A Controversial Tumor Marker: Is SM22 a Proper Biomarker for Gastric Cancer Cells? Na Li,†,‡ Jun Zhang,†,‡ Yumei Liang,§ Jianmin Shao,†,‡ Fuli Peng,†,‡ Maomao Sun,†,‡ Ningzhi Xu,†,‡ Xianghong Li,§ Rong Wang,*,†,‡,| Siqi Liu,*,†,‡ and Youyong Lu*,†,‡,⊥ Beijing Genomics Institute, Chinese Academy of Sciences, Beijing Airport Industrial Zone B-6, Shunyi, Beijing 101318, Peking University School of Oncology, Beijing Institute for Cancer Research, 52 Fucheng Road, Haidian District, Beijing 100036, Beijing Proteomics Institute, Beijing Airport Industrial Zone B-6, Shunyi, Beijing 101318, and Department of Pathology, General Hospital of PLA, 28 Fuxing Road, Beijing 100853, China, and Department of Genetics and Genomic Sciences, Mount Sinai School of Medicine, New York 10029 Received April 25, 2007
SM22, a dominant protein in smooth muscle cells (SMCs), has been widely reported to be abnormally expressed in many solid tumors. However, the expression patterns of SM22 are not consistent in all tumors, not even in the same ones. Whether SM22 should be considered a tumor biomarker is still debated in different laboratories. Herein, we have carried out a systematical investigation to validate SM22 expression in the primary tissues of gastric cancer (GC). Of eight cases, seven samples were found in the elevated expression of SM22 proteins through proteomic analysis. The observation was further verified by the approaches of Western blotting and quantitative RT-PCR. Surprisingly, the results achieved from tissue microarray in 126 GC cases appeared contrary to the proteomic conclusion, in which the highly expressed SM22 was mainly found in smooth muscle layers, blood vessels, and myofibroblasts. This suggested that the increased abundance of SM22 in the cancerous regions was not caused by the presence of the GC cells. Furthermore, the expression of SM22 was measured in different GC cell lines and SMCs with Western blotting and quantitative RT-PCR. The results revealed that SM22 expression in SMCs was dramatically higher than that of the GC cells, which indicates that SM22 is unlikely to be a proper biomarker for GC. Instead, it can be considered a potential indicator for the abnormal developments of smooth muscles, blood vessels, or myofibroblasts triggered by tumorigenesis. Keywords: gastric cancer • biomarker • SM22 • proteomics • tissue microarray
Introduction Gastric cancer (GC), one of the most commonly diagnosed cancers, is the second leading cause of cancer-related death in the world. More than 700000 people, approximately 42% patients in China, suffer from this disease every year.1-3 Like many other cancers, the mortality caused by GC is closely correlated with its development stages. With greater public awareness and earlier diagnostic intervention, the incidence and mortality of GC have declined gradually over the past several decades, particularly in developed countries. In the early stage of GC, 5 year survival rates can reach over 90%. However, due to the expansion of the aging population worldwide, it is predicted that GC will still be considered as a life-threatening * To whom correspondence should be addressed. Phone: +86-1080485325. Fax: +86-10- 80485324. E-mail:
[email protected] (Y.L.),
[email protected] (S.L.),
[email protected] (R.W.). † Chinese Academy of Sciences. ‡ Beijing Proteomics Institute. § General Hospital of PLA. | Mount Sinai School of Medicine. ⊥ Beijing Institute for Cancer Research.
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disease in the next few decades. The early diagnosis for GC with greatly improved sensitivity and accuracy is necessitated in clinical practice. Although endoscopic evaluation is viewed as a gold standard in the diagnosis of GC, it is an expensive and invasive technique. The endoscopic mucosal resection is also limited to lesions located at the gastric cardia or lesser curvature. In comparison, diagnostic techniques based on molecular biology are not only more effective but also much less invasive. A great effort has been made during the past decade to better define biological characteristics of GC. Using comparative genomic hybridization, Wu et al. analyzed the genetic imbalances in 62 primary gastric adenocarcinomas and found changes in the DNA copy number in 84% of those samples.4 Serial analysis of gene expression (SAGE) is a powerful means to explore new biomarkers of GC. After SAGE on 46 cases of GC, Oue et al. defined 27 genes and tags potentially involved in carcinogenesis, invasion, and metastasis.5 Even though these potential biomarkers are not of clinical relevance yet, they could still contribute to our understanding of the GC biological features. 10.1021/pr0702363 CCC: $37.00
2007 American Chemical Society
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Is SM22 a Proper Biomarker for Gastric Cancer?
It is well-known that proteins representing the preponderance of biologically active molecules corresponding with most cellular functions and the measurements of protein expression and modification may directly indicate the status of the disease. Recently, proteomic screening has been widely employed to discover cancer biomarkers, including GC. Using differential proteomic approaches, several investigators have discovered many cancer-related candidates that were not detected by other approaches. SM22 is one of them.6-17 This protein, also called transgelin, is a 22 kDa protein and has different isoforms.18-21 SM22 is abundantly expressed in all the tissues containing smooth muscle cells (SMCs), while its biological and physiological functions remain unclear. Through subtractive hybridization, Genini et al. first suggested that SM22 might have an important role in determining or maintaining the normal phenotype in human primary myoblasts. They also suggested that low expression of SM22 was possibly involved in the progression of malignancy.22 Furthermore, Shields et al. found that Ras was involved in the down-regulation of SM22 in human breast and colon carcinoma cell lines.23 Proteomic evidence revealed the abnormal expression of SM22 protein in varied cancer cells and tissues. For instance, Qi et al. reported that SM22 was up-regulated in esophageal squamous cell carcinoma,7 whereas Yeo et al. observed that SM22 expression was significantly reduced in human colon cancer.12 Intriguingly, SM22 was also found up-regulated in GC.9 However, assessment of SM22 as a new cancer biomarker is still a controversial issue, because several queries regarding SM22 expression and distribution in cells or tissues have not been clarified. The patterns for SM22 expression seem inconsistent with tumor variety. Even for the same cancer, different laboratories produced conflicting results. Friedman et al. utilized the technique of two-dimensional difference gel electrophoresis (2D-DIGE) to analyze six samples of colon cancer and found 2.5-fold up-regulated SM22 in the patient tissues.10 After analysis of 27 cases of colon cancer by using the same electrophoresis but with silver staining, Stulik et al. concluded a significantly decreased expression of SM22 in the patients.11 Moreover, Mazzanti et al. did not detect abnormal expression of SM22 in eight pairs of tissues of colon cancer.13 Were these diverse data derived from the tissue sources, the sample preparations, the differential determination, or the protein identification? At an early time, SM22 was considered as a unique gene expressed in SMCs. Lawson et al. systematically investigated SM22 expression in various tissues and confirmed that SM22 could express in most tissues even at different abundances.18 The tumor tissues are composed of complex cells, some of which may contribute more to the expression of SM22. For example, most vascular systems contain rich SMCs except the monolayer capillary vessel. Thus, in analyzing SM22 expression in a tissue, an issue should be carefully addressed, which cells significantly contribute to SM22 abundances in the tissues. In those previous documents, the cell sources of SM22 expression in the tumor tissues were not evidently clarified even though some laboratories employed Western blotting or immunohistochemistry (IHC) to identify SM22 in tissues. To answer the questions above, a systematical investigation of SM22 is necessitated with three distinct principles in proteomic analysis: (1) to detect its expression levels with differently valid approaches; (2) to enlarge the sample cases for the statistical estimation of SM22 expression; (3) to distinguish the expression levels of SM22 in different cells in certain tissues. In this study, GC was selected as a target to be screened
for SM22 expression. First, we used 2-DE and MS techniques to analyze eight pairs of tissues from GC patients and to ensure whether SM22 proteins were differently expressed in the GC tissues and their adjacent regions. The proteomic results were further confirmed by Western blotting and quantitative RTPCR. Then we applied the tissue microarray (TMA) technique to screen 126 cases of GC and 31 cases of adjacent regions with monoclonal anti-SM22 antibody. On the basis of the image data of IHC staining, we evaluated the SM22 expression in the different regions of gastric tissues. To extend the investigation, the SM22 expression in the tissues of esophagus and colon cancer were also evaluated with IHC. Finally, the expression of SM22 was determined in different gastric cell lines and smooth muscle cells with Western blotting. After a thorough analysis of the collected data, we came to the conclusion that SM22 is unlikely to be a proper biomarker for GC, but it could be a potential indicator for angiogenesis and myofibroblasts in the tumor tissues.
Materials and Methods Tissue Specimens. Tissue specimens were collected following the informed consent of all the patients and approval of the local research ethical committee. For proteomic analysis, eight pairs of gastric cancer and adjacent non-neoplastic tissues were provided by Beijing Cancer Hospital & Institute. Each pair of samples was obtained from the same GC patient. All the samples were carefully examined by pathological biopsy. The stages of GC progress were defined according to pathological classification of the International Union Against Cancer (UICC) modified with TNM methodology. These samples were frozen in liquid nitrogen within 30 min after surgery until they were used for the experiments. All the samples used for TMA were collected from Beijing Cancer Hospital & Institute. Prior to IHC treatment, the samples were formalin fixed and H&E counterstained. All the samples of gastric, esophagus, or colon cancer were examined by the senior pathologist. Cell Culture. Two GC cell lines were used in this study, BGC823 collected and provided by the Peking University School of Oncology and AGS from ATCC. Both cell lines were originated from gastric adenocarcinoma and cultured with DMEM containing 5% fetal bovine serum (FBS) or 1640 containing 10% FBS, respectively. The primary cells of rat smooth muscle in the aorta were a gift kindly presented by Dr. Xian Wang of the Peking University Health Science Center, which were cultured in DMEM containing 10% FBS. Sample Preparation for Protein Extraction. The TCAacetone precipitation method was adopted in the sample preparation of gastric tissues. Briefly, the frozen samples were crushed to a fine powder by a metal mortar in liquid nitrogen. The fine powder was suspended in precooled 10% TCA in acetone containing 10 mM DTT followed by sonication on ice for 5 min and incubation at -20 °C for 45 min. The precipitated proteins were obtained by centrifugation at 35000g at 4 °C for 15 min and then, after being washed with precooled acetone, dissolved in the lysis buffer containing 8 M urea, 4% CHAPS, 0.5% ampholyte (3-10NL), 20 mM Tris, pH 8.5, 2 mM EDTA, 1 mM PMSF, 60 mM DTT, and protease inhibitor cocktail. After sonication on ice for 5 min and centrifugation at 40000g for 30 min, the resulting supernatants were directly delivered for 2-DE or Western blotting. The protein concentrations were estimated by the Bradford method. Journal of Proteome Research • Vol. 6, No. 8, 2007 3305
research articles To extract the proteins from the cultured cells, the harvested cells were washed with PBS buffer and subsequently dissolved with lysis buffer followed by sonication with a probe sonicator for 5 min. After centrifugation at 40000g for 30 min and quantification of protein concentrations, the supernatants were directly delivered for Westerm blotting analysis. Two-Dimensional Electrophoresis. The extracted proteins were separated by 2-DE according to the manufacturer’s instructions. The isoelectric focusing steps were carried out with 18 cm (pH 3-10NL) at 20 °C. The prepared proteins (200 µg/gel) were mixed with rehydration buffer to 350 µL/strip. The strips were rehydrated without voltage for 4 h and with 50 V for 8 h. Then isoelectric focusing was performed at 50 kV h programmed in the gradient mode in an IPGphor. The strips with the focused proteins were subsequently treated with DTT reduction and iodoacetamide (IAM) alkylation in equilibrated buffer containing 6 M urea, 30% glycerol, 2% SDS, and 50 mM Tris-HCl, pH 8.8. The equilibrated strips were transferred onto 12% polyacrylamide gels, and SDS-PAGE was carried out at 15 W/gel in Ettan DALT II. The 2-DE spots were visualized by silver staining without addition of glutaraldehyde. The stained gels were scanned using an Amersham Imagescanner, and TIFF images were imported into an Amersham ImageMaster Platinum for image analysis. Protein Identification by MALDI TOF/TOF MS. The significantly differential 2-DE spots between GC portions and their adjacent regions were excised and transferred into Eppendorf tubes. The gel particles were treated by reduction of DTT and alkylation of IAM followed by thorough washing and drying with subsequent incubation in 25 mM NH4HCO3, 50% ACN, and 100% ACN, respectively. The dried particles were rehydrated in 2 µL of digestion solution containing trypsin (10 ng/ µL) with 25 mM NH4HCO3 on ice for 30 min, and then 8 µL of 25 mM NH4HCO3 was added followed by incubation overnight at 37 °C. The digested peptides from 2-DE gel spots were analyzed by a Bruker Ultraflex MALDI TOF/TOF mass spectrometer (Bremen, Germany). The tryptic digests were cocrystallized with a matrix of CHCA and spotted on the target wells. The signals of peptide mass fingerprinting (PMF) were acquired in the reflectron mode in the m/z range from 600 to 4000. Calibration was performed externally using standard peptide mixtures and internally using the peptide fragments of trypsin autolysis products. On the basis of the PMF signals, the three strongest peptides with higher accuracy and higher abundance were further analyzed in the MS/MS mode. The MS/MS data were exported in a suitable format and submitted to the database for searching for proteins with MASCOT software (Matrix Science, London, U.K.). The latest version of the human protein database in the NCBInr databases was used in the protein search. A mass accuracy tolerance was allowed within 100 ppm. It was taken into account for a positive identification, such as the significant peaks in the MS spectra matching with the theoretical values of the peptide masses, the region percentage of the amino acid sequence covered by the matched peptides, the MS/MS ion score, the number of missed cleavages, and the values of the predicted MW and pI. Western Blotting. Samples of 10 µg of the extracted proteins from tissues or cells were subjected to 12% SDS-PAGE at 20 mA until the bromophenol blue dye reached the bottom of the gels and electrotransferred onto PVDF membranes at 350 mA for 1 h. The PVDF membranes were incubated in blocking buffer containing 5% milk powder and 0.1% Tween 20 at room 3306
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temperature for 2 h. SM22 protein was recognized by a specific polyclonal antibody that was a gift from Dr. Julian Solway. The secondary antibody of goat IgG anti-mouse IgG conjugated with peroxidase was used to pick up the primary antibody. The immune-recognition results were visualized with an ECL kit (Amersham Biosciences, Uppsala, Sweden) according to the manufacturer’s instructions. The expression of GAPDH (Santa Cruz, CA) or β-actin (Santa Cruz, CA) was used to normalize the protein loading. Quantitative RT-PCR. Total RNAs were isolated from eight pairs of human gastric tissues, rat smooth muscle cells, and two GC cell lines. The first-strand cDNAs were synthesized by reverse transcriptase (Invitrogen, California) using the total RNAs as the templates. Quantification of gene expression of SM22 and GAPDH was conducted with an ABI PRISM 7300 system (Foster City, CA). The primers were designed to span the exon-intron junctions of the genes. PCR was carried out with programmed parameters, heating at 95 °C for 3 min followed by 40 cycles of a four-stage temperature profile of 95 °C for 15 s, 60 °C for 20 s, 72 °C for 15 s, and 80 °C for 30 s, to collect the fluorescent signals. The melting curves for each PCR reaction were carefully analyzed to avoid nonspecific amplifications in the PCR products. The SM22 expression of each sample was transformed using the ∆∆Ct formula and normalized with GAPDH expression. Immunohistochemistry. Using a monoclonal anti-human SM22 antibody generated in our laboratory, immunohistochemical staining of SM22 was performed via two approaches, TMA and whole section slides. To construct TMA, tissue cylinders with a diameter of 0.6 mm and a height of 1 mm were then punched from selected areas of each donor tissue block and brought into a recipient paraffin block using a custom-made precision instrument (Beecher Instruments, Silver Springs, MD). There was 1 mm between two adjacent tissue cylinders, and each sample was duplicated once. Sections of the resulting TMA block (4 µm) were transferred to glass slides and baked at 60 °C overnight. To create whole section slides, a representative set of paraffin blocks were selected that contained well-matched cancer and adjacent non-neoplastic tissue. In both approaches, H&E-stained sections were used for histological verification. Prior to immunostaining, the slides were deparaffinized with xylene and rehydrated in graded alcohol rinses. The antigen retrieval was performed by microwave oven heating with 1 mM EDTA, pH 8.0. Endogenous peroxidase activity was blocked by incubation of 3% H2O2 at room temperature for 10 m. The slides were incubated in 3% milk for 2 h, and immunostaining was performed with a mouse anti-SM22 monoclonal antibody at 1:100 at 4 °C overnight in a moist chamber. Following a thorough wash with PBS, the slides were incubated with antimouse antibody conjugated with peroxidase. After three washes with PBS, immunostaining was developed with a commercial DAB kit (Beijing Zhongshan Golden Bridge Biotechnology, China), yielding a brown-colored signal. The sections were counterstained with hematoxylin, dehydrated, and mounted. Double Fluorescent IHC Staining of SM22 and r-Actin. The paraffin-embedded samples for GC were examined for the protein expression of SM22 and R-actin. The preparation of tissue slides was the same as above. These slides were incubated with goat serum for 30 min and incubated at 4 °C overnight in the blocking solution containing 1:100 mouse antihuman R-actin antibody (Zymed, San Francisco, CA) and 1:400 rabbit anti-human SM22 antibody. Before analysis with a
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Is SM22 a Proper Biomarker for Gastric Cancer?
found with significant differences in spot volume between the tumor tissues and their adjacent portions after normalization. A total of 51 differential spots were excised and digested by trypsin and subjected to MALDI TOF/TOF MS, resulting in 37 spots identified as proteins. Of these identifications, 28 were unique, including 19 up-regulated and 9 down-regulated. The relevant information is summarized in Tables 1 and 2. Several proteins identified were documented to be carcinogenic candidates, such as CK8 and HSP27 (up-regulated) and CA11 and AMP-18 (down-regulated), which are summarized in Supporting Information Table 1. Figure 1. Representative 2-DE images of the proteins extracted from the gastric tissues. (A) GC tissues (T). (B) Adjacent regions (N).
fluorescence microscope, the slides were incubated with the blocking solution containing FITC-labeled goat anti-mouse IgG and TRITC-labeled goat anti-rabbit IgG (Jackson Immuno Research, Pennsylvania) in a dark room for 1 h. The fluorescent signals were monitored under an Olympus IX 70 inverted fluorescence microscope.
Results Proteomic Analysis of Differentially Expressed Proteins in the GC Tissues. The proteins extracted from the samples of GC portions and their adjacent regions were well resolved by 2-DE. The representative images of 2-DE are depicted in Figure 1. The quantitative comparison of the 2-DE spots for each pair of samples was conducted with a combination of software and a manual check. Although the distribution patterns of the 2-DE spots in these samples were quite close, several spots were
Identification of SM22 in Gastric Tissues. In the 2-DE images, a string of spots around pH 8.0-9.0 and a molecular mass of 22 kDa were significantly up-regulated in the GC tissues (seven out of eight pairs). The comparison of close-up 2-DE images is shown in Figure 2A. On average, the normalized spot volumes of these spots in GC were 10.99 ( 5.13-fold higher than that of adjacent non-neoplastic regions. These spots were all identified as SM22 by MALDI TOF/TOF MS. Totally, 14 mass signals matched well with the theoretical mass values of tryptic fragments of SM22, covering 59 ( 3% of the amino acid sequence of this protein. A typical MALDI TOF/TOF mass spectrum derived from the SM22 peptides is illustrated in Figure 2B. Another string of 2-DE spots with a molecular mass of 18 kDa was also identified as SM22 by MALDI TOF/TOF MS. However, this SM22 isoform exhibited an insignificant difference between GC tissues and their adjacent regions. Validation of SM22 Protein Expression by Western Blotting. To validate the changes of SM22 expression in GC, Western
Table 1. Up-Regulated Proteins in the GC Tissues Determined by 2-DE and Mass Spectrometry accession no.
description
score
sequence coverage (%)
MW
pI
fold difference
gi|386959 gi|1942639 gi|6573280 gi|179987 gi|5360679 gi|10880933 gi|61679604 gi|2492759 gi|2506903 gi|11275302 gi|662841 gi|2340833 gi|5542166 gi|4389275 gi|999893 gi|55956899 gi|181573 gi|350170 gi|1890020
migration inhibitory factor-related protein 8 (S100 A8) human lithostathine β-tropomyosin chlordecone reductase anti-Entamoeba histolytica immunoglobulin κ light chain prostate cancer susceptibility protein human hemoglobin 3-hydroxyacyl-CoA dehydrogenase type-2 (type II HADH) ES1 protein homolog, mitochondrial precursor anti-TNF-R antibody light-chain Fab fragment heat shock protein 27 SM22 R human platelet profilin human serum albumin triosephosphate isomerase (Tim) keratin 9 cytokeratin 8 histone H2B mutant keratin 9
145 107 81 78 73 67 144 155 92 67 142 146 193 105 275 171 163 79 76
74 63 28 40 50 16 90 80 59 37 42 62 78 25 77 44 42 44 55
10885 16607 29980 35640 23576 93441 15834 27134 28467 23787 22427 22653 15014 67988 26807 62178 53529 13767 25913
6.51 5.66 4.70 7.03 8.26 8.13 6.76 7.66 8.50 6.19 7.83 8.87 8.46 5.69 6.51 5.14 5.52 10.32 4.46
∞ ∞ ∞ ∞ ∞ ∞ 29.32 25.44 8.92 8.92 5.37 4.50 3.45 2.91 2.84 2.52 2.00 1.62 1.52
Table 2. Down-Regulated Proteins in the GC Tissues Determined by 2-DE and Mass Spectrometry accession no.
description
score
sequence coverage (%)
MW
pI
fold difference
gi|443135 gi|94538357 gi|7229101 gi|18999392 gi|15679996 gi|16975162 gi|296653 gi|4758038 gi|135773
carbonic anhydrase Ii mutant 18 kDa antrum mucosa protein CA11 protein, down-regulated in gastric cancer cytochrome c oxidase subunit Va unknown (protein for IMAGE:3934797) peroxiredoxin 5 Hp2-R cytochrome c oxidase subunit Va precursor thioredoxin (SASP)
148 74 72 70 84 196 79 81 81
49 27 25 28 39 87 20 26 80
28776 20546 22270 16923 23322 17060 42126 16938 12015
6.63 5.65 5.90 6.30 8.00 6.96 6.25 6.30 4.82
-∞ -∞ -∞ 0.14 0.16 0.22 0.24 0.41 0.46
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Figure 2. Identification of SM22 proteins in the GC tissues. (A) Comparison of close-up images for the 2-DE spots corresponding to the differentical SM22 expression in eight pairs of samples of the GC tissues and their adjacent regions. (B) MALDI-TOF/TOF mass spectra of SM22. Inset: A parent ion of 1210.5278 was selected for MS/MS analysis, and the amino acid sequence, REELEEDYK, was confirmed with the mass signals of b-ions and y-ions.
ization by the expression of GAPDH, the relative intensities of immunorecognition of SM22 were increased infinitely in the cancer tissues versus their adjacent regions. Western blotting on 2-DE gels was employed to identify the isoforms of SM22. The 2-DE patterns recognized by anti-SM22 antibody were quite similar to the distribution of 2-DE spots identified by MS (Figure 3B), indicating that only the 22 kDa SM22 isoform displayed the significant increases of spot intensities in the cancer tissues. Hence, Western blotting data were very consistent with the proteomic observations.
Figure 3. Differential expression of SM22 in the GC tissues identified by Western blotting and quantitative RT-PCR. (A) Identification of SM22 in three typical pairs of samples of GC tissues with Western blotting. GAPDH was used as an internal control. (B) Two isoforms of SM22 were identified by 2-DE (top panel) and 2-DE-Western blotting (bottom panel). (C) Distribution of relative abundances of SM22 mRNA among eight pairs of GC tissues. The Ct value of the SM22 gene was normalized by that of GAPDH. Data were analyzed according to relative gene expression by the 2-∆∆Ct method.
blotting with polyclonal antibody against SM22 was used to check the protein expression of SM22 in these clinical samples. As depicted in Figure 3A, which represents the results of Western blotting from three pairs of tissues, SM22 proteins at 22 kDa were found significantly up-regulated in the cancer tissues as compared with their adjacent regions. With normal3308
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mRNA Expression of SM22 in Gastric Tissues. The mRNA expression of SM22 in the gastric tissues was determined by quantitative RT-PCR. As shown in Figure 3C, the relative abundance of SM22 mRNA was normalized by GAPDH. All the samples contained a detectable amount of SM22 mRNA. There was a higher level of SM22 mRNA in six cases of the GC tissues as compared with their corresponding adjacent tissues. The statistical estimation revealed that SM22 at transcription level was up-regulated in the GC tissues. Protein Expression of SM22 in Gastric Tissues Examined by IHC. Although the data above supported the increased protein expression of SM22 in the tissues of GC, the question was not clearly addressed of where the enhanced expression occurred in vivo. The techniques of IHC were employed to further investigate in which tissue layer(s) SM22 was specifically expressed. First of all, 157 samples of the gastric tissues were loaded on the slides of TMA, and the expression abundance of SM22 was recognized by monoclonal anti-SM22 antibody. Similar to the observations of proteomics and Western blotting, many regions with positive staining recognized by SM22
Is SM22 a Proper Biomarker for Gastric Cancer?
Figure 4. Representative positive images of IHC staining in gastric tissues. (A, B) Smooth muscle fibers in which cancer cells have invaded were strongly positive for SM22. (A) is the cross section and (B) the vertical section. (C) The blood vessel walls showed strong immunoreactivity. (D) The microvessel walls in the tumor stroma showed positive expression of SM22. (E) Myofibroblasts in GC tissues were positive for SM22. (F) Myofibroblasts at periglandular locations in normal appearance gastric mucosa were positive for SM22.
antibody were found in the cancer regions, but a few of them in their adjacent areas, 72.7% of the GC tissues with positive rates of SM22 staining and only 48.9% of the noncancer tissues with positive staining. The statistical estimation with the Kruskal-Wallis nonparametric test revealed a significant difference between the two groups (p ) 0.006). Supporting Information Figure 1 represents the typical IHC images of SM22 in the gastric tissues. Furthermore, on the basis of specific localization of the SM22 stained regions in the gastric tissues, the histological analysis exhibited that this protein could be favorably expressed in some special portions of the stomach. In the 126 GC cases, positive staining of SM22 was not observed in the epithelial cells in the gastric mucosa, which were believed to be a primary place of gastric malignancy. As depicted in Figure 4, the IHC image analysis demonstrated that the heavy staining of SM22 in the tissues was generally distributed in three patterns, in smooth muscles fibers, blood vessel walls, and myofibroblasts. (1) SM22 in smooth muscle fibers (Figure 4A,B): when cancer cells invaded the muscularis propria, the fibers with positive staining of SM22 were surrounded by many malignant cells. (2) SM22 in blood vessel walls (Figure 4C,D): during tumor progression and metastasis, angiogenesis was usually accompanied by fastgrowing malignant cells, in which SM22 was highly expressed in the tumor vessel walls, even the microvessel in the GC stroma in Figure 4D. (3) SM22 in myofibroblasts (Figure 4E): increased myofibroblasts in the GC stroma might facilitate angiogenesis and cancer progression, which also showed strong staining of SM22 in the GC tissues. In some cases of the adjacent non-neoplastic mucosal regions, SM22 was stained at periglandular locations, not in the apical epithelium (Figure 4F). Taking the evidence of these IHC images together, the high abundance of SM22 in the tumor tissues unlikely resulted from
research articles the GC cells, but was contributed by other cells relevant to microvessels and smooth muscle layers. SM22, thus, may not be a specific protein marker tightly associated with the GC cells, and its high expression may partially reflect other cellular events during tumorigenesis. R-Actin is a specific biomarker for SMCs. If the deduction above upon IHC image analysis is correct, SM22 protein is expected to be colocalized with this protein. With double fluorescent IHC staining, the expression of SM22 and R-actin in the gastric tissues were monitored by different fluorescent signals. As shown in Supporting Information Figure 2, the staining regions of R-actin (green) merged well with those of SM22 (red). The two proteins were colocalized in the SMCs of the microvessel walls. The double fluorescent IHC images, hence, provide supportive data for the conclusion drawn from IHC, which attributes the high SM22 expression in the GC tissues to the portions containing SMCs. Analysis of IHC Images of Two Cancers in Human Gastroenterol Tissues. Considering the controversial reports regarding SM22 expression in tumors, two other typical cancer tissues from the esophagus and colon were chosen to identify SM22 abundances in the different tissue regions using IHC with monoclonal anti-SM22 antibody. As shown in Figure 5, no significant staining was found in the epithelia of normal esophagus and colon (Figure 5A,E), whereas the smooth muscle layers in the normal tissues were positively recognized by SM22 antibody (Figure 5E). The images in Figure 5B-D,F-H exhibit the IHC results to examine the tumor tissues of the esophagus and colon. In Figure 5B,F, the cells of esophagus or colon cancer invading the muscularis propria were not recognized by SM22 antibody, but these fibers or tissues were heavily stained. In Figure 5C,G, the blood vessel walls in the tumor tissues, either esophagus or colon, was obviously observed through immunoreaction staining, but not for the cancer cells. In Figure 5D,H, the myofibroblasts in the tissues of esophagus or colon cancer were immunostained, and the cancer cells in the same tissues were not recognized by SM22 antibody. All the IHC patterns were similar to those of GC described above. Moreover, the IHC approach was employed to analyze the slides which represented lung, breast, and ovarian cancer (data not shown). The IHC results led to conclusions similar to those of the GC cases, i.e., which expression of SM22 protein was a specific signal for smooth muscle cells in muscularis propia and blood vessel walls or myofibroblasts, but not for the relevant tumor cells. Comparison of SM22 Expression in SMCs and GC Cell Lines. The gastric tissues consist of many different types of cells. A question should be clearly addressed in this study, whether the expression efficiency of SM22 in SMCs is significantly different from that of other gastric cells. Three cell lines, SMC, AGS, and BGC82, were collected and examined for the expression of SM22 protein using Western blotting. The results shown in Figure 6A present a dramatically enhanced expression of SM22 in SMCs as compared with the other two GC cells. With short exposure in ECL, the density of the immunoreactive band of SM22 in SMCs was almost infinitely different from that of the other two cells. With extensive exposure in ECL, the immunorecognition signals of SM22 in the two GC cells could be detectable even at faint visualization (data not shown). Moreover, the quantitative RT-PCR data were very consistent with the Western blotting results (Figure 6B,C), which showed more than 10000-fold differences between SMCs and the other two GC cells at the SM22 mRNA level. Therefore, it is deducible Journal of Proteome Research • Vol. 6, No. 8, 2007 3309
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Figure 5. IHC images of the esophagus (A-D) and the colon (E-H) tissues using monoclonal antibody against SM22. (A, E) Normal tissues of the esophagus and colon. (B, F) The smooth muscle fibers were invaded by the tumor cells. (C, (G) Blood vessels in the two tumor tissues. (D, H) Myofibroblasts in the two tumor tissues.
Figure 6. Comparison of SM22 gene expression among rat SMCs and the GC cell lines. (A) SM22 proteins in rat SMCs, AGS, and BGC823 were determined with Western blotting. GAPDH was used as an internal control. (B) Agarose gle to check the PCR amplification products of SM22 mRNAs in rat SMCs, AGS, and BGC823 after 40 cycles of RT-PCR. GAPDH was used as an internal control. (C) Quantitative analysis of SM22 mRNAs in rat SMCs, AGS, and BGC823 measurd by quantitative RT-PCR with the ABI PRISM 7300 system.
that the expression of SM22 protein in SMCs is a dominant source in the GC tissues. Obviously, this deduction was highly matched with the experimental observations, IHC and double fluorescent IHC staining, described above.
Discussion With proteomic approaches, the high expression of SM22 protein was found in seven out of eight cases of the GC tissues. The proteomic observation was further validated either by Western blotting or by quantitative RT-PCR. The resulting data seemed to indicate that SM22 was a potential protein marker for GC. As matter of fact, several reports have claimed such discoveries on the basis of similar strategies and data. The experimental results, based upon a large scale of samples in TMA and a careful image analysis of SM22 staining regions, however, reached a contrary conclusion that SM22 might not serve as an ideal protein marker for the GC cells because its overexpression was mainly found in the tissue layers containing rich SMCs. This study, thus, gave a certain answer to the 3310
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questions raised in the introduction and achieved the conclusion that SMCs, but not epithelial cells, were the dominant sources of SM22 expression in gastric tissues. What did we learn from the series of experimental data? First, IHC plus TMA is a feasible and effective approach for validation of proteomic data obtained from the analysis of tissue proteomes. During IHC experiments, an antibody with high specificity and titer is a key element to have an accurate analysis of IHC images. Second, although it is well accepted that sample collection is a critical step in the proteomic analysis for tissues, the experimental protocols are being debated to achieve satisfactory results. For instance, the technique of laser capture microdissection (LCM)24,25 was reported to be more accurate to collect the cancer cells from the tissues. In practice, LCM faces some technical obstructions, in which the quantities of collected cells may be limited for further proteomic analysis, and some invaded tissues are not easily identified and separated. According to the experience in this study, IHC seems a better choice if the sample sizes are not restricted strictly. Third, combination of multiple techniques for proteomic analysis is necessary in exploring the protein biomarkers of tumors. Since there is a lack of a universal technique which favors all the proteomic analysis, advantages and disadvantages for these adopted proteomic techniques must be fully evaluated to avoid misleading conclusions. The experimental evidence collected through this study is helpful in clarifing some confusing data regarding SM22 as a tumor biomarker in other cancers. The images of IHC shown in Figure 6 suggested that in the esophagus and colon cancer samples the abundant SM22 proteins were mainly located at the regions possessing rich SMCs, but not at the tumor layers. As mentioned in the Introduction, the abnormal expression of SM22 protein in colon cancer was noticed by several laboratories. These laboratories performed careful and accurate analysis of tissue proteomes; nevertheless, it merits nothing since these investigators have not employed IHC to check the SM22 distribution in these colorectal tissues. Are the controversial data caused by the varied contents of SMCs in these samples? This investigation definitely addressed one of the answers to this question.
research articles
Is SM22 a Proper Biomarker for Gastric Cancer?
The observation has confirmed that SM22 is not a proper biomarker for GC cells, but its role in gastric tumorigenesis remains inconclusive. SM22 is not only expressed in SMCs but also distributed among other cells. The functions of SM22 in these cells, nevertheless, are poorly elucidated. Fu et al. employed site-directed mutagenesis and demonstrated that several sites located at the C-terminal of SM22 were the deciding factors of affinity association with actin and the stability of the SM22-actin complex.26 Tumor cells usually display irregularities on their surface and contain the disrupted cytoskeletons. Hence, a possible role of SM22 in carcinogenesis is its involvement in skeletal protein networks. On the other hand, SM22 has phosphorylation sites at the C-terminus, and its phosphorylated form could weaken the affinity to actin. Logically, the phosphorylation triggered by signal transduction may exert influences on the functions of SM22. For instance, Kaplan-Albuquerque et al. found there were two mechanisms in which the activity of PI3K and Akt affected SM22 expression.27,28 One mechanism was the phosphorylation inhibition of P38 MAPK and JNK, and the other was the direct phosphorylation of the transcription factor. As described above, a high abundance of SM22 was present in the regions with rich SMCs, such as smooth muscle fibers, blood vessel walls, and myofibroblasts. What are the proper explanantions for this phenomenon? The inner and outer linings of the stomach wall generally consist of four layers of smooth muscle fibers, mucosa, submucosa, muscularis propria, and serosa. Most GC originates in the mucosal layer. As the tumor progresses, those malignant cells are defined as advanced GC (AGC) as they invade the layer of the muscularis propria and beyond. These invading cancer cells are able to penetrate deeply into the smooth muscle fibers, thus making the cell mixtures difficult to be clearly dissected in a proteomic analysis. At this point, IHC provides an accurate way to detect the invasion structure after proteomic analysis. The accumulated evidence indicates that progressive tumor growth is dependent on angiogenesis, which creates the blood supply that speeds growth of the tumor.29-32 Several investigators have demonstrated that tumor angiogenesis is associated with metastasis and prognosis in GC. Maeda et al. reported for 124 GC specimens that increasing microvessel counts correlated with lymph node metastasis, hepatic metastasis, and poor prognosis.29,33 Sometimes the angiogenic phenotypes could be treated as a criterion to distinguish the different histological forms of GC. Except the monolayer capillary vessel, most vascular systems contain rich SMCs; unsurprisingly, heavy staining of SM22 was detected in the angiogenic regions in these gastric tissues. In addition, double fluorescent IHC was employed to examine the colocalization of two SMC-specific proteins, R-actin and SM22; the results revealed that overlapped fluorescence strongly appeared in the microvessels. Thus, it is doubtless that SM22 generated from angiogenesis in GC tissues occupies most of the contribution to the total of this protein. Fibroblasts are mesenchymal cells with many vital functions during development and in adult organisms. Ishizuya-Oka et al. observed that more differentiated fibroblasts tended to be localized closer to the epithelium.34 More intriguingly, a transformation and shape-sensitive actin gelling protein found in fibroblasts has a sequence of amino acids identical with that of SM22, even though this protein did not appear in all types of fibroblasts. The current study of SM22 provided evidence in which the IHC staining became visible in normal gastric tissue (Figure 4), suggesting first that the human gastric
fibroblast was able to express SM22. With the development of genetics and cell biology, researchers began to realize that tumor growth is not only determined by malignant cancer cells but also by the tumor stroma.35-37 Fibroblasts often represent the majority of the stromal cells within various types of human carcinomas, yet the specific contributions of these cells to tumor growth are poorly understood. Nejjari et al. pointed out that the stromal fibroblasts contributed to promotion of fibronectin-mediated local invasion by tumor cells in GC.38 Our observations in this study are in agreement with this point, and heavy SM22 staining was found in these regions with dense cells of the gastric tumor and fibroblasts, indicating that fibroblasts were indeed associated with GC progression. In summary, the diverse data of SM22 protein expression achieved from different tumor tissues or laboratories may partially result from the varied contents of SMCs in the samples. Moreover, on the basis of the current information, we should not excluded the possibility that SM22 is involved in the carcinogenesis of GC. Despite the lack of detailed information regarding molecular mechanisms, the IHC data in this study revealed that the regions with cancer invasion and angiogenesis appeared with positive staining of SM22 in the GC tissues.
Acknowledgment. We thank Dr. Julian Solway (University of Chicago, Illinois) for the generous gift of the antiSM22 polyclonal antibody. This work was supported by grants from the Science and Technology Project in Beijing (D0905001040331), International Partnership Program for Creative Research Teams of the Chinese Academy of Sciences, National Key Basic Research Program Project (2004CB518708), and National Bio-Tech 863 program (2002-BA711A11) of China. Supporting Information Available: Supporting Figures 1 and 2, Table 1, and references. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Parkin, D. M.; Bray, F.; Ferlay, J.; Pisani, P. CA Cancer J. Clin. 2005, 55, 74-108. (2) Crew, K. D.; Neugut, A. I. World J. Gastroenterol. 2006, 12, 354362. (3) Yang, L. World J. Gastroenterol. 2006, 12, 17-20. (4) Wu, C. W.; Chen, G. D.; Fann, C. S.; Lee, A. F.; Chi, C. W.; Liu, J. M.; Weier, U.; Chen, J. Y. Genes, Chromosomes Cancer 2002, 35, 219-231. (5) Oue, N.; Hamai, Y.; Mitani, Y.; Matsumura, S.; Oshimo, Y.; Aung, P. P.; Kuraoka, K.; Nakayama, H.; Yasui, W. Cancer Res. 2004, 64, 2397-2405. (6) Klade, C. S.; Voss, T.; Krystek, E.; Ahorn, H.; Zatloukal, K.; Pummer, K.; Adolf, G. R. Proteomics 2001, 1, 890-898. (7) Qi, Y.; Chiu, J. F.; Wang, L.; Kwong, D. L.; He, Q. Y. Proteomics 2005, 5, 2960-2971. (8) Zhou, G.; Li, H.; Gong, Y.; Zhao, Y.; Cheng, J.; Lee, P. Proteomics 2005, 5, 3814-3821. (9) Ryu, J. W.; Kim, H. J.; Lee, Y. S.; Myong, N. H.; Hwang, C. H.; Lee, G. S.; Yom, H. C. J. Korean Med. Sci. 2003, 18, 505-509. (10) Friedman, D. B.; Hill, S.; Keller, J. W.; Merchant, N. B.; Levy, S. E.; Coffey, R. J.; Caprioli, R. M. Proteomics 2004, 4, 793-811. (11) Stulik, J.; Koupilova, K.; Osterreicher, J.; Knizek, J.; Macela, A.; Bures, J.; Jandik, P.; Langr, F.; Dedic, K.; Jungblut, P. R. Electrophoresis 1999, 20, 3638-3646. (12) Yeo, M.; Kim, D. K.; Park, H. J.; Oh, T. Y.; Kim, J. H.; Cho, S. W.; Paik, Y. K.; Hahm, K. B. Proteomics 2006, 6, 1158-1165. (13) Mazzanti, R.; Solazzo, M.; Fantappie, O.; Elfering, S.; Pantaleo, P.; Bechi, P.; Cianchi, F.; Ettl, A.; Giulivi, C. Am. J. Physiol. Gastrointest. Liver Physiol. 2006, 290, G1329-1338. (14) Sitek, B.; Luttges, J.; Marcus, K.; Kloppel, G.; Schmiegel, W.; Meyer, H. E.; Hahn, S. A.; Stuhler, K. Proteomics 2005, 5, 2665-2679. (15) Kim, J.; Kim, S. H.; Lee, S. U.; Ha, G. H.; Kang, D. G.; Ha, N. Y.; Ahn, J. S.; Cho, H. Y.; Kang, S. J.; Lee, Y. J.; Hong, S. C.; Ha, W. S.; Bae, J. M.; Lee, C. W.; Kim, J. W. Electrophoresis 2002, 23, 41424156.
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