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Nuclear Nrf2 expression is related to a poor survival in pancreatic adenocarcinoma
Pathology – Research and Practice 210 (2014) 35–39
Contents lists available at ScienceDirect
Pathology – Research and Practice journal homepage: www.elsevier.com/locate/prp
Original Article
Nuclear Nrf2 expression is related to a poor survival in pancreatic adenocarcinoma Y. Soini a,∗ , M. Eskelinen b , P. Juvonen b , V. Kärjä a , K.M. Haapasaari c , A. Saarela d , P. Karihtala e a Department of Pathology and Forensic Medicine, Institute of Clinical Medicine, Pathology and Forensic Medicine, School of Medicine, University of Eastern Finland, Cancer Center of Eastern Finland, P.O. Box 1627, FI-70211 Kuopio, Finland b Department of Surgery, Kuopio University Hospital and School of Medicine, University of Eastern Finland, Kuopio, Finland c Department of Pathology, University of Oulu, Finland d Department of Surgery, Oulu University Hospital, Oulu, Finland e Department of Oncology and Radiotherapy, University of Oulu and Oulu University Hospital, Finland
a r t i c l e
i n f o
Article history: Received 1 March 2013 Received in revised form 31 July 2013 Accepted 4 October 2013 Keywords: Pancreas Carcinoma Oxidative Cancer Sulfiredoxin
a b s t r a c t The aim of this study was to investigate the expression of Nrf2, sulfiredoxin and DJ1 in pancreatic cancer. The expression of Nrf2, sulfiredoxin and DJ1 was studied immunohistochemically in a large set of pancreatic adenocarcinomas consisting of 103 cases. Eighty six percent of the cases showed cytoplasmic Nrf2 and 24% nuclear Nrf2 positivity. Sulfiredoxin positivity was observed in 54% and DJ1 positivity in all cases. Nuclear Nrf2 positivity had an association with sulfiredoxin (p = 0.019) and was associated with a poor survival (p = 0.010). Stage IV tumors tended to have a more nuclear Nrf2 expression (p = 0.080). DJ1 expression was more often found in well-differentiated tumors (p = 0.012), and DJ1 expression was associated with better survival (p = 0.020). According to the results, nuclear Nrf2 expression predicts a worse survival in pancreatic adenocarcinoma, which is in keeping with its protection of cells against oxidative or xenobiotic stress. In accordance with Nrf2’s regulation of the synthesis of sulfiredoxin, there was an association between them (p = 0.019). DJ1 had no association with Nrf2, and its expression predicted a better survival of patients. © 2013 Elsevier GmbH. All rights reserved.
Introduction Nrf2 (nuclear factor erythroid-derived 2-like 2) belongs to the cap‘n’collar (CNC) bZIP transcription factors [1,2]. It controls the expression of genes for antioxidant enzymes, metal-binding proteins, drug-metabolising enzymes, drug transporters, and molecular chaperones [1,2]. During oxidative stress, Nrf2 is released from a complex with Keap1 (Kelch-like ECH-associated protein 1) and moves from cell cytoplasm to the nucleus, where it complexes with a small maf protein and upregulates genes that have antioxidant response element (ARE) in their regulatory regions [1,2]. Under normal conditions, Nrf2 stays in a complex with Keap1, and its activity is restricted through a Keap1-dependent ubiquitylation by Cul3-Rbx1 complex, which targets the CNC-bZIP transcription factor for proteasomal degradation [1–4]. In the development of cancer, Nrf2 acts in two ways. In early carcinogenesis, Nrf2 protects cells from chemically induced mutagenesis by inducing the expression of ARE containing genes [1,2,5].
∗ Corresponding author. Tel.: +358 503224255. E-mail address: ylermi.soini@uef.fi (Y. Soini). 0344-0338/$ – see front matter © 2013 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.prp.2013.10.001
Disturbances in Nrf2 function may thus provoke cancer development [5]. In keeping with this, Nrf2 knockout mice develop cancer much more frequently than their wild type counterparts [5]. When cancer is present, Nrf2, on the other hand, protects tumor cells from oxidative damage induced by chemotherapeutic drugs and radiation [5]. Dysfunction of Nrf2 may thus be a cause of cancer, and its overexpression, on the other hand, a reason for therapy resistance in already developed cancer. The Nrf2-Keap1 system may also influence the metastatic tendency of tumors. Nrf2-deficient mice display a larger number of metastatic nodules in the lung compared to wild type mice, while Keap1-knockdown mutant mice displayed an increased resistance against the cancer cell metastasis to the lung [6]. Peroxiredoxins are antioxidative enzymes which, in addition to catalase and glutathione peroxidases, reduce hydrogen peroxide [7]. Peroxiredoxins I–IV may undergo reversible oxidation in their cysteine sites to sulfinic acid, rendering the molecules to degradation [7,8]. Sulfiredoxin catalyses the reduction of sulfinic acid formation, thus salvaging peroxiredoxins from destruction [8]. Sulfiredoxin is also involved in the deglutathionylation of proteins following nitrosative or oxidative stress [9]. In cell lines, overexpression of sulfiredoxin stimulated cell proliferation and apoptosis
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Y. Soini et al. / Pathology – Research and Practice 210 (2014) 35–39
induced by cisplatin, effects which were mediated by phosphorylation of cell cycle regulators and kinases [10]. Sulfiredoxin is induced by Nrf2 and AP-1, and protects the lung from tobacco mediated oxidative damage [11,12]. Increased sulfiredoxin has been linked with oncogenic transformation, and it is overexpressed in various skin cancers [13]. DJ1 is a protein sensing oxidative damage in neuronal cells, and mutations in DJ-1 gene lead to an early onset of autosomal recessive Parkinson’s disease [14]. Elevated levels of DJ-1 have also been detected in amyotrophic lateral sclerosis, a neurologic condition, due to mutation or dysfunction of CuZnSOD and in astrocytes in cerebral infarction [15]. DJ1 has been considered to harbor oncogenic properties, and elevated levels of the proteins and mRNAs of the gene have been detected in brain astrocytomas, in leukemia and in renal cell carcinoma [16–18]. DJ1 is known to control the PTEN tumor suppressor gene and to stimulate PI3K-AKT/PKB and ERK1/3 signaling, thus influencing cmyc and cellular proliferation [16,19]. It also protects cells from hypoxia-induced cell death [20]. sh-RNA blockage of DJ1 leads to apoptosis of cells due to oxidative stress, and downregulation of DJ1 leads to suppression of HIF-1 responsive genes [14,20]. The protective effects of DJ1 on apoptosis are associated with its capability of decreasing Bax level through inhibiting p53 transcriptional activity [21]. In DJ1 knockout mice, the mitochondrial respiration is impaired, and there is impaired lysosomal autophagy and accumulation of defective mitochondria [22]. DJ1 stabilizes Nrf2 by preventing the association with its inhibitor protein, Keap1, and Nrf2’s subsequent ubiquitination. Without intact DJ-1, Nrf2 protein is unstable, and transcriptional responses are thereby decreased both basally and after induction [23]. The expression of Nrf2 has not been extensively studied in pancreatic cancer, nor are there any investigations on tissue expression of DJ-1, the Nrf2 stabilator or sulfiredoxin, one of Nrf2’s target genes in pancreatic tumors. This study aimed to evaluate the importance of Nrf2, DJ1 and sulfiredoxin protein expression in these tumors. Materials and methods A total of 103 cases of operated pancreatic adenocarcinoma were gathered from the University Hospitals of Oulu, Finland. The cases were reviewed, and the diagnosis and grades of the tumors were determined according to the guidelines of the WHO classification [24]. The mean age was 65 years. There were 50 female and 53 male patients. There were 41 well-differentiated, 37 moderately and 25 poorly differentiated cases. The mean diameter of the tumors was 3.13 cm (range 1–10 cm). Seventy cases were located in the head, 18 in the body, eight in the tail, and the remaining cases were combined locations. Perineural invasion was detected in 22 cases. All the material had been fixed in buffered formalin and embedded in paraffin with a similar protocol. The study had been approved by the ethical committee of Oulu University Hospital. Immunohistochemistry Four-m-thick tissue sections were cut from the paraffinembedded blocks. After deparaffinization and rehydration, the sections were heated in a microwave oven for 2 min × 5 min in trisaminomethane–ethylenediaminetetraacetic acid (Tris–EDTA) buffer (pH 9.0), incubated in a Tris–EDTA buffer for 20 min and washed twice for 5 min in phosphate buffered saline (PBS). Hydrogen peroxide (5%, 5 min) was used to block endogenous peroxidase. Non-specific binding was blocked with 1.5% normal serum in PBS for 35 min at room temperature. The sections were incubated overnight at 4 ◦ C with the mouse monoclonal anti – Nrf2 (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), sulfiredoxin (Proteintech Group Inc., Chicago IL, USA) and DJ1 (Abcam Inc.,
Fig. 1. Nuclear Nrf2 positivity is associated with a worse survival in pancreatic adenocarcinoma (p = 0.010). 0 indicates negative and 1 Nrf2 positive tumors.
Cambridge, UK) antibodies (dilutions 1:1000, 1:500 and 1:500, respectively) [25]. The slides were then incubated with a biotinylated secondary antibody and avidin–biotin-peroxidase complex (ABC Vectastain Elite Kit, Vector Laboratories, Burlingame, CA, USA). Careful rinses were performed with PBS in each step of the immunostaining procedure. The color was developed with diaminobenzidine tetrahydrocloride (DAP) (Sigma, St. Louis, MO, USA). The slides were counterstained with Mayer’s hematoxylin, washed, dehydrated, cleared and mounted with Depex (BDH, Poole, UK). In negative controls, the primary antibody was omitted. The evaluation of Nrf2 and DJ1 immunostaining was performed separately in tumor cell nuclei and cytoplasm. For sulfiredoxin, only cytoplasmic immunoreactivity was evaluated. The results for Nrf2 and DJ1 were semiquantitated as follows; 0–5% = negative 6–25%= weak positivity 26–75% = intermediate positivity 76–100%= strong positivity. Statistical analysis The statistical analyses were performed with SPSS for Windows software (SPSS, Chicago, IL, USA). Continuous data were compared using analysis of variance (ANOVA). When ANOVA results indicated that groups differed, post hoc comparisons were performed using two-tailed t-tests. Categorical data were compared using Fisher’s exact test designed for small sample groups. Survival-data was analyzed using the Kaplan–Meier method with the use of the logrank, Breslow and Tarone-Ware test. p-Values less than 0.05 were considered statistically significant. Results The results of the immunostainings are compiled in Table 1. Eighty six percent of the cases showed cytoplasmic Nrf2 and 24% nuclear Nrf2 positivity (Figs. 1 and 2). Sulfiredoxin positivity was seen in 55% and DJ1 positivity in 100% of cases (Figs. 3 and 4). There was an inverse association between cytoplasmic Nrf2 positivity and sulfiredoxin positivity (p = 0.007). On the other hand, there was a positive association between nuclear Nrf2 and sulfiredoxin positivity (p = 0.019). Cytoplasmic and nuclear Nrf2 positivity had a strong inverse association with each other (p < 0.001). DJ1 (cut point weak positivity), on the other hand, did not associate
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Table 1 Expression of Nrf2, sulfiredoxin and DJ1 in pancreatic adenocarcinoma.
Nrf2 (cytoplasmic) Nrf2 (nuclear) Sulfiredoxin DJ1
Negative n (%)
Weak n (%)
Intermediate n (%)
Strong n (%)
14 (14) 76 (75) 46 (45) 0 (0)
35 (34) 12 (12) 38 (37) 41 (55)
23 (23) 10 (10) 12 (12) 22 (30)
30 (29%) 4 (4) 7 (6) 11 (15)
or nuclear Nrf2 expression (p = 0.968 and p = 0.875). Tumor size or patient age did not associate with these factors (see Table 2). Curiously, male patients showed lower cytoplasmic Nrf2 expression (p = 0.029). Also, stage IV tumors appeared to have a low cytoplasmic Nrf2 expression (p = 0.052). They also tended to have a higher nuclear Nrf2 expression (p = 0.080). Discussion
Fig. 2. Nrf2 expression in pancreatic adenocarcinoma. In this case, both nuclear and cytoplasmic expression could be seen.
with cytoplasmic or nuclear Nrf2 positivity (p = 0.448, p = 0.137, respectively). DJ1 and sulfiredoxin had a near significant inverse association (p = 0.073). Cytoplasmic Nrf2 positivity did not associate with patient survival (log-rank, p = 0.948). Also sulfiredoxin did not associate with survival (p = 0.81). Nuclear Nrf2 positivity was, however, associated with a worse prognosis of the patients (log-rank p = 0.010) (Fig. 1) and strong and moderate cytoplasmic DJ1 expression associated with a better survival (log-rank p = 0.020) but they did not have an independent prognostic value (p = 0.46, 0.31). Of the variables, tumor stage (p = 0.049) and tumor location (p = 0.020) had an independent prognostic value. Low DJ1 expression was associated with poorly or moderately differentiated tumors (p = 0.009). This was not seen with sulfiredoxin (p = 0.319), or with cytoplasmic
Fig. 3. In a case of a ductal pancreatic adenocarcinoma, cytoplasmic expression of sulfiredoxin can be observed.
Nrf2 is a gene that senses oxidative and xenobiotic stress in cells and protects them from damage by inducing several antioxidative and other protective genes through binding to the ARE elements in DNA [1,2]. It has been shown to be important in lung cancer development, and blockage of Nrf2 expression by siRNA leads to inhibition of tumor growth and greater sensitivity to chemotherapeutic drugs [26,27]. The importance of Nrf2 in combating cigarette smoke-induced oxidative stress is reflected in the fact that Nrf2 deficiency also promotes emphysema development in mice [28]. Pancreatic cancer is a dismal disease with a five-year survival of 5% [24]. The most common histological variant of pancreatic carcinoma is ductal carcinoma, representing about 90% of carcinoma cases [24]. A risk factor for the development of pancreatic carcinoma is smoking, which is a known cause for the induction of reactive oxidative species and Nrf2 activation. Additionally, chronic pancreatitis is another risk factor for pancreatic carcinoma development, and inflammation is another known cause for oxidative stress in tissues [29]. The results show that Nrf2, DJ1 and sulfiredoxin are amply expressed in pancreatic carcinoma. In head and neck carcinoma, Nrf2 was overexpressed in 91.5% of tumors compared to normal squamous epithelium [30]. In non-small cell lung carcinomas and breast carcinomas, nuclear Nrf2 expression was detected in 26–30% of cases, which is at the same level as we found in pancreatic adenocarcinoma [27]. In non-small cell carcinoma patients, Nrf2 expression was associated with poor survival [27], a finding we
Fig. 4. The neoplastic ducts of pancreatic adenocarcinoma show expression of DJ1 protein.
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Table 2 Association of sulfiredoxin, Nrf2 and DJ1 expression with different parameters of the pancreatic carcinoma cases (p-values).
SRX Nrfc Nrfn DJ * ** ***
Sex
Tumor site
Stage I–II vs. III–IV
T
N
M
Grade
Size
Perineural invasion
0.418 0.029* 0.807 0.128
0.483 0.004*** 0.247 0.324
0.894 0.573 0.231 0.177
0.166 0.811 0.336 0.922
0.446 0.550 0.984 0.945
0.508 0.304 0.529 0.327
0.397 0.968 0.875 0.009**
0.319 0.366 0.594 0.193
0.199 0.348 0.508 0.590
Female stronger. Poorly differentiated show stronger expression. More in body and tail region.
could also detect in pancreatic adenocarcinoma. Patients with moderate or strong nuclear Nrf2 expression had a poorer prognosis On the other hand, in patients with negative or low Nrf2 expression, 20% of the patients appear to be long-term survivors. This is in line with the known function of Nrf2, making tumor cells more resistant to oxidative stress (e.g. adjuvant chemotherapy), a finding also detected in lung carcinoma [27]. Sulfiredoxin is an enzyme that reduces hyperoxidant forms of peroxiredoxins 1–4, and thus its increased expression might be expected to worsen patient survival as well [8]. Sulfiredoxin was not, however, associated with poor survival. Expression of sulfiredoxin is induced by Nrf2, and in line with this, we found an association between nuclear Nrf2 expression and sulfiredoxin [10]. Interestingly, there was also an inverse association between cytoplasmic Nrf2 expression and sulfiredoxin. DJ1 has also been associated with poor prognosis in laryngeal carcinoma and in lung carcinoma [31,32]. In laryngeal carcinoma, DJ1 expression was found in 85% of the cases [31]. According to two recent studies, serum levels of DJ1 seem to be elevated in pancreatic cancer patients compared to those with chronic pancreatitis and healthy individuals [33,34]. Interestingly, DJ1 was found to be one of three markers for pancreatic adenocarcinoma along with MMP9 and alpha-1B glycoprotein precursor in a proteomic analysis of pancreatic juice [35]. This finding is in line with our results showing a high expression of DJ1 in pancreatic carcinoma tissue. We could not detect a similar finding of prognostic significance for DJ1 in pancreatic adenocarcinoma as detected in lung and laryngeal carcinoma. Low DJ1 expression was also associated with high grade tumors and worse survival. Interestingly, in two previous studies, upregulation of DJ1 in pancreatic juice and in serum was associated with better differentiation of tumors, which seems to be in line with our findings of DJ1 being lower in high grade tumors [33,34]. Moreover, some tumors like astrocytomas show a similar tendency for grade and survival as observed in pancreatic adenocarcinomas [18]. In conclusion, the study shows that Nrf2 protects pancreatic cancer cells from oxidative damage, which is reflected in the worse survival of patients having a higher extent of nuclear nrf expression in their tumors. In accordance with Nrf2’s role as a regulator of sulfiredoxin, nuclear Nrf2 and sulfiredoxin showed an association. DJ1 was associated with less aggressive types of pancreatic adenocarcinoma, and its expression was not associated with Nrf2. Targeting Nrf2 and its nuclear translocation could be one option to treat pancreatic adenocarcinoma in the future. Acknowledgements Thelma Mäkikyrö Foundation, The Orion-Farmos Foundation, The Finnish Cultural Foundation, The Finnish Anti-Tuberculosis Association and The Finnish Cancer Society are acknowledged for their financial support References [1] K. Itoh, J. Mimura, M. Yamamoto, Discovery of the Negative Regulator of Nrf2, Keap1: a historical overview, Antioxid. Redox Signal. 13 (2010) 1665–1678.
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Contents lists available at ScienceDirect
Pathology – Research and Practice journal homepage: www.elsevier.com/locate/prp
Original Article
Nuclear Nrf2 expression is related to a poor survival in pancreatic adenocarcinoma Y. Soini a,∗ , M. Eskelinen b , P. Juvonen b , V. Kärjä a , K.M. Haapasaari c , A. Saarela d , P. Karihtala e a Department of Pathology and Forensic Medicine, Institute of Clinical Medicine, Pathology and Forensic Medicine, School of Medicine, University of Eastern Finland, Cancer Center of Eastern Finland, P.O. Box 1627, FI-70211 Kuopio, Finland b Department of Surgery, Kuopio University Hospital and School of Medicine, University of Eastern Finland, Kuopio, Finland c Department of Pathology, University of Oulu, Finland d Department of Surgery, Oulu University Hospital, Oulu, Finland e Department of Oncology and Radiotherapy, University of Oulu and Oulu University Hospital, Finland
a r t i c l e
i n f o
Article history: Received 1 March 2013 Received in revised form 31 July 2013 Accepted 4 October 2013 Keywords: Pancreas Carcinoma Oxidative Cancer Sulfiredoxin
a b s t r a c t The aim of this study was to investigate the expression of Nrf2, sulfiredoxin and DJ1 in pancreatic cancer. The expression of Nrf2, sulfiredoxin and DJ1 was studied immunohistochemically in a large set of pancreatic adenocarcinomas consisting of 103 cases. Eighty six percent of the cases showed cytoplasmic Nrf2 and 24% nuclear Nrf2 positivity. Sulfiredoxin positivity was observed in 54% and DJ1 positivity in all cases. Nuclear Nrf2 positivity had an association with sulfiredoxin (p = 0.019) and was associated with a poor survival (p = 0.010). Stage IV tumors tended to have a more nuclear Nrf2 expression (p = 0.080). DJ1 expression was more often found in well-differentiated tumors (p = 0.012), and DJ1 expression was associated with better survival (p = 0.020). According to the results, nuclear Nrf2 expression predicts a worse survival in pancreatic adenocarcinoma, which is in keeping with its protection of cells against oxidative or xenobiotic stress. In accordance with Nrf2’s regulation of the synthesis of sulfiredoxin, there was an association between them (p = 0.019). DJ1 had no association with Nrf2, and its expression predicted a better survival of patients. © 2013 Elsevier GmbH. All rights reserved.
Introduction Nrf2 (nuclear factor erythroid-derived 2-like 2) belongs to the cap‘n’collar (CNC) bZIP transcription factors [1,2]. It controls the expression of genes for antioxidant enzymes, metal-binding proteins, drug-metabolising enzymes, drug transporters, and molecular chaperones [1,2]. During oxidative stress, Nrf2 is released from a complex with Keap1 (Kelch-like ECH-associated protein 1) and moves from cell cytoplasm to the nucleus, where it complexes with a small maf protein and upregulates genes that have antioxidant response element (ARE) in their regulatory regions [1,2]. Under normal conditions, Nrf2 stays in a complex with Keap1, and its activity is restricted through a Keap1-dependent ubiquitylation by Cul3-Rbx1 complex, which targets the CNC-bZIP transcription factor for proteasomal degradation [1–4]. In the development of cancer, Nrf2 acts in two ways. In early carcinogenesis, Nrf2 protects cells from chemically induced mutagenesis by inducing the expression of ARE containing genes [1,2,5].
∗ Corresponding author. Tel.: +358 503224255. E-mail address: ylermi.soini@uef.fi (Y. Soini). 0344-0338/$ – see front matter © 2013 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.prp.2013.10.001
Disturbances in Nrf2 function may thus provoke cancer development [5]. In keeping with this, Nrf2 knockout mice develop cancer much more frequently than their wild type counterparts [5]. When cancer is present, Nrf2, on the other hand, protects tumor cells from oxidative damage induced by chemotherapeutic drugs and radiation [5]. Dysfunction of Nrf2 may thus be a cause of cancer, and its overexpression, on the other hand, a reason for therapy resistance in already developed cancer. The Nrf2-Keap1 system may also influence the metastatic tendency of tumors. Nrf2-deficient mice display a larger number of metastatic nodules in the lung compared to wild type mice, while Keap1-knockdown mutant mice displayed an increased resistance against the cancer cell metastasis to the lung [6]. Peroxiredoxins are antioxidative enzymes which, in addition to catalase and glutathione peroxidases, reduce hydrogen peroxide [7]. Peroxiredoxins I–IV may undergo reversible oxidation in their cysteine sites to sulfinic acid, rendering the molecules to degradation [7,8]. Sulfiredoxin catalyses the reduction of sulfinic acid formation, thus salvaging peroxiredoxins from destruction [8]. Sulfiredoxin is also involved in the deglutathionylation of proteins following nitrosative or oxidative stress [9]. In cell lines, overexpression of sulfiredoxin stimulated cell proliferation and apoptosis
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induced by cisplatin, effects which were mediated by phosphorylation of cell cycle regulators and kinases [10]. Sulfiredoxin is induced by Nrf2 and AP-1, and protects the lung from tobacco mediated oxidative damage [11,12]. Increased sulfiredoxin has been linked with oncogenic transformation, and it is overexpressed in various skin cancers [13]. DJ1 is a protein sensing oxidative damage in neuronal cells, and mutations in DJ-1 gene lead to an early onset of autosomal recessive Parkinson’s disease [14]. Elevated levels of DJ-1 have also been detected in amyotrophic lateral sclerosis, a neurologic condition, due to mutation or dysfunction of CuZnSOD and in astrocytes in cerebral infarction [15]. DJ1 has been considered to harbor oncogenic properties, and elevated levels of the proteins and mRNAs of the gene have been detected in brain astrocytomas, in leukemia and in renal cell carcinoma [16–18]. DJ1 is known to control the PTEN tumor suppressor gene and to stimulate PI3K-AKT/PKB and ERK1/3 signaling, thus influencing cmyc and cellular proliferation [16,19]. It also protects cells from hypoxia-induced cell death [20]. sh-RNA blockage of DJ1 leads to apoptosis of cells due to oxidative stress, and downregulation of DJ1 leads to suppression of HIF-1 responsive genes [14,20]. The protective effects of DJ1 on apoptosis are associated with its capability of decreasing Bax level through inhibiting p53 transcriptional activity [21]. In DJ1 knockout mice, the mitochondrial respiration is impaired, and there is impaired lysosomal autophagy and accumulation of defective mitochondria [22]. DJ1 stabilizes Nrf2 by preventing the association with its inhibitor protein, Keap1, and Nrf2’s subsequent ubiquitination. Without intact DJ-1, Nrf2 protein is unstable, and transcriptional responses are thereby decreased both basally and after induction [23]. The expression of Nrf2 has not been extensively studied in pancreatic cancer, nor are there any investigations on tissue expression of DJ-1, the Nrf2 stabilator or sulfiredoxin, one of Nrf2’s target genes in pancreatic tumors. This study aimed to evaluate the importance of Nrf2, DJ1 and sulfiredoxin protein expression in these tumors. Materials and methods A total of 103 cases of operated pancreatic adenocarcinoma were gathered from the University Hospitals of Oulu, Finland. The cases were reviewed, and the diagnosis and grades of the tumors were determined according to the guidelines of the WHO classification [24]. The mean age was 65 years. There were 50 female and 53 male patients. There were 41 well-differentiated, 37 moderately and 25 poorly differentiated cases. The mean diameter of the tumors was 3.13 cm (range 1–10 cm). Seventy cases were located in the head, 18 in the body, eight in the tail, and the remaining cases were combined locations. Perineural invasion was detected in 22 cases. All the material had been fixed in buffered formalin and embedded in paraffin with a similar protocol. The study had been approved by the ethical committee of Oulu University Hospital. Immunohistochemistry Four-m-thick tissue sections were cut from the paraffinembedded blocks. After deparaffinization and rehydration, the sections were heated in a microwave oven for 2 min × 5 min in trisaminomethane–ethylenediaminetetraacetic acid (Tris–EDTA) buffer (pH 9.0), incubated in a Tris–EDTA buffer for 20 min and washed twice for 5 min in phosphate buffered saline (PBS). Hydrogen peroxide (5%, 5 min) was used to block endogenous peroxidase. Non-specific binding was blocked with 1.5% normal serum in PBS for 35 min at room temperature. The sections were incubated overnight at 4 ◦ C with the mouse monoclonal anti – Nrf2 (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), sulfiredoxin (Proteintech Group Inc., Chicago IL, USA) and DJ1 (Abcam Inc.,
Fig. 1. Nuclear Nrf2 positivity is associated with a worse survival in pancreatic adenocarcinoma (p = 0.010). 0 indicates negative and 1 Nrf2 positive tumors.
Cambridge, UK) antibodies (dilutions 1:1000, 1:500 and 1:500, respectively) [25]. The slides were then incubated with a biotinylated secondary antibody and avidin–biotin-peroxidase complex (ABC Vectastain Elite Kit, Vector Laboratories, Burlingame, CA, USA). Careful rinses were performed with PBS in each step of the immunostaining procedure. The color was developed with diaminobenzidine tetrahydrocloride (DAP) (Sigma, St. Louis, MO, USA). The slides were counterstained with Mayer’s hematoxylin, washed, dehydrated, cleared and mounted with Depex (BDH, Poole, UK). In negative controls, the primary antibody was omitted. The evaluation of Nrf2 and DJ1 immunostaining was performed separately in tumor cell nuclei and cytoplasm. For sulfiredoxin, only cytoplasmic immunoreactivity was evaluated. The results for Nrf2 and DJ1 were semiquantitated as follows; 0–5% = negative 6–25%= weak positivity 26–75% = intermediate positivity 76–100%= strong positivity. Statistical analysis The statistical analyses were performed with SPSS for Windows software (SPSS, Chicago, IL, USA). Continuous data were compared using analysis of variance (ANOVA). When ANOVA results indicated that groups differed, post hoc comparisons were performed using two-tailed t-tests. Categorical data were compared using Fisher’s exact test designed for small sample groups. Survival-data was analyzed using the Kaplan–Meier method with the use of the logrank, Breslow and Tarone-Ware test. p-Values less than 0.05 were considered statistically significant. Results The results of the immunostainings are compiled in Table 1. Eighty six percent of the cases showed cytoplasmic Nrf2 and 24% nuclear Nrf2 positivity (Figs. 1 and 2). Sulfiredoxin positivity was seen in 55% and DJ1 positivity in 100% of cases (Figs. 3 and 4). There was an inverse association between cytoplasmic Nrf2 positivity and sulfiredoxin positivity (p = 0.007). On the other hand, there was a positive association between nuclear Nrf2 and sulfiredoxin positivity (p = 0.019). Cytoplasmic and nuclear Nrf2 positivity had a strong inverse association with each other (p < 0.001). DJ1 (cut point weak positivity), on the other hand, did not associate
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Table 1 Expression of Nrf2, sulfiredoxin and DJ1 in pancreatic adenocarcinoma.
Nrf2 (cytoplasmic) Nrf2 (nuclear) Sulfiredoxin DJ1
Negative n (%)
Weak n (%)
Intermediate n (%)
Strong n (%)
14 (14) 76 (75) 46 (45) 0 (0)
35 (34) 12 (12) 38 (37) 41 (55)
23 (23) 10 (10) 12 (12) 22 (30)
30 (29%) 4 (4) 7 (6) 11 (15)
or nuclear Nrf2 expression (p = 0.968 and p = 0.875). Tumor size or patient age did not associate with these factors (see Table 2). Curiously, male patients showed lower cytoplasmic Nrf2 expression (p = 0.029). Also, stage IV tumors appeared to have a low cytoplasmic Nrf2 expression (p = 0.052). They also tended to have a higher nuclear Nrf2 expression (p = 0.080). Discussion
Fig. 2. Nrf2 expression in pancreatic adenocarcinoma. In this case, both nuclear and cytoplasmic expression could be seen.
with cytoplasmic or nuclear Nrf2 positivity (p = 0.448, p = 0.137, respectively). DJ1 and sulfiredoxin had a near significant inverse association (p = 0.073). Cytoplasmic Nrf2 positivity did not associate with patient survival (log-rank, p = 0.948). Also sulfiredoxin did not associate with survival (p = 0.81). Nuclear Nrf2 positivity was, however, associated with a worse prognosis of the patients (log-rank p = 0.010) (Fig. 1) and strong and moderate cytoplasmic DJ1 expression associated with a better survival (log-rank p = 0.020) but they did not have an independent prognostic value (p = 0.46, 0.31). Of the variables, tumor stage (p = 0.049) and tumor location (p = 0.020) had an independent prognostic value. Low DJ1 expression was associated with poorly or moderately differentiated tumors (p = 0.009). This was not seen with sulfiredoxin (p = 0.319), or with cytoplasmic
Fig. 3. In a case of a ductal pancreatic adenocarcinoma, cytoplasmic expression of sulfiredoxin can be observed.
Nrf2 is a gene that senses oxidative and xenobiotic stress in cells and protects them from damage by inducing several antioxidative and other protective genes through binding to the ARE elements in DNA [1,2]. It has been shown to be important in lung cancer development, and blockage of Nrf2 expression by siRNA leads to inhibition of tumor growth and greater sensitivity to chemotherapeutic drugs [26,27]. The importance of Nrf2 in combating cigarette smoke-induced oxidative stress is reflected in the fact that Nrf2 deficiency also promotes emphysema development in mice [28]. Pancreatic cancer is a dismal disease with a five-year survival of 5% [24]. The most common histological variant of pancreatic carcinoma is ductal carcinoma, representing about 90% of carcinoma cases [24]. A risk factor for the development of pancreatic carcinoma is smoking, which is a known cause for the induction of reactive oxidative species and Nrf2 activation. Additionally, chronic pancreatitis is another risk factor for pancreatic carcinoma development, and inflammation is another known cause for oxidative stress in tissues [29]. The results show that Nrf2, DJ1 and sulfiredoxin are amply expressed in pancreatic carcinoma. In head and neck carcinoma, Nrf2 was overexpressed in 91.5% of tumors compared to normal squamous epithelium [30]. In non-small cell lung carcinomas and breast carcinomas, nuclear Nrf2 expression was detected in 26–30% of cases, which is at the same level as we found in pancreatic adenocarcinoma [27]. In non-small cell carcinoma patients, Nrf2 expression was associated with poor survival [27], a finding we
Fig. 4. The neoplastic ducts of pancreatic adenocarcinoma show expression of DJ1 protein.
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Y. Soini et al. / Pathology – Research and Practice 210 (2014) 35–39
Table 2 Association of sulfiredoxin, Nrf2 and DJ1 expression with different parameters of the pancreatic carcinoma cases (p-values).
SRX Nrfc Nrfn DJ * ** ***
Sex
Tumor site
Stage I–II vs. III–IV
T
N
M
Grade
Size
Perineural invasion
0.418 0.029* 0.807 0.128
0.483 0.004*** 0.247 0.324
0.894 0.573 0.231 0.177
0.166 0.811 0.336 0.922
0.446 0.550 0.984 0.945
0.508 0.304 0.529 0.327
0.397 0.968 0.875 0.009**
0.319 0.366 0.594 0.193
0.199 0.348 0.508 0.590
Female stronger. Poorly differentiated show stronger expression. More in body and tail region.
could also detect in pancreatic adenocarcinoma. Patients with moderate or strong nuclear Nrf2 expression had a poorer prognosis On the other hand, in patients with negative or low Nrf2 expression, 20% of the patients appear to be long-term survivors. This is in line with the known function of Nrf2, making tumor cells more resistant to oxidative stress (e.g. adjuvant chemotherapy), a finding also detected in lung carcinoma [27]. Sulfiredoxin is an enzyme that reduces hyperoxidant forms of peroxiredoxins 1–4, and thus its increased expression might be expected to worsen patient survival as well [8]. Sulfiredoxin was not, however, associated with poor survival. Expression of sulfiredoxin is induced by Nrf2, and in line with this, we found an association between nuclear Nrf2 expression and sulfiredoxin [10]. Interestingly, there was also an inverse association between cytoplasmic Nrf2 expression and sulfiredoxin. DJ1 has also been associated with poor prognosis in laryngeal carcinoma and in lung carcinoma [31,32]. In laryngeal carcinoma, DJ1 expression was found in 85% of the cases [31]. According to two recent studies, serum levels of DJ1 seem to be elevated in pancreatic cancer patients compared to those with chronic pancreatitis and healthy individuals [33,34]. Interestingly, DJ1 was found to be one of three markers for pancreatic adenocarcinoma along with MMP9 and alpha-1B glycoprotein precursor in a proteomic analysis of pancreatic juice [35]. This finding is in line with our results showing a high expression of DJ1 in pancreatic carcinoma tissue. We could not detect a similar finding of prognostic significance for DJ1 in pancreatic adenocarcinoma as detected in lung and laryngeal carcinoma. Low DJ1 expression was also associated with high grade tumors and worse survival. Interestingly, in two previous studies, upregulation of DJ1 in pancreatic juice and in serum was associated with better differentiation of tumors, which seems to be in line with our findings of DJ1 being lower in high grade tumors [33,34]. Moreover, some tumors like astrocytomas show a similar tendency for grade and survival as observed in pancreatic adenocarcinomas [18]. In conclusion, the study shows that Nrf2 protects pancreatic cancer cells from oxidative damage, which is reflected in the worse survival of patients having a higher extent of nuclear nrf expression in their tumors. In accordance with Nrf2’s role as a regulator of sulfiredoxin, nuclear Nrf2 and sulfiredoxin showed an association. DJ1 was associated with less aggressive types of pancreatic adenocarcinoma, and its expression was not associated with Nrf2. Targeting Nrf2 and its nuclear translocation could be one option to treat pancreatic adenocarcinoma in the future. Acknowledgements Thelma Mäkikyrö Foundation, The Orion-Farmos Foundation, The Finnish Cultural Foundation, The Finnish Anti-Tuberculosis Association and The Finnish Cancer Society are acknowledged for their financial support References [1] K. Itoh, J. Mimura, M. Yamamoto, Discovery of the Negative Regulator of Nrf2, Keap1: a historical overview, Antioxid. Redox Signal. 13 (2010) 1665–1678.
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