Cyclin-Dependent Kinase 5 Is Amplified and Overexpressed in Pancreatic Cancer and Activated by Mutant K-Ras

Published OnlineFirst August 8, 2011; DOI: 10.1158/1078-0432.CCR-10-2288

Clinical Cancer Human Cancer Biology Research

John P. Eggers1, Paul M. Grandgenett1, Eric C. Collisson5, Michelle E. Lewallen2, Jarrod Tremayne1, Pankaj K. Singh1, Benjamin J. Swanson1,3, Judy M. Andersen1, Thomas C. Caffrey1, Robin R. High4, Michel Ouellette1, and Michael A. Hollingsworth1,2

Abstract Purpose: To evaluate the nature of cyclin-dependent kinase 5 (CDK5) hyperactivity in pancreatic cancer progression. Experimental Design: We used genetic, biochemical, and molecular biology methods to investigate the nature and function of overexpression of CDK5 and its activators p35 and p39 during the progression of pancreatic cancer. Results: Amplification of the CDK5 gene or either of its main activators, p35 and p39, was observed in 67% of human pancreatic ductal adenocarcinoma (PDAC). CDK5, p35, and p39 were rarely expressed in pancreatic ducts whereas more than 90% of PDACs had increased levels of CDK5 and p35. Increased levels of CDK5, p35, and p39 protein were observed in several pancreatic cancer cell lines. Inhibition of CDK5 kinase activity using a CDK5 dominant-negative mutant or the drug roscovitine significantly decreased the migration and invasion of pancreatic cancer cells in vitro. Increased CDK5 kinase activity was also observed in immortalized human pancreatic nestin-expressing (HPNE) cells expressing a mutant form of K-Ras (G12D) compared with HPNE cells expressing native K-Ras. G12D K-Ras increased cleavage of p35 to p25, a stable and greater activator of CDK5, thus implicating a role for CDK5 in early progression of PDAC. Inhibition of the signaling cascade downstream of mutant K-Ras (G12D) that involves mitogen-activated protein/extracellular signal– regulated kinase, phosphoinositide 3-kinase, or CDK5 decreased p25 protein levels. Conclusion: These results suggest that mutant K-Ras acts in concert with CDK5 and its activators to increase malignant progression,migration,andinvasionof pancreatic cancer cells. Clin Cancer Res; 17(19); 1–11. 2011 AACR.

Introduction nearly universal in PDACs and common in precursor pancreatic intraepithelial neoplasia lesions (1). The identi- Pancreatic ductal adenocarcinomas (PDAC) are lethal fication of effectors downstream of K-Ras that modulate tumors that result in a median survival of less than growth, cell migration, and invasion will help under- 6 months from time of diagnosis and a 5-year survival stand the process of disease progression and enable the rate of less than 5% (http://emedicine.medscape.com/ development of novel therapeutic targets for patients with article/280605-overview). The poor survival is largely at- PDACs. tributed to aggressive growth, advanced stage at diagnosis, Perineural invasion (PNI) is a hallmark of pancreatic and local recurrence. Activating mutations in K-Ras are cancer (2, 3). Pancreatic innervation includes enteric ner- vous connections to spinal cord and brain that mediate pancreatic exocrine and endocrine functions (4). An exten- Authors' Affiliations: 1Eppley Institute for Research in Cancer and Allied 2 3 sive neural network surrounding the pancreas coupled with Diseases; Departments of Biochemistry and Molecular Biology, Pathol- ogy and Microbiology, and 4Biostatistics, College of Public Health, Uni- the frequent PNI observed in pancreatic cancer led us to versity of Nebraska Medical Center, Omaha, Nebraska; and 5Department investigate molecules expressed in pancreatic cancer cells of Hematology & Oncology, University of California, San Francisco, California that are also involved in cellular migration during neural development. Cyclin-dependent kinase 5 (CDK5) is nec- Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/). essary for proper cortical neuronal migration and pattern- –/– Corresponding Author: Michael A. Hollingsworth, University of Nebraska ing. CDK5 (deficient) mice die in the perinatal period Medical Center, 985950 Nebraska Medical Center, Omaha, NE 68198. because of the congenital loss of 4 of 6 cortical layers in the Phone: 402-559-8343; Fax: 402-559-2813; E-mail: [email protected] brain (5). CDK5-catalyzed phosphorylation of p21-activat- doi: 10.1158/1078-0432.CCR-10-2288 ed kinase (PAK1) and focal adhesion kinase (FAK) is 2011 American Association for Cancer Research. required for proper neuronal migration (6, 7). PAK1 and

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Translational Relevance mAB CDK5 (Upstate); and the horseradish peroxidase– conjugated goat a-mouse and a-rabbit (Pierce) were We show that cyclin-dependent kinase (CDK5) or its used at concentrations recommended by the manufac- R coactivators p35 and p39 are significantly overexpressed turer. The CDK5 inhibitor -roscovitine was purchased in more than 90%, 94%, and 75% of pancreatic ductal from Calbiochem. adenocarcinoma (PDAC), respectively, compared with normal pancreases, in part, because of genomic ampli- Comparative genomic hybridization fication, as genes for CDK5, p35, or p39 were amplified We selected formalin-fixed, paraffin-embedded blocks in 67% (n ¼ 39) of tumors analyzed. CDK5 is hyper- from pancreaticoduodenectomy specimens carried out activated by signaling downstream of mutant K-Ras, for PDAC at University of California, San Francisco, resulting in increased tumor migration and invasive between 1993 and 2004. Tissue blocks were reviewed properties. CDK5 represents a novel and unexplored by a gastrointestinal pathologist and confirmed to be therapeutic target for both early- and late-stage PDACs PDAC. Dissection was carried out; genomic DNA was and should be investigated in solid tumors that harbor isolated by a proprietary phenol-chloroform–based ex- K-Ras mutations including those of pancreas, lung, and traction procedure at Response Genetics. Extracted geno- colon. mic DNA (70 ng) was subjected to genome-wide allele- specific copy number analysis on a molecular inversion probe platform as described (23). Molecular inversion probe copy number was assessed by using the Nexus FAK are also implicated in regulating cytoskeletal organi- Software (BioDiscovery). zation during tumor cell migration (8–10). Recently, CDK5 kinase activity has been correlated with increased invasion Cell culture in prostate carcinomas and glioblastoma multiforme All cell lines were maintained in RPMI supplemented (11, 12). with 10% heat-inactivated FBS and ciprofloxacin. Stably CDK5 is the only known CDK to become activated transfected/transduced cell lines were maintained with the through binding noncyclin molecules (13). The best char- appropriate selection as indicated in the next section. acterized CDK5 activators, p35 and p39, have C-terminal regions that contain cyclin box folds that bind and activate Plasmids and stable transfections and transductions CDK5 (14). In neural cells, calpain-mediated cleavage of The reverse tetracycline transactivator (rtTA, "tet-on," p35 and p39 to p25 and p29, respectively, yields activators kindly provided by Kay Uwe Wagner, University of with greater stability, increased CDK5 kinase activation, Nebraska Medical Center; ref. 24) was transfected into and altered localization and substrate specificity as com- S2-013 cells and selected with G418 at 600 mg/mL. After pared with the full-length p35 and p39 forms (15, 16). In stable selection with G418, the pBI (Clontech) vector with this report, we investigated the expression and function of a zeocin-resistant gene (kindly provided by Barry Nelkin, CDK5 and 2 of its activators, p35 and p39, during the Johns Hopkins University School of Medicine, Baltimore, pathogenesis of PDACs. MD) was transfected, and stable expressing cell lines were selected with zeocin at 200 mg/mL (11). Clones expressing Materials and Methods the CDK5 dominant-negative (CDK5DN) insert were selected if there was more than a 2-fold increase in the Cells, antibodies, and reagents CDK5 protein expression after addition of 3 mg/mL doxy- S2-013 cells are a cloned subline of a human pancreatic cycline, thus indicating induced expression of CDK5DN. tumor cell line SUIT-2 derived from a liver metastasis (17). The clones were sorted for enhanced green fluorescent HPAF2 is a human pancreatic adenocarcinoma cell line protein to isolate a high level CDK5DN-expressing derived from peritoneal ascitic fluid (18). T3M4 cells are a population. human carcinoembryonic antigen–producing cell line All stable transfections used Genporter2 (Genlantis) as established from a primary PDAC transplanted into nude per the manufacturer’s recommendations. mice and then cultured (18). FG cells are a fast-growing clone established from the Colo-357 PDAC cell line (19). Western blotting and immunoprecipitation Human pancreatic nestin-expressing (HPNE) cells are a Briefly and as previously described (25), cell lysate human telomerase reverse transcriptase (hTERT; active proteins were resolved on 12% or 4% to 20% Novex telomerase)-immortalized HPNE ductal progenitor cell Tris-Glycine denaturing polyacrylamide gels (Invitrogen) line (20). tHPNE cells are HPNE cells transduced with in a 1 SDS-PAGE buffer. Blocking and primary antibody mutant K-Ras (G12D), E6/E7, and SV40 small t antigen incubation was carried out overnight at 4C or for 1 hour at (20–22). room temperature. Secondary antibodies were incubated The rabbit polyclonal antibodies to p35, p39, and for 1 hour at room temperature. Primary and secondary CDK5 (Santa Cruz Biotechnology); p44/p42 MAPK antibody incubations were followed with five 3-minute monoclonal antibodies (Cell Signaling); FAK, CDK5, washes in PBS. All incubations were carried out in PBS and phospho PAK1 Abs (Abcam); mAB b-Actin (Sigma); containing 3% dried milk.

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Immunohistochemistry significant. The data were analyzed with PROC GLIMMIX CDK5, p35, and p39 were investigated for frequency of of SAS, version 9.2. Data entered directly as percentages expression in PDACs, metastases from PDACs, and normal (migration) were evaluated with a regression model on the pancreases. Briefly and as previously described (26), basis of the b distribution. 5 mmol/L thick, paraffin-embedded tissue sections were immunostained. The tissue sections in paraffin were rehy- Results drated through incubations in xylene and then an alcohol gradient from 100% to 70% followed by an H O incuba- 2 CDK5, p35, and p39 expression in nontransformed tion. Antigen retrieval using Antigen Unmasking Solution pancreas (Vector Labs) was carried out according to the man- We determined the expression pattern of CDK5, p35, ufacturer’s instructions. The sections were blocked in and p39 in 9 different normal pancreatic tissue samples. Dako Blocking Solution (Dako) for 30 minutes at Consistent with previous reports, CDK5, p35, and p39 were 24C. The primary antibodies were incubated for 45 highly expressed in islet b cells (28, 29). We extend this minutes at 24C. CDK5 (Abcam), p35 (Santa Cruz Bio- finding to show that CDK5, p35, and p39 were rarely technology), p39 (Santa Cruz Biotechnology), and rabbit visualized in ductal cells (Fig. 1A). Only 2 of 9 samples IgG (Sigma) were used at 1:70, 1:100, 1:100, and 2 mg/mL, expressed detectable CDK5 in a few ductal cells and weak respectively, and the secondary goat anti-rabbit IgG staining in adjoining acinar cells. Ductal cells positive for (Dako) for 30 minutes at 24Cwith3PBSwashes CDK5 also expressed p35, which was independently between each Ab/staining step. Positive staining was expressed sporadically in more than half of the normal visualized with a 3,30-diaminobenzidine substrate solu- samples; however, only 3 of 9 samples showed more than tion (Vector Labs) for 2 to 5 minutes and then slides were 25% of the ducts staining positive for p35 (Table 1). p39 counterstained in hematoxylin. All slides were analyzed was only expressed in 38% of normal ducts, and only 1 by 2 individuals trained in immunohistochemistry and sample had greater than 10% of the ducts staining positive interpretations confirmed by a pathologist. for p39 (Table 1). Migration assay Cells were grown to 90% confluence on 6-well plates CDK5 and two of its activators, p35 and p39, are and placed in RPMI containing 0.5% FBS. Twenty-four expressed in pancreatic adenocarcinoma cell lines, hours later, a scratch was made with either a p20 or p1000 tumors, and metastases pipette tip. Using a colony marker, areas of similar cell CDK5, p25 (the cleavage product of p35 with higher density and scratch characteristics were marked and capacity for activating CDK5), and p29 (the cleavage prod- photographed. S2-013 cells were grown for 24 hours uct of p39 with a higher capacity for activating CDK5) were and HPNE cells for 18 hours and then photographed expressed in 10 of 10 PDAC cell lines tested, ranging from again in the same field. The photographs were imported poorly to well differentiated (Fig. 1B), all expressing mu- K Ras into Microsoft PowerPoint and analyzed for percentage tant - except T3M4 cells (18, 30, 31). Thus, the pre- migration into the scratch. The results are reported in fold dominant forms of p35 and p39 observed in these cell lines migration relative to that of the control. TScratch (27) was were their activated N-terminal cleavage products, p25 and used to analyze the total area of migration by HPNE cells. p29 (15, 16). Results are reported in total area covered by migrated Examination of CDK5, p35, and p39 expression in cells. Indicated cell treatments were initiated at time of primary PDACs and liver and lung metastases by immu- making the scratch. nohistochemistry revealed CDK5 expression in more than 90% of primary PDACs with similar expression of p35 and Matrigel invasion assay less for p39 (Fig. 1C and Table 2). CDK5, p35, and p39 In vitro analysis of invasion was measured by seeding cells were observed in all 6 examples of PNI that were examined P < on the upper chamber of a Matrigel-coated Boyden chamber (Fig. 1C and Table 2). CDK5 was significantly ( 0.0005) in serum-free media with the lower chamber containing overexpressed in primary PDACs compared with normal media with 10% FBS. T3M4, HPAF2, and S2-013 cells were pancreases (Tables 1and 2). CDK5, p35, and p39 expres- seeded at 5 104 cells per filter. tHPNE and FG cells were sion was especially evident at the leading front of tumors seeded at 1 104 and 1 106 cells per filter, respectively. (Fig. 1C). Roscovitine (10 mmol/L) was added just before cell seeding. After a 24-hour incubation using 10% FBS as the chemoat- Genomic amplification of CDK5, p35, and p39 in tractant in the lower chamber, the upper chamber was PDACs cleaned. The filter was stained with Diff-Quick staining kit CDK5, p35, and p39 were individually amplified in (Allegiance), and the invading cells were counted. approximately 33% of PDACs (n ¼ 39; Fig. 1D). Interest- ingly, 67% of 39 pancreatic tumors examined exhibited Statistical analysis amplification of at least one of these genes (Fig. 1D). A few Statistical analysis was conducted using paired Student’s deletions were noted (approximately 10%, data not t test or c2 test as indicated with P < 0.05 being statistically shown); however, almost all (3 of 4) tumor samples with

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A H&E CDK5 p35 IgG Islet

Normal pancreas Nontransformed duct

Nontransformed ducts Normal pancreas

Figure 1. CDK5 and p35 expression in normal pancreas Capan1 Capan2 CFPAC1Colo-357 FG HPAF Panc1 S2-007 S2-013 T3M4 tHPNE HPNE.krasHPNE and PDACs. A, most normal Phospho-S732 FAK pancreatic ductal cells are negative for CDK5 and p35 in FAK most ducts (row 1). p35 detected within a pancreatic duct whereas p25 an adjacent duct is negative for p35 p35 and the corresponding staining for CDK5 is negative in p29 both ducts (row 2). B, CDK5 and 2 of its activators, p35 and p39, and p39 their cleavage products, p25 and CDK5 p29, respectively, are expressed in multiple PDAC cell lines. β-Actin Increased levels of S732 FAK (CDK5 phosphorylation site) were observed in the PDAC cell lines. C, C H&E CDK5 immunohistochemical analysis of primary pancreatic cancer Invading samples shows expression of PDAC CDK5, p35, and p39 in a primary pancreatic cancer exhibiting PNI. Nerve Nerve

p35 p39

Invading PDAC

Invading Nerve PDAC Nerve

deletions in either p35 or p39 had a concomitant ampli- (HPNE; ref. 20), which was transduced independently and fication in the reciprocal gene (p35 or 39). in combination with retroviruses expressing mutant active K-Ras (G12D; kras), human papillomavirus E6 and E7 K-Ras increased CDK5 kinase activity (E6/7) to inhibit Rb and p53, and SV40 small t antigen We investigated the expression and activity of CDK5, to inhibit protein phosphatase 2A (St; refs. 20, 21). The p35, and p39 during the premalignant and malignant utility of these cells for investigating relevant signaling transformation of pancreatic epithelial cells by using an pathways downstream of mutant K-Ras has been estab- immortalized human pancreatic ductal progenitor cell line lished (32). HPNE.kras cells had slightly higher p35 mRNA

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Table 1. Expression of CDK5, p35, or p39 in D cdk5 pathwaygeneamplification normal pancreatic ducts 70 Normal pancreases % of ducts in sample with 60 at least 1 cell positive

50 Protein % samples 01–10 10–25 25–50 >50 40 positive in ducts 30 %change 20 CDK5 (n ¼ 9) 22 78 11 0 11 0 P35 (n ¼ 9) 67 44 22 11 33 0 10 P39 (n ¼ 8) 38 75 13 0 0 13 0 cdk5 p35 p39 cdk5,p35,orp39 Geneamplified tide-3-kinase (PI3K; LY290027), and CDK5 (roscovitine)

Figure 1. (Continued ) D, comparative genomic hybridization of PDACs inhibited p35 cleavage to p25 (Fig. 2C). Inhibition of (n ¼ 39) shows genomic amplification for CDK5, p35, and p39. H&E, calpain had a minimal decrease in p35 cleavage to p25 hematoxylin and eosin. whereas other protease inhibitors including MG132, protease inhibitor 1, and lactacystin had no effect on p25 levels (Fig. 2C). All HPNE cells with mutated K-Ras had similar and protein levels than HPNE parental cells (Fig. 3, data levels of p35 protein, indicating that the changes in p25 not shown). K-Ras–transformed cells showed significantly protein expression are likely the result of proteolytic pro- elevated levels of the p35 cleavage product, p25, (Fig. 2A) cessing. Therefore, cleavage of p35 to p25 in the context of and enhanced CDK5 kinase activation, as evidenced by mutant active K-Ras is influenced by MEK, PI3K, and CDK5 increased phosphorylation of the CDK5 substrates FAK at signaling and regulatory pathways (Fig. 2C). serine 732 (6) and PAK1 at threonine 212 (ref. 7; Fig. 2A). Inhibiting CDK5 kinase activity with R-roscovitine (>4-fold CDK5 kinase activity increases cell migration specificity for CDK5 compared with CDK2; ref. 33) for 24 S2-013 cells, a cloned pancreatic tumor cell line derived hours in serum-containing media abolished S732 FAK from a PDAC liver metastasis, displayed significantly re- phosphorylation in HPNE.kras cells and markedly reduced duced migration when treated with the CDK5 inhibitor S732 phosphorylation in HPNE.kras.E6/7.St cells (Fig. 2B). roscovitine (Fig. 3A). S2-013 cells expressing the rtTA, tet- Inhibition of CDK5 kinase activity also decreased S732 on, and the CDK5DN construct under the tetracycline- phosphorylation in S2-013, HPAF, tHPNE, and HPNE.kras responsive element had significantly reduced migration cells when introduced under growth factor stimulation compared with the S2-013 control cell lines when expres- during a 6-hour stimulation with 10% FBS after an 18- sion was induced by treatment with doxycycline (Fig. 3A). hour serum starvation (Fig. 2D). We did not observe a The effects of CDK5 kinase activity on cellular motility difference in p29 (the cleavage product of p39) expression in the context of mutant K-Ras activity were investigated at in concert with expression of mutant K-Ras (Fig. 2A); thus, the different stages of transformation in HPNE cells. we focused on the correlation between mutant K-Ras ex- Expression of mutant K-Ras in HPNE.kras and HPNE. pression and the subsequent increased p25 expression and kras.E6/7.St significantly increased migration compared CDK5 kinase activity. with HPNE.E6/7.St cells lacking mutant K-Ras (Fig. 3B), We investigated other signaling pathways that may reg- thus confirming prior reports of mutant K-Ras increasing ulate p35 cleavage to p25 in HPNE.kras cells. Inhibition cellular migration (34, 35). CDK5 inhibition with roscov- (24 hours) of mitogen-activated protein/extracellular itine in HPNE.kras and HPNE.kras.E6/7.St cells resulted in signal–regulated kinase (MEK; U0126), phosphoinosi- a similar reduction of total migration, 18% of the total area,

Table 2. Expression of CDK5, p35, or p39 in pancreatic tumors, liver and lung metastases, and during PNI

CDK5 CDK5 % p35 p35 % p39 p39 % positive negative positive positive negative positive positive negative positive

Primary pancreatic tumor 22 2 92 15 1 94 3 1 75 Liver metastases 7 3 70 5 1 83 2 3 40 Lung metastases 6 1 86 7 0 100 3 2 60 PNI 6 0 100 6 0 100 5 0 100

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A C l t Proteaseinhibitors Calpain inhibitors 7

h 1

e 6 in inh 3 HPNE HPNE.krasHPNE.kras.E6/HPNE.kras.smallHPNE.kras.E6/7.small t HPNE.E6/7.smal t ALLN CDK5 icle + protease in veh p35 .kras + LY290027

p25 HPNE + HPNE.krasHPNE.kras + vehicleHPNE.kras + roscovitineHPNE + U012HPNE.krasHPNE.k + caspasrasHPNE.kras + MG132HPNE.kras HPNE.kra + lactacystinHPNE.krass + HPN + calpeptinE.krasHPNE.kr + calpaasHPNE.kras + PD + EST p35 Phospho-T212PAK1 p25 PAK1 CDK5 Phospho-S732FAK β-Actin FAK p29 β-Actin B D s + roscovitine

E + vehicle + vehicle PAF.kra S2-013 + vehicleS2-013 + roscovitineHPAF + vehicleH tHPN tHPNE + roscovitineHPNE.krasHPNE.kras + vehicle + roscovitine p25 HPNE.kras HPNE.kras + roscovitine tHPNE + vehicletHPNE + roscovitine CDK5 p35 p25 Phospho-S732FAK

Phospho-S732FAK FAK FAK CDK5 p29 β‐Actin β-Actin

Figure 2. Mutant K-Ras increases CDK5 kinase activity. A, HPNE cells with the indicated oncogenic insults (K-Ras, E6/E7, and small t) were examined for expression of CDK5, p35, p25, p29, phospho-T212PAK1, PAK1, phospho-S732 FAK, FAK, and b-actin as a loading control. B, HPNE.kras and tHPNE (HPNE.kras.E6/7.St) cells were investigated for expression of CDK5, p25, phospho-S732 FAK, FAK, p29, and b-actin with and without inhibition of CDK5 kinase activity by treatment with roscovitine for 24 hours in serum-containing media. C, CDK5, p35, p25, and b-actin expression in HPNE.kras cells after incubation for 24 hours with vehicle, CDK5 inhibitor (roscovitine), MEK inhibitor (U0126), PI3K inhibitor (LY290027), caspase inhibitor, protease inhibitors (MG132, protease inh 1, lactacystin, and ALLN), and calpain inhibitors (calpeptin, calpain inh 3, PD, EST) compared with HPNE cells without mutant active K-Ras. D, S2-013, HPAF, tHPNE, and HPNE.kras cells were serum starved for 18 hours followed by stimulation with 10% FBS for 6 hours with roscovitine or the vehicle control: the results show levels of phospho S732 FAK, FAK, p25, p35, CDK5, and b-actin expression as determined by Western blotting.

in both cell lines and correlated to a 38% and 35% decrease by 60%, 37%, 49%, 60%, 47%, and 47%, respectively in total migration with CDK5 inhibition, respectively (Fig. 4A). (Fig. 3B). HPNE.E6/7.St cells had significantly less migration than cells containing mutant K-Ras, and in these cells, Inhibition of CDK5 kinase alters cell morphology in inhibition of CDK5 kinase with roscovitine did not signif- HPNE cells expressing mutant K-Ras icantly affect migration into the total area (3.6% reduction) We sought to investigate the role of CDK5 kinase as compared with both cell lines tested expressing mutant activity on cell morphology in the context of mutant K-Ras (18% reduction; Fig. 3B). The smaller decrease in K-Ras expression in HPNE cells. Parental HPNE cells HPNE.E6/7.St migration following roscovitine treatment is treated with roscovitine for 3 days showed no observable due to minimal CDK5 activity in these cells, which changes in cellular morphology (Fig. 4B). HPNE.kras expresses p35 and very little p25 (Fig. 2A). cells treated with roscovitine for 3 days displayed enhanced spreading upon culture in plastic, as evidenced CDK5 kinase activity increases invasion in vitro by a more flattened appearance with shorter cellular through a Matrigel-coated Boyden chamber processes compared with cells with the vehicle control Inhibition of CDK5 kinase activity with roscovitine (Fig. 4C). HPNE.kras cells transduced with the in PDAC cell lines S2-013, T3M4, FG, HPAF2, HPNE. CDK5DN construct had a cellular phenotype similar kras.E6/7.St (tHPNE), and HPNE.kras cells reduced to HPNE.kras cells treated with roscovitine (Fig. 4D). invasion through a Matrigel-coated Boyden chamber Thus, inhibition of CDK5 kinase activity increased

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with normal pancreatic ducts (Fig. 1A and Table 1; A P < 0.0005). Primary human PDACs exhibit frequent 1.5 * amplification of the genes encoding p35, p39, or CDK5. ** Each of these genes is individually amplified in about 33% of tumors, and collectively one or more of these genes is 1.0 amplified in approximately 67% of tumor samples (Fig. 1D, n ¼ 39). Results presented by Harada and colleagues (that did not address or discuss CDK5, p35, and 0.5 p39 directly) are consistent with our results, except p39 was not as frequently amplified (36). Overexpression of any of control migration

Experiment migration/ these (p35, p39, or CDK5) is predicted to result in 0.0 enhanced activation of CDK5 kinase activity. This supports the hypothesis that activation of CDK5, in part, through gene amplification and commensurate overexpression of CDK5, p35, or p39, contributes to the progression of pancreatic cancer. Our results also shed light on the types S2-013 + vehicle S2-013.pbi + dox S2-013.pbi + 0 dox of cells within the pancreas that express CDK5, which is S2-013 + roscovitine widely reported to be "ubiquitously" expressed in all

S2-013.CDK5DN28 + dox organs. Within the pancreas, expression of CDK5 is highest S2-013.CDK5DN28 + 0 dox in islets, but we find that there is little or only sporadic B expression of CDK5, p35, or p39 in normal acinar or ductal Vehicle cells in the pancreas. Roscovitine The known contribution of CDK5 to neuronal migra- 60 * * tion and patterning (5) suggested to us a link between CDK5 expression and PNI, a prominent feature of PDACs. Sustained activation of phospho-Erk1/2 was achieved through stimulation by nerve growth factor in rat pheo- 40 chromocytoma PC12 cells, which resulted in Egr1 binding and activation of the p35 promoter and a subsequent increase of p35 transcript and protein (37). Nerve growth 20 factor–induced upregulation of p35 protein levels may % scratch closure explain, in part, the high p35 levels observed in PNI (Fig.1CandTable2),andweproposethatthemicro- 0 environment around the nerves, where physiologically t s S ra normal CDK5 expression is highest (38, 39), induces or /7. PNE 6 H selects for increased CDK5 and p39 levels in PDACs NE.k t E.E N HP undergoing PNI. P H We sought to determine whether CDK5 activation con- tributed to pancreatic cancer progression in the context of known early transforming events (40). Analysis of variants Figure 3. CDK5 kinase activity increases migration. A, inhibition of CDK5 in S2-013 cells, with roscovitine or under a doxycycline (dox)-inducible of HPNE, a normal pancreatic cell line immortalized with CDK5DN construct, significantly decreased cell migration as determined hTERT, that was subsequently transduced with retroviruses with scratch assays. B, quantification of the total area of migration using expressing mutant active K-Ras (G12D; HPNE.kras) TScratch, a program that analyzes area occupied by cells, for each HPNE showed higher p35 or p39 mRNA and protein levels in permutation and treatment to assess the differences in migration response to activated K-Ras whereas CDK5 levels remained conveyed through mutant K-Ras and CDK5 kinase activity. Inhibition of CDK5 kinase activity, significantly reduced migration in HPNE.kras and constant in HPNE.kras cells (Fig. 3, data not shown). tHPNE (HPNE.kras.E6/7.St) cells while showing minimal differences in Differences in CDK5 expression between normal pancreas cells lacking mutant K-Ras, HPNE.E6/7.St. (column 4 or 6 compared (Fig. 1 and Table 1) and immortalized but nontransformed P < P < with 1). *, 0.005; **, 0.02. HPNE cells (Fig. 2A) could be attributed to the expression of nestin, which HPNE cells express, as nestin expression has been correlated to CDK5 expression in differentiating spreading of HPNE cells but only in the context of mutant myoblasts and in regenerating muscle tissue (41). Alterna- K-Ras. tively, CDK5 may be expressed in pancreatic ductal progenitor cells, which would also explain CDK5 expression in Discussion HPNE cells, which are derived from progenitors of ductal cells (20). We report for the first time that CDK5 or its activators are There were increased p25 levels in HPNE.kras cells, a overexpressed in PDACs (Fig. 1B–D and Table 2) compared stable cleavage product of p35 that is highly activating for

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A 600 * Vehicle Roscovitine

** *** 400

* 200 ** # cells invading/filter 0

FG T3M4 S2-013 tHPNE HPAF2 HPNE.kras Figure 4. CDK5 kinase activity B increases in vitro invasion and HPNEparentalcells enhances cell spreading in the context of mutant K-Ras. A, invasion in vitro through a Matrigel-coated Boyden chamber by S2-013, tHPNE, FG, HPAF2, T3M4, and HPNE.kras cells following inhibition of CDK5 kinase activity with roscovitine compared with the vehicle control. B, HPNE cells incubated with roscovitine for 72 hours did not result in altered cellular Vehicle Roscovitine(CDK5inhibitor) morphology. C, HPNE.kras cells incubated with roscovitine for 72 hours resulted in enhanced C spreading as evidenced by a more flattened appearance with shorter HPNE.kras cells cellular processes. D, HPNE.kras cells transduced with a CDK5DN construct, HPNE.kras.CDK5DN, also displayed enhanced spreading compared with the control cell line, HPNE.kras. Control. *, P < 0.007; **, P < 0.05; ***, P < 0.02.

Vehicle Roscovitine

HPNE.kras.control HPNE.kras.CDK5DN

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CDK5 (Fig. 2A). p25 lacks the N-terminal myristoylation systems that were evaluating morphologic changes associ- sequence of p35, which causes relocalization from the cell ated with senescence, which were not investigated in our membrane to cytoplasm and potentiates its capacity to experimental system. bind and activate CDK5 (13). p25 expression is likely Along with increased p25 expression and CDK5 kinase responsible for increased CDK5 kinase activity in HPNE. activity, mutant K-Ras increased migration of HPNE.kras kras cells compared with HPNE parental cells (Fig. 2A). and HPNE.kras.E6/7.St cells as compared with HPNE.E6/7. Similar to SH-SY5Y neuroblastoma cells (42) and in con- St cells lacking mutant K-Ras (Fig. 3B). The increase of trast to normal neuronal cells (43), we found that CDK5 migration in the HPNE.kras.E6/7.St cells relative to HPNE. kinase activity in the context of mutant K-Ras (HPNE.kras kras cells suggested that the transforming insults intro- cells) increased p25 levels (Fig. 2B) whereas inhibition duced by E6/7 and small t antigen had little effect on of CDK5 kinase had no effect on p25 levels in the context migration (Fig. 3B). Inhibition of CDK5 kinase activity of the additional oncogenic effects of E6/7 and small significantly reduced migration of HPNE cells expressing t antigen (Fig. 2B). This supports the hypothesis that mutant K-Ras (HPNE.kras and HPNE.kras.E6/7.St cells) CDK5 hyperactivation contributes to tumor progression whereas it had little effect on cells lacking mutant K-Ras especially in the context of early mutant K-Ras expression. (HPNE.E6/7.St cells; Fig. 3B). Inhibiting CDK5 kinase We further show that signaling through MEK and PI3K activity with a CDK5 dominant-negative construct or ros- increased p25 expression (Fig. 2C). This leads us to pro- covitine also significantly decreased migration of S2-013 pose that signaling from activated mutant K-Ras (G12D) cells (Fig. 3A). through MEK and PI3K enhance p25 expression, which CDK5 inhibition with roscovitine reduced invasion by in turn increases CDK5 kinase activity (Fig. 3 and Supple- 37% to 60% in S2-013, tHPNE, FG, HPAF2, and T3M4 mentary Fig. S1). PDAC cell lines (Fig. 4A). These results are consistent with Our results complement and extend recently published previous findings in neuroblastoma, prostate cancer, and findings by Feldman and colleagues (44), which showed other pancreatic cancer cell lines, where inhibition of that inhibition of CDK5 in pancreatic cancer cell lines by CDK5 kinase activity reduced invasion in vitro (11, 12). the dominant-negative construct decreased signaling In summary, we show that CDK5 or its activators p35 through the RalA and RalB pathways that are downstream and p39 are overexpressed in more than 90% PDACs of activated Ras, and that commensurate inhibition of MEK (Table 2) compared with normal pancreases (Table 1). and PI3K pathways reinforced these effects. The Ral path- CDK5, p35, and p39 expression in PDACs can be attrib- ways were shown to contribute to anchorage-independent uted, in part, to genomic amplification, as CDK5, p35, or growth and tumorigenicity of these tumor cells lines, and p39 was amplified in 67% (n ¼ 39) of tumors analyzed their results suggest that these effects are downstream of (Fig. 1D). Furthermore, CDK5 is hyperactivated down- CDK5. Our results are consistent with these findings and stream of mutant K-Ras signaling, resulting in increased extend them by showing that K-Ras signaling enhances the phosphorylation of CDK5 substrates, increased p35 activity of CDK5 by increasing the steady-state levels of p25 mRNA and protein expression, increased p35 cleavage to upstream of the Ral pathways. p25 (which is dependent on MEK, PI3K, calpain, and The influence of mutant K-Ras and CDK5 kinase activity CDK5 signaling), decreased cell spreading, and increased on migration was investigated in HPNE cells bearing cell migration (Figs. 3 and 5 and Supplementary Fig. S1, selected mutations and transforming insults. CDK5 is nec- data not shown). We confirm prior reports that CDK5 essary for proper neuronal cortical layering, a process that inhibition can decrease migration and invasion in vitro requires CDK5-mediated phosphorylation of FAK at S732 (Fig. 4A). and probably PAK1 at T212 (6, 7). We found that increased CDK5 represents a novel and unexplored therapeutic CDK5 activity and p25 expression in HPNE cells expressing target for both early- and late-stage PDACs and should be mutant K-Ras increased phosphorylation of S732 FAK and investigated in other solid tumors that harbor K-Ras muta- T212 PAK1 (Fig. 2A). We propose that PDACs have tions including those of lung and colon. R-roscovitine appropriated these phosphorylation events to alter cell (Selicilib) has been investigated in phase I clinical trials, morphology and increase cellular migration. Inhibition with minimal toxicity at lower doses, supporting the fea- of CDK5 with roscovitine or the CDK5 dominant-negative sibility of using a CDK5 inhibitor in the clinic (47). More construct enhanced spreading in HPNE.kras cells, as evi- potent CDK5 kinase inhibitors have been developed as denced by a more flattened appearance with shorter cel- potential therapies for Alzheimer’s disease, in an attempt lular processes than in the respective controls (Fig. 4C and to downregulate hyperactive CDK5 kinase activity associ- D) whereas there was no change in cellular morphology in ated with p25 expression that is observed in that disease parental HPNE cells with CDK5 inhibition (Fig. 4B). Our (15). We are currently investigating the use of such inhi- results support the proposal that CDK5 has a role in bitors in xenograft and other animal models of pancreatic modulating cell morphology and cytoskeletal reorganiza- cancer. It is also possible that CDK5 inhibitors may reduce tion that has been presented by Mao and Hinds (45) and pain in pancreatic cancer patients with PNI. CDK5 has Alexander and colleagues (46), even though the precise previously been shown to reduce thermal afferent nocicep- morphologic findings are not entirely congruent because tive pain signaling (48). Type II diabetes mellitus (T2DM) is they were obtained in different cell types and experimental observed in the majority of PDACs (49), and increased

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Eggers et al.

CDK5 kinase activity is known to increase glucotoxicity Grant Support and independently decrease insulin synthesis and secretion (50, 51). Thus, inhibiting CDK5 kinase activity with This work was supported by grants from the NIH (R01CA57362, U01CA111294, and P50 CA127297), National Cancer Institute (NCI) training molecular inhibitors may also improve the associated grant (CA09476), and student assistantship awards from the University of T2DM in PDAC. Nebraska and NCI Cancer Center Grant P30CA36727. 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. Disclosure of Potential Conflicts of Interest Received September 7, 2010; revised July 18, 2011; accepted July 24, No potential conflicts of interest were disclosed. 2011; published OnlineFirst August 8, 2011.

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Cyclin-Dependent Kinase 5 Is Amplified and Overexpressed in Pancreatic Cancer and Activated by Mutant K-Ras

John P. Eggers, Paul M. Grandgenett, Eric C. Collisson, et al.

Clin Cancer Res Published OnlineFirst August 8, 2011.

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