The progression from precursor lesions of pancreatic cancer, including pancreatic intraepithelial neoplasia and intraductal papillary mucinous neoplasm (IPMN), to invasive disease is characterized by stepwise accumulation of genetic alterations. However, it remains unclear whether additional alterations are required for the progression of high-grade neoplasms to invasive pancreatic carcinoma. We compared the genomic profiles of paired noninvasive and invasive carcinoma tissues collected from patients with IPMN. We demonstrate that the frequency of genomic copy-number aberrations significantly increased during the course of invasion, and the loss of 8p11.22-ter was more often associated with invasive tissues. Expression profiling in pancreatic cancer cell lines with and without 8p11.22-ter revealed that DUSP4, an MAPK phosphatase, was significantly downregulated in cells lacking 8p11.22-ter as well as in invasive carcinomas due to genomic loss. Restoration of DUSP4 expression in pancreatic cancer cells significantly suppressed invasiveness and anoikis resistance via ERK inactivation. Accordingly, we found that blockade of ERK signaling by MEK inhibition was effective in an orthotopic xenograft model and significantly extended survival. Collectively, our findings demonstrate a genetic mechanism by which pancreatic precursor lesions progress to invasive carcinomas and highlight DUSP4 as a novel invasion suppressor that can be therapeutically exploited through manipulation of ERK signaling. Cancer Res; 76(9); 2612–25. ©2016 AACR.

Pancreatic cancer remains a highly lethal disease despite advances in surgical care and adjuvant chemotherapy with new anticancer drugs, including molecular targeting drugs. The overall survival rate remains poor, only 5% of patients surviving for 5 years (1).

Most invasive pancreatic cancers are believed to arise from histologically well-defined precursor lesions referred to as pancreatic intraepithelial neoplasia (PanIN) or intraductal papillary mucinous neoplasm (IPMN), because these precursor lesions and invasive pancreatic cancers share critical genetic abnormalities, including mutation of the KRAS, TP53, p16/CDKN2A, and SMAD4 genes (2–10). In low-grade precursors, such as PanIN-1A, -1B, and IPMN with low-grade dysplasia, KRAS activation due to genomic mutation and telomere shortening are predominant (11). On the other hand, in high-grade precursors such as PanIN-3 and IPMN with high-grade dysplasia, inactivation of p16/CDKN2A, TP53, and SMAD4 due to mutation or genetic loss are frequently detected in addition to the genomic alterations found in low-grade precursors. Thus, stepwise accumulation of genetic alterations during the progression of pancreatic precursors has been well established (12). However, it remains to be determined whether additional gene alterations are required for progression from high-grade precursors to invasive pancreatic cancers.

In this study, our aim was to identify gene abnormalities necessary for the transformation from carcinoma in situ (CIS) of the pancreas to invasive pancreatic cancer. We compared the genomic profiles of paired samples from the invasive region and the CIS region of the identical IPMN with an associated invasive carcinoma (referred to hereafter as "IPMN-inv"; ref. 13).

Reagents

Human recombinant EGF, blasticidin and puromycin were purchased from Invitrogen. Anisomycin, JNK inhibitor (SP600125), and p38 inhibitor (SB239063) were purchased from Calbiochem. ERK inhibitor (SCH772984) was purchased from ChemieTek. MEK inhibitors used in in vitro experiments (U0126) and xenograft experiments (PD325901) were purchased from Calbiochem and LC Laboratories, respectively. The proliferation inhibitor mitomycin C was purchased from Nacalai tesque. G418, hydroxypropyl methylcellulose and Tween 80 were purchased from Sigma.

Patients and tissues

Tissue samples were obtained from 10 (case 14, 20, 25–31, and 40) and 19 (case 1–12, 16–19, 22, 23, and 24) patients with IPMN-inv and IDC (Supplementary Tables S1 and S3), respectively, who underwent surgical resection at Oita University Hospital (Oita, Japan). The specimens were fixed in 10% formalin and embedded in paraffin (FFPE). Serial sections cut from paraffin-embedded blocks were used for histopathology, array CGH, mutation analysis of the KRAS gene, and IHC. To obtain genomic DNA, serial sections 10-μm thick were prepared and stained with toluidine blue (Wako). CIS and invasive regions in each individual case of IPMN-inv were diagnosed and distinguished microscopically by two surgical pathologists (N. Hijiya and T. Uchida), and were collected separately using LCM (Arcturus Engineering). Cancer cells of IDC were also collected using LCM. All samples included a proportion of cancer cells exceeding 80% of the total. Genomic DNA was extracted according to the standard proteinase K digestion method, followed by phenol/chloroform extraction and ethanol precipitation. As the reference DNA for array CGH, genomic DNA from normal renal cortex was prepared as described previously (14). Genomic analyses in this study were approved by the Ethics Committee of Oita University Hospital (Approval number: P-07-05 and 437) in accordance with the Ethical Guidelines for Clinical Research of the Japanese Ministry of Health, Labor, and Welfare, 2008 (http://www.mhlw.go.jp/english/). Written informed consent was obtained from all patients.

Cell lines

The human pancreatic cancer cell lines AsPC-1 and BxPC-3 were obtained from the ATCC. KP4-1, PK-45H, PK-59, MIA Paca2, PANC-1, and PK-1 were obtained from RIKEN BRC through the National Bio-Resource Project of MEXT, Japan. SUIT-2, TCC-Pan2, and KP-1-NL were obtained from the Japanese Collection of Research Bioresources. PK-9 was obtained from the Cell Resource Center for Biomedical Research Institute of Development, Tohoku University. Two human pancreatic duct cell lines, hTERT-HPNE immortalized by transduction with hTERT (referred to as “HPNE”) and hTERT-HPNE E6/E7/K-RasG12D/st developed by transduction of HPNE cells with human papillomavirus E6 and E7 oncogenes, the G12D mutant of the human K-Ras gene and SV40 small t antigen (referred to as “HPNE-T”), were purchased from the ATCC. All cell lines were authenticated by the providers for growth, morphology, short-tandem repeat profile, and isoenzymology. All cell lines were propagated and frozen immediately after arrival. The cells revived from the frozen stock were used within less than 10 weeks in accordance with the supplier's instructions. Genomic DNA for array CGH was extracted from pancreatic cancer cell lines using the same procedure as that described above, and total RNA for expression analysis was extracted using an RNeasy Mini kit (Qiagen). As the reference DNA for array CGH, genomic DNA was prepared from human peripheral blood mononuclear cells of 8 healthy donors.

Array CGH analysis

Array CGH analysis was performed using 244 k and 44 k oligonucleotide CGH arrays (Agilent Technologies) for genomic DNA from cancer tissues and cancer cell lines, respectively, as described previously (14). Labeling and hybridization were performed in accordance with the manufacturer's instructions. Briefly, 1 to 2 μg of cancer DNA and an equal amount of control DNA were digested with AluI and RsaI (Promega). The digested DNAs were labeled with Cy5-dUTP and Cy3-dUTP, respectively, using a Genomic DNA Labeling Kit Plus (Agilent Technologies), and purified with Microcon YM-30 filters (Millipore). Equal amounts of DNAs were subsequently pooled and mixed with human Cot-1 DNA, dissolved in hybridization buffer (Agilent Technologies), denatured, and hybridized to the 244 k- and 44 k-CGH array at 65°C for 40 and 24 hours, respectively. Glass slides were washed and then scanned in accordance with the manufacturer's instructions.

Expression microarray analysis

Three hundred nanograms of total RNAs extracted from 12 pancreatic cancer cell lines were subjected to expression microarray analysis in accordance with the manufacturer's instructions, as described previously (15). Briefly, Cy3-labeled cRNA targets were generated using a Quick Amp Labeling Kit, one-color (Agilent Technologies). A human 44 K oligoarray was used for hybridization, in accordance with the manufacturer's recommendations (Agilent Technologies). A laser confocal scanner (Agilent Technologies) was used to measure signal intensities in the expression microarray analysis. Feature Extraction software (Version 9.1, Agilent Technologies) with the manufacturer's recommended settings was applied for the microarray image analysis. Analysis of the microarray images was performed with Genespring 7.3.1 Software (Agilent Technologies). All the data obtained in the expression microarray analysis are available on the GEO database, under accession number GSE65176 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=uvgjcmcmthujzsz&acc=GSE65176). For comparison among multiple arrays, probe set data were median-normalized per chip, followed by filtering based on signal intensity and flagged values. The genes downregulated due to loss at 8p11.22-ter were identified using the following filters: located at 8p11.22-ter, showing more than a 2-fold increase relative to normalized data in cell lines without loss at 8p11.22-ter compared with those showing loss at 8p11.22-ter, and with more than 1,000 of raw data in cell lines without loss at 8p11.22-ter.

Lentiviruses expressing candidate genes

Human Gateway entry clones containing full-length cDNAs of BIN3 (FLJ13918AAAF), FBXO25 (FLJ84754AAAF), PLEKHA2 (FLJ75843AAAF), PLAT (FLJ93864AAAF), FDFT1 (FLJ33164AAAF), RAB11FIP1 (FLJ34977AAAF), ZNF395 (FLJ11188AAAF), DUSP4 (FLJ95698AAAF), and ZDHHC2 (FLJ83050AAAF) were purchased from NBRC, NITE, Kisarazu, Japan. Using these clones with the Gateway system (Invitrogen), lentiviruses encoding each of these cDNA and those encoding no cDNAs for transient transduction were generated as described previously (15) and designated LV-BIN3, LV-FBXO25, LV-PLEKHA2, LV-PLAT, LV-FDFT1, LV-RAB11FIP1, LV-ZNF395, LV-DUSP4, LV-ZDHHC2, and LV-cont, respectively. Transduction of pancreatic cancer cell lines was performed at an optimized multiplicity of infection of 5 with Polybrene at a final concentration of 6.0 μg/mL. Forty-eight hours after transduction, the cells were used for the following experiments. For establishing stable clones re-expressing DUSP4, lentivirus encoding both DUSP4 and the blasticidin resistance gene was generated (15).

Establishment of stable clones

PANC-1 clones stably reexpressing DUSP4 (PANC-1/DUSP4-1, 2) were established by transduction with the lentivirus encoding both DUSP4 and the blasticidin resistance gene, and selection with blasticidin. As a control, PANC-1 transduced with lentivirus encoding only the blasticidin resistance gene was prepared (PANC-1/cont). PANC-1 stably expressing the dominant negative form of MEK1 (PANC-1/MEK1dn) and a control clone (PANC-1/vec) were established by transfection with an expression plasmid containing MEK1dn cDNA (16) and an empty plasmid, respectively, followed by selection with G418. PK-59 clones stably expressing DUSP4 shRNA (PK-59/shDUSP4-1, 2) were established by transduction with the lentivirus encoding both DUSP4 shRNA and the puromycin resistance gene (MISSION shRNA Lentiviral Transduction Particles, human DUSP4; Sigma), and selection with puromycin. As a control, PK-59 transduced with lentivirus encoding non-target shRNA and the puromycin resistance gene (MISSION TRC2 Control Transduction Particle puro Non-target shRNA#1; Sigma) was prepared (PK-59/shCont).

Statistical analysis

Statistical analyses were performed using JMP 11.0 (SAS Institute). Comparison of the number of probes showing CNA between the CIS region of IPMN-inv and paired invasive region of IPMN-inv for each case was determined by the paired t test. Chromosomal imbalances frequently detected in invasive region of IPMN-inv compared with in CIS region of IPMN-inv were analyzed by the McNemar test. Comparison of frequent chromosomal aberrations in IDC with those in the invasive region of IPMN-inv was determined by the Fisher exact test. The association between DUSP4 expression in cancer cells and the status of their invasiveness or 8p genotype was determined by the Fisher exact test. Survival curves were calculated using the Kaplan–Meier method, and differences were analyzed with the log-rank test. Comparison of the median of the concordance rates between paired or non-paired CISs and invasive regions of IPMN-inv was determined by the Mann–Whitney U test. All other measurements were analyzed using ANOVA and Student t test. P values of <0.05 were considered statistically significant.

Loss at 8p11.22-ter is unique in the invasive region of IPMN-inv

To identify the copy-number aberrations (CNA) responsible for the invasiveness of IPMN-inv, we compared the genomic profiles between paired samples of CIS and the invasive region from each of the 10 patients with IPMN-inv (Fig. 1A and Supplementary Table S1). The pattern of CNAs was quite similar between the two regions, such as gains at 1q and 8q, and losses at 6q, 9p, 12q, 14q, 17p, and 18q (Fig. 1B). The significance of this similarity was confirmed by calculating the concordance rates of probes showing CNA between the CIS region of IPMN-invs and paired or non-paired invasive regions of IPMN-invs (Supplementary Fig. S1A). As shown in Supplementary Fig. S1B, the concordance rate was highest between the CIS and the paired invasive region in 8 of the 10 cases. The median of the concordance rate between paired samples was significantly higher than that between non-paired samples (P = 0.002), indicating that genomic CNAs of the paired samples from IPMN-invs are closely related. Meanwhile, the number of probes showing CNA was significantly increased in the invasive region of IPMN-inv as compared with that in the paired CIS (Fig. 1C), suggesting that genomic CNAs accumulate in IPMN-inv. Among the chromosomal loci showing CNA, only 8p11.22-ter showed more significant loss in the invasive region of IPMN-inv than in the CIS region of IPMN-inv (P = 0.031, Supplementary Table S2). Interestingly, the pattern of the averaged frequencies of CNAs in invasive ductal carcinoma (IDC) closely resembled that in the invasive region of IPMN-inv (Fig. 1B and Supplementary Fig. S2; and Supplementary Tables S3 and S4). Furthermore, loss at 8p11.22-ter was also found frequently in IDC (Supplementary Table S4), suggesting that this aberration may be involved in the invasiveness of cancer cells in both IPMN-inv and IDC.

Figure 1.

Accumulation of additional genomic alterations during progression of IPMN to invasive carcinoma. A, histologic image of a representative case of IPMN-inv. Both the CIS and invasive regions of IPMN-inv were collected separately using LCM. B, averaged frequencies of CNAs of each chromosome in 10 cases of IPMN-inv. Horizontal lines show the chromosome number and vertical lines show frequencies (%) of CNA for the 10 patients. C, comparison of the number of probes showing CNA between the CIS region of IPMN-inv and paired invasive region of IPMN-inv for each case. Probes showing CNA were significantly increased in the invasive region of IPMN-inv as compared with those in the paired CIS. The P value was determined by the paired t test; scale bars, 500 μm (top photograph in A) and 100 μm (bottom photographs in A).

Figure 1.

Accumulation of additional genomic alterations during progression of IPMN to invasive carcinoma. A, histologic image of a representative case of IPMN-inv. Both the CIS and invasive regions of IPMN-inv were collected separately using LCM. B, averaged frequencies of CNAs of each chromosome in 10 cases of IPMN-inv. Horizontal lines show the chromosome number and vertical lines show frequencies (%) of CNA for the 10 patients. C, comparison of the number of probes showing CNA between the CIS region of IPMN-inv and paired invasive region of IPMN-inv for each case. Probes showing CNA were significantly increased in the invasive region of IPMN-inv as compared with those in the paired CIS. The P value was determined by the paired t test; scale bars, 500 μm (top photograph in A) and 100 μm (bottom photographs in A).

Close modal

Next, to identify genes downregulated due to loss at 8p11.22-ter, we compared the expression profiles of pancreatic cell lines with and without loss at 8p11.22-ter (Supplementary Fig. S3A and S3B). Among all the annotated genes assigned on the array CGH platform at 8p11.22-ter, 9 genes—BIN3, FBXO25, PLEKHA2, PLAT, FDFT1, RAB11FIP1, ZNF395, DUSP4, and ZDHHC2—were found to be significantly downregulated in the cell lines with loss at 8p11.22-ter in comparison with those without such loss (Fig. 2A and Supplementary Fig. S4).

Figure 2.

Invasiveness of pancreatic cancer cells is dependent on DUSP4 downregulation due to 8p loss. A, candidate genes whose expressions were significantly downregulated in pancreatic cancer cell lines with 8p loss compared with those without 8p loss. B, inhibitory effects of candidate genes on proliferation or invasiveness of PANC-1. The proliferation and invasiveness of PANC-1 transduced with each indicated lentivirus were represented as indices relative to those transduced with control lentivirus (LV-cont), which were each set to 1. C, inhibitory effects of DUSP4 on proliferation or invasiveness of KP4-1 and MIA Paca2. The proliferation and invasiveness of KP4-1 or MIA Paca2 transduced with LV-cont were each set to 1. D, macroscopic views of NOD-SCID mice 10 weeks after transplantation of PANC-1/cont or PANC-1/DUSP4-1 to the pancreas. Pancreatic tumors (yellow arrowheads) and bloody ascites (red arrows) are indicated. E, pancreatic tumors (top) and their weights (bottom). F, disseminated tumor nodules in the mesenterium (top, arrowheads) and the number of nodules disseminated in the peritoneal cavity (bottom). G, metastatic pulmonary tumors (top, arrows) and the number of metastatic foci in the lungs (bottom). Horizontal bars represent mean values. H, survival curves of mice bearing PANC-1/cont, PANC-1/DUSP4-1, or PBS only. The P value was determined by the log-rank test. The data in B, C, E, and F are represented as the mean ± SD; *, P < 0.05; **, P < 0.005, and ***, P < 0.0005 by ANOVA and the Student t test (B, C, E–G); scale bars, 10 mm (D–F) and 100 μm (G). All data except A are representative of three independent experiments.

Figure 2.

Invasiveness of pancreatic cancer cells is dependent on DUSP4 downregulation due to 8p loss. A, candidate genes whose expressions were significantly downregulated in pancreatic cancer cell lines with 8p loss compared with those without 8p loss. B, inhibitory effects of candidate genes on proliferation or invasiveness of PANC-1. The proliferation and invasiveness of PANC-1 transduced with each indicated lentivirus were represented as indices relative to those transduced with control lentivirus (LV-cont), which were each set to 1. C, inhibitory effects of DUSP4 on proliferation or invasiveness of KP4-1 and MIA Paca2. The proliferation and invasiveness of KP4-1 or MIA Paca2 transduced with LV-cont were each set to 1. D, macroscopic views of NOD-SCID mice 10 weeks after transplantation of PANC-1/cont or PANC-1/DUSP4-1 to the pancreas. Pancreatic tumors (yellow arrowheads) and bloody ascites (red arrows) are indicated. E, pancreatic tumors (top) and their weights (bottom). F, disseminated tumor nodules in the mesenterium (top, arrowheads) and the number of nodules disseminated in the peritoneal cavity (bottom). G, metastatic pulmonary tumors (top, arrows) and the number of metastatic foci in the lungs (bottom). Horizontal bars represent mean values. H, survival curves of mice bearing PANC-1/cont, PANC-1/DUSP4-1, or PBS only. The P value was determined by the log-rank test. The data in B, C, E, and F are represented as the mean ± SD; *, P < 0.05; **, P < 0.005, and ***, P < 0.0005 by ANOVA and the Student t test (B, C, E–G); scale bars, 10 mm (D–F) and 100 μm (G). All data except A are representative of three independent experiments.

Close modal

Downregulation of DUSP4 is associated with invasiveness, but not with cell proliferation

To determine which of the 9 genes are involved in the invasiveness of pancreatic cancer cells, we transduced PANC-1 cells, in which the whole of 8p had been hemizygously lost and the expression levels of all the 9 genes were low, with lentiviruses encoding each of the 9 genes (Supplementary Fig. S5A and S5B). As shown in Fig. 2B, the growth of PANC-1 cells was significantly suppressed by re-expression of ZNF395, RAB11FIP1, or PLEKHA2, whereas the invasiveness of PANC-1 was significantly suppressed only when DUSP4 was re-expressed. Similar results were obtained in experiments using the other pancreatic carcinoma cell lines, KP4-1, in which the DUSP4 gene was hemizygously lacking and its expression was downregulated, and MIA Paca2, in which DUSP4 was downregulated without loss of the DUSP4 gene (Fig. 2C), suggesting that DUSP4 suppresses the invasiveness of pancreatic cancer cells but does not affect their proliferation in vitro.

Re-expression of DUSP4 leads to suppression of tumor growth and metastasis in vivo

We next analyzed the effect of DUSP4 re-expression on an orthotopic xenograft model of pancreatic cancer. In mice that had received transplanted PANC-1 cells stably re-expressing DUSP4 (PANC-1/DUSP4-1; see Supplementary Fig. S6A–S6C), bloody ascites was rarely seen and the tumor mass was relatively small, whereas mice that had received transplanted PANC-1/cont exhibited significant bloody ascites and had large tumor masses in the abdominal cavity (Fig. 2D). The weights of primary tumors and the number of disseminated tumors in the peritoneal cavity were reduced in PANC-1/DUSP4-1–bearing mice (Fig. 2E and F). Micrometastases in the lungs were also significantly reduced in PANC-1/DUSP4-1–bearing mice (Fig. 2G). Furthermore, PANC-1/DUSP4-1–bearing mice showed a longer survival time after transplantation (Fig. 2H) than PANC-1/cont–bearing mice. These data were reproducible when we used another stable clone, PANC-1/DUSP4-2 (Supplementary Fig. S7A–S7D), suggesting that DUSP4 downregulation is involved not only in the invasiveness but also the growth of tumor cells in vivo, although DUSP4 did not affect cell proliferation in vitro (Fig. 2B and Supplementary Fig. S6B). Despite the difference in the level of DUSP4 expression between the two clones (Supplementary Fig. S6A), there were few differences between the phenotypes observed for each clone (Fig. 2E and F, Supplementary Fig. S7C and S7D), suggesting that both of them might have expressed a sufficient amount of DUSP4 to restore the cellular functions of DUSP4, which were abolished by 8p loss.

Downregulation of DUSP4 induces resistance to anoikis as well as enhanced invasiveness

To clarify the inconsistency between the in vitro and in vivo results in terms of cell growth (Fig. 2B, D, and E, Supplementary Fig. S6B and S7C), we next tested the possible involvement of DUSP4 downregulation in anoikis resistance of tumor cells. As shown in Fig. 3A, under conventional culture conditions, cell proliferation did not differ between PANC-1/DUSP4-1 or PANC-1/DUSP4-2 cells and PANC-1/cont. However, when cultured on ultra-low attachment plates to induce anoikis, PANC-1 cells re-expressing DUSP4 exhibited markedly reduced growth and increased apoptosis in comparison with PANC-1/cont (Fig. 3B and C), suggesting that reexpression of DUSP4 in PANC-1 cells induces anoikis. Furthermore, we found that secretion of matrix metalloproteinase (MMP)-2, which plays a significant role in tumor invasion in pancreatic cancer (17), was suppressed by reexpression of DUSP4 in PANC-1 cells, leading to reduced production of active–MMP-2 in the cell supernatants (Fig. 3D). In addition, the motility of PANC-1 cells was suppressed by DUSP4 re-expression (Fig. 3E). Conversely, knockdown of DUSP4 by shRNA or pooled siRNA mixture targeting different sequences of DUSP4 mRNA enhanced anoikis resistance, invasiveness, and cell motility in PK-59 cells, which express DUSP4 (Fig. 3F–J). These findings suggest that DUSP4 downregulation may lead to enhancement of both tumor growth and invasiveness via augmentation of anoikis resistance, MMP-2 production, and cell motility in vivo.

Figure 3.

Invasiveness and resistance to anoikis are significantly suppressed by re-expression of DUSP4. A and B, proliferation of PANC-1/cont, PANC-1/DUSP4-1, and PANC-1/DUSP4-2 cells cultured in conventional adherent plates (n = 6; A) or in ultra-low attachment plates (n = 6; B). C, enhanced apoptosis of PANC-1/DUSP4-1 and PANC-1/DUSP4-2 cells cultured in ultra-low attachment plates for 4 days (n = 3). D, gelatin zymography for detection of pro-MMP-2 and MMP-2 in PANC-1, PANC-1/cont, PANC-1/DUSP4-1, and PANC-1/DUSP4-2 cells. Representative gel image is shown in the top. The intensity of each band was calculated using ImageJ software, and the MMP-2 activity in each sample is represented as the intensity of active MMP-2 relative to that of pro-MMP-2 in the bottom bar graph. E, suppression of cell motility in PANC-1/DUSP4-1 and PANC-1/DUSP4-2 cells. Representative images of pre (1 hour after seeding)- and post (72 hours after seeding)-migration wells are shown in the top. The white dotted circle in each picture represents the margin of adhering cells at 1 hour after seeding. In the bottom graph, the motility at each time point is shown as the percentage area closed by migrating cells (n = 3). F–J, knockdown experiments using PK-59 cells. Knockdown of DUSP4 in PK-59 cells enhanced their anoikis resistance (F–H), invasiveness (I), and cell motility (J). The data in A–C and E–J are represented as the mean ± SD; *, P < 0.05; **, P < 0.005 and ***, P < 0.0005 by ANOVA and the Student t test. All data are representative of three independent experiments.

Figure 3.

Invasiveness and resistance to anoikis are significantly suppressed by re-expression of DUSP4. A and B, proliferation of PANC-1/cont, PANC-1/DUSP4-1, and PANC-1/DUSP4-2 cells cultured in conventional adherent plates (n = 6; A) or in ultra-low attachment plates (n = 6; B). C, enhanced apoptosis of PANC-1/DUSP4-1 and PANC-1/DUSP4-2 cells cultured in ultra-low attachment plates for 4 days (n = 3). D, gelatin zymography for detection of pro-MMP-2 and MMP-2 in PANC-1, PANC-1/cont, PANC-1/DUSP4-1, and PANC-1/DUSP4-2 cells. Representative gel image is shown in the top. The intensity of each band was calculated using ImageJ software, and the MMP-2 activity in each sample is represented as the intensity of active MMP-2 relative to that of pro-MMP-2 in the bottom bar graph. E, suppression of cell motility in PANC-1/DUSP4-1 and PANC-1/DUSP4-2 cells. Representative images of pre (1 hour after seeding)- and post (72 hours after seeding)-migration wells are shown in the top. The white dotted circle in each picture represents the margin of adhering cells at 1 hour after seeding. In the bottom graph, the motility at each time point is shown as the percentage area closed by migrating cells (n = 3). F–J, knockdown experiments using PK-59 cells. Knockdown of DUSP4 in PK-59 cells enhanced their anoikis resistance (F–H), invasiveness (I), and cell motility (J). The data in A–C and E–J are represented as the mean ± SD; *, P < 0.05; **, P < 0.005 and ***, P < 0.0005 by ANOVA and the Student t test. All data are representative of three independent experiments.

Close modal

Constitutive activation of MAPKs due to downregulation of DUSP4 is responsible for enhanced resistance to anoikis and invasiveness of pancreatic carcinomas

It has been reported that DUSP4 is located in the nucleus and dephosphorylates MAPKs, including ERKs, p38s, and JNKs (18–20). Therefore, we hypothesized that the augmented invasiveness and anoikis resistance caused by DUSP4 downregulation might be mediated via constitutively activated MAPKs. To identify which MAPKs are specifically activated by DUSP4 downregulation in pancreatic cells, we first analyzed whether the phosphorylation levels of MAPKs are affected by DUSP4. As shown in Fig. 4A, the phosphorylation levels of ERKs, p38s, and JNKs were shown to be reduced in PANC-1 cells by re-expression of DUSP4. Therefore, we next analyzed the effects of MAPKs inhibition on acquisition of resistance to anoikis, invasiveness, and augmented cell motility by using inhibitors. As shown in Supplementary Fig. S8A to S8E, treatment of PANC-1 cells with an MEK inhibitor (U0126), a p38 inhibitor (SB239063), and a JNK inhibitor (SP600125) led to suppression of proliferation (Supplementary Fig. S8A), anoikis resistance (Supplementary Fig. S8B and S8C), invasiveness (Supplementary Fig. S8D), and cell motility (Supplementary Fig. S8E). In addition, combined treatment with MEK and JNK inhibitors exerted additive effects, especially on invasiveness and motility (Supplementary Fig. S8D and S8E). Furthermore, similar to MEK inhibition, direct inhibition of ERK with SCH772984 also exerted suppressive effects on PANC-1 cells (Supplementary Fig. S8F–S8J). Next, we analyzed the effect of DUSP4 downregulation on the phosphorylation levels of ERKs, p38s, and JNKs in hTERT-HPNE E6/E7/K-RasG12D/st (referred to as “HPNE-T”; ref. 21) cells, derived from an immortalized pancreatic ductal cell line hTERT-HPNE (referred to as “HPNE”; ref. 22). Because knockdown of DUSP4 did not affect the phosphorylation levels of MAPKs in HPNE-T cells cultured under conventional conditions (data not shown), we analyzed the effects of DUSP4 knockdown on transiently activated MAPKs in HPNE-T cells cultured under serum-starved conditions. As shown in Fig. 4B, DUSP4 expression was markedly increased in both the parental and control siRNA-transfected HPNE-T cells (HPNE-T/siCont) by EGF stimulation, followed by rapid dephosphorylation of activated ERKs. On the other hand, in DUSP4 siRNA-transfected HPNE-T cells (HPNE-T/siDUSP4), in which DUSP4 expression was undetectable, dephosphorylation of ERKs was delayed relative to that in HPNE-T or HPNE-T/siCont cells (Fig. 4B), suggesting that the induced expression of DUSP4 is related to the suppression of transiently activated ERKs. By contrast, dephosphorylation of both p38s and JNKs, which had been activated by anisomycin stimulation, was not delayed in HPNE-T/siDUSP4 relative to HPNE-T or HPNE-T/siCont (Fig. 4B). However, we noticed that DUSP4 expression in HPNE-T or HPNE-T/siCont was not induced, but rather suppressed by stimulation with anisomycin (Fig. 4B), suggesting that activation of both p38s and JNKs might not be associated with the induction of DUSP4. Therefore, we were unable to determine whether p38 and JNK might be substrates of DUSP4 in pancreatic ductal cells at this stage.

Figure 4.

Downregulation of DUSP4 is related to the activation of ERKs. A and B, the effects of DUSP4 re-expression or DUSP4 knockdown on the phosphorylation levels of MAPKs in PANC-1 transduced with the indicated lentivirus (A) or HPNE-T transfected with the indicated siRNA (B), respectively. The cells were serum-starved overnight and then stimulated with 10 ng/mL EGF or 5 μmol/L anisomycin for the indicated periods.

Figure 4.

Downregulation of DUSP4 is related to the activation of ERKs. A and B, the effects of DUSP4 re-expression or DUSP4 knockdown on the phosphorylation levels of MAPKs in PANC-1 transduced with the indicated lentivirus (A) or HPNE-T transfected with the indicated siRNA (B), respectively. The cells were serum-starved overnight and then stimulated with 10 ng/mL EGF or 5 μmol/L anisomycin for the indicated periods.

Close modal

DUSP4 expression is upregulated in intraepithelial neoplasms, but lost in invasive carcinomas

Next, we immunohistochemically analyzed the expression levels of DUSP4 in IPMN-inv cases. DUSP4 was rarely detectable in normal pancreatic duct cells, but was strongly expressed in nuclei in the CIS region of IPMN-inv, in which KRAS mutations were detected in many cases (Fig. 5A and B). Because DUSP4 was found to be upregulated by EGF stimulation or introduction of mutated KRAS in an immortalized pancreatic ductal cell line (Supplementary Fig. S9A–S9C), probably through a negative feedback mechanism against ERK activation (19, 23–26), the induced expression of DUSP4 in CIS may represent a physiologic response against the aberrant ERK signaling caused by KRAS activation (Fig. 5C). In contrast, in invasive cancer cells of IPMN-inv, nuclear expression of DUSP4 was very low or undetectable (Fig. 5A and B). These results reveal that the level of DUSP4 expression in cancer cells is significantly correlated with the status of their invasiveness and 8p genotype (Fig. 5D). Importantly, further immunohistochemical analysis of IDCs accompanying PanINs revealed that DUSP4 was induced more frequently in higher-grade PanINs, in which KRAS mutations are frequently detected (27), but was significantly downregulated in invasive pancreatic carcinoma cells (Supplementary Fig. S10A and S10B), indicating that in IDCs, DUSP4 might be downregulated in a similar way to IPMN-inv. Thus, immunohistochemical analyses suggest that, during the progression of pancreatic carcinogenesis, expression of DUSP4 in invasive pancreatic cancers may be regulated in a coordinated manner by sequentially accumulated genetic aberrations such as mutated KRAS and 8p loss.

Figure 5.

DUSP4 expression is upregulated in intraepithelial neoplasms but lost in invasive carcinomas. A, representative images of immunohistochemical analysis of DUSP4 expression in IPMN-inv cases. B, the status of 8p loss, KRAS mutation, and DUSP4 expression in IPMN-inv cases. C, the regulatory feedback loop between ERK and DUSP4. Activated ERK phosphorylates transcription factors and DUSP4, leading to induction of DUSP4 mRNA expression and inhibition of DUSP4 protein degradation in the proteasome, respectively. As a result, phosphatase activity of DUSP4 is upregulated, and consequently ERK is dephosphorylated. D, significant correlation of DUSP4 expression in pancreatic cancer cells with both their invasiveness and 8p genotype. The P values were determined by the Fisher exact probability test; scale bars, 100 μm (A).

Figure 5.

DUSP4 expression is upregulated in intraepithelial neoplasms but lost in invasive carcinomas. A, representative images of immunohistochemical analysis of DUSP4 expression in IPMN-inv cases. B, the status of 8p loss, KRAS mutation, and DUSP4 expression in IPMN-inv cases. C, the regulatory feedback loop between ERK and DUSP4. Activated ERK phosphorylates transcription factors and DUSP4, leading to induction of DUSP4 mRNA expression and inhibition of DUSP4 protein degradation in the proteasome, respectively. As a result, phosphatase activity of DUSP4 is upregulated, and consequently ERK is dephosphorylated. D, significant correlation of DUSP4 expression in pancreatic cancer cells with both their invasiveness and 8p genotype. The P values were determined by the Fisher exact probability test; scale bars, 100 μm (A).

Close modal

Blockade of the ERK signaling pathway as a potentially effective therapeutic strategy for pancreatic cancer

We next analyzed the effect of PD325901, a potent MEK1 inhibitor in in vivo experiments (28), in an orthotopic xenograft model of pancreatic cancer. After confirming that the anoikis resistance of PANC-1 cells was significantly suppressed by PD325901 (Fig. 6A), as well as by U0126 shown in Supplementary Fig. S8B and S8G, we administrated PD325901 orally to mice bearing PANC-1. As shown in Fig. 6B, phosphorylation of ERK in transplanted PANC-1 cells was strongly suppressed by oral administration of PD325901, although phosphorylation of p38, JNK and AKT was unchanged. We also found that tumor growth and metastasis in mice transplanted with PANC-1 cells were significantly suppressed by administration of PD325901, resulting in improved survival (Fig. 6C–E). These observations closely matched the results obtained from mice bearing transplanted PANC-1 cells re-expressing DUSP4 (Fig. 2D–F and H).

Figure 6.

Activation of the ERK signaling pathway due to DUSP4 downregulation is an effective therapeutic target for pancreatic cancer. A, the effect of PD325901 on proliferation of PANC-1 cultured in conventional adherent plates (top) or ultra-low attachment plates (bottom). B–E, the orthotopic xenograft model with oral administration of PD325901. B, phosphorylation levels of MAPKs in pancreatic tumors determined by immunohistochemistry (top) and Western blot analyses (bottom). C, NOD-SCID mice after 6 weeks of treatment. Pancreatic tumors (yellow arrowheads) and bloody ascites (red arrows) are indicated. D, pancreatic tumors (top), their weights (middle), and disseminated nodules in the mesenterium (bottom, arrowheads). E, the effects of MEK inhibitor on survival of PANC-1–bearing mice. The P value was determined by the log-rank test. F, the effect of MEK1dn on ERK phosphorylation. G, the effect of MEK1dn on proliferation of PANC-1 cultured in conventional adherent plates (top) or in ultra-low-attachment plates (bottom). H, the effect of MEK1dn on invasiveness of PANC-1. Representative images of invaded cells (top) and their number (bottom) are shown. I, the effect of MEK1dn on survival of mice bearing orthotopic xenografts. The P value was determined by the log-rank test. J, model of pancreatic cancer invasion resulting from activation of ERK due to DUSP4 loss. The data in A, D, G, and H are represented as the mean ± SD; *, P < 0.05; **, P < 0.005, and ***, P < 0.0005 by ANOVA and the Student t test; scale bars, 10 mm (C and D). All data are representative of three independent experiments.

Figure 6.

Activation of the ERK signaling pathway due to DUSP4 downregulation is an effective therapeutic target for pancreatic cancer. A, the effect of PD325901 on proliferation of PANC-1 cultured in conventional adherent plates (top) or ultra-low attachment plates (bottom). B–E, the orthotopic xenograft model with oral administration of PD325901. B, phosphorylation levels of MAPKs in pancreatic tumors determined by immunohistochemistry (top) and Western blot analyses (bottom). C, NOD-SCID mice after 6 weeks of treatment. Pancreatic tumors (yellow arrowheads) and bloody ascites (red arrows) are indicated. D, pancreatic tumors (top), their weights (middle), and disseminated nodules in the mesenterium (bottom, arrowheads). E, the effects of MEK inhibitor on survival of PANC-1–bearing mice. The P value was determined by the log-rank test. F, the effect of MEK1dn on ERK phosphorylation. G, the effect of MEK1dn on proliferation of PANC-1 cultured in conventional adherent plates (top) or in ultra-low-attachment plates (bottom). H, the effect of MEK1dn on invasiveness of PANC-1. Representative images of invaded cells (top) and their number (bottom) are shown. I, the effect of MEK1dn on survival of mice bearing orthotopic xenografts. The P value was determined by the log-rank test. J, model of pancreatic cancer invasion resulting from activation of ERK due to DUSP4 loss. The data in A, D, G, and H are represented as the mean ± SD; *, P < 0.05; **, P < 0.005, and ***, P < 0.0005 by ANOVA and the Student t test; scale bars, 10 mm (C and D). All data are representative of three independent experiments.

Close modal

To rule out any unexpected effect of the MEK inhibitor, we established PANC-1 cells stably expressing the dominant negative form of MEK1 (PANC-1/MEK1dn), in which the phosphorylation level of ERK was relatively reduced (Fig. 6F). As shown in Fig. 6G and H, anoikis resistance and invasiveness in vitro were significantly suppressed in PANC-1/MEK1dn, similarly to the experiment in which MEK inhibitors were used (Supplementary Fig. S8B, S8D, S8G, and S8I, and Fig. 6A). In in vivo xenograft experiments with PANC-1/MEK1dn, we obtained results similar to those obtained using PD325901 (Fig. 6E and I), suggesting that MEK1 inhibition may be an efficient strategy for treatment of pancreatic cancer.

Accumulation of genetic aberrations during the development of pancreatic precursor lesions has been well documented (12). However, the molecular mechanisms by which precursor lesions progress to invasive cancers remain largely unknown. By comparison of genomic profiles between invasive and noninvasive cancer cells from the same IPMN-inv patients, we demonstrated that 8p loss was associated with the invasive phenotype of IPMN-inv. Interestingly, the genomic profiles of IDC, in which 8p loss was also frequently detected, showed a similar pattern to those of the invasive region of IPMN-inv, suggesting that 8p loss also contributes to the progression of PanIN to IDC. From the genes located on chromosome 8p, we identified DUSP4, which was one of the genes showing downregulation along with copy-number loss, as an invasion suppressor gene in pancreatic cancer, because re-expression of DUSP4 suppressed the invasiveness of pancreatic cancer cells in vitro and also in vivo, with a decrease of pulmonary metastasis and peritoneal dissemination, in an orthotopic model of pancreatic cancer.

DUSP4 is a member of the MAPK phosphatases and negatively regulates ERK, p38, and JNK in the nucleus (18–20, 29). Consistent with the previous reports, ectopic expression of DUSP4 caused dephosphorylation of all three MAPKs in pancreatic cancer cells. By knockdown assay, we showed that the transiently activated ERKs induced DUSP4 expression, which in turn suppressed the activated ERKs. We also showed that re-expression of DUSP4 inhibited MMP-2 production, cell motility, and anoikis resistance of pancreatic cancer cells. Because involvement of ERK activation in MMP-2 activation (30), cell motility (31), and anoikis (32) has been already reported, we hypothesized that inhibition of invasiveness by DUSP4 may be mediated by ERK inactivation. As expected, our observations in the DUSP4 reexpression experiments were well phenocopied by treatment of pancreatic cancer cells with an MEK inhibitor. Furthermore, inhibition of MEK activation by either the dominant negative form or the pharmacologic inhibitor improved the survival of mice bearing orthotopic pancreatic cancer xenografts. Taken together, our findings suggest that ERK activation induced by DUSP4 downregulation may cause MMP-2 production, cell motility, and anoikis resistance, leading to enhanced invasiveness of pancreatic cancer cells.

In this study, we found that mutant KRAS caused persistent upregulation of DUSP4 in HPNE-T cells, probably through feedback against activated ERK. Knockdown of the upregulated DUSP4 resulted in prolonged activation of ERK, suggesting that KRAS mutation and DUSP4 downregulation may activate ERK in HPNE-T cells in a coordinated manner. Furthermore, we found that expression of DUSP4 was rarely detectable in normal epithelium of the pancreatic duct, was strongly induced in the CIS, and again decreased in the invasive region of IPMN-inv and IDC. Considering that the majority of IPMN-inv cases harbor KRAS mutation (33), induction of DUSP4 in the CIS region of IPMN-inv is probably due to negative feedback for activation of KRAS/ERK. In addition, it is possible that downregulation of DUSP4 along with 8p loss might lead to persistent ERK activation by breakdown of the feedback loop in pancreatic tumor cells, thereby contributing to acquisition of invasiveness (see Fig. 6J). Thus, the pattern of DUSP4 expression in clinical tissues strongly suggests that DUSP4 functions as a tumor suppressor mainly by regulating the activation of ERK during the progression of pancreatic precursors to invasive carcinomas, although we were unable to completely exclude the possibility that DUSP4 loss contributes to activation of p38 and JNK, both of which were shown to be related to the invasiveness and anoikis resistance of pancreatic cancer cells. Our hypothetical model is summarized in Fig. 6J.

Similarly to the distribution pattern of DUSP4 in pancreatic cancer tissues, DUSP6, another member of the dual-specificity phosphatase family, has been reported to show decreased expression in invasive pancreatic cancers (34, 35). Expression of DUSP6 is also induced by mutant KRAS, and specifically dephosphorylates and inactivates ERK (36, 37). By combining the data for DUSP4 obtained by our group and that for DUSP6 obtained in previous studies, it can be speculated that ERK activation caused by dysregulation of ERK-specific phosphatases may play an important role in the invasiveness of pancreatic cancer cells.

Our observations suggest that ERK activation caused by KRAS mutations and dysfunction of ERK phosphatases play a pivotal role in the development of invasive pancreatic cancers, leading us to speculate that targeting of ERK activities by MEK inhibitors may be a reasonable approach for treatment of pancreatic cancers. However, two phase II studies of MEK inhibitors have failed to demonstrate any significant survival advantage for patients with pancreatic cancer (38, 39). Furthermore, although MEK inhibition conferred a significant survival advantage in the present orthotopic pancreatic cancer xenograft model, the effect was somewhat limited (median survival of 82 days vs. 101 days for vehicle vs. PD325901, respectively). One plausible explanation for these limitations may be that oncogenic pathway(s) other than RAS–MEK–ERK may also contribute to progression of early invasive cancers to aggressive malignant cancers, such as locally disseminated or distant metastatic cancers. Indeed, the two previous phase II studies of MEK inhibitors enrolled patients with metastatic or refractory pancreatic cancers (38, 39). Furthermore, PANC-1 cells, from which we developed our orthotopic models, were derived from locally disseminated pancreatic cancers (40). Therefore, to achieve useful therapeutic effects of MEK inhibitors against such aggressive malignant cancers, identification and targeting of additional genetic alterations responsible for the aggressive phenotype in combination with MEK inhibition would be required. Otherwise, MEK inhibitors may be effective for the management of patients with early invasive pancreatic cancers.

In conclusion, by focusing on the differences in genomic alterations between invasive and noninvasive tumor cells from the same IPMN-inv patients, we have identified a novel invasion suppressor gene, DUSP4, that functions in pancreatic carcinogenesis. It has been reported that promoter hypermethylation is related to the downregulation of DUSP4 in glioblastoma and lymphoma (41, 42). However, despite hemizygous loss of the DUSP4 gene in invasive pancreatic cancers, the expression level of DUSP4 was markedly decreased and failed to recover upon treatment with the epigenetic modifier, 5-aza-2-deoxycytidine or trichostatin A in PANC-1 cells (data not shown), suggesting that a mechanism other than epigenetic silencing might be involved in the downregulation of DUSP4 expression. Downregulation of DUSP4 has been reported to be involved in the malignant phenotypes of various tumors, including ovarian cancer (43), lung cancer (44), glioblastoma (41), breast cancer (45), colorectal cancer (46), and lymphoma (42). We showed for the first time that DUSP4 loss contributes to pancreatic cancer progression. To our knowledge, DUSP4 is the first invasion suppressor gene shown to be involved in the genetic progression of pancreatic cancer, because most of the genetic events involved in pancreatic cancer progression, including activation of KRAS and deficiency of TP53, CDKN2A, SMAD4, and DUSP6, contribute mainly to the proliferation or survival of tumor cells (Fig. 6J; refs. 34, 47–50).

On the basis of our present data and previous clinical trials (38, 39), it would appear difficult to achieve molecular targeted therapy using MEK inhibitors alone. However, in addition to the mechanisms involved in early invasion, an understanding of the mechanisms responsible for malignant transformation or resistance to molecular targeting drugs would facilitate the development of combination therapy that includes the use of MEK inhibitors. Thus, our results may shed new light on not only the genetic progression model, but also new avenues for clinical treatment of pancreatic cancers.

No potential conflicts of interest were disclosed.

Conception and design: N. Hijiya, H. Mori, M. Moriyama

Development of methodology: N. Hijiya, C. Nakada, M. Seto

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): N. Hijiya, C. Nakada, T. Kai, K. Shibata, Y. Yamada, M. Moriyama

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): N. Hijiya, Y. Tsukamoto, C. Nakada, L.T. Nguyen, K. Matsuura, M. Inomata, T. Uchida, Y. Yamada, I. Takeuchi, M. Seto, M. Aoki, M. Takekawa, M. Moriyama

Writing, review, and/or revision of the manuscript: N. Hijiya, Y. Tsukamoto, C. Nakada, L.T. Nguyen, K. Matsuura, A. Tokunaga, M. Seto, M. Aoki, M. Takekawa, M. Moriyama

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): Y. Tsukamoto, C. Nakada, T. Kai, M. Inomata, A. Tokunaga, K. Amada, M. Takekawa

Study supervision: Y. Tsukamoto, C. Nakada, K. Shirao, M. Seto, M. Moriyama

The authors thank M. Kimoto, T. Iwao, Y. Miyanari, F. Kawamura, and M. Takeda for technical assistance.

This study was supported by Japan Society for the Promotion of Science (JSPS) KAKENHI grant numbers 22590335 and Research Fund at the Discretion of the President, Oita University.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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