摘要
背景：染色体3p21.3区域的异常,与肺,肾,头颈部,胸部,子宫颈癌,胃肠道等肿瘤的形成密切相关。这些异常包括纯合性缺失和杂合性丢失及蛋白表达的异常,提示该区域肿瘤抑制基因的存在。肿瘤抑制基因的存在为肿瘤的分子治疗提供了机会,在过去几十年,人们投入了大量的精力,以明确3p21.3区候选的肿瘤抑制基因并对其生物学功能进行研究。3p21.3着丝粒的关重要区域边界(也叫LUCA)大约120kb处现已发现9个肿瘤抑制基因：HYAL2, Hyall, FUS1, RASSF1, BLU, NPRL1,101F6, p16和CACNA2DA基因。这些基因的产物广泛参与生物学过程,包括细胞的增殖、细胞周期动力学、信号转导、离子交换和转运、细胞凋亡,其功能已得到很好的阐明。3p21.3还包含卢卡区以外的其他候选肿瘤抑制基因,目前还没有得到很好的研究。为了探索潜在的3p21.3区域的肿瘤抑制基因,Kiss etal.通过基因组测序发现并克隆了Leucine Zipper Transcription Factor Like 1 (LZTFL1).LZTFL1距3p21.3末端卢卡区约5 Mb处。Northern blot分析表明LZTFL1 mRNA广泛表达于人和老鼠各类组织。序列分析表明人类和小鼠LZTFL1具有90.6%的同源性。LZTFL1包含299氨基酸残基,分子量大小约为34.6KD,并含有一个基本结构域,一个螺旋-螺旋结构域,一个亮氨酸拉链结构域,提示LZTFL1在功能上可能是一个肿瘤抑制基因。然而,目前关于LZTFL1是肿瘤抑制基因缺乏生物学和遗传学上的证据。
肿瘤的发展往往与肿瘤细胞分化的缺失相关,但根本原因和机制仍知之甚少。人类实体肿瘤的绝大部分都是属于不同类型的起源上皮细胞癌。E-cadherin是维持正常上皮完整性和极性的跨膜糖蛋白,是以嗜同性方式(同种分子间拉链式结合)介导同型细胞-细胞间黏附的细胞黏附分子,其细胞浆尾段通过catenin包括α-catenin和β-catenin及其他相关蛋白结合间接与细胞骨架肌动蛋白互相作用,因此,粘合连接相关的蛋白质,对维持上皮细胞分化的状态和极性至关重要。黏附连接破坏可以通过上皮间质转化(epithelial-mesenchymal transition, EMT)的过程产生侵袭性的间质细胞,此过程可以使极性上皮细胞转化为失去极化具有侵袭性间质细胞。EMT被认为是潜在的肿瘤形成的机制之一。质膜E-cadherin的丢失可以使细胞胞浆池β-catenin的水平上升,接着β-catenin可以易位到细胞核,激活基因,促进细胞增殖和EMT。
本研究为了证实LZTFL1是否具有肿瘤抑制基因的功能,我们提出3个问题：首先,在肿瘤组织中,LZTFL1的表达是否下调,LZTFL1表达的下调是否具有任何临床意义?第二,肿瘤细胞重新获得LZTFL1的功能是否能够抑制肿瘤细胞的生长?最后,LZTFL1抑制肿瘤生长的潜在机制是什么?
方法和结果：
1. LZTFL1在人肿瘤组织中的表达及临床意义
为了研究LZTFL1的功能,首先我们制备并纯化了LZTFL1兔多克隆抗体,我们利用此抗体,用免疫组化方法,检测了组织芯片中正常组织及相应匹配的肿瘤组织LZTFLl的表达水平。在正常的乳腺,食道,胰腺,胃,卵巢,前列腺,肺,结肠,甲状腺,肾脏,膀胱,肝脏的正常组织上皮细胞,可见较高水平的LZTFL1表达,而在几乎所有相应的浸润性癌组织样本中,只有弥散的、低水平LZTFL1的表达。对组织芯片每一类正常的和相应癌组织样本多个病例的分析表明,LZTFL1的表达在上述人肿瘤组织明显下调。
为了进一步阐明在癌症患者中LZTFL1下调的临床意义,我们用免疫组化的方法检测了84名胃癌患者肿瘤组织中的LZTFL1的表达水平。肿瘤组织样本LZTFL1表达下降程度与TNM分期呈显著负相关,并与淋巴结转移个数呈显著负相关,LZTFL1表达水平与病人的生存时间显著相关,中等或较强表达LZTFL1肿瘤病人总的生存期明显好于那些无或弱表达LZTFL1肿瘤病人
2.体内外实验对LZTFL1生物学功能的研究
为了明确LZTFL1在肿瘤发生中起的作用,我们进行了获得性功能的研究,是否在肿瘤细胞中过表达LZTFL1可以抑制肿瘤细胞生长。我们使用了诱导表达系统Hela-Tet-on,加入强力霉素后,诱导培养的Hela-Tet-on细胞能持续表达LZTFL1.我们发现在锚定非依赖性的情况下(软琼脂集落形成实验),LZTFL1对肿瘤细胞的生长有抑制作用。实验结果显示,Hela-Tet-on细胞4周后形成大量的克隆,在Dox诱导后,LZTFL1表达的细胞株克隆形成数目显著减少。作为对照的Hela-Tet-on细胞加或不加强力霉素,Hela-Tet-on-EGFP细胞加或不加强力霉素和Hela-Tet-on-LZTFL1-EGFP细胞不加强力霉素克隆形成数目差异不大,提示LZTFL1确实能够特异性的抑制锚定非依赖性的肿瘤细胞的生长。我们还在HT-29细胞中测试了是否过表达LZTFL1能抑制肿瘤细胞的克隆形成,实验结果证实在这此细胞中LZTFL1有类似的抑制作用。
由于在胃癌病人组织LZTFL1的表达下调与肿瘤的转移有显著相关性,我们进一步检测了LZTFL1在细胞迁移上的作用。Hela-Tet-on-LZTFL1-EGFP在强力霉素诱导下上调表达LZTFL1后,穿过Transwell小室的细胞数目显著降低。在阴性对照,强力霉素诱导后并未对Hela-Tet-on细胞或是Hela-Tet-on-EGFP细胞的迁移产生影响,通过Transwells小室的迁移细胞数在Hela-Tet-on细胞或Hela-Tet-on-EGFP细胞有或无强力霉素诱导,及Hela-Tet-on-LZTFL 1-EGFP细胞无强力霉素诱导,穿过Transwell小室的细胞数目无明显区别。
为了进一步证实是否在体内过表达LZTFL1也能抑制肿瘤细胞的生长,克隆Hela-Tet-on-LZTFL 1-EGFP细胞及Hela-Tet-on细胞皮下注射到裸鼠左后肢。5周后,注射Hela-Tet-on细胞的裸鼠形成很大的肿瘤,而在含强力霉素水喂养的诱导表达LZTFL1的注射Hela-Tet-on-LZTFL 1-EGFP细胞的裸鼠,相比较未予强力霉素水喂养裸鼠,肿瘤的大小显著缩小。我们的结果表明,LZTFL1在体内明显抑制肿瘤生长。
3. LZTFL1抑制肿瘤细胞生长的分子机制的研究
大量研究表明,在肿瘤中,许多抑癌基因的失活是由于抑癌基因启动子区CpG岛异常甲基化引起的表观基因的沉默,或是由于组蛋白去乙酰化酶过表达引起的,所以为了初步研究LZTFL1在肿瘤细胞中失活的机制,我们分别用DNA甲基化酶抑制剂5’-氮杂2’-脱氧胞苷和丁酸钠(组蛋白去乙酰化酶抑制剂,HDAC抑制剂)处理人肠癌细胞HT-29。在5’-氮杂2’-脱氧胞苷处理和未处理的细胞之间,LZTFL1的表达水平没有变化,而丁酸钠处理后LZTFL1表达显著增高。其他的HDAC抑制剂具有相似上调HT-29的LZTFL1表达的效果。这些结果提示LZTFL1在HT-29细胞中失活是由于染色体结构的改变所引起的。丁酸钠是肠道内自然产生的复合物,它能诱导肠上皮细胞的分化。丁酸钠处理后,HT-29细胞LZTFL1表达上调提示LZTFL1的表达水平与细胞的分化状态相关。为了在体内验证这个假设,我们用免疫组化染色的方法用LZTFL1抗体研究了小鼠小肠隐窝-绒毛轴LZTFL1的表达情况。小肠上皮处于持续自我更新的过程,在隐窝处的干细胞处于快速增殖和分化的状态,并不断向肠绒毛的顶端的迁徙。实验结果表明,沿着隐窝-绒毛轴,LZTFL1的表达水平逐渐递升,在隐窝处,LZTFL1表达水平最低,在绒毛的顶端,LZTFL1的表达最强,进一步,我们用共聚焦荧光显微镜进行了LZTFL1和E-cadherin/β-catenin共定位研究。在正常分化的肠上皮细胞中,LZTFL1的表达与E-cadherin在质膜上重叠,而在结肠肿瘤中,由于LZTFL1的表达缺失,不具有共定位的特征。
LZTFL1和E-cadherin的共定位提示LZTFL1可能可以稳定E-cadherin介导的黏附连接。的确我们的实验结果表明,用phorbol 12-myristate 13-acetate (PMA)处理细胞(PMA是一种已知的能够破坏上皮细胞紧密连接的分散因子),相比较EGFP-HT-29或是HT-29细胞,表达LZTFL1的HT-29细胞能更耐受PMA诱导的细胞分散。
结论：我们的研究结果表明,LZTFL1是一个肿瘤抑制因子,LZTFL1表达缺失有显著的临床意义。胃癌患者中,LZTFL1表达水平可作为一个独立的判断预后的指标。我们推测,LZTFL1通过促进肿瘤细胞的分化,从而抑制肿瘤的生长,通过抑制上皮-间质转化从而抑制肿瘤细胞转移。在肿瘤细胞中上调表达LZTFLl可能对癌症分化治疗具有临床价值。
Background:Chromosomal abnormalities at the 3p21.3 region are frequent and early events in the formation of human tumors of the lung, kidney, head and neck, breast, cervix, and gastrointestinal tract. These abnormalities range from homozygous deletions and loss of heterozygosity to loss of protein expression, suggesting the presence of tumor suppressor genes (TSG) in this region. As TSGs offer opportunities for molecular cancer therapy, there have been intense efforts during the past decades to identify candidate 3p21.3 TSGs and to characterize their biological functions. A critical region of～120 kb in the 3p21.3 centromeric border (also called LUCA region) has been identified and contains nine candidate TSGs:HYAL2, HYAL1, FUS1, RASSF1, BLU, NPRL1,101F6, PL6 and CACNA2DA. The functions of many of these gene products are being elucidated and they seem to be involved in a wide spectrum of biological processes, including cell proliferation, cell cycle kinetics, signaling transduction, ion exchange and transportation, and cell death. The 3p21.3 region also contains other candidate TSGs outside the LUCA region, but they are less well studied. In an effort to discover potential TSGs in the 3p21.3 region, Kiss and colleagues identified and cloned leucine zipper transcription factor-like 1 (LZTFLl) through an elimination test and subsequent genomic sequencing and cDNA cloning. LZTFL1 is located-5 Mb from the LUCA region on the telomeric end of the 3p21.3 region. Northern blot analysis indicates that LZTFL1 mRNA is expressed ubiquitously in both human and mouse. The open reading frame from human and mouse cDNAs revealed a protein of 299 amino acids with a molecular weight of 34.6 kDa. The sequence analysis suggested that LZTFL1 shares 90.6% sequence identity between human and mouse. LZTFL1 contains a basic region, a coilcoil domain, and a leucine zipper domain, it was suggested that LZTFL1 may function as a tumor suppressor. However, the biochemical and genetic evidence of LZTFL1 as a tumor suppressor gene is lacking.
The loss of differentiation in cancer cells is often associated with tumor progression, but the underlying causes and mechanisms remain poorly understood. The majority of human solid tumors are carcinomas that originated from various epithelial cell types. Differentiated carcinomas are composed of cohesive polarized epithelial cells connected to one another by intercellular adherens junctions. E-cadherin is the core molecule of adherens junctions. The cytoplasmic tail of E-cadherin is indirectly linked to the actin cytoskeleton through catenins, includingα-andβ-catenin, and other associated proteins. The attachments of E-cadherin to the cytoskeleton, hence associated proteins in the adherens junction, are essential for maintaining the differentiated state of epithelial cells and the apico-basal polarity of the epithelium. Disruption of the adherens junction can generate invasive mesenchymal cells through a process called epithelial-mesenchymal transition (EMT), which converts polarized, immotile epithelial cells to motile invasive mesenchymal cells. EMT has been proposed to be a potential mechanism for carcinoma metastases. Loss of membranous E-cadherin can also increase the cytoplasmic pool ofβ-catenin, which can then translocate to the nucleus and activate genes that promote cell proliferation and EMT.
In the present study, we sought to test whether LZTFL1 functions as a tumor suppressor. We asked three experimental questions. First, is LZTFL1 expression downregulated in tumors and does the loss of LZTFL1 expression have any clinical significance? Second, can LZTFL1 gain-of function inhibit tumor growth? Finally, what is the potential mechanism(s) behind LZTFL1 inhibition of tumor cell growth?
Methods and Findings:
1. The expression of LZTFL1 in human tumors and its clinical significance
In order to study the biological function of LZTFL1, we first generated and affinity-purified a rabbit polyclonal antibody against LZTFL1. We next surveyed the expression of LZTFL1 in various normal human tissues and their corresponding cancer samples by immunohistochemical analysis of tissue microarrays. Intense LZTFL1 staining was visible in epithelial cells of normal tissues of breast, esophagus, pancreas, stomach, ovary, prostate, lung, colon, thyroid, kidney, bladder, and liver. In almost all the corresponding invasive carcinoma samples, only diffused, low levels of LZTFL1 staining were observed. Analysis of multiple cases in each individual type of normal and matched cancer samples in the tissue microarray showed that LZTFL1 was significantly downregulated in the aforementioned human tumors.
To address the clinical significance of downregulation of LZTFL1 in cancers, tissue samples from a cohort of 84 patients diagnosed with the stomach cancer were screened by immunohistochemistry for LZTFL1 expression. Loss of LZTFL1 expression was found to have significant inverse correlations with TNM stages of the tumor and with the number of metastasized lymph nodes, and LZTFL1 expression level correlated significantly with the survival time as well, The overall survival was significantly better for patients with tumors demonstrating moderate or strong LZTFL1 expression than those whose tumors showed negligible or weak expression.
2. The biological function of LZTFL1 in vitro and in vivo
In order to determine whether LZTFL1 plays a role in the tumorigenesis, we performed gain-of-function studies to test whether increased level of LZTFL1 expression in tumor cells can inhibit tumor cell growth. We used an inducible expression system to induce LZTFL1 expression through addition of doxycycline (Dox) in cultured Hela-Tet-on cells that constitutively produce the reverse tetracycline transactivator. We found that LZTFL1 has effect on tumor cell growth under anchorage-independent conditions in soft agar assays. A large number of colonies of Hela-Tet-on cells were visible within 4 weeks, The LZTFL1-expressing cells upon addition of Dox showed dramatically reduced number of colonies. As controls, the numbers of colonies in Hela-Tet-on cells with and without Dox, Hela-Tet-on-EGFP cells with and without Dox, and in Hela-LZTFL1-EGFP cells without Dox were similar, suggesting that LZTFL1 indeed specifically inhibited anchorage-independent growth of tumor cells. We also tested whether overexpression of LZTFL1 inhibits the colony formation ability of intestinal epithelial carcinoma cell HT-29 and observed similar inhibitory effect of LZTFL1 in this cell.
As downregulation of LZTFL1 in human gastric tumors correlated with tumor metastases, we next investigated a role for LZTFL1 in cell migration. Upregulation of LZTFL1 in Hela-Tet-on-LZTFL-EGFP upon Dox induction significantly reduced the migration properties of Hela cells in Transwell assays, In negative controls, Dox had no effect on migration of Hela-Tet-on cells. The number of cells migrated through the Transwells are similar among Hela-Tet-on cells, Hela-Tet-on-EGFP cells and Hela-Tet-on-LZTFL1-EGFP cell without Dox.
To further test whether overexpression of LZTFL1 results in suppression of tumor growth in vivo, we injected subcutaneously Hela-Tet-on and Hela-Tet-on-LZTFL1-EGFP cells into the flank of nude mice. As expected, at the end of 5 weeks, mice injected with Hela-Tet-on cells developed large tumors. The tumor size in mice with Hela-Tet-on-LZTFLl-EGFP cells in the presence of Dox in the drinking water for induction of LZTFL1 were significantly reduced compared to those in the absence of Dox. Our results suggest that LZTFLl inhibited tumor growth significantly in vivo.
3. The mechanism of LZTFL1 inhibit tumor cell growth
It has been demonstrated that many TSGs are inactivated in cancer by epigenetic silence induced by aberrant methylation of CpG island in the promoter region of the TSG or by overexpression of histone deacetylases(HDACs). To understand the mechanism of LZTFL1 inactivation in tumor cells, we treated HT-29 cells with 5'-aza-2'-deoxycytidine, a DNA methylation inhibitor, and Sodium butyrate (NaB), a HDAC inhibitor, respectively. No difference of LZTFL1 expression was observed between 5'-aza-2'-deoxycytidine treated and untreated cells whereas NaB treatment increased the level of LZTFL1 expression. Other HDAC inhibitors had similar effects on the upregulation of LZTFL1 expression in HT-29 cells, These results suggest that LZTFL1 is inactivated in HT-29 cells by alterations in chromatin structure.
NaB is a naturally-occurring compound in the intestine and induces differentiation of epithelial cells in culture, Upregulation of LZTFL1 in NaB treated HT-29 cells suggests that the expression level of LZTFL1 may be correlated with the differentiation status of the cell. To test this hypothesis in vivo, we stained the mouse small intestine with anti-LZTFL1 antibody along the crypt-villus axis. The intestinal epithelium undergoes constant self-renewing processes. A graded expression of LZTFL1 along the crypt-villus axis was observed with a minimal staining of LZTFLl in the crypt and maximum staining at the apex of the villus. Next, we performed co-localization studies of LZTFL1 with E-cadherin/β-catenin using confocal immunofluorescence microscopy. Expression of LZTFLl overlaps with that of E-cadherin at the plasma membrane in differentiated normal colonic epithelial cells, This co-localization was absent in colorectal carcinomas due to a loss of LZTFL1 protein expression.
LZTFL1 and E-cadherin co-localization suggest LZTFL1 may be able to stabilize E-cadherin-mediated adhesion junctions. Indeed, we observed that, when treated with phorbol 12-myristate13-acetate(PMA), a known scatter factor to break down the epithelial tight junction, the LZTFL1 expressing HT-29 cells are more resistant to PMA-induced cell scattering than EGFP-expressing or parental HT-29 cells.
Conclusions:Our results indicate that LZTFL1 is a tumor suppressor and loss of LZTFL1 expression has significant clinical outcomes.LZTFL1 expression may serve as an independent prognostic marker for survival outcome of gastric cancer patients. We hypothesize that LZTFL1 may inhibit tumor cell growth by promoting tumor cell differentiation and tumor metastasis through suppression of epithelail-mesenchymal transition. Upregulation of LZTFL1 expression in tumors may have therapeutic value in differentiation therapy against cancer.
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