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GRIM-19 expression is a potent prognostic marker in colorectal cancer
Human Pathology (2015) xx, xxx–xxx
www.elsevier.com/locate/humpath
Original contribution
GRIM-19 expression is a potent prognostic marker in colorectal cancer☆,☆☆ Miao Hao PhD a , Zhenbo Shu MD b , Hongyan Sun MD c , Ran Sun MD a , Yuqian Wang PhD a , Tie Liu MD c , Degang Ji MD b , Xianling Cong MD c,⁎ a
Science Research Center, China-Japan Union Hospital of Jilin University, Changchun 130033, China Department of Surgery, China-Japan Union Hospital of Jilin University, Changchun 130033, China c Tissue Bank, China-Japan Union Hospital of Jilin University, Changchun 130033, China b
Received 13 May 2015; revised 21 July 2015; accepted 23 July 2015
Keywords: GRIM-19; Colorectal cancer; Immunohistochemistry; Prognostic biomarker; Tumor suppressor
Summary Retinoid-interferon-induced mortality-19 (GRIM-19), a recently discovered cell death regulatory gene, may function as a tumor suppressor in many human malignancies. However, the expression of GRIM-19 in and its prognostic value for patients with colorectal cancer (CRC) have not been well investigated to date. Here, GRIM-19 expression was measured immunohistochemically in 94 colon samples and by quantitative real-time reverse transcriptase polymerase chain reaction in 15 paired CRC tissues and adjacent normal tissues. The prognostic significance was assessed using Kaplan-Meier survival estimates and log-rank tests. Our results showed that GRIM-19 mRNA and protein levels in adenoma tissues were similar to those in adjacent normal tissues. However, GRIM-19 expression was severely depressed in carcinomas compared to matched normal tissues (P = .000). Additionally, we found GRIM-19 to be located in both the cytoplasm and nucleus in normal tissues but only in the cytoplasm in CRC tissues. Alteration in GRIM-19 expression occurs early in the pathogenesis of CRC; moreover, low GRIM-19 expression was associated with poor tumor differentiation (P = .013), the presence of lymph nodes (P = .000), metastasis to other organs (P = .045) and vascular invasion (P = .010). During a mean period of 40 months follow-up, patients without GRIM-19 had a statistically significantly lower rate of recurrence/metastasis (P b .05) and a shorter overall survival time (P b .01) than the patients with GRIM-19 expression. Taken together, GRIM-19 expression is closely associated with CRC progression and might be a very promising prognostic biomarker for CRC patients. © 2015 Elsevier Inc. All rights reserved.
1. Introduction ☆
Funding/Support: This work was supported by the National Natural Science Foundation of China (Grant Nos. 81371595 and 81401966), the research project of Jilin Provincial Science and Technology Department (Nos. 20130206001YY, 20140414061GH and 20150204082SF). ☆☆ The authors have no conflict of interest. ⁎ Corresponding author. Tissue Bank, China-Japan Union Hospital of Jilin University, 126 Xiantai Street, Changchun, Jilin 130033, China. E-mail address: [email protected] (X. Cong). http://dx.doi.org/10.1016/j.humpath.2015.07.020 0046-8177/© 2015 Elsevier Inc. All rights reserved.
Colorectal cancer (CRC) is the third most commonly diagnosed cancer in males and the second in females. In 2008, more than 1.2 million people were diagnosed with CRC, and more than 600 000 died from the disease [1]. Indeed, despite improvements in surgical techniques and drug therapy, the 5-year survival rate of patients with CRC
2 remains low [2,3]. Because of development after resection, tumor recurrence and metastasis are virtually inevitable and are a major cause of death in CRC patients. In addition to diagnosis and treatment, prognosis is an important matter for cancer patients. Therefore, it is urgent to identify possible therapeutic or prognostic biomarkers and to elucidate their relationship with CRC while considering relevant clinicopathological correlations [4]. GRIM-19 (Gene associated with Retinoid-Interferon induced Mortality–19), which is present in both the nuclear and cytoplasmic compartments, is a subunit of complex I of the mitochondrial membrane respiratory chain [5,6]. Recently, the GRIM19 gene has been identified as an apoptosis-related gene that represents a novel type of tumor suppressor [7,8]. The GRIM-19 protein binds to signal transducer and activator of transcription–3 to inhibit its transcriptional activity [9,10]. It has been reported that the overexpression of GRIM-19 suppresses hepatocellular carcinoma growth [11] and induces apoptosis in human breast carcinoma cell line MCF-7 [7]. Conversely, GRIM-19 expression is lost or significantly reduced in many human malignancies, such as lung cancer [12], cervical cancer [13], kidney cancer [14], hepatocellular carcinoma [15] and gliomas [16]. Regarding colorectal carcinoma, only Gong et al have demonstrated that GRIM-19 is severely reduced at the mRNA level [17]. However, the prognostic significance of GRIM-19 expression and its association with clinicopathologic features have not yet been reported in CRC. In this study, we used immunohistochemistry (IHC) and quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) analyses to evaluate the expression of GRIM-19 in patients with colorectal carcinoma and to systematically elucidate the prognostic relevance of GRIM-19 expression and correlations with clinicopathologic features. Our results indicated that GRIM-19 might serve as a prognostic biomarker in colorectal carcinoma.
2. Materials and methods
M. Hao et al. demographic and clinicopathologic data of all patients, including age, gender, tumor size, tumor location, histologic differentiation, depth of invasion, lymph node stage, and vascular invasion, were collected from the patients' medical records. This study design, including the use of all archival tissues, was approved by the ethical committee of the China-Japan Union Hospital of Jilin University. The effect of GRIM-19 expression on the time to recurrence/metastasis and the overall survival (OS) time was calculated by the Kaplan-Meier method and analyzed by the log-rank test [18,19]. Follow-up data were summarized at the end of December 2014, with a median follow-up of 40 months (range, 1-42 months). Time to recurrence/metastasis and OS were considered to be the primary endpoints. Time to recurrence/metastasis was calculated from the date of resection to the date when tumor recurrence/metastasis was diagnosed. Overall survival was calculated from the date of resection to the date of death or last follow-up.
2.2. qRT-PCR Total RNA was isolated from the tissue samples using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Reverse transcription was carried out using a Goscript Reverse Transcription System Kit (A5000; Promega, Madison, Wisconsin, USA) according to the manufacturer's instructions. mRNA levels were quantified by qRT-PCR with the Quantitect SYBR Green PCR Kit (Roche, Mannheim, Germany) and normalized to Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) using the following primers: GRIM-19-F, 5′ACCGGAAGTGTGGGATACTG-3′, and GRIM-19-R, 5′-GCTCACGGTTCCACTTCATT-3′; GAPDH-F, 5′AGAAGGCTGGGGCTCATTTG-3′, and GAPDH-R, 5′-AGGGGCCATCCACAGTCTTC-3′. The cycling parameters were 40 cycles of 95°C for 15 seconds, 60°C for 15 seconds, and 72°C for 30 seconds. Relative gene expression was normalized to the geometric mean of the housekeeping gene GAPDH and calculated according to the Livak method (2−ΔΔCt) [20]. The experiments were independently repeated three times.
2.1. Patients, tissue specimens and follow-up 2.3. Immunohistochemical analysis Pairs of primary CRC tissues and adjacent normal colorectal tissues were obtained from 94 patients; adenoma tissues and adjacent normal tissues were obtained from 3 patients undergoing curative surgical operations at the China-Japan Union Hospital of Jilin University (Changchun, China). No patients received chemotherapy or radiotherapy prior to surgery. The matched normal colorectal tissues were obtained from a segment of the resected specimens that was N5 cm away from the tumor. All tissues were fixed in 10% neutral-buffered formalin and embedded in paraffin blocks for IHC. In addition, 15 of the paired tissues obtained immediately after surgery were snap-frozen in liquid nitrogen and kept at −80°C for further analysis. The
Expression of GRIM-19 and K-ras was determined by IHC staining of the tissue sections using mouse anti– GRIM-19 monoclonal antibody (ab110240, Abcam, Hong Kong, China) and rabbit anti–K-ras polyclonal antibody (RAB-0638; Maxim, China). All specimens were fixed with 4% formaldehyde, dehydrated, embedded, and cut into 4-μm serial sections. Endogenous peroxidase activity was blocked by incubation with 3% hydrogen peroxide in methanol for 10 minutes. For high-pressure antigen retrieval, the sections were washed 3 times with phosphate-buffered saline and incubated in 0.01 mmol/L sodium citrate buffer (pH 6.0) for 3 minutes. The sections were then incubated with the primary
GRIM-19 serves as a prognostic biomarker for colorectal cancer antibody (1:300 dilution) at 4°C overnight. After 3 rinses with phosphate-buffered saline, the sections were incubated with the corresponding secondary antibody for 10 minutes at room temperature. The antigen-antibody complexes were detected by the streptavidin-biotin-peroxidase method using diaminobenzidine as the chromogenic substrate (Maxim, Fuzhou, China). Finally, the sections were counterstained with 10% hematoxylin, dehydrated, mounted, and examined by light microscopy. In all cases, negative controls were performed by omitting the primary antibody.
2.4. Evaluation of immunohistochemical staining Immunohistochemical staining was blindly scored by two pathologists. The GRIM-19 protein was assessed in both the cytoplasm and nucleus. The overall amount of staining was determined by the staining intensity and the proportion/extent of stained tumor cells according to published scoring methods [21,22]. The mean percentage of positive tumor cells was determined in at least five areas at 400× magnification and assigned to one of the following categories: 0, ≤5%; 1, 5%-25%; 2, 25%-50%; 3, 50%-75%; 4, ≥75%. The average estimated intensity of staining in positive cells was scored as 1 (weak), 2 (moderate) or 3 (intense). The total score was
3
calculated by multiplying the scores for intensity and the extent of staining. A staining score of higher than 2 for GRIM-19 expression was considered positive, whereas a score below 2 was considered negative.
2.5. Statistical analysis All the statistical analyses were carried out using SPSS 17.0 Software (SPSS, Chicago, IL). The results are expressed as the mean ± SD. The recurrence/metastasis rates and survival rates were calculated by the Kaplan-Meier method and compared using the log-rank test. Categorical data between GRIM-19 expression and clinicopathologic features were analyzed by the χ2 test or Fisher's exact test. P b .05 was considered to be significant.
3. Results 3.1. Differential expression of GRIM-19 in normal colorectal tissues and CRC tissues To investigate the role of GRIM-19 in colorectal carcinoma, we evaluated its expression by immunohistochemical analysis
Fig. 1 IHC analysis of GRIM-19 expression (A, C, E, G: original magnification ×200; B, D, F, H: ×400; I: ×100). The expression of GRIM-19 in adjacent normal tissues (score 2: A and B; score 6: C and D) and tumor tissues (score 2: E and F; score 6: G and H). Differential expression of GRIM-19 in colon adenoma and carcinoma tissues (I; adenoma tissue: red arrow; adjacent adenocarcinoma tissue: black arrow).
4
M. Hao et al.
Fig. 2 Relative expression levels of GRIM-19 mRNA in 15 paired adjacent normal colorectal tissues and CRC tissues (A) and 3 paired adjacent normal tissues and adenoma tissues (B), as shown by real-time PCR assay. The relative expression data were analyzed by the 2−ΔΔCt method. GAPDH was used as an internal control. *P b .05, **P b .01.
Table Correlation between expression of GRIM-19 and clinicopathologic features of colorectal carcinoma Characteristic
n
GRIM-19 (%)
n = 94 Positive (n = 21) Age, y b65 ≥65 Gender Male Female Tumor size, cm b5 ≥5 Tumor location Colon Rectum Histologic differentiation Well and Moderate Poor Depth of invasion Tis-T2 T3-T4 Lymph node metastasis Yes No Metastasis to other organs Present Absent Vascular invasion Yes No K-ras Positive Negative ⁎ P b .05; ⁎⁎ P b .01.
P
Negative (n = 73)
56 38
16 (76.2) 40 (54.8) .078 5 (23.8) 33 (45.2)
61 33
18 (85.7) 43 (58.9) .023 ⁎ 3 (14.3) 30 (41.1)
39 55
6 (28.6) 33 (45.2) .173 15 (71.4) 40 (54.8)
48 46
9 (42.9) 39 (53.4) .393 12 (57.1) 34 (46.6)
70 24
20 (95.2) 50 (68.5) .013 ⁎ 1 (4.8) 23 (31.5) .011
3 91
1 (4.8) 2 (2.7) .536 20 (95.2) 71 (97.3)
60 34
6 (28.6) 54 (74.0) .000 ⁎⁎ 15 (71.4) 19 (26.0)
45 49
6 (28.6) 39 (53.4) .045 ⁎ 15 (71.4) 34 (46.6)
41 53
4 (19.0) 37 (50.7) .010 ⁎ 17 (81.0) 36 (49.3)
28 66
2 (9.5) 26 (35.6) .021 ⁎ 19 (90.5) 47 (64.4)
in 94 patients. Among the 94 paired tissues, GRIM-19 expression was detected in 80 normal tissues (85.1%) and 21 tumor tissues (22.3%). IHC revealed a more frequent absence of GRIM-19 expression in CRC tissues than in adjacent nontumorous tissues (P = .000). We first confirmed the location of GRIM-19 in colon tissues. As shown in Fig. 1, the GRIM-19 protein was primarily located in the cytoplasm, with slightly weak staining in the nucleus (Fig. 1D), but was found only in the cytoplasm in CRC tissues (Fig. 1H). We also observed that GRIM-19 was highly expressed in non-tumor colonic epithelial cells (Fig. 1C and D). However, most CRC tissues showed a lack of GRIM-19 expression (Fig. 1E and F). In addition, we found that GRIM-19 was also highly expressed in adenoma tissue (Fig. 1F, red arrow), with weak staining in adjacent adenocarcinoma tissue (Fig. 1F, black arrow). Our qRT-PCR analysis further confirmed that in CRC patients, GRIM-19 mRNA was significantly decreased in tumor tissues in 73.3% (11/15) compared with matched non-tumor tissues (Fig. 2A). In addition, GRIM-19 mRNA expression in adenoma tissues was similar to that in adjacent normal tissue (Fig. 2B). These data are in line with the results of IHC analysis.
3.2. Correlation of GRIM-19 expression levels with clinicopathologic features of CRC The potential association between GRIM-19 expression and clinicopathologic features in 94 CRC patients is presented in the Table. In female patients, negative GRIM-19 expression was more common than positive GRIM-19 expression (41.1% versus 14.3%; P = .023). Moreover, a lack of GRIM-19 expression was significantly associated with poor tumor differentiation (P = .013), lymph node metastasis (P = 0), metastasis to other organs (P = .045) and vascular invasion (P = .010). Expression of K-ras was more common in cancers without GRIM-19 expression (P = .021). No significant correlation with GRIM-19 expression was found for age, tumor size, tumor location or depth of invasion (Table).
GRIM-19 serves as a prognostic biomarker for colorectal cancer
5
Fig. 3 Kaplan-Meier analysis of the association between GRIM-19 expression and recurrence/metastasis (A) as well as the survival (B) of patients with colorectal carcinoma. The patients without GRIM-19 expression (n = 73) had a significantly higher recurrence rate and lower overall survival rate than those with positive GRIM-19 expression (n = 21).
3.3. Lower GRIM-19 expression is associated with poor prognosis of CRC To further evaluate the prognostic value of GRIM-19 expression in colorectal carcinoma, we used Kaplan-Meier analysis to calculate the effect of GRIM-19 expression on the time to recurrence/metastasis and OS time. As shown in Fig. 3A, CRC patients without GRIM-19 expression had a higher recurrence/metastasis rate (P b .05, log-rank test) than those with positive GRIM-19 expression. Moreover, as shown in Fig. 3B, there was a trend of better OS for patients with GRIM-19 expression compared with those without GRIM-19, and the difference between the two survival curves was statistically significant (P b .01, log-rank test). These results indicate that GRIM-19 might be a very promising prognostic biomarker for colorectal carcinoma.
4. Discussion CRC is a malignant tumor in humans, and the poor 5-year survival in CRC patients after curative resection is mainly due to the high rate of recurrence and/or metastasis. Thus, it is important for researchers to screen biomarkers and to clarify the potential mechanisms underlying CRC recurrence/metastasis. GRIM-19 was recently reported to be a potential tumor suppressor [8]. More recently, various reports describe a loss or decrease in GRIM-19 expression in colorectal, prostate, breast and renal cell carcinomas. For example, Gong et al observed much lower mRNA expression of GRIM-19 in 23 colorectal tissues compared to normal tissues [17]. In a study by Zhang et al [23], the authors demonstrated by immunostaining that GRIM-19 expression
levels were also reduced in primary prostate cancer. Similar results were found in breast carcinomas [24] and renal cell carcinomas [8,25]. In the present study, our results confirmed that the mRNA and protein expression of GRIM-19 were both significantly decreased in CRC tissues. These data are strongly consistent with a previous publication by Gong et al [17]. However, the relationship between GRIM-19 expression and its prognostic value in CRC remains unclear. Our data suggest that a lack of GRIM-19 expression is common in female patients, a finding that should be further investigated in a large-scale, multicenter clinical study. Moreover, our results indicate that GRIM-19 expression is down-regulated in recurrent/ metastatic CRC patients compared with non-recurrent/ metastatic patients. A clinicopathological association analysis indicates that low GRIM-19 expression in tissue sections is strongly associated with poor tumor differentiation, the presence of lymph nodes, metastasis to other organs and vascular invasion. K-ras is a well-known oncogene considered to participate in the progression of CRC [26]. In this study, we found a highly significant relationship between reduced expression of GRIM-19 and overexpression of K-ras, suggesting that GRIM-19 might be a tumor suppressor in CRC. In addition, we provided evidence that lower GRIM-19 expression levels in CRC patients is significantly associated with a poorer 5-year survival and a high rate of recurrent/ metastatic disease. Taken together, these results indicate that GRIM-19 might be a potential prognosis marker for predicting recurrence/metastasis in CRC patients. Currently, there is controversy concerning the cellular localization of GRIM-19. Originally, it was observed primarily in the nucleus of HeLa cells [7]. However, recent IHC studies have reported that GRIM-19 in both the nucleus and cytoplasm in hepatocellular carcinoma [15], breast carcinoma [24], and
6 lung carcinoma [12]. The results of the present study showed for the first time that GRIM-19 is predominantly found in the cytoplasm and nucleus in normal and adenoma tissues but only in the cytoplasm in CRC tissues (Fig. 1). Lufei et al proposed that GRIM-19 may be distributed among different organelles depending on the cell type and that its cellular location may be also regulated by phosphorylation and acetylation [9]. Colorectal cancer frequently occurs in pre-existing adenomas, and its carcinogenesis is well known as a normaladenoma-carcinoma sequence. Many studies have suggested that the differential expression of certain genetic factors between normal and carcinoma tissues might provide insight into molecular carcinogenesis, such as that of galecin-4 in hepatocellular carcinoma [18] and GATA4 in breast carcinoma [27]. However, surprisingly little expression profiling data have been reported in adenoma tissues to date. Our results suggest that mRNA and protein levels of GRIM-19 in adenoma tissues are similar to those in normal tissues but higher than those in tumor tissues. Thus, expression changes of GRIM-19 in the adenoma-carcinoma sequence might play an important role in the carcinogenesis of CRC and deserve further investigation. In conclusion, our study confirms that GRIM-19 expression level is significantly reduced in CRC tissues, with lower GRIM-19 expression in CRC patients being associated with more aggressive characteristics of CRC. These results indicate that GRIM-19 may serve as a novel potential prognosis biomarker for CRC.
References [1] Jemal A, Bray F, Center MM, et al. Global cancer statistics. CA Cancer J Clin 2011;61:69-90. [2] Cunningham D, Atkin W, Lenz HJ, et al. Colorectal cancer. Lancet 2010;20:1030-47. [3] Brenner H, Kloor M, Pox CP. Colorectal cancer. Lancet 2014;26: 1490-502. [4] Mylonas CC, Lazaris AC. Colorectal cancer and basement membranes: clinicopathological correlations. Gastroenterol Res Pract 2014;2014:1-7. [5] Chen Y, Lu H, Liu Q, et al. Function of GRIM-19, a mitochondrial respiratory chain complex I protein, in innate immunity. J Biol Chem 2012;287:27227-35. [6] Lu H, Cao X. GRIM-19 is essential for maintenance of mitochondrial membrane potential. Mol Biol Cell 2008;19:1893-902. [7] Angell JE, Lindner DJ, Shapiro PS, et al. Identification of GRIM-19, a novel cell death-regulatory gene induced by the interferon-β and retinoic acid combination, using a genetic approach. J Biol Chem 2000;275:33416-26. [8] Moreira S, Correia M, Soares P, et al. GRIM-19 function in cancer development. Mitochondrion 2011;11:693-9.
M. Hao et al. [9] Lufei C, Ma J, Huang G, et al. GRIM‐19, a death‐regulatory gene product, suppresses Stat3 activity via functional interaction. EMBO J 2003;22:1325-35. [10] Tammineni P, Anugula C, Mohammed F, et al. The import of the transcription factor STAT3 into mitochondria depends on GRIM-19, a component of the electron transport chain. J Biol Chem 2013;288:4723-32. [11] Kong D, Zhao L, Du Y, et al. Overexpression of GRIM-19, a mitochondrial respiratory chain complex I protein, suppresses hepatocellular carcinoma growth. Int J Clin Exp Pathol 2014;7:7459-507. [12] Fan XY, Jiang ZF, Cai L, et al. Expression and clinical significance of GRIM-19 in lung cancer. Med Oncol 2012;29:3183-9. [13] Zhou Y, Li M, Wei Y, et al. Down-regulation of GRIM-19 expression is associated with hyperactivation of STAT3-induced gene expression and tumor growth in human cervical cancers. J Interferon Cytokine Res 2009;29:695-703. [14] He X, Cao X. Identification of alternatively spliced GRIM-19 mRNA in kidney cancer tissues. J Hum Genet 2010;55:507-11. [15] Li F, Ren W, Zhao Y, et al. Downregulation of GRIM-19 is associated with hyperactivation of p-STAT3 in hepatocellular carcinoma. Med Oncol 2012;29:3046-54. [16] Zhang Y, Hao H, Zhao S, et al. Downregulation of GRIM-19 promotes growth and migration of human glioma cells. Cancer Sci 2011;102:1991-9. [17] Gong LB, Luo XL, Liu SY, et al. Correlations of GRIM-19 and its target gene product STAT3 to malignancy of human colorectal carcinoma. Ai Zheng 2007;26:683-7. [18] Cai Z, Zeng Y, Xu B, et al. Galectin-4 serves as a prognostic biomarker for the early recurrence/metastasis of hepatocellular carcinoma. Cancer Sci 2014;105:1510-7. [19] You J, Liu WY, Zhu GQ, et al. Metabolic syndrome contributes to an increased recurrence risk of non-metastatic colorectal cancer. Oncotarget 2015;6:1-12. [20] Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Method 2001;25:402-8. [21] Konno R, Yamakawa H, Utsunomiya H, et al. Expression of survivin and Bcl-2 in the normal human endometrium. Mol Hum Reprod 2000; 6:529-34. [22] Lu C-D, Altieri DC, Tanigawa N. Expression of a novel antiapoptosis gene, survivin, correlated with tumor cell apoptosis and p53 accumulation in gastric carcinomas. Cancer Res 1998;58:1808-12. [23] Zhang L, Gao L, Li Y, et al. Effects of plasmid-based Stat3-specific short hairpin RNA and GRIM-19 on PC-3 M tumor cell growth. Clin Cancer Res 2008;14:559-68. [24] Zhou T, Chao L, Rong G, et al. Down-regulation of GRIM-19 is associated with STAT3 overexpression in breast carcinomas. HUM PATHOL 2013;44:1773-9. [25] Alchanati I, Nallar SC, Sun P, et al. A proteomic analysis reveals the loss of expression of the cell death regulatory gene GRIM-19 in human renal cell carcinomas. Oncogene 2006;25:7138-47. [26] Bozkurt O, Inanc M, Turkmen E, et al. Clinicopathological characteristics and prognosis of patients according to recurrence time after curative resection for colorectal cancer. Asian Pac J Cancer Prev 2014;15:9277-81. [27] Takagi K, Moriguchi T, Miki Y, et al. GATA4 immunolocalization in breast carcinoma as a potent prognostic predictor. Cancer Sci 2014; 105:600-7.
www.elsevier.com/locate/humpath
Original contribution
GRIM-19 expression is a potent prognostic marker in colorectal cancer☆,☆☆ Miao Hao PhD a , Zhenbo Shu MD b , Hongyan Sun MD c , Ran Sun MD a , Yuqian Wang PhD a , Tie Liu MD c , Degang Ji MD b , Xianling Cong MD c,⁎ a
Science Research Center, China-Japan Union Hospital of Jilin University, Changchun 130033, China Department of Surgery, China-Japan Union Hospital of Jilin University, Changchun 130033, China c Tissue Bank, China-Japan Union Hospital of Jilin University, Changchun 130033, China b
Received 13 May 2015; revised 21 July 2015; accepted 23 July 2015
Keywords: GRIM-19; Colorectal cancer; Immunohistochemistry; Prognostic biomarker; Tumor suppressor
Summary Retinoid-interferon-induced mortality-19 (GRIM-19), a recently discovered cell death regulatory gene, may function as a tumor suppressor in many human malignancies. However, the expression of GRIM-19 in and its prognostic value for patients with colorectal cancer (CRC) have not been well investigated to date. Here, GRIM-19 expression was measured immunohistochemically in 94 colon samples and by quantitative real-time reverse transcriptase polymerase chain reaction in 15 paired CRC tissues and adjacent normal tissues. The prognostic significance was assessed using Kaplan-Meier survival estimates and log-rank tests. Our results showed that GRIM-19 mRNA and protein levels in adenoma tissues were similar to those in adjacent normal tissues. However, GRIM-19 expression was severely depressed in carcinomas compared to matched normal tissues (P = .000). Additionally, we found GRIM-19 to be located in both the cytoplasm and nucleus in normal tissues but only in the cytoplasm in CRC tissues. Alteration in GRIM-19 expression occurs early in the pathogenesis of CRC; moreover, low GRIM-19 expression was associated with poor tumor differentiation (P = .013), the presence of lymph nodes (P = .000), metastasis to other organs (P = .045) and vascular invasion (P = .010). During a mean period of 40 months follow-up, patients without GRIM-19 had a statistically significantly lower rate of recurrence/metastasis (P b .05) and a shorter overall survival time (P b .01) than the patients with GRIM-19 expression. Taken together, GRIM-19 expression is closely associated with CRC progression and might be a very promising prognostic biomarker for CRC patients. © 2015 Elsevier Inc. All rights reserved.
1. Introduction ☆
Funding/Support: This work was supported by the National Natural Science Foundation of China (Grant Nos. 81371595 and 81401966), the research project of Jilin Provincial Science and Technology Department (Nos. 20130206001YY, 20140414061GH and 20150204082SF). ☆☆ The authors have no conflict of interest. ⁎ Corresponding author. Tissue Bank, China-Japan Union Hospital of Jilin University, 126 Xiantai Street, Changchun, Jilin 130033, China. E-mail address: [email protected] (X. Cong). http://dx.doi.org/10.1016/j.humpath.2015.07.020 0046-8177/© 2015 Elsevier Inc. All rights reserved.
Colorectal cancer (CRC) is the third most commonly diagnosed cancer in males and the second in females. In 2008, more than 1.2 million people were diagnosed with CRC, and more than 600 000 died from the disease [1]. Indeed, despite improvements in surgical techniques and drug therapy, the 5-year survival rate of patients with CRC
2 remains low [2,3]. Because of development after resection, tumor recurrence and metastasis are virtually inevitable and are a major cause of death in CRC patients. In addition to diagnosis and treatment, prognosis is an important matter for cancer patients. Therefore, it is urgent to identify possible therapeutic or prognostic biomarkers and to elucidate their relationship with CRC while considering relevant clinicopathological correlations [4]. GRIM-19 (Gene associated with Retinoid-Interferon induced Mortality–19), which is present in both the nuclear and cytoplasmic compartments, is a subunit of complex I of the mitochondrial membrane respiratory chain [5,6]. Recently, the GRIM19 gene has been identified as an apoptosis-related gene that represents a novel type of tumor suppressor [7,8]. The GRIM-19 protein binds to signal transducer and activator of transcription–3 to inhibit its transcriptional activity [9,10]. It has been reported that the overexpression of GRIM-19 suppresses hepatocellular carcinoma growth [11] and induces apoptosis in human breast carcinoma cell line MCF-7 [7]. Conversely, GRIM-19 expression is lost or significantly reduced in many human malignancies, such as lung cancer [12], cervical cancer [13], kidney cancer [14], hepatocellular carcinoma [15] and gliomas [16]. Regarding colorectal carcinoma, only Gong et al have demonstrated that GRIM-19 is severely reduced at the mRNA level [17]. However, the prognostic significance of GRIM-19 expression and its association with clinicopathologic features have not yet been reported in CRC. In this study, we used immunohistochemistry (IHC) and quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) analyses to evaluate the expression of GRIM-19 in patients with colorectal carcinoma and to systematically elucidate the prognostic relevance of GRIM-19 expression and correlations with clinicopathologic features. Our results indicated that GRIM-19 might serve as a prognostic biomarker in colorectal carcinoma.
2. Materials and methods
M. Hao et al. demographic and clinicopathologic data of all patients, including age, gender, tumor size, tumor location, histologic differentiation, depth of invasion, lymph node stage, and vascular invasion, were collected from the patients' medical records. This study design, including the use of all archival tissues, was approved by the ethical committee of the China-Japan Union Hospital of Jilin University. The effect of GRIM-19 expression on the time to recurrence/metastasis and the overall survival (OS) time was calculated by the Kaplan-Meier method and analyzed by the log-rank test [18,19]. Follow-up data were summarized at the end of December 2014, with a median follow-up of 40 months (range, 1-42 months). Time to recurrence/metastasis and OS were considered to be the primary endpoints. Time to recurrence/metastasis was calculated from the date of resection to the date when tumor recurrence/metastasis was diagnosed. Overall survival was calculated from the date of resection to the date of death or last follow-up.
2.2. qRT-PCR Total RNA was isolated from the tissue samples using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Reverse transcription was carried out using a Goscript Reverse Transcription System Kit (A5000; Promega, Madison, Wisconsin, USA) according to the manufacturer's instructions. mRNA levels were quantified by qRT-PCR with the Quantitect SYBR Green PCR Kit (Roche, Mannheim, Germany) and normalized to Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) using the following primers: GRIM-19-F, 5′ACCGGAAGTGTGGGATACTG-3′, and GRIM-19-R, 5′-GCTCACGGTTCCACTTCATT-3′; GAPDH-F, 5′AGAAGGCTGGGGCTCATTTG-3′, and GAPDH-R, 5′-AGGGGCCATCCACAGTCTTC-3′. The cycling parameters were 40 cycles of 95°C for 15 seconds, 60°C for 15 seconds, and 72°C for 30 seconds. Relative gene expression was normalized to the geometric mean of the housekeeping gene GAPDH and calculated according to the Livak method (2−ΔΔCt) [20]. The experiments were independently repeated three times.
2.1. Patients, tissue specimens and follow-up 2.3. Immunohistochemical analysis Pairs of primary CRC tissues and adjacent normal colorectal tissues were obtained from 94 patients; adenoma tissues and adjacent normal tissues were obtained from 3 patients undergoing curative surgical operations at the China-Japan Union Hospital of Jilin University (Changchun, China). No patients received chemotherapy or radiotherapy prior to surgery. The matched normal colorectal tissues were obtained from a segment of the resected specimens that was N5 cm away from the tumor. All tissues were fixed in 10% neutral-buffered formalin and embedded in paraffin blocks for IHC. In addition, 15 of the paired tissues obtained immediately after surgery were snap-frozen in liquid nitrogen and kept at −80°C for further analysis. The
Expression of GRIM-19 and K-ras was determined by IHC staining of the tissue sections using mouse anti– GRIM-19 monoclonal antibody (ab110240, Abcam, Hong Kong, China) and rabbit anti–K-ras polyclonal antibody (RAB-0638; Maxim, China). All specimens were fixed with 4% formaldehyde, dehydrated, embedded, and cut into 4-μm serial sections. Endogenous peroxidase activity was blocked by incubation with 3% hydrogen peroxide in methanol for 10 minutes. For high-pressure antigen retrieval, the sections were washed 3 times with phosphate-buffered saline and incubated in 0.01 mmol/L sodium citrate buffer (pH 6.0) for 3 minutes. The sections were then incubated with the primary
GRIM-19 serves as a prognostic biomarker for colorectal cancer antibody (1:300 dilution) at 4°C overnight. After 3 rinses with phosphate-buffered saline, the sections were incubated with the corresponding secondary antibody for 10 minutes at room temperature. The antigen-antibody complexes were detected by the streptavidin-biotin-peroxidase method using diaminobenzidine as the chromogenic substrate (Maxim, Fuzhou, China). Finally, the sections were counterstained with 10% hematoxylin, dehydrated, mounted, and examined by light microscopy. In all cases, negative controls were performed by omitting the primary antibody.
2.4. Evaluation of immunohistochemical staining Immunohistochemical staining was blindly scored by two pathologists. The GRIM-19 protein was assessed in both the cytoplasm and nucleus. The overall amount of staining was determined by the staining intensity and the proportion/extent of stained tumor cells according to published scoring methods [21,22]. The mean percentage of positive tumor cells was determined in at least five areas at 400× magnification and assigned to one of the following categories: 0, ≤5%; 1, 5%-25%; 2, 25%-50%; 3, 50%-75%; 4, ≥75%. The average estimated intensity of staining in positive cells was scored as 1 (weak), 2 (moderate) or 3 (intense). The total score was
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calculated by multiplying the scores for intensity and the extent of staining. A staining score of higher than 2 for GRIM-19 expression was considered positive, whereas a score below 2 was considered negative.
2.5. Statistical analysis All the statistical analyses were carried out using SPSS 17.0 Software (SPSS, Chicago, IL). The results are expressed as the mean ± SD. The recurrence/metastasis rates and survival rates were calculated by the Kaplan-Meier method and compared using the log-rank test. Categorical data between GRIM-19 expression and clinicopathologic features were analyzed by the χ2 test or Fisher's exact test. P b .05 was considered to be significant.
3. Results 3.1. Differential expression of GRIM-19 in normal colorectal tissues and CRC tissues To investigate the role of GRIM-19 in colorectal carcinoma, we evaluated its expression by immunohistochemical analysis
Fig. 1 IHC analysis of GRIM-19 expression (A, C, E, G: original magnification ×200; B, D, F, H: ×400; I: ×100). The expression of GRIM-19 in adjacent normal tissues (score 2: A and B; score 6: C and D) and tumor tissues (score 2: E and F; score 6: G and H). Differential expression of GRIM-19 in colon adenoma and carcinoma tissues (I; adenoma tissue: red arrow; adjacent adenocarcinoma tissue: black arrow).
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Fig. 2 Relative expression levels of GRIM-19 mRNA in 15 paired adjacent normal colorectal tissues and CRC tissues (A) and 3 paired adjacent normal tissues and adenoma tissues (B), as shown by real-time PCR assay. The relative expression data were analyzed by the 2−ΔΔCt method. GAPDH was used as an internal control. *P b .05, **P b .01.
Table Correlation between expression of GRIM-19 and clinicopathologic features of colorectal carcinoma Characteristic
n
GRIM-19 (%)
n = 94 Positive (n = 21) Age, y b65 ≥65 Gender Male Female Tumor size, cm b5 ≥5 Tumor location Colon Rectum Histologic differentiation Well and Moderate Poor Depth of invasion Tis-T2 T3-T4 Lymph node metastasis Yes No Metastasis to other organs Present Absent Vascular invasion Yes No K-ras Positive Negative ⁎ P b .05; ⁎⁎ P b .01.
P
Negative (n = 73)
56 38
16 (76.2) 40 (54.8) .078 5 (23.8) 33 (45.2)
61 33
18 (85.7) 43 (58.9) .023 ⁎ 3 (14.3) 30 (41.1)
39 55
6 (28.6) 33 (45.2) .173 15 (71.4) 40 (54.8)
48 46
9 (42.9) 39 (53.4) .393 12 (57.1) 34 (46.6)
70 24
20 (95.2) 50 (68.5) .013 ⁎ 1 (4.8) 23 (31.5) .011
3 91
1 (4.8) 2 (2.7) .536 20 (95.2) 71 (97.3)
60 34
6 (28.6) 54 (74.0) .000 ⁎⁎ 15 (71.4) 19 (26.0)
45 49
6 (28.6) 39 (53.4) .045 ⁎ 15 (71.4) 34 (46.6)
41 53
4 (19.0) 37 (50.7) .010 ⁎ 17 (81.0) 36 (49.3)
28 66
2 (9.5) 26 (35.6) .021 ⁎ 19 (90.5) 47 (64.4)
in 94 patients. Among the 94 paired tissues, GRIM-19 expression was detected in 80 normal tissues (85.1%) and 21 tumor tissues (22.3%). IHC revealed a more frequent absence of GRIM-19 expression in CRC tissues than in adjacent nontumorous tissues (P = .000). We first confirmed the location of GRIM-19 in colon tissues. As shown in Fig. 1, the GRIM-19 protein was primarily located in the cytoplasm, with slightly weak staining in the nucleus (Fig. 1D), but was found only in the cytoplasm in CRC tissues (Fig. 1H). We also observed that GRIM-19 was highly expressed in non-tumor colonic epithelial cells (Fig. 1C and D). However, most CRC tissues showed a lack of GRIM-19 expression (Fig. 1E and F). In addition, we found that GRIM-19 was also highly expressed in adenoma tissue (Fig. 1F, red arrow), with weak staining in adjacent adenocarcinoma tissue (Fig. 1F, black arrow). Our qRT-PCR analysis further confirmed that in CRC patients, GRIM-19 mRNA was significantly decreased in tumor tissues in 73.3% (11/15) compared with matched non-tumor tissues (Fig. 2A). In addition, GRIM-19 mRNA expression in adenoma tissues was similar to that in adjacent normal tissue (Fig. 2B). These data are in line with the results of IHC analysis.
3.2. Correlation of GRIM-19 expression levels with clinicopathologic features of CRC The potential association between GRIM-19 expression and clinicopathologic features in 94 CRC patients is presented in the Table. In female patients, negative GRIM-19 expression was more common than positive GRIM-19 expression (41.1% versus 14.3%; P = .023). Moreover, a lack of GRIM-19 expression was significantly associated with poor tumor differentiation (P = .013), lymph node metastasis (P = 0), metastasis to other organs (P = .045) and vascular invasion (P = .010). Expression of K-ras was more common in cancers without GRIM-19 expression (P = .021). No significant correlation with GRIM-19 expression was found for age, tumor size, tumor location or depth of invasion (Table).
GRIM-19 serves as a prognostic biomarker for colorectal cancer
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Fig. 3 Kaplan-Meier analysis of the association between GRIM-19 expression and recurrence/metastasis (A) as well as the survival (B) of patients with colorectal carcinoma. The patients without GRIM-19 expression (n = 73) had a significantly higher recurrence rate and lower overall survival rate than those with positive GRIM-19 expression (n = 21).
3.3. Lower GRIM-19 expression is associated with poor prognosis of CRC To further evaluate the prognostic value of GRIM-19 expression in colorectal carcinoma, we used Kaplan-Meier analysis to calculate the effect of GRIM-19 expression on the time to recurrence/metastasis and OS time. As shown in Fig. 3A, CRC patients without GRIM-19 expression had a higher recurrence/metastasis rate (P b .05, log-rank test) than those with positive GRIM-19 expression. Moreover, as shown in Fig. 3B, there was a trend of better OS for patients with GRIM-19 expression compared with those without GRIM-19, and the difference between the two survival curves was statistically significant (P b .01, log-rank test). These results indicate that GRIM-19 might be a very promising prognostic biomarker for colorectal carcinoma.
4. Discussion CRC is a malignant tumor in humans, and the poor 5-year survival in CRC patients after curative resection is mainly due to the high rate of recurrence and/or metastasis. Thus, it is important for researchers to screen biomarkers and to clarify the potential mechanisms underlying CRC recurrence/metastasis. GRIM-19 was recently reported to be a potential tumor suppressor [8]. More recently, various reports describe a loss or decrease in GRIM-19 expression in colorectal, prostate, breast and renal cell carcinomas. For example, Gong et al observed much lower mRNA expression of GRIM-19 in 23 colorectal tissues compared to normal tissues [17]. In a study by Zhang et al [23], the authors demonstrated by immunostaining that GRIM-19 expression
levels were also reduced in primary prostate cancer. Similar results were found in breast carcinomas [24] and renal cell carcinomas [8,25]. In the present study, our results confirmed that the mRNA and protein expression of GRIM-19 were both significantly decreased in CRC tissues. These data are strongly consistent with a previous publication by Gong et al [17]. However, the relationship between GRIM-19 expression and its prognostic value in CRC remains unclear. Our data suggest that a lack of GRIM-19 expression is common in female patients, a finding that should be further investigated in a large-scale, multicenter clinical study. Moreover, our results indicate that GRIM-19 expression is down-regulated in recurrent/ metastatic CRC patients compared with non-recurrent/ metastatic patients. A clinicopathological association analysis indicates that low GRIM-19 expression in tissue sections is strongly associated with poor tumor differentiation, the presence of lymph nodes, metastasis to other organs and vascular invasion. K-ras is a well-known oncogene considered to participate in the progression of CRC [26]. In this study, we found a highly significant relationship between reduced expression of GRIM-19 and overexpression of K-ras, suggesting that GRIM-19 might be a tumor suppressor in CRC. In addition, we provided evidence that lower GRIM-19 expression levels in CRC patients is significantly associated with a poorer 5-year survival and a high rate of recurrent/ metastatic disease. Taken together, these results indicate that GRIM-19 might be a potential prognosis marker for predicting recurrence/metastasis in CRC patients. Currently, there is controversy concerning the cellular localization of GRIM-19. Originally, it was observed primarily in the nucleus of HeLa cells [7]. However, recent IHC studies have reported that GRIM-19 in both the nucleus and cytoplasm in hepatocellular carcinoma [15], breast carcinoma [24], and
6 lung carcinoma [12]. The results of the present study showed for the first time that GRIM-19 is predominantly found in the cytoplasm and nucleus in normal and adenoma tissues but only in the cytoplasm in CRC tissues (Fig. 1). Lufei et al proposed that GRIM-19 may be distributed among different organelles depending on the cell type and that its cellular location may be also regulated by phosphorylation and acetylation [9]. Colorectal cancer frequently occurs in pre-existing adenomas, and its carcinogenesis is well known as a normaladenoma-carcinoma sequence. Many studies have suggested that the differential expression of certain genetic factors between normal and carcinoma tissues might provide insight into molecular carcinogenesis, such as that of galecin-4 in hepatocellular carcinoma [18] and GATA4 in breast carcinoma [27]. However, surprisingly little expression profiling data have been reported in adenoma tissues to date. Our results suggest that mRNA and protein levels of GRIM-19 in adenoma tissues are similar to those in normal tissues but higher than those in tumor tissues. Thus, expression changes of GRIM-19 in the adenoma-carcinoma sequence might play an important role in the carcinogenesis of CRC and deserve further investigation. In conclusion, our study confirms that GRIM-19 expression level is significantly reduced in CRC tissues, with lower GRIM-19 expression in CRC patients being associated with more aggressive characteristics of CRC. These results indicate that GRIM-19 may serve as a novel potential prognosis biomarker for CRC.
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