LTF抑制AKT通路影响鼻咽癌生长与转移
摘要
乳转铁蛋白(lactotransferrin, LTF)属于转铁蛋白家族,它是一种具有多种重要生物学功能的糖蛋白。LTF具有参与铁代谢、抗菌、抗真菌、抗病毒、免疫调节和抗炎、抗氧化、抗肿瘤等多种生物学功能。近年来大量的体内外研究显示LTF具有明显的抗肿瘤作用。目前关于LTF对肿瘤细胞直接的作用的机制并不清楚。研究发现,LTF能诱导肿瘤细胞G1/S周期阻滞,并且能调控细胞周期相关蛋白如p21、p27、Rb,LTF也能诱导某些肿瘤细胞发生凋亡。
我们前期研究利用湖南鼻咽癌高发家系,通过连锁分析及单体型分析,发现染色体3p21为鼻咽癌易感基因区。而LTF基因正好位于此区域。前期研究发现LTF在鼻咽癌中显著下调或缺失,且其表达水平与鼻咽癌的临床分期、转移及患者预后密切相关。初步功能研究发现,LTF明显抑制鼻咽癌细胞增殖,将细胞阻滞于Go/G1期,并且LTF抑制MAPK信号传导通路,下调cyclin D1、pRb、p21、p27等周期相关蛋白。有研究报道,在鼻咽癌组织和细胞系中LTF基因位点存在杂合性丢失和启动子区域的高甲基化。这些证据提示,LTF在鼻咽癌是一个潜在的抑瘤基因,它在鼻咽癌发生发展中可能发挥重要的作用。
在本课题中,为了进一步阐述LTF抑制鼻咽癌生长转移的分子机制,以及LTF在鼻咽癌中表达下调机制,我们进行了以下研究,并取得了相应的结果：
1.LTF通过抑制MAPK通路下调PDKl表达,导致AKT活性下调
首先,我们通过功能获得和缺失方法证实了LTF具有抑制鼻咽癌生长转移作用。为了研究LTF基因的抑瘤分子机制,采用全基因组表达谱芯片分析及qRT-PCR和Western blot等实验发现,LTF能显著下调PDK1(3-phosphoinositide-dependent protein kinase1,PDPK1or PDK1)的mRNA及蛋白表达水平。接下来,我们确定LTF能否影响PDK1的转录活性。我们将PDK1启动子克隆入荧光素酶报告载体与LTF表达质粒或LTF shRNA共转染细胞,发现LTF能抑制PDK1的转录活性。因为c-Jun能激活PDK1转录,而以往的研究显示LTF能抑制MAPK通路活性和c-Jun的表达。为了确定LTF抑制PDK1表达是否与抑制MAPK信号通路和c-Jun的表达有关,我们分别用P13K抑制剂LY294002、MEK抑制剂PD98059、JNK抑制剂SP600125处理LTF基因封闭的细胞,观察PDK1表达变化情况。我们发现MAPK抑制剂能拮抗LTF基因封闭细胞的c-Jun表达,导致PDK1显著下调,然而P13K抑制剂处理的细胞,PDK1表达无明显变化。这表明MAPK信号通路参与LTF抑制PDK1过程。随后,我们在LTF基因沉默细胞中转染c-Jun siRNA证实了抑制c-Jun表达确实能下调PDK1表达。这些结果表明,LTF能通过抑制MAPK活性下调PDK1的表达。
AKT是PDK1最经典的靶分子,因此我们检测LTF对AKT通路的影响。我们发现过表LTF明显抑制AKT通路激活,而封闭LTF表达则促进AKT通路激活。AKT的充分活化需要PDK1磷酸化其Thr308和mTORC2磷酸化其Ser473。PDK1只负责磷酸化AKT蛋白的Thr308,与Ser473的磷酸化无关。我们意外地发现,AKT Ser473磷酸化被LTF下调的程度相似于甚至高于LTF下调Thr308磷酸化的程度。更重要的是,无论是野生型PDK1还是PDK1活性形式(PDK1-A280V)都能拯救LTF对AKT Thr308磷酸化的影响,但是两者都不能拯救其Ser473的磷酸化(图1-18)。这些结果表明,LTF能通过下调PDK1而抑制AKT活性,并且也提示,LTF还能通过其他的机制抑制AKT活性。
2.LTF通过与Keratin18相互作用阻止Keratin18与14-3-3蛋白结合,进而阻止Keratin18促进AKT通路的激活
为探讨LTF抑制AKT活性的新机制,我们寻找新的LTF交互作用蛋白。通过免疫共沉淀结合质谱分析,我们鉴定出3个LTF相互作用蛋白,分别是Keratin18(K18)、Keratin16(K16)、Keratin10(K10)。随后,通过免疫共沉淀-Western blot证实LTF与K18存在交互作用。并通过免疫荧光检测发现,LTF与K18在细胞浆内存在共定位现象。
K18是细胞骨架中间丝蛋白中的角蛋白(Keratin)家族成员,新近研究显示,Keratin家族成员K17能以磷酸化依赖形式与adaptor蛋白14-3-3σ蛋白结合,K17与其结合后能作为一个支架蛋白募集信号蛋白激活AKT并且促进上皮细胞的生长。以往的研究显示Ser33磷酸化的K18也能与14-3-3蛋白,并且K18与14-3-3蛋白与有丝分裂期14-3-3蛋白在细胞内的重新分布有关。本研究我们也证实了K18能以磷酸化依赖形式与14-3-3σ相互作用,与K18结合是14-3-3蛋白滞留在胞浆内所必需的。并且我们也发现,K18能像其家族成员K17一样通过将14-3-3蛋白滞留在胞浆内而激活AKT通路,从而促进鼻咽癌细胞生长增殖。另外,抑制K18表达不影响MAPK通路和PDK1的表达,提示LTF抑制MAPK活性及下调PDK1表达与K18无关。
为了明确LTF是否可通过与K18交互作用影响K18与14-3-3蛋白结合从而影响AKT通路激活,我们在过表达LTF的鼻咽癌细胞系中检测K18与14-3-3蛋白的结合。结果显示,LTF过表达在不影响K18磷酸化情况下阻止K18与14-3-3结合。通过免疫荧光发现过表达LTF能隔离14-3-3蛋白于胞核内。随后我们发现,LTF过表达能阻止K18促进AKT通路的激活及解除K18促进鼻咽癌细胞生长作用。
3.LTF可通过AKT通路抑制鼻咽癌细胞生长转移
LTF能通过两种不同的机制抑制P13K/AKT通路活性。为了确定LTF抑制鼻咽癌细胞生长转移的功能是否通过AKT通路途径实现,我们使用P13K/AKT特异性抑制剂处理LTF基因沉默的鼻咽癌细胞发现,与未处理的LTF基因沉默细胞相比,P13K/AKT特异性抑制剂处理的LTF基因沉默细胞生长速度减慢,软琼脂集落形成率减少,细胞侵袭能力下降。这些结果表明LTF确实可通过抑制AKT通路影响鼻咽癌细胞生长转移。
采用组织芯片通过免疫组化检测鼻咽癌组织中LTF与AKT磷酸化表达情况。结果显示：与正常鼻咽上皮组织相比,LTF在鼻咽癌组织中表达明显下调；而AKT的磷酸化表达的则与之相反,与正常鼻咽组织相比,pAKT Thr308及pAKT Ser473在鼻咽癌组织中表达明显上调。在鼻咽癌中LTF与pAKT Thr308和pAKT Ser473呈负相关。并且,pAKT Thr308与pAKT Ser473呈正相关。这进一步支持LTF负调控AKT通路。
4.miR-214通过靶向抑制LTF影响AKT通路,从而促进鼻咽癌细胞增殖侵袭能力
前期研究中我室多次大样本量证实,LTF在鼻咽癌中显著下调或缺失。为确定LTF在鼻咽癌中下调是否与miRNA异常有关,我们研究LTF与miRNA关系。通过在线软件Targetscan预测及荧光素酶活性检测发现,miR-214能特异地与LTF3'UTR结合。并且miR-214能抑制LTF mRNA及蛋白表达。这说明miR-214直接靶向调控LTF。体内外研究显示,miR-214具有促进鼻咽癌生长转移的作用。因LTF能抑制AKT活性,所以我们检测miR-214对AKT通路的影响。结果显示,miR-214能促进AKT激活,并且过表达LTF能拮抗miR-214激活AKT,表明miR-214通过靶向抑制LTF影响AKT通路。接下来我们也通过细胞生长曲线和细胞侵袭实验证实,miR-214通过靶向抑制LTF,影响AKT通路,促进鼻咽癌细胞增殖侵袭能力。
5.鼻咽癌中LTF低表达与miR-214高表达相关
我们运用qRT-PCR在临床标本中检测miR-214与LTF表达情况。结果显示,与正常鼻咽上皮组织相比,miR-214在鼻咽癌组织表达普遍上调,并且在有转移的鼻咽癌组织明显高于无淋巴结转移的鼻咽癌组织。而LTF在组织中的表达模式与miR-214正好相反,与正常鼻咽上皮组织相比,LTF在鼻咽癌组织表达明显下调,并且在有转移的鼻咽癌组织明显低于无淋巴结转移的鼻咽癌组织。通过相关性分析发现鼻咽癌组织中LTF低表达与miR-214高表达相关。
综上所述,LTF能通过两种不同机制抑制AKT通路激活：一是LTF通过抑制MAPK通路下调转录因子c-Jun,而c-Jun是PDK1转录激活因子,c-Jun下调导致PDK1表达下调,最终导致AKT活性下调；二是LTF通过与K18相互作用阻止K18与14-3-3蛋白结合,使14-3-3聚积于细胞核内,导致AKT活性下调。而LTF可通过抑制AKT通路发挥其抑制鼻咽癌生长转移作用。我们通过研究LTF与miRNA关系发现,鼻咽癌中LTF的表达受到miR-214的调控,miR-214通过靶向LTF影响AKT通路,从而促进鼻咽癌细胞生长侵袭能力。鼻咽癌组织中miR-214高表达是导致LTF低表达的机制之一。
Human lactotransferrin (LTF) is a member of the transferrinn family of iron-binding glycoprotein. LTF has the wide range of functions including iron homeostasis, anti-microbial and immunomodulatory effects, as well as antitumor activity. LTF has been shown to reduce tumor growth and metastasis both in vitro and in vivo. The mechanisms by which LTF has direct effect on tumor cell growth have yet not been established. It has been reported that LTF results in tumor cell growth arrest at the G1to S transition of the cell cycle and affects the expression and/or activity of critical cell cycle regulatory proteins including p21, p27, and retinoblastoma protein (pRb). LTF also induces apoptosis in several types of cancerous cells.
In our previous study, we collected samples from18families at high risk of NPC from the Hunan province in southern China and identified chromosome3p21as a novel locus for susceptibility genes candidates by linkage analysis. We previously demonstrated that LTF was strongly downreguated in in nasopharyngeal carcinoma (NPC) and negatively associated with the progression, metastasis and prognosis in NPC. We also showed that LTF inhibits NPC cell proliferation, induces cell cycle G1/S arrest, and modulates the MAPK signaling pathway and cyclin D1-related proteins such as cyclin D1, phos-Rb, p21, p27. Furthermore, downregulation of LTF relates to loss of heterozygosity (LOH) at the LTF locus and hypermethylation of LTF promoter region. These data suggest direct evidence of the important role of LTF in NPC tumorigenesis and progression. In this study, we have identified LTF as a negative regulator of AKT signaling pathway in NPC, providing an important mechanism by which LTF suppresses the growth and metastasis of NPC, and showed that the overerexpression of miR-214is cause relationship to LTF downregulation.
1. LTF downregulats PDK1via repression of MAPK pathway, leading to inhibition of AKT activity.
In our study, we further confirmed that LTF may suppress tumorigenesis and metastasis in NPC. To explore the mechanisms by which LTF inhibits NPC tumorigenesis, we revealed that LTF can suppress PDK1expression at mRNA and protein levels by microarray analysis followed by validation of RT-PCR and Western blot. We next assessed whether LTF downregulates PDK1expression by decreasing its transcription. Luciferase reporter assays indicated that overexpression of LTF in both5-8F and CNE2cells suppressed PDK1promoter activity, while inhibiting endogenous LTF in HONE1cells increased it. Because the transcript factor c-Jun serves to activate transcription of PDK1while LTF downregulates MAPK pahthway and reduce c-Jun expression in NPC cells, we speculated that LTF-induced PDK1downregulation depended on the repression of MAPK pahthway and c-Jun. Thus, we utilized several kinase inhibitors including PD98059(MEK inhibitor), SP600125(JNK inhibitor) and LY294002(PI3K inhibitor) to investigate whether MAPK pathway and c-Jun are involved in LTF-induced PDK1downregulation. As expected, knockdown of LTF induced c-Jun and PDK1expression, whereas treatment of the LTF shRNA/HONE1cells with the MAPKs inhibitor either PD98059or SP600125resulted in reduction of c-Jun, further leading to reduced PDK1level. However, no obvious alteration to c-Jun or PDK1was found in cells treated with PI3K (LY294002) inhibitor. To confirm that c-Jun regulates PDK1level, we inhibited the expression level of c-Jun. On treatment with c-Jun siRNA, LTF-depleted cells showed a decrease in the expression of c-Jun associated with a reduction in the expression of PDK1. These results suggest that the MAPK/c-JUN pathway is involved in LTF-induced PDK1downregulation in NPC cells at least partly.
Because AKT is the most classical target of PDK1, we analyzed the effect of LTF on AKT signalling. Overexpression of LTF markedly inhibited the activation of AKT pathway. Alternatively, knockdown of LTF showed a significant increase in the activated AKT. AKT for full activation requires phosphorylation at Thr308and Ser473by PDK1and mTORC2, respectively. Much to our surprise, AKT Ser473phosopharylation was reduced to an extent greater than that of Thr308, the site directly phosphorylated by PDK1on AKT (Fig.3A). Importantly, overexpression of either WT PDK1or PDK1-A280V (a constitutively active form of PDK1in LTF-overexpressing5-8F cells efficiently restored downregulation of AKT phosphorylation at Thr308but not at Ser473. The aforementioned results suggest that LTF can inhibit AKT activation via PDK1downregulation, but PDK1downregulation is not sufficient for inhibiting full activation of AKT signaling in NPC cells.
2. LTF by interaction with K18blocks the ability of K18to bind to14-3-3and to stimulate AKT activity.
To investigate the mechanisms underlying LTF-mediated inhibition of AKT activity aside from PDK1downregulation, we sought to identify LTF-interacting proteins. By coimmunoprecipitation combined with mass spectrometric sequencing, we found three LTF-interacted proteins, namely Keratin18, Keratin16and Keratin1. Keratin-18was subsequently confirmed by coimmunoprecipitation-Western blot analysis. Moreover, immunofluorescence staining showed that LTF colocalizes with K18in the cytoplasm of5-8F cells stably expressing LTF
K18is a member of Keratin family. A recent study showed that the family member K17acts as a scaffold protein to bring together the necessary signaling proteins to trigger the PI3K/AKT by binding the adaptor protein14-3-3σ in a phosphorylation-dependent manner and retaining14-3-3in the cytoplasm, and thereby promotes epithelial cell growth. K18also can bind to14-3-3proteins when K18Ser33is phosphorylated, and this binding may partially modulate hepatocyte mitotic progression, in association with redistribution of14-3-3proteins during mitosis. Indeed, here we further confirmed that K18binds14-3-3σ in a phosphorylation-dependent manner in NPC cells, and K18is required for the retention of14-3-3in the cytoplasm. Moreover, K18, like K17, contributes to trigger the PI3K/AKT pathway by the retention of14-3-3in the cytoplasm, in turn promotes the proliferation and growth in NPC cells. However, K18knockdown has little effect on the activity of MAPK pathway and PDK1level, indicating the inhibition of MAPK signaling and PDK1downreguation by LTF seems to be K18-independent.
Given our data showing that LTF can associate with K18, we next examined whether this association influenced ability of K18to bind14-3-3. As a result, overexpression of LTF causing induction of LTF-K18binding blocked the interactions of K18and14-3-3under the condition that K18phosophorylation remained unaffected and thereby influenced subcellular localization of14-3-3in NPC cells. Indeed, introduction of LTF expression plasmid in K18overexpression cells abolished the effect of K18on AKT pathway. In addition, overexpression of LTF also inhibited the abilities of K18to enhance the growth and soft-agar colony formation of5-8F cells. These results demonstrated that LTF suppressed AKT signaling and thereby reduced tumorigenesis partially by its interaction with K18.
3. LTF inhibits NPC cell proliferation and invasion via AKT pathway.
This current study revealed LTF can repress AKT signaling through multiple mechanisms. To clarify whether LTF suppresses NPC cells proliferation and invasiveness through inhibiting AKT signaling, we employed PI3K/AKT specific inhibitor Wortmannin and LY294002to treating the NPC cells in which endogenous LTF had been silenced through shRNA technique. We observed either Wortmannin or LY294002treatment significantly decreased cell proliferation rates, inhibited anchorage-independent growth as well as invasiveness ability in LTF absent HONE1cells. These results suggest that LTF's tumor suppressor function in NPC cells are partly through inhibiting AKT signaling.
We further examined the expression levels of LTF, pAKT (Thr308or Ser473) and total AKT by immunohistochemically analyzing tissue microarrays containing normal nasopharyngeal epithelia(n=33), normal epithelia adjacent to NPC (n=23), and NPC (n=138). The results revealed that the levels of LTF expression were significantly downregulated, but the phosopharylation levels of AKT at Thr308or Ser473were upregulated in the majority of NPC tissues compared with those normal nasopharyngeal epithelium tissues. Meanwhile, the expression levels of total AKT were unchanged in NPCs relative to normal nasopharyngeal epithelium tissues. Statistical analysis revealed that LTF expression negatively correlated with pAKT Thr308(R=-0.225, P=0.008) and pAKT Ser473(R=-0.345, P=0.000) in NPC tissues. Moreover, the additional effects that pAKT Thr308positively correlated with pAKT Ser473(r=0.413, P=0.000) in NPC samples were observed. Taken together, our results reveal an inverse relationship between low LTF expression and hyperactivation of AKT in human NPC, further supporting a model of LTF as a negative regulator of AKT signaling.
4. MiR-214targets LTF and subsequently suppresses AKT pahthway, resulting in promotion of NPC cell proliferation and invasion.
We previously showed that LTF is downregulated or absent in NPC. To determine whether decreased expression LTF correlates with aberrant miRNAs, we investigate the relationship between LTF and miRNAs. We demonstrated miR-214could bind to the3'untranslated region (UTR) of LTF by TargetScan software and luciferase reporter assays and inhibited its expression at mRNA and protein levels, suggesting that miR-214directly targets LTF. Both in vitro and in vivo assays revealed that miR-214may trigger growth and metastasis in NPC. Given that LTF inhibits AKT pahthway, we assessed the effect of miR-214on AKT activity. As a result, overexpression of LTF blocked miR-214-mediated AKT activation, which indicates that LTF suppresses AKT pahthway by targeting LTF. In vitro assays revealed miR-214promotes NPC cell proliferation and invasion through inhibiting LTF and in turn AKT pathway.
5. Decreased LTF expression correlates with increased miR-214expression in NPC.
In this study, we used qRT-PCR to measure miR-214and LTF expression levels in NPC tissue samples. MiR-214consistently showed increased expression levels in NPC tissues with respect to nasopharyngeal epithelial tissues and positively associated with tumor metastasis. However, LTF showed an inverse expression model, that is, LTF is downregulated in NPC samples compared with nasopharyngeal epithelial tissues. Moreover, the expression of LTF negatively correlates with tumor metastasis. Importantly, decreased LTF expression correlates with increased miR-214expression in NPC by correlation analysis.
In conclusion, LTF can repress AKT signaling through two mechanisms. Firstly, LTF reduces PDK1level by suppressing MAPK pathway and downregulating c-Jun level, leading to the inhibition of AKT activity. Secondly, LTF by interaction with K18blocks the formation of K18-14-3-3complex, which causes the sequestering of14-3-3in nucleus, and subsequently results in decreased AKT activity. Moreover, we have shown that LTF plays an important role in suppressing tumorigenesis of NPC partially through inhibition of AKT pathway. In addition, miR-214promoted NPC cell proliferation and invasion through inhibiting LTF and in turn AKT pathway. We have demonstrate that LTF is regulated by miR-214in NPC, namely, decreased LTF expression is partially due to increased miR-214expression in NPC.
我们前期研究利用湖南鼻咽癌高发家系,通过连锁分析及单体型分析,发现染色体3p21为鼻咽癌易感基因区。而LTF基因正好位于此区域。前期研究发现LTF在鼻咽癌中显著下调或缺失,且其表达水平与鼻咽癌的临床分期、转移及患者预后密切相关。初步功能研究发现,LTF明显抑制鼻咽癌细胞增殖,将细胞阻滞于Go/G1期,并且LTF抑制MAPK信号传导通路,下调cyclin D1、pRb、p21、p27等周期相关蛋白。有研究报道,在鼻咽癌组织和细胞系中LTF基因位点存在杂合性丢失和启动子区域的高甲基化。这些证据提示,LTF在鼻咽癌是一个潜在的抑瘤基因,它在鼻咽癌发生发展中可能发挥重要的作用。
在本课题中,为了进一步阐述LTF抑制鼻咽癌生长转移的分子机制,以及LTF在鼻咽癌中表达下调机制,我们进行了以下研究,并取得了相应的结果：
1.LTF通过抑制MAPK通路下调PDKl表达,导致AKT活性下调
首先,我们通过功能获得和缺失方法证实了LTF具有抑制鼻咽癌生长转移作用。为了研究LTF基因的抑瘤分子机制,采用全基因组表达谱芯片分析及qRT-PCR和Western blot等实验发现,LTF能显著下调PDK1(3-phosphoinositide-dependent protein kinase1,PDPK1or PDK1)的mRNA及蛋白表达水平。接下来,我们确定LTF能否影响PDK1的转录活性。我们将PDK1启动子克隆入荧光素酶报告载体与LTF表达质粒或LTF shRNA共转染细胞,发现LTF能抑制PDK1的转录活性。因为c-Jun能激活PDK1转录,而以往的研究显示LTF能抑制MAPK通路活性和c-Jun的表达。为了确定LTF抑制PDK1表达是否与抑制MAPK信号通路和c-Jun的表达有关,我们分别用P13K抑制剂LY294002、MEK抑制剂PD98059、JNK抑制剂SP600125处理LTF基因封闭的细胞,观察PDK1表达变化情况。我们发现MAPK抑制剂能拮抗LTF基因封闭细胞的c-Jun表达,导致PDK1显著下调,然而P13K抑制剂处理的细胞,PDK1表达无明显变化。这表明MAPK信号通路参与LTF抑制PDK1过程。随后,我们在LTF基因沉默细胞中转染c-Jun siRNA证实了抑制c-Jun表达确实能下调PDK1表达。这些结果表明,LTF能通过抑制MAPK活性下调PDK1的表达。
AKT是PDK1最经典的靶分子,因此我们检测LTF对AKT通路的影响。我们发现过表LTF明显抑制AKT通路激活,而封闭LTF表达则促进AKT通路激活。AKT的充分活化需要PDK1磷酸化其Thr308和mTORC2磷酸化其Ser473。PDK1只负责磷酸化AKT蛋白的Thr308,与Ser473的磷酸化无关。我们意外地发现,AKT Ser473磷酸化被LTF下调的程度相似于甚至高于LTF下调Thr308磷酸化的程度。更重要的是,无论是野生型PDK1还是PDK1活性形式(PDK1-A280V)都能拯救LTF对AKT Thr308磷酸化的影响,但是两者都不能拯救其Ser473的磷酸化(图1-18)。这些结果表明,LTF能通过下调PDK1而抑制AKT活性,并且也提示,LTF还能通过其他的机制抑制AKT活性。
2.LTF通过与Keratin18相互作用阻止Keratin18与14-3-3蛋白结合,进而阻止Keratin18促进AKT通路的激活
为探讨LTF抑制AKT活性的新机制,我们寻找新的LTF交互作用蛋白。通过免疫共沉淀结合质谱分析,我们鉴定出3个LTF相互作用蛋白,分别是Keratin18(K18)、Keratin16(K16)、Keratin10(K10)。随后,通过免疫共沉淀-Western blot证实LTF与K18存在交互作用。并通过免疫荧光检测发现,LTF与K18在细胞浆内存在共定位现象。
K18是细胞骨架中间丝蛋白中的角蛋白(Keratin)家族成员,新近研究显示,Keratin家族成员K17能以磷酸化依赖形式与adaptor蛋白14-3-3σ蛋白结合,K17与其结合后能作为一个支架蛋白募集信号蛋白激活AKT并且促进上皮细胞的生长。以往的研究显示Ser33磷酸化的K18也能与14-3-3蛋白,并且K18与14-3-3蛋白与有丝分裂期14-3-3蛋白在细胞内的重新分布有关。本研究我们也证实了K18能以磷酸化依赖形式与14-3-3σ相互作用,与K18结合是14-3-3蛋白滞留在胞浆内所必需的。并且我们也发现,K18能像其家族成员K17一样通过将14-3-3蛋白滞留在胞浆内而激活AKT通路,从而促进鼻咽癌细胞生长增殖。另外,抑制K18表达不影响MAPK通路和PDK1的表达,提示LTF抑制MAPK活性及下调PDK1表达与K18无关。
为了明确LTF是否可通过与K18交互作用影响K18与14-3-3蛋白结合从而影响AKT通路激活,我们在过表达LTF的鼻咽癌细胞系中检测K18与14-3-3蛋白的结合。结果显示,LTF过表达在不影响K18磷酸化情况下阻止K18与14-3-3结合。通过免疫荧光发现过表达LTF能隔离14-3-3蛋白于胞核内。随后我们发现,LTF过表达能阻止K18促进AKT通路的激活及解除K18促进鼻咽癌细胞生长作用。
3.LTF可通过AKT通路抑制鼻咽癌细胞生长转移
LTF能通过两种不同的机制抑制P13K/AKT通路活性。为了确定LTF抑制鼻咽癌细胞生长转移的功能是否通过AKT通路途径实现,我们使用P13K/AKT特异性抑制剂处理LTF基因沉默的鼻咽癌细胞发现,与未处理的LTF基因沉默细胞相比,P13K/AKT特异性抑制剂处理的LTF基因沉默细胞生长速度减慢,软琼脂集落形成率减少,细胞侵袭能力下降。这些结果表明LTF确实可通过抑制AKT通路影响鼻咽癌细胞生长转移。
采用组织芯片通过免疫组化检测鼻咽癌组织中LTF与AKT磷酸化表达情况。结果显示：与正常鼻咽上皮组织相比,LTF在鼻咽癌组织中表达明显下调；而AKT的磷酸化表达的则与之相反,与正常鼻咽组织相比,pAKT Thr308及pAKT Ser473在鼻咽癌组织中表达明显上调。在鼻咽癌中LTF与pAKT Thr308和pAKT Ser473呈负相关。并且,pAKT Thr308与pAKT Ser473呈正相关。这进一步支持LTF负调控AKT通路。
4.miR-214通过靶向抑制LTF影响AKT通路,从而促进鼻咽癌细胞增殖侵袭能力
前期研究中我室多次大样本量证实,LTF在鼻咽癌中显著下调或缺失。为确定LTF在鼻咽癌中下调是否与miRNA异常有关,我们研究LTF与miRNA关系。通过在线软件Targetscan预测及荧光素酶活性检测发现,miR-214能特异地与LTF3'UTR结合。并且miR-214能抑制LTF mRNA及蛋白表达。这说明miR-214直接靶向调控LTF。体内外研究显示,miR-214具有促进鼻咽癌生长转移的作用。因LTF能抑制AKT活性,所以我们检测miR-214对AKT通路的影响。结果显示,miR-214能促进AKT激活,并且过表达LTF能拮抗miR-214激活AKT,表明miR-214通过靶向抑制LTF影响AKT通路。接下来我们也通过细胞生长曲线和细胞侵袭实验证实,miR-214通过靶向抑制LTF,影响AKT通路,促进鼻咽癌细胞增殖侵袭能力。
5.鼻咽癌中LTF低表达与miR-214高表达相关
我们运用qRT-PCR在临床标本中检测miR-214与LTF表达情况。结果显示,与正常鼻咽上皮组织相比,miR-214在鼻咽癌组织表达普遍上调,并且在有转移的鼻咽癌组织明显高于无淋巴结转移的鼻咽癌组织。而LTF在组织中的表达模式与miR-214正好相反,与正常鼻咽上皮组织相比,LTF在鼻咽癌组织表达明显下调,并且在有转移的鼻咽癌组织明显低于无淋巴结转移的鼻咽癌组织。通过相关性分析发现鼻咽癌组织中LTF低表达与miR-214高表达相关。
综上所述,LTF能通过两种不同机制抑制AKT通路激活：一是LTF通过抑制MAPK通路下调转录因子c-Jun,而c-Jun是PDK1转录激活因子,c-Jun下调导致PDK1表达下调,最终导致AKT活性下调；二是LTF通过与K18相互作用阻止K18与14-3-3蛋白结合,使14-3-3聚积于细胞核内,导致AKT活性下调。而LTF可通过抑制AKT通路发挥其抑制鼻咽癌生长转移作用。我们通过研究LTF与miRNA关系发现,鼻咽癌中LTF的表达受到miR-214的调控,miR-214通过靶向LTF影响AKT通路,从而促进鼻咽癌细胞生长侵袭能力。鼻咽癌组织中miR-214高表达是导致LTF低表达的机制之一。
Human lactotransferrin (LTF) is a member of the transferrinn family of iron-binding glycoprotein. LTF has the wide range of functions including iron homeostasis, anti-microbial and immunomodulatory effects, as well as antitumor activity. LTF has been shown to reduce tumor growth and metastasis both in vitro and in vivo. The mechanisms by which LTF has direct effect on tumor cell growth have yet not been established. It has been reported that LTF results in tumor cell growth arrest at the G1to S transition of the cell cycle and affects the expression and/or activity of critical cell cycle regulatory proteins including p21, p27, and retinoblastoma protein (pRb). LTF also induces apoptosis in several types of cancerous cells.
In our previous study, we collected samples from18families at high risk of NPC from the Hunan province in southern China and identified chromosome3p21as a novel locus for susceptibility genes candidates by linkage analysis. We previously demonstrated that LTF was strongly downreguated in in nasopharyngeal carcinoma (NPC) and negatively associated with the progression, metastasis and prognosis in NPC. We also showed that LTF inhibits NPC cell proliferation, induces cell cycle G1/S arrest, and modulates the MAPK signaling pathway and cyclin D1-related proteins such as cyclin D1, phos-Rb, p21, p27. Furthermore, downregulation of LTF relates to loss of heterozygosity (LOH) at the LTF locus and hypermethylation of LTF promoter region. These data suggest direct evidence of the important role of LTF in NPC tumorigenesis and progression. In this study, we have identified LTF as a negative regulator of AKT signaling pathway in NPC, providing an important mechanism by which LTF suppresses the growth and metastasis of NPC, and showed that the overerexpression of miR-214is cause relationship to LTF downregulation.
1. LTF downregulats PDK1via repression of MAPK pathway, leading to inhibition of AKT activity.
In our study, we further confirmed that LTF may suppress tumorigenesis and metastasis in NPC. To explore the mechanisms by which LTF inhibits NPC tumorigenesis, we revealed that LTF can suppress PDK1expression at mRNA and protein levels by microarray analysis followed by validation of RT-PCR and Western blot. We next assessed whether LTF downregulates PDK1expression by decreasing its transcription. Luciferase reporter assays indicated that overexpression of LTF in both5-8F and CNE2cells suppressed PDK1promoter activity, while inhibiting endogenous LTF in HONE1cells increased it. Because the transcript factor c-Jun serves to activate transcription of PDK1while LTF downregulates MAPK pahthway and reduce c-Jun expression in NPC cells, we speculated that LTF-induced PDK1downregulation depended on the repression of MAPK pahthway and c-Jun. Thus, we utilized several kinase inhibitors including PD98059(MEK inhibitor), SP600125(JNK inhibitor) and LY294002(PI3K inhibitor) to investigate whether MAPK pathway and c-Jun are involved in LTF-induced PDK1downregulation. As expected, knockdown of LTF induced c-Jun and PDK1expression, whereas treatment of the LTF shRNA/HONE1cells with the MAPKs inhibitor either PD98059or SP600125resulted in reduction of c-Jun, further leading to reduced PDK1level. However, no obvious alteration to c-Jun or PDK1was found in cells treated with PI3K (LY294002) inhibitor. To confirm that c-Jun regulates PDK1level, we inhibited the expression level of c-Jun. On treatment with c-Jun siRNA, LTF-depleted cells showed a decrease in the expression of c-Jun associated with a reduction in the expression of PDK1. These results suggest that the MAPK/c-JUN pathway is involved in LTF-induced PDK1downregulation in NPC cells at least partly.
Because AKT is the most classical target of PDK1, we analyzed the effect of LTF on AKT signalling. Overexpression of LTF markedly inhibited the activation of AKT pathway. Alternatively, knockdown of LTF showed a significant increase in the activated AKT. AKT for full activation requires phosphorylation at Thr308and Ser473by PDK1and mTORC2, respectively. Much to our surprise, AKT Ser473phosopharylation was reduced to an extent greater than that of Thr308, the site directly phosphorylated by PDK1on AKT (Fig.3A). Importantly, overexpression of either WT PDK1or PDK1-A280V (a constitutively active form of PDK1in LTF-overexpressing5-8F cells efficiently restored downregulation of AKT phosphorylation at Thr308but not at Ser473. The aforementioned results suggest that LTF can inhibit AKT activation via PDK1downregulation, but PDK1downregulation is not sufficient for inhibiting full activation of AKT signaling in NPC cells.
2. LTF by interaction with K18blocks the ability of K18to bind to14-3-3and to stimulate AKT activity.
To investigate the mechanisms underlying LTF-mediated inhibition of AKT activity aside from PDK1downregulation, we sought to identify LTF-interacting proteins. By coimmunoprecipitation combined with mass spectrometric sequencing, we found three LTF-interacted proteins, namely Keratin18, Keratin16and Keratin1. Keratin-18was subsequently confirmed by coimmunoprecipitation-Western blot analysis. Moreover, immunofluorescence staining showed that LTF colocalizes with K18in the cytoplasm of5-8F cells stably expressing LTF
K18is a member of Keratin family. A recent study showed that the family member K17acts as a scaffold protein to bring together the necessary signaling proteins to trigger the PI3K/AKT by binding the adaptor protein14-3-3σ in a phosphorylation-dependent manner and retaining14-3-3in the cytoplasm, and thereby promotes epithelial cell growth. K18also can bind to14-3-3proteins when K18Ser33is phosphorylated, and this binding may partially modulate hepatocyte mitotic progression, in association with redistribution of14-3-3proteins during mitosis. Indeed, here we further confirmed that K18binds14-3-3σ in a phosphorylation-dependent manner in NPC cells, and K18is required for the retention of14-3-3in the cytoplasm. Moreover, K18, like K17, contributes to trigger the PI3K/AKT pathway by the retention of14-3-3in the cytoplasm, in turn promotes the proliferation and growth in NPC cells. However, K18knockdown has little effect on the activity of MAPK pathway and PDK1level, indicating the inhibition of MAPK signaling and PDK1downreguation by LTF seems to be K18-independent.
Given our data showing that LTF can associate with K18, we next examined whether this association influenced ability of K18to bind14-3-3. As a result, overexpression of LTF causing induction of LTF-K18binding blocked the interactions of K18and14-3-3under the condition that K18phosophorylation remained unaffected and thereby influenced subcellular localization of14-3-3in NPC cells. Indeed, introduction of LTF expression plasmid in K18overexpression cells abolished the effect of K18on AKT pathway. In addition, overexpression of LTF also inhibited the abilities of K18to enhance the growth and soft-agar colony formation of5-8F cells. These results demonstrated that LTF suppressed AKT signaling and thereby reduced tumorigenesis partially by its interaction with K18.
3. LTF inhibits NPC cell proliferation and invasion via AKT pathway.
This current study revealed LTF can repress AKT signaling through multiple mechanisms. To clarify whether LTF suppresses NPC cells proliferation and invasiveness through inhibiting AKT signaling, we employed PI3K/AKT specific inhibitor Wortmannin and LY294002to treating the NPC cells in which endogenous LTF had been silenced through shRNA technique. We observed either Wortmannin or LY294002treatment significantly decreased cell proliferation rates, inhibited anchorage-independent growth as well as invasiveness ability in LTF absent HONE1cells. These results suggest that LTF's tumor suppressor function in NPC cells are partly through inhibiting AKT signaling.
We further examined the expression levels of LTF, pAKT (Thr308or Ser473) and total AKT by immunohistochemically analyzing tissue microarrays containing normal nasopharyngeal epithelia(n=33), normal epithelia adjacent to NPC (n=23), and NPC (n=138). The results revealed that the levels of LTF expression were significantly downregulated, but the phosopharylation levels of AKT at Thr308or Ser473were upregulated in the majority of NPC tissues compared with those normal nasopharyngeal epithelium tissues. Meanwhile, the expression levels of total AKT were unchanged in NPCs relative to normal nasopharyngeal epithelium tissues. Statistical analysis revealed that LTF expression negatively correlated with pAKT Thr308(R=-0.225, P=0.008) and pAKT Ser473(R=-0.345, P=0.000) in NPC tissues. Moreover, the additional effects that pAKT Thr308positively correlated with pAKT Ser473(r=0.413, P=0.000) in NPC samples were observed. Taken together, our results reveal an inverse relationship between low LTF expression and hyperactivation of AKT in human NPC, further supporting a model of LTF as a negative regulator of AKT signaling.
4. MiR-214targets LTF and subsequently suppresses AKT pahthway, resulting in promotion of NPC cell proliferation and invasion.
We previously showed that LTF is downregulated or absent in NPC. To determine whether decreased expression LTF correlates with aberrant miRNAs, we investigate the relationship between LTF and miRNAs. We demonstrated miR-214could bind to the3'untranslated region (UTR) of LTF by TargetScan software and luciferase reporter assays and inhibited its expression at mRNA and protein levels, suggesting that miR-214directly targets LTF. Both in vitro and in vivo assays revealed that miR-214may trigger growth and metastasis in NPC. Given that LTF inhibits AKT pahthway, we assessed the effect of miR-214on AKT activity. As a result, overexpression of LTF blocked miR-214-mediated AKT activation, which indicates that LTF suppresses AKT pahthway by targeting LTF. In vitro assays revealed miR-214promotes NPC cell proliferation and invasion through inhibiting LTF and in turn AKT pathway.
5. Decreased LTF expression correlates with increased miR-214expression in NPC.
In this study, we used qRT-PCR to measure miR-214and LTF expression levels in NPC tissue samples. MiR-214consistently showed increased expression levels in NPC tissues with respect to nasopharyngeal epithelial tissues and positively associated with tumor metastasis. However, LTF showed an inverse expression model, that is, LTF is downregulated in NPC samples compared with nasopharyngeal epithelial tissues. Moreover, the expression of LTF negatively correlates with tumor metastasis. Importantly, decreased LTF expression correlates with increased miR-214expression in NPC by correlation analysis.
In conclusion, LTF can repress AKT signaling through two mechanisms. Firstly, LTF reduces PDK1level by suppressing MAPK pathway and downregulating c-Jun level, leading to the inhibition of AKT activity. Secondly, LTF by interaction with K18blocks the formation of K18-14-3-3complex, which causes the sequestering of14-3-3in nucleus, and subsequently results in decreased AKT activity. Moreover, we have shown that LTF plays an important role in suppressing tumorigenesis of NPC partially through inhibition of AKT pathway. In addition, miR-214promoted NPC cell proliferation and invasion through inhibiting LTF and in turn AKT pathway. We have demonstrate that LTF is regulated by miR-214in NPC, namely, decreased LTF expression is partially due to increased miR-214expression in NPC.
引文
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