EVI1 oncogene promotes KRAS pathway through suppression of microRNA-96 in pancreatic carcinogenesis


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).

Figure 1

Expression of EVI1 oncoprotein in pancreatic neoplastic tissues and pancreatic-lineage cell lines. (a-f), Immunohistochemical analysis of EVI1 in pancreatic tissue ((a, c, e) hematoxylin and eosin (H&E) staining; (b, d, f) EVI1 immunostaining). (a, b) Non-neoplastic pancreas. Normal pancreatic ducts (black arrowhead) and islet (black star) do not express EVI1. Acinar cells (black arrow) weakly express EVI1. (c, d) PanINs. In the PanIN-1 lesion (low-grade PanIN), EVI1 is expressed strongly in the nucleus. (e, f) PDACs. In well-differentiated PDACs, EVI1 is expressed strongly. (g, h) Expression of EVI1 mRNA (g) and EVI1 protein (h) in several pancreatic-lineage cell lines. These cells express three major variants of EVI1, MDS1/EVI1, EVI1 and EVI1s. See also Supplementary Figure 1.

Table 1 EVI1 expression in normal, metaplastic and neoplastic pancreatic ductal lesions

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.

Figure 2

Oncogenic functions of EVI1 in proliferation and migration of pancreatic cancer cells. (a) Two siRNA targeted EVI1, siEVI1-#1 and siEVI1-#2, effectively downregulate the expression of EVI1 in PK-8 and HPDE cells. (b) Effects of EVI1 knockdown on proliferation of EVI1-positive pancreatic-lineage cells. Growth assay was carried out in 96-well plates, where cells were plated at 3000 cells per well and grown in 0.5% fetal bovine serum medium. Experiments were carried out in triplicate. *P<0.01. (c) Effects of EVI1 knockdown on cellular migration of pancreatic-lineage cells. Migration activity was examined in normal medium in HPDE cells and in 0.5% fetal bovine serum medium in PK-8 cells (HPDE cells; photos, PK-8 cells; bar graph). Experiments were carried out in triplicate. *P<0.01. (d, e) EVI1 knockdown leads to G1 arrest. Fluorescence-activated cell-sorting analysis in PK-8 cells transfected with control siRNA or siEVI1 in the absence (d) or presence (e) of nocodazole (100 ng/ml). See also Supplementary Figure 2.

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).

Figure 3

Silencing of EVI1 suppresses the expression of KRAS protein and modulates ERK and p27Kip1. (ac) EVI1 knockdown inhibits KRAS-ERK pathway and upregulates p27Kip1. After transfection with control siRNA or siEVI1 in PK-8 cells and HPDE cells, immunoblot analysis was performed using the indicated antibodies. (d) Upregulation of p27Kip1 by the treatment with ERK inhibitor U0126 in PK-8 cells. (e) U0126 causes cell cycle G1 arrest in PK-8 cells. PK-8 cells were treated for 24 h with dimethyl sulfoxide or U0126 (5, 25 or 100 μM). Analysis of the cell cycle distribution was conducted using propidium iodide staining. (f) EVI1 controls cellular growth via the KRAS-ERK pathway. PK-8 cells were transfected with siRNAs and treated with various doses of U0126 48 h after transfection. Cell viability was determined using WST-8 assay in triplicate after 24 h. Data are shown as absolute proliferation. **P<0.05, NS: not significant.

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).

Figure 4

EVI1 transcriptionally suppresses miR-96 and miR-181a in PK-8 cells. (a) Schematic diagram of human KRAS 3′UTR and potential miRNA target sites. (b) Expression changes of potential KRAS-targeting miRNAs by EVI1 knockdown. Mature miRNAs in PK-8 cells were analyzed using qRT–PCR after EVI1 knockdown (72 h). (c) Transcriptional upregulation of miR-96 and miR-181a by EVI1 knockdown. Primary miRNA-96 (pri-miR-96) and the host gene of miR-181a (RP11-31E23.1-001, MIR181A2) were measured after EVI1 knockdown (72 h). *P<0.01, **P<0.05. (d) Schematic representation of the genomic regions around human miR-96 and miR-181a-1, with putative EVI1-binding sites predicted by rVISTA 2.0 (http://rvista.dcode.org/). (e) Chromatin immunoprecipitation analysis for EVI1 in PK-8 cells using primer sets amplifying the putative EVI1-binding sites indicated in d.

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

Figure 5

miR-96 is a potent regulator of KRAS with bona fide tumor-suppressive functions. (a) miR-96 inhibits KRAS expression. After transfection with negative control or synthetic miRNAs (20 nM) in PK-8 cells and HPDE cells, immunoblot analysis was performed. The graph shows miR-96 and miR-181a expression measured by qRT–PCR after transfection to PK-8 cells, as a representative for the experiments. (b) miR-96 inhibits ERK phosphorylation in PK-8 cells. (c) Luciferase activity in HEK293T cells cotransfected with control vector or KRAS 3′UTR luciferase reporters and miR-96 or miR-181a. Data are presented as mean±s.e.m. and normalized to vector control. (df) miRNA-96 decreased proliferation and migration and induced G1 arrest in PK-8 cells, examined as in Figures 2a–c. *P<0.01.

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.

Figure 6

miR-96 participates in EVI1-mediated growth control. (a) Inhibition of miR-96 by TuD-RNA slightly attenuates growth inhibition by EVI1 knockdown. PK-8 cells were transduced with TuD-RNA vectors, transfected with siRNAs and then subjected to WST-8 proliferation assay. (b) Involvement of miR-96 in EVI1-mediated KRAS regulation. PK-8 cells were transduced with TuD-RNA vectors, transfected with siRNAs and then subjected to immunoblot analysis. (c) Effects of miR-96 introduction on EVI1 expression. After transfection with negative control or synthetic miRNAs (20 nM) in PK-8 cells, immunoblot analysis was performed. (d) Correlation between KRAS and EVI1 expression in clinical samples of pancreatic cancer patients (GSE17891). (e) Summary of EVI1-miR-96-KRAS axis in pancreatic carcinogenesis. QM-PADC, quasi-mesenchymal PDAC.

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).

Cell lines

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.

Western blotting

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

Chromatin immunoprecipitation analysis was performed in PK-8 cells according to a previous report.45 The primer sequences used are given in Supplementary Table 3.

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.

Microarray analysis

Microarray data for PDAC were obtained through accession no. GSE17891 from the National Center for Biotechnology Information’s Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/).37

Statistical analysis

Statistical analysis was performed using Student’s t-test. P<0.05 was considered to be statistically significant (*P<0.05).

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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).

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Correspondence to M Fukayama.

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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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  • pancreatic cancer
  • PanIN
  • EVI1
  • KRAS
  • microRNA

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