LTF Suppresses Tumor Growth And Metastasis By Inhibiting AKT Pathway In Nasopharyngeal Carcinoma

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 LTFK18is 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.