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The Ubiquitin-Proteasome Pathway Mediates Gelsolin Protein Downregulation in Pancreatic Cancer

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The Ubiquitin-Proteasome Pathway Mediates Gelsolin Protein Downregulation in Pancreatic Cancer The Ubiquitin-Proteasome Pathway Mediates Gelsolin Protein Downregulation in Pancreatic Cancer Xiao-Guang Ni,1 Lu Zhou,2 Gui-Qi Wang,1 Shang-Mei Liu,3 Xiao-Feng Bai,4 Fang Liu,5 Maikel P Peppelenbosch,2 and Ping Zhao4 1Department of Endoscopy, Cancer Institute and Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China; 2Department of Cell Biology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands; 3Department of Pathology, 4Department of Abdominal Surgery, and 5State Key Laboratory of Molecular Oncology, Cancer Institute and Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China A well-known observation with respect to cancer biology is that transformed cells display a disturbed cytoskeleton. The under- lying mechanisms, however, remain only partly understood. In an effort to identify possible mechanisms, we compared the pro- teome of pancreatic cancer with matched normal pancreas and observed diminished protein levels of gelsolin—an actin fila- ment severing and capping protein of crucial importance for maintaining cytoskeletal integrity—in pancreatic cancer. Additionally, pancreatic ductal adenocarcinomas displayed substantially decreased levels of gelsolin as judged by Western blot and immunohistochemical analyses of tissue micoarrays, when compared with cancerous and untransformed tissue from the same patients (P < 0.05). Importantly, no marked downregulation of gelsolin mRNA was observed (P > 0.05), suggesting that post- transcriptional mechanisms mediate low gelsolin protein levels. In apparent agreement, high activity ubiquitin-proteasome path- way in both patient samples and the BxPC-3 pancreatic cancer cell line was detected, and inhibition of the 26s proteasome sys- tem quickly restored gelsolin protein levels in the latter cell line. The status of ubiquitinated gelsolin is related to lymph node metastasis of pancreatic cancer. In conclusion, gelsolin levels are actively downregulated in pancreatic cancer and enhanced targeting of gelsolin to the ubiquitin-proteasome pathway is an important contributing factor for this effect. Online address: http://www.molmed.org doi: 10.2119/2008-00020.Ni INTRODUCTION Among the mechanisms that mediate and even silencing of the gelsolin gene Pancreatic cancer is one of the most deviant cytoskeletal structure in cancer has been documented in many types of virulent malignances with an overall cells is an aberrant expression of gelsolin. human cancers, including bladder, five-year survival rate of only 3–5% and Gelsolin is a Ca2+- and polyphosphoinosi- breast, lung, prostate, gastric, and ovar- a median survival time after diagnosis tide 4, 5-bisphosphate (PIP2)-regulated ian cancers (5–10). Strikingly, forced ex- of < 6 months (1). Despite this immense actin filament severing and capping pression of gelsolin can cause cancer cell clinical problem, pancreatic cancer biol- protein (2). Gelsolin was first isolated morphological reversion and greatly re- ogy remains poorly understood in com- from rabbit lung macrophages as a mod- duce colony-forming ability in vitro, as parison with other cancers. For in- ulator of the cytoplasmic actin gel-sol well as tumorigenicity in vivo in various stance, although it has been recognized transformation (3), and is generally con- tumor types (11). However, downregula- for almost 40 years that transformed sidered one of the most important regu- tion of gelsolin protein levels in pancre- cells display a disturbed actin cyto- lators of the actin cytoskeleton (4). Thus, atic cancer has not been demonstrated skeletal structure, the mechanisms me- gelsolin is an obvious candidate for ex- convincingly, nor have the molecular diating disturbed actin filament organi- plaining altered cytoskeletal reorganiza- mechanisms that could mediate such zation in pancreatic cancer remain tion in pancreatic cancer. In support for downregulation been identified. largely obscure. such a notion, diminished expression In the present study, we provide con- vincing evidence that gelsolin protein levels become severely downregulated in Address correspondence and reprint requests to Ping Zhao, Department of Abdominal pancreatic cancer. This downregulation Surgery, Cancer Institute and Hospital, Chinese Academy of Medical Sciences and differs from most other tumor types be- Peking Union Medical College, NO.17 Panjiayuan, Chaoyang District, P.O. Box 2258, Beijing cause the downregulation is not mirrored 100021, P.R.China.Phone: 86-10-87711782; Fax: 86-10-87711782; E-mail: nixiaoguang@ at the mRNA level, apparently excluding yahoo.com.cn. pretranslational mechanisms. In apparent Submitted February 15, 2008; Accepted for publication June 6, 2008; Epub (www.molmed. agreement, we see that gelsolin is specifi- org) ahead of print June 11, 2008. cally routed into the ubiquitination- 582 | NI ET AL. | MOL MED 14(9-10)582-589, SEPTEMBER-OCTOBER 2008 RESEARCH ARTICLE proteasome system and that inhibition of treated with 0.1% DMSO as a vehicle Primary data collection and analysis this system, at least in vitro, is sufficient control. were carried out using the GenePix Pro to restore gelsolin protein levels. Thus, 4.0 (Axon Instruments) software pack- downregulation of gelsolin is an intrinsic Antibody Microarray Detection age. The antibody spot was excluded part of the pancreatic cancerous process Procedure and Data Analysis from further analysis if it had a signal: which, unique to this type of cancer, Antibody microarrays (BD Clontech background ratio of < 1.5 in both chan- seems to be partly mediated by ubiquitin- 380, Cat. K1847-1, Lot. 2070683) were nels. For each spot, the median of ratios proteasome machinery. obtained from BD Biosciences Clontech was used in subsequent analysis. The (Palo Alto, CA, USA), and sample prepa- data were imported into the BD Clontech MATERIALS AND METHODS ration and processing procedures were Ab Microarray Analysis Workbook. The performed as described in the antibody relative fluorescence units of C-Cy5/N-Cy3 Patients and Specimens microarray user manual. Briefly, to re- from slide 1 (ratio 1) and N-Cy5/C-Cy3 Fresh tissue samples of 11 pancreatic duce the influence of biological heteroge- from slide 2 (ratio 2) were calculated ductal adenocarcinomas and their corre- neity among tumor samples derived and an internally normalized ratio (INR) sponding distant normal counterparts from different patients, 200 mg from each was obtained by computing the square were obtained at the time of resection of seven pancreatic ductal adenocarci- root of ratio 1/ratio 2. This value repre- with informed consent from Cancer In- noma samples were pooled together and sents the abundance of an antigen in stitute and Hospital (CIH), Chinese pulverized using liquid nitrogen. An pancreatic cancer relative to that of nor- Academy of Medical Sciences (CAMS), equivalent pool was made from matched mal pancreas. The value of INR > 1.5 or and Peking Union Medical College normal pancreas. Total protein was ex- < 0.7 was considered as a significantly (PUMC) between November 2001 and tracted from 200 mg of pooled cancer different protein expression level be- March 2003. The samples were snap- sample or pooled normal pancreas using tween the two samples. frozen in liquid nitrogen before storage extraction/labeling buffer, and then at –80° C. None of them received preop- mixed with Cy3 or Cy5 (Amersham Bio- Measurement of Gelsolin mRNA erative radiotherapy or chemotherapy. sciences, Piscataway, NJ, USA) sepa- Expression by Semiquantitative Formalin-fixed paraffin-embedded tis- rately. To prevent skewing of the array Reverse Transcriptase-PCR sue blocks containing pancreatic cancer results, cancer sample (C) and normal Total RNA was isolated from pancre- and normal pancreatic tissue were col- sample (N) were each split into two atic cancer specimens (n = 9) and their lected from the archives of the Depart- equal portions. Each portion was then la- corresponding distant normal counter- ments of Surgery at CIH, CAMS, and beled with either Cy3 or Cy5 to produce parts and cell line BxPC-3 using RNeasy PUMC between January 1991 and Au- four samples: C-Cy3, C-Cy5, N-Cy3, and MinElute cleanup kit (QIAGEN, Valen- gust 2002, and subjected to tissue mi- N-Cy5. There are two same slides in the cia, CA, USA) according to the manufac- croarray construction. The relevant kit. C-Cy5 (40 µg) and N-Cy3 (40 µg) turer’s instruction. RNA quality was as- ethical committee approved of the inves- were mixed and added to slide 1, and sessed on agarose gel electrophoresis tigations performed. N-Cy5 (40 µg) and C-Cy3 (40 µg) were and spectrophotometric analysis. Re- mixed and added to slide 2. Following verse transcription (RT) reactions were Cell Culture and Drug Treatment 30 min incubation at room temperature, performed on 5 µg of total RNA using Human pancreatic adenocarcinoma the slides were washed, dried, and SuperScript First-Strand synthesis for cell line BxPC-3 was obtained from scanned using the GenePix 4000B Mi- RT-PCR II kit (Invitrogen, Carlsbad, CA, American Type Culture Collection croaray Scanner (Axon Instruments, USA) at 42° C for 80 min, and 0.5–1 µg (Manassas, VA, USA). BxPC-3 cells Union City, CA, USA). There are 768 aliquots of the cDNA then were sub- were cultured in RPMI 1640 medium spots in this antibody microarray. Each jected to RT-PCR. The specific gelsolin supplemented with 10% heat-inacti- antibody is arrayed by duplicate spots primer sequences were used as follows: vated fetal bovine serum, 100 U mL–1 side-by-side on the slide. Besides the an- 5’-CCACTGCGTCGCGGGG-3’ (sense) penicillin, and 100 µg/mL streptomycin tibody capture-dependent spots, there and 5’-GGCAGCCAGCTCAGCCATG-3’ at 37° C in 5% CO2. Specific proteasome are four paired spots pre-labeled with (antisense). The PCR step was performed inhibitor lactacystin (Sigma-Aldrich, St. Cy3 and Cy5 bovine serum albumin using Taq DNA polymerase (Invitrogen). Louis, MO, USA) was dissolved in di- (BSA) located near the outermost corners As an internal control, GAPDH was am- methyl sulfoxide (DMSO). Cells were of the printed area that serve as orienta- plified to ensure cDNA quality and seeded on 100 mm plates.
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