Neuropilin 1 (NRP1) is a novel tumor marker in hepatocellular carcinoma
Introduction
HCC is a major health problem worldwide. It is the sixth most common cancer in the world, with more than half a million new cases annually [1]. TEAD family members share a highly conserved DNA binding domain called the TEA domain [2]. TEAD family members direct the transactivation of a wide variety of genes, perform as the major intracellular mediators of the Hippo-YAP pathway and are essential for HCC cell proliferation [3, 4]. The dominant-negative mutation TEAD1-Y406H has been identified as the causative mutation in Sveinsson's choriorentinal atrophy [5]. Zhou et al. produced this point mutation in HCC and found that it could abolish the YAP-TEAD interaction, likely by disrupting the hydrogen bond network between YAP and TEAD1 [6]. This mutation also hindered YAP-activated reporter gene expression [6, 7]. Moreover, the TEAD1-Y406H mutation also reduces the tumor growth rate in an HCC xenograft model [6], suggesting that TEAD1 could be a critical proto-oncoprotein in HCC, which is activated by dysregulation of Hippo/YAP pathway. In addition, TEAD mRNA levels are significantly up-regulated in HCC compared to those in normal liver tissues [8]. These results suggest that TEAD plays essential roles in HCC development. However, the target membrane proteins that reflect the TEAD function in HCC remain largely unknown. Furthermore, membrane proteins are promising candidates for HCC tumor markers because these proteins are derived from tumor lesions and are easily released into the peripheral blood. Whether target membrane proteins of TEAD are valuable HCC tumor markers needs further investigation.
Neuropilin 1 (NRP1) is type I transmembrane glycoprotein that was originally found to play a role in neuronal axon guidance and embryonic angiogenesis [9, 10]. NRP1 can promote various aspects of tumorigenesis, such as angiogenesis, cell survival, migration, invasion and chemo-resistance [11, 12]. In recent years, NRP1 was revealed to be a pivotal barrier for anti-tumor immunity. NRP1 interacts with immune cell-expressed ligand semaphorin-4a (Sema4a) to potentiate intratumoral regulatory T cell (Tregs) function and survival and then limit anti-tumor immune responses [13]. Using a NRP1-deficient mouse model of melanoma, Overacre-Delgoffe et al. observed that NRP1-deficient Tregs produce interferon-γ (IFNγ), which drives the fragility of the surrounding wild-type Tregs, boosts anti-tumor immunity, and facilitates tumor clearance [14]. Moreover, deletion of NRP1 from microglia or bone marrow-derived macrophages slows glioma progression [15]. In addition, emerging studies have reported that NRP1 may be a target of cancer treatment. MNRP1685A, a monoclonal antibody to NRP1, has been reported to exhibit good effects in clinical trials [16, 17]. Small molecule inhibitors of NRP1 such as EG00229, also slow down tumor progression [18, 19]. In the previous study, we performed ChIP-seq analysis and the data revealed that NRP1 may be a novel direct target of TEAD in cancer cells [20]. However, whether NRP1 stimulates tumorigenesis and performs as a novel tumor marker in HCC remains unknown.
Here, we validated that NRP1 was a novel TEAD target and required for the tumorigenesis of HCC. Furthermore, serum levels of NRP1 were specifically elevated in HCC patients compared to healthy individuals and patients with other diseases, suggesting that NRP1 could be a specific diagnostic marker for HCC. Compared to AFP, NRP1 has a better AUC-ROC (0.971) with a cutoff value of 68 pg/ml (sensitivity: 93.7%, specificity: 98.7%). NRP1 may be a substitute for AFP for HCC early diagnosis in the clinic.
Section snippets
Blood samples
All patients diagnosed with HCC (mean age ± SD, 55.37 ± 8.63 years; male: female ratio, 2.35:1), hepatitis B (mean age ± SD, 38.66 ± 6.44 years; male: female ratio, 1.35:1), hepatitis C (mean age ± SD, 57.45 ± 11.03 years; male: female ratio, 1.2:1), cirrhosis (mean age ± SD, 58.04 ± 9.66 years; male: female ratio, 2.83:1), colon cancer (mean age ± SD, 59.24 ± 12.06 years; male: female ratio, 3.75:1), gastric cancer (mean age ± SD, 55.66 ± 11.44 years; male: female ratio, 3.75:1), lung cancer
qPCR and Luciferase reporter assay
Total RNA was isolated from cells using Trizol (Ambion, Carlsbad, CA, USA) and reversed transcribed using the PrimeScript™ RT reagent Kit (Perfect Real Time) (TaKaRa, Dalian, China). Resulting cDNAs were analyzed by quantitative PCR using SYBR premix Ex Taq (TaKaRa). Luciferase reporter vectors were stably co-transfected with a Renilla luciferase expression plasmid into Bel-7402 and SMMC-7721 cells. Luciferase activities were detected using the dual-luciferase reagent (Promega). All the primers
NRP1 is the direct target of TEAD
Previous ChIP-seq analysis predicted that NRP1 could be the direct target of TEAD [20]. Therefore, we performed a series of experiments to further determine whether NRP1 was directly regulated by TEAD. qPCR data showed that mRNA levels of NRP1 were increased by TEAD overexpression and decreased by TEAD knockdown in two HCC cell lines, Bel-7402 and SMMC-7721,which have shown high carcinogenic properties in our previous studies [21, 22] (Fig. 1A). Similarly, protein levels of NRP1 were also
Discussion
TEAD is an important stimulator of liver tumorigenesis [[27], [28], [29]] and performs its oncogenic role mainly in the nucleus. For example, it is well known that the transcription cofactor YAP directly binds to TEAD and stimulates the transcription of a series of downstream genes in the nucleus [30, 31]. Our study also reported the interaction between TEAD and another nuclear factor, AP-1 [9], which is consistent with findings from other researchers [25, 32]. However, whether TEAD-related
Acknowledgements
This study was supported by the National Natural Science Foundation of China (Grants 81570055 and 81201913).
References (40)
- et al.
Regulation of the hippo pathway transcription factor TEAD
Trends Biochem. Sci.
(2017) - et al.
Neuropilin-1 is expressed by endothelial and tumor cells as an isoform-specific receptor for vascular endothelial growth factor
Cell
(1998) - et al.
Tead and AP1 coordinate transcription and motility
Cell Rep.
(2016) - et al.
TFCP2 is required for YAP-dependent transcription to stimulate liver malignancy
Cell Rep.
(2017) - et al.
Validation of the AFP model as a predictor of HCC recurrence in patients with viral hepatitis-related cirrhosis who had received a liver transplant for HCC
J. Hepatol.
(2017) - et al.
TRIB2 acts downstream of Wnt/TCF in liver cancer cells to regulate YAP and C/EBPalpha function
Mol. Cell
(2013) - et al.
YAP activation is an early event and a potential therapeutic target in liver cancer development
J. Hepatol.
(2014) - et al.
Merlin/NF2 loss-driven tumorigenesis linked to CRL4(DCAF1)-mediated inhibition of the hippo pathway kinases Lats1 and 2 in the nucleus
Cancer Cell
(2014) - et al.
Impeding macrophage entry into hypoxic tumor areas by Sema3A/Nrp1 signaling blockade inhibits angiogenesis and restores antitumor immunity
Cancer Cell
(2013) - et al.
Neuropilin-1 is upregulated in hepatocellular carcinoma and contributes to tumour growth and vascular remodelling
J. Hepatol.
(2011)
Serum DKK1 as a protein biomarker for the diagnosis of hepatocellular carcinoma: a large-scale, multicentre study
Lancet Oncol.
Serum CD166: a novel hepatocellular carcinoma tumor marker
Clin. Chim. Acta
Global cancer statistics, 2012
CA Cancer J. Clin.
Characterization of the transcription activation function and the DNA binding domain of transcriptional enhancer factor-1
EMBO J.
Yes-associated protein/TEA domain family member and hepatocyte nuclear factor 4-alpha (HNF4alpha) repress reciprocally to regulate hepatocarcinogenesis in rats and mice
Hepatology
A novel TEAD1 mutation is the causative allele in Sveinsson's chorioretinal atrophy (helicoid peripapillary chorioretinal degeneration)
Hum. Mol. Genet.
Targeting hippo pathway by specific interruption of YAP-TEAD interaction using cyclic YAP-like peptides
FASEB J.
TEAD mediates YAP-dependent gene induction and growth control
Genes Dev.
Genome-wide molecular profiles of HCV-induced dysplasia and hepatocellular carcinoma
Hepatology
Growth-associated expression of a membrane protein, neuropilin, in Xenopus optic nerve fibers
Dev. Neurosci.
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These two authors contributed equally to this study.