Elsevier

Clinica Chimica Acta

Volume 485, October 2018, Pages 158-165
Clinica Chimica Acta

Neuropilin 1 (NRP1) is a novel tumor marker in hepatocellular carcinoma

https://doi.org/10.1016/j.cca.2018.06.046Get rights and content

Highlights

  • The −1848 ~ −1841 region in the NRP1 promoter is critical for TEAD binding.

  • NRP1 is specifically up-regulated in the serum of HCC patients.

  • Serum NRP1 is significantly associated with AFP and other liver function markers.

  • The AUC-ROC for serum NRP1 0.971 which is better than AFP.

Abstract

Background

TEA domain transcription factor (TEAD) has an oncogenic role in hepatocellular carcinoma (HCC). However, whether a membrane protein can serve not only as a tumor marker that reflects TEAD function but also as a therapeutic target that stimulates tumorigenesis in HCC remains unknown.

Methods

Tissue NRP1 was measured using immunohistochemistry. Cell viability, colony formation and caspase3/7 activity were assessed using MTT, soft agar and caspase 3/7 Glo assays, respectively. Serum NRP1 was examined using ELISA and Western blotting.

Results

NRP1 expression was up-regulated by TEAD. We also identified a conserved TEAD-binding motif in the NRP1 promoters, which was essential for the TEAD-NRP1 interaction. NRP1 was upregulated in HCC tissues and cell lines, and knockdown of NRP1 inhibited the transformative phenotypes of HCC cells. Notably, the concentrations of serum NRP1 in the HCC patients were much higher than those of hepatitis B, hepatitis C, cirrhosis, breast cancer, colon cancer, gastric cancer and lung cancer patients. Moreover, serum NRP1 was significantly associated with AFP, γ-GT, Alb, bile acid, ALT, AST, ALP and pre-Alb. The area under the receiver operating characteristic curve (AUC-ROC) for serum NRP1 was 0.971, presenting better diagnostic performance compared to AFP.

Conclusions

NRP1 is a novel tumor marker in HCC.

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).

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    These two authors contributed equally to this study.

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