【作者】 朱颖；

【导师】 朱文；

【作者基本信息】 四川大学 ， 生物与医药， 2023， 博士

【摘要】 背景和目的肺癌是全球死亡率第一的恶性肿瘤,严重威胁人类健康。非小细胞肺癌(Non-small cell lung cancer,NSCLC)是肺癌的主要病理类型,约占肺癌总数的85%,主要包括肺腺癌(Lung adenocarcinoma,LUAD)和肺鳞癌(Lung squamous cell carcinoma,LUSC),其中,LUAD是NSCLC的主要类型。由于肺癌早期阶段临床症状较为隐匿,大部分患者确诊时已处于晚期,晚期(Ⅳ期)肺癌患者五年生存率仅约5%。因此,深入揭示NSCLC起源发生及发展的分子机制将有助于肺癌的早期诊断和治疗。肺是一个开放性器官,是人体与外界环境进行气体交换的主要场所。肺泡约占肺表面积的99%,是肺部气体交换的主要部位。因此,肺泡极易受到外界环境的直接损伤。成熟的肺泡由肺泡I型上皮细胞(Alveolar type Ⅰ epithelial cells,AT1)和肺泡Ⅱ型上皮细胞(Alveolar type Ⅱ epithelial cells,AT2)组成。AT1细胞呈扁平状,约占肺泡上皮表面积的95%,主要分布于毛细血管周围,通过提供较大的肺泡表面积促进气体交换。AT2细胞呈立方状,位于肺泡角,具有特异的层状体结构和顶端微绒毛,约占肺泡上皮细胞表面积的5%,通过分泌表面活性物质,维持肺泡表面张力,防止肺泡塌陷。高度特征化的AT2细胞不仅在气体交换中发挥重要作用,在肺泡的自我更新和损伤修复过程中也至关重要。目前,已在小鼠模型以及人源肺泡类器官模型中证实AT2细胞是肺泡干细胞。肿瘤干细胞(Cancer Stem Cells,CSCs)是肿瘤的起源细胞,其可来源于正常干细胞的异常恶性转化。NSCLC是一组具有明显异质性的恶性肿瘤,肺部存在多种干细胞被认为与其异质性密切相关。近年来,肺部AT2细胞与NSCLC起源发生的关系引起了更多关注。由于AT2细胞具有多功能干细胞性质,这使得异常的AT2细胞具有可恶性转化成为NSCLC细胞的潜能。近年来多个研究已在多种肺癌基因工程小鼠模型(Genetically engineered mouse models,GEMMs)中证实异常的AT2细胞是NSCLC的起源细胞之一。在人NSCLC中,较早期的研究仅在病理组织学水平发现NSCLC细胞可表达AT2细胞标志物,且含有AT2细胞特有的细胞器层状体,提示人NSCLC细胞可能来源于AT2细胞的恶性转化。早在2014年一项发表于Nature杂志的研究指出小鼠AT2细胞中EGFR(Epidermal growth factor receptor)或KRAS(Kirsten rat sarcoma)激活突变可驱动AT2细胞自我复制形成AT2样细胞,并进一步促进肺部肿瘤形成,但是相关分子机制尚未明晰。最近的研究利用单细胞测序技术在处于不同时期的25例临床病例组织的52个样本(3个不典型增生组织样本、5个原位腺癌组织样本、9个微浸润腺癌组织样本、17个肺腺癌IA期组织样本以及18个癌旁非肿瘤组织样本)中发现了一群与AT2细胞高度相似的细胞群(称作AT2样细胞)出现在具有恶性特征的IA期LUAD组织细胞中,且AT2样细胞的转录谱已开始异于AT2细胞。该结果表明异常的AT2细胞是人LUAD的起源细胞,也进一步提示肺部AT2细胞中的基因表达异常可能与NSCLC起源发生相关。但是,目前对AT2细胞特异性基因的表达异常在NSCLC起源发生及发展中的分子机制鲜见报道。SLC34A2基因属于溶质转运蛋白家族,该基因定位于人类基因组4p15.2,编码钠依赖的磷酸盐协同转运蛋白Napi2b,该蛋白表达于多种上皮组织细胞的细胞膜。已有研究通过在多种组织中检测SLC34A2的表达发现其在肺和胚胎肺中表达量最高。特别地,SLC34A2被认为在肺部特异性表达于AT2细胞。此外,在体外诱导人多能干细胞分化为成熟且具有功能的AT2细胞的过程中,SLC34A2表达逐渐上调,因此,SLC34A2也可作为人肺部AT2细胞成熟的标志之一。特别是,最新的研究在人胚胎肺部芽尖祖细胞构建的类器官模型中发现,表达AT2细胞标志基因SFTPC的肺泡谱系类器官组织在分化成为肺泡类器官的过程中,SLC34A2与SFTPC基因的表达均逐渐上调,表明AT2细胞参与了肺泡类器官的形成过程,更进一步提示SLC34A2在维持人肺部AT2细胞的功能中扮演重要角色。同时,SLC34A2基因编码的Napi2b蛋白在人和小鼠之间的同源性高达76%,其主要生理功能是在细胞外低磷状态时可调控无机磷离子由细胞外向细胞内逆浓度梯度主动运输,以促进细胞对无机磷离子的吸收,从而维持细胞对无机磷离子的生理需求。特别地,在肺部AT2细胞中,Napi2b蛋白是由SLC34家族基因编码的表达量最高的无机磷离子转运蛋白。无机磷酸盐(Inorganic phosphate,Pi)是人体内一种丰富的元素,参与维持细胞正常结构和功能,进而调控体内多种关键的生命过程。已有研究报道Pi稳态失调与包括肺癌在内的多种肿瘤发生密切相关。但是SLC34A2基因表达异常与体内Pi稳态失调和肺癌起源发生的具体调节机制目前尚不清楚。SLC34A2在肺部AT2细胞中承担重要作用,其表达异常势必会影响AT2细胞的正常功能。既往研究发现,SLC34A2的表达在人和大鼠胚胎肺发育过程中逐渐上调,直至肺发育成熟后维持恒定,表明SLC34A2在肺发育和肺稳态维持中至关重要。但与胚胎肺发育过程相反,相较于癌旁非肿瘤组织,SLC34A2的表达量在15例临床NSCLC组织中显著下调了10倍。一致地,我们的前期研究也证实了,相较于癌旁非肿瘤组织和肺上皮细胞,SLC34A2的表达水平在NSCLC组织和NSCLC细胞株中均显著下调。我们通过进一步的生物学功能研究证明,过表达SLC34A2可在体外显著抑制NSCLC细胞增殖、迁移和侵袭,在体内显著抑制裸鼠NSCLC皮下移植瘤细胞生长。在初步的机制研究中,我们发现过表达SLC34A2抑制NSCLC恶性表型可能与抑制MEK/ERK和PI3K/AKT/mTOR信号通路关键蛋白的表达相关,但是尚未阐明具体分子机制。此外,我们前期研究通过基因芯片检测还发现,过表达SLC34A2可显著上调硒结合蛋白1(Selenium binding protein 1,SELENBP1)的表达。最近,我们的研究证实,相较于对应癌旁非肿瘤组织,SELENBP1在45/59例NSCLC组织中表达显著下调;同时,SELENBP1蛋白与AT2细胞标志蛋白SP-C定位一致,均表达于肺泡角,证明了SELENBP1同样显著表达于肺部AT2细胞。通过进一步的机制研究,我们还证明过表达SELENBP1可通过抑制PI3K/AKT/mTOR通路抑制NSCLC发生发展。综上研究结果提示SLC34A2可能通过上调SELENBP1的表达抑制PI3K/AKT/mTOR通路进而抑制NSCLC发生发展。因此,基于我们的前期研究基础,本研究将从临床NSCLC组织、人NSCLC细胞系以及裸鼠NSCLC皮下移植瘤模型三个方面深入探究SLC34A2抑制NSCLC发生发展的潜在分子机制。这不仅可以为临床肺癌早期诊断和治疗靶点的选择提供理论依据,也为今后深入理解肺部AT2细胞的异常在NSCLC起源发生和发展中的分子机制奠定科学基础。研究方法和结果1.为了进一步在大规模临床样本中获取SLC34A2在癌旁非肿瘤组织和NSCLC组织中的表达差异,首先,我们通过对TCGA数据库中513例LUAD组织和59例癌旁非肿瘤组织、223例LUSC组织和17例癌旁非肿瘤组织以及GEO数据库中53例LUAD组织和11例癌旁非肿瘤组织的分析表明,相较于癌旁非肿瘤组织,SLC34A2的表达水平在NSCLC组织中显著下调(P<0.05)。紧接着,我们通过qRT-PCR在收集的84对临床NSCLC组织样本中进一步证实,相较于对应癌旁非肿瘤组织,SLC34A2的表达在NSCLC组织中的确显著下调(P<0.05)。此外,我们通过IHC(n=11)和Western blot(n=6)在收集的临床样本的蛋白水平同样证实,与对应癌旁非肿瘤组织相比,Napi2b(SLC34A2编码)蛋白的表达水平在NSCLC组织中也显著下调(P<0.05)。由此,我们在TCGA、GEO数据库和收集的临床组织样本中证实,相较于癌旁非肿瘤组织,SLC34A2的表达水平在NSCLC组织中显著下调。接下来,为了探究SLC34A2的临床应用价值,我们在TCGA数据库和GEO数据库中分析了其表达水平与临床信息的关联性。TCGA数据库分析结果初步表明,SLC34A2的表达水平在NSCLC(LUAD和LUSC)不同临床分期之间无显著差异(P>0.05);同时,GEO数据库两个分别含有50例LUAD样本分期信息的数据集的分析也得到了一致的结果。由于我们收集的LUSC样本例数较少,因此,仅在收集的73例LUAD组织中分析了不同临床分期之间SLC34A2的表达差异。我们的分析结果与TCGA和GEO数据库分析结果一致,在收集的73例LUAD样本中,SLC34A2的表达水平在I期与Ⅱ期、Ⅱ期与ⅡI期之间无显著差异(P>0.05)。此外,对GEO数据库的51例LUAD样本的分析结果显示,SLC34A2的表达水平在不同组织分化程度中无显著差异(P>0.05)。一致地,我们收集的59例LUAD组织样本中SLC34A2的表达水平在不同组织分化程度间也无显著差异。以上结果表明,SLC34A2的表达水平在NSCLC不同临床分期和分化程度之间均无显著差异,提示了SLC34A2可能是NSCLC(特别是LUAD)早期起源发生相关的基因。随后,根据SLC34A2基因的表达水平,我们对TCGA数据库中240例LUAD样本和242例LUSC样本进行生存分析。根据SLC34A2表达水平的中位数分为SLC34A2相对低表达组和SLC34A2相对高表达组,分析结果显示,在LUAD中,与SLC34A2相对高表达的患者相比,SLC34A2相对低表达的患者总体生存率明显降低(P<0.005);但是,与LUAD不同,在LUSC中,SLC34A2相对高表达预示患者总体生存率降低(P<0.005)。同时,我们对GEO数据库的51例LUAD样本的分析结果与TCGA数据库LUAD样本的结果相一致。另外,在我们收集的73例LUAD样本中,相较于无淋巴结转移的病例组,有淋巴结转移的病例组患者SLC34A2的表达水平显著降低(P<0.05)。由此,上述结果表明,LUAD组织中,SLC34A2相对低表达与临床预后不良相关,提示SLC34A2在LUAD中具有一定的预后诊断价值。2.基于本课题组前期研究已经证明过表达SLC34A2可以显著抑制NSCLC细胞增殖、迁移和侵袭,且过表达SLC34A2可以显著上调SELENBP1的表达。另外,我们最近的研究证实过表达SELENBP1也可以显著抑制NSCLC细胞增殖、迁移和侵袭的恶性表型。为了探究过表达SLC34A2抑制NSCLC发生发展的潜在分子机制是否与SELENBP1相关,首先,我们分析了SLC34A2与SELENBP1的表达相关性。在TCGA数据库检索的483例LUAD和486例LUSC样本中,SLC34A2与SELENBP1的m RNA表达量均呈显著正相关(P<0.05)。同时,GEO数据库中两个分别含有53例和51例LUAD样本的数据集的分析也得到了一致的结果。随后,在我们收集的65例临床NSCLC组织中,Spearman相关分析进一步表明SLC34A2和SELENBP1的m RNA表达量显著正相关(P<0.05);且相较于癌旁非肿瘤组织,在45例SLC34A2下调的临床LUAD组织中,35/45例(77.78%)LUAD组织的SELENBP1的m RNA水平也显著下调表达。接下来,在这35例SLC34A2和SELELNBP1均显著下调的LUAD组织中随机挑选了11例,通过Western blot(n=10)和IHC(n=11)实验更进一步证实,与对应癌旁非肿瘤组织相比,这11例临床LUAD组织中Napi2b(SLC34A2)、SELENBP1蛋白均也显著下调表达,两者呈正向表达关系,且在癌旁非肿瘤组织中,Napi2b和SELENBP1蛋白的表达均定位于肺泡角AT2细胞。最后,在过表达SLC34A2的NSCLC细胞系中,我们发现过表达SLC34A2可以显著上调SELENBP1的m RNA和蛋白表达水平(P<0.05)。由此,我们在TCGA、GEO数据库和收集的临床NSCLC组织样本中证明了SLC34A2与SELENBP1的表达量显著正相关,同时在收集的临床NSCLC组织样本和过表达SLC34A2的人NSCLC细胞系中证明了SLC34A2与SELENBP1的正向表达关系。3.基于我们前期研究已经证明过表达SELENBP1可通过抑制PI3K/AKT/mTOR通路抑制NSCLC细胞增殖、迁移和侵袭,为了进一步探究过表达SLC34A2能否通过上调SELENBP1的表达抑制PI3K/AKT/mTOR通路进而抑制NSCLC细胞增殖、迁移和侵袭,首先,我们通过qRT-PCR和Western blot实验明确过表达SLC34A2的NSCLC细胞中SELENBP1已被成功敲低。接下来,CCK-8实验结果显示,过表达SLC34A2的NSCLC细胞中敲低SELENBP1可显著增强NSCLC细胞增殖能力(P<0.01)。克隆形成实验结果表明,过表达SLC34A2的NSCLC细胞中敲低SELENBP1可显著促进NSCLC细胞克隆形成能力(P<0.01)。划痕愈合实验结果说明,过表达SLC34A2的NSCLC细胞中敲低SELENBP1后细胞的横向迁移能力显著增强(P<0.005)。Millicell实验结果显示,过表达SLC34A2的NSCLC细胞中敲低SELENBP1可以显著促进NSCLC细胞纵向迁移能力(P<0.005)。Transwell实验结果表明,过表达SLC34A2的NSCLC细胞中敲低SELENBP1后细胞的侵袭能力显著增强(P<0.005)。接下来,通过Western blot实验,我们发现过表达SLC34A2的NSCLC细胞中敲低SELENBP1可显著上调p-PI3K、p-AKT以及p-mTOR的蛋白表达水平(P<0.05)。以上结果证明过表达SLC34A2可以通过上调SELENBP1的表达抑制PI3K/AKT/mTOR信号通路进而抑制NSCLC细胞增殖、迁移和侵袭。4.由于Napi2b(SLC34A2)蛋白位于细胞膜,无法直接调控SELENBP1的表达。已有研究表明转录因子TTF-1(Thyroid transcription factor-1)可以与SELENBP1的启动子结合,进而上调SELENBP1的表达。在进一步的机制研究中,为了阐明NSCLC细胞中过表达SLC34A2能否通过TTF-1上调SELENBP1的表达,首先,我们分析了SLC34A2与TTF-1的表达相关性。TCGA数据库NSCLC组织中SLC34A2与TTF-1表达水平的相关性分析显示,在483例LUAD中,SLC34A2与TTF-1的m RNA表达水平显著正相关(P<0.05);但是,在486例LUSC中获得了相反的结果(P<0.005)。随后,结合前述收集的临床LUAD组织中SELENBP1的表达情况,在收集的35例SLC34A2、SELENBP1相较于癌旁非肿瘤组织均下调表达的LUAD组织中统计发现,与癌旁非肿瘤组织相比,22/35(62.86%)例LUAD组织中TTF-1的m RNA表达水平也显著下调。接下来,在上述22例SLC34A2、SELELNBP1、TTF-1均显著下调的LUAD组织中随机挑选了11例,通过Western blot(n=6)和IHC(n=11)实验共同证实收集的临床LUAD组织中Napi2b(SLC34A2)、SELENBP1和TTF-1蛋白均也显著下调表达,呈正向表达关系;同时,在癌旁非肿瘤组织中,Napi2b、SELENBP1和TTF-1蛋白的表达均定位于AT2细胞。最后,在过表达SLC34A2的NSCLC细胞系中,我们发现过表达SLC34A2可以显著上调TTF-1的m RNA和蛋白表达水平(P<0.05)。至此,我们通过TCGA数据库证明NSCLC特别是LUAD中SLC34A2、SELENBP1、TTF-1的表达显著正相关,同时在收集的临床NSCLC组织和过表达SLC34A2的人NSCLC细胞系中证明了SLC34A2、SELENBP1、TTF-1的正向表达关系。接下来,为了探究NSCLC细胞中过表达SLC34A2通过TTF-1上调SELENBP1表达的分子机制,我们首先通过JASPAR在线数据库预测得到转录因子TTF-1可能与SELENBP1启动子区域的5个位置以及TTF-1启动子区域的3个位置结合。再通过CUT&TAG实验结果证明,在NSCLC细胞中,过表达SLC34A2可以显著促进TTF-1与SELENBP1启动子区域的5个结合位置结合;同时也显著促进与TTF-1启动子区域其中2个结合位置的结合(P<0.05)。由此,以上结果表明,在NSCLC细胞中,过表达SLC34A2可以显著促进转录因子TTF-1与SELENBP1和TTF-1的启动子结合。转录因子发挥作用受到其转录活性的调节。已有研究证实激活MEK/ERK通路可增加TTF-1在Ser327位点的磷酸化水平,进而抑制TTF-1转录活性。为了进一步揭示NSCLC细胞中过表达SLC34A2是否通过抑制MEK/ERK通路增强TTF-1转录活性,我们通过Western blot实验发现,NSCLC细胞中过表达SLC34A2可以显著降低TTF-1(Ser327)磷酸化水平,使用MEK/ERK抑制剂的对照组细胞中TTF-1(Ser327)磷酸化水平也显著下调(P<0.05)。结合前述结果已证明NSCLC细胞中过表达SLC34A2可显著上调SELENBP1和TTF-1的m RNA和蛋白表达水平。由此,综上结果证明,NSCLC细胞中过表达SLC34A2可通过抑制MEK/ERK信号通路降低TTF-1(Ser327)磷酸化水平进而增强TTF-1转录活性。随后,qRT-PCR和Western blot实验结果进一步表明敲低TTF-1可以显著降低A549-SLC34A2和H1299-SLC34A2中SELENBP1的m RNA和蛋白表达水平(P<0.05)。至此,该部分实验结果证明,过表达SLC34A2可以通过抑制MEK/ERK信号通路降低TTF-1(Ser327)磷酸化水平,进而通过增强TTF-1转录活性介导SELENBP1的表达上调。5.此外,在进一步探究过表达SLC34A2抑制MEK/ERK信号通路的分子机制研究中,我们关注到RAS蛋白是经典MEK/ERK信号轴上游的重要蛋白。RAS蛋白作为RAS家族的小GTPase,在信号转导过程中作为二元开关发挥作用,通过接收上游受体酪氨酸激酶信号,RAS开关调节RAS蛋白与GDT结合的失活状态和与GTP结合的激活状态之间的转化。与RAS蛋白相似,LRRK2蛋白也是一个可以与GDP/GTP结合的分子开关,且LRRK2在肺中主要表达于AT2细胞。因此,我们推测LRRK2可能参与SLC34A2调控MEK/ERK通路的过程。为了进一步探究NSCLC细胞中过表达SLC34A2能否通过LRRK2抑制MEK/ERK信号通路,首先,我们通过TCGA数据库中的分析发现,在483例LUAD和486例LUSC中,SLC34A2与LRRK2的表达量均显著正相关(P<0.05);同时,我们对GEO数据库中两个分别含有53例和51例LUAD样本的数据集的分析也获得了一致的结果。紧接着,相较于癌旁非肿瘤组织,在收集的29例SLC34A2下调表达的LUAD中统计发现,与对应癌旁非肿瘤组织相比,23/29(79.31%)例LUAD组织中LRRK2的m RNA表达也显著下调。接下来,在这23例SLC34A2、LRRK2均显著下调的LUAD组织中随机挑选了6例,通过Western blot和IHC实验进一步证明,相对于癌旁非肿瘤组织,Napi2b(SLC34A2)显著下调的LUAD组织中LRRK2蛋白水平也显著下调,呈正向表达关系;且在癌旁非肿瘤组织中,Napi2b(SLC34A2)与LRRK2蛋白的表达均定位于AT2细胞。最后,在过表达SLC34A2的NSCLC细胞中,我们还发现过表达SLC34A2可以显著上调LRRK2的m RNA和蛋白表达水平(P<0.05)。由此,我们通过对TCGA、GEO数据库中信息的分析证明NSCLC中SLC34A2与LRRK2的表达正相关,同时在收集的临床NSCLC组织和过表达SLC34A2的人NSCLC细胞系中均证明了SLC34A2与LRRK2的正向表达关系。接下来,Western blot实验结果表明,在过表达SLC34A2的NSCLC细胞中,敲低LRRK2后可以显著上调MEK/ERK信号通路中关键蛋白MEK和ERK1/2的磷酸化水平(P<0.05),从而逆转了过表达SLC34A2对MEK/ERK信号通路的抑制作用。此外,NSCLC细胞中过表达SLC34A2还可以显著上调LRRK2(T1503)的磷酸化水平(P<0.05)。至此,我们的研究结果证明,NSCLC中过表达SLC34A2可通过上调LRRK2的表达抑制MEK/ERK信号通路。6.最后,为了更进一步在体内验证过表达SLC34A2可上调SELENBP1、TTF-1和LRRK2的表达以及过表达SLC34A2抑制NSCLC细胞生长与抑制MEK/ERK和PI3K/AKT/mTOR通路相关,我们构建了裸鼠NSCLC皮下移植瘤模型,结果显示A549-SLC34A2组肿瘤体积显著小于A549-Control组(P<0.05);且相较于A549-Control组移植瘤组织,A549-SLC34A2组移植瘤组织中Ki-67蛋白表达显著下调(P<0.001)。接下来,IHC和Western blot实验结果表明,与过表达SLC34A2的NSCLC细胞株中体外实验结果相一致,A549-SLC34A2组移植瘤组织中SELENBP1、TTF-1、LRRK2的蛋白表达均显著高于A549-Control组移植瘤(P<0.01)。最后,IHC和Western blot实验结果共同证明,与A549-Control组移植瘤组织相比,A549-SLC34A2组皮下移植瘤组织中p-MEK、p-ERK1/2、p-PI3K、p-AKT和p-mTOR蛋白表达均显著下调(P<0.01)。至此,我们在裸鼠NSCLC皮下移植瘤模型中证明,过表达SLC34A2可上调SELENBP1、TTF-1和LRRK2的蛋白表达水平,且过表达SLC34A2抑制NSCLC细胞生长与抑制MEK/ERK和PI3K/AKT/mTOR通路激活相关。结论综上,本研究首次从临床NSCLC组织、人NSCLC细胞系、裸鼠NSCLC皮下移植瘤模型三个方面深入探究了SLC34A2抑制人NSCLC发生发展的分子机制。研究结果发现,在癌旁非肿瘤组织中,Napi2b(SLC34A2)、SELENBP1、TTF-1和LRRK2蛋白均明显表达于肺部AT2细胞。并且,相较于对应癌旁非肿瘤组织,SLC34A2、SELENBP1、TTF-1和LRRK2的m RNA和蛋白在NSCLC组织中均显著下调表达,且SLC34A2与SELENBP1、TTF-1和LRRK2的表达水平显著正相关。分子机制研究中证明了,在NSCLC中,过表达SLC34A2可通过上调LRRK2的表达抑制MEK/ERK信号通路激活,进而降低TTF-1(Ser327)磷酸化水平,增强TTF-1转录活性介导SELENBP1的表达上调,进一步通过SELENBP1抑制PI3K/AKT/mTOR信号通路的激活最终抑制NSCLC发生发展。本研究建立了SLC34A2/LRRK2/TTF-1/SELENBP1信号轴在NSCLC发生发展中的调控网络,这不仅为临床NSCLC早期诊断及分子治疗策略提供新靶点,也为今后深入理解肺部AT2细胞异常在NSCLC起源发生及发展中的分子机制奠定了科学基础。更多还原

【Abstract】 Background and ObjectiveLung cancer（LC）,the deadliest malignant tumor worldwide,severely threatens to human health.Non-small cell lung cancer（NSCLC）constitutes the predominant histological subtype of LC,accounting for approximately 85%of all LC cases.Lung adenocarcinoma（LUAD）and Lung squamous cell carcinoma（LUSC）are two predominant histological phenotypes of NSCLC.LUAD represents the main pathological entity of NSCLC.Due to the lack of typically asymptomatic at early disease,most cases were diagnosed at an advanced stage,and the 5-year survival rate of patients with stage Ⅳ disease was only approximately 5%.Therefore,further elucidation of the underlying molecular mechanisms in origin and development of LC will contribute to diagnosis and treatment of LC.The most essential function of mammalian lung is to facilitate gas exchange between the human body and external environment.As alveoli represents 99%of the surface area of the lung,and serves as the main site for gas exchange in lung,thus the alveolar is also continually exposed to direct damage from external environment.The mature alveolar is constituted by alveolar type Ⅰ（AT1）and alveolar type Ⅱ（AT2）epithelial cells.AT1 cells are flat,occupy about 95%of the alveolar epithelial surface and primarily cover the peripheral capillaries,thereby promote gas exchange through providing an expansive alveolar surface area.AT2 cells are cuboidal and located in the alveolar corners,characterized with lamellar body and apical microvilli.AT2 cells make up about 5%of alveolar epithelial cells and secrete surfactant to maintain alveolar surface tension,preventing alveolar collapse.The highly characteristic AT2 cells play an important role not only in gas exchange but also during the process of alveolar self-renewal and injury repair.Currently,in both mouse models and human-derived alveolar organoids,AT2 cells has been identified as alveolar stem cells.Cancer stem cells（CSCs）are the progenitor cells responsible for tumor initiation,deriving from malignant transformation of the normal stem cells.NSCLC represents a highly heterogeneous group of malignant tumors,with the existence of multiple stem cell populations in the lung closely linked to this heterogeneity.In recent years,growing attention has been focused on the relationship between AT2 cells and the origin of NSCLC.Since AT2 cells have the properties of stem cells,there is a possibility that they undergo malignant transformation into NSCLC cells.Over the years,several studies have demonstrated that abnormal AT2 cells were one of the origin cells of NSCLC in various genetically engineered mouse models（GEMMs）of lung cancer.In human NSCLC,the early researches have only discovered that the AT2 cell markers were expressed and the lamellar bodies also were observed in the NSCLC cells,which indicated that human NSCLC might originate from malignant transformation of AT2cells.Moreover,previous studies revealed that mutation of EGFR or KRAS within AT2cells in mice could drive the self-replication of AT2 cells,leading to formation of AT2-like cell and promoting the tumorigenesis of pulmonary tumors.However,the underlying molecular mechanisms remain still unknown.A recent study employed single-cell sequencing（sc RNA-Seq）techniques to identify a group of cells which were highly similar to AT2 cells（named AT2-like cells）in the stage of IA lung adenocarcinoma tissues.Notably,the gene expression pattern of these AT2-like cells was different with AT2 cells.It suggests that the abnormal AT2 cells may serve as the original cell of human lung adenocarcinoma,which further advised that aberrant gene expression of AT2 cells may associated with the origin of NSCLC.However,the molecular mechanisms governing these abnormal genes of AT2 cells especially specific gene during the origin and development are not fully understand.The solute carrier family 34 member A2（SLC34A2）,located on human chromosome4p15.2,belongs to the solute carrier families and encodes the sodium-dependent phosphate cotransporter protein Napi2b.Napi2b is expressed in the cell membranes of a variety of epithelial tissue cells.Previous studies have examined the expression of SLC34A2 in a variety of tissues and found that SLC34A2 exhibits its highest expression levels in lung and embryonic lung tissues.Specially,SLC34A2 was a specific gene in AT2 cells.In addition,the expression of SLC34A2 was gradually increased during the human pluripotent stem cell differentiation into mature and functional AT2 cells in vitro.Therefore,SLC34A2 could also be used as a marker for human mature AT2 cells.In particular,the latest research has also revealed that the expression of SLC34A2 and SFTPC were progressively upregulated during the differentiation of alveolar lineage organoid tissues expressing the AT2 cell marker gene SFTPC into alveolar organoid tissues.This result indicated that AT2 cells were involved in the formation of alveolar organoid tissues,and further underscored that the SLC34A2 played an important role in maintaining the function of human AT2 cells.Meanwhile,the Napi2b protein shares76%homology between humans and mice.The primary physiological function of Napi2b is to regulate the active transport of inorganic phosphorus ions from the extracellular environment to the intracellular space against the concentration gradient,particularly in a low-phosphorus state.This promotes the cellular uptake of inorganic phosphorus ions and helps maintain the physiological level.Specifically,within the inorganic phosphate ion transport protein encoded by the SLC34 family,Napi2b protein is the most highly expressed in lung AT2 cells.In the human body,inorganic phosphate（Pi）is an abundant element involved in maintaining normal cellular structure and function,which in turn regulates a variety of critical biological processes in vivo.It has been reported that Pi homeostasis disorders were closely related to the development of various tumors,including lung cancer.However,the precise mechanism underlying the relationship between the abnormal expression of SLC34A2and Pi homeostasis,Pi homeostasis and the origin of lung cancer are reminds to be explored.Since SLC34A2 played an important role in lung AT2 cells,thus any abnormality in its expression will inevitably affect the normal function of AT2 cells.Previous studies have found that the expression of SLC34A2 progressively upregulated during human and rat embryonic lung development until it reaches a constant level after lung maturation.This demonstrated that the SLC34A2 played an importance action in lung development and maintenance of lung homeostasis.However,in contrast to the process of embryonic lung development,the SLC34A2 gene exhibited a significantly 10-fold downregulation in 15 clinical NSCLC tissues compared to paired adjacent non-tumor tissues.These findings were consistent with our previous study,which confirmed that the expression of SLC34A2 was significantly decreased in NSCLC tissues and NSCLC cell lines compared to paired adjacent non-tumor tissues and lung epithelial cells,respectively.Further biological function studies demonstrated that stable overexpression of SLC34A2 significantly inhibited NSCLC cell proliferation,migration and invasion in vitro,and it also suppressed subcutaneous transplantation tumor cell growth in vivo.Preliminary mechanistic studies have identified that the inhibition of NSCLC malignant progression by overexpression of SLC34A2 gene may be associated with the inhibition of the expression of key proteins of MEK/ERK and PI3K/AKT/mTOR signaling pathways,However,the specific regulatory mechanisms have not been elucidated.In addition,our previous study also found that overexpression of SLC34A2 gene led to a significantly increase in the m RNA expression of selenium binding protein 1（SELENBP1）as gene microarray analysis.Recently,our study confirmed that the expression of SELENBP1 was significantly down-regulated in 45out of 59 NSCLC tissues compared with corresponding adjacent non-tumor tissues.The SELENBP1 protein was found to be localize in the alveolar corners of the lungs,along with the AT2 cell marker protein SP-C.The result demonstrated that SELENBP1could also be expressed in lung AT2 cells.Further mechanistic studies indicated that overexpression of SELENBP1 could inhibit the malignant progression of NSCLC by suppressing the PI3K/AKT/mTOR pathway.In summary,the above findings suggested that overexpression of SLC34A2 may inhibit the PI3K/AKT/mTOR pathway by upregulating SELENBP1 expression,thereby impeding the malignant process of NSCLC.Therefore,building upon our established research foundation,our study aims to deeply investigate the potential molecular mechanism of SLC34A2 to inhibit the malignant progression of NSCLC.We conduct investigations using clinical NSCLC tissues,human NSCLC cell lines,and nude mice NSCLC subcutaneous graft tumor model.This research not only provide a theoretical framework for the early lung cancer diagnosis and the identification of therapeutic targets,but also establish a scientific foundation for in-depth comprehension of the malignant transformation of AT2 cells into NSCLC cells in the future.Methods and Results1.To further examine the expression differences of SLC34A2 between NSCLC tissues and adjacent non-tumor tissues in large-scale clinical samples,firstly,we investigated the expression differences of SLC34A2 using data from the Cancer Genomics Atlas（TCGA）and Gene Expression Omnibus（GEO）datasets.We observed that the expression of SLC34A2 was significantly down-regulated in NSCLC tissues compared to adjacent non-tumor tissues in LUAD（n=513）tissues and adjacent non-tumor tissues（n=59）,LUSC（n=223）tissues and adjacent non-tumor tissues（n=17）from the TCGA database,as well as LUAD（n=53）tissues and adjacent non-tumor tissues（n=11）from the GEO database.And it was further confirmed that the expression levels of SLC34A2 were indeed significantly decreased in collected NSCLC tissues compared to the corresponding adjacent non-tumor tissues（n=84）by qRT-PCR.In addition,the expression of Napi2b（encoded by SLC34A2）protein was also significantly down-regulated in collected NSCLC tissues compared to the corresponding adjacent tissues by IHC（n=11）and Western blot（n=6）.Collectively,the above results demonstrated that the expression of SLC34A2 was significantly decreased in NSCLC tumor tissues compare to adjacent non-tumor tissues.Next,we found that the expression of SLC34A2 had no significant correlation between stage I and stage Ⅱ,stage I and stage ⅡI,stage I and stage Ⅳ,stage Ⅱ and stage ⅡI,stage Ⅱ and stage Ⅳ,stage ⅡI and stage Ⅳ in LUAD,the similar correlation was presented in LUSC,except stage I and stage Ⅱ.Meanwhile,the similar results were observed in LUAD samples from two GEO database GES140343（n=50）and GSE166720（n=50）.Because few LUSC tissue samples were collected,we chose collected LUAD samples（n=73）for further investigation.Consistent with the results from TCGA and GEO database,the expression of SLC34A2 had no obvious difference between stage I and stage Ⅱ,stage Ⅱ and stage ⅡI.Furthermore,there was no significant difference in the level of SLC34A2 between LUAD patients with poor and moderate differentiation,as well as those with poor and well differentiation in LUAD samples（n=51）from GSE140343.Consistently,our results showed that there was no significant difference in the expression of SLC34A2 between low and medium-low differentiation,as well as between low and medium differentiation in our collected LUAD tissue samples.Thus,these results indicated that the expression level of SLC34A2 had no significant difference in NSCLC patients with different clinical stages and differentiation degrees,suggesting that SLC34A2 may be involved in the origin of NSCLC,particularly in LUAD.Subsequently,we found that lower expression of SLC34A2 was associated with shorter overall survival（OS）in LUAD（n=240）,while the opposite result was observed in LUSC（n=242）from TCGA database.We used the datasets of LUAD samples（n=51）from GES140343 to further confirm the above result from the TCGA database.In addition,in our collection of LUAD samples（n=73）,the expression of SLC34A2 was significantly lower in LUAD patients with lymph node metastasis compared to those without.Thus,the above results demonstrated that the relatively low expression of SLC34A2 was associated with poor prognosis,suggesting that SLC34A2 may also has a certain prognostic diagnostic value in LUAD.2.Given that overexpression of SLC34A2 could inhibited NSCLC malignant characteristics in our previous studies.And SELENBP1 was listed as one of the significantly upregulated genes of SLC34A2 overexpressing-regulated by microarray analysis in our previous research.Importantly,our published data also uncovered that overexpression of SELENBP1 could inhibit the malignant progression of NSCLC.To further explore whether the underlying molecular mechanism in tumor inhibition effect of SLC34A2 was associated with SELENBP1,we first detected the expression correlation between SLC34A2 and SELENBP1,firstly,a visible positive correlation was respectively presented between the expression of SLC34A2 and SELENBP1 in LUAD（n=483）and LUSC（n=486）patient samples from the TCGA database.Meanwhile,similar results were also present in LUAD samples from two datasets GES166720（n=53）and GSE140343（n=51）,respectively.Secondly,consistent with the above data,our analysis also indicated that the expression of SLC34A2 was positive correlated with SELENBP1 in our collected clinical NSCLC tissues（n=65）.Furthermore,the m RNA expression levels of SELENBP1 were decreased in the 35 out of 45（77.78%）collected LUAD tissues,in which the expression of SLC34A2 was downregulated.The expression of Napi2b and SELENBP1 in tumors were significantly lower than that in paired adjacent non-tumors by Western blot（n=11）and IHC（n=11）.Most importantly,the protein expression of Napi2b and SELENBP1 were located in AT2 cells of adjacent non-tumor tissues.In addition,overexpression of SLC34A2 noticeably promoted the expression of SELENBP1 at both m RNA and protein levels.As a result,a significant positive correlation between the expression of SLC34A2 and SELENBP1 was demonstrated in the TCGA and GEO databases and in the collected clinical NSCLC tissue samples,as well as in the collected clinical NSCLC tissue samples and human NSCLC cell lines overexpressing SLC34A2.3.To further confirmed that SELENBP1 was involved in SLC34A2 overexpressing-mediated tumor inhibition effect,si RNA was used to decreased the expression of SELENBP1 in SLC34A2 overexpressing NSCLC cells.The si RNA-mediated knockdown of SELENBP1 in A549-SLC34A2 and H1299-SLC34A2 were confirmed first by qRT-PCR and Western blot.The results showed that the expression of SELENBP1 was decreased in A549-SLC34A2 and H1299-SLC34A2 cells at m RNA and protein levels after treating with si RNA,respectively.Subsequently,as determined by CCK-8,knockdown of SELENBP1 promoted the proliferation of A549-SLC34A2and H1299-SLC34A2 cells.Then colony-forming assay revealed that knockdown of SELENBP1 significantly promoted the clone formation ability of A549-SLC34A2 and H1299-SLC34A2 cells.Moreover,the cell migration ability was evaluated by wound healing assay and Millicell assay,our results uncovered that the cell migration prominently facilitated in A549-SLC34A2 and H1299-SLC34A2 cells after knockdown of SELENBP1.Transwell assays showed that knockdown of SELENBP1 enhanced invasion ability of A549-SLC34A2 and H1299-SLC34A2 cells.Thus,the above results demonstrated that knockdown of SELENBP1 expression reversed the inhibitory effect of overexpression of SLC34A2 gene on the proliferation,migration and invasion ability of NSCLC cells.As overexpression of SELENBP1 has been proved to inhibit malignant progression of NSCLC cells through inactivating the PI3K/AKT/mTOR pathway,we subsequently evaluated the expression of components of the PI3K/AKT/mTOR signaling pathway.Notably,the most of essential members of the PI3K/AKT/mTOR signaling pathway,such as p-PI3K,p-AKT and p-mTOR,were increased in both A549-SLC34A2 and H1299-SLC34A2 after knockdown of SELENBP1,indicating that knockdown of SELENBP1 could invert the SLC34A2 overexpressing-mediated inactivation of PI3K/AKT/mTOR.Taken together,these observations demonstrated that overexpression of SLC34A2 suppressed malignant characteristics of NSCLC cells via increasing the expression levels of SELENBP1 in vitro.4.As the Napi2b（SLC34A2）protein is located in the cell membrane,it cannot directly regulate the expression of SELENBP1.Based on that TTF-1（also known as NKx2.1）enrichment at the promoter region of SELENBP1 was confirmed by Ch IP-seq in Kras^G12D-driven murine lung tumors and human lung adenocarcinoma cell line H441.And TTF-1 could increase the expression of SELENBP1 in murine cells.To further verify that if the overexpression of SLC34A2 up-regulated the levels of SELENBP2 by TTF-1,firstly,in this study,we found that the expression levels of SLC34A2 was significantly positively correlated with TTF-1 in LUAD（n=483）from TCGA database.However,the opposite result was obtained in LUSC（n=486）.Furthermore,the m RNA expression levels of TTF-1 were decreased in the 22 out of 35（62.86%）collected LUAD tissues compared with the adjacent non-tumor tissues,in which the expression of SLC34A2 and SELENBP1 were downregulated.Next,the expression of Napi2b and SELENBP1 in tumors were significantly lower than that in paired adjacent non-tumors by Western blot（n=6）and IHC（n=11）.Specially,the protein expression of Napi2b,SELENBP1 and TTF-1 were colocalized in AT2 cells of adjacent non-tumor tissues,and the protein expression of Napi2b showed a positive expression relationship with SELENBP1 and TTF-1.Moreover,overexpression of SLC34A2 significantly increased the m RNA and protein expression levels of TTF-1 in NSCLC cells.These results demonstrated a significant positive correlation between the expression of SLC34A2,SELENBP1,and TTF-1 in NSCLC especially in LUAD from the TCGA database,as well as a positive expression relationship of SLC34A2,SELENBP1,and TTF-1 in the collected clinical NSCLC tissues and human NSCLC cell lines overexpressing SLC34A2.Next,to investigate whether overexpression of SLC34A2 gene upregulates SELENBP1 expression through TTF-1 in NSCLC cells,TTF-1 binding sites in promoter of SELENBP1 and TTF-1 were predicted through the online dataset JASPAR.Meanwhile,the transcription factors,binding to the promoter of SELENBP1 and TTF-1 were checked using the UCSC database and the Human TFDB online website.Not surprisingly,TTF-1 was present.We next performed the cleavage under targets and tagmentation（CUT&TAG）assay using A549-SLC34A2 and A549-Control cells,the result showed that the overexpression of SLC34A2 could increase TTF-1 binding with SELENBP1 in the 5 predicted promoter regions,meanwhile,overexpression of SLC34A2 also promoted TTF-1 binging with itself promoter except in the P3 fragment of TTF-1.Thus,the above results demonstrated that overexpression of SLC34A2significantly promotes the binding of the transcription factor TTF-1 to the promoters of SELENBP1 and TTF-1 in NSCLC cells.The function of transcription factor is regulated by transcriptional activity.Next,we further explored the molecular mechanism by SLC34A2 overexpressing regulating the transcriptional activity of TTF-1.The inactivation of MEK/ERK signaling pathway was found to increase the transcriptional activity of TTF-1 in rat FRTL-5 cells.Coincidently,our previous study preliminarily suggested that overexpression of SLC34A2 significantly suppressed the activation of MEK/ERK pathway in NSCLC cells.Ras inhibited transcriptional function of TTF-1 directly through ERK,which phosphorylated the serine residues（such as Ser S327）of TTF-1.Based on the above researches,we speculated that if overexpression of SLC34A2 increased the transcriptional activity of TTF-1 by inhibition of MEK/ERK signaling pathway.A549-Control and H1299-Control cells were respectively treated with appropriate U0126,a specific MEK inhibitor,which mimic the role of overexpressed SLC34A2 in A549-SLC34A2 and H1299-SLC34A2 cells.The phosphorylation level of TTF-1 on Ser327was decreased in A549-Control and H1299-Control cells after treated with U0126,although overexpression of SLC34A2 could not fully reproduce the extent of suppression obtain with control cells treated with U0126.Combined with the previous results it has been demonstrated that overexpression of SLC34A2 in NSCLC cells significantly upregulated the m RNA and protein expression levels of SELENBP1 and TTF-1.The above results suggested that overexpression of SLC34A2 elevated the transcriptional activity of TTF-1 by inhibition of MEK/ERK signaling pathway in NSCLC cells.Moreover,we silenced TTF-1 with si RNAs in A549-SLC34A2 and H1299-SLC34A2cells,and found that the expression of SELENBP1 at m RNA and protein level were downregulated following silencing of TTF-1.Taken together,these results indicated that overexpression of the SLC34A2 could inhibit the phosphorylation level of TTF-1（Ser327）by suppressing the MEK/ERK signaling pathway and then further up-regulate the expression of SELENBP1 through the enhancement of TTF-1 transcriptional activity.5.In addition,to further explore the molecular mechanism by which SLC34A2overexpression inhibits MEK/ERK signaling,we were concerned that fundamental components of MEK/ERK pathway include the RAS family of small GTPases,Ras protein（including KRAS,NRAS and HRAS）act as a binary switch that cycle between active GTP-loaded and inactive GDP-loaded forms.Similar to RAS,Leucine-rich repeat kinase 2（LRRK2）is also a molecular switch that binds to GDP/GTP.LRRK2 is expressed in a variety of tissue cells,specially,it is primarily present in AT2 cells of lung.Therefore,we speculated that overexpression of SLC34A2 might inhibit the MEK/ERK pathway through LRRK2.To prove this hypothesis,firstly,the correlation between SLC34A2 and LRRK2 was proved.Notably,the expression of LRRK2 was positively associated with SLC34A2 in LUAD（n=483）and LUSC（n=486）patients from TCGA database.The expression of LRRK2 also showed the significantly correlation with SLC34A2 in LUAD（n=51）tissues in GEO datasets（GSE166720）.Moreover,in our collected clinical tissues,79.31 percent of SLC34A2 downregulation samples bear the decreased level of LRRK2 by qRT-PCR.Consistently,the protein expression of Napi2b and LRRK2 were decreased in tumors compared with paired adjacent tissues by western blot and IHC,respectively.We further uncovered that LRRK2 and Napi2b were also colocalized in AT2 cells in serial section of collected adjacent non-tumor tissues by IHC.In addition,overexpression of SLC34A2 markedly upregulated the expression of LRRK2 at both m RNA and protein levels.These results indicated a positive expression correlation between the expression of SLC34A2 and LRRK2 in TCGA and GEO databases,and overexpression of SLC34A2 could increase the expression levels of LRRK2 in NSCLC cells.Subsequently,to confirm if overexpression of SLC34A2 suppressed activation of MEK/ERK pathway by LRRK2,we silenced LRRK2 with si RNAs in A549-SLC34A2and H1299-SLC34A2 cells,and found that two essential members of the activating MEK/ERK signaling pathway,such as p-MEK and p-ERK1/2 were upregulated following silencing of LRRK2.In addition,overexpression of SLC34A2 in NSCLC cells also significantly upregulated the phosphorylation level of LRRK2（T1503）.Taken together,these results demonstrated that overexpression of SLC34A2 inactivated the MEK/ERK signaling pathway via increasing the expression of LRRK2.6.Finally,to further verify in vivo that overexpression of SLC34A2 upregulated the expression of SELENBP1,TTF-1 and LRRK2,and overexpression of SLC34A2inhibiting NSCLC cell growth was associated with the inhibition of MEK/ERK and PI3K/AKT/mTOR pathways,we established tumor xenograft model to assess tumor formation in athymic mice.We observed that overexpression of SLC34A2 markedly decreased tumor size and obviously suppressed tumor growth.The tumor tissue samples were collected for further analyses.H&E staining illustrated that retrieved subcutaneous tumor tissues did not have any obvious histologic changes in both A549-SLC34A2 and A549-Control tumors.Notably,the cell proliferation protein Ki-67 was prominently decreased in A549-SLC34A2 tumors compared with A549-Control tumors.Moreover,consistent with the results in vitro,overexpression of SLC34A2 could significantly increase the protein level of SELENBP1,TTF-1 and LRRK2 in retrieved subcutaneous tumor by IHC and western blot.Furthermore,subcutaneous tumor tissues were also used to verify the influence of SLC34A2 in MEK/ERK and PI3K/AKT/mTOR pathway.In accordance with the results in vitro,the levels of p-MEK and p-ERK1/2 were both markedly inhibited in A549-SLC34A2 tumors compared to A549-Control tumors.Meanwhile,we also found that overexpression of SLC34A2 decreased the expression of p-PI3K,p-AKT and p-mTOR.In total,overexpression of SLC34A2 could elevate the expression of SELENBP1,TTF-1 and LRRK2,and overexpression of SLC34A2 inhibiting growth of NSCLC cells was associated with the inactivation of MEK/ERK and PI3K/AKT/mTOR signaling pathway in vivo.ConclusionIn summary,our results revealed that the protein expression of Napi2b,SELENBP1,TTF-1 and LRRK2 were located in AT2 cells of adjacent non-tumor tissues.However,the expression of SLC34A2,SELENBP1,TTF-1 and LRRK2 were significantly decreased in NSCLC tissues compared with their paired adjacent non-tumor tissues,and their m RNA expression have obvious positive correlation.Mechanistically,the present study elucidated for the first time that overexpression of SLC34A2 could inhibit the activation of MEK/ERK signaling pathway through up-regulating the expression of LRRK2,and subsequently suppressed the expression of p-TTF-1（Ser327）,which upregulated the expression of SELENBP1 by regulating TTF-1 transcriptional activity.Ultimately,overexpression of SLC34A2 depressed the activation of PI3K/AKT/mTOR signaling pathway via up-regulating the expression of SELENBP1,which significantly inhibited the malignant characteristics of NSCLC.Our current research established the regulatory network of SLC34A2/LRRK2/TTF-1/SELENBP1 signaling axis in the development of NSCLC,which not only provides a new target for early diagnosis and treatment of NSCLC,but also make contribution for further understanding the molecular mechanisms of AT2 cells on origin and development of NSCLC.更多还原