Oncogene (2004) 23, 71–81 & 2004 Nature Publishing Group All rights reserved 0950-9232/04 $25.00 www.nature.com/onc 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