Expression and Functional Significance of CDC25B in Human Pancreatic

Expression and functional signiﬁcance of CDC25B in human pancreatic ductal adenocarcinoma

Junchao Guo1,2,Jo¨ rg Kleeff1, Junsheng Li1, Jiayi Ding3,Ju¨ rgen Hammer3, Yupei Zhao2, Thomas Giese4, Murray Korc5, Markus W Bu¨ chler1 and Helmut Friess*,1

1Department of General Surgery, University of Heidelberg, Im Neuenheimer Feld 110, 69120 Heidelberg, Germany; 2Department of General Surgery, Peking Union Medical College Hospital, Beijing, China; 3Roche Genomic and Information Sciences, Hoffmann-La Roche Inc., Nutley, NJ, USA; 4Institute of Immunology, University of Heidelberg, Heidelberg, Germany; 5Division of Endocrinology, Diabetes, and Metabolism, Department of Medicine, Biological Chemistry and Pharmacology, University of California, Irvine, CA, USA

Pancreatic ductal adenocarcinoma (PDAC) is one of the pancreatic cancer cases in the US was 30 300, while the leading causes of cancer-related deaths. Deregulation of estimated number of deaths was 29 700 (Jemal et al., cell-cycle control is thought to be a crucial event in 2002). PDAC is characterized by late diagnosis, early malignant transformation, and CDC25 phosphatases are recurrence after potential curative resection, and relative a familyof cyclin-dependent kinase activators, which act unresponsiveness to standard oncological therapies such at different points of the cell cycle, including G1–S and as radiotherapy and chemotherapy (DiMagno et al., G2–M transition. Here, we investigated the expression 1999). The overall 5-year survival rate is less than 5%, and functional significance of CDC25s in PDAC. and more than 85% of the tumors are diagnosed when CDC25B mRNA expression levels in human pancreatic the tumor has infiltrated into adjacent organs or when tissue samples were analysed by cDNA array, quantitative distant metastases are present (Gudjonsson, 1987). PCR and Northern blotting. Immunohistochemistrywas Furthermore, even after potential curative resection, carried out to localize and quantifyCDC25B expression. the 5-year survival rate is only around 20% in large Two specific CDC25B inhibitors were utilized to deter- series (Neoptolemos et al., 2001a, b). Numerous studies mine the functional relevance of CDC25B. Byquantitative have documented different genetic and epigenetic RT–PCR, CDC25B mRNA was overexpressed in pan- alterations in PDAC, including mutations of the proto- creatic cancer (7.5-fold) in comparison to the normal oncogene K-ras, and tumor-suppressor gene mutations pancreas. Strong nuclear CDC25B immunoreactivitywas such as those of p53, Smad4/DPC4 and p16/CDKN2A present in both pancreatic and metastatic cancer samples, (Bardeesy and DePinho, 2002). In addition, overexpres- and there was a marked increase of the percentage of sion of growth factors and growth factor receptors is positive cells in primarycancer (48.6 716.3%) and present in a significant proportion of these tumors, and metastatic tissues (71.773.1%) compared to normal PDACs display a number of different alterations in samples (8.371.8%). Two CDC25B inhibitors reduced apoptosis, invasion and metastasis-regulating pathways. the growth of pancreatic cancer cell lines, resulting in the These changes combine to give PDAC cells a distinct accumulation of phosphorylated CDC2 and G2/M arrest. growth advantage (Kleeff et al., 1998; Koliopanos et al., These findings demonstrate an important role of CDC25B 2001; Liao et al., 2001; Ozawa et al., 2001). in cell-cycle progression, raising the possibility that Deregulation of cell-cycle control is thought to be a inhibition of CDC25B mayhave therapeutic potential in crucial event in the malignant transformation of cells pancreatic cancer. (Tsai and McKay, 2002). Alterations in cell-cycle Oncogene (2004) 23, 71–81. doi:10.1038/sj.onc.1206926 regulatory genes are commonly found in human cancers (Sherr, 1996), and cell proliferation that reflects cell- Keywords: cell cycle, cyclin-dependent kinase, CDC25B, cycle progression is an influential factor contributing to pancreatic ductal adenocarcinoma the aggressive growth behavior of cancer cells. Two critical checkpoints have been characterized, and are located at the G1–S phase and G2–Mphase transitions. Introduction Central components in the cell-cycle machinery are cyclins and their corresponding cyclin-dependent Pancreatic ductal adenocarcinoma (PDAC) is the fourth kinases (CDKs). The complexes of CDK/cyclin are leading cause of cancer-related deaths in the United activated and inactivated sequentially to regulate States according to the latest statistical analysis (Jemal different phases of the cell cycle. Several gene mutations et al., 2002). In 2002, the estimated number of new are involved in deregulated cell-cycle progression in PDAC, such as mutations of the tumor-suppressor gene *Correspondence: H Friess; E-mail: [email protected] p53, which result in antiapoptotic responses and anti- berg.de G1–S phase arrest; mutations of K-ras, which result in Received 16 May 2003; revised 28 June 2003; accepted 1 July 2003 continued cell proliferation and survival (Bardeesy and CDC25B in pancreatic cancer J Guo et al 72 DePinho, 2002); and mutations of CDKN2A, which pancreas, respectively. Furthermore, in metastatic encodes two tumor suppressors – INK4a and ARF – lesions, CDC25B levels were increased 2.2- and 4.1-fold that disrupt retinoblastoma (RB) and p53 pathways, in comparison with primary cancer and normal pan- subsequently resulting in uncontrolled cell-cycle pro- creatic samples, respectively. In contrast, there was no gression (Lal et al., 2000). significant change in CDC25A and CDC25C mRNA CDC25 phosphatases, consisting of CDC25A, B and levels between pancreatic cancer, chronic pancreatitis C, are a novel family of CDK phosphatases, which and normal pancreatic samples. activate CDK/cyclin complexes by dephosphorylation These DNA array results were confirmed by Northern of CDKs at different checkpoints of the cell cycle blot analysis and quantitative RT–PCR. Northern blot (Galaktionov and Beach, 1991; Sebastian et al., 1993). analysis of total RNA isolated from 48 PDAC and 24 CDC25A is expressed in the late G1 phase and controls normal pancreatic tissues revealed absent or weak G1/S transition by activating the CDK2/Cyclin E expression of the approximately 3.0 kb CDC25B mRNA complex (Hoffmann et al., 1994), while CDC25B and transcript (Wu and Wolgemuth, 1995) in the normal C are essential for G2 and G2/Mtransition (Hoffmann samples. In contrast, in 40 of 48 (83%) PDAC samples, et al., 1993; Lammer et al., 1998). CDC25A and the CDC25B mRNA transcript was clearly detectable. CDC25B, but not CDC25C, exhibit oncogenic potential In 26 (54%) of these samples there was strong since they can transform primary murine fibroblasts in expression, whereas in 14 (29%) samples there was low cooperation with either activated H-ras or loss of RB1 to moderate expression of this mRNA moiety in (Galaktionov et al., 1995). Overexpression of CDC25A comparison to normal pancreatic tissues. An example or CDC25B has been reported in different human of a Northern blot depicting eight PDAC and four tumors, including non-Hodgkin’s lymphoma, neuro- normal tissue samples is shown in Figure 1a (upper blastoma, head and neck cancer, lung cancer, esopha- panel). Previously, different splicing variants have been geal cancer, gastric cancer, colorectal cancer and described for CDC25B (Kudo et al., 1997). Therefore, prostate cancer (Gasparotto et al., 1997; Kudo et al., we also characterized splice variants of CDC25B in 1997; Hernandez et al., 2000, 2001; Miyata et al., 2001; cancerous and normal pancreatic tissues by RT–PCR Sasaki et al., 2001; Sato et al., 2001; Ito et al., 2002; using a pair of primers that selectively amplifies four Ngan et al., 2003). The expression and functional role of variants yielding fragments of 627 bp (CDC25B4), CDC25B phosphatases in PDAC has so far not been 522 bp (CDC25B3), 480 bp (CDC25B1) and 399 bp analysed. Therefore, in the present study we investigated (CDC25B2). The CDC25B3 isoform was detectable in the expression of CDC25s in PDAC in order to virtually all cases, but at relatively higher levels in elucidate the role of these genes as modulators of cell- cancerous than in normal tissues. The other isoforms cycle progression in this disease. In addition, CDC25B were below the level of detection (Figure 1a, lower inhibitors were used to study the functional role of these panel). It is currently not known whether these splice phosphatases in the pathogenesis of PDAC. variants are present only at low levels in pancreatic tissues, or alternatively whether there are other less abundant splice variants. Changes in CDC25B mRNA levels were quantified by Results real-time QRT–PCR. In pancreatic cancer samples, the median expression level of CDC25B mRNA was 7.5- CDC25B expression in human pancreatic cancer and 3.9-fold higher than in the normal and chronic We first screened the expression of the three CDC25 pancreatitis tissue samples, respectively (Table 1b). The family members (CDC25A, B and C) in pancreatic expression of the CDC25B3 splicing variant displayed a cancer, chronic pancreatitis and the normal pancreas similar pattern. Thus, CDC25B3 mRNA levels were 6.3- utilizing DNA arrays. This analysis revealed that and 3.2-fold increased in PDAC in comparison with the CDC25B, but not CDC25A or CDC25C, was signifi- normal pancreas and chronic pancreatitis, respectively cantly overexpressed in pancreatic cancer compared to (Figure 1b). This analysis also revealed that the either chronic pancreatitis or normal pancreatic tissues CDC25B3 isoform accounts for approximately 43% of (Table 1a). Thus, CDC25B mRNA expression levels total CDC25B mRNA expression in pancreatic cancer were increased 1.4- and 1.9-fold in pancreatic cancer samples, and approximately 50% in both CP and compared with chronic pancreatitis and the normal normal pancreatic samples (Figure 1b). Since different

Table 1a Expression of CDC25 family members in the normal pancreas, chronic pancreatitis and pancreatic cancer as determined by DNA array Mean values Fold changes

No CP PDAC Mx Ca vs No Mx vs No Mx vs Ca

CDC25A 10.2# 3.7 5.7 6.5 0.6 0.6 1.1 CDC25B 14.5 19.3 27.5 59.1 1.9* 4.1* 2.2* CDC25C 6.8 7.7 3.3 4.9 0.5 0.7 1.5

PDAC: pancreatic ductal adenocarcinoma; CP: chronic pancreatitis; Mx: metastasis; No: normal pancreas; *Po0.05; #arbitrary units

Oncogene CDC25B in pancreatic cancer J Guo et al 73

Figure 1 CDC25B expression analysis in pancreatic tissues and cell lines. Total RNA (15 mg) isolated from the indicated pancreatic tissues (a, upper panel) and cell lines (c, upper panel) was subjected to Northern blot analysis and hybridized with the labeled CDC25B cDNA probe and rehybridized with the 7S cDNA probe to verify equivalent RNA loading as described in the Materials and methods section. Size markers are indicated on the left. RT–PCR analysis of CDC25B splice variants in seven normal and seven cancerous tissues (a, lower panel) as well as seven pancreatic cancer cell lines (c, lower panel) as described in the Materials and methods section. The 522 and 399 bp RT–PCR products representing the CDC25B3 and CDC25B2 splice forms, respectively, are indicated on the right. Size markers are indicated on the left. Quantitative PCR for CDC25B and CDC25B3 (b) in the normal pancreas, chronic pancreatitis and pancreatic cancer tissues was carried out as described in the Materials and methods section. Results are presented as mean7s.e.m. Immunoblot analysis (d) of CDC25B in pancreatic cancer cell lines. Protein lysates (20 mg) were subjected to immunoblot analysis using an anti- CDC25B antibody as described in the Materials and methods section. The molecular size markers (in kDa) are indicated on the left

Table 1b Expression of CDC25B and the B3 splice variant in the Nonetheless, our RT–PCR as well as QRT–PCR data normal pancreas, chronic pancreatitis and pancreatic cancer as both point to a predominance of the CDC25B3 splice determined by quantitative RT–PCR variant in pancreatic tissues. Mean values Fold changes

No CP PDAC Ca vs No Ca vs CP CDC25B expression in human pancreatic cancer cell lines

CDC25B 80# 155 597 7.5* 3.9* The expression of CDC25B was next analysed in seven CDC25B3 41 82 259 6.3* 3.2* human pancreatic cancer cell lines (Figure 1c). Six pancreatic cancer cell lines exhibited high levels of PDAC: pancreatic ductal adenocarcinoma; CP: chronic pancreatitis; CDC25B mRNA expression, whereas BxPc-3 cells # Mx: metastasis; No: normal pancreas; *Po0.05; copy number/ml exhibited weak CDC25B expression. RT–PCR analysis revealed predominant expression of the CDC25B3 splice primers might result in distinct efﬁciencies of the QRT– variant in six cell lines. The CDC25B2 splice variant was PCR, these numbers represent only an estimate of the also detectable, but at lower levels (Figure 1c). Western relative abundance of the CDC25B3 splice variant. blot results generally conﬁrmed the mRNA expression

Oncogene CDC25B in pancreatic cancer J Guo et al 74 data obtained by Northern blot analysis. Thus, Mia- 1999c), TGF-b reduced CDC25B mRNA levels PaCa-2 and PANC-1 had the highest expression levels (Figure 2a) and increased p21 mRNA levels of CDC25B, while BxPc-3 exhibited absent to weak (Figure 2b). In the other pancreatic cancer cell lines expression. In contrast, in Capan-1 cells, CDC25B that are resistant to the growth inhibitory effects of mRNA was present at a moderate level, while protein TGF-b due to Smad4 mutations/deletions (Capan-1, levels were below the level of detection by Western blot BxPc-3) (Schutte et al., 1996; Kleeff et al., 1999c), or analysis (Figure 1c). absent TGF-b receptor type II expression (Mia-PaCa-2) (Freeman et al., 1995), TGF-b did not alter CDC25B Regulation of CDC25B expression in human pancreatic mRNA expression levels (data not shown). cancer cell lines To determine the mechanisms of increased expression of CDC25B localization in human pancreatic tissues CDC25B in pancreatic cancer cell lines, we next To delineate the signiﬁcance of CDC25B overexpression determined the effects of TGF-b on CDC25B mRNA in PDAC further, immunohistochemistry was carried levels, since it has been shown that CDC25B can be out next. In the normal pancreas, nuclear CDC25B transcriptionally regulated by TGF-b (Ulloa and staining was observed in some ductal, acinar and islet Tabibzadeh, 2001). QRT–PCR analysis revealed that cells. Calculation of the percentage of positive cells in the TGF-b-responsive cell line Colo-357 (Kleeff and revealed that in the normal pancreas, 8.371.8% of cells Korc, 1998), TGF-b led to a marked but short-term were CDC25B positive (Figure 3a, b). In contrast, in reduction of CDC25B mRNA expression (Figure 2a), PDAC, 48.6716.3% of the cancer cells displayed whereas there was a sustained increase in the mRNA positive nuclear CDC25B immunoreactivity (Figure expression of the known TGF-b target gene, the CDK 3f–h). Interestingly, in addition to the nuclear localiza- inhibitor p21 (Figure 2b) (Kleeff and Korc, 1998). tion of CDC25B, some cancer cells also exhibited Similarly, in Panc-1 pancreatic cancer cells, which are cytoplasmic staining (Figure 3h). In pancreatic intrae- also growth inhibited by TGF-b although to a lesser pithelial neoplasia (PanIN) lesions that were present in extent when compared to Colo-357 cells (Kleeff et al., some cancer samples, a comparable percentage of CDC25B-positive cells could be observed (Figure 3c). In addition, in areas surrounding the tumor, consisting of tubular complexes, morphological normal as well as dedifferentiating acinar cells, 20.877.5% of these cells displayed positive nuclear CDC25B staining (Figure 3d, e). Fibroblasts in the tissue surrounding the tumor also displayed CDC25B immunoreactivity in the majority of cases (Figure 3h). We also examined the expression of CDC25B in liver and lymph node metastases of PDAC samples. Ap- proximately 71.773.1% of the tumor cells in the metastatic lesions were CDC25B positive (Figure 3i). In contrast, liver or lymph node tissues only occasion- ally exhibited CDC25B-positive cells (Figure 3i, inset). Statistical analysis revealed a signiﬁcant difference of the percentage of CDC25B-positive cell between normal tissues, areas next to cancer, pancreatic cancer and metastatic lesions. In contrast, there was no difference in the percentage of CDC25B-positive cells between stage I/II and III/IV tumors, between grade 1/2 and 3 PDACs (Figure 3k) as well as between lymph node-negative and -positive tumors. In addition, there was no correlation between the percentage of CDC25B-positive cells and patient age and gender.

Functional role of CDC25B in human pancreatic cancer cells To assess the functional role of CDC25B further, two Figure 2 Regulation of CDC25B mRNA expression by TGF-b. CDC25B-speciﬁc inhibitors were utilized (Lazo et al., Colo-357 (solid circle) and Panc-1 (open triangle) cell lines were 2001). These inhibitors exerted a dose-dependent incubated in the absence (0) or presence of 200 pMTGF- b1 for the growth-inhibitory effect in six of seven pancreatic cancer indicated time. RNA was extracted as described and QRT-PCR for CDC25B (a) and p21CIP1 (b) was performed. The respective control cell lines (Figure 4a). In BxPc-3 cells, there was a slight mRNA expression levels were set to 100%. Results are presented as effect at low concentrations that did not increase with mean7s.e.m. of three independent experiments higher dosages. Calculation of the growth inhibition of

Oncogene CDC25B in pancreatic cancer J Guo et al 75

Figure 3 CDC25B localization in pancreatic tissues. Immunohistochemistry using a CDC25B-speciﬁc antibody was carried out in the normal pancreas (a, b), pancreatic cancer (c–h) and liver metastasis (i). C depicts a PanIN-Ia lesion observed in a PDAC sample. D and E depict areas surrounding the tumor mass containing degenerating acinar cells as well as tubular complexes. The inset in (i) depicts normal liver tissue next to the metastatic lesion. Percentage of positive cells in normal, next to cancer, cancer, metastasis as well as in pancreatic cancer according to stage and grade (k). Data are expressed as mean7s.e.m.

50% value (GI50) revealed GI50 values of 2.070.5 mm to 48 h, the percentage of cells in the G0/G1, S and G2/M 5.470.8 mm for NSC663284 and 1.670.4 mm to phases was determined by portioning the DNA histo- 6.271.8 mm for NSC668394 in the six responsive cell gram based on the control G1 and G2/Mpeaks. The lines. In contrast, GI50 values for both inhibitors were results revealed a shift to the G2 phase and G2/M more than 10 mm in BxPc-3 cells, which exhibited the accumulation in Capan-I, Colo-357, Mia-PaCa-2, Panc- lowest expression levels of CDC25B (Table 2a). 1 and T3M4 (Table 2b). In Mia-PaCa-2 cells, for To determine the effects of CDC25B inhibitors on example, the population of G2/Mincreased about 130% cell-cycle progression in pancreatic cancer cells, FACS following addition of the CDC25B inhibitor analysis was carried out next. Cells were treated with the NSC668394. In contrast, in BxPc-3 cells, no signiﬁcant GI50 dose of the respective CDC25B inhibitor. After change in cell-cycle distribution was observed after

Oncogene CDC25B in pancreatic cancer J Guo et al 76

Figure 4 Effects of CDC25B inhibitors in human pancreatic cancer cells. Pancreatic cancer cells (5000/well) were seeded in 96-well plates and incubated for 48 h with different concentrations of the CDC25B inhibitors. Cell growth was determined by the MTT assay (a). Data are expressed as mean7s.e.m. of triplicate determinations of three independent experiments. (b) Exemplary FACS analysis of Mia-PaCa-2 (left) and BxPc-3 (right). Measurement of DNA content for 20 000 cells in each sample was performed and the DNA histogram was interpreted using CellQuest Pro software as described in the Materials and methods section. Control (violet), NSC663284 (pink) and NSC668394 (green). (c, d) Two pancreatic cancer cell lines, BxPc-3 and Mia-PC-2, which showed the lowest and highest expression levels of CDC25B, respectively, were treated with NSC663284 and NSC668394 at the GI50 dose for the indicated time. Immunoblot analysis was carried out with the anti-phospho-cdc2 and the control cdc2 antibody as described in the Materials and methods section. Representative blots from two independent experiments are shown

Table 2a Summary of GI50 values for two speciﬁc CDC25B inhibitors in cultured human pancreatic cancer lines Aspc-1 Bxpc-3 Capan-1 Colo-357 Mia-PaCa-2 Panc-1 T3M4 NCI data*

NSC663284 (mm) 5.470.8 410.0 3.470.4 2.470.2 2.270.1 4.571.2 2.070.5 1.570.6 NSC668394 (mm) 6.271.8 410.0 2.270.4 1.670.4 1.870.1 2.770.8 2.270.8 2.172.0

*Average GI50 values of a panel of 60 human tumor cell lines (http://www.dtp.nci.nih.gov/)

Oncogene CDC25B in pancreatic cancer J Guo et al 77 Table 2b Changes in G2/Mpopulation in human pancreatic cancer In the present study, we have investigated the cell lines induced by CDC25B inhibitors expression and functional role of CDC25B in PDAC. NSC663284 (%) NSC668394 (%) At the mRNA level, cDNA array revealed that CDC25B expression levels were increased in pancreatic Aspc-1 +2.6 À2.7 cancer compared with chronic pancreatitis and the Bxpc-3 +1.3 À11.4 Capan-1 +58.4 +81.8 normal pancreas, especially in metastatic lesions, in Colo-357 +93.1 +123.8 which CDC25B mRNA levels were 2.2- and 4.1-fold Mia-PaCa-2 +129.0 +130.2 increased in comparison to primary cancer and normal Panc-1 +58.2 +84.7 pancreatic samples, respectively. In contrast, CDC25A T3M4 +50.7 +63.4 and CDC25C showed no significant difference between normal and cancerous samples. These results are in The change in G2/Mpopulation was calculated by test G2/M percentage minus control G2/Mpercentage/control G2/Mpercentage agreement with previous observations in other tumors. For example, in gastric cancer, CDC25B is expressed at high levels compared to both CDC25A and CDC25C incubation with both CDC25B inhibitors (Table 2b). An (Kudo et al., 1997). Quantitative PCR confirmed these exemplary FACS analysis of Mia-PaCa-2 and BxPc-3 findings, demonstrating that CDC25B mRNA levels in cells is shown in Figure 4b. pancreatic cancer samples were increased compared to To determine further the mechanism of G2/M the normal pancreas and chronic pancreatitis. arrest induced by CDC25B inhibitors, we investigated Interestingly, TGF-b led to transcriptional repression the phosphorylation of the CDC25B substrate cdc2. of CDC25B mRNA expression in two TGF-b-respon- In Mia-PaCa-2 cells, the level of phosphorylated sive pancreatic cancer cell lines, but was without effect in cdc2 increased time dependently following addition three other pancreatic cancer cell lines with Smad4 of the CDC25B inhibitors, with maximal effects mutations or other alterations in the TGF-b signaling occurring after 12 h of incubation (Figure 4c). In pathway. Since pancreatic cancers in vivo frequently contrast, in BxPc-3 cells there was no difference in exhibit alterations of the TGF-b signaling pathway such CDC2 phosphorylation following addition of the as Smad4 mutations (Hahn et al., 1996), low expression CDC25B inhibitors (Figure 4d). of the type I TGF-b receptor (Wagner et al., 1998) or Smad6/Smad7 overexpression (Kleeff et al., 1999a, b), these results provide another evidence of how these tumors might benefit from disturbances in the TGF-b Discussion signaling pathway, that is, by becoming resistant to the repressive effects of TGF-b on CDC25B expression. The eucaryotic cell cycle is controlled by a family of Immunohistochemistry revealed that in PDAC, protein kinases named CDKs, whose activation and 48.6716.3% of the cancer cells displayed positive inactivation is regulated by CDK inhibitors (e.g., p16) nuclear CDC25B immunoreactivity, whereas in the and activators (e.g., CDC25s). The G1–S phase and the normal pancreas only 8.371.8% cells exhibited positive G2–Mphase checkpoints of the cell cycle are precisely nuclear staining. In addition to the nuclear localization controlled by numerous protein–protein interactions of CDC25B, some cancer cells also exhibited cytoplas- such as between the 14-3-3 protein, Cdk2 and chk-1 and mic staining. There are conflicting reports regarding the others (van Hemert et al., 2001). Cell-cycle deregulation localization of CDC25B in other tissues and cells. Ito can lead to uncontrolled growth and contributes to and coworkers as well as other research groups have tumor formation. Defects in a variety of molecules that reported cytoplasmic accumulation of CDC25B (Gab- regulate the cell cycle such as p53, cdk inhibitors (e.g., rielli et al., 1996; Gasparotto et al., 1997; Broggini et al., p15, p16, p18, p21 and p27) and Rb have been 2000; Takemasa et al., 2000; Hernandez et al., 2001; implicated in tumorigenesis. Sato et al., 2001). Karlsson et al. (1999) reported that CDC25s have been characterized as dual-specificity CDC25B is located in the nucleus in the G1 phase and phosphatases that activate cyclin/CDKs complexes at exported to the cytoplasm during the S phase. Davezac different points of the cell cycle (Galaktionov and et al. (2000) demonstrated that the localization of Beach, 1991; Sebastian et al., 1993). Overexpression of CDC25B in the cytoplasm or in the nucleus depends CDC25A and CDC25B has been documented in many on the combined effects of a nuclear localization signal, human malignancies, suggesting their involvement in the a nuclear export signal and on the interaction with 14-3- pathogenesis and progression of malignant transforma- 3 proteins. This difference in the observed localization in tion (Gasparotto et al., 1997; Kudo et al., 1997; pancreatic tissues might be due to different, tissue- Hernandez et al., 2000, 2001; Ito et al., 2002; Miyata specific transportation and trafficking of the protein. et al., 2001; Sasaki et al., 2001; Sato et al., 2001; Ngan The functional consequences of this observation, how- et al., 2003). PDAC is characterized by uncontrolled ever, are currently not known. cell-cycle progression, resistance to cell-cycle arrest and In the present study, the percentage of CDC25B- apoptosis, in part due to alterations involving mutations positive cells was significantly higher in PDAC and of the proto-oncogene K-ras and p53, Smad4/DPC4 metastatic lesions in comparison to the normal pancreas and p16/CDKN2A tumor-suppressor gene mutations (Po0.01). However, there was no correlation between (Ozawa et al., 2001; Bardeesy and DePinho, 2002). the percentage of CDC25B-positive cells and stage or

Oncogene CDC25B in pancreatic cancer J Guo et al 78 grade of the tumor. In addition, PanIN lesions exhibited Materials and methods a comparable percentage of CDC25B-positive cells, suggesting that overexpression of CDC25B is an early Patients and tissue samples event in the pathogenesis of PDAC. In line with this Samples from 48 human primary PDACs (30 male, 18 female; hypothesis, in areas surrounding the tumor, the percen- median age 66 years; range 38–84 years), 10 cases of liver and/ tage of CDC25B-positive cells was significantly in- or lymph node metastasis (seven males, three females; median creased in comparison to normal pancreatic tissues. age 64 years; range 48–83 years), and samples of 34 chronic Although a correlation between CDC25B expression pancreatitis (28 males, six females; median age 50 years; range and stage or grade of the tumor has been observed in 16–64 years) were collected from patients who underwent several human malignances such as non-small-cell lung pancreatic resection in the Department of Visceral and Transplantation Surgery (University of Bern, Switzerland) carcinoma, ovarian cancer, breast cancer and colorectal and the Department of General Surgery (University of cancer (Galaktionov et al., 1995; Broggini et al., 2000; Heidelberg, Germany). In all, 24 normal human pancreatic Takemasa et al., 2000; Sasaki et al., 2001), we could not tissue samples were obtained from previously healthy organ detect such a correlation. Our results instead suggest donors (14 males, 10 females; median age 42 years; range 14– that in PDAC, increased CDC25B expression occurs 73 years) through a donor program. According to the TNM early in the process of malignant transformation. classification of the Union Internationale Contre le Cancer Recent studies have shown that the role of CDC25B (UICC), there were 10 stage I ductal adenocarcinomas, 10 in tumor pathogenesis and progression is dependent on stage II, 24 stage III and four stage IV ductal adenocarcino- post-transcriptional regulation. One important post- mas. There were five grade 1, 23 grade 2 and 20 grade 3 transcriptional alteration is splicing, and five CDC25B tumors. Freshly removed samples were snap frozen in liquid nitrogen and stored at À801C until analysis. In addition, a splicing variants have been identified so far. The portion of the tissue samples were fixed in 5% paraformalde- CDC25B3 splice variant is the main isoform in normal hyde solution for 12–24 h and paraffin embedded for and cancerous colorectal tissues. In contrast, CDC25B2 histological analysis. The study was approved by the Ethics seems to be important in the pathogenesis of non- Committees of the University of Bern (Switzerland) and the Hodgkin’s lymphomas (Kudo et al., 1997; Hernandez University of Heidelberg (Germany) and by the Human et al., 2000), and CDC25B1 in regulating the initiation Subjects Committee of the University of California (Irvine, of the mammalian mitosis (Baldin et al., 2002). In the CA, USA). present study, the CDC25B3 isoform was predominant in normal as well as cancerous tissues, suggesting that RNA extraction this isoform, and not other splice variants, plays an Total RNA from human pancreatic tissues and pancreatic important role under physiological and pathophysiolo- cancer cell lines was isolated either by the single-step gical conditions in the pancreas. guanidinium thiocyanate method as described previously or Since CDC25s are important components of the cell- MagNAPure LC mRNA isolation kit II (Roche Diagnostic cycle machinery and since their expression is frequently GmbH, Mannheim, Germany) (Friess et al., 1993; Kleeff et al., altered in human malignancies, efforts have been made 1998; Koliopanos et al., 2001). to inhibit their activity specifically. Recently, novel CDC25B inhibitors were utilized to inhibit tumor cell cDNA synthesis growth (Lazo et al., 2001). In our study, two CDC25B cDNA was synthesized from total RNA by reverse transcrip- inhibitors – NSC663284 and NSC668394 – dose tion using a cDNA kit according to the manufacturer’s dependently inhibited pancreatic cancer cell growth. instructions (First Strand cDNA Synthesis Kit, Roche Three lines of evidence suggest that their effects could Diagnostic GmbH, Mannheim, Germany). specifically be attributed to CDC25B inhibition. First, FACS analysis revealed that the two compounds cDNA array resulted in G2/Marrest and G2 accumulation. Second, s the levels of phosphorylated cdc2 increased time The GeneChip HG-U95Av2 array used in this study was dependently after addition of CDC25B inhibitors, fabricated by Affymetrix Inc. (Santa Clara, CA, USA). It suggesting that these inhibitors indeed blocked CDC25B contains about 10 000 full-length human genes from the UniGene database (Build 95). Poly (A) þ RNA isolation, phosphatase activity. Third, Mia-PaCa-2 cells that cDNA synthesis and cRNA in vitro transcription was carried displayed the highest expression levels of CDC25B were out as reported previously (Friess et al., 2001; Tureci et al., most sensitive to the CDC25B inhibitors, whereas BxPc- 2003). The in vitro transcription product was purified and 3 cells that expressed only low levels of CDC25B were fragmented as described (Friess et al., 2001; Tureci et al., resistant to the inhibitors. 2003). Hybridization of the fragmented in vitro transcription In conclusion, our findings demonstrate that product to oligonucleotide arrays was performed as suggested CDC25B is overexpressed in PDAC and metastatic by the manufacturer (Affymetrix Inc.). lesions, providing a novel aspect in the pathogenesis of this disease. CDC25B inhibitors act to inhibit pancreatic Quantitative RT–PCR cancer cell growth by blocking G2/Mphase transition Quantitative PCR was performed with the LightCycler via inhibition of dephosphorylation of cdc2. Together, FastStart DNA SYBR Green kit (Roche Applied Science, our findings raise the possibility that inhibition of Mannheim, Germany). The primer sets were designed for CDC25B phosphatase may ultimately have a therapeu- CDC25B (forward 5-GGG CAA GTT CAG CAA CAT CGT tic role in this disorder. GGA-3, reverse 5-GTA GCC GCC TTT CAG GAT ATA

Oncogene CDC25B in pancreatic cancer J Guo et al 79 CAT C-3) and the CDC25B3 splice variant (forward 5-GAA DAB chromogen mixture for 20 s. Finally, sections were ACC CCA AAG AGT CAG GTA G-3, reverse 5-GAG TTG counterstained with Mayer’s hematoxylin and mounted in GTG ATG TTC CGA AGC AC-3). To control for specificity permanent mounting medium (Friess et al., 1993; Kleeff et al., of the amplification products, a melting curve analysis was 1998). To calculate the percentage of CDC25B positive cells, performed. The number of transcripts was calculated from a 10 high-power fields were selected at random for each section, standard curve obtained by plotting known input concentra- and at least 500 cells were evaluated. tions of four different representative plasmids (log dilutions) to the PCR cycle number at which the detected fluorescence Protein extraction intensity reaches a fixed value (z). Data of two independent analyses for each sample and parameter were averaged. The Cells and tissues were lysed in 0.5 ml lysis buffer containing copy number of the different CDC25B and variant B3 was 50 mm Tris-HCl (pH 7.5), 150 mm NaCl, 2 mm EDTA (pH 8.0) normalized by the housekeeping genes HPRT and cyclophillin and 1% SDS with proteinase inhibitors (one tablet/10 ml, B (Search-LC GmbH, Heidelberg, Germany). Roche Molecular Biochemicals, Basel, Switzerland). Protein concentration was determined by the BCA protein assay Conventional RT–PCR (Pierce Chemical Co., Rockford, IL, USA).

RT–PCR was carried out under standard conditions, using the Immunoblot following primer pairs: forward 5-CTC ATT AGT GCC CCA CTG GT-3 and reverse 5-ATC AGC TCT CGG TGG TCA Total protein (20 mg/lane) was size fractionated by SDS– CT-3 for CDC25B for subcloning; forward 5-GCT TCC TCG polyacrylamide gel electrophoresis (SDS/PAGE) and trans- CCG GTC ACC AC-3 and reverse 5-CCT GCG GCT GGC ferred onto nitrocellulose membranes (BioRad Laboratories, CCA CTC-3 for the CDC25B for splicing variants; forward 5- Hercules, CA, USA). Blots were blocked in TBS with Tween- CTG GCC GGG ACC TGA CTG ACT-3 and reverse 5-CTA 20 (0.1%) at pH 7.5 containing 5% nonfat milk for 1 h and GAA GCA TTT GCG GTG GAC GAT-3 for beta-actin as an then incubated overnight with the indicated antibody internal control. Products were separated by electrophoresis in (CDC25B, CDC2 and phospho-CDC2). Following three 1% agarose-TBE gels, and bands were visualized by ethidium washes with TBS-T, membranes were incubated with the bromide staining (24). secondary antibody for 1 h at 231C. Antibody detection was performed with an enhanced chemiluminescence (ECL) reac- Subcloning tion system (Amersham Life Sciences, Amersham, UK).

A 305 RT–PCR fragment of the human CDC25B gene was Cell culture subcloned into the pGEMT-easy vector (Promega Biotechnol- ogy, Madison, WI, USA) using standard conditions. Authen- Pancreatic cancer cell lines were cultured in RPMI1640 (AsPc- ticity was confirmed by sequencing (QIAGEN Genomic 1, BxPc-3, Capan-1 and T3M4) or DMEM (Colo-357, Mia- services, Hilden, Germany). PaCa-2 Panc-1) with 10% fetal bovine serum, 100 U/ml penicillin and 100 mg/ml streptomycin and maintained at 1 Northern blot analysis 37 Cin5%CO2. For TGF-b induction experiments, cells were serum starved overnight. Subsequently, cells were Total RNA (15 mg/lane) was size fractionated on 1.2% incubated in a serum-free medium in the absence or presence agarose/1.8 m formaldehyde gels. Gels were stained with of 200 pMTGF- b1 (R&D Systems Inc., Minneapolis, MN, ethidium bromide for verification of RNA integrity and USA) for the indicated time. After 48 h, RNA was extracted loading equivalency. Fractionated RNA was transferred onto and QRT–PCR was carried out as described. Genescreen membranes (NENt Life Science Products, Boston, MA, USA) and crosslinked by UV irradiation. Blots MTT assay were then prehybridized for 12 h at 421C in 50% formamide, 1% SDS, 5 Â Denhardt’s, 100 mg/ml salmon sperm DNA, 50 mm Two CDC25B inhibitors (NSC663284 and NSC668394) were Na2PO4, pH 7.4, 10% dextran, 75 mm NaCl and 5 mm EDTA. the kind gift of Robert J Schultz (Drug Synthesis & Chemistry Blots were then hybridized for 24 h at 421C in the presence of Branch, Developmental Therapeutics Program, Division of an a32P dCTP (Amersham Pharmacia Biotech, Freiburg, Cancer Treatment and Diagnosis, National Cancer Institute). Germany) labeled cDNA probe for CDC25B (106 cpm/ml) or Both compounds were identified from 140 000 compounds in 7S (105 cpm/ml), rinsed twice with 2 Â SSC and washed twice the NCI Compound Repository, as specific inhibitors of with 0.2 Â SSC/2%SDS at 50 and 551C for 20 min, respec- CDC25B (Lazo et al., 2001). Determination of the GI50 was tively. All blots were exposed at À801C to Kodak BiomaxMS carried out as described (http://www.dtp.nci.nih.gov/). Briefly, films with Kodak-intensifying screens (Friess et al., 1993; 5 Â 103 cells/well were seeded in 96-well plates. After incuba- Kleeff et al., 1998, 1999a). tion for 48 h with different concentrations of NSC663284 or NSC668394, cell growth was determined by the MTT assay. Immunohistochemistry GI50 was calculated from [(TiÀTz)/(CÀTz)] Â 100 ¼ 50 (Tz: time zero, C: control growth, and Ti: test growth in the Paraffin-embedded tissue sections (3 mm thick) were deparaffi- presence of drug at different concentration levels). nized and rehydrated. Slides were covered with 3% H2O2 for 10 min and 10% normal goat serum for 30 min at room Cell-cycle analysis by flow cytometry temperature before incubation overnight with a rabbit anti- CDC25B antibody (Cell Signaling Technology, Beverly, MA, Cells seeded in 12-well plates (1 Â 105 cells/well) were incubated USA) at 41C. After washing with TBS þ 0.1%BSA, slides were for 48 h at the GI50 concentration. Cells were next harvested incubated with an HRP-linked anti-rabbit secondary antibody and washed with PBS, then resuspended in 1 ml of hypotonic (DAKO Envision Kit, Carpenteria, CA, USA) for 45 min, then PI buffer: 50 mg/ml PI, 0.1% Triton X-100 and 0.1% washed three times with TBS þ 0.1%BSA, followed by NaCitrate) and stored overnight at 41C (Nicoletti et al., incubation with a buffered substrate for liquid DAB/liquid 1991). Measurement of DNA content for 20 000 cells in each

Oncogene CDC25B in pancreatic cancer J Guo et al 80 sample was performed using FACScan (Becton Dickinson and independent experiments, unless indicated otherwise. For Company, New Jersey, USA). statistical analysis, the Mann–Whitney U-test was used. Po0.05 was taken as the level of signiﬁcance. Statistics Acknowledgements Patient data are expressed as median and range. Experimental This work was supported, in part, by US Public Health Service results are expressed as mean7s.e.m. of at least three Grant CA-40162 to MKorc.

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