Preliminary Studies Of Effect And Signal Pathway Of P21-activated Kinase1(PAK1) Of Proliferation In Colorectal Carcinoma

P21-activated protein kinasel (PAK1), a main downstream effectors of small Rho GTPases, Rac1 and Cdc42, plays an important role in the regulation of cell morphogenesis, motility, mitosis, and angiogenesis. Despite its importance, the molecular mechanisms of PAK1 contributed to colorectal carcinogenesis remain unclear. In the present study, we showed that PAK1 knockdown decreased proliferation and delayed the G1/S cell-cycle transition, increased apoptosis in vivo and in vitro. In addition, PAK1 knock-down downregulated c-Jun amino terminal kinases (JNK) activity and the levels of CyclinDl, CDK4/6. Inhibition of the JNK activity by chemical inhibitor (SP600125) significantly reduced the effects of PAK1 on CRC proliferation. In conclusion, our results demonstrate that the expression of PAK1 is up-regulated in CRCs, which played an important role in the metastasis of CRC. The mechanism by which PAK1 induced cancer growth might involve activation of JNK. Targeting PAK1 might promise to be a novel treatment strategy for colorectal carcinoma.Materials and MethodsReagentsAnti-JNK, anti-phospho-JNK antibodies, U0126 (Cell Signaling Technology), Rabbit anti-PAK1 and anti-GAPDH,ant-CDK4,anti-CDK6 antibodies (Santa Cruz,USA), Rabbit anti-PTEN antibodies (Mlipore Corporation).SB203580 and SP600125,5-fluorouracil (Sigma Aldrich) were obtained from the indicated sources. ContructsDouble-strand oligonucleotides corresponding to the target sequences were cloned into pGPU6/GFP/Neo plasmid (Shanghai GenePharma Co.China). In this study, the target sequences for human PAK1 cDNA are AAGATTGGACAAGGTGCTTCA. Mammalian expression plasmids for PAK1 and control empty plasmids were generous gifts from Pr Hongquan Zhang (Peking University Health Science Center, Beijing)Tissue culture and stable clone selectionThe SW480, LoVo cells from the American Type Culture Collection (Manassas, VA) were cultured in the RPMI 1640 media supplemented with 10% fetal bovine serum and 100 units each of penicillin and streptomycin. Cells were transfected with 4μg DNA constructsusing LipofectAMINE 2000 reagent (Invitrogen, Carlsbad, CA) as per protocols recommended by the manufacturer. For constructing the stable transfectant, pGPU6/GFP/Neo and pGPU6/GFP/Neo shPakl were separately transfected into LoVo. Forty-eight hours after transfection, G418 at 1.0 mg/mL (Merck) was added for the selection of stable clones.Cell proliferation assaysStable LoVo clones were obtained by transfection with the PAK1 shRNA expression vector and empty vector, followed by selection in 1 mg/mL G418 media. To measure the effects of knockdown on cell proliferation, PAK1 shRNA-transfected cells were seeded at concentrations of 2×103 per well in 96-well plates. After 0,1,2,3d, cell proliferation was measured by cell counting kit-8 (CCK-8) (Beyotime Institute of biotechnology) according to the manufacturer’s instructions.SW480 cells were transfected in 6-well plate.24 hours late, the transfected cells were trypsinized and plated in 96-well plates with 1.0×103 cells in 100μl of the medium and allowed to attach for 24 h before initiating treatment. Either 20 mM SP600125 or DMSO alone was added to fresh media then used to culture cells. After 48h, CCK-8 (10μl) was added to each well at different time points and incubated at 37℃for 1.5 h and absorbance at 450 nm was measured after 1 h of incubation, following the manufacturer’s instructions. Colony Forming Efficiency in Soft AgarTo examine the anchorage-independent growth, One milliliter of 0.5% agarose in high-fertility culture medium was allowed to solidify in 35-mm culture dishes. These were overlaid with 1 ml of 0.33% agarose containing 1×103 cells/ml in middle fertility culture medium. One milliliter of low-fertility culture medium was added to each well the next day. Cells from different stably transfected cell strains were incubated for 2 weeks at 37℃.The cell colonies (more than 50 cells) were counted under the microscope. The average of three independent experiments was shown.Serum-Starvation and Analysis of Apoptosis1×106 vector-shPAK1 or shPAK1 LoVo cells were grown to confluence in 6-well plates in 10% FBS for 24h, when media was replaced to 0.2% FBS for an additional 48h.Apoptosis was measured simultaneously by staining cells with Annexin-V-PE (BioVision) and 7AAD (BD Pharmingen) according to the manufacturer’s protocol. After culture at different time points, samples were harvested (including detached cells), suspended in PBS and fixed in 70% ethanol, and their DNA content was evaluated after propidium iodide staining. Fluorescence-activated cell sorting analysis was carried out using a FACScan flow cytometer (Beckton Dickinson, San Diego, CA) and CellQuest software. Cell debris and fixation artifacts were gated out.Western blot analysisTotal cell lysates were extracted by PBSTDS (100 ml 10 3 PBS,10 ml 100% Triton X 100,5 g nadeoxycholate,1 g SDS,1 nM EDTA, up to 1,000 ml/day H2O) on ice and were centrifuged at 20,000 rpm for 30 min at 4℃. Concentration of the protein was measured. Protein (50 ug) was mixed with loading buffer with final concentration of 1×. After boiled for 8 min, proteins were separated by running in SDS-polyacrylamide gel. Proteins were then transferred to a nitrocellulose membrane and then blocked for 1 h at room temperature in TBS-T [50 mmol/L Tris-HCl (pH 7.5),150 mmol/L NaCl,0.1% Tween 20 buffer containing 5% nonfat milk. Membranes were then incubated overnight at 4℃or 1 h at room temperature with the respective primary antibodies:(phospho-JNK(1:500), JNK(1:500), cyclinD1(1:500), CDK4(1:500),CDK6(1:500),GAPDH(1:500),PTEN (1:500).Anti-mouse IgG-HRP, anti-rabbit IgG-HRP antibody (Santa Cruz) and the blots were developed with a Pierce ECL Western Blotting Substrate (PIERCE, Rockford, USA)) don’t need to describe in such details.Drug Treatment, Irradiation and Adenovirus InfectionCells were seeded in 6-well plates at 20-30% density 48 h before treatment. For in vitro studies, unless noted, the final concentrations used for MAPK inhibitors were 20μM.Tumor cell xenotransplantationLoVo cells stably expressing PAK1 shRNA and empty vector controls were s.c. injected into the lower flanks of NCr nu/nu mice (Taconic Farms) at a dose of 5×106 viable tumor cells. Mouse tumor growth was measured with digital caliper and calculated by using the formula of a rotational ellipsoid:V=π/6×A×B2, where V is volume, A is the longest tumor axis, and B is the perpendicular shorter tumor axis. All animal experiments were done according to the institutional guidelines of Lifespan Animal Welfare Committee of Nanfang Hospital.ResultsEffect of PAK1 expression on cell proliferation, apoptosisTo assess the role of PAK1 in CRC cells proliferation, we knocked down PAK1 expression using short hairpin RNAs, which effectively downregulated PAK1 expression in LoVo cells.Growth of LoVo cells was obviously decreased after transfection of PAK1-shRNA compared with transfected empty vector.There was statistical difference between PAKl-shRNA and empty vector after 48h (P<0.05).shPAK1 transfectants were 3-fold more apoptotic when compared with vector (P<0.05). Next, we analyzed the effect of PAK1 knockdown on the tumorigenic potential of LoVo cells. LoVo shPAKl presented significantly inhibited anchorage-independent growth in soft-agar compared with empty vector.PAK1 knockdown induced a delay in the G1 Phase of the cell cycle and decreased the expression of cyclinDl, CDK4/6To investigate the effects of PAK1 on the cell cycle, we analyzed the number and distribution of cells in the phases of the cell cycle by flow cytometry.PAKl knockdown significantly increased the number of cells at the G1 transition.PAKl knockdown resulted in almost 12% increase in the number of cells in G1 compared with empty vector-transfected cells(P<0.05).We next investigated the effect of PAK1 on crucial G1 checkpoint proteins. For the cell lysate LoVo PAK1-shRNA and vector controls were analyzed for the level of cyclinDl, CDK4/6. As shown in Fig.1G, knockdown of PAK1 in LoVo cells reduced the expression of cyclinDl, CDK4/6.PAK1 effects on cell proliferation and G1 transition required activity of JNKRecently, Ching et al reports that direct phosphorylation of JNK at Ser178 by PAK1 plays an important role in the regulation of in the metastasis of HCC. In light of this, we next tested the hypothesis that PAK1 might promote colon tumorigenesis by regulating JNK in CRC. Knockdown of PAK1 effectively decreased the expression of phosphorylated JNK. To investigate whether the effects of PAK1 on cell proliferation and transition from the G1 phase to mitosis necessitated JNK activity, we employed SP600125, an inhibitor of JNK activity by a pharmacologic JNK inhibitor, significantly reduced proliferation, the number of cells in G1 cells and the level of CDK4/6,CyclinDl in SW480 cells with PAK1 upregulation. U0126 or SB20358 had no effects on proliferation transition.PAK1 knockdown decreased proliferation and tumorigenicity in vivoWe generated s.c. xenografts in NCr nu/nu mice by using LoVo shPAK1 cells compared with empty vector controls. Once xenografts reached an average volume of at least 100 mm3, animals were divided into 2 groups:vector, shPAK1. Xenograft tumors were monitored for 30 days. Tumor growth was significantly suppressed in PAK1 shRNA group compared to that in empty vector group (P＜0.05). These data suggest that PAK1-knockdown might suppress tumor growth in vivo.DiscussionIt has been reported that the expression of PAK1 expression was increased significantly with malignant progression of human colorectal carcinoma, suggesting that PAK1 might play an important role in the carcinogenesis and progression of colorectal cancer. However, direct demonstration of PAK1’s function in colon cancer tumorigenesis is currently lacking. To elucidate the role that PAK1 may have in CRC progression, we used small interfering RNA technique to knock down PAK1 in colorectal cancer cell lines. A major novel finding presented in our study was that PAK1 downregulation in CRC cancer cells greatly decreased their proliferative capacity. Targeted downregulation of PAK1 in LoVo cells resulted in significant reduction of mammary tumor size (P<0.05). These observations suggested that colorectal cancer cells can become dependent on PAK1 for survival.Next, we found that PAK1 knockdown prolonged the doubling time of CRC cells and caused an arrest at the G1 transition of the cell cycle. It has been reported that entry into G1 from a quiescent state (GO) is associated with the expression and activation of Cyclin D:Cdk4/Cdk6, which phosphorylate and inactivate the retinoblastoma protein (Rb) during the first half of G1. This hypophosphorylated form of Rb is now capable of binding to E2F family members, which is necessary for G1/S phase transition and cell cycle progression. As expected, Pakl could regulate the expression of Cyclin D:Cdk4/Cdk6. JNK has been shown to modulate the expression of cyclinDl/Cdk4 in many cells.The JNK/c-Jun pathway is a critical component of the proliferative response and induces Go to G1 cell cycle progression in many cell types. The JNK pathway has also been shown to be closely linked to apoptosis. Activation of the JNK pathway leads to regulation of cellular survival or cellular apoptosis, and the decision appears to be cell- type specific as well as stimulating signal specific. Recently, it has been shown that c-Jun contributes to the promotion of cellular survival by regulating the expression of PTEN. Several studies report that Pakl induces cancer carcinogenesis may involve activation of JNK. In this study, we showed that the level of phosphorylated JNK was decreased in LoVo shPAK1 cells. We hypothesized PAK1 induces transcription of cyclinDl, CDK4/6 depend of JNK pathway activity. Indeed, we found that the observed effects of PAK1 downregulation on LoVo cells proliferation and G1 transition require JNK, as inhibition of c-Jun amino terminal kinases (JNK) by SP600125, the first selective JNK inhibitor, abolished PAK1-induced cell proliferation in CRC cells. U0126 or SB20358 had no effects on cell proliferation regulated by PAK1 upregulation. PAK downregulation has been shown to downregulate several important pro-apoptotic pathways. In this study, we also showed that the apoptosis rate was increased in LoVo shPAK1.In conclusion, we provided the convincing evidences that knockdown of PAK1 in colorectal cancer cells attenuated their tumorigenic properties. Our study implied that PAK1 knockdown decreased tumor growth in vivo and in vitro by arresting the transition from G1 phase to mitosis and promoting apoptosis. We provided the first functional link between PAK1 and JNK and showed that PAK1 knockdown leaded to JNK downregulation and subsequently decreased colon cancer proliferation and progression through G1. We thus provided a new lead toward future development of specific strategies to downregulate PAK1 levels and function, and for possible prevention of colorectal cancer.