Comparative proteomic study for profiling differentially expressed proteins between Chinese left- and right-sided colon cancers Hong Shen,1,2 Jinlin Huang,1 Haiping Pei,2 Shan Zeng,3 Yiming Tao,1 Liangfang Shen,3 Liang Zeng4 and Hong Zhu3,5 1 Institute of Medical Sciences, Departments of 2Gastrointestinal Surgery, 3Oncology, Xiangya Hospital, Central South University, 4Department of Pathology, Tumor Hospital of Hunan Province, Changsha, Hunan, China
(Received June 7, 2012 ⁄ Revised September 6, 2012 ⁄ Accepted September 14, 2012 ⁄ Accepted manuscript online September 24, 2012 ⁄ Article first published online November 17, 2012)
The aim of the present study is to profile differentially expressed protein markers between left-sided colon cancer (LSCC) and right-sided colon cancer (RSCC). Fresh tumor tissue samples from LSCC (n = 7) and RSCC (n = 7) groups were analyzed by two-dimensional electrophoresis coupled with MALDITOF-MS, followed by Western blotting. In 50 paraffin embedded samples from each group, levels of four differentially expressed proteins (identified by proteomics analysis) were measured by tissue microarray with immunohistochemistry staining to compare the different protein markers between LSCC and RSCC. Sixteen proteins were found to be differentially expressed between LSCC and RSCC. Ten proteins including HSP60 and PDIA1 were identified to be highly expressed in LSCC (P < 0.01 or P < 0.05), while the expression of six proteins including EEF1D and HSP-27 were higher in RSCC (P < 0.01 or P < 0.05). Virtually all of the indentified proteins were involved in cellular energy metabolism, protein folding/unfolding, and/or oxidative stress. Human colon tumors at various locations have different proteomic biomarkers. Differentially expressed proteins associated with energy metabolism, protein folding/unfolding and oxidative stress contribute to different tumorigenesis, tumor progression, and prognosis between left- and right-sided colon cancer. (Cancer Sci 2013; 104: 135–141)
(FAP) dominates in the left hemicolon, but the tumorigenesis of hereditary nonpolyposis colon cancer (HNPCC), which lacks evidence of adenoma existence, is mainly located in the right hemicolon.(7) Besides the different clinical manifestations, the disparities of microenvironment, embryonic origin, and distribution of immunocytes between the left and right hemicolons suggest that there might be various molecular mechanisms contributing to the tumorigenesis of colon cancers at different locations. The profiling and identification of specific protein signatures associated with location-specific colon cancers may provide new clues for understanding the molecular mechanisms of colon cancer progression. Such proteins may serve as novel biomarkers and therapeutic targets enabling earlier detection and improved treatment strategies for colon cancer. Using two-dimensional electrophoresis (2-DE) coupled with mass spectrometry (MS) and tissue microarray, the aim of this study is to identify protein markers of right- and leftsided colon cancers, so as to better understand the different mechanisms underlying the genesis, clinical manifestations and outcomes between left- and right-sided hemicolon cancers. Materials and Methods
C
olon cancer is one of the most common causes of cancerrelated deaths worldwide. In the United States alone, an estimated 101 340 new cases and 49 380 deaths due to colon cancer were recorded in 2011.(1) The molecular mechanisms of carcinogenesis and colon cancer progression are therefore of major clinical importance. With the splenic flexure as the boundary, the colon is anatomically divided into left and right sides. The left-sided colon includes the splenic flexure, descending and sigmoid colons, while the right side includes the ileocecal junction, ascending and transverse colons. Existing evidence indicates that leftsided colon cancer (LSCC) differs importantly from right-sided colon cancer (RSCC) in terms of epidemiology, risk factors, clinical manifestations, and metastatic characteristics. The prevalence of colon cancer location (i.e. left or right) changes with age in both sexes. Older patients are more likely to have RSCC, which grow larger than left-sided lesions.(2) Also while males have a higher prevalence of tumors, women tend to present with right-sided tumor development.(3) Konopke et al.(4) reported that most liver metastases of the right hemicolon were located in the right lobe, while left colonic carcinomas tended to spread homogeneously to both liver lobes. Right-sided colon cancer (RSCC) also shows fewer adenomatous remnants than LSCC.(5) RSCC is typically exophytic and mainly follows an adenoma-carcinoma pattern.(6) The malignant transformation of familial adenomatous polyposis doi: 10.1111/cas.12029 © 2012 Japanese Cancer Association
Patients and tissue preparation. To screen differently expressed protein markers in a 2-DE/MS assay, fresh surgical specimens from seven cases of left-sided and seven cases of right-sided colon adenocarcinoma (Suppl. Table S1) were randomly collected from Xiangya Hospital, Central South University, China. For tissue microarray to confirm the significance of suspected protein markers, 100 colon adenocarcinoma cases receiving radical resection were randomly collected and divided into LSCC and RSCC groups with 50 cases in each group. For tissue microarray, 10% formalin fixed and paraffin embedded colon cancer specimens were used. The research protocol and methods are detailed in the Supplementary Patients and Methods. IPG-2-DE and image analysis. A total of 400 lg protein was loaded onto an IPG strip for first-dimensional isoelectric focusing (IEF). Protein separation in the second dimension (SDS-PAGE) was carried out following the manufacturer’s instructions (Bio-Rad Laboratories, Hercules, CA, USA). The silver nitrate-stained gels were scanned by an Imagescanner (Amersham Biosciences, Piscataway, NJ, USA) and PD-
5 To whom correspondence should be addressed. E-mail:
[email protected]
Cancer Sci | January 2013 | vol. 104 | no. 1 | 135–141
QuestTM 7.3.1 (Bio-Rad Laboratories) software was used for the image analysis.(8) In-gel trypsin digestion of target protein. The differential spots between LSCC and RSCC groups were excised from the gels and digested as previously prescribed.(8) MALDI-TOF-MS. The extracted and eluted tryptic peptide mixture was analyzed using a Voyager-DETM STR BiospectrometryTM Workstation System 4307 (Applied Biosystems, Foster City, CA, USA) in positive ion-reflector mode. A list of the corrected mass peaks was the output of peptide mass fingerprinting (PMF).(9) Protein identification and database analysis. Protein identification using PMF was performed by protein query MASCOT software (http://www.marrixscience.com) against the nonredundant protein databases of SwissProt (Version 57.11). The searching parameters and the criteria for positive identification of proteins were set up as previously described.(9) Reverse transcriptase polymerase chain reaction. Total RNA was isolated from the fresh tumor tissue, and the synthesized cDNA was amplified by PCR using an Advantage PCR Kit (BD Biosciences, Palo Alto, CA, USA) as previously prescribed.(10) The specific primers for human EEF1D (eukaryotic translation elongation factor 1 delta), heat shock protein beta-1 (heat shock protein-27, HSP-27), CH60 (heat shock protein-60, HSP-60), PDIA1 (protein disulfide-isomerase A1), and the housekeeping gene of GAPDH are listed in Table S2. Western blot. Fifty micrograms of total protein extracted from tumor tissue was separated on 10% SDS-polyacrylamide gels and the transferred membranes were incubated with rabbit polyclonal antibodies against EEF1D, HSP-27, HSP-60, and PDIA1 (Abcam, Cambridge, MA, USA), followed by a subsequent incubation with the secondary antibody of HRP-conjugated sheep anti-rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Bands were visualized by using the ECL kit (Santa Cruz).(10) Tissue microarray and immunohistochemistry. The tumor fragments were sampled from the dense tumor areas and tissue microarray was performed using an ATA-27 Automated Arrayer (Beecher Instruments, Sun Prairie, WI, USA). The 5-lm slides were deparaffinized with xylene and ethanol using the DAKO EnVision System (Dako Diagnostics, Zug, Switzerland). Antigen retrieval was carried out by pressure-cooking in 6.5 mM citrate buffer and endogenous peroxidase activity was blocked with 2.5% hydrogen peroxide.(9) Following incubation with the primary antibody against the target protein, peroxidase labeled polymer and substrate-chromogen were then applied to visualize the protein.(8) Following hematoxylin counterstaining, tumor specimens were scored in a semiquantitative manner as previously described.(11) Statistical analysis. Chi-squared tests were performed for numerical variables and t-tests were applied for measurement variables using StatView (Version 5.0) software (SAS Institute, Cary, NC, USA). The differences with P-values < 0.05 were considered to be significant. Results Proteome differential expression between LSCC and RSCC. The 2-D gels displayed 881 ± 48 and 890 ± 64 protein spots in LSCC and RSCC groups, respectively. In accordance with the screening standard of | score(d) | 2 (the fold change of protein expression 2), the image comparison identified 55 proteins expressed differently between LSCC and RSCC gels in five parallel experiments. The 55 ‘differential spots’ were excised from the gels and digested by TPCK-trypsin for PMF using MALDI-TOF-MS. Based on a comprehensive comparison of matched mass number, sequence coverage rates and protein scores for protein
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matching, 16 non-redundant proteins were finally verified to be differentially expressed between LSCC and RSCC groups (Fig. 1). Compared with the expressed proteins in fresh RSCC tissue, LSCC tissue exhibited 10 highly-expressed proteins including protein disulfide-isomerase A1 (PDIA1, Fig. 2D), 78 kDa glucose-regulated protein, HSP-60 (Fig. 2C), thioredoxin domain-containing protein-5, prohibitin, t-complex protein 1 subunit epsilon, HSP-70, isocitrate dehydrogenase, protein disulfide-isomerase A3, and macrophage-capping protein, as well as six lowly-expressed proteins including ATP synthase subunit beta, elongation factor 1-delta (EEF1D, Fig. 2A), heat shock protein beta-1 (HSP-27, Fig. 2B), apolipoprotein A-I, transthyretin, and heat shock protein beta-6 (Table 1). Confirmation of differentially expressed mRNA. Based on biological function and involvement in cancer genesis and development, three proteins having the highest MASCOT scores (EEF1D, HSP-60, and PDIA1) as well as HSP-27 were selected for further investigation. As shown in Figure 3 (A–D), RT-PCR demonstrated that mRNA expression of HSP-27 and EEF1D was higher, while that of HSP-60 and PDIA1 was lower in RSCC as compared to LSCC tumor tissues. Respective mRNA expression of HSP27, EEF1D, HSP-60, and PDIA1 in the LSCC group was 0.60, 0.47, 2.70, and 2.48 times that in RSCC tissue (Fig. 3A). Differences in mRNA expression levels between LSCC and RSCC groups were statistically significant in all cases (P < 0.05). Confirmation of differentially expressed proteins. Western blotting confirmed our MALDI-TOF-MS findings that protein expression of EEF1D and HSP-27 was higher in RSCC, while that of HSP-60 and PDIA1 was higher in fresh LSCC tissue. As shown in Fig. 4 (A–D), the respective expression levels of EEF1D, HSP-27, HSP-60, and PDIA1 proteins in LSCC tissue was 0.53, 0.48, 2.04, and 3.06 times that in RSCC tissue. Consistent with the significant differences in mRNA expression, these differences in protein expression between LSCC and RSCC groups were also statistically significant (Fig. 3B; P < 0.05). Immunohistochemistry analysis in LSCC and RSCC. Figure 4 depicts a representative example of immunohistochemistry staining following tissue microarray of each suspected protein for both LSCC and RSCC samples. Staining for all four proteins of interest was mainly located in the cytoplasm. The respective percentages of those cases with positive expression of EEF1D, HSP-27, HSP-60, or PDIA1 was 48% (24/50), 28% (14/50), 72% (36/50), and 76% (38/50) in the LSCC group, and 68% (34/50), 60% (30/50), 42% (21/50), and 56% (28/50) in the RSCC group. Statistical analysis demonstrated that positive protein expression rates of EEF1D and HSP-27 were significantly higher in RSCC, while that of HSP-60 and PADI1 were significantly higher in LSCC (Table 2, P < 0.05). Discussion
With the application of proteomic techniques combined with other molecular information platforms, scientists attempt to establish a ‘biosignature’ profile of human cancers for classifying tumors into distinct, clinically relevant subtypes and predicting prognoses.(12) In recent years, proteomics has been applied to discover novel biomarkers for screening, early detection, diagnosis, and prognosis of colorectal cancer (CRC).(13–15) The frequency of BRAF mutation and microsatellite instability high phenotype (MSI-H) are significantly higher in proximal colon cancers. There is a significant difference in the regional expression of 10 tumor-associated markers (CDX2, CD44v6, CD44s, TOPK, nuclear beta-catenin, pERK, APAF-1, E-cadherin, p21 and bcl2) and four immune response markers (CD68, CD163, FoxP3 and TIA-1).(16) p53 doi: 10.1111/cas.12029 © 2012 Japanese Cancer Association
(A)
(B)
(C)
(D)
Fig. 1. The upper figures are representative 2-DE maps of human left- and right-sided colon cancers from five independent experiments. The numbered spots represent proteins differentially expressed more than two times between left-sided colon cancer (LSCC) and right-sided colon cancer (RSCC) groups. The lower one is peptide mass fingerprinting (PMF) of protein spots numbered 12, 13, 3, and 1 on 2-D gels, which correspond to EEF1D (A), HSP-27 (B), HSP-60 (C), and PDIA1 (D) respectively. HSP-60 and PDIA1 are highly expressed in LSCC, while EEF1D and HSP-27 are highly expressed in RSCC (results from five independent experiments) (MASCOT scores are 92, 132, 110, and 65, P < 0.05). Ions score is -10*Log(P), where P is the probability that the observed match is a random event.
overexpression is found more often in RSCC than in cases of LSCC.(17,18) Immunological analysis of 35 nuclear protein preparations indicated expression of p36 antigen in nine of 11 right-sided (81.8%) and 21 of 24 (87.5%) left-sided colorectal tumor cases, but not in any control tissue samples.(19) Proteomic techniques can yield volumes of biological information that are not easily managed by a single laboratory. Also, variability stemming from different patient populations and methodological inconsistencies between laboratories may contribute to variation in reported findings. So, further investigations and a global proteomic profile explaining the significantly different phenotypes of LSCC and RSCC will benefit the scientists and clinicians. To further define the molecular mechanisms involved in the different manifestations of location-associated colon tumors, we identified 16 meaningful and non-redundant protein biomarkers differentially expressed between LSCC and RSCC. Proteins associated with cellular energy metabolism, protein folding/unfolding and oxidative stress–particularly a group of HSP family members–are the most important markers of the differing generative and developmental mechanisms that distinguish LSCC and RSCC. This is consistent with previous findings that demonstrate a correlation between energy metabolism, abnormal antioxidant status and colorectal cancer.(20) Therefore, there is a need for large-scale, appropriately controlled multicenter study of these promising candidate Shen et al.
markers in order to develop a more detailed understanding of the involvement of each protein in tumorigenesis, progression and prognosis in LSCC and RSCC. Interestingly, all 10 highly expressed proteins in LSCC – PDIA1, PDIA3, 78 kDa glucose-regulated protein (GRP78), HSP-60, thioredoxin domain-containing protein 5 (TXNDC5), prohibitin (PHB), T-complex protein 1 subunit epsilon (TCP1-e), HSP-70, isocitrate dehydrogenase (IDH) subunit alpha, and macrophage-capping protein (CapG)–are molecular chaperones involved in the regulation of cellular metabolism, protein folding/unfolding, and oxidative stress. Protein disulfide is an enzyme in the eukaryotic endoplasmic reticulum (ER) that catalyzes the formation and breakage of disulfide bonds between cysteine residues during protein folding. The oxidoreductase behavior of PDI impedes fold maturation of endoplasmic reticulum-processed proteins in the pivotal structure-coupled step of oxidative folding.(21) It was found that PDI was overexpressed in gastric cancer(22) and was related to the invasive properties of malignant glioma.(23) Moreover, PDI was reported to be associated with the proliferation and differentiation of the CRC cell line Caco-2.(24) Our data indicate that two PDI subtypes of PDIA1 and PDIA3 are highly expressed in LSCC, although their particular roles in LSCC are as yet unknown. TXNDC5 is induced by hypoxia and protects hypoxic cells from apoptosis. It has a protein disulphide isomeraseCancer Sci | January 2013 | vol. 104 | no. 1 | 137 © 2012 Japanese Cancer Association
(A)
(B)
(C)
(D)
like domain, which plays an important role in protein folding, chaperone activity, and protection against oxidative stress induced ER stress. A proteomic study demonstrated 138
Fig. 2. mRNA expression of EEF1D, HSP-27, HSP60, and PDIA1 in fresh left-sided colon cancer (LSCC) and right-sided colon cancer (RSCC) tissues identified by reverse transcription-polymerase chain reaction (RT-PCR) (A,B). Samples of No. 1–7 represent LSCC tissue and that of No. 8–14 represent RSCC tissue. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is used as the internal loading control. Indicated by the optical density value (OD) of the electrophoresis band and calibrated by GAPDH mRNA expression, the relative mRNA expression level of each interested gene is represented in the paired bar graph on the right side, which demonstrates that the mRNA expression levels of EEF1D and HSP-27 are higher in RSCC tissues, while that of HSP-60 and PDIA1 are higher in LSCC group. Protein expression of EEF1D, HSP-27, HSP-60, and PDIA1 in fresh LSCC and RSCC tissues identified by Western blotting (C,D). Samples of No. 1–7 represent LSCC tissues and that of No. 8–14 represent RSCC tissues. Calibrated by the internal loading control of GAPDH, the paired bar graph on the right side indicates the relative protein expression level of each suspected gene. The Western gel, representative of five independent experiments, demonstrates that the protein expression of EEF1D and HSP-27 are higher in RSCC tissues, while that of HSP-60 and PDIA1 are higher in the LSCC group.
that TXNDC5 was significantly upregulated in colorectal adenoma and cancer tissues as compared with that in normal mucosa. Therefore, our finding that TXNDC5 was doi: 10.1111/cas.12029 © 2012 Japanese Cancer Association
Table 1. Differentially expressed proteins between two groups of left-sided colon carcinoma (LSCC) and right-sided colon carcinoma (RSCC) No. Accession
Mr (Da)
1 PDIA1_HUMAN 2 GRP78_HUMAN 3 CH60_HUMAN 4 TXND5_HUMAN 5 PHB_HUMAN 6 TCPE_HUMAN 7 GRP75_HUMAN 8 IDH3A_HUMAN 9 PDIA3_HUMAN 10 CAPG_HUMAN 11 ATPB_HUMAN 12 EEF1D_HUMAN 13 HSPB1_HUMAN 14 APOA1_HUMAN 15 TTHY_HUMAN 16 HSPB6_HUMAN
57480 72402 61187 48283 29843 60089 73920 40022 57146 38784 56525 31217 22826 30759 15991 17182
pI value 4.76 5.07 5.7 5.63 5.57 5.45 5.87 6.47 5.98 5.88 5.26 4.9 5.98 5.56 5.52 5.95
Sequence Coverage (%) 43 35 25 28 40 49 28 12 52 20 60 40 29 38 73 69
Protein Score
Matched/ mass no
Not matched/ mass no.
Description
132 184 92 108 110 153 136 72 190 69 222 110 65 64 125 122
15 19 11 10 9 20 20 5 27 6 19 9 5 9 9 9
15 7 7 9 7 17 13 0 33 1 9 10 5 28 10 11
Protein disulfide - isomerase A1 78 kDa glucose-regulated protein Heat shock protein 60 Thioredoxin domain-containing protein 5 Prohibitin T-complex protein 1 subunit epsilon Stress-70 protein, mitochondrial Isocitrate dehydrogenase subunit alpha Protein disulfide-isomerase A3 Macrophage-capping protein ATP synthase subunit beta Elongation factor 1-delta Heat shock protein beta-1, HSP-27 Apolipoprotein A-1 Transthyretin Heat shock protein beta-6
As compared to RSCC tissue, 1–10 refers to the highly expressed proteins in LSCC, 11–16 refers to the lowly-expressed proteins in LSCC. pI represents isoelectric point. The protein score, which is calculated by the Mascot search engine for each protein matched from the MS peak list, is calculated on the probability that peptide mass matches are non-random events. Protein Score = 10Log P.
(A)
(B)
Fig. 4. Immunohistochemical detection of EEF1D, HSP-27, HSP-60, and PDIA1 in left-sided colon cancer (LSCC) and right-sided colon cancer (RSCC) tissues. The expression of all four target proteins stained in colon glandular epithelium is increased in colon cancer tissue (T) than that in the paired adjacent non-tumor tissue (NT) ( 9 400 magnification).
Fig. 3. Statistical analysis of mRNA and protein expression levels. The optical densities (ODs) of the targeted electrophoresis bands on reverse transcription-polymerase chain reaction (RT-PCR) and Western Blotting gels were calibrated by glyceraldehyde 3-phosphate dehydrogenase (GAPDH) ODs and statistically compared. “h” and “&” represents the experimental groups of LSCC and RSCC, respectively. “*” indicates statistical significance between left-sided colon cancer (LSCC) and right-sided colon cancer (RSCC) groups (P < 0.05). Error bars represent standard error of means.
unregulated in cancerous tissue further implicates endogenous TXNDC5 overexpression in the early development of CRC.(25) Shen et al.
Prohibitin and TCP-1-e are two molecular chaperones highly expressed in LSCC. The former is conserved evolutionarily and mainly present on mitochondrial membranes, and is essential for metabolism, mitochondrial function, cell proliferation and development. The absence of PHB leads to increased generation of reactive oxygen species (ROS), disorganized mitochondrial nucleoids, abnormal cristae morphology and an increased sensitivity towards stimuli-elicited apoptosis.(26) Chen et al.(27) found an association between PHB overexpression and the transformation from adenoma to CRC, implicating PHB as a potential diagnostic and differentiation biomarker of CRC. In an ATP-dependent manner, TCP-1-e assists the folding of actin and tubulin proteins upon ATP hydrolysis. It is documented that TCP-1-e is overexpressed in CRC and is correlated with its progression.(28) Isocitrate dehydrogenase plays a crucial role in lipid metabolism, glucose sensing, and in protection against reactive oxygen Cancer Sci | January 2013 | vol. 104 | no. 1 | 139 © 2012 Japanese Cancer Association
Table 2. Positive protein expression rates of EEF1D, HSP-27, HSP-60, and PDIA1 in left-sided colon carcinoma (LSCC) and right-sided colon carcinoma (RSCC) EEF1D Group LSCC RSCC v2 value P-value
HSP-27
HSP-60
PDIA1
Case 50 50
N
P
Rate
N
P
Rate
N
P
Rate
N
P
Rate
26 16
24 34 10.390 0.001
48% 68%
36 20
14 30 4.150 0.043
28% 60%
14 29
36 21 9.180 0.002
72% 42%
12 22
38 28 4.456 0.035
76% 56%
v2 and P-values represent the statistic analysis results between LSCC and RSCC groups. N, negative protein expression; P, positive protein expression. Rate indicates the percentage of the cases with positive expression of target protein.
species generated during lipid oxidation and other processes. Isocitrate dehydrogenase deficiency leads to increased lipid peroxidation, oxidative DNA damage, intracellular peroxide generation, and decreased survival after oxidant exposure. Cellular IDH levels are associated with protection from apoptosis after exposure to ROS or singlet oxygen species and with protection from cell death. IDH1 Arg132 mutations have been identified in CRC.(29) CapG may bind DNA to regulate cytoplasmic and/or nuclear structures through potential interactions with actin. It was reported that the expressions of CapG could be significantly induced by hypoxia in cancer cells.(30) Heat shock proteins (HSPs) are a class of functionally related proteins involved in protein–protein interactions such as folding/unfolding, the establishment of proper protein conformation and prevention of unwanted protein aggregation. Their expression is induced by a range of environmental and pathophysiological stimuli, such as increased temperature and oxidative stress.(31) Evidence indicating the importance of HSPs in colorectal tumorigenesis is increasing. HSP-60 and HSP-70 are overexpressed during colorectal carcinogenesis,(32,33) and are significantly correlated with low tumoral differentiation, pathohistological characteristics,(34) and CRC metastasis.(35,36) As a molecular chaperone required for endoplasmic reticulum integrity and stress-induced autophagy, GRP78 is a member of the HSP family and plays a central role in regulating: the unfolded protein response, protein binding and bridging, as well as anti-apoptosis. GRP78 promotes tumor proliferation, metastasis, and resistance to a wide variety of therapies. An elevated GRP78 level generally correlates with higher pathologic grade, recurrence, and poor patient survival in human colon and gastric cancers.(37) Two smaller HSPs of HSP-27 and HSP-b6 were highly expressed in RSCC, while three larger HSPs of HSP-60, HSP70 and GRP78 were highly expressed in LSCC. This finding suggests that the HSPs in different molecular weights may play various roles in the development of LSCC and RSCC. HSP-27 enhanced the tumorigenicity of immunogenic rat colon carcinoma cell clones(38) and elevated incidence of lymph node metastasis in CRC.(8) Due to the potent effects of HSPs on chemosensitivity, the differentially expressed HSPs between LSCC and RSCC suggest that colon cancer at different locations may have distinct sensitivities to the same chemotherapy regimen. The other four highly expressed proteins in RSCC were: ATP synthase subunit beta (ATPases-b), EEF1D, apolipoprotein A-I (Apo A1), and transthyretin (TTR). ATPases-b is an important mitochondrial membrane-bound enzyme that provides energy for cells through the synthesis of adenosine triphosphate (ATP): required for cellular energy provision and for efficient execution of apoptosis. It has been shown that in colon carcinomas, mitochondrial cellular activity is limited by the selective repression of ATPases-b gene expression. Moreover,
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the metabolic state of the cells provided a bioenergetic signature of carcinogenesis and prognostic value in colorectal carcinomas.(39) ATPases-b expression was found to be lower in 5Fluorouracil-resistant cells with a decreased ATP synthase activity, which might lead to cellular events responsible for 5FU resistance.(40) EEF1D is responsible for the enzymatic delivery of aminoacyl tRNAs to the ribosome and functions as guanine nucleotide exchange factor. A higher expression of EEF1D in tumor tissue suggests that malignant transformation in vivo requires an increase in translation factor mRNA and protein synthesis for entry into and transition through the cell cycle.(40) In human esophagus cancer and primary hepatocarcinoma, the expression of EEF1D was upregulated in tumor tissue and has been shown to be correlated with the lymph node metastases, advanced stage, poorer prognosis, and tumor differentiation.(41,42) The differential expression of EEF1D reported here, suggests that gene transcription regulated by EEF1D may contribute to the different proteomic profiles of LSCC and RSCC. Apo A1, a major protein component of high density lipoprotein (HDL) in the blood, has a major role in lipid metabolism, specifically the clearance cholesterol. Chylomicrons secreted from the intestinal enterocyte also contain ApoA1, but it is quickly transferred to HDL in the bloodstream. A previous proteomic study has implicated Apo A1 as a marker of colon cancer progression.(43) Transthyretin is a serum and cerebrospinal fluid carrier of the thyroid hormone thyroxine and retinol. The concentrations of TTR in the blood more closely reflect recent dietary intake as opposed to overall nutritional status. Transthyretin serum concentration has been identified as an independent marker capable of distinguishing control subjects from colorectal adenoma and colorectal cancer patients. Moreover, TTR had been patented as a biomarker for colorectal adenoma and/or carcinoma.(44) In conclusion, human colon tumors at various locations have different proteomic biomarkers. Differentially expressed proteins associated with energy metabolism, protein folding/ unfolding and oxidative stress contribute to different tumorigenesis, tumor progression, and prognostic outcomes between left- and right-sided colon cancer. Acknowledgements This study was supported by the grants from the Natural Science Foundation of China (No: 30770971, 30872463, 81070362, and 81172470), the grants from Natural Science Foundation of Hunan Province (No: 06JJ4119 & 11JJ2049), a grant from the Department of Science and Technology of Hunan Province (No: 2009JT1052).
Disclosure Statement The authors have no conflicts of interest.
doi: 10.1111/cas.12029 © 2012 Japanese Cancer Association
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Supporting Information Additional Supporting Information may be found in the online version of this article: Table S1. Summary of the clinicopathological features of 14 colon cancer cases in the screening cohort. Table S2. The primers and reaction conditions of regular RT-PCR. Data S1. Supplementary Patients and Methods.
Shen et al.
Cancer Sci | January 2013 | vol. 104 | no. 1 | 141 © 2012 Japanese Cancer Association
