Increased P21-activated Kinase-1 Expression Is Associated With Progression And Metastatic Behavior In Human Colorectal Carcinoma

Background and objectivesColorectal cancer(CRC) is one of the major malignancies in the world. The prognosis of CRCs is poor, due to frequent metastasis and tumor recurrence. Worldwide almost one million new cases occur annually, amounting to 492000 related deaths^[1-3]. With the many changes having taken place in people's diet and lifestyle, CRCs has become the third most common type of digestive tumor in China, and the number of new cases arising each year is still increasing. The overall incidence is identical in men and women, with the risk beginning at age 40 and increasing with age. Thus colorectal cancer ranks as the frequent cause of cancer deaths among China. Despite the rate of improvements in surgery, radiotherapy and chemotherapy, unfortunately, the prognosis of CRCs has not been gained progress over the past decades, with an overall five-year survival rate of around 40%- 50%^[4]. Therefore, novels diagnose and treatments need to be developing in order to enrich the therapeutic armamentarium. In recent years, molecular biology has applied to the study of colonic carcinoma, both in the human and in the experimental animal. The data obtained have enriched our understanding of colonic carcinogenesis and are of potential interest for CRCs diagnosis and prevention.PAK1 (P21-activated kinase-1), a member of a family of serine/threonine protein kinase, was the first PAK to be cloned^[5] . Moreover, PAK1, which plays an essential role in embryonic development and tissue growth, and is also necessary for the spread and growth of tumor cells, such as breast and ovarian cancers^[6]. PAK1 is a direct target of the small GTPases Cdc42 and Racl, and binding of GTPases to PAK1 stimulates its kinase activity via autophosphorylation[7]. PAK1 contains an N-terminal regulatory domain and a C-terminal kinase domain, its N-terminal regulatory domain contains GTPase binding domain to mediate the binding of PAK1 to Rac/Cdc42^[8]. Among normal tissues, PAK1 is highly expressed in the brain, muscle, and spleen. Accumulating evidence indicates that PAK1 is important for a variety of cellular functions including cell morphogenesis, motility, survival, mitosis, cell cycle and angiogenesis. PAK1 are involved in the regulation of cellular function via phosphorylating a number of downstream target protein. Many evidences showed that Pakl activation occurs during the process of tumorigenesis, and PAK1 is likely to provide insight into the role of PAKs in human cancers. Despite the recent reports of the involvement of PAK1 in signaling cascades in human cancer cells and the fact of PAK1 is downstream of the small GTPases, Cdc42 and Rac1, the relationship of PAK1 to malignant progression of CRCs and the role of the PAK1 pathway in the biology of human colorectal cancer cells remain unknown. In this study, detect PAK1 expression in colon cancer patients and colorectal cancer cell lines and its molecular mechanism of action in transfected colorectal cancer cell to provide basis for further underscoring the link between PAK1 expression and CRCs progression..Material and methodsThe potential role of PAK1 protein expression in colorectal carcinomasImmunohistochemistry of normal, benign colon polypus, colon adenoma, primary, and metastatic human tumor specimens was to detect the relationship of PAK1 expression with the pathological features. The study-included patients with colorectal carcinoma, according to the Dukes classification of malignant tumors, were divided as Dukes A, Dukes B, Dukes C, and Dukes D. Formalin-fixed paraffin sections were stained for PAK1 (1: 200 dilutions) using the Streptavidin-preoxidase (SP) technique. Antigen retrieval was achieved by microwave treatment with citrate buffer at pH 6.0 at 95℃for10 min. The immunohistochemical staining was scored in the following grades according to the percentage of positive cells:0,＜5% positive; 1, 5% to 25% positive; 2, 26% to 50% positive; and 3,＞51% positive. Morphological and immunohistochemical results were to correlate with clinicopathologic parameters.The influences on the phenotypes of SW480 cell mediated by changing PAK1 expression1. Cell Culture SW480(ATCC) cells were maintained in RPMI-1640(Gibco) supplemented with 10% fetal bovine serum (FBS) (Hycone), penicillin (100units/ml), and streptomycin(100mg/ml)). Cells were passaged using 0.25%Trypsin-EDTA and maintained in culture for 48h before performing experiments.2. Construction recombinant eukaryotic PAK1 expression vector Primers, targeted to PAK1 (GenBank: NM₀02576) code sequence(CDS) and containing EcoRâ… and BamHâ… sites at the ends, were designed. The polymerase chain reaction(PCR) products of CDS and pEGFP-C1 were both digested with EcoRâ… and BamHâ… and subsequently ligated for 4h at 16℃using T4 ligase. The recombinant plasmids was digested with EcoRâ… and BamHâ… followed by electrophoresis with 1% agarose and sequence analysis to identify.3. Construction of recombinant PAKI shRNA expression vector Designing three pairs of small interfering RNAs(siRNAs) of PAK1 mRNA(GenBank: NM₀02576), and six corresponding single-strand short hairpin RNAs(shRNAs), containing BamHâ… and Hindâ…¢sites and 9nt hairpin structure, were synthesized and annealed. The annealed products and the linear pRNAT6.1/Neo plasmid, digested with BamHâ… and Hindâ…¢, were ligated for 4h at 16℃using T4 ligase. The recombinant plasmids were digested with BamHâ… and Hindâ…¢followed by electrophoresis with agarose and sequence analysis to identify.4. Transfection and selection of stably transfected cell clones The recombinant plasmids and control plasmids were transfected into SW480 cells using lipofectamine2000^TM reagent (Invitrogen) according to the manufacturer's protocol. Forty-eight hours after the addition of DNA, the transfected cells were selected in growth medium containing 0.8 mg/ml Geneticin (G418; Life Technologies, Inc.). Colonies resistant to G418 were isolated. After 3-4 wk of culture, visible colonies were picked up and expanded. The stably transfected clone cells were observed to show green fluorescence under fluorescent microscope and the clones without expression of the transfected gene did not show green fluorescence5. PAK1 exprssion detecting The expression changes of PAK1 were detected using reverse transcription polymerase chain reaction (RT-PCR), western-blotting, and immunofluorescence staining at mRNA and protein levels.6. Cell proliferation and apoptosis To observe the changes of cell growth after regulating expression of PAK1, cell proliferation was analyzed with MTT assay and 5-Fu induced cell apoptosis was detected by DNA fragment assays.7. Cell-matrix adhesion analysis Cell-matrix adhesion assays were employed, as previously described^[9, 10]8. Cell migration assays Migration of SW480, PAK1-overexpressng SW480(SW480^PAK1) and PAK1-lowexpressing SW480(SW480^shRNA1) cells was studied using 6.5mm Transwell chambers with 8μl pores (Costar) as previously described^[11].9. Cell culture wound healing assay^[12]. Wounds were created in confluent cells using a pipette tip. The cells were then washed with medium to remove any free-floating cells and debris, and culture plates were incubated at 37℃. Wound healing was observed at 0, 24, 48, and 72 hours within the scrape line, and representa- tive scrape lines for each cell line were photographed.10. Cell invasion assays Cell invasion assays were performed as described for the cell migration assays, except that the Transwell filters were additionally coated on the upper side with 30 mg of Matrigel.11 Anchorage-independent growth Assays Anchorage-independent growth in soft agar was performed as described previously^[13]. In brief, the cells (10⁵) were plated on 60-mm dishes. After 21 days, colonies were scored after staining the dishes with 0.5 mg/ml 3-(4, 5-dirnethylthiazol-2-y1)-2, 5-diphenyltetrazolium bromide (Sigma) overnight at 37℃.12. Gelatin zymography^[14] Cells were plated at a density of 2×10⁵ in 24-well plates. After 16 hours, cells washed with 0.01M PBS and incubated in 500μl of serum-free medium for 24 hours, were performed as described previously.13. Tumorigenesis in nude mice Cells were suspended at the density of 2×10⁷ cells in 200μl of RPMI-1640, and injected subcutaneously into the flank region of athymic nude mice. Twelve mice were to distribute to PAK1-shRNA group, vector control group, 6 per group at random. The animals were to inspecte at regular intervals for the appearance of visible tumors to measure the time of first appearance. All animals were observed for up to 30 days following the injection, the mice were sacrificed and the tumors were carefully removed by blunt dissection. The tumors were to weigh and their average growth rates were measured.Statistical analysisFor immunohistochemistry results, Kruskal-Wallis Test was used, and the results of RT-PCR, Western blotting and MTT assays were expressed as mean±SD of at least three separate experiments, were analyzed by one-way analysis of variance (ANOVA). P values of=0.05 was considered statistically significant.Results1. PAK1 expression increases during human eolorectal cancer progression and metastasis. Immunohistochemistry was performed to examine PAK1 expression levels in paraffin-embeded tissue from normal colon mucosa, benign polypi and adenomas to primary colon cancers and metastasis colon cancers, which is a typical progression pathway during colon carcinogenensis. Representative shows that PAK1 expression is negative in benign polypi, begins to increase in adenoma but is still weak compared to the carcinoma, and over-expressed in primary colon adenocarcinoma but is absent or barely detectable in paired normal mucosa beside the cancer (P＜0.001). Furthermore, PAK1 expression is extremely much higher in lymph node metastasis and liver metastasis in contrast to primary colon carcinoma. However, the expression of PAK1 was not correlated with the histological differentiation(P.＞0.05) and Dukes' classification (P＞0.05).2. The effects of changing PAK1 expression on cellular biological activity of SW480 cell.(1) Constructed human PAK1 eukaryotic expression vector and human PAK1 hairpin siRNA eukaryotic expression vectors successfully.The DNA oligonucleotides encoding PAK1 mRNA and short haipin siRNA against PAK1 were synthesized, and cloned to construct recombinant plasmid respectively, which were identified by restriction enzyme digestion analysis and DNA sequencing.(2) Geneticin-resistant cell lines were screened after the recombinant plasmids were transfected into SW480 cells.Following to the instruction of lipofectamine^TM2000 the transfection were performed, plasmids served as negative control, SW480 as blank control. The RT-PCR results showed that mRNA level of PAK1 in screened transfected cell lines SW480^shnRNA1was lowest, mRNA of PAK1 in transfected cell line SW480^shRNA2-3was significantly decreased also, its were confirmed in protein level by Western Blotting.Six kinds of selected Geneticin-resistant colonies were respectively named as: SW480/pEGFP/C1-PAK1 (to abbreviate SW480^PAK1), SW480/pEGFP/C1(to abbreviate SW480^Vector), SW480/pRNAT-U6.1/Neo-PAK1^shRNA1-3(to abbreviate non specific 8W480 ^shRNA1-3), and SW480/pRNAT-U6.1/Neo-PAK1^non-specific construct (to abbreviate SW480^shRNA-N) of PAK1. This laid sound basis for the following research of PAK1.(3)After 24h incubation, there were notable differences compared the growth velocity of SW480 cells in the group of 5W480^vector, 5W480^PAK1, SW480^shRNA-Nand 8W480^shRNA1 (F=11.006, P=O.O00＜0.001). The growth velocity of SW480^shRNA1 was reduced obviously to contrast with SW480å’ŒSW480^shRNA-N(0.400±0.004 vs 0.532±0.0696, P= 0.016＜0.05; 0.400±0.004 vs0.533±0.038, P= 0.015＜0.05)., and 5W480^PAK1 was increased significantly to compare with SW480 and SW480^vector(0.719±0.125 vs 0.532±0.070, P=0.002＜0.01; 0.719±0.125 vs0.530±0.039, P=0.001＜0.01).(4) Compared with SW480å’ŒSW480^shRNA-N, treatment with 5-Fu(15μg/ml) for 12h resulted in a significant increase of apoptosis in SW480^shRNA1 cells, no significant apoptosis was observed in SW480^PAK1 cells induced with 5-Fu (30μg/ml) compared with with SW480 and 8W480^vector cells.(5) Alloplasm adhesiveness (cell-matrix adhesion) tests showed that there was a significant difference between the adhesive cell numbers of these three cell lines. The adhesive cell number of SW480^shRNA1 was significantly less than that of SW480 (P＜0.05) and SW480^shRNA-N (P＜0.001), while the last two groups had no significant difference, suggesting that compared with SW480 and SW480^shRNA-N, the cell-matrix adhesiveness of 5W480^shRNA1 was decreased notably. As well as the adhesive cell number of SW480^PAK1 was significantly more than that of SW480 (P＜0.001) and SW480^vector (P＜0.001),(6) A monolayer wound-healing assay revealed almost no migration in SW480^shRNA cells compared with SW480^control cells, but is not of SW480^PAK1, and Cell migration assays showed homoioplastic result too.(7) Using a Boyden chamber invasion assay, we observed a significant decrease in the invasive capacity of SW480^shRNA cells, but increace of SW480^PAK1.(8)A gelatin zymogram for MMP activation demonstrated a decrease in MMP-2 activity in SW480^shRNA1 compared with SW480 ^shRNA-N cells. However, a significant decrease of MMP- 9 activity was not observed.(9)Tumorigenicity assay showed the tumor weight of SW480^shRNA group(0.1075±0.06571)g were less than control group (0.9517±0.72323) (P＜0.001). No lymph node and distant organ metastasis were found in all tumors.ConclusionsTaken to gether, the results of immunohistochemistry show that PAK1 expression is increased with progression through the adenoma to carcinoma sequence, with the most dramatic increases in invasive and metastatic CRCs, suggesting PAK1 might play an important role in colorectal tumorigenesis. With molecular biology technology, cell biology technology, and RNA interference technique, the role of PAK1 in cell-matrix adhesion, cell migration, cell proliferation and cell apoptosis was studied in the model colorectal cancer SW480 cells. Experimental results indicate that PAK1 plays an important role in regulation of cell-matrix adhesion, cell migration and cell apoptosis in colorectal cancer cells and further has an effect on tumor invasion and metastasis. Increased expression of PAK1 mainly helps colorectal cancer metastasis in the distance, enhances the ability of anti-apoptosis in colorectal cancer SW480 cells and has significant effects to promote proliferation of SW480 cells. It not only provide the basis for the further study in the mechanisms of colorectal cancer invasion and metastasis but also suggest that signal pathway mediated by PAK1- targeted for therpeutic intervention in colorectal cancer. These data implicate PAK1 as an exciting target for therapy of colorectal carcinoma.