PAK4在乳腺癌发病机制和诊断中的作用研究
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摘要
研究背景与目的
乳腺癌发病率居女性恶性肿瘤首位,占全身各种恶性肿瘤的7%-10%,是危害妇女健康的主要肿瘤。全世界每年约有120～140万妇女发生乳腺癌,约有50万妇女死于该病。北美、北欧是乳腺癌的高发地区,其发病率约为亚、非、拉美地区的4倍。我国虽是乳腺癌的低发地区,但其发病率逐年上升,每年递增3%左右,尤其沪、京、津及沿海地区是我国乳腺癌的高发地区,以上海最高,1972年上海的乳腺癌标化发病率为17／10万,1995年则为38／10万,为女性恶性肿瘤发病率中的第1位。如何有效地预防和治疗乳腺癌成为人们关注的焦点。
随着现代分子生物学的发展,研究者们逐渐认识到在真核细胞信号传导过程中,通路蛋白的磷酸化和去磷酸化修饰是信号通路调控的重要方式,两者之间的平衡在细胞正常信号转导及肿瘤发生、发展过程中起着非常重要的作用。p21-activated kinase (PAKs)即p21小GTP酶活化激酶,是一组丝氨酸／苏氨酸蛋白质磷酸化激酶家族。分子量为21 kD、Rho家族的小GTP酶即Cdc42和Rac1是PAKs的上游分子(即PAKs为Cdc42和Rac1的效应分子)。在分子结构上,PAK N-末端为激酶的调节区,C-末端为激酶的底物结合区,具有激酶活性。PAK4-3构成第一组PAK,分子量为62-64 kD,其激活需要Cdc42和Rac。PAK4-6构成第二组PAK,分子量为65-68 Kd。除调节胚胎发育、正常的细胞生长,分化和凋亡外,PAK家族成员还在人类疾病中起重要作用。PAK4是PAK家族中与人类肿瘤关系密切的成员,其在细胞骨架修复、延长正常细胞寿命和正向调节一些正常生理代谢等方面都起重要作用。PAK4为第二组PAK中最先别报道者,能在大多被检组织中检测到,但在乳腺癌和卵巢癌细胞中PAK4的表达远高于其它肿瘤细胞,而且PAK4表达与乳腺癌和卵巢癌细胞的增殖和侵袭转移呈正相关。但PAK4在乳腺癌的发生发展、演化和转移中的作用机制尚未明确,因此本课题在系统地检测PAK4在乳腺癌旁正常组织—乳腺囊性增生病—乳房纤维腺瘤—原发性乳腺癌组织—乳腺癌转移瘤组织中的表达差异和分布特点基础上,同时构建PAK4真核表达载体和AK4 shRNA真核表达载体,转染乳腺癌MDA-MB-231细胞,观察上调和下调乳腺癌细胞PAK4表达后对乳腺癌细胞增殖、凋亡,细胞粘附、运动、侵袭和致瘤等生物学活性的影响,以期深入了解PAK4在乳腺癌细胞演化和转移中的作用和机制,并探讨PAK4作为乳腺癌转移标志和作为潜在治疗靶点的可能性。研究方法
PAK4在良性及恶性乳腺病变中的表达检测及意义
用免疫组织化学S-P法检测PAK4在乳腺癌癌旁正常组织、乳腺囊性增生病、乳房纤维腺瘤、乳腺癌和乳腺癌转移瘤组织中的表达,并分析PAK4表达与乳腺癌临床病理特征的关系。
二、PAK4对乳腺癌细胞表型的影响
利用基因重组技术分别构建PAK4真核表达载体和PAK4 shRNA真核表达载体,并以乳腺癌细胞MDA-MB-231作为模型,转染重组载体后观测对乳腺癌MDA-MB-231细胞生物学活性的影响。
1、PAK4真核表达重组质粒的构建按照GenBank上公布的PAK4 mRNA序列,在其编码区(CDS)设计两端带有EcoRI和BamHⅠ酶切位点的引物,利用PCR扩增PAK4 CDS区。EcoRI和BamHⅠ双酶切纯化的PCR产物和真核表达载体pEGFP/C1, T4连接酶连接酶切纯化后的PCR产物和酶切纯化后的pEGFP/C1,转化到乳腺杆菌DH5a中。挑单菌落培养扩增,提取重组质粒后行酶切鉴定,选择插入片段正确的克隆测序鉴定。
2、PAK4 shRNA重组载体的构建根据siRNA设计的原则,在PAK4 mRNACDS设计3对siRNA核苷酸序列,并设计一对非相关核苷酸序列作为对照,经GenBank在线BLAST确定确定与其它人类基因无高度同源后,分布在所设计的siRNA两端分别加上带有BamHⅠ和EcoRI酶切位点的shRNAs单链送公司合成。复性合成双链,按常规操作把shRNA序列克隆到pRNAT-U6.1/Neo载体中,重组质粒酶切和测序鉴定。
3、转染细胞和建立稳定转染细胞克隆采用Invintrogen公司的Lipofectamine2000TM脂质体进行细胞转染。细胞转染48h后用分别用G418筛选阳性细胞克隆,挑取单克隆细胞扩增培养。
4、PAK4表达调控效应检测RT-PCR和免疫印迹、免疫细胞化学与激光共聚焦显微镜检测PAK4 mRNA和蛋白质水平表达改变。
5、细胞增殖与凋亡MTT法检测PAK4对乳腺癌细胞MDA-MB-231增殖的影响；采用DNA片段法检测PAK4对5-Fu诱导的细胞凋亡的影响。
6、细胞基质粘附实验参照文献的方法研究PAK4对乳腺癌细胞MDA-MB-231粘附的影响。
7、单层细胞划痕愈伤实验分析单层细胞划痕愈伤实验分析PAK4对乳腺癌细胞移行的影响。
8、侵袭实验观察改变PAK4表达对乳腺癌细胞侵袭能力的影响。
9、软琼脂糖集落形成实验观察RNAi沉默PAK4表达对MDA-MB-231细胞锚着独立性生长的影响。
10.裸鼠成瘤实验观察PAK4表达对MDA-MB-231细胞裸鼠成瘤的影响,包括成瘤瘤体大小、侵袭转移部位差异。
三、对PAK4在乳腺癌恶性程度诊断及预后中的作用进行初步研究
选择乳腺癌中的一个特殊部分即三阴乳腺癌(TNBC)进行研究,探讨PAK4在TNBC的发病机制中是否起了特殊作用；并对乳腺癌组织中PAK4表达不同的患者复发、转移的情况进行对照,
研究结果
一、PAK4在良、恶性乳腺病变中的表达及意义
1.乳腺癌、乳房纤维腺瘤等组织的PAK4阳性产物主要位于胞浆,尤以核周明显,细胞基质未见明显染色。
2.正常乳腺组织(或乳腺囊性增生病)、乳房纤维腺瘤、乳腺癌和乳腺癌转移瘤组织中的PAK4表达总体阳性率依次升高；提示PAK4与乳腺癌演进和转移密切相关。
3.在非浸润性癌、早期浸润癌、浸润癌三种不同病理分期的乳腺癌组织中PAK4的表达呈明显依次升高趋势,各组两两比较均有显著性差异。
二、PAK4对乳腺癌细胞生物学活性的影响
1.成功构建了PAK4真核表达载体pEGFP-C1/PAK4,并经酶切和测序鉴定证实；成功构建了PAK4 shRNA真核表达载体pRNA6.1/Neo-shRNA1、pRNA6.1/Neo-shRNA2、pRNA6.1/Neo-shRNA3和PAK4 shRNA非相关序列载体pRNA6.1/Neo-shRNA-N,并经酶切和测序鉴定证实插入序列与设计的完全一致。
2.重组载体转染MDA-MB-231细胞,经G418筛选得到稳定细胞克隆株。
3.与对照pRNA6.1/Neo-shRNA-N和pRNA6.1/Neo相比,pRNA6.1/Neo-shRNA1、pRNA6.1/Neo-shRNA2和pRNA6.1/Neo-shRNA3可以有效下调MDA-MB-231细胞内源性PAK4 mRNA和蛋白的表达；pEGFP-C1/PAK4在MDA-MB-231细胞浆中稳定表达,提示MDA-MB-231PAK4建株成功。
4.PAK4高表达可以促进大肠癌MDA-MB-231细胞增殖,下调PAK4表达则可以抑制大肠癌细胞的增殖；PAK4高表达可以增强大肠癌细胞的异质粘附,下调PAK4表达则抑制MDA-MB-231细胞的异质粘附；PAK4增强大肠癌细胞的非锚定依赖式生长,下调PAK4表达则抑制大肠癌细胞的集落形成能力；PAK4增强大肠癌细胞移行能力,下调PAK4表达则明显抑制大肠癌细胞的移行能力；PAK4增强大肠癌细胞对基质的侵袭能力,下调PAK4表达则可以减弱大肠癌细胞的侵袭力。
5.PAK4可以抗5-Fu诱导的大肠癌凋亡,抑制PAK4表达可以增强大肠癌细胞对5-Fu的敏感性。PAK4高表达可以促进大肠癌细胞分泌基质金属蛋白酶2,提示PAK4增加大肠癌细胞的转移潜能可能与增强基质金属蛋白酶2的活性有关。
6.血管内皮生长因子可以诱导PAK4在大肠癌细胞中的表达,提示VEGF可能通过PAK4影响大肠癌细胞的侵袭和转移；裸鼠成瘤实验显示,抑制PAK4基因的表达,可以负向调节大肠癌细胞的增殖和转移。
三、PAK4在乳腺癌诊断中的作用的初步研究对PAK4在乳腺癌恶性程度诊断及预后中的作用进行的研究显示：
1.三阴乳腺癌患者PAK4的表达率及表达量均明显高于非三阴乳腺癌患者。
2.三阴乳腺癌患者中PAK4表达高者复发及转移率高于表达低者。
3.PAK4有可能是三阴乳腺癌发病机制中的重要因素之一。
结论
1、PAK4在不同的乳腺病变中表达有显著性差异。
2、上下调PAK4表达可以明显改变乳腺癌细胞MDA-MB-231的多种生物学活性。
3、PAK4有可能是三阴乳腺癌发病机制中的重要因素之一
Background and objectives
Breast 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 methods
The potential role of PAK4 protein expression in breast cancer
Immunohistochemistry 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 expression
1.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 fluorescence
5. 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 described
8.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 described
9. 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.
Results
1. 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.
Conclusions
Taken 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.
乳腺癌发病率居女性恶性肿瘤首位,占全身各种恶性肿瘤的7%-10%,是危害妇女健康的主要肿瘤。全世界每年约有120～140万妇女发生乳腺癌,约有50万妇女死于该病。北美、北欧是乳腺癌的高发地区,其发病率约为亚、非、拉美地区的4倍。我国虽是乳腺癌的低发地区,但其发病率逐年上升,每年递增3%左右,尤其沪、京、津及沿海地区是我国乳腺癌的高发地区,以上海最高,1972年上海的乳腺癌标化发病率为17／10万,1995年则为38／10万,为女性恶性肿瘤发病率中的第1位。如何有效地预防和治疗乳腺癌成为人们关注的焦点。
随着现代分子生物学的发展,研究者们逐渐认识到在真核细胞信号传导过程中,通路蛋白的磷酸化和去磷酸化修饰是信号通路调控的重要方式,两者之间的平衡在细胞正常信号转导及肿瘤发生、发展过程中起着非常重要的作用。p21-activated kinase (PAKs)即p21小GTP酶活化激酶,是一组丝氨酸／苏氨酸蛋白质磷酸化激酶家族。分子量为21 kD、Rho家族的小GTP酶即Cdc42和Rac1是PAKs的上游分子(即PAKs为Cdc42和Rac1的效应分子)。在分子结构上,PAK N-末端为激酶的调节区,C-末端为激酶的底物结合区,具有激酶活性。PAK4-3构成第一组PAK,分子量为62-64 kD,其激活需要Cdc42和Rac。PAK4-6构成第二组PAK,分子量为65-68 Kd。除调节胚胎发育、正常的细胞生长,分化和凋亡外,PAK家族成员还在人类疾病中起重要作用。PAK4是PAK家族中与人类肿瘤关系密切的成员,其在细胞骨架修复、延长正常细胞寿命和正向调节一些正常生理代谢等方面都起重要作用。PAK4为第二组PAK中最先别报道者,能在大多被检组织中检测到,但在乳腺癌和卵巢癌细胞中PAK4的表达远高于其它肿瘤细胞,而且PAK4表达与乳腺癌和卵巢癌细胞的增殖和侵袭转移呈正相关。但PAK4在乳腺癌的发生发展、演化和转移中的作用机制尚未明确,因此本课题在系统地检测PAK4在乳腺癌旁正常组织—乳腺囊性增生病—乳房纤维腺瘤—原发性乳腺癌组织—乳腺癌转移瘤组织中的表达差异和分布特点基础上,同时构建PAK4真核表达载体和AK4 shRNA真核表达载体,转染乳腺癌MDA-MB-231细胞,观察上调和下调乳腺癌细胞PAK4表达后对乳腺癌细胞增殖、凋亡,细胞粘附、运动、侵袭和致瘤等生物学活性的影响,以期深入了解PAK4在乳腺癌细胞演化和转移中的作用和机制,并探讨PAK4作为乳腺癌转移标志和作为潜在治疗靶点的可能性。研究方法
PAK4在良性及恶性乳腺病变中的表达检测及意义
用免疫组织化学S-P法检测PAK4在乳腺癌癌旁正常组织、乳腺囊性增生病、乳房纤维腺瘤、乳腺癌和乳腺癌转移瘤组织中的表达,并分析PAK4表达与乳腺癌临床病理特征的关系。
二、PAK4对乳腺癌细胞表型的影响
利用基因重组技术分别构建PAK4真核表达载体和PAK4 shRNA真核表达载体,并以乳腺癌细胞MDA-MB-231作为模型,转染重组载体后观测对乳腺癌MDA-MB-231细胞生物学活性的影响。
1、PAK4真核表达重组质粒的构建按照GenBank上公布的PAK4 mRNA序列,在其编码区(CDS)设计两端带有EcoRI和BamHⅠ酶切位点的引物,利用PCR扩增PAK4 CDS区。EcoRI和BamHⅠ双酶切纯化的PCR产物和真核表达载体pEGFP/C1, T4连接酶连接酶切纯化后的PCR产物和酶切纯化后的pEGFP/C1,转化到乳腺杆菌DH5a中。挑单菌落培养扩增,提取重组质粒后行酶切鉴定,选择插入片段正确的克隆测序鉴定。
2、PAK4 shRNA重组载体的构建根据siRNA设计的原则,在PAK4 mRNACDS设计3对siRNA核苷酸序列,并设计一对非相关核苷酸序列作为对照,经GenBank在线BLAST确定确定与其它人类基因无高度同源后,分布在所设计的siRNA两端分别加上带有BamHⅠ和EcoRI酶切位点的shRNAs单链送公司合成。复性合成双链,按常规操作把shRNA序列克隆到pRNAT-U6.1/Neo载体中,重组质粒酶切和测序鉴定。
3、转染细胞和建立稳定转染细胞克隆采用Invintrogen公司的Lipofectamine2000TM脂质体进行细胞转染。细胞转染48h后用分别用G418筛选阳性细胞克隆,挑取单克隆细胞扩增培养。
4、PAK4表达调控效应检测RT-PCR和免疫印迹、免疫细胞化学与激光共聚焦显微镜检测PAK4 mRNA和蛋白质水平表达改变。
5、细胞增殖与凋亡MTT法检测PAK4对乳腺癌细胞MDA-MB-231增殖的影响；采用DNA片段法检测PAK4对5-Fu诱导的细胞凋亡的影响。
6、细胞基质粘附实验参照文献的方法研究PAK4对乳腺癌细胞MDA-MB-231粘附的影响。
7、单层细胞划痕愈伤实验分析单层细胞划痕愈伤实验分析PAK4对乳腺癌细胞移行的影响。
8、侵袭实验观察改变PAK4表达对乳腺癌细胞侵袭能力的影响。
9、软琼脂糖集落形成实验观察RNAi沉默PAK4表达对MDA-MB-231细胞锚着独立性生长的影响。
10.裸鼠成瘤实验观察PAK4表达对MDA-MB-231细胞裸鼠成瘤的影响,包括成瘤瘤体大小、侵袭转移部位差异。
三、对PAK4在乳腺癌恶性程度诊断及预后中的作用进行初步研究
选择乳腺癌中的一个特殊部分即三阴乳腺癌(TNBC)进行研究,探讨PAK4在TNBC的发病机制中是否起了特殊作用；并对乳腺癌组织中PAK4表达不同的患者复发、转移的情况进行对照,
研究结果
一、PAK4在良、恶性乳腺病变中的表达及意义
1.乳腺癌、乳房纤维腺瘤等组织的PAK4阳性产物主要位于胞浆,尤以核周明显,细胞基质未见明显染色。
2.正常乳腺组织(或乳腺囊性增生病)、乳房纤维腺瘤、乳腺癌和乳腺癌转移瘤组织中的PAK4表达总体阳性率依次升高；提示PAK4与乳腺癌演进和转移密切相关。
3.在非浸润性癌、早期浸润癌、浸润癌三种不同病理分期的乳腺癌组织中PAK4的表达呈明显依次升高趋势,各组两两比较均有显著性差异。
二、PAK4对乳腺癌细胞生物学活性的影响
1.成功构建了PAK4真核表达载体pEGFP-C1/PAK4,并经酶切和测序鉴定证实；成功构建了PAK4 shRNA真核表达载体pRNA6.1/Neo-shRNA1、pRNA6.1/Neo-shRNA2、pRNA6.1/Neo-shRNA3和PAK4 shRNA非相关序列载体pRNA6.1/Neo-shRNA-N,并经酶切和测序鉴定证实插入序列与设计的完全一致。
2.重组载体转染MDA-MB-231细胞,经G418筛选得到稳定细胞克隆株。
3.与对照pRNA6.1/Neo-shRNA-N和pRNA6.1/Neo相比,pRNA6.1/Neo-shRNA1、pRNA6.1/Neo-shRNA2和pRNA6.1/Neo-shRNA3可以有效下调MDA-MB-231细胞内源性PAK4 mRNA和蛋白的表达；pEGFP-C1/PAK4在MDA-MB-231细胞浆中稳定表达,提示MDA-MB-231PAK4建株成功。
4.PAK4高表达可以促进大肠癌MDA-MB-231细胞增殖,下调PAK4表达则可以抑制大肠癌细胞的增殖；PAK4高表达可以增强大肠癌细胞的异质粘附,下调PAK4表达则抑制MDA-MB-231细胞的异质粘附；PAK4增强大肠癌细胞的非锚定依赖式生长,下调PAK4表达则抑制大肠癌细胞的集落形成能力；PAK4增强大肠癌细胞移行能力,下调PAK4表达则明显抑制大肠癌细胞的移行能力；PAK4增强大肠癌细胞对基质的侵袭能力,下调PAK4表达则可以减弱大肠癌细胞的侵袭力。
5.PAK4可以抗5-Fu诱导的大肠癌凋亡,抑制PAK4表达可以增强大肠癌细胞对5-Fu的敏感性。PAK4高表达可以促进大肠癌细胞分泌基质金属蛋白酶2,提示PAK4增加大肠癌细胞的转移潜能可能与增强基质金属蛋白酶2的活性有关。
6.血管内皮生长因子可以诱导PAK4在大肠癌细胞中的表达,提示VEGF可能通过PAK4影响大肠癌细胞的侵袭和转移；裸鼠成瘤实验显示,抑制PAK4基因的表达,可以负向调节大肠癌细胞的增殖和转移。
三、PAK4在乳腺癌诊断中的作用的初步研究对PAK4在乳腺癌恶性程度诊断及预后中的作用进行的研究显示：
1.三阴乳腺癌患者PAK4的表达率及表达量均明显高于非三阴乳腺癌患者。
2.三阴乳腺癌患者中PAK4表达高者复发及转移率高于表达低者。
3.PAK4有可能是三阴乳腺癌发病机制中的重要因素之一。
结论
1、PAK4在不同的乳腺病变中表达有显著性差异。
2、上下调PAK4表达可以明显改变乳腺癌细胞MDA-MB-231的多种生物学活性。
3、PAK4有可能是三阴乳腺癌发病机制中的重要因素之一
Background and objectives
Breast 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 methods
The potential role of PAK4 protein expression in breast cancer
Immunohistochemistry 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 expression
1.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 fluorescence
5. 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 described
8.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 described
9. 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.
Results
1. 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.
Conclusions
Taken 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.
引文
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