非小细胞肺癌TGF-βⅡ型受体基因突变和表达研究
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摘要
转化生长因子β(transforming growth factor beta,TGF-β)是一种具有多种功能的细胞因子,TGF-β信号传导途径可介导多种上皮细胞生长抑制效应,调控细胞的生长、分化、凋亡、迁移及细胞外基质的形成,并且与肿瘤的发生、发展有密切关系。该信号传导通路由配体(TGF-β等)、受体(包括II型受体TGFBR2及I型受体TGFBR1)、Smads蛋白、转录调节的靶基因等组成。目前已证实,肿瘤细胞逃避TGF-β负调控的常见机制之一是TGF-β受体表达受抑制。TGFBR2是TGF-β信号的直接接受者,TGF-β首先与细胞膜上的TGFBR2结合,以启动下游通路,该受体在调控TGF-β诱导的细胞捕获和凋亡中发挥重要作用。在多种恶性肿瘤细胞中,包括非小细胞肺癌(NSCLC),TGFBR2等蛋白的异常表达可导致TGF-β信号传导紊乱,使细胞生长分化失去控制,从而促进了癌症的发生与发展。然而,虽然在NSCLC中TGFBR2的表达常有改变,但其编码区域的突变却非常少见。该受体表达的改变是由于基因突变等遗传因素改变引起,还是由于表观遗传因素所致,目前尚无定论。这促使我们对TGFBR2的编码区域进行突变检测,并进一步研究检测到的TGFBR2突变对TGF-β信号传导通路的影响和在NSCLC发生中的作用,以及探讨TGFBR2的表达改变与临床病理因素的关系。
第一部分非小细胞肺癌TGFBR2基因的突变检测
目的:1、检测各类型肺癌细胞和NSCLC组织中TGFBR2的编码区域突变情况。2、检测新发现的GCC(肺巨细胞癌)细胞TGFBR2纯合突变对其mRNA和蛋白表达的影响。
方法:1、对17株各类型肺癌细胞、2株正常人胚肺成纤维细胞、115对NSCLC组织及其相应的正常肺组织(对照组),用PCR-DGGE(多聚酶链式反应-变性梯度凝胶电泳)及直接测序法,进行TGFBR2全部7个外显子基因突变检测:(1)用蛋白酶K消化和酚-氯仿法抽提样本的基因组DNA;(2)用PCR-DGGE检测样本TGFBR2基因的全部7个外显子;(3)纯化PCR产物及直接测序法进行基因测序分析。2、用RT-PCR的方法检测突变GCC细胞的mRNA:用TRIzol试剂抽提总RNA,用SUPERSCRIPTTM II RNase HˉReverse Transcriptase Kit逆转录成cDNA;设计两套引物分别扩增TGFBR2 1-4号外显子和4-7号外显子,GAPDH(甘油醛-3-磷酸脱氢酶)cDNA作为内参照;8%聚丙烯酰胺凝胶电泳检测PCR产物大小。3、采用免疫共沉淀和Western blotting方法检测含有截断突变(c.492_507del)的GCC细胞TGFBR2蛋白大小,并用细胞免疫荧光染色法观察其在细胞上的表达。Western blotting一抗为鼠抗人TGFBR2细胞外区域单克隆抗体,二抗为HRP标记的羊抗鼠抗体;细胞免疫荧光染色一抗同前,二抗为FITC标记的羊抗鼠抗体。
结果:1、在两株来源于同一GCC母细胞系的细胞95D和PG中发现了TGFBR2一种新的纯合截断突变(c.492_507del),并且在其相应的肺癌组织中得到证实。另外,在该患者其他组织(肝、肾等)以及其他15例GCC和LCC(大细胞癌)患者肺癌组织中发现种系杂合型截断突变(c.492_507del),其突变率分别为2/2 GCC和7/13 LCC,而在100例其他类型NSCLC(包括腺癌、鳞癌和腺鳞癌)及所有正常对照细胞和组织中未发现上述突变。2、正常人胚肺成纤维细胞HFL1的TGFBR2外显子RT-PCR扩增产物分别为717 bp和805 bp,而突变GCC细胞扩增产物分别为701 bp和805 bp;3、Western blotting显示HFL1细胞TGFBR2蛋白为全长,而GCC细胞为截断蛋白。细胞免疫荧光染色显示突变的TGFBR2蛋白仍能在细胞膜上表达。
结论:1、新发现的TGFBR2截断突变(c.492_507del)只存在于GCC细胞和GCC、LCC组织中,基因突变造成TGFBR2的失活可能在GCC和LCC的发病机制中起重要作用。2、缺失16 bp的TGFBR2截断突变没有影响到mRNA的转录和剪切,但导致翻译时提前出现终止密码子,产生TGFBR2截断蛋白。3、TGFBR2突变产生的截断蛋白细胞外区完整,发生改变的跨膜区并未使蛋白从细胞表面释放,蛋白仍能在胞膜上表达。
第二部分TGFBR2截断突变对TGF-β信号传导通路的影响
目的:验证GCC细胞的TGFBR2截断突变在TGF-β信号传导通路失活中起了重要作用。
方法:1、突变细胞对外源性TGF-β1刺激的敏感性试验:(1)生长抑制试验:用不同浓度(0, 0.2, 1, 5 ng/ml)的外源性TGF-β1刺激突变细胞,用MTS比色法进行细胞计数来评价细胞的生长状况,确定TGF-β1对突变细胞有无生长抑制效应,TGF-β1浓度为5 ng/ml时细胞增殖降低40%,认为对TGF-β1敏感;(2)外源性TGF-β1对突变细胞纤连蛋白(FN)的mRNA表达的影响:对携带有突变的GCC细胞分别施加TGF-β1浓度为0和5 ng/ml的无血清培养液,培养48 hr后,用实时定量PCR和△△Ct法检测TGF-β1处理前后FN的mRNA相对表达量变化,内参照基因为GAPDH。2、野生型TGFBR2基因转染及TGF-β1诱导的转录激活效应检测:将携带野生型TGFBR2基因的质粒pcDNA3-TGFBR2(或空质粒pcDNA3)和TGF-β诱导的萤光素酶报告质粒p3TP-lux共同转染突变细胞,再以含5 ng/ml TGF-β1的培养液或不含TGF-β1的培养液处理细胞,观测在外源性TGF-β1作用前后细胞裂解液萤光素酶相对活性的变化,以检测突变细胞在转染了野生型TGFBR2基因后,是否恢复了对TGF-β1的敏感性。Renilla萤光素酶报告基因pRL-SV40作为内对照报告基因,瞬时共同转染培养细胞,校正不同的转染效率。用Berthold Lumat LB 9507化学发光检测仪测定萤光素酶活性。
结果:1、突变的GCC细胞失去了对TGF-β1的敏感性,在外源性TGF-β1作用下,突变细胞未能显示出TGF-β1的生长抑制效应和促细胞外基质合成的作用。2、当突变细胞转染了野生型TGFBR2基因后,细胞恢复了对TGF-β1的敏感性。
结论:GCC细胞中的TGFBR2突变在TGF-β信号传导通路失活中起了重要作用。
第三部分TGFBR2在NSCLC中的表达及其临床病理意义
目的:1、检测TGFBR2蛋白在LCC(包括GCC)和非LCC(包括腺癌、鳞癌和腺鳞癌)中的表达,分析其表达的高低与NSCLC的发生的相关性及与病理类型、患者年龄、性别、肿瘤大小、淋巴结转移、TNM分期等的关系。2、分析LCC中TGFBR2表达与截断突变(c.492_507del)的关系。
方法:取115对NSCLC样本(癌组织及其相应的正常肺组织)以及1例人正常细支气管样本(阳性对照)分别制成90点、90点和53点的三块组织芯片,用组织免疫化学方法(两步法)检测TGFBR2蛋白在NSCLC各病理类型中的表达。石蜡切片常规脱蜡和脱水,3%H2O2阻断内源性过氧化物酶,0.01M柠檬酸盐缓冲液(PH 6.0)中煮沸20 min抗原修复,滴加封闭用正常羊血清封闭,加一抗(鼠抗人TGFBR2单克隆抗体)室温湿盒中孵育,加辣根过氧化物酶标记的羊抗鼠/兔二抗,DAB显色,同时以PBS代替一抗设立阴性对照,人正常细支气管作为阳性对照。染色结果判定标准:阳性信号呈棕黄色颗粒状,定位于细胞膜和胞浆。先根据肿瘤细胞染色强度分为四级:0(未着色);1(浅染);2(较深);3(深染);再根据肿瘤细胞着色分布(阳性细胞占肿瘤细胞的百分比)分为四级:0(0～25%细胞着色);1(26～50%细胞着色);2(51～75%细胞着色);3(76～100%细胞着色)。最终结果为两者的乘积,0为表达缺失(阴性表达),1～9为阳性表达,如癌组织评分等于其对照组织,认为是表达正常,如低于其对照组织,认为是表达下调。
结果:1、对照组115例正常肺泡上皮和细支气管上皮细胞均为全部细胞强染色,评分为9分;而在115例肺癌标本中,表达缺失2例(1.7%),其余113例为阳性表达。表达缺失或下调占47.8%(55/115),表达正常52.2%(60/115),TGFBR2在NSCLC与正常对照组织中的表达差别显著(P<0.0001)。其中,腺癌、鳞癌、腺鳞癌表达下调分别占其总数的44%(22/50)、43.2%(19/44)和33.3%(2/6),而大细胞癌表达下调高达80.0%(12/15),有别于其他病理类型(P<0.05)。2、TGFBR2的异常表达与患者年龄、性别、肿瘤大小和局部侵袭(T)、淋巴结侵袭(N)、转移(M)、临床分期在统计学上无差异。2、突变型和野生型LCC在TGFBR2表达上的差别也无统计学意义。
结论:1、TGFBR2在NSCLC与正常对照组织中的表达差别显著,提示TGFBR2的表达下调可能与NSCLC的发生或发展相关。2、在TGFBR2表达水平上,LCC与非LCC差别也有显著的统计学意义。3、LCC的TGFBR2表达下调与其截断突变并无关系。4、TGFBR2在NSCLC的表达变化与患者年龄、性别、肿瘤大小和局部侵袭(T)、淋巴结侵袭(N)、转移(M)、临床分期在统计学上无差别。
全文总结
1、在两株来源于同一GCC母细胞系的肺癌细胞95D和PG中发现了TGFBR2一种新的纯合截断突变(c.492_507del),并且在该患者相应的肺癌组织中得到证实。另外,在该患者的肝、肾等组织以及其他15例GCC和LCC患者肺癌组织中发现有种系杂合型截断突变(c.492_507del),而在100例其他类型NSCLC(包括腺癌、鳞癌和腺鳞癌)中未发现上述突变,所有正常对照细胞和组织中也未发现该突变。
2、GCC细胞TGFBR2的16 bp的纯合截断突变(c.492_507del)导致在cDNA的第590-592核苷酸过早形成终止密码子,使翻译提前终止于紧随跨膜区后的位置,所产生的TGFBR2截断蛋白只含有191个氨基酸残基,其细胞外区完整,跨膜区发生改变,胞质激酶区丧失,导致携有纯合突变的细胞失去对TGF-β的敏感性。突变细胞转染野生型TGFBR2基因后,TGF-β信号传导通路得以恢复。
3、NSCLC与正常对照组织中TGFBR2的表达差别显著,提示TGFBR2的表达下调可能与NSCLC的发生或发展相关。TGFBR2的表达异常与病理类型有关,LCC与非LCC的表达水平有显著差别,但LCC的TGFBR2表达下调与其截断突变并无关系。TGFBR2的表达变化与患者年龄、性别、肿瘤大小和局部侵袭(T)、淋巴结侵袭(N)、转移(M)、临床分期在统计学上无差别。
4、作为抑癌基因,TGFBR2的截断突变和表达下调共同使TGF-β信号传导通路失活,导致相应的细胞失去生长抑制并促进肿瘤形成。以往的LCC(包括GCC)与非LCC区分是依靠表型来决定的,我们的研究为区分LCC与非LCC提供了基因型和表达上的证据。LCC不仅在分化程度上,而且在TGFBR2的基因突变和表达上都与腺癌和鳞癌等其他类型NSCLC有所不同。
Transforming growth factor beta (TGF-β) is a multifunctional cytokine that plays a central role in the signaling networks that regulate cell growth, differentiation, adhesion, apoptosis, migration and modification of the microenvironment in which the cells reside. The TGF-βsignal is transduced through a pair of transmembrane serine-threonine kinase receptors named receptor type II and type I and Smad proteins. Many malignant tumor cells including non-small lung carcinoma (NSCLC) cells are frequently resistant to TGF-β-mediated signal transduction, suggesting that alterations of the TGF-βsignaling pathway maybe involved in tumorigenesis. TGF-βbinds directly to TGF-βreceptor type II (TGFBR2), a constitutively active transmembrane serine/threonine kinase, and is then recognized by TGF-βreceptor type I, which is phosphorylated and activated by TGFBR2. Therefore, inactivation of TGFBR2 can result in TGF-βresistance, and TGFBR2 has been shown to function as a tumor suppressor in many solid tumors. Variants of the TGFBR2 gene are rarely seen in primary NSCLC cells and tissues, even though reduction of TGFBR2 expression has been found frequently. Whether the changes of the protein in NSCLC is genetic changes or epigenetic changes, however, remains unclear. The importance of TGF-βsignaling pathway in suppressing tumorigenesis, together with the high frequency of TGFBR2 mutations in many other types of cancers, directed us to screen for TGFBR2 mutations in NSCLC and to explore further the effect of the identified mutations on TGF-βsignal transduction and their role in tumorigenesis. The correlation between the expression and the mutation status of TGFBR2 were also analyzed.
Part 1 Mutational analyses of the transforming growth factor beta typeⅡreceptor gene in primary non-small cell lung cancer
Objectives: 1. To detect the mutation status of TGFBR2 in 17 human lung carcinoma cell lines, two normal human fetal lung fibroblast cell lines and 115 pairs of NSCLC tissues. 2. To investigate the effect of the mutated gene on TGFBR2 mRNA and protein expressions in giant cell carcinoma (GCC) cell lines.
Methods: 1. Mutation of TGFBR2 was detected by polymerase chain reaction-denatured gradient gel electrophoresis (PCR-DGGE) and direct sequencing in 17 human lung carcinoma cell lines, 2 normal human fetal lung fibroblast cell lines and 115 pairs of NSCLC tissues. (1)Genomic DNA was extracted from the cell lines, tumor and corresponding normal tissue samples by standard proteinase K digestion and phenol-chloroform extraction. (2)All seven exons of the TGFBR2 gene were assayed by PCR-DGGE. (3)DNA sequencing analysis: The PCR products were separated on agarose gels. Bands of the expected size were excised and purified and then sequenced directly on an automatic DNA sequencer. 2. The expression of mutated TGFBR2 on the mRNA level in GCC cell lines was investigated by RT-PCR. Total RNA from the cells was prepared by use of TRIzol reagent and was reverse transcribed with the SUPERSCRIPTTM II RNase HˉReverse Transcriptase Kit. Two sets of primers were designed so that the amplified fragments spanned exons 1 to 4 and 4 to 7 with several nucleotides overlapping allowing to check the integrity of the mRNA expression. Glyceralaldehyde 3-phosphate dehydrogenase (GAPDH) cDNA was also amplified as an internal control. The RT-PCR products were separated on 8% PAGE. 3. Expression of TGFBR2 in GCC cell lines was detected using immunoprecipitation and Western blotting and confirmed by immunocytochemical analysis. Immunoprecipitation was performed using the antibody targeted to the extracellular domain. Western blot analysis was performed using monoclonal antibody against the extracellular domain of TGFBR2 and horseradish peroxidase-conjugated secondary antibody. For immunocytochemical analysis, the cells were incubated with monoclonal antibody and stained with FITC-conjugated goat antimouse antibody.
Results: 1. A novel homozygous microdeletion (c.492_507del) was identified in two cell lines(95D and PG) derived from the same GCC and was confirmed in the corresponding tumor tissues. Furthermore, a heterozygous c.492_507del was found in the germ-line of one patient, as well as in the other GCC cases and some large cell carcinomas (LCC). The total frequency of the mutation was 2/2 GCC and 7/13 LCC. No mutation was detected in any of the other 100 NSCLC samples, including the subtypes of AdC, SqC and Ad-SqC, and no mutation was detected in any of the corresponding normal lung tissues and other cell lines. 2. HFL1 cells had amplified the two products of 717 bp and 805 bp; whereas the expression of both fragments of 701 bp and 805 bp were detected from the TGFBR2 mutated GCC cells. 3. The truncated protein was identified in GCC cells and the full length TGFBR2 was detected in normal HFL1 cells. The mutant protein could be seen on the membrane of the GCC cells.
Conclusions: 1. A Novel Microdeletion(c.492_507del) was found in two cell lines derived from the same giant cell carcinoma (GCC), as well as in the GCC cases and some large cell carcinomas (LCC) but not in other subtypes of NSCLC. The mutated TGFBR2 seems to play an important role in the abrogation of TGF-βsignal transduction and tumorigenesis of GCC and LCC. 2. The16 bp microdeletion in TGFBR2 has not affected the integrity of transcription and splicing of the mRNA. 3. The c.492_507del mutation causes a premature stop of translation which results in a truncated protein. The alteration in transmembrane domain had not made the protein to be released from the cell surface.
Part 2 Effect of the mutated TGFBR2 on TGF-βsignal transduction in the GCC Cells
Objective: To confirm the mutated TGFBR2 plays an important role in the abrogation of TGF-βsignal transduction and tumorigenesis of GCC.
Methods: 1. For assessing the sensitivity to exogenous TGF-βof GCC cells carrying microdeletion in TGFBR2: (1)Growth inhibition assay was used to assess the antiproliferation effect of TGF-β1 on GCC cells carrying microdeletion in TGFBR2. The cell numbers were determined in response to increasing concentrations of exogenous TGF-β1 (0, 0.2, 1 or 5 ng/ml) by MTS assay. Cells that showed over 40% reduction of cell proliferation by TGF-β1 (5 ng/ml) were determined to be TGF-β1 responsive. (2)To study the effect of exogenous TGF-β1 on fibronectin (FN) expression of GCC cells carrying microdeletion in TGFBR2, the relative quantification of fibronectin mRNA levels from cells treated with or without 5 ng/ml of TGF-β1 for 48 h was performed using real-time, fluorescence quantitative PCR and the△△C t method. The FN gene expression was presented relative to the housekeeping gene GAPDH. 2. To investigate whether the abrogated TGF-βsignal transduction is solely due to the inactivation of TGFBR2, we sought to restore TGF-β1 responsiveness in GCC cells by introducing wild-type TGFBR2. A TGF-β1 responsive luciferase reporter construct, p3TP-Lux, was used to cotransfect the GCC cells with the vector pcDNA3-TGFBR2 which expressed wild-type TGFBR2 or empty vector pcDNA3. Cells were treated with or without 5 ng/ml of TGF-β1 in medium. Renilla luciferase reporter gene pRL-SV40 was cotransfected to normalize the transfection efficiency. Luciferase activity in cell lysate was determined by the Dual-luciferase Reporter Assay System.
Results: 1. The TGFBR2-defective GCC cells showed loss of sensitivity to TGF-β1 induction both in growth inhibition and stimulation of extracellular matrix proteins. 2. After the transfection of wild-type TGFBR2 into the mutant cells, the sensitivity to TGF-β1 was restored.
Conclusions: The mutant TGFBR2 plays an important role in the abrogation of the TGF-βpathway in GCC cells.
Part 3 Expression of TGFBR2 in NSCLC and its possible implications
Objectives: 1. To examine the expression level of TGFBR2 both in LCC (including GCC) and non-LCC tumors, which include adenocarcinoma (AdC), squamous cell carcinoma (SqC) and adenosquamous carcinoma (Ad-SqC). Analyses were also conducted to determine the associations between TGFBR2 expression and clinical features, such as pathological types, age, gender, tumor size, nodal involvement, metastasis and stage. 2. The correlation between the expression and the mutation status of TGFBR2 in LCC samples were also analyzed.
Methods: 115 pairs of NSCLC samples (tumor tissues and their corresponding normal tissues) and one case of human normal bronchial epithelium were accumulated and made into three tissue microarrays which containing 90 dots, 90 dots and 53 dots respectively. Expression of TGFBR2 was detected by immunohistochemistry assay using the monoclonal antibody against the extracellular domain of TGFBR2 via the two-step immunohistochemical staining with the MaxVision system. After the paraffin sections being deparaffinized and hydrated, sections were placed into a 3% hydrogen peroxide solution for 10 min to block endogenous peroxide activity. For antigen retrieval, the sections were treated with boiling 0.01 mol/L citrate buffer for 20 min and were then incubated with 10% goat serum for 20 min at room temperature. After the blocking serum was removed, the sections were incubated with the primary antibody (1:50) at room temperature for 1 h. The sections were then incubated with the working solution of horseradish peroxidase-labeled goat anti-mouse immunoglobulin for 30 min. Finally, the peroxidase activity was developed with 3,3’-diaminobenzidine tetrahydrochloride and hydrogen peroxide. Human normal bronchial epithelium was used as a positive control. Negative control for each specimen has been done by treating the sections with PBS instead of the primary antibody. All sections were examined by using standard light microscopy and scored semi-quantitatively on the basis of the percentage of cells that displayed immunoreactivity and on the intensity of the staining reaction. The expression of TGFBR2 was predominantly on the membrane and in the cytoplasm of the epithelial cells. All the normal tissues stained strongly in over 75% of cells and their staining scores were 9.The samples were first grouped into four grades according to the staining intensity: 0 (negative staining, the same with that of negative control), 1 (weak staining), 2 (medium staining) and 3 (strong staining). The percentages of positive staining cells were assigned as 0 for 0~25%, 1 for 26~50%, 2 for 51~75% and 3 for 76~100%, respectively. The final score was determined as the product of the proportion and intensity scores and will be 0~9. The sample was assigned as negative staining if the final score was 0 or positive staining when the final score was 1~9. Moreover, a cancer sample was assigned as a preserved (Pr) or a reduced type (Re) of TGFBR2 expression if its final score was the same with or less than that of its corresponding normal lung tissue.
Results: 1. All the normal tissues stained strongly in over 75% of cells and their staining scores were 9. According to the criteria to classify the tumor samples, 2 cases showed negative TGFBR2 expression (1.7%) and other 113 cases showed positive expression. Furthermore, 47.8% (55/115) of tumor tissues showed reduced or loss of TGFBR2 expression and 52.2% (60/115) of tumors had preserved TGFBR2 expression. In case of adenocarcinoma, squamous cell carcinoma and adenosquamous cell carcinoma, the frequency of reduced type were 44%(22/50), 43.2%(19/44) and 33.3%(2/6) respectively, while in case of large cell carcinoma, the frequency of reduced type was extremely higher(80.0%, 12/15), indicating that most of the LCC cells had reduced TGFBR2 expression. No significant association was found between TGFBR2 expression and pathological types, age, gender, tumor size and local invasion, nodal involvement, metastasis or stage. 2. No correlation was found between TGFBR2 expression and c.492 507del mutation in LCC tissue sections.
Conclusions: 1. The differences in the expression level of TGFBR2 between NSCLC samples and corresponding normal lung tissues suggest that the defective TGFBR2 expression might contribute to the carcinogenesis and/or development of NSCLC. 2. There is significant difference on the TGFBR2 expression level between the LCC and non-LCC subtypes. 3. No correlation was found between TGFBR2 expression and c.492 507del mutation in LCC tissue sections. 4. No significant association was found between TGFBR2 expression and age, gender, tumor size and local invasion, nodal involvement, metastasis or stage.
Summary
1. A novel homozygous TGFBR2 microdeletion (c.492_507del) was identified in two cell lines derived from the same giant cell carcinoma (GCC) and was confirmed in the corresponding tumor tissues. Furthermore, a heterozygous c.492_507del was found in the germ-line of one patient, as well as in the other GCC cases and some large cell carcinomas (LCC) but not in other subtypes of NSCLC.
2. The 16 bp-microdeletion introduced a premature stop codon at positions 590–592 of the cDNA, resulting in a truncated TGFBR2 protein with a mutated transmembrane domain and loss of kinase domain. The GCC cells were characterized as being unresponsive to TGF-βinduction both in growth inhibition and stimulation of extracellular matrix protein. Moreover, after the reconstitution of wild-type TGFBR2 expression, the sensitivity to TGF-βwas restored. Therefore, mutated TGFBR2 seems to play an important role in the abrogation of TGF-βsignal transduction in GCC cells.
3. The differences in the expression level of TGFBR2 between NSCLC samples and corresponding normal lung tissues suggest that the defective TGFBR2 expression might contribute to the carcinogenesis and/or development of NSCLC. There is significant difference on the TGFBR2 expression level between the LCC and non-LCC subtypes, but no correlation was found between TGFBR2 expression and c.492 507del mutation in LCC tissue sections. No significant association was found between TGFBR2 expression and age, gender, tumor size and local invasion, nodal involvement, metastasis or stage.
4. The reduced expression of TGFBR2 and the existence of c.492_507del mutation inactivate the TGF-βsignal transduction, leading to the loss of growth inhibition of the corresponding cells and acceleration of tumor formation. Our findings in LCC provided genotypic and expression supports in the classification of LCC and non-LCC subtypes which was previously classified solely by phenotypes. LCC is distinct from AdC and SqC not only in differentiation, but also in the pattern of TGFBR2 expression and TGFBR2 mutation.
第一部分非小细胞肺癌TGFBR2基因的突变检测
目的:1、检测各类型肺癌细胞和NSCLC组织中TGFBR2的编码区域突变情况。2、检测新发现的GCC(肺巨细胞癌)细胞TGFBR2纯合突变对其mRNA和蛋白表达的影响。
方法:1、对17株各类型肺癌细胞、2株正常人胚肺成纤维细胞、115对NSCLC组织及其相应的正常肺组织(对照组),用PCR-DGGE(多聚酶链式反应-变性梯度凝胶电泳)及直接测序法,进行TGFBR2全部7个外显子基因突变检测:(1)用蛋白酶K消化和酚-氯仿法抽提样本的基因组DNA;(2)用PCR-DGGE检测样本TGFBR2基因的全部7个外显子;(3)纯化PCR产物及直接测序法进行基因测序分析。2、用RT-PCR的方法检测突变GCC细胞的mRNA:用TRIzol试剂抽提总RNA,用SUPERSCRIPTTM II RNase HˉReverse Transcriptase Kit逆转录成cDNA;设计两套引物分别扩增TGFBR2 1-4号外显子和4-7号外显子,GAPDH(甘油醛-3-磷酸脱氢酶)cDNA作为内参照;8%聚丙烯酰胺凝胶电泳检测PCR产物大小。3、采用免疫共沉淀和Western blotting方法检测含有截断突变(c.492_507del)的GCC细胞TGFBR2蛋白大小,并用细胞免疫荧光染色法观察其在细胞上的表达。Western blotting一抗为鼠抗人TGFBR2细胞外区域单克隆抗体,二抗为HRP标记的羊抗鼠抗体;细胞免疫荧光染色一抗同前,二抗为FITC标记的羊抗鼠抗体。
结果:1、在两株来源于同一GCC母细胞系的细胞95D和PG中发现了TGFBR2一种新的纯合截断突变(c.492_507del),并且在其相应的肺癌组织中得到证实。另外,在该患者其他组织(肝、肾等)以及其他15例GCC和LCC(大细胞癌)患者肺癌组织中发现种系杂合型截断突变(c.492_507del),其突变率分别为2/2 GCC和7/13 LCC,而在100例其他类型NSCLC(包括腺癌、鳞癌和腺鳞癌)及所有正常对照细胞和组织中未发现上述突变。2、正常人胚肺成纤维细胞HFL1的TGFBR2外显子RT-PCR扩增产物分别为717 bp和805 bp,而突变GCC细胞扩增产物分别为701 bp和805 bp;3、Western blotting显示HFL1细胞TGFBR2蛋白为全长,而GCC细胞为截断蛋白。细胞免疫荧光染色显示突变的TGFBR2蛋白仍能在细胞膜上表达。
结论:1、新发现的TGFBR2截断突变(c.492_507del)只存在于GCC细胞和GCC、LCC组织中,基因突变造成TGFBR2的失活可能在GCC和LCC的发病机制中起重要作用。2、缺失16 bp的TGFBR2截断突变没有影响到mRNA的转录和剪切,但导致翻译时提前出现终止密码子,产生TGFBR2截断蛋白。3、TGFBR2突变产生的截断蛋白细胞外区完整,发生改变的跨膜区并未使蛋白从细胞表面释放,蛋白仍能在胞膜上表达。
第二部分TGFBR2截断突变对TGF-β信号传导通路的影响
目的:验证GCC细胞的TGFBR2截断突变在TGF-β信号传导通路失活中起了重要作用。
方法:1、突变细胞对外源性TGF-β1刺激的敏感性试验:(1)生长抑制试验:用不同浓度(0, 0.2, 1, 5 ng/ml)的外源性TGF-β1刺激突变细胞,用MTS比色法进行细胞计数来评价细胞的生长状况,确定TGF-β1对突变细胞有无生长抑制效应,TGF-β1浓度为5 ng/ml时细胞增殖降低40%,认为对TGF-β1敏感;(2)外源性TGF-β1对突变细胞纤连蛋白(FN)的mRNA表达的影响:对携带有突变的GCC细胞分别施加TGF-β1浓度为0和5 ng/ml的无血清培养液,培养48 hr后,用实时定量PCR和△△Ct法检测TGF-β1处理前后FN的mRNA相对表达量变化,内参照基因为GAPDH。2、野生型TGFBR2基因转染及TGF-β1诱导的转录激活效应检测:将携带野生型TGFBR2基因的质粒pcDNA3-TGFBR2(或空质粒pcDNA3)和TGF-β诱导的萤光素酶报告质粒p3TP-lux共同转染突变细胞,再以含5 ng/ml TGF-β1的培养液或不含TGF-β1的培养液处理细胞,观测在外源性TGF-β1作用前后细胞裂解液萤光素酶相对活性的变化,以检测突变细胞在转染了野生型TGFBR2基因后,是否恢复了对TGF-β1的敏感性。Renilla萤光素酶报告基因pRL-SV40作为内对照报告基因,瞬时共同转染培养细胞,校正不同的转染效率。用Berthold Lumat LB 9507化学发光检测仪测定萤光素酶活性。
结果:1、突变的GCC细胞失去了对TGF-β1的敏感性,在外源性TGF-β1作用下,突变细胞未能显示出TGF-β1的生长抑制效应和促细胞外基质合成的作用。2、当突变细胞转染了野生型TGFBR2基因后,细胞恢复了对TGF-β1的敏感性。
结论:GCC细胞中的TGFBR2突变在TGF-β信号传导通路失活中起了重要作用。
第三部分TGFBR2在NSCLC中的表达及其临床病理意义
目的:1、检测TGFBR2蛋白在LCC(包括GCC)和非LCC(包括腺癌、鳞癌和腺鳞癌)中的表达,分析其表达的高低与NSCLC的发生的相关性及与病理类型、患者年龄、性别、肿瘤大小、淋巴结转移、TNM分期等的关系。2、分析LCC中TGFBR2表达与截断突变(c.492_507del)的关系。
方法:取115对NSCLC样本(癌组织及其相应的正常肺组织)以及1例人正常细支气管样本(阳性对照)分别制成90点、90点和53点的三块组织芯片,用组织免疫化学方法(两步法)检测TGFBR2蛋白在NSCLC各病理类型中的表达。石蜡切片常规脱蜡和脱水,3%H2O2阻断内源性过氧化物酶,0.01M柠檬酸盐缓冲液(PH 6.0)中煮沸20 min抗原修复,滴加封闭用正常羊血清封闭,加一抗(鼠抗人TGFBR2单克隆抗体)室温湿盒中孵育,加辣根过氧化物酶标记的羊抗鼠/兔二抗,DAB显色,同时以PBS代替一抗设立阴性对照,人正常细支气管作为阳性对照。染色结果判定标准:阳性信号呈棕黄色颗粒状,定位于细胞膜和胞浆。先根据肿瘤细胞染色强度分为四级:0(未着色);1(浅染);2(较深);3(深染);再根据肿瘤细胞着色分布(阳性细胞占肿瘤细胞的百分比)分为四级:0(0～25%细胞着色);1(26～50%细胞着色);2(51～75%细胞着色);3(76～100%细胞着色)。最终结果为两者的乘积,0为表达缺失(阴性表达),1～9为阳性表达,如癌组织评分等于其对照组织,认为是表达正常,如低于其对照组织,认为是表达下调。
结果:1、对照组115例正常肺泡上皮和细支气管上皮细胞均为全部细胞强染色,评分为9分;而在115例肺癌标本中,表达缺失2例(1.7%),其余113例为阳性表达。表达缺失或下调占47.8%(55/115),表达正常52.2%(60/115),TGFBR2在NSCLC与正常对照组织中的表达差别显著(P<0.0001)。其中,腺癌、鳞癌、腺鳞癌表达下调分别占其总数的44%(22/50)、43.2%(19/44)和33.3%(2/6),而大细胞癌表达下调高达80.0%(12/15),有别于其他病理类型(P<0.05)。2、TGFBR2的异常表达与患者年龄、性别、肿瘤大小和局部侵袭(T)、淋巴结侵袭(N)、转移(M)、临床分期在统计学上无差异。2、突变型和野生型LCC在TGFBR2表达上的差别也无统计学意义。
结论:1、TGFBR2在NSCLC与正常对照组织中的表达差别显著,提示TGFBR2的表达下调可能与NSCLC的发生或发展相关。2、在TGFBR2表达水平上,LCC与非LCC差别也有显著的统计学意义。3、LCC的TGFBR2表达下调与其截断突变并无关系。4、TGFBR2在NSCLC的表达变化与患者年龄、性别、肿瘤大小和局部侵袭(T)、淋巴结侵袭(N)、转移(M)、临床分期在统计学上无差别。
全文总结
1、在两株来源于同一GCC母细胞系的肺癌细胞95D和PG中发现了TGFBR2一种新的纯合截断突变(c.492_507del),并且在该患者相应的肺癌组织中得到证实。另外,在该患者的肝、肾等组织以及其他15例GCC和LCC患者肺癌组织中发现有种系杂合型截断突变(c.492_507del),而在100例其他类型NSCLC(包括腺癌、鳞癌和腺鳞癌)中未发现上述突变,所有正常对照细胞和组织中也未发现该突变。
2、GCC细胞TGFBR2的16 bp的纯合截断突变(c.492_507del)导致在cDNA的第590-592核苷酸过早形成终止密码子,使翻译提前终止于紧随跨膜区后的位置,所产生的TGFBR2截断蛋白只含有191个氨基酸残基,其细胞外区完整,跨膜区发生改变,胞质激酶区丧失,导致携有纯合突变的细胞失去对TGF-β的敏感性。突变细胞转染野生型TGFBR2基因后,TGF-β信号传导通路得以恢复。
3、NSCLC与正常对照组织中TGFBR2的表达差别显著,提示TGFBR2的表达下调可能与NSCLC的发生或发展相关。TGFBR2的表达异常与病理类型有关,LCC与非LCC的表达水平有显著差别,但LCC的TGFBR2表达下调与其截断突变并无关系。TGFBR2的表达变化与患者年龄、性别、肿瘤大小和局部侵袭(T)、淋巴结侵袭(N)、转移(M)、临床分期在统计学上无差别。
4、作为抑癌基因,TGFBR2的截断突变和表达下调共同使TGF-β信号传导通路失活,导致相应的细胞失去生长抑制并促进肿瘤形成。以往的LCC(包括GCC)与非LCC区分是依靠表型来决定的,我们的研究为区分LCC与非LCC提供了基因型和表达上的证据。LCC不仅在分化程度上,而且在TGFBR2的基因突变和表达上都与腺癌和鳞癌等其他类型NSCLC有所不同。
Transforming growth factor beta (TGF-β) is a multifunctional cytokine that plays a central role in the signaling networks that regulate cell growth, differentiation, adhesion, apoptosis, migration and modification of the microenvironment in which the cells reside. The TGF-βsignal is transduced through a pair of transmembrane serine-threonine kinase receptors named receptor type II and type I and Smad proteins. Many malignant tumor cells including non-small lung carcinoma (NSCLC) cells are frequently resistant to TGF-β-mediated signal transduction, suggesting that alterations of the TGF-βsignaling pathway maybe involved in tumorigenesis. TGF-βbinds directly to TGF-βreceptor type II (TGFBR2), a constitutively active transmembrane serine/threonine kinase, and is then recognized by TGF-βreceptor type I, which is phosphorylated and activated by TGFBR2. Therefore, inactivation of TGFBR2 can result in TGF-βresistance, and TGFBR2 has been shown to function as a tumor suppressor in many solid tumors. Variants of the TGFBR2 gene are rarely seen in primary NSCLC cells and tissues, even though reduction of TGFBR2 expression has been found frequently. Whether the changes of the protein in NSCLC is genetic changes or epigenetic changes, however, remains unclear. The importance of TGF-βsignaling pathway in suppressing tumorigenesis, together with the high frequency of TGFBR2 mutations in many other types of cancers, directed us to screen for TGFBR2 mutations in NSCLC and to explore further the effect of the identified mutations on TGF-βsignal transduction and their role in tumorigenesis. The correlation between the expression and the mutation status of TGFBR2 were also analyzed.
Part 1 Mutational analyses of the transforming growth factor beta typeⅡreceptor gene in primary non-small cell lung cancer
Objectives: 1. To detect the mutation status of TGFBR2 in 17 human lung carcinoma cell lines, two normal human fetal lung fibroblast cell lines and 115 pairs of NSCLC tissues. 2. To investigate the effect of the mutated gene on TGFBR2 mRNA and protein expressions in giant cell carcinoma (GCC) cell lines.
Methods: 1. Mutation of TGFBR2 was detected by polymerase chain reaction-denatured gradient gel electrophoresis (PCR-DGGE) and direct sequencing in 17 human lung carcinoma cell lines, 2 normal human fetal lung fibroblast cell lines and 115 pairs of NSCLC tissues. (1)Genomic DNA was extracted from the cell lines, tumor and corresponding normal tissue samples by standard proteinase K digestion and phenol-chloroform extraction. (2)All seven exons of the TGFBR2 gene were assayed by PCR-DGGE. (3)DNA sequencing analysis: The PCR products were separated on agarose gels. Bands of the expected size were excised and purified and then sequenced directly on an automatic DNA sequencer. 2. The expression of mutated TGFBR2 on the mRNA level in GCC cell lines was investigated by RT-PCR. Total RNA from the cells was prepared by use of TRIzol reagent and was reverse transcribed with the SUPERSCRIPTTM II RNase HˉReverse Transcriptase Kit. Two sets of primers were designed so that the amplified fragments spanned exons 1 to 4 and 4 to 7 with several nucleotides overlapping allowing to check the integrity of the mRNA expression. Glyceralaldehyde 3-phosphate dehydrogenase (GAPDH) cDNA was also amplified as an internal control. The RT-PCR products were separated on 8% PAGE. 3. Expression of TGFBR2 in GCC cell lines was detected using immunoprecipitation and Western blotting and confirmed by immunocytochemical analysis. Immunoprecipitation was performed using the antibody targeted to the extracellular domain. Western blot analysis was performed using monoclonal antibody against the extracellular domain of TGFBR2 and horseradish peroxidase-conjugated secondary antibody. For immunocytochemical analysis, the cells were incubated with monoclonal antibody and stained with FITC-conjugated goat antimouse antibody.
Results: 1. A novel homozygous microdeletion (c.492_507del) was identified in two cell lines(95D and PG) derived from the same GCC and was confirmed in the corresponding tumor tissues. Furthermore, a heterozygous c.492_507del was found in the germ-line of one patient, as well as in the other GCC cases and some large cell carcinomas (LCC). The total frequency of the mutation was 2/2 GCC and 7/13 LCC. No mutation was detected in any of the other 100 NSCLC samples, including the subtypes of AdC, SqC and Ad-SqC, and no mutation was detected in any of the corresponding normal lung tissues and other cell lines. 2. HFL1 cells had amplified the two products of 717 bp and 805 bp; whereas the expression of both fragments of 701 bp and 805 bp were detected from the TGFBR2 mutated GCC cells. 3. The truncated protein was identified in GCC cells and the full length TGFBR2 was detected in normal HFL1 cells. The mutant protein could be seen on the membrane of the GCC cells.
Conclusions: 1. A Novel Microdeletion(c.492_507del) was found in two cell lines derived from the same giant cell carcinoma (GCC), as well as in the GCC cases and some large cell carcinomas (LCC) but not in other subtypes of NSCLC. The mutated TGFBR2 seems to play an important role in the abrogation of TGF-βsignal transduction and tumorigenesis of GCC and LCC. 2. The16 bp microdeletion in TGFBR2 has not affected the integrity of transcription and splicing of the mRNA. 3. The c.492_507del mutation causes a premature stop of translation which results in a truncated protein. The alteration in transmembrane domain had not made the protein to be released from the cell surface.
Part 2 Effect of the mutated TGFBR2 on TGF-βsignal transduction in the GCC Cells
Objective: To confirm the mutated TGFBR2 plays an important role in the abrogation of TGF-βsignal transduction and tumorigenesis of GCC.
Methods: 1. For assessing the sensitivity to exogenous TGF-βof GCC cells carrying microdeletion in TGFBR2: (1)Growth inhibition assay was used to assess the antiproliferation effect of TGF-β1 on GCC cells carrying microdeletion in TGFBR2. The cell numbers were determined in response to increasing concentrations of exogenous TGF-β1 (0, 0.2, 1 or 5 ng/ml) by MTS assay. Cells that showed over 40% reduction of cell proliferation by TGF-β1 (5 ng/ml) were determined to be TGF-β1 responsive. (2)To study the effect of exogenous TGF-β1 on fibronectin (FN) expression of GCC cells carrying microdeletion in TGFBR2, the relative quantification of fibronectin mRNA levels from cells treated with or without 5 ng/ml of TGF-β1 for 48 h was performed using real-time, fluorescence quantitative PCR and the△△C t method. The FN gene expression was presented relative to the housekeeping gene GAPDH. 2. To investigate whether the abrogated TGF-βsignal transduction is solely due to the inactivation of TGFBR2, we sought to restore TGF-β1 responsiveness in GCC cells by introducing wild-type TGFBR2. A TGF-β1 responsive luciferase reporter construct, p3TP-Lux, was used to cotransfect the GCC cells with the vector pcDNA3-TGFBR2 which expressed wild-type TGFBR2 or empty vector pcDNA3. Cells were treated with or without 5 ng/ml of TGF-β1 in medium. Renilla luciferase reporter gene pRL-SV40 was cotransfected to normalize the transfection efficiency. Luciferase activity in cell lysate was determined by the Dual-luciferase Reporter Assay System.
Results: 1. The TGFBR2-defective GCC cells showed loss of sensitivity to TGF-β1 induction both in growth inhibition and stimulation of extracellular matrix proteins. 2. After the transfection of wild-type TGFBR2 into the mutant cells, the sensitivity to TGF-β1 was restored.
Conclusions: The mutant TGFBR2 plays an important role in the abrogation of the TGF-βpathway in GCC cells.
Part 3 Expression of TGFBR2 in NSCLC and its possible implications
Objectives: 1. To examine the expression level of TGFBR2 both in LCC (including GCC) and non-LCC tumors, which include adenocarcinoma (AdC), squamous cell carcinoma (SqC) and adenosquamous carcinoma (Ad-SqC). Analyses were also conducted to determine the associations between TGFBR2 expression and clinical features, such as pathological types, age, gender, tumor size, nodal involvement, metastasis and stage. 2. The correlation between the expression and the mutation status of TGFBR2 in LCC samples were also analyzed.
Methods: 115 pairs of NSCLC samples (tumor tissues and their corresponding normal tissues) and one case of human normal bronchial epithelium were accumulated and made into three tissue microarrays which containing 90 dots, 90 dots and 53 dots respectively. Expression of TGFBR2 was detected by immunohistochemistry assay using the monoclonal antibody against the extracellular domain of TGFBR2 via the two-step immunohistochemical staining with the MaxVision system. After the paraffin sections being deparaffinized and hydrated, sections were placed into a 3% hydrogen peroxide solution for 10 min to block endogenous peroxide activity. For antigen retrieval, the sections were treated with boiling 0.01 mol/L citrate buffer for 20 min and were then incubated with 10% goat serum for 20 min at room temperature. After the blocking serum was removed, the sections were incubated with the primary antibody (1:50) at room temperature for 1 h. The sections were then incubated with the working solution of horseradish peroxidase-labeled goat anti-mouse immunoglobulin for 30 min. Finally, the peroxidase activity was developed with 3,3’-diaminobenzidine tetrahydrochloride and hydrogen peroxide. Human normal bronchial epithelium was used as a positive control. Negative control for each specimen has been done by treating the sections with PBS instead of the primary antibody. All sections were examined by using standard light microscopy and scored semi-quantitatively on the basis of the percentage of cells that displayed immunoreactivity and on the intensity of the staining reaction. The expression of TGFBR2 was predominantly on the membrane and in the cytoplasm of the epithelial cells. All the normal tissues stained strongly in over 75% of cells and their staining scores were 9.The samples were first grouped into four grades according to the staining intensity: 0 (negative staining, the same with that of negative control), 1 (weak staining), 2 (medium staining) and 3 (strong staining). The percentages of positive staining cells were assigned as 0 for 0~25%, 1 for 26~50%, 2 for 51~75% and 3 for 76~100%, respectively. The final score was determined as the product of the proportion and intensity scores and will be 0~9. The sample was assigned as negative staining if the final score was 0 or positive staining when the final score was 1~9. Moreover, a cancer sample was assigned as a preserved (Pr) or a reduced type (Re) of TGFBR2 expression if its final score was the same with or less than that of its corresponding normal lung tissue.
Results: 1. All the normal tissues stained strongly in over 75% of cells and their staining scores were 9. According to the criteria to classify the tumor samples, 2 cases showed negative TGFBR2 expression (1.7%) and other 113 cases showed positive expression. Furthermore, 47.8% (55/115) of tumor tissues showed reduced or loss of TGFBR2 expression and 52.2% (60/115) of tumors had preserved TGFBR2 expression. In case of adenocarcinoma, squamous cell carcinoma and adenosquamous cell carcinoma, the frequency of reduced type were 44%(22/50), 43.2%(19/44) and 33.3%(2/6) respectively, while in case of large cell carcinoma, the frequency of reduced type was extremely higher(80.0%, 12/15), indicating that most of the LCC cells had reduced TGFBR2 expression. No significant association was found between TGFBR2 expression and pathological types, age, gender, tumor size and local invasion, nodal involvement, metastasis or stage. 2. No correlation was found between TGFBR2 expression and c.492 507del mutation in LCC tissue sections.
Conclusions: 1. The differences in the expression level of TGFBR2 between NSCLC samples and corresponding normal lung tissues suggest that the defective TGFBR2 expression might contribute to the carcinogenesis and/or development of NSCLC. 2. There is significant difference on the TGFBR2 expression level between the LCC and non-LCC subtypes. 3. No correlation was found between TGFBR2 expression and c.492 507del mutation in LCC tissue sections. 4. No significant association was found between TGFBR2 expression and age, gender, tumor size and local invasion, nodal involvement, metastasis or stage.
Summary
1. A novel homozygous TGFBR2 microdeletion (c.492_507del) was identified in two cell lines derived from the same giant cell carcinoma (GCC) and was confirmed in the corresponding tumor tissues. Furthermore, a heterozygous c.492_507del was found in the germ-line of one patient, as well as in the other GCC cases and some large cell carcinomas (LCC) but not in other subtypes of NSCLC.
2. The 16 bp-microdeletion introduced a premature stop codon at positions 590–592 of the cDNA, resulting in a truncated TGFBR2 protein with a mutated transmembrane domain and loss of kinase domain. The GCC cells were characterized as being unresponsive to TGF-βinduction both in growth inhibition and stimulation of extracellular matrix protein. Moreover, after the reconstitution of wild-type TGFBR2 expression, the sensitivity to TGF-βwas restored. Therefore, mutated TGFBR2 seems to play an important role in the abrogation of TGF-βsignal transduction in GCC cells.
3. The differences in the expression level of TGFBR2 between NSCLC samples and corresponding normal lung tissues suggest that the defective TGFBR2 expression might contribute to the carcinogenesis and/or development of NSCLC. There is significant difference on the TGFBR2 expression level between the LCC and non-LCC subtypes, but no correlation was found between TGFBR2 expression and c.492 507del mutation in LCC tissue sections. No significant association was found between TGFBR2 expression and age, gender, tumor size and local invasion, nodal involvement, metastasis or stage.
4. The reduced expression of TGFBR2 and the existence of c.492_507del mutation inactivate the TGF-βsignal transduction, leading to the loss of growth inhibition of the corresponding cells and acceleration of tumor formation. Our findings in LCC provided genotypic and expression supports in the classification of LCC and non-LCC subtypes which was previously classified solely by phenotypes. LCC is distinct from AdC and SqC not only in differentiation, but also in the pattern of TGFBR2 expression and TGFBR2 mutation.
引文
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