastric carcinoma. However, the practicality of implementing these techniques to screen patient populations has yet to be determined.

Beyond the diagnostic implications, proteomic studies have also shed light on the carcinogenesis, metastatic potential, and drug resistance of gastric carcinoma. It is well known that the pathogenesis of gastric carcinoma starts with chronic gastritis, which then progresses to atrophy and dysplasia. One of the major causes of chronic gastritis is Helicobacter pylori infection, which eventually may result in clinically divergent outcomes of gastric carcinoma and duodenal ulcer.

Previous molecular studies have shown that virulence factors such as cytotoxin-associated gene A (CagA) produced increased Interleukin (IL)-8, resulting in an increased level of host inflammation, and are associated with an increased risk of gastric carcinoma.10 Wu et al.11 compared acid-glycine extract of H. pylori probed with serum samples from 15 patients with gastric carcinoma and 15 patients with duodenal ulcer. Among H. pylori protein antigens, which showed higher frequency in the gastric carcinoma group, cochaperonin GroES was identified as the dominant gastric carcinoma-related antigen, with significantly higher seropositivity in gastric carcinoma samples (64.2%, n = 95) than in gastritis (30.9%, n = 95) and duodenal ulcer samples (35.5%, n = 124). Furthermore, Wu et al.11 also showed that GroES stimulated production of IL-8 in mononuclear inflammatory cells and induced cellular proliferation; up-regulation of c-jun, c-fos, and cyclin D1; and down-regulation of p27 (Kip1). In a proteomic study by Chan et al.,12 the alteration in expressed proteins profile in the H. pylori-infected gastric epithelial AGS cell line was characterized. Eight of the proteins showing the greatest variation in H. pylori-infected gastric epithelial AGS cells were then shown to be more upregulated in gastric carcinoma tissues than in nonneoplastic gastric tissues (Table 16-1). These proteins include a promoter of NFκB











inhibitor IkBα degradation and an inhibitor of apoptotic cell death, valosin-containing protein; molecular chaperones, T-complex protein 1, heat shock 70-kDa protein, and mitochondrial matrix protein P1; promoter of cell adhesion and migration, FK506-binding protein 4 (FKBP4); Laminin γ-1; enolase a; and cell cycle regulator and DNA repair protein, 14-3-3 β. These studies provide further evidence of H. pylori’s role in the carcinogenesis of gastric carcinoma.








Table 16-1A Summary of proteomic studies in gastric cancer














































































































































































































































































































































































































































































































































































































































































































































































































































Proteins categorized according to Cell Cycle and Cell Proliferation Regulation, Cell Migration and Metastasis, Cell Structure and Motility, DNA Repair Immunity Defenses, Oncoprotein, Signal Transduction, and Transcription and Translation (d: downregulated; u: upregulated)


Studies: 1. Zhang et al.7 2. Chen et al.13 3. He et al.19 4. Yoshihara et al.20 5. Nishigaski et al.6 6. Kon et al.8 7. Chan et al.12 8. Chen et al.14 9. Yang et al.1710. Wang et al.16

1

2

3

4

5

6

7

8

9

10


Sample size (N)


84

44

10

5

14

24

10




Specimen type (T: tissue, J: gastric juice, C: cell line)

T

T

T

T

T

J

C/T

C

C

C


Protein

Function











Cell Cycle and Cell Proliferation Regulation


14-3-3 β/α

Regulator of cell cycle







u




Cell division control protein 42 homologue

Regulates cadherin-mediated cell-cell adhesion








d



Cell division kinase 6

Related to cell proliferation, tumor heterogeneity, invasion, and metastasis








u



Foveolin precursor FOV (gastrokine-1)

Growth factor




d







Microtubule-associated protein, RP/EB family, member 1

Regulation of cell cycle, cell proliferation, microtubule-binding, APC protein C-terminus binding





u






Mitotic checkpoint protein isoform MAD1a

Cell proliferation, mitotic spindle checkpoint, centrosome





d






Prohibitin

Inhibits cellular proliferation



u








Proteasome activator PA28 b-chain

Immunoproteasome assembly and antigen processing


u









SEPT 2 protein

Cytokinesis, mitosis

u










S-phase kinase-associated protein 1A

Mediates the ubiquitination of proteins involved in cell cycle progression, signal transduction, and transcription








d



T-complex protein beta

Related to p53 and activates DNA damage checkpoints








d



Tumor RMS cell line RD specific product (CYR61)

Insulin-like growth factor binding, extracellular





d






Cell Migration and Metastasis


Annexin I

Regulates cell proliferation, promotes membrane fusion, calcium-dependent phospholipid binding, inhibition of phospholipase A2

d







d

u


Catechol O-methyltransferase

Cancer progression and lymph node metastasis








u



Galectin-1

Promotes cancer cell invasion and metastasis








u



High mobility group protein 1

Interacts with transcription factors and regulates transcription related to tumor growth and invasion








u



Laminin γ-1 chain precursor

Induces collagenase IV, matrix metalloproteinase (MMP-9)







u




Platelet-derived endothelial cell growth factor

Angiogenesis


u









S100 calcium-binding protein A11

Calcium-binding, actin filament bundle formation, and cell motility







u

u





Tropomyosin (1: isoform; 2:3/4)

Stabilizes and binds actin filaments

u


u1





d2



Vimentin

Intermediate filament in mesenchymal cells related to migration








u



Cell Structure and Motility


ACTG1 protein

Cytoskeleton

u










Actin alpha2

Structural protein

u










Alpha-actinin

Anchors actin to intracellular structures








u



Cytokeratin 8

Intermediate filaments




u







Cytokeratin 20

Intermediate filaments




d







Cytoskeletal 5

Epidermis development, cytoskeleton organization and biogenesis, cellular morphogenesis








u



Cytoskeletal 17

Marker of basal cell differentiation








d



Tubulin alpha 6

Microtubules


u









WDR1 protein

Induces disassembly of actin filaments


u









DNA Repair


Uracil DNA glycosylase

Excise uracil residues from the DNA


u









Manganese superoxide dismutase (MnSOD)

Protection of DNA from oxidative damage


d


u







Immunity Defenses


α defensin

Microbicidal peptides






u





FK506-binding protein 4 (FKBP4)

Immunoregulation, protein folding/trafficking, binds FK506/rapamycin







u




MHC class I antigen

Inhibits evasion of the immune system and enhances tumor growth








d



Oncoprotein


18-kDa Antrum mucosa protein (AMP-18)

Human stomach specific, epithelial cell mitogen




d







Signal Transduction


Actin filament-binding protein (frabin)

Signal transduction










u


Catenin, 120ctn

Interacts with cadherin to regulate cell adhesion properties








u



Catenin, alpha-1

Actin crosslinking at adherens junctions








d



Catenin, beta

Modulates of cytoskeletal dynamics and cell proliferation








u



Chloride intracellular channel protein1 (CLIC1)

Signal transduction, Ion homeostasis, cell volume regulation, transepithelial transport.


u








d


Eukaryotic translation initiation factor 5A

Signal transduction









u


Integrin alpha 6/beta 4

Cell-matrix adhesion








u



N-myc downstream regulated gene 1

Signal transduction









d


Peroxiredoxin 5

High antioxidant efficiency to effect cell differentiation and apoptosis








u



Raf kinase inhibitor protein

Suppresses metastasis, angiogenesis, and vascular invasion








d



Ras GTPase-activating-like protein IQGAP1

Actin cytoskeleton assembly and E-cadherin-mediated cell adhesion








d



Rho-related GTP-binding protein RhoG

Small GTPase-mediated signal transduction, positive regulation of cell proliferation, actin cytoskeleton organization








d



RMD5 homolog B (RMND5B)

Signal transduction










d


Tyrosine 3/tryptophan 5-monooxygenase activation protein

Activates protein kinase C and Ca2+/calmodulin-dependent protein kinase II


u









Vinculin

Mediates the interactions between integrins and the actin








d



Transcription and Translation


Elongation factor 2

Transcription & translation









d


HnRNP A2

RNA trafficking, telomere maintenance

u










HnRNP-E1

RNA-binding protein, RNA trafficking

d










MADS box transcription enhancer factor 2, polypeptide C

Regulation of transcription, transcription from Pol II promoter, nucleus




u







Nucleophosmin

Ribosome assembly and transport









u


Translation elongation factor EF-Tu (EF-Tu)

Translation factor, cell growth, chaperone activity


u
















Table 16-1B Summary of proteomic studies in gastric cancer



































































































































































































































































































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May 22, 2016 | Posted by in ONCOLOGY | Comments Off on Proteomics, Morphoproteomics, and Targeted Therapy of Gastric Carcinoma

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Metabolic Proteins (d: downregulated; u: upregulated)


Studies: 1. Zhang et al.7 2. Chen et al.13 3. He et al.19 4. Yoshihara et al.20 5. Nishigaski et al.6 6. Melle et al.21 7. Lee et al.22 8. Kon et al.8 9. Chan et al.12 10. Chen et al.14 11. Yang et al.17 12. Wang et al.16

1

2

3

4

5

6

7

8

9

10

11

12


Sample size (N)


84

44

10

5

14

21

30

24

10




Specimen type (T: tissue, J: gastric juice, C: cell line)

T

T

T

T

T

T

J

J

C/T

C

C

C


Protein

Function













Carbohydrate Metabolism


Cytosolic malate dehydrogenase

Citric acid cycle


d











Glyceraldehyde-3-phosphate dehydrogenase

Glycolysis


u











Isocitrate dehydrogenase

Metabolic enzyme, citric acid cycle

d

u











L-lactate dehydrogenase B chain

Metabolic enzyme











d


MPI Mannose-6-phosphate isomerase

Metabolic enzyme






d







Phosphoglycerate kinase 1 (PGK-1)

Glycolysis





u








Phosphoglycerate mutase 1

Metabolic enzyme, glycolysis


u

u










Pyruvate kinase (PK)

Glycolysis


u

u










Pyruvate kinase3, isoform1

Glycolysis

d












TPI

Glycolytic enzyme



u









d


α enolase

Glycolysis, plasminogen receptor


u

u







u



Lipid, Fatty Acid, and Steroid Metabolism


Acyl-CoA dehydrogenase

Mitochondrial fatty acid β-oxidation

d












ApoA-I-binding protein

Regulation of lipid transport, metabolism of HDL particles




d









Apolipoprotein A-1 (ApoA1)/precursor

Cholesterol metabolism, acute phase protein


d

d










Fatty acid-binding protein

Transportation











d