Ubiquitin-specific peptidase 22 overexpression may promote cancer progression and poor prognosis in human gastric carcinoma YAN HE1, YIN-JI JIN1, YU-HUA ZHANG, HONG-XUE MENG, BAO-SHAN ZHAO, YANG JIANG, JI-WEI ZHU, GUAN-YING LIANG, DAN KONG, and XIAO-MING JIN HARBIN AND DAQING, CHINA
Ubiquitin-specific peptidase 22 (USP22) was recently identified as a new tumor cell marker, and previous studies demonstrated its expression in a variety of tumors and its correlation with tumor progression. Because tumor progression plays an important role in cancer, researchers are paying more attention to the correlation between USP22 expression and metastatic potential, resistance to chemotherapy, and patient prognosis. This study showed that USP22 is highly expressed in gastric cancer tissues, and significant differences in USP22 expression (P , 0.01) were identified between different types of gastric cancer (the highest expression was found in poorly differentiated adenocarcinomas). In addition USP22 expression was found to be correlated with the promotion of cancer evolution, tumor invasion, and lymph node metastasis. The C-myc protein was also shown to have synergistic effects with USP22 in gastric cancer tissue. On the basis of the results, USP22 expression may play an important role in gastric carcinoma tissue, particularly in precancerous lesions during the gastric cancer evolution process. (Translational Research 2015;165:407–416) Abbreviations: AH ¼ atypical hyperplasia; DUB ¼ deubiquitinating enzyme; IF ¼ immunofluorescent; IgG ¼ immunoglobulin G; IM ¼ intestinal metaplasia; PL ¼ precancerous lesion; qRT-PCR ¼ quantitative real-time polymerase chain reaction; UCH ¼ ubiquitin carboxy-terminal hydrolase; USP22 ¼ ubiquitin-specific peptidase 22; W-B ¼ Western blot
INTRODUCTION
G
astric carcinoma is the second leading cause of cancer-related deaths in the world. Some epidemiologic evidence suggests that chronic gastritis and intestinal metaplasia (IM) may lead to the development of the precursors of gastric cancer. Deubiquitinating enzymes (DUBs) regulate a number of cellular mechanisms, including preimplantation,
1
Yan He and Yin-Ji Jin contributed equally to this work.
From the Department of Pathology, Harbin Medical University, Harbin, China; Department of Pathology, Harbin Medical University (Daqing), Daqing, China; Department of Forensic Medicine, Harbin Medical University, Harbin, China; Department of Gynecology, Third Affiliated Hospital of Harbin Medical University, Harbin, China. Submitted for publication March 24, 2014; revision submitted August 24, 2014; accepted for publication September 9, 2014.
growth, differentiation, oncogenesis, cell cycle progression, transcriptional activation, and signal transduction.1 Recently, Lee et al2 demonstrated that a novel human DUB gene, ubiquitin-specific protease 22 (USP22), is the core of a multitude of physiological and pathologic processes. USP22 forms part of a polycomb-cancer stem cell signature that uniformly exhibits a marked propensity toward metastatic
*Reprint requests: Xiao-Ming Jin, Department of Pathology, Harbin Medical University, 157 Baojian Road, Nangang District, Harbin, China and Dan kong, Department of Gynecology, Third Affiliated Hospital of Harbin Medical University, Harbin, China; e-mail: [email protected] or [email protected] 1931-5244/$ - see front matter Ó 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.trsl.2014.09.005
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AT A GLANCE COMMENTARY He Y, et al. Background
Gastric carcinoma is the second leading cause of cancer-related deaths in the world. Ubiquitinspecific peptidase 22 (USP22) was recently identified as a new tumor cell marker that may be correlated with cancer metastatic potential, resistance to chemotherapy, and patient prognosis. Translational Significance
Our results indicate that increased USP22 expression is positively correlated with gastric cancer differentiation, infiltration depth, and lymph node metastasis. The C-myc protein was also shown to have synergistic effects with USP22 on gastric cancer tissues. USP22 expression may play an important role in gastric carcinoma tissue, particularly in precancerous lesions during the gastric cancer evolution process. Therefore, USP22 may function as al marker of tumor evolution in the presence of other tumor cell metastatic markers.
dissemination and a therapy-resistance phenotype.3,4 The polycomb genes play an important role in tumor development and occurrence in terms of not only tumor growth but also the potential for cancer metastasis and chemotherapy drug resistance. These genes are closely related to patient prognosis and are therefore considered tumor cell marker genes. USP22 and the C-myc oncogene have a crucial partnership: USP22 is required for Myc-driven transcription. The depletion of USP22 in cells decreases the ability of Myc to activate the transcription of its targets.5-7 USP22 is expressed in some normal human tissues, such as myocardial tissue, skeletal muscle, and the lung, but its expression is significantly upregulated in malignant tumors.2 High USP22 expression often indicates that the tumor is about to spread throughout the body, and this phenomenon is called a ‘‘cancer stem cell signal.’’ However, the relationship between the expression of USP22 and gastric histology has not been previously studied. In the present study, we investigated the protein and messenger RNA (mRNA) expression levels of USP22 and C-myc in different precancerous lesion (PL) tissues through immunofluorescent (IF) staining, Western blot (W-B) analysis, and quantitative real-time polymerase chain reaction (qRT-PCR) and analyzed the correlation between the USP22
and C-myc levels and clinicopathologic features, invasion, metastasis, histologic subtypes, and relevance to prognosis. MATERIALS AND METHODS Patient and tissue samples. A total of 90 gastric adenocarcinoma tissue samples (here in after referred to as 90 cases) were obtained from the surgery department of the Fourth Affiliated Hospital Medical University between October 2007 and December 2010. Each sample contained cancer tissue, adjacent tissues (2.5–3.0 cm away from the cancer tissue), and distant tissue (6.0–8.0 cm away from the cancer tissue), resulting in a total of 270 fresh tissue samples. The specimens were snap-frozen in liquid nitrogen and stored at 280 C. These samples were subjected to qRT-PCR, W-B, and IF staining analyses to assess the USP22 and C-myc transcript abundance and protein expression. All the tissues were also fixed with formalin, paraffin embedded, and prepared for hematoxylin and eosin staining. To further analyze the relationship between the USP22 and C-myc levels and the cancer infiltration depth, additional 150 paraffin-embedded specimens (here in after referred to as 150 cases) were collected from gastric cancer patients undergoing surgery at Hei Long Jiang Province Hospital between October 2007 and December 2010 for immunohistochemistry to assess USP22 and C-myc protein expression and histopathology only. Two histologic analyses (WHO8 and Lauren et al9) criteria were used to evaluate the classification of all samples. None of the patients received preoperative chemotherapy or radiotherapy. This study was approved by the Institutional Review Board of Harbin Medical University Medical Center and received ethical approval from the Ethics Committee. All the subjects provided written informed consent and were assured of their anonymity and the confidentiality of the data. Immunohistochemical and Immunofluorescent analysis. The formalin-fixed, paraffin embedded, 4-mm
thick sections were dewaxed and rehydrated in a graded series of ethanol solutions. The endogenous peroxidase activity was quenched with 3% H2O2 for 15 minutes. After washing with phosphate-buffered saline (PBS), the sections were incubated with an antiUSP22 antibody (1:200, immunoglobulin G [IgG]; Abcam, Cambridge, California) or an anti-C-myc antibody (1:100, Y69, IgG; MaiXin Bio-technology, FZ, China) overnight at 4 C. The sections were incubated with peroxidase-conjugated streptavi-din for 30 minutes, and the reaction products were visualized using diaminobenzidine as a chromogen and counterstained with commercial hematoxylin. Specific
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isotype control PBS (in the absence of the primary antibodies) was used as a negative control. USP22 and C-myc were expressed in the nuclei of gastric cancer cell. An IF analysis of USP22 and C-myc was performed as previously described.10 Frozen 5-mm thick sections from each case were fixed with acetone, and the nonspecific binding was blocked through a 15-minute incubation with secondary antibody animal serum. The sections were incubated with primary antibodies against USP22 (1:200) and C-myc (1:100, 9E10, IgG; Santa Cruz Biotechnology, Dallas, California) overnight at 4 C and then with either Fluorescein isothiocyanateconjugated goat anti-rabbit or horse anti-mouse antibodies (1:200; Vector Laboratories) for 1 hour at room temperature. After washing with PBS, the specimens were counterstained with 40 ,6-diamidino-2phenylindole (Vector Laboratories) and observed with a biological research microscope (E-800; Nikon Eclipse, Tokyo, Japan). No positive staining was observed in the samples that were not incubated with the primary antibody, which served as the negative control. USP22 was expressed in the nuclei of gastric cancer cell, and C-myc was expressed in the cytoplasm. For each sample, 10 random fields were examined at 3400 magnification. To calculate the ratio of positive cells, the positive cells of USP22 and C-myc were counted and the numbers were divided by the total cell number in each field.11 W-B analysis. The total protein contents of the frozen tumor, adjacent, and matched distant tissues were extracted by suspending the sample in a lysis buffer consisting of 20 mM Tris-HCl (pH 7.5), 2 mM EDTA, 150 mM NaCl, 1% Triton X-100, and protease inhibitors. The protein concentration in the supernatant was determined using the Bradford method (Bio-Rad, Hercules, California). Equal amounts of protein were separated by 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto a nitrocellulose membrane. The membranes were then blocked with Tris Buffered Saline with Tween-20 containing 1% bovine serum albumin (Promega, Madison, Wisconsin) overnight and incubated with a rabbit anti-human USP22 (66 kDa) polyclonal antibody (1:500; Abcam) or a mouse anti-human C-myc (55 kDa) mono-clonal antibody (1:300; Santa Cruz Biotechnology) for 2 hours at 37 C. After washing with Tris Buffered Saline with Tween-20, the membrane was incubated with a secondary antibody against rabbit or mouse IgG (antimouse IgG, DyLight 680; anti-rabbit IgG, DyLight 800; Cell Signaling, Boston) at a 1:10,000 dilution for 1 hour. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Santa Cruz Biotechnology) was used as an internal control. The proteins were visualized using an
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infrared imaging system (Odyssey; LI-COR Bioscience, Ltd, Kent, UK). Quantitative real-time polymerase chain reaction. The total RNA from the frozen tissue specimens was extracted with TRIzol reagent (Invitrogen, Carlsbad, California) and RNeasy columns, which were used according to the manufacturers’ recommendations. The first-strand complementary DNA synthesis was performed with 2 mg of total RNA using HighCapacity Complementary DNA Reverse Transcription Kits (Applied Biosystems, Foster City, California). The qRT-PCR analysis was performed on the ABI PRISM 7000 Sequence Detection System using the TaqMan1 Universal PCR Master Mix (Applied Biosystems) and the TaqMan1 Gene Expression Assays probe and primer mix (Applied Biosystems) according to the manufacturer’s specifications. The assay identification numbers were as follows: USP22, Hs00392758_m1; GAPDH, Hs02758991_g1. The human GAPDH gene was used as an endogenous control. All the reactions were performed in triplicate. The relative mRNA levels were calculated based on the Ct values and corrected with the GAPDH expression level using the following equation: 2-DCt [DCt Ct (USP22) -Ct (GAPDH)]. Statistical analysis. All results were analyzed using a 2-tailed Student t test (adjusted for SAS Institute Inc., Cary, North Carolina). In all the analyses, P , 0.05 was deemed significant.
RESULTS Patient characteristics. All fresh frozen samples (270) from the 90 cases and 150 paraffin-embedded samples were examined histologically to confirm the diagnosis of gastric cancer. Forty-three of the 90 patients contained atypical hyperplasia (AH) or intestinal metaplasia (IM) features in the tissue adjacent to the cancer tissue. Commonly, AH and IM were both considered as PLs. The adjacent tissues were considered as AH if following change displayed: AH glands are covered with crowded and distended cells, the nuclei arrayed as pseudostratified layers with scarce mucus or without mucus. Mucosa cells exhibit considerable pleomorphism and often possess hyperchromatic nuclei that are abnormally large for the size of the cell. Mitotic figures are more abundant than usual. Frequently the mitoses appear in abnormal locations within the epithelium.12 The noninvolved epithelium showed normal gastric histology. Therefore, only the 43 cases that contained cancer, PL (AH/IM) locations and noninvolved epithelium (normal gastric mucosa) were used to evaluate the
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Fig 1. USP22 expression in 90 fresh cases. (A–C) Expression of USP22 was observed by immunofluorescent assay. Dotted or granular staining was observed in the cancer cell nucleus, part of the duct, and a few scattered cancer cells. (D and E) The expression of USP22 in various differentiated types, metastasis, and nonmetastasis groups of gastric adenocarcinomas. (F) The expression of USP22 was observed in various differentiated types of gastric cancer by W-B assay. (G) The messenger RNA level of USP22 was observed by quantitative real-time polymerase chain reaction. *P , 0.05, **P , 0.01. DAPI, 40 ,6-diamidino-2-phenylindole; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; H&E, hematoxylin and eosin; Well, well-differentiated gastric adenocarcinomas; Moderate, moderately differentiated gastric adenocarcinomas; Poor, poorly differentiated gastric adenocarcinomas; USP22, ubiquitin-specific peptidase 22; W-B, Western blot.
relationship of expression of USP22, C-myc vs the different locations of the samples. Increased expression of USP22 in gastric cancer. The USP22 staining was localized within the nuclei of gastric cancer cells. USP22 was expressed in 63.37% of the fresh gastric cancer tissues and in 54.46% of the paraffin-embedded gastric cancer tissues. The expression level of USP22 was higher in the poorly differentiated gastric adenocarcinomas than in the well-differentiated adenocarcinomas (P , 0.05; Figs 1, A–D and 3, C and E, Table I). In addition, USP22 level was higher in the diffuse type of gastric adenocarcinomas than in the intestinal type (P , 0.01; Table I). The W-B and qRT-PCR
assays revealed that USP22 was highly expressed in the poorly differentiated gastric adenocarcinomas (P , 0.05; Fig 1, F and G, Table I). We also found no significant correlation between the USP22 mRNA and protein levels and the tested clinicopathologic parameters, which included the patient’s sex, age, and tumor size. However, the expression of USP22 was found to be correlated with differentiation, infiltration depth, and lymph node metastasis of the gastric cancer samples (P , 0.05; Figs 1, E and 3, F and G, Table I). The gastric adenocarcinoma groups of the poorly differentiated histology type, the deeper infiltration, and lymph node metastasis were inclined to express more USP22.
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Fig 2. C-myc expression in 90 fresh cases. (A–C) Expression of C-myc was observed by immunofluorescent assay. C-myc is visible in part of the gland cell cytoplasm and scattered throughout the cancer cell cytoplasm and nuclear membrane. (D and E) The expression of C-myc in various differentiated types and groups of metastasis and nonmetastasis. (F) The C-myc expression in various differentiated types by Western blot assay. * P , 0.05, **P , 0.01. Well, well-differentiated gastric adenocarcinomas; Moderate, moderately differentiated gastric adenocarcinomas; Poor, poorly differentiated gastric adenocarcinomas; DAPI, 40 ,6-diamidino-2phenylindole; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Increased expression of C-myc in gastric cancer.
C-myc was primarily expressed in the cytoplasm (fresh gastric cancer tissues) or the nuclei of gastric cancer cells (paraffin-embedded cancer tissues). In addition, C-myc was found to be expressed in 50.63% of the fresh gastric cancer tissues and in 42.30% of the paraffin-embedded gastric cancer tissues. The expression of C-myc was found higher in the group of poorly differentiated gastric adenocarcinomas than in the group of well-differentiated adenocarcinoma (P , 0.05; Figs 2, A–D and 3, D and H, Table I). In addition, it was found higher in the diffuse type than in the intestinal type (Table I). The W-B analysis revealed that C-myc was highly expressed in the poorly differentiated gastric adenocarcinomas (Fig 2, F). We also found no significant correlation between the C-myc protein levels and the tested clinico-
pathologic parameters, which included the patient’s sex, age, tumor size, and infiltration depth. However, C-myc expression was found correlated with differentiation and lymph node metastasis (Figs 2, E and 3, I, Table I). The gastric adenocarcinoma groups of poorly differentiated and lymph node metastasis were inclined to express more C-myc. Increased expression of USP22 and C-myc in 43 fresh gastric cancer cases. Forty-three of the 90 fresh gastric
cancer cases were confirmed to be gastric cancer with AH or IM (PL) and exhibited normal gastric mucous membranes (noninvolved gastric mucosa, normal) in the transition area. The USP22 and C-myc levels in the 43 patients progressively decreased in different parts (cancer tissue, PL, and noninvolved gastric mucosa) (Fig 4, A and B). USP22 and C-myc were found high in PL and noninvolved mucosa tissue adjacent to
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Fig 3. USP22 and C-myc expressions in 150 paraffin-embedded gastric tissues. (A and B) Morphology of representative gastric carcinoma tissue. Hematoxylin and eosin staining: (A) magnification, 3100, (B) magnification, 3400. (C and D) Immunohistochemistry results (magnification, 3400). (E–G) The expression of USP22 in various histology types, infiltrate depth, metastasis, and nonmetastasis groups. (H and I) The expression of C-myc in various histology types, metastasis, and nonmetastasis groups. There was no statistical difference in various infiltrate depth groups for the expression of C-myc.*P , 0.05, **P , 0.01. Well, well-differentiated gastric adenocarcinomas; Moderate, moderately differentiated gastric adenocarcinomas; Poor, poorly differentiated gastric adenocarcinomas; USP22, ubiquitin-specific peptidase 22.
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Table I. Positive cell percentage of USP22 and C-myc with clinical parameters USP22 (%)
Variable
Sex Male (n 5 68 in 90 cases; n 5 95 in 150 cases) Female (n 5 22 in 90 cases; n 5 55 in 150 cases) Age #55/59 (45/90; 77/150) .55/59 (45/90; 73/150) Tumor grade Well-differentiated (18/90; 4/150) Moderately differentiated (24/90; 38/150) Poorly differentiated (48/90; 108/150) Lauren type Intestinal type (49/90; 63/150) Diffuse type (41/90; 87/150) Infiltration M (4/90; 8/150) SM (2/90; 13/150) MP (18/90; 24/150) SE (66/90; 105/150) Metastasis (lymph node) Yes (73/90; 96/150) No (17/90; 54/150)
C-myc (%)
n 5 90
n 5 150
n 5 90
n 5 150
Mean 6 SD
Mean 6 SD
Mean 6 SD
Mean 6 SD
64.69 6 19.05 62.05 6 22.14
54.88 6 12.69 54.04 6 12.26
51.96 6 19.16 49.32 6 21.25
43.64 6 12.58 40.96 6 13.81
63.22 6 19.86 64.87 6 19.84
63.95 6 13.06 55.23 6 11.94
50.82 6 19.56 51.80 6 19.8
42.52 6 12.91 42.81 6 13.32
43.39 6 17.49*,† 58.25 6 14.03*,‡ 74.69 6 15.36*,§
27.00 6 9.20*,† 44.68 6 11.08*,‡ 59.07 6 9.44*,§
31.28 6 17.42*,† 45.04 6 14.48*,‡ 61.96 6 15.02*,§
19.50 6 5.26*,† 31.13 6 10.69*,‡ 47.57 6 10.29*,§
54.65 6 18.69*,i 75.27 6 14.56
48.62 6 14.31*,i 58.59 6 8.86
41.80 6 18.66*,i 62.69 6 13.91
36.13 6 13.86*,i 47.39 6 10.18
56.75 6 24.24{,# 71.00 6 1.41{,** 65.28 6 18.93 63.94 6 20.21
45.25 6 18.45{,# 48.00 6 18.61{,** 52.88 6 12.42 56.49 6 10.51
44.50 6 23.56 60.00 6 7.07 52.44 6 19.62 51.15 6 19.83
41.75 6 13.71 39.08 6 15.83 38.17 6 14.28 44.20 6 12.22
66.95 6 18.11{ 51.59 6 22.18
56.73 6 10.15{ 50.74 6 15.21
54.19 6 18.17{ 38.94 6 21.23
44.10 6 11.70{ 40.09 6 14.98
Abbreviations: M, mucosa; MP, muscularis propria; SD, standard deviation; SE, serosa; SM, submucosa. *P ＜ 0.01. † For the comparison of well-differentiated vs moderately differentiated. ‡ For the comparison of moderately vs poorly differentiated. § For the comparison of well-differentiated vs poorly differentiated. i For the comparison of intestinal vs diffuse type. { P ＜ 0.05. # For the comparison of M vs SE. **For the comparison of SM vs SE.
cancer tissues, which were usually diagnosed as poor differentiated adenocarcinoma (Fig 4, C and D). In contrast, low or no USP22/C-myc expression was found in noninvolved gastric mucosa when the tumor was diagnosed as well-differentiated adenocarcinoma. The expression of the 2 proteins was positively correlated. Correlations were observed between USP22
and C-myc expression and the clinicopathologic data. First, the USP22 and C-myc expression levels in the gastric cancer tissues were positively correlated (Fig 5, A and B). In addition, the USP22 and C-myc expression levels and the WHO and Lauren’s histologic classifications of gastric cancer were compared and related to the clinical and pathologic data shown in Table I. DISCUSSION
Sequence analysis has revealed that USP22, a novel DUB, belongs to a large family of proteins with ubiquitin hydrolase activity. C-myc is a nuclear oncogene that functions in protein phosphorylation and exhibits spe-
cific DNA binding, thereby affecting multiple aspects of cell growth, differentiation, apoptosis, and cell cycle progression.13,14 C-myc has dual functions in cells, that is, it stimulates cell proliferation and promotes cell apoptosis. The C-myc protein expression increases significantly in cancerous cells, making the cell exempt from the limits of normal growth and giving the cell a malignant phenotype.15 USP22 is required for the activation of target gene transcription by Myc. Most importantly, the depletion of USP22 compromises Myc function, including transformation. Consistent with its critical role in cell cycle progression, USP22 depletion results in a specific G1 arrest. Previous studies have suggested that the C-myc gene and the tumorsuppressor gene p53 have an important impact on the transcriptional activation activity of Spt–Ada–Gcn5– acetyltransferase.16,17 The USP22-Myc partnership6 is present in most tumor cells. Because the Myc protein controls the expression of thousands of other genes, the depletion of cellular USP22 can inhibit Myc function, inhibiting the invasive growth of cancer cells.
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Fig 4. (A and B) The correlation of the expression of USP22 and C-myc between the locations of tumor, PL, and noninvolved gastric epithelium (normal) in 43 cases. The expression of USP22 and C-myc decreased by the order of tumor, PL, and noninvolved gastric mucosa. *P , 0.05. (C and D) Categorized by the various differentiated histology types, the expressions of USP22 and C-myc were analyzed in various locations of 43 gastric cancer samples. *P , 0.05, **P , 0.01. Well, well-diferentiated gastric adenoarcinoas; Moderate, moderately differentiated gastric adenocarcinomas; Poor, poorly differentiated gastric adenocarcinomas; PL, precancerous lesion; Poor, USP22, ubiquitin-specific peptidase 22.
Fig 5. Relevance of USP22 and C-myc expression. (A) The positive expression of USP22 and C-myc in 90 fresh gastric cancer samples. (B) The relevance of USP22 and C-myc expression is shown (r 5 0.971, P 5 0.00, P , 0.01). Well, well-differentiated gastric adenocarcinomas; Moderate, moderately differentiated gastric adenocarcinomas; Poor, poorly differentiated gastric adenocarcinomas; USP22, ubiquitin-specific peptidase 22.
Thus, the Myc-mediated gene transcription process requires USP22, and USP22 completely controls its transcription. The present study found that USP22 expression is significantly higher in poorly differentiated adenocarcinoma (P , 0.01) and that the promotion of cancer evolution, gastric cancer invasion depth, and gastric cancer
lymph node metastasis are correlated with USP22 expression. The USP22 level progressively increased from pericarcinomatous tissue to cancer tissue. Some researchers18,19 have shown that the USP22 expression increases significantly from normal mucosa to carcinomas, which suggests that USP22 activation increases during colorectal and bladder
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carcinogenesis. USP22 expression and colon cancer metastasis to the liver are correlated with poor prognosis.19 We demonstrated that USP22 is highly expressed in normal mucosa tissue adjacent to cancer tissue, which were usually diagnosed as poorly differentiated adenocarcinoma. In contrast, low or no USP22 expression was found in the normal mucosa when the tumor was diagnosed as well-differentiated adenocarcinoma. This may indicate that USP22 played a role in the process of precancerous gastric tissue progress into the cancer. In addition, USP22 may play a direct functional role in the regulation of cell differentiation. USP22 depletion causes cells to accumulate in the G1 phase of the cell cycle and results in concomitant decreases in the percentage of cells in the synthesis and second gap/ mitosis phases.20-23 A high risk of gastric cancer could be predicted if USP22 is highly expressed in the PLs. These results are consistent with the results obtained by other researchers in different tumor tissues, such as colorectal cancer, bladder cancer, and papillary thyroid carcinoma.19,24-27 The examination of both C-myc and USP22 expression revealed that USP22 and C-myc have a synergistic effect on gastric cancer tissues. Thus, USP22 may be a new cancer stem cell marker worthy of further study. The USP22 gene encodes a cancer-related protein that can control the expression of a large variety of genes; however, USP22 is also very vulnerable to the effects of some drugs. McMahon6 suggested that USP22 may become a potential novel target for cancer drugs. Because of the role of USP22 in cell cycle regulation in tumor cells, the design of an USP22-specific inhibitory factor will likely be valuable for cancer treatment. The discovery of USP22 inhibitors using the findings obtained through experimental research and early clinical applications will likely lead to improvements in cancer research. Therefore, efforts to identify more efficient molecular markers for the detection and prognosis of gastric cancer are of great clinical importance. ACKNOWLEDGMENTS
Conflicts of Interest: The authors have read the journal’s policy on disclosure of potential conflicts of interest and have none to declare. This work was supported by grants from the National Natural Science Foundation of China (no. 81101735), grants from the Program for New Century Excellent Talents in Heilongjiang Provincial University (1253– NCET–015), grants from the Harbin Scientific and Technological Talents Research Program (2012RFLX S013).This work was supported by the Heilongjiang Provincial Science and Technology Innovation Team
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in Higher Education Institutes for Infection and Immunity, Harbin Medical University. The authors thank Elsevier Language Editing Services from Elsevier Ltd for excellent editing of this manuscript. All authors have read the journal’s authorship agreement and agree with the statements. REFERENCES
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22. Reyes-Turcu FE, Ventii KH, Wilkinson KD. Regulation and cellular roles of ubiquitin-specific deubiquitinating enzymes. Annu Rev Biochem 2009;78:363–97. 23. Nijman SM, Luna-Vargas MP, Velds A, et al. A genomic and functional inventory of deubiquitinating enzymes. Cell 2005;123: 773–86. 24. Liu YL, Yang YM, Xu H, Dong XS. Increased expression of ubiquitin-specific protease 22 can promote cancer progression and predict therapy failure in human colorectal cancer. J Gastroenterol Hepatol 2010;25:1800–5. 25. Lv L, Xiao XY, Gu ZH, et al. Silencing USP22 by asymmetric structure of interfering RNA inhibits proliferation and induces cell cycle arrest in bladder cancer cells. Mol Cell Biochem 2011;346:11–21. 26. Schrecengost RS, Dean JL, Dedeurwaerder S, et al. USP22 regulates oncogenic signaling pathways to drive lethal cancer progression. Cancer Res 2014;74:272–85. 27. Wang H, Li YP, Chen JH, et al. Prognostic significance of USP22 as an oncogene in papillary thyroid carcinoma. Tumor Biol 2013; 34:1635–9.
Ubiquitin-specific peptidase 22 (USP22) was recently identified as a new tumor cell marker, and previous studies demonstrated its expression in a variety of tumors and its correlation with tumor progression. Because tumor progression plays an important role in cancer, researchers are paying more attention to the correlation between USP22 expression and metastatic potential, resistance to chemotherapy, and patient prognosis. This study showed that USP22 is highly expressed in gastric cancer tissues, and significant differences in USP22 expression (P , 0.01) were identified between different types of gastric cancer (the highest expression was found in poorly differentiated adenocarcinomas). In addition USP22 expression was found to be correlated with the promotion of cancer evolution, tumor invasion, and lymph node metastasis. The C-myc protein was also shown to have synergistic effects with USP22 in gastric cancer tissue. On the basis of the results, USP22 expression may play an important role in gastric carcinoma tissue, particularly in precancerous lesions during the gastric cancer evolution process. (Translational Research 2015;165:407–416) Abbreviations: AH ¼ atypical hyperplasia; DUB ¼ deubiquitinating enzyme; IF ¼ immunofluorescent; IgG ¼ immunoglobulin G; IM ¼ intestinal metaplasia; PL ¼ precancerous lesion; qRT-PCR ¼ quantitative real-time polymerase chain reaction; UCH ¼ ubiquitin carboxy-terminal hydrolase; USP22 ¼ ubiquitin-specific peptidase 22; W-B ¼ Western blot
INTRODUCTION
G
astric carcinoma is the second leading cause of cancer-related deaths in the world. Some epidemiologic evidence suggests that chronic gastritis and intestinal metaplasia (IM) may lead to the development of the precursors of gastric cancer. Deubiquitinating enzymes (DUBs) regulate a number of cellular mechanisms, including preimplantation,
1
Yan He and Yin-Ji Jin contributed equally to this work.
From the Department of Pathology, Harbin Medical University, Harbin, China; Department of Pathology, Harbin Medical University (Daqing), Daqing, China; Department of Forensic Medicine, Harbin Medical University, Harbin, China; Department of Gynecology, Third Affiliated Hospital of Harbin Medical University, Harbin, China. Submitted for publication March 24, 2014; revision submitted August 24, 2014; accepted for publication September 9, 2014.
growth, differentiation, oncogenesis, cell cycle progression, transcriptional activation, and signal transduction.1 Recently, Lee et al2 demonstrated that a novel human DUB gene, ubiquitin-specific protease 22 (USP22), is the core of a multitude of physiological and pathologic processes. USP22 forms part of a polycomb-cancer stem cell signature that uniformly exhibits a marked propensity toward metastatic
*Reprint requests: Xiao-Ming Jin, Department of Pathology, Harbin Medical University, 157 Baojian Road, Nangang District, Harbin, China and Dan kong, Department of Gynecology, Third Affiliated Hospital of Harbin Medical University, Harbin, China; e-mail: [email protected] or [email protected] 1931-5244/$ - see front matter Ó 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.trsl.2014.09.005
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AT A GLANCE COMMENTARY He Y, et al. Background
Gastric carcinoma is the second leading cause of cancer-related deaths in the world. Ubiquitinspecific peptidase 22 (USP22) was recently identified as a new tumor cell marker that may be correlated with cancer metastatic potential, resistance to chemotherapy, and patient prognosis. Translational Significance
Our results indicate that increased USP22 expression is positively correlated with gastric cancer differentiation, infiltration depth, and lymph node metastasis. The C-myc protein was also shown to have synergistic effects with USP22 on gastric cancer tissues. USP22 expression may play an important role in gastric carcinoma tissue, particularly in precancerous lesions during the gastric cancer evolution process. Therefore, USP22 may function as al marker of tumor evolution in the presence of other tumor cell metastatic markers.
dissemination and a therapy-resistance phenotype.3,4 The polycomb genes play an important role in tumor development and occurrence in terms of not only tumor growth but also the potential for cancer metastasis and chemotherapy drug resistance. These genes are closely related to patient prognosis and are therefore considered tumor cell marker genes. USP22 and the C-myc oncogene have a crucial partnership: USP22 is required for Myc-driven transcription. The depletion of USP22 in cells decreases the ability of Myc to activate the transcription of its targets.5-7 USP22 is expressed in some normal human tissues, such as myocardial tissue, skeletal muscle, and the lung, but its expression is significantly upregulated in malignant tumors.2 High USP22 expression often indicates that the tumor is about to spread throughout the body, and this phenomenon is called a ‘‘cancer stem cell signal.’’ However, the relationship between the expression of USP22 and gastric histology has not been previously studied. In the present study, we investigated the protein and messenger RNA (mRNA) expression levels of USP22 and C-myc in different precancerous lesion (PL) tissues through immunofluorescent (IF) staining, Western blot (W-B) analysis, and quantitative real-time polymerase chain reaction (qRT-PCR) and analyzed the correlation between the USP22
and C-myc levels and clinicopathologic features, invasion, metastasis, histologic subtypes, and relevance to prognosis. MATERIALS AND METHODS Patient and tissue samples. A total of 90 gastric adenocarcinoma tissue samples (here in after referred to as 90 cases) were obtained from the surgery department of the Fourth Affiliated Hospital Medical University between October 2007 and December 2010. Each sample contained cancer tissue, adjacent tissues (2.5–3.0 cm away from the cancer tissue), and distant tissue (6.0–8.0 cm away from the cancer tissue), resulting in a total of 270 fresh tissue samples. The specimens were snap-frozen in liquid nitrogen and stored at 280 C. These samples were subjected to qRT-PCR, W-B, and IF staining analyses to assess the USP22 and C-myc transcript abundance and protein expression. All the tissues were also fixed with formalin, paraffin embedded, and prepared for hematoxylin and eosin staining. To further analyze the relationship between the USP22 and C-myc levels and the cancer infiltration depth, additional 150 paraffin-embedded specimens (here in after referred to as 150 cases) were collected from gastric cancer patients undergoing surgery at Hei Long Jiang Province Hospital between October 2007 and December 2010 for immunohistochemistry to assess USP22 and C-myc protein expression and histopathology only. Two histologic analyses (WHO8 and Lauren et al9) criteria were used to evaluate the classification of all samples. None of the patients received preoperative chemotherapy or radiotherapy. This study was approved by the Institutional Review Board of Harbin Medical University Medical Center and received ethical approval from the Ethics Committee. All the subjects provided written informed consent and were assured of their anonymity and the confidentiality of the data. Immunohistochemical and Immunofluorescent analysis. The formalin-fixed, paraffin embedded, 4-mm
thick sections were dewaxed and rehydrated in a graded series of ethanol solutions. The endogenous peroxidase activity was quenched with 3% H2O2 for 15 minutes. After washing with phosphate-buffered saline (PBS), the sections were incubated with an antiUSP22 antibody (1:200, immunoglobulin G [IgG]; Abcam, Cambridge, California) or an anti-C-myc antibody (1:100, Y69, IgG; MaiXin Bio-technology, FZ, China) overnight at 4 C. The sections were incubated with peroxidase-conjugated streptavi-din for 30 minutes, and the reaction products were visualized using diaminobenzidine as a chromogen and counterstained with commercial hematoxylin. Specific
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isotype control PBS (in the absence of the primary antibodies) was used as a negative control. USP22 and C-myc were expressed in the nuclei of gastric cancer cell. An IF analysis of USP22 and C-myc was performed as previously described.10 Frozen 5-mm thick sections from each case were fixed with acetone, and the nonspecific binding was blocked through a 15-minute incubation with secondary antibody animal serum. The sections were incubated with primary antibodies against USP22 (1:200) and C-myc (1:100, 9E10, IgG; Santa Cruz Biotechnology, Dallas, California) overnight at 4 C and then with either Fluorescein isothiocyanateconjugated goat anti-rabbit or horse anti-mouse antibodies (1:200; Vector Laboratories) for 1 hour at room temperature. After washing with PBS, the specimens were counterstained with 40 ,6-diamidino-2phenylindole (Vector Laboratories) and observed with a biological research microscope (E-800; Nikon Eclipse, Tokyo, Japan). No positive staining was observed in the samples that were not incubated with the primary antibody, which served as the negative control. USP22 was expressed in the nuclei of gastric cancer cell, and C-myc was expressed in the cytoplasm. For each sample, 10 random fields were examined at 3400 magnification. To calculate the ratio of positive cells, the positive cells of USP22 and C-myc were counted and the numbers were divided by the total cell number in each field.11 W-B analysis. The total protein contents of the frozen tumor, adjacent, and matched distant tissues were extracted by suspending the sample in a lysis buffer consisting of 20 mM Tris-HCl (pH 7.5), 2 mM EDTA, 150 mM NaCl, 1% Triton X-100, and protease inhibitors. The protein concentration in the supernatant was determined using the Bradford method (Bio-Rad, Hercules, California). Equal amounts of protein were separated by 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto a nitrocellulose membrane. The membranes were then blocked with Tris Buffered Saline with Tween-20 containing 1% bovine serum albumin (Promega, Madison, Wisconsin) overnight and incubated with a rabbit anti-human USP22 (66 kDa) polyclonal antibody (1:500; Abcam) or a mouse anti-human C-myc (55 kDa) mono-clonal antibody (1:300; Santa Cruz Biotechnology) for 2 hours at 37 C. After washing with Tris Buffered Saline with Tween-20, the membrane was incubated with a secondary antibody against rabbit or mouse IgG (antimouse IgG, DyLight 680; anti-rabbit IgG, DyLight 800; Cell Signaling, Boston) at a 1:10,000 dilution for 1 hour. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Santa Cruz Biotechnology) was used as an internal control. The proteins were visualized using an
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infrared imaging system (Odyssey; LI-COR Bioscience, Ltd, Kent, UK). Quantitative real-time polymerase chain reaction. The total RNA from the frozen tissue specimens was extracted with TRIzol reagent (Invitrogen, Carlsbad, California) and RNeasy columns, which were used according to the manufacturers’ recommendations. The first-strand complementary DNA synthesis was performed with 2 mg of total RNA using HighCapacity Complementary DNA Reverse Transcription Kits (Applied Biosystems, Foster City, California). The qRT-PCR analysis was performed on the ABI PRISM 7000 Sequence Detection System using the TaqMan1 Universal PCR Master Mix (Applied Biosystems) and the TaqMan1 Gene Expression Assays probe and primer mix (Applied Biosystems) according to the manufacturer’s specifications. The assay identification numbers were as follows: USP22, Hs00392758_m1; GAPDH, Hs02758991_g1. The human GAPDH gene was used as an endogenous control. All the reactions were performed in triplicate. The relative mRNA levels were calculated based on the Ct values and corrected with the GAPDH expression level using the following equation: 2-DCt [DCt Ct (USP22) -Ct (GAPDH)]. Statistical analysis. All results were analyzed using a 2-tailed Student t test (adjusted for SAS Institute Inc., Cary, North Carolina). In all the analyses, P , 0.05 was deemed significant.
RESULTS Patient characteristics. All fresh frozen samples (270) from the 90 cases and 150 paraffin-embedded samples were examined histologically to confirm the diagnosis of gastric cancer. Forty-three of the 90 patients contained atypical hyperplasia (AH) or intestinal metaplasia (IM) features in the tissue adjacent to the cancer tissue. Commonly, AH and IM were both considered as PLs. The adjacent tissues were considered as AH if following change displayed: AH glands are covered with crowded and distended cells, the nuclei arrayed as pseudostratified layers with scarce mucus or without mucus. Mucosa cells exhibit considerable pleomorphism and often possess hyperchromatic nuclei that are abnormally large for the size of the cell. Mitotic figures are more abundant than usual. Frequently the mitoses appear in abnormal locations within the epithelium.12 The noninvolved epithelium showed normal gastric histology. Therefore, only the 43 cases that contained cancer, PL (AH/IM) locations and noninvolved epithelium (normal gastric mucosa) were used to evaluate the
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Fig 1. USP22 expression in 90 fresh cases. (A–C) Expression of USP22 was observed by immunofluorescent assay. Dotted or granular staining was observed in the cancer cell nucleus, part of the duct, and a few scattered cancer cells. (D and E) The expression of USP22 in various differentiated types, metastasis, and nonmetastasis groups of gastric adenocarcinomas. (F) The expression of USP22 was observed in various differentiated types of gastric cancer by W-B assay. (G) The messenger RNA level of USP22 was observed by quantitative real-time polymerase chain reaction. *P , 0.05, **P , 0.01. DAPI, 40 ,6-diamidino-2-phenylindole; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; H&E, hematoxylin and eosin; Well, well-differentiated gastric adenocarcinomas; Moderate, moderately differentiated gastric adenocarcinomas; Poor, poorly differentiated gastric adenocarcinomas; USP22, ubiquitin-specific peptidase 22; W-B, Western blot.
relationship of expression of USP22, C-myc vs the different locations of the samples. Increased expression of USP22 in gastric cancer. The USP22 staining was localized within the nuclei of gastric cancer cells. USP22 was expressed in 63.37% of the fresh gastric cancer tissues and in 54.46% of the paraffin-embedded gastric cancer tissues. The expression level of USP22 was higher in the poorly differentiated gastric adenocarcinomas than in the well-differentiated adenocarcinomas (P , 0.05; Figs 1, A–D and 3, C and E, Table I). In addition, USP22 level was higher in the diffuse type of gastric adenocarcinomas than in the intestinal type (P , 0.01; Table I). The W-B and qRT-PCR
assays revealed that USP22 was highly expressed in the poorly differentiated gastric adenocarcinomas (P , 0.05; Fig 1, F and G, Table I). We also found no significant correlation between the USP22 mRNA and protein levels and the tested clinicopathologic parameters, which included the patient’s sex, age, and tumor size. However, the expression of USP22 was found to be correlated with differentiation, infiltration depth, and lymph node metastasis of the gastric cancer samples (P , 0.05; Figs 1, E and 3, F and G, Table I). The gastric adenocarcinoma groups of the poorly differentiated histology type, the deeper infiltration, and lymph node metastasis were inclined to express more USP22.
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Fig 2. C-myc expression in 90 fresh cases. (A–C) Expression of C-myc was observed by immunofluorescent assay. C-myc is visible in part of the gland cell cytoplasm and scattered throughout the cancer cell cytoplasm and nuclear membrane. (D and E) The expression of C-myc in various differentiated types and groups of metastasis and nonmetastasis. (F) The C-myc expression in various differentiated types by Western blot assay. * P , 0.05, **P , 0.01. Well, well-differentiated gastric adenocarcinomas; Moderate, moderately differentiated gastric adenocarcinomas; Poor, poorly differentiated gastric adenocarcinomas; DAPI, 40 ,6-diamidino-2phenylindole; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Increased expression of C-myc in gastric cancer.
C-myc was primarily expressed in the cytoplasm (fresh gastric cancer tissues) or the nuclei of gastric cancer cells (paraffin-embedded cancer tissues). In addition, C-myc was found to be expressed in 50.63% of the fresh gastric cancer tissues and in 42.30% of the paraffin-embedded gastric cancer tissues. The expression of C-myc was found higher in the group of poorly differentiated gastric adenocarcinomas than in the group of well-differentiated adenocarcinoma (P , 0.05; Figs 2, A–D and 3, D and H, Table I). In addition, it was found higher in the diffuse type than in the intestinal type (Table I). The W-B analysis revealed that C-myc was highly expressed in the poorly differentiated gastric adenocarcinomas (Fig 2, F). We also found no significant correlation between the C-myc protein levels and the tested clinico-
pathologic parameters, which included the patient’s sex, age, tumor size, and infiltration depth. However, C-myc expression was found correlated with differentiation and lymph node metastasis (Figs 2, E and 3, I, Table I). The gastric adenocarcinoma groups of poorly differentiated and lymph node metastasis were inclined to express more C-myc. Increased expression of USP22 and C-myc in 43 fresh gastric cancer cases. Forty-three of the 90 fresh gastric
cancer cases were confirmed to be gastric cancer with AH or IM (PL) and exhibited normal gastric mucous membranes (noninvolved gastric mucosa, normal) in the transition area. The USP22 and C-myc levels in the 43 patients progressively decreased in different parts (cancer tissue, PL, and noninvolved gastric mucosa) (Fig 4, A and B). USP22 and C-myc were found high in PL and noninvolved mucosa tissue adjacent to
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Fig 3. USP22 and C-myc expressions in 150 paraffin-embedded gastric tissues. (A and B) Morphology of representative gastric carcinoma tissue. Hematoxylin and eosin staining: (A) magnification, 3100, (B) magnification, 3400. (C and D) Immunohistochemistry results (magnification, 3400). (E–G) The expression of USP22 in various histology types, infiltrate depth, metastasis, and nonmetastasis groups. (H and I) The expression of C-myc in various histology types, metastasis, and nonmetastasis groups. There was no statistical difference in various infiltrate depth groups for the expression of C-myc.*P , 0.05, **P , 0.01. Well, well-differentiated gastric adenocarcinomas; Moderate, moderately differentiated gastric adenocarcinomas; Poor, poorly differentiated gastric adenocarcinomas; USP22, ubiquitin-specific peptidase 22.
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Table I. Positive cell percentage of USP22 and C-myc with clinical parameters USP22 (%)
Variable
Sex Male (n 5 68 in 90 cases; n 5 95 in 150 cases) Female (n 5 22 in 90 cases; n 5 55 in 150 cases) Age #55/59 (45/90; 77/150) .55/59 (45/90; 73/150) Tumor grade Well-differentiated (18/90; 4/150) Moderately differentiated (24/90; 38/150) Poorly differentiated (48/90; 108/150) Lauren type Intestinal type (49/90; 63/150) Diffuse type (41/90; 87/150) Infiltration M (4/90; 8/150) SM (2/90; 13/150) MP (18/90; 24/150) SE (66/90; 105/150) Metastasis (lymph node) Yes (73/90; 96/150) No (17/90; 54/150)
C-myc (%)
n 5 90
n 5 150
n 5 90
n 5 150
Mean 6 SD
Mean 6 SD
Mean 6 SD
Mean 6 SD
64.69 6 19.05 62.05 6 22.14
54.88 6 12.69 54.04 6 12.26
51.96 6 19.16 49.32 6 21.25
43.64 6 12.58 40.96 6 13.81
63.22 6 19.86 64.87 6 19.84
63.95 6 13.06 55.23 6 11.94
50.82 6 19.56 51.80 6 19.8
42.52 6 12.91 42.81 6 13.32
43.39 6 17.49*,† 58.25 6 14.03*,‡ 74.69 6 15.36*,§
27.00 6 9.20*,† 44.68 6 11.08*,‡ 59.07 6 9.44*,§
31.28 6 17.42*,† 45.04 6 14.48*,‡ 61.96 6 15.02*,§
19.50 6 5.26*,† 31.13 6 10.69*,‡ 47.57 6 10.29*,§
54.65 6 18.69*,i 75.27 6 14.56
48.62 6 14.31*,i 58.59 6 8.86
41.80 6 18.66*,i 62.69 6 13.91
36.13 6 13.86*,i 47.39 6 10.18
56.75 6 24.24{,# 71.00 6 1.41{,** 65.28 6 18.93 63.94 6 20.21
45.25 6 18.45{,# 48.00 6 18.61{,** 52.88 6 12.42 56.49 6 10.51
44.50 6 23.56 60.00 6 7.07 52.44 6 19.62 51.15 6 19.83
41.75 6 13.71 39.08 6 15.83 38.17 6 14.28 44.20 6 12.22
66.95 6 18.11{ 51.59 6 22.18
56.73 6 10.15{ 50.74 6 15.21
54.19 6 18.17{ 38.94 6 21.23
44.10 6 11.70{ 40.09 6 14.98
Abbreviations: M, mucosa; MP, muscularis propria; SD, standard deviation; SE, serosa; SM, submucosa. *P ＜ 0.01. † For the comparison of well-differentiated vs moderately differentiated. ‡ For the comparison of moderately vs poorly differentiated. § For the comparison of well-differentiated vs poorly differentiated. i For the comparison of intestinal vs diffuse type. { P ＜ 0.05. # For the comparison of M vs SE. **For the comparison of SM vs SE.
cancer tissues, which were usually diagnosed as poor differentiated adenocarcinoma (Fig 4, C and D). In contrast, low or no USP22/C-myc expression was found in noninvolved gastric mucosa when the tumor was diagnosed as well-differentiated adenocarcinoma. The expression of the 2 proteins was positively correlated. Correlations were observed between USP22
and C-myc expression and the clinicopathologic data. First, the USP22 and C-myc expression levels in the gastric cancer tissues were positively correlated (Fig 5, A and B). In addition, the USP22 and C-myc expression levels and the WHO and Lauren’s histologic classifications of gastric cancer were compared and related to the clinical and pathologic data shown in Table I. DISCUSSION
Sequence analysis has revealed that USP22, a novel DUB, belongs to a large family of proteins with ubiquitin hydrolase activity. C-myc is a nuclear oncogene that functions in protein phosphorylation and exhibits spe-
cific DNA binding, thereby affecting multiple aspects of cell growth, differentiation, apoptosis, and cell cycle progression.13,14 C-myc has dual functions in cells, that is, it stimulates cell proliferation and promotes cell apoptosis. The C-myc protein expression increases significantly in cancerous cells, making the cell exempt from the limits of normal growth and giving the cell a malignant phenotype.15 USP22 is required for the activation of target gene transcription by Myc. Most importantly, the depletion of USP22 compromises Myc function, including transformation. Consistent with its critical role in cell cycle progression, USP22 depletion results in a specific G1 arrest. Previous studies have suggested that the C-myc gene and the tumorsuppressor gene p53 have an important impact on the transcriptional activation activity of Spt–Ada–Gcn5– acetyltransferase.16,17 The USP22-Myc partnership6 is present in most tumor cells. Because the Myc protein controls the expression of thousands of other genes, the depletion of cellular USP22 can inhibit Myc function, inhibiting the invasive growth of cancer cells.
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Fig 4. (A and B) The correlation of the expression of USP22 and C-myc between the locations of tumor, PL, and noninvolved gastric epithelium (normal) in 43 cases. The expression of USP22 and C-myc decreased by the order of tumor, PL, and noninvolved gastric mucosa. *P , 0.05. (C and D) Categorized by the various differentiated histology types, the expressions of USP22 and C-myc were analyzed in various locations of 43 gastric cancer samples. *P , 0.05, **P , 0.01. Well, well-diferentiated gastric adenoarcinoas; Moderate, moderately differentiated gastric adenocarcinomas; Poor, poorly differentiated gastric adenocarcinomas; PL, precancerous lesion; Poor, USP22, ubiquitin-specific peptidase 22.
Fig 5. Relevance of USP22 and C-myc expression. (A) The positive expression of USP22 and C-myc in 90 fresh gastric cancer samples. (B) The relevance of USP22 and C-myc expression is shown (r 5 0.971, P 5 0.00, P , 0.01). Well, well-differentiated gastric adenocarcinomas; Moderate, moderately differentiated gastric adenocarcinomas; Poor, poorly differentiated gastric adenocarcinomas; USP22, ubiquitin-specific peptidase 22.
Thus, the Myc-mediated gene transcription process requires USP22, and USP22 completely controls its transcription. The present study found that USP22 expression is significantly higher in poorly differentiated adenocarcinoma (P , 0.01) and that the promotion of cancer evolution, gastric cancer invasion depth, and gastric cancer
lymph node metastasis are correlated with USP22 expression. The USP22 level progressively increased from pericarcinomatous tissue to cancer tissue. Some researchers18,19 have shown that the USP22 expression increases significantly from normal mucosa to carcinomas, which suggests that USP22 activation increases during colorectal and bladder
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carcinogenesis. USP22 expression and colon cancer metastasis to the liver are correlated with poor prognosis.19 We demonstrated that USP22 is highly expressed in normal mucosa tissue adjacent to cancer tissue, which were usually diagnosed as poorly differentiated adenocarcinoma. In contrast, low or no USP22 expression was found in the normal mucosa when the tumor was diagnosed as well-differentiated adenocarcinoma. This may indicate that USP22 played a role in the process of precancerous gastric tissue progress into the cancer. In addition, USP22 may play a direct functional role in the regulation of cell differentiation. USP22 depletion causes cells to accumulate in the G1 phase of the cell cycle and results in concomitant decreases in the percentage of cells in the synthesis and second gap/ mitosis phases.20-23 A high risk of gastric cancer could be predicted if USP22 is highly expressed in the PLs. These results are consistent with the results obtained by other researchers in different tumor tissues, such as colorectal cancer, bladder cancer, and papillary thyroid carcinoma.19,24-27 The examination of both C-myc and USP22 expression revealed that USP22 and C-myc have a synergistic effect on gastric cancer tissues. Thus, USP22 may be a new cancer stem cell marker worthy of further study. The USP22 gene encodes a cancer-related protein that can control the expression of a large variety of genes; however, USP22 is also very vulnerable to the effects of some drugs. McMahon6 suggested that USP22 may become a potential novel target for cancer drugs. Because of the role of USP22 in cell cycle regulation in tumor cells, the design of an USP22-specific inhibitory factor will likely be valuable for cancer treatment. The discovery of USP22 inhibitors using the findings obtained through experimental research and early clinical applications will likely lead to improvements in cancer research. Therefore, efforts to identify more efficient molecular markers for the detection and prognosis of gastric cancer are of great clinical importance. ACKNOWLEDGMENTS
Conflicts of Interest: The authors have read the journal’s policy on disclosure of potential conflicts of interest and have none to declare. This work was supported by grants from the National Natural Science Foundation of China (no. 81101735), grants from the Program for New Century Excellent Talents in Heilongjiang Provincial University (1253– NCET–015), grants from the Harbin Scientific and Technological Talents Research Program (2012RFLX S013).This work was supported by the Heilongjiang Provincial Science and Technology Innovation Team
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in Higher Education Institutes for Infection and Immunity, Harbin Medical University. The authors thank Elsevier Language Editing Services from Elsevier Ltd for excellent editing of this manuscript. All authors have read the journal’s authorship agreement and agree with the statements. REFERENCES
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