Study On The Role Of P21-activated Kinase-4 In Pathogenesy And Diagnosis Of Breast Cancer

Background and objectivesBreast cancer (BC) is one of the major malignancies in the world. The prognosis of BCs is poor, due to frequent metastasis and tumor recurrence. Worldwide almost one million new cases occur annually, amounting to 500000 related deaths.With the many changes having taken place in people's diet and lifestyle, BCs has become the third most common type of female tumor in China, and the number of new cases arising each year is still increasing. Despite the rate of improvements in surgery, radiotherapy and chemotherapy, unfortunately, the prognosis of BCs has not been gained progress over the past decades, with an overall five-year survival rate of around 40%-50%. 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 breastic carcinoma, both in the human and in the experimental animal. The data obtained have enriched our understanding of breastic carcinogenesis and are of potential interest for BCs diagnosis and prevention.PAK4 (P21-activated kinase-4), a member of a family of serine/threonine protein kinase, was the first PAK to be cloned. Moreover, PAK4, 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.PAK4 is a direct target of the small GTPases Cdc42 and Racl, and binding of GTPases to PAK4 stimulates its kinase activity via autophosphorylation. PAK4 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. Among normal tissues, PAK4is highly expressed in the brain, muscle, and spleen. Accumulating evidence indicates that PAK4 is important for a variety of cellular functions including cell morphogenesis, motility, survival, mitosis, cell cycle and angiogenesis. PAK4 are involved in the regulation of cellular function via phosphorylating a number of downstream target protein. Many evidences showed that PAK4 activation occurs during the process of tumorigenesis, and PAK4 is likely to provide insight into the role of PAKs in human cancers. Despite the recent reports of the involvement of PAK4 in signaling cascades in human cancer cells and the fact of PAK4 is downstream of the small GTPases, Cdc42 and Racl, the relationship of PAK4 to malignant progression of BCs and the role of the PAK4 pathway in the biology of human breast cancer cells remain unknown. In this study, detect PAK4 expression in breast cancer patients and breast cancer cell lines and its molecular mechanism of action in transfected breast cancer cell to provide basis for further underscoring the link between PAK4 expression and BCs progression.Material and methodsThe potential role of PAK4 protein expression in breast cancerImmunohistochemistry of normal, benign breast polypus, breast adenoma, primary, and metastatic human tumor specimens was to detect the relationship of PAK4 expression with the pathological features. Formalin-fixed paraffin sections were stained for PAK4 (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℃for 10 min. The immunohistochemical staining was scored in the following grades according to the percentage of positive cells. Morphological and immunohistochemical results were to correlate with clinicopathologic parameters.The influences on the phenotypes of MDA-MB-231 cell mediated by changing PAK4 expression1.Cell Culture MDA-MB-231 cells were maintained in RPMI-1640 supplemented with 10% fetal bovine serum, 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 PAK4 expression vector Primers, targeted to PAK4 code sequence(CDS) and containing EcoRⅠand BamHⅠsites at the ends, were designed. The polymerase chain reaction(PCR) products of CDS and pEGFP-C1 were both digested with EcoRⅠand BamHⅠand subsequently ligated for 4h at 16℃using T4 ligase. The recombinant plasmids was digested with EcoR I and BamHⅠfollowed by electrophoresis with 1% agarose and sequence analysis to identify.3. Construction of recombinant PAK4 shRNA expression vector Designing three pairs of small interfering RNAs(siRNAs) of PAK4 mRNA, and six corresponding single-strand short hairpin RNAs(shRNAs),containing BamHⅠand HindⅢsites and 9nt hairpin structure, were synthesized and annealed. The annealed products and the linear pRNAT6.1/Neo plasmid, digested with BamHⅠand HindⅢ,were ligated for 4h at 16℃using T4 ligase. The recombinant plasmids were digested with BamHⅠand HindⅢfollowed 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 MDA-MB-231 cells using lipofectamine2000TMreagent (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.). Breasties resistant to G418 were isolated. After 3-4 wk of culture, visible breasties 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. PAK4 exprssion detecting The expression changes of PAK4 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 PAK4, 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 described8.Cell migration assays Migration of MDA-MB-231,PAK4-overexpressng MDA-MB-231 (MDA-MB-231PAK4) and PAK4-lowexpressing MDA-MB-231 (MDA-MB-231shRNAI) cells was studied using 6.5mm Transwell chambers with 8μl pores (Costar) as previously described9. Cell culture wound healing assay. 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 representative scrape lines for each cell line were photographed.lO.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' In brief, the cells (105) were plated on 60-mm dishes. After 21 days, breasties were scored after staining the dishes with 0.5 mg/ml 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Sigma) overnight at 37℃.11.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 PAK4-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.Results1. PAK4 expression increases during human breast cancer progression and metastasis.Immunohistochemistry was performed to examine PAK4 expression levels in paraffin-embeded tissue from normal breast mucosa, benign polypi and adenomas to primary breast cancers and metastasis breast cancers, which is a typical progression pathway during breast carcinogenensis. Representative shows that PAK4 expression is negative in benign polypi, begins to increase in adenoma but is still weak compared to the carcinoma, and over-expressed in primary breast adenocarcinoma but is absent or barely detectable in paired normal mucosa beside the cancer (P<0.001). Furthermore, PAK4 expression is extremely much higher in lymph node metastasis and liver metastasis in contrast to primary breast carcinoma. However, the expression of PAK4 was not correlated with the histological differentiation(P>0.05) and Dukes' classification (P>0.05).2. The effects of changing PAK4 expression on cellular biological activity of MDA-MB-231 cell.(1) Constructed human PAK4 eukaryotic expression vector and human PAK4 hairpin siRNA eukaryotic expression vectors successfully.(2) Geneticin-resistant cell lines were screened after the recombinant plasmids were transfected into MDA-MB-231 cells.(3)After 24h incubation, there were notable differences compared the growth velocity of MDA-MB-231 cells in the group of MDA-MB-231vector, MDA-MB-231PAK4, MDA-MB-231shRNA-Nand MDA-MB-231shRNAI(F=11.006, P=0.000<0.001).(4) Compared with MDA-MB-231和MDA-MB-231shRNA-N, treatment with 5-Fu (15μg/ml) for 12h resulted in a significant increase of apoptosis in MDA-MB-231shRNAI 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.(6) A monolayer wound-healing assay revealed almost no migration in MDA-MB-231shRNA cells compared with MDA-MB-231control cells, but is not of MDA-MB-231PAK4, 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 MDA-MB-231shRNA cells, but increace of MDA-MB-231PAK4(8)A gelatin zymogram for MMP activation demonstrated a decrease in MMP-2 activity in MDA-MB-231shRNAI compared with MDA-MB-231shRNA-N cells. However, a significant decrease of MMP-9 activity was not observed. (9)Tumorigenicity assay showed the tumor weight of MDA-MB-231shRNA 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 PAK4 expression is increased with progression through the adenoma to carcinoma sequence, with the most dramatic increases in invasive and metastatic BCs, suggesting PAK4 might play an important role in breast tumorigenesis. It not only provide the basis for the further study in the mechanisms of breast cancer invasion and metastasis but also suggest that signal pathway mediated by PAK4-targeted for therpeutic intervention in breast cancer. These data implicate PAK4 as an exciting target for therapy of breast carcinoma.