Med Oncol (2012) 29:3046–3054 DOI 10.1007/s12032-012-0234-8

ORIGINAL PAPER

Downregulation of GRIM-19 is associated with hyperactivation of p-STAT3 in hepatocellular carcinoma Feifei Li • Wanhua Ren • Yanda Zhao • Zhaoqing Fu • Yongjian Ji • Yuhua Zhu Chengyong Qin

•

Received: 22 March 2012 / Accepted: 28 March 2012 / Published online: 11 April 2012 Ó Springer Science+Business Media, LLC 2012

Abstract Accumulating evidence has implicated that constitutive activation of signal transducer and activator of transcription protein 3 (STAT3) may be a major oncogenic factor involved in hepatocellular carcinoma (HCC) development. Gene associated with retinoid-interferon-induced mortality-19 (GRIM-19) has been shown to be a tumor suppressor associated with growth control and suppression of STAT3 activity. The downregulation of GRIM-19 expression has been shown in a number of human tumor types, and it has been correlated with hyperactivation of STAT3. However, the role of GRIM-19 in the pathogenesis of HCC has not been evaluated. The aim of our study was to evaluate GRIM-19 expression levels and investigate their correlation with phosphorylated STAT3 (p-STAT3) levels in HCC. GRIM-19 and p-STAT3 expression levels were analyzed in HCC and adjacent nontumorous liver tissues (ANLT) by immunohistochemistry, western blot analysis, and RT-PCR. GRIM-19 protein expression was predominantly located in the cytoplasm with weak staining in the nucleus in ANLT, but only located in the cytoplasm in HCC tissues. HCC samples exhibited low levels of GRIM-19 and moderate to high levels of p-STAT3 F. Li  W. Ren  Y. Zhao  Y. Zhu  C. Qin (&) Department of Gastroenterology, Provincial Hospital Affiliated to Shandong University, 324 Jingwu Weiqi Road, Jinan 250021, China e-mail: [email protected] Z. Fu Department of Pathology, PLA No. 456 Hospital, 25 Wuyingshan Road, Jinan 250031, China Y. Ji Department of Pathology, Provincial Hospital Affiliated to Shandong University, 324 Jingwu Weiqi Road, Jinan 250021, China

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expression. In contrast, ANLT was characterized by high levels of GRIM-19 and low levels of p-STAT3 expression. Downregulation of GRIM-19 was closely correlated with increased histological grade in HCC. GRIM-19 expression is closely correlated with histological grading and p-STAT3 in HCC. Thus, the potential role of GRIM-19 in HCC development may be through these correlations. Keywords GRIM-19  p-STAT3  Phosphorylated signal transducer and activator of transcription protein 3  Hepatocellular carcinoma

Introduction Hepatocellular carcinoma (HCC) is the fifth most common malignancy in the world, and it is estimated to cause approximately half a million deaths annually [1]. Although the underlying mechanisms of hepatocellular carcinogenesis remain to be investigated, accumulating evidence has indicated that signal transducer and activator of transcription (STAT) proteins, in particular STAT3, may be directly or indirectly involved in HCC development [2, 3]. Constitutively activated STAT3, through its phosphorylation at tyrosine residue 705, has been reported in many human cancers, including carcinomas of the breast, lung, prostate, ovary, and melanoma [4–8]. In general, activated STAT3 participates in carcinogenesis by promoting angiogenesis or stimulating cell proliferation [9–11]. Recently, activated STAT3 has been shown in the progression of viral hepatitis and oncogenesis associated with hepatitis B virus (HBV) and hepatitis C virus (HCV) in the liver [12–17]. In addition, STAT3 activity is also increased in chemicallyinduced HCC [18]. Taken together, these findings suggest that constitutively activated STAT3 may play a crucial role

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in HCC development. However, the consequences of constitutive phosphorylation of STAT3 and the mechanisms that trigger this constitutive activation require further investigation. In recent years, several studies have evaluated the gene associated with retinoid-interferon-induced mortality-19 (GRIM-19). GRIM-19 is a novel gene product involved in interferon-b/retinoic acid (IFN-b/RA)-induced apoptosis, and it suppresses STAT3-induced gene expression [19]. In addition, GRIM-19 plays a major role in the control of cell growth through regulation of STAT3. Lufei et al. [20] have reported that GRIM-19 binds to STAT3 and inhibits its transcriptional activation function. Inhibition of STAT3 activation by GRIM-19 appears to be an important step in oncogenesic suppression [21]. Furthermore, constitutive activation of STAT3 promotes anti-apoptotic gene expression to support tumor survival [22]. Low expression levels of GRIM-19 have been shown in renal, prostate, esophageal, and colonic tumors [23]. However, GRIM-19 expression levels and the associated pathologies have not been reported for liver tumors. In the current study, we demonstrate a significant downregulation of GRIM-19 protein levels in HCC that was associated with increased expression of p-STAT3 and downstream target genes. In addition, we present novel findings that downregulation of GRIM-19 and hyperactivation of p-STAT3 expression in HCC lesions were closely correlated with an increased histological grading in HCC.

Materials and methods Tissue specimens Liver tissue specimens from 55 HCCs, removed by surgical resection, and four nonneoplastic, noncirrhotic liver wedge biopsies were obtained from the Provincial Hospital affiliated with Shandong University, Shandong, China. Freshly excised tissues were frozen at -80 °C for RNA and protein analyses. For pathological diagnosis, a portion of the tissue was paraffin-embedded and subsequently cut into 3 um thick sections. One section from each specimen was stained with hematoxylin and eosin (H&E) staining and examined in a double-blinded study by two pathologists (Fu ZQ and Ji YJ) to confirm the diagnosis of HCC. The adjacent nontumorous liver tissue (ANLT) specimen was taken from liver tissue that was more than 5 cm from the edge of the tumor and contained no tumor cells as confirmed by pathological examination. Of the 55 HCC patients, 43 were men and 12 were women. Their ages ranged from 38 to 80 years with a median age of 58 years. The etiology of the subjects was as follows: 39 were hepatitis B virus-positive, six were hepatitis C virus-positive, 8 had alcoholic

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cirrhosis, and two were cryptogenic. The tumor size ranged between 0.6–10.5 cm with an average diameter of 3.8 cm. Among the HCC specimens, 13 were positive for lymph node metastasis, and 42 had no lymph node metastasis. The tumors were graded according to the WHO criteria and included 12 well-differentiated (grade 1), 34 moderately differentiated (grade II), and nine poorly differentiated (grade III) HCC. None of the patients underwent chemotherapy or radiotherapy prior to the surgical removal of the tumor. The Investigation and Ethics Committee of the Provincial Hospital affiliated with Shandong University approved the study and informed consent was obtained from each patient. Immunohistochemistry Immunohistochemistry was performed as previously described [24]. All specimens were fixed with 4 % formaldehyde, dehydrated, embedded, and cut into 3 um serial sections. Endogenous peroxidase activity was blocked by treatment with 3 % hydrogen peroxide for 15 min. The sections were washed three times with phosphate buffer solution (PBS) followed by incubation with the appropriate dilutions of various antibodies overnight at 4 °C. Mouse anti-human GRIM-19 antibody (Abcam, Hong Kong) and mouse anti-human p-STAT3 (Tyr705) antibody (Cell Signaling, Beverly, MA, USA) were utilized. Sections were washed three times in PBS and then incubated with a biotinylated secondary antibody for 20 min at room temperature. Antigen–antibody complexes were detected by the avidin–biotin–peroxidase method using diaminobenzidine as the chromogenic substrate (Dako, Carpinteria, CA, USA). Finally, the sections were counterstained with hematoxylin, dehydrated, mounted, and examined by light microscopy. Negative controls were performed in all cases by omitting the primary antibodies. Western blot analysis Tissues were collected and lysed. The protein concentration was determined with the BCA reagent (Pierce, Rockford, IL, USA). Equal amounts of protein were subjected to electrophoresis on an 8–15 % SDS-PAGE gel and then transferred to a nitrocellulose membrane (Schleicher & Schuell BioScience GmbH, Germany). After blocking with 5 % nonfat milk (for GRIM-19, STAT3, and VEGF) or 5 % BSA (for p-STAT3) in 10 mM TrisCl (pH 8.0), 150 mM NaCl, and 0.05 % Tween 20 (TBST) for 1 h, the membranes were incubated with various antibodies overnight at 4 °C. The following antibodies were used: mouse polyclonal anti-human GRIM-19 antibody (Abcam, Hong Kong), mouse monoclonal anti-human STAT3 (Abcam, Hong Kong), mouse monoclonal anti-human p-STAT3

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(Tyr705) (Cell Signaling, Beverly, MA, USA), and rabbit polyclonal anti-human VEGF (Abcam, Hong Kong). The membranes were washed three times (10 min each) with PBS containing 0.1 % Tween 20 and incubated with secondary antibody (IgG) conjugated with horseradish peroxidase (Amersham International). Laminin (Abcam, Hong Kong) and b-actin (Santa Cruz, CA, USA) were used as a control to demonstrate equal loading and protein transfer. Protein bands were visualized with the enhanced chemiluminescence (ECL) detection system (Amersham Life Science Inc., Arlington Heights, IL, USA). Reverse transcription-PCR analysis Total RNA was isolated by using the Trizol reagent (Invitrogen) and purified by acid-phenol. The reverse transcription was performed by incubating 2–3 ug of total RNA, 2.5 uM random primer, and 200 units of Superscript II reverse transcriptase (Invitrogen) in a 20 ul reaction volume for 1 h at 42 °C. PCR amplification was performed using two pairs of primers in the same reaction: one primer pair, 50 -GACTACAAACGGAAC TTGCC-30 and 50 GAGCCTCCTCTGTGGTGC-30 , was used to amplify a 343 bp particular gene fragment (GRIM-19), and another primer pair, 50 -CTGGGACGACATGGAGAAAA-30 and 50 - AAGGAAGGCTGGAAGAGTGC-30 , was used to amplify a 564 bp fragment that served as an internal control (b-actin). For semiquantitative results, the PCR cycle number was adjusted to anywhere from 28 to 35. The PCR products were visualized on 2 % agarose gel containing 0.5 lg/ml ethidium bromides.

Fig. 1 GRIM-19 protein expression was predominantly located in the cytoplasm. a Weak staining was also noticeable in the nucleus in ANLT. b Negative staining for GRIM-19 in the nucleus in HCC tissues

Statistical analysis SPSS10.0 software was used for all statistical analyses. All data are expressed as mean ± SEM. Differences between groups were analyzed by the Student’s unpaired t-test when two groups were analyzed. Correlations between protein expression levels were evaluated by the Spearman rankorder correlation coefficient. A value of p \ 0.05 was considered significant.

located in the cytoplasm in HCC tissues. To confirm this observation, pure nuclear protein was extracted separately from HCC tissues and ANLT, and GRIM-19 protein expression was determined by western blot analysis. Consistent with the immunohistochemistry results, GRIM-19 expression levels in the nucleus was significantly higher in ANLT compared to HCC tissues (p \ 0.001). Data from two representative samples are presented in Fig. 5a.

Results

Lower expression levels of GRIM-19 in HCC compared to adjacent nontumorous liver tissue

Cellular localization of GRIM-19 in liver tissue We first analyzed the localization of GRIM-19 in liver tissues using a purified GRIM-19 antibody and an immunohistochemistry assay. As shown in Fig. 1, GRIM-19 protein expression was predominantly located in the cytoplasm with weak staining in the nucleus in ANLT, but only

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Since GRIM-19 was identified as a potential tumor suppressor in recent studies, we evaluated the expression status of GRIM-19 in primary liver cancers using immunohistochemistry. Briefly, the percentage of cells positively stained in each section were categorized as follows: negative (samples with B5 % positive cells), low (5–25 % positive cells), moderate (25–50 % positive cells), and

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Fig. 2 Scoring systems for GRIM-19 and p-STAT3 immunostaining. The percentage of cells positively stained in each section were categorized as follows: negative (samples with B 5 % positive cells), low (5–25 % positive cells), moderate (25–50 % positive cells), and

strong (50–100 % positive) were indicated as -, ?, ??, and ???. Sample classification: a and g well-differentiated HCC tissue, b and c nonneoplastic, noncirrhotic liver tissue, and (d, e, f, and h nonneoplastic but cirrhotic liver tissues. Original magnification 4009

strong (50–100 % positive) were indicated as -, ?, ??, and ???, respectively, as shown in Fig. 2. GRIM-19-positive staining was found in 3/4 (75 %) of the normal liver tissues, 40/55 (72.7 %) of the adjacent nontumorous liver tissues, and 25/55 (45.5 %) of the HCC tissues. GRIM-19 expression in the ANLT tissues was not significantly different between the nontumorous, noncirrhotic specimens (12/16, 75 %) (Fig. 3a) and the nontumorous but cirrhotic specimens (28/39, 71.8 %) (Fig. 3b). In HCC tissues, however, GRIM-19 expression was extremely reduced (Fig. 3c), and in some cases, even absent (Fig. 3d). In the cases with positive GRIM-19 expression, 23/65 (35.4 %) were determined to be ?? to ??? (by the ranking system described in the methods section); and among them, 20/23 (87 %) were ANLT. Thus, GRIM-19 protein expression was extremely lower in HCC tissues than in the ANLT. To verify this finding, we examined the expression of GRIM-19 mRNA using RT-PCR. Notably, the GRIM-19 mRNA expression levels were significantly lower in HCC tissues compared to the ANLT (p \ 0.001). Data from two representative samples are presented in Fig. 5b, c.

factor known to be inhibited by GRIM-19 binding. To explore the relationship between GRIM-19 and p-STAT3 expression in HCC, we evaluated the expression status of p-STAT3 in HCC samples. In HCC tissues, immunopositive p-STAT3 was confined to the nuclei of cancerous cells. Expression of p-STAT3 in nonneoplastic, noncirrhotic liver was absent (Fig. 4a), while positive staining was found in 14/39 (35.9 %) (Fig. 4b) of the cases with liver cirrhosis and in 47/55 (85.5 %) of the HCC samples (Fig. 4c, d). In the positively stained cases, 40/61 (65.6 %) were determined to be ?? to ???, and among them, 35/40 (87.5 %) cases were HCC tissues. These findings indicate that p-STAT3 protein expression is highly elevated in HCC tissues compared to the ANLT. These results suggest that an inverse correlation existed between the expression of GRIM-19 and p-STAT3 in HCC tissues and the ANLT. To investigate this relationship further, we examined the expression of GRIM-19 and p-STAT proteins in HCC tissues (n = 55) and the ANLT (n = 55) by western blot analysis. The expression of GRIM-19 protein was significantly decreased in all grades of HCC tissues (p \ 0.05), and the expression of p-STAT3 protein was significantly increased in all grades of HCC tissues (p \ 0.05) (Fig. 5d) compared to the b-actin control. Next, we examined the expression of total STAT3 and the p-STAT3 target protein VEGF in HCC tissues (n = 20) and the ANLT (n = 20). Consistent with the reduced GRIM-19 expression and the

Downregulation of GRIM-19 was associated with increased p-STAT3 in HCC tissue GRIM-19 is known to play an important role in the control of cell growth exerted through STAT3, which is a transcription

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Fig. 3 Expression of GRIM-19 in HCC and the ANLT. A and a Positive expression of GRIM-19 in nonneoplastic, noncirrhotic liver. B and b Increased cytoplasmic immunostaining of GRIM-19 in liver cirrhosis. C and c Low cytoplasmic expression of GRIM-19 in a

well-differentiated HCC. D and d Negative GRIM-19 expression in a poorly differentiated HCC. A, B, C, D: Original magnification 100c, a, b, c, d: Original magnification 4009

increased p-STAT3 expression that were found, total STAT3 expression (p = 0.008) and VEGF expression (p = 0.012) were significantly upregulated in the HCC tissues compared to the ANLT. Data from three representative samples are presented in Fig. 5e. Together, these results suggest that downregulation of GRIM-19 may be associated with increased p-STAT3 activity in HCC tissues.

Correlation of GRIM-19 expression levels with clinicopathological parameters

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We evaluated possible correlations of GRIM-19 mRNA expression in HCC tissues between different patient subgroups according to various clinicopathological parameters (Table 1). Two significant correlations of GRIM-19

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Fig. 4 P-STAT3 is overexpressed in HCC. a Negative p-STAT3 expression in nonneoplastic, noncirrhotic liver. b Positive nuclear expression of p-STAT3 in liver cirrhosis. Increased nuclear

immunostaining of p-STAT3 in cases of c well-differentiated HCC and d poorly differentiated HCC. Original magnification 4009

expression were observed for vascular invasion (p = 0.032) and histological grade (p = 0.001). No significant correlation of GRIM-19 mRNA expression was found for age, gender, HBV infection, presence of cirrhosis, tumor size, serum a-fetoprotein levels, or local lymph node metastasis (Table 1).

GRIM-19 expression may increase after interferon stimulation, and it may be released rapidly from the mitochondria into the cytoplasm and translocate to the nucleus. Lufei et al. [20] proposed that the distribution of GRIM-19 in mitochondria or nuclei may depend on the type of cells, and its location may be influenced by phosophorylation or acetylation. GRIM-19 may also have different subtypes that are located at different sites in cells. In our study, the expression levels of GRIM-19 mRNA and protein in the HCC tissues were significantly lower than in the ANLT. Similar to our findings, decreased GRIM-19 mRNA and protein expression has been previously demonstrated in renal cell and carcinoma research reported by Alchanati et al. [23] and in colorectal cancer research reported by Gong et al. [26]. GRIM-19 is a novel gene product involved in interferon-b/retinoic acid (IFN-b/ RA)-induced apoptosis, and it suppresses STAT3-induced gene expression [19]. In addition, GRIM-19 plays a major role in the control of cell growth through regulation of STAT3 [27]. We also showed that the HCC lesions exhibited a significantly higher nuclear expression of p-STAT3 compared to the ANLT. Most importantly, we found an inverse correlation between GRIM-19 and p-STAT3 expression in HCC tissues relative to the ANLT. Similar findings have been reported in human cervical cancers [28]. Constitutive

Discussion The importance of GRIM-19 in tumor growth regulation has been recently recognized. Since low expression of GRIM-19 occurs in renal, esophageal, prostate, and colonic tumors [23], we examined the status of GRIM-19 expression in HCC tissues and ANLT. The results from this study for the first time showed that the GRIM-19 protein expression was predominantly located in the cytoplasm with weak staining in the nucleus in ANLT, but only located in the cytoplasm in HCC tissues. Currently, there is controversy concerning the intracellular location of GRIM19. GRIM-19 expression was initially reported to be predominantly located in the nuclei of IFN-b and RA-treated HeLa cells [19]. However, a more recent immunofluorescence study discovered that GRIM-19 was mainly located at the mitochondrial inner membrane and was a component of mitochondrial complex I [25]. They proposed that

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Fig. 5 a GRIM-19 protein nuclear expression in the hepatocellular carcinoma tissues was lower than in ANLT. b and c Expression of GRIM-19 mRNA in the hepatocellular carcinoma tissues was lower than in ANLT. d GRIM-19 expression decreased in the grades of

HCC tissues and p-STAT3 increased. e Downregulation of GRIM-19 was associated with increased p-STAT3, total STAT3, and p-STAT3 target protein VEGF. *p \ 0.05 versus respective control. N nontumorous tissue, T HCC tissue

tyrosyl phosphorylation of STAT3, which is required for its DNA binding activity in many cases, is activated by a number of cellular oncogenes known to promote tumor growth and transform cells [29]. Although several mechanisms may account for p-STAT3 overexpression in cancer, the in vivo regulators of p-STAT3 in hepatocarcinogenesis are largely unknown. We demonstrated overexpression of p-STAT3 in HCC according to previous studies, and we additionally showed a significant correlation of activated STAT3 with GRIM-19 expression. Consistent with our findings, several in vitro studies have implicated loss or reduction of GRIM-19 expression results in robust upregulation of STAT3-regulated genes in some tumors, and restoration of GRIM-19 suppresses the growth-promoting activities of STAT3 [23, 27–29]. We also found that increased levels of activated STAT3 were associated with increased expression of the downstream VEGF, which is a key factor involved in tumor

angiogenesis. Several studies have suggested that STAT3 could participate in oncogenesis through upregulation of VEGF [30–32]. Altered STAT3 activation has been shown to enhance VEGF production and maintain angiogenic phenotypes in primary gastric cancer [33], breast cancer [34], and ovarian cancer [35]. In addition, we demonstrated a significant correlation of GRIM-19 expression with tumor angiogenesis and histological grading, which may be related to tumor cell migration, which is consistent with findings by Kalvakolanu et al. [36] that indicate that GRIMs may be a family of new tumor suppressor genes. In previous studies, Hu et al. [37] reported that some oncogenic proteins, such as the vIRF1 of human herpes virus-8 and the E6 oncogenes of high-risk human papilloma viruses, bind to GRIM-19 and inactivate it. Similarly, GW112, a protein found in human esophageal cancers, has been shown to inhibit GRIM-19-mediated apoptosis [38]. These findings suggest that GRIM-19 may be a target

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Med Oncol (2012) 29:3046–3054 Table 1 GRIM-19 mRNA expression in human hepatocellular carcinoma and correlation with clinicopathological parameters

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Cases

GRIM-19/b-actin (x ± s)

B60

31

0.44 ± 0.17

[60

24

0.46 ± 0.23

Male

43

0.52 ± 0.10

Female

12

0.26 ± 0.19

Negative

16

0.45 ± 0.18

Positive

39

0.31 ± 0.11

Item Age (years)

0.968

Gender

0.522

HBV

0.128

Serum a-fetoprotein (ng/ml)

0.323

B20

34

0.66 ± 0.22

[20

21

0.30 ± 0.26

16

0.57 ± 0.21

39

0.33 ± 0.17

Fibrosis Absent Present Tumor size (cm)

0.504

0.659

B5 cm

35

0.56 ± 0.13

[5 cm

20

0.39 ± 0.15 0.032a

Vascular invasion Absent

46

0.51 ± 0.21

Present

9

0.16 ± 0.13

Absent

42

0.46 ± 0.12

Present

13

0.42 ± 0.29

Lymph node metastasis

0.923

0.001a

Histological grade

a

p value

Grade I

12

0.91 ± 0.23

Grade II

34

0.48 ± 0.11

Grade III

9

0.01 ± 0.05

Statistically significant

of certain viral proteins [39, 40]. In the present study, although hepatocarcinogenesis is known to be associated with cirrhosis and viral hepatitis, we found no significant correlation between GRIM-19 expression and hepatitis B virus (HBV)-associated HCC. Meanwhile, there was no significant correlation of tumorous GRIM-19 expression with the presence of fibrosis in the ANLT or serum fetoprotein levels. Taken together, our results indicate that GRIM-19 may play a role in HCC development. However, the degree of fibrosis as well as HBV infection status may not directly influence the potential role of GRIM-19 in HCC. In conclusion, our results for the first time showed that GRIM-19 protein expression was predominantly located in the cytoplasm with weak staining in the nucleus in ANLT, but only located in the cytoplasm in HCC tissues. GRIM19 is suppressed in HCC tissues, with a corresponding increment in p-STAT3 activity. Furthermore, the level of GRIM-19 expression correlates with the vascular invasion and histological grading of human HCC tissues. All of

these indicate that GRIM-19 may play a role in HCC development and the potential roles of GRIM-19 and p-STAT3 in the pathogenesis of HCC warrants further investigation. Acknowledgments This work was supported by Grant No. 2007BS03005 from the Shandong Science and Technology Committee of China. We also thank Medjaden Bioscience Limited for assisting in the preparation of this manuscript. Conflict of interest

None.

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