Despite frequent KRAS mutation, the early molecular mechanisms of pancreatic ductal adenocarcinoma (PDAC) development have not been fully elucidated. By tracking a potential regulator of another feature of PDAC precursors, acquisition of foregut or gastric epithelial gene signature, we herein report that aberrant overexpression of ecotropic viral integration site 1 (EVI1) oncoprotein, which is usually absent in normal pancreatic duct, is a widespread marker across the full spectrum of human PDAC precursors and PDAC. In pancreatic cancer cells, EVI1 depletion caused remarkable inhibition of cell growth and migration, indicating its oncogenic roles. Importantly, we found that EVI1 upregulated KRAS expression through suppression of a potent KRAS suppressor, miR-96, in pancreatic cancer cells. Collectively, the present findings suggest that EVI1 overexpression and KRAS mutation converge on activation of the KRAS pathway in early phases of pancreatic carcinogenesis and propose EVI1 and/or miR-96 as early markers and therapeutic targets in this dismal disease.
Pancreatic ductal adenocarcinoma (PDAC) is the most common pancreatic malignancy with the worst prognosis of all cancers. Three distinct epithelial lesions, pancreatic intraepithelial neoplasias (PanINs), intraductal papillary mucinous neoplasms (IPMNs) and mucinous cystic neoplasms (MCNs), are recognized as PDAC precursors.1 Early detection and intervention are important for PDAC management and it is indispensable to identify the mechanisms of pancreatic carcinogenesis.
Recently, several lines of evidence have indicated that the acquisition of extrapancreatic foregut or gastric epithelial markers occurs during an early phase of pancreatic carcinogenesis. A previous study demonstrated that human PanIN represented abnormal epithelial differentiation toward gastric epithelial phenotype.2 The foregut or gastric epithelium-like molecules, such as a gastric-specific tight junction molecule, claudin-18 (CLDN18) and MUC5AC, are expressed in about 80–95% of PanIN-1.3, 4, 5
During pancreatic carcinogenesis, activating point mutation of KRAS is an early event in PDAC development.1, 6 In low-grade PanIN (PanIN-1) lesions, two distinct genetic changes, telomere shortening and KRAS mutation, were observed.1, 7 KRAS mutation was detected in about 40% of PanIN-1 lesions by either plain PCR for KRAS without modification or a mutation-enriched PCR procedure.1 Accordingly, several reports have shown that pancreas-specific expression of mutant KRAS in mouse models resulted in the development of PanINs.8, 9 These studies suggest that KRAS mutation may be a key regulator of initiation and progression of pancreatic carcinogenesis. However, relatively low prevalence of mutant KRAS in low-grade PanINs and increasing frequency in high-grade PanINs and PDACs (36% of PanIN-1A, 44% of PanIN-1B and 87% of PanIN-2/3 lesions) may imply that activating mutations of KRAS are more involved after PanIN initiation.1 The murine pancreatic carcinogenesis models showed that only a small subset of cells with mutant KRAS expression formed mouse PanIN lesions, which did not affect the entire organ.9, 10 In this context, it has been postulated that alteration of other signaling pathways such as epidermal growth factor, Notch and Hedgehog signaling may contribute to PanIN formation and PDAC progression in parallel or synergistically with KRAS mutation.6, 10 Therefore, additional events besides KRAS mutation may be required for the initiation of PDAC precursors, although details still remain elusive.
Taking these findings into account, the above-mentioned phenotypic changes of pancreatic epithelium in human PanIN may provide promising routes to understand the background mechanism of PDAC precursors. By tracking a potential transcriptional regulator of gastric epithelial genes in pancreatic neoplasms, we herein report that overexpression of ecotropic viral integration site 1 (EVI1) oncoprotein marks the full spectrum of human PDAC precursors and PDAC. EVI1 is an oncogenic transcriptional factor that is frequently implicated in hematological malignancies and in some solid cancers.11, 12 EVI1 is usually absent in normal pancreatic duct, but is expressed in normal gastric epithelium and diffusely expressed in the majority of PDAC precursors and PDAC, suggesting that EVI1 represents a key regulator involved in the development of PDAC precursors. We further revealed that suppression of EVI1 attenuated KRAS expression and that EVI1 transcriptionally suppressed a potent KRAS regulatory microRNA (miRNA), miRNA-96.
Widespread overexpression of EVI1 oncoprotein in pancreatic neoplasms
The acquisition of the gastric phenotype was detected in the early phase of pancreatic carcinogenesis. To identify potential regulator of this phenotype and marker involved in early phases of pancreatic tumorigenesis, we extracted gastric epithelial genes consisting of approximately 10 genes, which are highly and specifically expressed in gastric tissues, from a microarray database of various normal tissues13 and searched for potential transcription factor binding to these genes using the MatInspector program.14, 15 These gastric epithelial genes included ATP4B, GIF, LIPF and so on (Supplementary Table 1), and potential binding of many transcription factors to promoter regions of at least eight genes was found. We also noticed a correlation between these transcription factors and CLDN18, a gastric marker in PanIN, and extracted certain transcription factors, such as EVI1, GATA6 and FOXA2 (Supplementary Table 2). GATA6 and FOXA2 were previously shown to be involved in a subset of pancreatic cancer.16, 17 As EVI1 has an important oncogenic role in some malignancies, we tried to examine the expression and the role of EVI1 in a clinical cohort of pancreatic neoplasms (Figure 1, Table 1 and Supplementary Figure 1).
Immunohistochemical analysis showed that ductal epithelial cells, endocrine cells and interstitial fibroblasts did not express EVI1 in normal pancreatic tissue, but only acinar cells weakly expressed EVI1 (Figures 1a and b). Normal gastric tissues were used as positive controls, as we confirmed strong expression of EVI1 in normal gastric epithelium. Ductal metaplasia of acinar cells also showed weak or absent expression of EVI1 (Supplementary Figures 1A and B). In contrast, EVI1 was expressed strongly and diffusely in the nucleus of neoplastic cells in nearly all cases of three PDAC precursors: PanIN (100%, 32/32; PanIN-1: Figures 1c and d, PanIN-3: Supplementary Figures 1C and D), IPMN (96.9%, 63/65; Supplementary Figures 1E–H) and MCN (100%, 5/5; Table 1). In PDACs, over 90% of well-differentiated and moderately differentiated cancers and about 70% of poorly differentiated cancers demonstrated EVI1 overexpression (Figures 1e and f, and Supplementary Figures 1I and J).
Oncogenic activity of EVI1 in pancreatic cancer cells
EVI1 encodes a transcriptional regulator containing a repressor domain and an acidic domain with proximal and distal zinc finger motifs. Proximal zinc finger domain recognizes consensus 5′-IndexTermTGATATGAATAGATA-3′-like or 5′-IndexTermACAAGATAA-3′-like sequences.11, 12 EVI1 has several splice variants such as Delta324 EVI1 (EVI1s) and Myelodysplastic Syndrome1/EVI1 (MDS1/EVI1).11, 12 We examined the expression of EVI1 using quantitative reverse transcription–PCR (qRT–PCR) and western blotting in nine pancreatic cancer cells (PANC-1, PK-1, PK-8, PK-9, PK-45H, PK-45P, KLM-1, MIA PaCa-2, Capan-1) and an immortal normal human pancreatic ductal epithelial cell line, HPDE cells (Figures 1g and h). In five pancreatic cancer cells and HPDE cells, three EVI1 variants, EVI1, MDS1/EVI1 and EVI1s, were expressed. In two pancreatic cancer cells (PK-45H cells and PK-45P cells), MDS1/EVI1 was expressed. In two pancreatic cancer cells (MIA PaCa-2 cells and PANC-1 cells), no expression of EVI1 was observed. The correlative tendency of expression between EVI1 and CLDN18 was observed in HPDE cells (data not shown).
On the basis of the previously reported oncogenic roles of EVI1 in hematological malignancies,11, 12 we explored whether EVI1 has a tumor-promoting role in pancreatic-lineage cells. Two small interfering RNAs (siRNAs) against EVI1 were designed: siEVI1-#1 targeted a sequence conserved in three major variants, MDS1/EVI1, EVI1 and EVI1s; siEVI1-#2 targeted a sequence present only in MDS1/EVI1 and EVI1, although targeting of three variants was not as strict as theoretically expected. We confirmed that similar results were obtained for both siRNAs (Figure 2a). In PK-8 pancreatic cancer cells and HPDE cells highly expressing EVI1, downregulation of EVI1 by siRNA significantly inhibited proliferation (Figure 2b and Supplementary Figure 2A). The same tendency was observed in other EVI1 highly expressed pancreatic cancer cells, KLM-1 (Supplementary Figure 2B). In addition, downregulation of EVI1 significantly suppressed the migration of HPDE, PK-8 and PK-1 cells (Figure 2c and Supplementary Figure 2C). Furthermore, silencing of EVI1 resulted in increase in G1 fraction in fluorescence-activated cell-sorting analysis (Figure 2d). Nocodazole trap experiment showed that EVI1 knockdown prevented the nocodazole-induced G2/M block (Figure 2e), suggesting that EVI1 downregulation induces G1 arrest. These results thus indicate that EVI1 functions as an oncogenic factor in pancreatic cancer.
EVI1 modulates KRAS-extracellular signal-regulated protein kinase (ERK)-p27Kip1 pathway in pancreatic cancer cells
EVI1 has been previously shown to be associated with transforming growth factor-β,18, 19 c-Jun N-terminal kinase20 and class III phosphatidylinositol 3-kinase/Akt signaling pathways,21, 22 thereby modulating cell growth capacity. In HPDE cells, which upregulate PAI-1 mRNA expression in response to transforming growth factor-β treatment, EVI1 knockdown did not affect the transforming growth factor-β-induced upregulation of PAI-1 mRNA expression (Supplementary Figure 3A). In addition, EVI1 knockdown had little or no effect on the phosphorylation status of p38 MAPK, Akt and c-Jun N-terminal kinase in HPDE, PK-8 and BxPC-3 cells (Supplementary Figure 3B). These results suggest that other signaling pathway underlies EVI1-mediated regulation of cell proliferation.
KRAS was shown to be a major regulator of pancreatic carcinogenesis, so we investigated a potential link between EVI1 and KRAS. We found that downregulation of EVI1 significantly decreased the expression level of KRAS protein in PK-8 cells (Figure 3a). In addition, the same tendency was also observed in pancreatic-lineage cell lines with wild-type KRAS, HPDE cells and BxPC-3 cells (Figure 3a and Supplementary Figure 3C). The downregulation of KRAS mRNA was also observed under EVI1 knockdown in PK-8 cells (Supplementary Figure 3D). Phosphorylation of ERK,23 a downstream target of KRAS, was also decreased by knockdown of EVI1 in PK-8 cells, HPDE cells and BxPC-3 cells (Figure 3b and Supplementary Figure 3E). A previous study reported that a cyclin-dependent kinase inhibitor, p27Kip1, lay beneath the RAS-activated RAF-MAP/ERK kinase (MEK)-ERK pathway and that MEK inhibition induced G1 arrest along with the upregulation of p27Kip1 in pancreatic cancer cells.23 Consistent with these findings, EVI1 knockdown resulted in elevated p27Kip1 expression in PK-8 cells, HPDE cells and BxPC-3 cells (Figure 3c and Supplementary Figure 3F).
In accordance with the previous findings, the treatment with ERK1/2 inhibitor, U0126, induced the upregulation of p27Kip1 and G1 arrest in PK-8 cells (Figures 3d and e). To test the involvement of the KRAS-ERK-p27 pathway in EVI1-mediated growth control, PK-8 cells were in combination treated with EVI1 siRNA and U0126. U0126 treatment inhibited cell growth dose dependently, irrespective of the presence or absence of EVI1 siRNAs. As expected, growth inhibition by EVI1 silencing was attenuated in U0126-treated pancreatic cancer cells and became undetectable by high-dose U0126 treatment. These results indirectly suggest that EVI1 controls cellular growth via the KRAS-ERK pathway (Figure 3f).
Regulation of potential KRAS modifiers, microRNA-96 and microRNA-181a, by EVI1
miRNAs are pivotal post-transcriptional regulators of gene expression and elicit tumor-suppressive or oncogenic functions by targeting oncogenes or tumor suppressors, respectively.24, 25 As for regulation of the KRAS pathway, the association of several miRNAs and KRAS has been shown:26 miR-143 and miR-181 target KRAS in colon cancer and oral squamous cell carcinoma, respectively.27, 28 Furthermore, a recent study reported that EVI1 suppresses miR-143 and thus modulates KRAS expression in colon cancer cells,29 although downregulation of miR-143 is not consistently observed in clinical samples of pancreatic cancer.30 On the basis of these findings, we explored the involvement of miRNAs in EVI1-mediated KRAS regulation. Among various miRNA species that were potentially regulated by EVI1 or deregulated in pancreatic cancer,12, 29, 31, 32, 33, 34, 35 certain miRNAs, such as miR-96, -143, -181, -155, -216a and -216b, were predicted to target KRAS 3′-untranslated region (3′UTR) by using a number of target prediction algorithms (Figure 4a).
We examined whether EVI1 knockdown could change the expression of these mature miRNAs by qRT–PCR analysis. The expression levels of mature miR-96 and miR-181a were upregulated upon EVI1 silencing in PK-8 cells (Figure 4b). The upregulation of miR-96 was also observed in HPDE cells (Supplementary Figure 3G). miR-96 is located in the intergenic region on 7q32 and miR-181a arises from two primary miRNA clusters, which are located in the intron of two host genes, RP11-31E23.1-001 on 1q32 and MIR181A2HG-001 on 9q33. Quantitative analysis of primary miRNA or host genes showed that EVI1 knockdown induced the expression of pri-miR-96, RP11-31E23.1-001 and MIR181A2HG-001 (Figure 4c), suggesting that EVI1 controls these miRNAs through transcriptional regulation of pri-miRNAs. The genomic regions of ∼10-kb base pairs around these miRNAs were analyzed with rVISTA 2.0 to identify EVI1-binding sites (http://rvista.dcode.org/). With the use of a matrix similarity threshold of 0.80, one putative binding site was predicted in the case of miR-96, and two putative binding sites were predicted in the case of miR-181a-1 (Figure 4d). Chromatin immunoprecipitation analysis in PK-8 cells demonstrated that EVI1 binds to the potential binding site around miR-96 and miR-181a-1 (Figure 4e).
miRNA-96 potently suppresses KRAS and serves as tumor suppressor in pancreatic cancer
To verify the activity of miR-96 and miR-181a as KRAS modifiers, we compared the effects of miR-96 and miR-181a introduction on KRAS expression. miR-96 introduction more potently suppressed the levels of KRAS protein and ERK phosphorylation than miR-181a in PK-8 cells (Figures 5a and b). miR-96 introduction also suppressed the level of KRAS protein in HPDE cells and BxPC-3 cells (Figure 5a and Supplementary Figure 3H). This was confirmed by the luciferase reporter assay examining connections between KRAS 3′UTR and miR-96/-181a (Figure 5c). Our results were also consistent with a recent report that miR-96 was downregulated in pancreatic cancer and suppressed KRAS.36
Further functional analysis delineated potential tumor-suppressive functions of miR-96 and miR-181a. miR-96 introduction into PK-8 cells retarded cell proliferation and phenocopied the effects of EVI1 knockdown, whereas miR-181a did not suppress cell growth (Figure 5d). This clearly contrasted with the tumor-suppressive function of miR-181a previously reported in oral squamous cell carcinoma, suggesting context-dependent roles of this miRNA family. In wound closure analysis, both miR-96 and miR-181a inhibited cell motility of PK-8 cells, but the effect of miR-96 was more potent than that of miR-181a (Figure 5e). In addition, we confirmed that introduction of miR-96, but not miR-181a, resulted in cell cycle arrest in the G1 phase in PK-8 cells (Figure 5f). Therefore, these results indicate that miR-96, but not miR-181a, is a potent regulator of KRAS with tumor-suppressive functions in pancreatic cancer cells.
Involvement of miRNA-96 in EVI1-mediated proliferation control
To confirm the functional role of endogenous miR-96 in EVI1-mediated proliferative control, we established efficient suppression of these miRNAs through the decoy RNA system, in which tough decoy RNA (TuD RNA) acts as a decoy against specific miRNA. As shown in Figures 6a and b, inhibition of endogenous miR-96 by TuD-RNA showed a tendency to attenuate growth inhibition and KRAS suppression by silencing of EVI1, suggesting the possibility that miR-96 is at least partially involved in EVI1-driven pancreatic oncogenicity. In addition, during a series of experiments, we found that miR-96 potently suppressed the expression level of EVI1 in accordance with the presence of miR-96-binding site within EVI1 3′UTR (Figure 6c). This result suggests that EVI1 and miR-96 are linked in a reciprocal feedback loop.
Finally, we examined the correlation between EVI1 and KRAS in human pancreatic cancer data set using a publicly available microarray dataset (GSE17891).37 This analysis confirmed the positive correlation between EVI1 and KRAS mRNA expression (Figure 6d), and revealed that the expression levels of EVI1 and KRAS were higher in classical PDAC subtype than in exocrine-like PDAC and quasi-mesenchymal PDAC subtypes, which were previously defined by gene expression profiling in pancreatic cancer.37
In the present study, we revealed the association between EVI1 oncoprotein, miR-96 tumor suppressor and KRAS, a key mediator of pancreatic carcinogenesis (Figure 6e). The oncogenic role of EVI1 has been reported in some malignancies.11, 12, 29, 38 Our findings indicate that EVI1 serves as an important oncogenic factor by inducing deregulation of the balance between KRAS and miRNA-96 in pancreatic cancer. Transforming growth factor-β pathway and phosphatidylinositol 3-kinase/AKT/mammalian target of rapamycin (mTOR) pathway have been reported as pathways related to cell proliferation regulated by EVI1.18, 19, 21, 22 EVI1 was also reported as being related to programmed cell death through modulation of c-Jun N-terminal kinase or phosphatidylinositol 3-kinase/AKT pathway.20, 22 Although these pathways might be related to oncogenic role of EVI1 in pancreatic carcinogenesis, we were unable to find strong relation between EVI1 and the molecules composed of these pathways. We found that EVI1 modulated KRAS expression through miR-96 regulation in pancreatic lineage cells. This is supported by the positive relationship between EVI1 and KRAS expression in microarray data of pancreatic cancers (Figure 6d). In accordance with this, Szafranska et al.30 and Yu et al.36 showed that miR-96 was downregulated in pancreatic cancer, compared with that in normal pancreatic tissue. On the other hand, in pancreatic cancers, we could not confirm the EVI1-miR-143-KRAS axis reported in colon cancer.29 Therefore, EVI1 might regulate several tumor-suppressive miRNAs in a tissue-specific manner. Considering that miR-181 has been reported to be a KRAS regulator in oral squamous cell carcinoma, the relationship between miRNAs and KRAS might also depend on cancer types.27
It was previously shown that increased expression of EVI1 was caused partly by the high copy number in ovarian cancers,38 but we could not find amplification within EVI1-coding regions in pancreatic cancers (data not shown). As other mechanisms, previous reports suggested that methylation of EVI1 promoter regions or EVI1 acetylation could engender expression of EVI1.34, 39 Deregulation of the balance between miR-96 and EVI1 might also cause EVI1 overexpression, as we found that EVI1 and miR-96 were linked in a reciprocal feedback loop (Figures 6c and e). The previous studies of miRNA expression profiles in pancreatic cancer have mainly focused on comparison between PDACs and normal pancreas or chronic pancreatitis. Thus, more detailed miRNA profiling including that in PDAC precursors might shed light on the mechanisms of EVI1 upregulation and the timing of miR-96 downregulation during pancreatic tumorigenesis. Although further analyses are needed, some risk factors for pancreatic cancer, such as chronic pancreatitis and smoking, might also be associated with EVI1 upregulation in PDAC precursors.
In this study, immunohistochemical analysis revealed that EVI1 was expressed in almost all pancreatic neoplastic lesions, although EVI1 was not expressed in the normal pancreatic duct. This expression pattern of EVI1 raises hopes for clinical application as a tumor marker and suggests that EVI1 is involved in early pancreatic tumorigenic transformation.
Thus far, on the grounds of mutational analyses of human pancreatic neoplasm and establishment of mutant KRAS-driven pancreatic cancer mouse models, many researchers have regarded oncogenic KRAS as being critical for pancreatic carcinogenesis. However, only one-third of human PanIN-1 possesses oncogenic KRAS and only a small subset of cells with mutant KRAS expression can develop into PanINs in mouse models. Both findings leave it unclear whether KRAS mutation is solely responsible for initiation of PDAC precursors.6, 9, 10 Most recently, by means of pyrosequencing and high-resolution melt-curve analysis, Kanda et al. demonstrated that virtually all PanINs (92.0% of PanIN-1A and 92.3% of PanIN-1B) harbored KRAS mutation.40 However, they simultaneously found that KRAS mutations were generally present in only a fraction of the cells comprising the lesion in the earliest PanIN lesions and that many low-grade PanINs contained mixtures of mutant and wild-type KRAS cells. In their report, PanIN-1 lesions had low mutant KRAS concentrations (about 20% of alleles), suggesting that the driving force behind the expansion of all cells within PanIN lesions is not solely mutant KRAS, at least in a cell-autonomous manner. Moreover, Ji et al. reported that the degree of KRAS activity controlled the development of pancreatic disease besides the presence of KRAS mutation.41, 42 In a mouse model, activity of mutant KRAS differed among non-transformed tissue, PanIN and PDAC, although all of these lesions harbored KRAS mutation. These observations give rise to a hypothesis that other molecular mechanisms than KRAS mutation contribute to the expansion of PanIN cells and the constitutive activation of KRAS pathway. Our results suggest that EVI1 overexpression mediates altered cell expansion in PanIN and activation of KRAS pathway in pancreatic cancer cells with wild-type or mutant KRAS as one of these potential mechanisms, providing a scientific basis for this hypothesis.
In addition, the contribution of oncogenic KRAS to pancreatic carcinogenesis is controversial in other preneoplastic lesions, IPMN and MCN. Comparison of microdissected tissue from IPMN and PDAC showed that the total number of KRAS mutations was lower in IPMN than in PDAC.43 It has also been reported that the incidence of KRAS mutation was lower in IPMNs than in PanINs. In MCNs, oncogenic mutation of KRAS is observed in low-grade dysplasia, but studies of MCNs have reported a range in the prevalence of KRAS mutations.44 Because of the low penetration of KRAS mutation in IPMNs and MCNs, an oncogenic driver other than KRAS mutation might be needed. Our results that oncogenic EVI1 was widely expressed in both IPMN and MCN suggested that EVI1 might contribute to the initiation of carcinogenesis in these neoplastic lesions through modification of KRAS activity.
The expression pattern of EVI1 in normal digestive organs may also have some developmental implications in pancreatic carcinogenesis. Expression of extrapancreatic foregut or gastric epithelial markers is observed in most PDAC precursors.2, 5 Considering that EVI1 is usually expressed in gastric tissue and absent in normal pancreatic duct, misexpression of EVI in pancreatic lineage cells could drive phenotypic shift of pancreatic lineage cells toward gastric phenotype.
In conclusion, the present study demonstrated that EVI1 has an important oncogenic role through deregulation of the balance between KRAS and miR-96 in pancreatic carcinogenesis. Overexpression of EVI1 oncoprotein marks the full spectrum of human pancreatic cancer precursors and PDAC as a useful early marker of pancreatic neoplasms. Furthermore, inhibition of KRAS is an important therapeutic target in pancreatic cancer, thereby indicating the potential of EVI1 and/or miR-96as therapeutic targets. These findings might open a new avenue for our understanding of pancreatic carcinogenesis and the development of new therapeutic options.
Materials and methods
We reviewed the University of Tokyo Hospital pathology archives for the period from 1986 to 2009 and analyzed 224 cases of pancreatic ductal neoplasms, including 156 PDACs, 64 IPMNs and 5 MCNs.5 Thirty-two PanINs were also selected from these specimens. Investigations were performed in accordance with ethical standards authorized by the Research Ethics Committee of the University of Tokyo.
Histopathological and immunohistochemical examination
For each case, tumor tissues were processed and embedded in paraffin, and all the tissue slides were examined as previously described. Tissue microarrays for PDACs were constructed as previously described. The histological diagnosis was based on the World Health Organization classification and the textbook from the Armed Forces Institute of Pathology.1, 7 Immunohistochemical staining in surgically resected specimens was performed using the Ventana BenchMark automated immunostainer. EVI1 expression was evaluated according to the staining pattern and intensity. The immunostaining of EVI1 was evaluated as being negative, weakly positive, moderately positive or strongly positive. The labeling of EVI1 was regarded as strong (3+) if more than 80% of the neoplastic cells were labeled at a strong intensity; moderate (2+) if 25–80% of the neoplastic cells were labeled at any intensity or if more than 80% of these cells were labeled at a moderate or weak intensity; weak (1+) if 1–25% of the neoplastic cells were labeled at any intensity; and negative (0) if less than 1% of the neoplastic cells were labeled. Normal gastric epithelium was used as a positive control for EVI1 immunostaining. The immunohistochemical specimens were blindly and independently evaluated by two pathologists (MT and JS).
HPDE cell line was a kind gift from Dr Ming Tsao, Ontario Cancer Institute. One pancreatic cancer cell line, BxPC-3, was purchased from American Type Culture Collection. The other nine pancreatic cancer cell lines were obtained from Genome Science Division, RCAST, the University of Tokyo. Among pancreatic cancer cell lines, eight cell lines, PANC-1, PK-1, PK-8, PK-9, PK-45H, PK-45P, KLM-1 and BxPC-3, were cultured in RPMI 1640 medium (Nacalai Tesque Inc. Kyoto, Japan), and two cell lines, MIA PaCa-2 and Capan-1, were cultured in Dulbecco’s modified Eagle’s medium (Nacalai Tesque Inc.) supplemented with 10% fetal bovine serum, penicillin (40 U/ml) and streptomycin (50 μg/ml). HPDE cells were cultured in keratinocyte serum-free medium with 0.2 ng/ml epidermal growth factor and 30 μg/ml bovine pituitary extract (Invitrogen, Carlsbad, CA, USA). HEK293T cells were maintained in Dulbecco’s modified Eagle’s medium (Nacalai Tesque Inc.) supplemented with 10% fetal bovine serum.
Antibodies and reagents
The antibodies used for immunohistochemical examination, western blotting and chromatin immunoprecipitation were as follows: EVI1 C50E12 (Cell Signaling Technology, Danvers, MA, USA), KRAS F234 Santa Cruz Biotechnologies Inc. (Santa Cruz, CA, USA), ERK1 SC-94 (Santa Cruz), phospho-p44/42 MAPK (T262/Y204) #9102 (Cell Signaling), Akt1/2N-19 (Santa Cruz), phospho-Akt (Ser473) #9271 (Cell Signaling), phospho-SMAD3 (Ser423/425) C25A9 (Cell Signaling), p38 #9212 (Cell Signaling), phospho-p38 MAPK (Thr180/Try182) #9211 (Cell Signaling), c-jun #9165 (Cell Signaling), phospho-c-jun (Ser73) #9164 (Cell Signaling), p27 K25020 (BD Biosciences, San Jose, CA, USA) and β-actin I-19 (Santa Cruz). MEK inhibitor U0126 was purchased from Wako.
siRNA and miRNA duplex
siRNAs and synthetic miRNA duplex were purchased from Invitrogen and Qiagen (Hilden, Germany; miRNA precursor), respectively, and introduced at 20 nM using Lipofectamine RNAi MAX (Invitrogen). Control siRNAs were purchased from Invitrogen (Stealth RNAi) or Qiagen (AllStars Negative Control). The transfected cells were used for subsequent experiments after 72 h. Details of siRNAs are given in Supplementary Table 3.
Cultured cells were lysed in a lysis buffer containing 10 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM EDTA, 1.0% Triton X-100, 1.0% sodium deoxycholate, 0.1% SDS, 1 mM phenylmethanesulfonylfluoride, protease inhibitor cocktail (Sigma-Aldrich, St Louis, MO, USA), and phosphatase inhibitor EDTA-free (Nacalai Tesque). Ten to forty μg of protein per lane was loaded and fractionated on a 7–15% SDS polyacrylamide gel. After transfer onto a polyvinylidene difluoride membrane, probing was conducted. The membranes were visualized using the ECL Plus Western Blotting Detection System (GE Healthcare, Buckinghamshire, UK).
RNA isolation and qRT–PCR
For detection of mRNAs, RNA was extracted using RNeasy Mini Kit (Qiagen) and subjected to reverse transcription using the PrimeScript 1st strand cDNA Synthesis kit (Invitrogen). qRT–PCR was performed with iCycler (Bio-Rad, Hercules, CA, USA) and analyzed using the absolute standard curve method. For detection of miRNA, total RNA was extracted using mirVana miRNA Isolation kit (Ambion, Austin, TX, USA). The expression levels of mature miRNAs were determined using TaqMan MicroRNA assay kit (Applied Biosystems, Foster City, CA, USA) or miScript PCR system (Qiagen). Data analysis was carried out by the comparative CT method. Results were normalized to β-actin for mRNA detection, and U6 snRNA or RNU44 snoRNA for evaluation of mature miRNA. The primer sequences used are given in Supplementary Table 3.
Growth assay, migration assay and cell cycle analysis
Cells (3 × 103 cells) were seeded in 96-well plates and transfected with siRNAs. After 0, 24, 48, 72 and 96 h, cell viability was quantified by colorimetric assay using WST-8 (Nacalai Tesque). In an in vitro wound-healing assay, cells were grown to confluence and then damaged with a plastic pipette tip. The wound area was then photographed and at >15 h thereafter. The distance of migration was determined using computer-driven image analysis. Cell cycle profiling was performed using FACScan flow cytometer (Beckman Coulter, Brea, CA, USA). For nocodazole treatment, cells were collected after 17 h incubation with nocodazole (100 ng/ml).
Chromatin immunoprecipitation analysis
Luciferase reporter assay
The 3′UTR segment of KRAS was cloned into the XhoI and NotI sites of the 3′UTR of the luciferase gene in the Psicheck 2 dual luciferase reporter vector (Promega, Fitchburg, WI, USA). Cells were transfected with each reporter construct with miRNA precursor using Lipofectamine 2000 (Invitrogen), and luciferase activity was measured 48 h after transfection using the Dual-Luciferase Reporter Assay System (Promega).
Inhibition of miRNA by TuD RNA
TuD RNA against miR-96 was designed and transferred into CSII-EF-RfA-EG lentivirus vector (a kind gift from H Miyoshi, RIKEN, Tsukuba, Japan) as previously described.45 The lentiviral particles were produced according to a previous report, concentrated using Lenti-X Concentrator Kit (Takara Bio, Shiga, Japan), and introduced into cultured cells.
Statistical analysis was performed using Student’s t-test. P<0.05 was considered to be statistically significant (*P<0.05).
Hruban R, Boffetta P, Hiraoka N, Iacobuzio-Donahue C, Kato Y, Kern S et al. Tumours of the pancreas. In: Bosman F, Carneiro F, Hruban R, Theise N eds). WHO Classification of Tumours of the Digestive System 4th edn International Agency for Research on Cancer (IARC), Lyon, 2010, pp 279–337.
Prasad NB, Biankin AV, Fukushima N, Maitra A, Dhara S, Elkahloun AG et al. Gene expression profiles in pancreatic intraepithelial neoplasia reflect the effects of Hedgehog signaling on pancreatic ductal epithelial cells. Cancer Res 2005; 65: 1619–1626.
Kim GE, Bae HI, Park HU, Kuan SF, Crawley SC, Ho JJ et al. Aberrant expression of MUC5AC and MUC6 gastric mucins and sialyl Tn antigen in intraepithelial neoplasms of the pancreas. Gastroenterology 2002; 123: 1052–1060.
Yonezawa S, Higashi M, Yamada N, Yokoyama S, Goto M . Significance of mucin expression in pancreatobiliary neoplasms. J Hepatobiliary Pancreat Sci 2010; 17: 108–124.
Tanaka M, Shibahara J, Fukushima N, Shinozaki A, Umeda M, Ishikawa S et al. Claudin-18 is an early-stage marker of pancreatic carcinogenesis. J Histochem Cytochem 2011; 59: 942–952.
Morris JPt, Wang SC, Hebrok M . KRAS, Hedgehog, Wnt and the twisted developmental biology of pancreatic ductal adenocarcinoma. Nat Rev Cancer 2010; 10: 683–695.
Hruban R, Pitman M, Klimstra D . Tuomrs of the pancreas. In: Silverberg S (ed). AFIP Atlas of Tumor Pathology. American Registry of Pathology, Washington DC, 2007, pp 51–164.
Guerra C, Mijimolle N, Dhawahir A, Dubus P, Barradas M, Serrano M et al. Tumor induction by an endogenous K-ras oncogene is highly dependent on cellular context. Cancer Cell 2003; 4: 111–120.
Hingorani SR, Petricoin EF, Maitra A, Rajapakse V, King C, Jacobetz MA et al. Preinvasive and invasive ductal pancreatic cancer and its early detection in the mouse. Cancer Cell 2003; 4: 437–450.
De La OJ, Emerson LL, Goodman JL, Froebe SC, Illum BE, Curtis AB et al. Notch and Kras reprogram pancreatic acinar cells to ductal intraepithelial neoplasia. Proc Natl Acad Sci USA 2008; 105: 18907–18912.
Goyama S, Kurokawa M . Pathogenetic significance of ecotropic viral integration site-1 in hematological malignancies. Cancer Sci 2009; 100: 990–995.
Yoshimi A, Kurokawa M . Evi1 forms a bridge between the epigenetic machinery and signaling pathways. Oncotarget 2011; 2: 575–586.
Ge X, Yamamoto S, Tsutsumi S, Midorikawa Y, Ihara S, Wang SM et al. Interpreting expression profiles of cancers by genome-wide survey of breadth of expression in normal tissues. Genomics 2005; 86: 127–141.
Cartharius K, Frech K, Grote K, Klocke B, Haltmeier M, Klingenhoff A et al. MatInspector and beyond: promoter analysis based on transcription factor binding sites. Bioinformatics 2005; 21: 2933–2942.
Quandt K, Frech K, Karas H, Wingender E, Werner T . MatInd and MatInspector: new fast and versatile tools for detection of consensus matches in nucleotide sequence data. Nucleic Acids Res 1995; 23: 4878–4884.
Song Y, Washington MK, Crawford HC . Loss of FOXA1/2 is essential for the epithelial-to-mesenchymal transition in pancreatic cancer. Cancer Res 2010; 70: 2115–2125.
Kwei KA, Bashyam MD, Kao J, Ratheesh R, Reddy EC, Kim YH et al. Genomic profiling identifies GATA6 as a candidate oncogene amplified in pancreatobiliary cancer. PLoS Genet 2008; 4: e1000081.
Izutsu K, Kurokawa M, Imai Y, Maki K, Mitani K, Hirai H . The corepressor CtBP interacts with Evi-1 to repress transforming growth factor beta signaling. Blood 2001; 97: 2815–2822.
Kurokawa M, Mitani K, Irie K, Matsuyama T, Takahashi T, Chiba S et al. The oncoprotein Evi-1 represses TGF-beta signalling by inhibiting Smad3. Nature 1998; 394: 92–96.
Kurokawa M, Mitani K, Yamagata T, Takahashi T, Izutsu K, Ogawa S et al. The evi-1 oncoprotein inhibits c-Jun N-terminal kinase and prevents stress-induced cell death. EMBO J 2000; 19: 2958–2968.
Yoshimi A, Goyama S, Watanabe-Okochi N, Yoshiki Y, Nannya Y, Nitta E et al. Evi1 represses PTEN expression and activates PI3K/AKT/mTOR via interactions with polycomb proteins. Blood 2011; 117: 3617–3628.
Liu Y, Chen L, Ko TC, Fields AP, Thompson EA . Evi1 is a survival factor which conveys resistance to both TGFbeta- and taxol-mediated cell death via PI3K/AKT. Oncogene 2006; 25: 3565–3575.
Gysin S, Lee SH, Dean NM, McMahon M . Pharmacologic inhibition of RAF–;>MEK–;>ERK signaling elicits pancreatic cancer cell cycle arrest through induced expression of p27Kip1. Canceer Res 2005; 65: 4870–4880.
Suzuki HI, Miyazono K . Dynamics of microRNA biogenesis: crosstalk between p53 network and microRNA processing pathway. J Mol Med (Berl) 2010; 88: 1085–1094.
Suzuki HI, Miyazono K . Emerging complexity of microRNA generation cascades. J Biochem 2011; 149: 15–25.
Suzuki HI, Yamagata K, Sugimoto K, Iwamoto T, Kato S, Miyazono K . Modulation of microRNA processing by p53. Nature 2009; 460: 529–533.
Shin KH, Bae SD, Hong HS, Kim RH, Kang MK, Park NH . miR-181a shows tumor suppressive effect against oral squamous cell carcinoma cells by downregulating K-ras. Biochem Biophys Res Commun 2011; 404: 896–902.
Chen X, Guo X, Zhang H, Xiang Y, Chen J, Yin Y et al. Role of miR-143 targeting KRAS in colorectal tumorigenesis. Oncogene 2009; 28: 1385–1392.
Gao JS, Zhang Y, Tang X, Tucker LD, Tarwater PM, Quesenberry PJ et al. The Evi1, microRNA-143, K-Ras axis in colon cancer. FEBS Lett 2011; 585: 693–699.
Szafranska AE, Davison TS, John J, Cannon T, Sipos B, Maghnouj A et al. MicroRNA expression alterations are linked to tumorigenesis and non-neoplastic processes in pancreatic ductal adenocarcinoma. Oncogene 2007; 26: 4442–4452.
Gomez-Benito M, Conchillo A, Garcia MA, Vazquez I, Maicas M, Vicente C et al. EVI1 controls proliferation in acute myeloid leukaemia through modulation of miR-1-2. Br J Cancer 2010; 103: 1292–1296.
De Weer A, Van der Meulen J, Rondou P, Taghon T, Konrad TA, De Preter K et al. EVI1-mediated down regulation of MIR449A is essential for the survival of EVI1 positive leukaemic cells. Br J Haematol 2011; 154: 337–348.
Nagaraja AK, Creighton CJ, Yu Z, Zhu H, Gunaratne PH, Reid JG et al. A link between mir-100 and FRAP1/mTOR in clear cell ovarian cancer. Mol Endocrinol 2010; 24: 447–463.
Senyuk V, Premanand K, Xu P, Qian Z, Nucifora G . The oncoprotein EVI1 and the DNA methyltransferase Dnmt3 co-operate in binding and de novo methylation of target DNA. PLoS One 2011; 6: e20793.
Dickstein J, Senyuk V, Premanand K, Laricchia-Robbio L, Xu P, Cattaneo F et al. Methylation and silencing of miRNA-124 by EVI1 and self-renewal exhaustion of hematopoietic stem cells in murine myelodysplastic syndrome. Proc Natl Acad Sci USA 2010; 107: 9783–9788.
Yu S, Lu Z, Liu C, Meng Y, Ma Y, Zhao W et al. miRNA-96 suppresses KRAS and functions as a tumor suppressor gene in pancreatic cancer. Cancer Res 2010; 70: 6015–6025.
Collisson EA, Sadanandam A, Olson P, Gibb WJ, Truitt M, Gu S et al. Subtypes of pancreatic ductal adenocarcinoma and their differing responses to therapy. Nat Med 2011; 17: 500–503.
Nanjundan M, Nakayama Y, Cheng KW, Lahad J, Liu J, Lu K et al. Amplification of MDS1/EVI1 and EVI1, located in the 3q26.2 amplicon, is associated with favorable patient prognosis in ovarian cancer. Cancer Res 2007; 67: 3074–3084.
Vazquez I, Maicas M, Cervera J, Agirre X, Marin-Bejar O, Marcotegui N et al. Down-regulation of EVI1 is associated with epigenetic alterations and good prognosis in patients with acute myeloid leukemia. Haematologica 2011; 96: 1448–1456.
Kanda M, Matthaei H, Wu J, Hong SM, Yu J, Borges M et al. Presence of somatic mutations in most early-stage pancreatic intraepithelial neoplasia. Gastroenterology 2012; 142: 730–733 e739.
Logsdon CD, Ji B . Ras activity in acinar cells links chronic pancreatitis and pancreatic cancer. Clin Gastroenterol Hepatol 2009; 7: S40–S43.
Ji B, Tsou L, Wang H, Gaiser S, Chang DZ, Daniluk J et al. Ras activity levels control the development of pancreatic diseases. Gastroenterology 2009; 137: 1072–1082 1082 e1-6.
Kitago M, Ueda M, Aiura K, Suzuki K, Hoshimoto S, Takahashi S et al. Comparison of K-ras point mutation distributions in intraductal papillary-mucinous tumors and ductal adenocarcinoma of the pancreas. Int J Cancer 2004; 110: 177–182.
Jimenez RE, Warshaw AL, Z’Graggen K, Hartwig W, Taylor DZ, Compton CC et al. Sequential accumulation of K-ras mutations and p53 overexpression in the progression of pancreatic mucinous cystic neoplasms to malignancy. Ann Surg 1999; 230: 501–509 discussion 509-511.
Matsuyama H, Suzuki HI, Nishimori H, Noguchi M, Yao T, Komatsu N et al. miR-135b mediates NPM-ALK-driven oncogenicity and renders IL-17-producing immunophenotype to anaplastic large cell lymphoma. Blood 2011; 118: 6881–6892.
Sentani K, Oue N, Tashiro T, Sakamoto N, Nishisaka T, Fukuhara Tea . Immunohistochemical staining of Reg IV and claudin-18 is useful in the diagnosis of gastrointestinal signet ring cell carcinoma. Am J Surg Pathol 2008; 32: 1182–1189.
We thank all members of the Department of Pathology, University of Tokyo. This work was supported by the Industrial Technology Research Grant Program (2008) from the New Energy and Industrial Technology Development Organization (NEDO) of Japan (SI), the Grant-in-Aid for Scientific Research on Innovative Areas (SI),46 the Grant-in-Aid for Young Scientists (A),46 the GCOE Program for ‘Integrative Life Science Based on the Study of Biosignaling Mechanisms’ (HIS, KM), and for ‘Chemical Biology of the Diseases’ (MF and MK) from the Ministry of Education, Culture, Sports, Science and Technology of Japan and the Cell Science Research Foundation (HIS, KM).
The authors declare no conflict of interest.
Supplementary Information accompanies this paper on the Oncogene website
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Tanaka, M., Suzuki, H., Shibahara, J. et al. EVI1 oncogene promotes KRAS pathway through suppression of microRNA-96 in pancreatic carcinogenesis. Oncogene 33, 2454–2463 (2014). https://doi.org/10.1038/onc.2013.204
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