Comprehensive analysis and immunohistochemistry localization of NRP1 expression in pancancer and normal individual tissues in relation to SARS-COV-2 susceptibility
JIEWEN FU1,2*, JIAYUE HE1*, LIANMEI ZHANG1,3%, JINGLIANG CHENG1, PENGFEI ZHANG4, CHUNLI WEI1, JUNJIANG FU1 and DABING LI1,2
1Key Laboratory of Epigenetics and Oncology, The Research Center for Preclinical Medicine, Southwest Medical University;
2School of Basic Medical Sciences, Southwest Medical University, Luzhou, Sichuan 646000; 3Department of Pathology, The Affiliated Huai’an No. 1 People’s Hospital of Nanjing Medical University, Huai’an, Jiangsu 223300;
ANational Health Commission Key Laboratory of Cancer Proteomics, Department of Oncology, Xiangya Hospital, Central South University, Changsha, Hunan 410008, P.R. China
Received July 16, 2023; Accepted November 20, 2023
DOI: 10.3892/etm.2023.12340
Abstract. Neuropilin 1 (NRP1/CD304) is a typical membrane- bound co-receptor for vascular endothelial growth factor, semaphorin family members and viral severe acute respi- ratory syndrome coronavirus 2 (SARS-CoV-2). However, NRP1 expression levels across cancer types and the potential role of SARS-CoV-2 infection in patients with cancer are not clear. Online databases, such as The Cancer Genome Atlas database of Human Protein Atlas, Gene Expression Profiling Interactive Analysis and cBioPortal were used for the expres- sion analysis in this study. Immunohistochemical (IHC) staining for NRP1 was performed in the tissues of patients with non-small cell carcinoma. As a result, it was found that NRP1 mRNA and protein expression levels were highest in the female reproductive tissues and the respiratory system, specifically in the nasopharynx, bronchus and fallopian tube, as well as in adipocytes, hepatic stellate cells, Sertoli cells, endothelial cells and dendritic cells. IHC showed that the NRP1 protein was mainly localized to the cytoplasm and membrane in the tissues of patients with non-small cell carcinoma, demonstrating its role in lung infection by SARS-CoV-2, due to invasion of cell membranes by the virus. Levels of NRP1 mRNA were significantly increased in
Correspondence to: Professor Junjiang Fu or Professor Dabing Li, Key Laboratory of Epigenetics and Oncology, The Research Center for Preclinical Medicine, Southwest Medical University, 3-319 Zhongshan Road, Luzhou, Sichuan 646000, P.R. China E-mail: fujunjiang@swmu.edu.cn
E-mail: lidabing@swmu.edu.cn
*Contributed equally
Key words: NRP1/CD304, expression, coronavirus disease 2019, cancer, target
lymphoid neoplasm diffuse large B-cell lymphoma, esopha- geal carcinoma, glioblastoma multiforme, head and neck squamous cell carcinoma, kidney renal clear cell carcinoma (KIRC), pancreatic adenocarcinoma, stomach adenocarci- noma and thymoma, and significantly decreased in cervical squamous cell carcinoma and endocervical adenocarcinoma, kidney chromophobe, lung squamous cell carcinoma, ovarian serous cystadenocarcinoma, uterine corpus endo- metrial carcinoma and uterine carcinosarcoma, compared with corresponding healthy tissues in pancancer, indicating roles for viral invasion in most cancer types. Moreover, low NRP1 expression was significantly associated with long overall survival (OS) time in adrenocortical carcinoma, brain lower grade glioma, stomach adenocarcinoma and uveal melanoma, but with short OS time in KIRC only. The ENST00000374867.6 (NRP1-202) isoform is most highly expressed in most cancer types and thus could be involved in tumorigenesis and SARS-CoV-2 invasion in cancer patients. NRP1 may be involved in SARS-CoV-2 invasion in patients with cancer, including those with lung cancer.
Introduction
Neuropilin 1 (NRP1; OMIM: 602069), also known as CD304/VEGF165R/NRP/vascular endothelial cell growth factor 165 receptor, is a typical membrane-bound co-receptor both for members of the semaphorin family and vascular endothelial growth factor (VEGF) (1-4). The NRP1 gene is located at human chromosome 10p11.22. NRP1 encodes a deduced 923-amino acid protein with a molecular mass of 103,134 Da (NM_003873.7, NP_003864.5) containing an N-terminal signal sequence, a transmembrane region, an ectodomain and a cytoplasmic domain, consistent with the structure of cell surface receptors (5). These specific domains participate in different signaling pathways and have versatile roles in controlling survival, migration, invasion, angiogenesis and axon guidance through ligands binding to co-receptors, including VEGF and semaphorin family members (3,4).
Two landmark papers by Cantuti-Castelvetri et al (6) and Daly et al (7) found that NRP1 can act as a receptor to facilitate severe acute respiratory syndrome coronavirus 2 (SARS CoV-2) invasion into host cells (8). Mutation of novel NRP1 interac- tion sites located in the vestigial plasminogen-apple-nematode (PAN) domain was recently reported to reduce SARS-CoV-2 S-protein internalization (9). SARS-CoV-2 causes severe coronavirus disease 2019 (COVID-19), which has been the leading global pandemic since outbreaks began at the end of 2019. Unlike the S-protein of SARS-CoV-1, the S-protein of SARS-CoV-2 has a polybasic sequence domain (Arg-Arg-Ala-Arg) (the C-end rule) at the S1-S2 boundary that facilitates cleavage by furin (10), an enzyme convertase that catalyzes conversion of a substance to its active state. Thus, SARS-CoV-2 can easily enter host cells with the aid of NRP1, promoting its infectivity and tropism (11). In addi- tion, cells from bronchoalveolar lavage fluid of patients with COVID-19, but not uninfected cells, show an increase in NRP1 RNA expression in SARS-CoV-2-positive cells (6), further enhancing SARS-CoV-2 entry. Wang et al (12) reported that NRP1 is highly expressed in macrophages and dendritic cells (DCs) of myeloid lineage but not in CD4+ T cells, acting as an inhibitor of human immunodeficiency virus-1 infec- tivity. Targeting NRP1 is a potential approach to preventing SARS-CoV-2 entry (13,14) and developing potential antitumor drugs (15,16), with peptide-based inhibition of angiogenesis, proliferation and migration in tumor cells (17). In addition, NRP1 facilitates the invasion and replication of other various viruses, such as herpesvirus Epstein-Barr virus (EBV) (18), pseudorabies virus (19), mouse cytomegalovirus (20), and the human T-cell lymphotropic virus-1 (HTLV-1) and HTLV-2 retroviruses (21).
Small-molecule inhibitors of the S-protein of SARS-CoV-2 may bind to NRP1 (22). In silico analysis has revealed that natural product small molecules that interfere with SARS-CoV-2 binding to NRP1 are potential candidate novel antiviral agents (23-26). Folic acid, leucovorin and alimemazine may have the potential to prevent SARS-CoV-2 internaliza- tion by interacting with the S-protein/NRP1 complex (27,28). Targeting NRP1 with small molecules thus has the potential to interfere with SARS-CoV-2 invasion (29). However, the poten- tial of NRP1 expression in SARS-CoV-2-infected patients with all types of cancer is not clear. It is essential to identify novel small molecules from natural products or traditional Chinese medicine with antitumor functions that can modulate expres- sion of host cell entry regulators to interfere with SARS-CoV-2 entry (11,22,30). The present study analyzed NRP1 expres- sion, DNA mutations and prognosis with different levels of NRP1 expression across cancer types, and susceptibility to SARS-CoV-2 invasion.
Materials and methods
Online databases. Human NRP1 gene expression in normal tissues and cancer was analyzed in The Cancer Genome Atlas (TCGA) database of the Human Protein Atlas (HPA) (https://www.proteinatlas. org/ENSG00000099250-NRP1/tissueand(https://www.protein- atlas.org/ENSG00000099250-NRP1/pathology) (31,32), and the association between NRP1 gene expression and survival in
patients with cancer was analyzed by Gene Expression Profiling Interactive Analysis (GEPIA 2; http://gepia2.cancer-pku. cn/#analysis) (33,34) in TCGA and GTEx data. Mutation and survival analyses for NRP1 across multiple cancer types were conducted using cBioPortal (https://www.cbioportal. org/results/cancerTypesSummary?case_set_id=all&gene_ list=NRP1&cancer_study_list=5c8a7d55e4b046111fee2296) (35-38). For these analyses, the term ‘NRP1’ was used in the online systems.
Antibodies and reagents. NRP1 antibody was purchased from Santa Cruz Biotechnology, Inc. (cat. no. sc-5307). 3,3’-Diaminobenzidine (DAB Substrate System; cat. no. ZLI-9017) was purchased from Origene Technologies, Inc.
Immunohistochemistry (IHC) for NRP1. The non-small cell carcinoma tissues were collected from a resection specimen from a 78-year-old male patient treated in Affiliated Huai’an No. 1 People’s Hospital of Nanjing Medical University Huai’an City, with informed consent. The tissues from patients with lung cancer were fixed in 10% neutral formalin for 24 h at room temperature. Paraffin sections (5 pm) were deparaf- finized in xylene and rehydrated using a descending alcohol series (100, 95, 85 and 70%). The sections were immersed in 10 uM sodium citrate buffer, and heated at 98℃ for 12 min for antigen retrieval. Following washing in 1X PBS, slides were incubated with 3% hydrogen peroxide for 10 min, washed in PBS again, and covered with blocking serum (5% bovine serum albumin) for 30 min at room temperature prior to incubation overnight at 4℃ with the primary antibody diluted at 1:200. The sections were sequentially washed in PBS, incubated with the biotin-conjugated secondary antibody (cat. no. SP-9000; ready-to-use; Origene Technologies, Inc.) for 60 min, incu- bated with the streptavidin-conjugated horseradish peroxidase (HRP) for 10 min and finally with the DAB Substrate System. The sections were then counterstained with hematoxylin for 20 sec at room temperature, dehydrated using an ascending alcohol series (70, 85, 95 and 100%), cleared with xylene and mounted with neutral balsam. Images were captured under a light microscope (30).
Statistical analysis. Statistical analysis was performed using an unpaired t-test (two groups) with SPSS v25.0 software, and data are expressed as the mean ± standard deviation. P<0.01 was considered to indicate a statistically significant difference. Kaplan-Meier curves and the log-rank test were also used (http://gepia2.cancer-pku.cn/#survival).
Results
NRP1 expression in human normal tissues, including immune cells. The expression levels for viral receptors might play important roles in SARS-CoV-2 susceptibility in normal indi- vidual tissues. The present study analyzed NRP1 expression levels using the HPA and the results are shown in Fig. 1. NRP1 mRNA and protein were mainly expressed in tissues of the female reproductive tract, followed by the respiratory system, muscle tissues, bone marrow and lymphoid tissues, endocrine tissues, liver and gallbladder, kidney and urinary bladder, male reproductive tissues, gastrointestinal tract and pancreas,
A
RNA AND PROTEIN EXPRESSION SUMMARY
B
Organ
Expression Alphabetical
RNA expression (nTPM)’ Protein expression (score)”
Score
Brain
High-
Cerebral cortex
Eye
Medium
Endocrine tissues
Low
Colon
Respiratory system
Proximal digestive
Not.
detected
Hippocampus
tract
Cerebral cortex
Cerebellum
Caudate
Thyroid gland
Parathyroid gland
Adrenal gland
Nasopharynx
Bronchus
Lung
Oral mucosa
Salivary gland
Esophagus
Stomach
Duodenum
Small intestine
Colon
Rectum
Liver
Gallbladder
Pancreas
Kidney
Urinary bladder
Testis
Epididymis
Seminal vesicle
Prostate
Vagina
Ovary
Fallopian tube
Endometrium
Cervix
Placenta
Breast
Heart muscle
Smooth muscle
Skeletal muscle
Soft tissue
Adipose tissue
Skin
Appendix
Spleen
Lymph node
Tonsil
Bone marrow
Liver
Gastrointestinal tract
Liver & Gallbladder
Kidney
Expression
Detection
All organs
Pancreas
C
Consensus dataset
RNA tissue specificity: Low tissue specificity
Organ
Expression
Alphabetical
Kidney & Urinary
bladder
nTPM
Testis
140
Male reproductive
120
tissues
100
Female reproductive
tissues
Lymph node
80
60
Muscle tissues
40
Connective & Soft
20
tissue
0
Skin
Cerebral cortex
Cerebellum
Choroid plexus
Olfactory bulb
Basal ganghe
halamus
Hypothalamus
Midbrain
Pons
Medulla oblongata
Spinal cord
Hippocampal formation
White matter
Amygdala
Retina
Thyroid gland
Parathyroid gland
Adrenal gland
Pituitary gland
Lung
Salivary gland
Esophagus
Tongue
Stomach
Duodenum
Small intestine
Colon
Rectum
Gallbladder
Pancreas
Kidney
Urinary bladder
Jestis
Epididymis
Seminh
Seminal vesicle
Prostate
vagina
Ovary
Fallopian tube Endometrium
Cervix
Placenta
breast
Breast
Heart muscle
Smooth muscle
Skeletal muscle
Adipose tissue
SKIN
Appendix
Spleen
Lymph node
Tonsil
Bone marrow
Thymus
Bone marrow &
Lymphoid tissues
D
HPA dataset nTPM
Immune cell type specificity: Immune cell enriched (plasmacytoid DC)
Lineage
Expression Alphabetical
40
30
Granulocytes
E
Monocytes
20
-Colls B-cells
Singer cell types nTPM
RNA singer cell types specificity: Cell type enhanced (Adipocytes, Sertoli cells, Hepatic stellate cells, Endothelial cells)
Group
Expression Alphabetical
10
Dendritic cells NK-cells
Total PBMC
400
Glandular epithelial cels Squamous epithelial cells Specialized epithelial cells
0
Basophil
Eosinophil
Neutrophil
Classical monocyte
Non-classical monocyte
Intermediate monocyte
T-reg
GdT-cell
MAIT T-cell
Memory CD4 T-cell
Naive CD4 T-cell
Memory CD8 T-cell
Naive CD8 T-cell
Memory B-cell
Naive B-cell
Plasmacytoid DC
Myeloid DC
Nk-cell
300
Total PBMC
Endocrine cells
200
Global cells
Trophoblast
100
Endothe al cells
Muscle ces
Bo pocytes cells
0
Mesenchymal cells
Basal respiratory cells
Exocrine glandular cells
Basal Prosc
Brast glandular
Breast myoepithelial c
Endometrial ciated
Glandular aflevering
Suprabasal keratinocytes
Undifferentiated cells
Blood & immune cells
Basal squamous preub
Squamous epithelial cell
cel5 De
Alveolar ce & Ve
Cholangiocyle
roximal
Distal tubular o
Collegrothelal cell
Uroina an
Sertoli cell
Enteroendocrine c
Pancreatic onguyding
Cone photoreceptor C
Early spermatid
Gastric mucus-secrating col
steenvilous Local
mouth muscle
Smooth mio
Endometrial stromal cell
Undifferentiated
Plasma Co
Panetti ce
Macropray
Langerhans cells
Erythrcia wane
Intestinal goblet cells
with low or no expression in other tissues. For connective and soft tissue, and the brain, the NRP1 mRNA levels were high whereas the protein levels were relatively low (Fig. 1A). Specifically, the NRP1 protein expression was highest in the nasopharynx and bronchus (respiratory system), and fallopian tube (female reproductive tissue) (Fig. 1B), whereas the NRP1 mRNA expression was highest in the placenta [133.7 normal- ized transcript per million (nTPM), female reproductive tissue] and adipose tissue (125.2 nTPM, connective and soft tissue) (Fig. 1C). The NRP1 mRNA expression in immune cells was analyzed and found to be enriched in plasmacytoid DCs (39.3 nTPM, dendritic cells); other cells had no or very low levels of NRP1 mRNA expression (Fig. 1D). Finally, the NRP mRNA levels in single cell types were enhanced, including adipocytes (366.0 nTPM), Sertoli cells (282.7 nTPM), hepatic stellate cells (184.6 nTPM) and endothelial cells (174.4 nTPM) (Fig. 1E). Overall, NRP1 is most highly expressed in the female reproductive tissues and the respiratory system, specifically in the nasopharynx, bronchus and fallopian tube, as well as in adipocytes, hepatic stellate cells, Sertoli cells, endothelial cells and dendritic cells.
NRP1 expression in cancer tissues and corresponding healthy tissues of different cancer types. Patients with malignant cancer are more vulnerable to SARS-CoV-2 attack, leading to high mortality rates (39-42). Expression levels of NRP1
between tumor tissues and corresponding healthy tissues among different cancer types were analyzed using GEPIA 2, and the results are shown in Fig. 2. The levels of NRP1 were significantly increased in lymphoid neoplasm diffuse large B-cell lymphoma (DLBC), esophageal carcinoma (ESCA), glioblastoma multiforme (GBM), head and neck squamous cell carcinoma (HNSC), kidney renal clear cell carcinoma (KIRC), pancreatic adenocarcinoma (PAAD), stomach adeno- carcinoma (STAD) and thymoma (THYM), and significantly decreased in cervical squamous cell carcinoma and endocer- vical adenocarcinoma (CESC), kidney chromophobe (KICH), lung squamous cell carcinoma (LUSC), ovarian serous cyst- adenocarcinoma (OV), uterine corpus endometrial carcinoma (UCEC) and uterine carcinosarcoma (UCS) compared with matched healthy tissues in different cancer types (TCGA normal and GTEx data) (Fig. 2A-C). These findings indicate roles for viral invasion in most cancer types, especially in DLBC, ESCA, GBM, HNSC, KIRC, PAAD, STAD and THYM.
IHC results for non-small cell carcinoma stained by NRP1. IHC was performed to assess NRP1 expression and localiza- tion in the tissues of patients with non-small cell carcinoma, and representative results are shown in Fig. 3. The NRP1 protein was mainly localized to the cytoplasm and membrane (Fig. 3A and B). Panel C shows the control without NRP1
UCS, uterine carcinosarcoma; UVM, uveal melanoma.
lary cell carcinoma; LAML, acute myeloid leukemia; LGG, brain lower grade glioma; LIHC, liver hepatocellular carcinoma; LUAD, lung adenocarcinoma; LUSC, lung squamous cell carcinoma; MESO, mesothelioma; OV, ovarian serous cystadenocarcinoma; PAAD, pancreatic adenocarcinoma; PCPG, pheochro- mocytoma and paraganglioma; PRAD, prostate adenocarcinoma; READ, rectum adenocarcinoma; SARC, sarcoma; SKCM, skin cutaneous melanoma; STAD, stomach adenocarcinoma; TGCT, testicular germ cell tumors; THCA, thyroid carcinoma; THYM, thymoma; UCEC, uterine corpus endometrial carcinoma;
Figure 2. NRP1 expression comparison between cancer tissues and matched healthy tissues. (A) NRP1 expression comparison between human tumor tissues and matched healthy tissues among different cancer types. Red colors indicate increase of expression while green colors indicate decrease of expression in tumor tissues in The Cancer Genome Atlas database. (B) NRP1 expression increases significantly in cancer tissues compared with that in matched healthy tissues among different cancer types. (C) NRP1 expression is significantly decreased in cancer tissues compared with that in matched healthy tissues among different cancer types. “P<0.01. N, normal; T, tumor; NRP1, neuropilin 1; TPM, transcripts per million. ACC, adrenocortical carcinoma; BLCA, bladder urothelial carcinoma; BRCA, breast invasive carcinoma; CESC, cervical squamous cell carcinoma and endocervical adenocarcinoma; CHOL, cholangio- carcinoma; COAD, colon adenocarcinoma; DLBC, lymphoid neoplasm diffuse large B-cell lymphoma; ESCA, esophageal carcinoma; GBM, glioblastoma multiforme; HNSC, head and neck squamous cell carcinoma; KICH, kidney chromophobe; KIRC, kidney renal clear cell carcinoma; KIRP, kidney renal papil-
A
TPM
Expression -log2 (TPM+1)
0
2
4
6
8
C
Expression -log2 (TPM+1)
0
2
4
6
8
100
200
300
400
500
600
T (n=77)
ACC
N (n=128)
T (n=404)
BLCA
1
T (n=1085)
BRCA
T (n=306)
CESC
N (n=13)
T (n=36) N (n=9)
CHOL
Y
T (n=275)
COAD
T (n=47)
DLBC
T (n=182)
ESCA
N (n=286)
T (n=163)
GBM
N (n=207)
T (n=519)
HNSC
T (n=66)
KICH
N (n=53)
T (n=523)
KIRC
N (n=100)
T (n=286)
N (n=60)
KIRP LAML
T (n=173) N (n=70)
T (n=518)
LGG
N (n=207)
T (n=369)
LIHC
N (n=160)
T (n=483)
N (n=347)
T (n=486) N (n=338)
LUSC MESO OV
N (n=88)
T (n=179)
N (n=171)
T (n=182) N (n=3)
PCPG
T (n=492)
PRAD
N (n=152)
T (n=92)
READ
N (n=318)
T (n=262)
SARC
N (n=2)
T (n=461)
N (n=558)
T (n=408)
N (n=211)
T (n=137)
N (n=165)
T (n=512)
THCA
N (n=337)
T (n=118) N (n=339)
THYM
T (n=174) N (n=91)
UCEC
T (n=57)
N (n=78)
T (n=79)
UCS UVM
| (T=306, N=13) CESC | (T=47, N=337) (T=182, DLBC ESCA | ||
| (T=56, N=153) KICH | 2 . | N=286) (T=163, N=207) GBM | |
| (T=486, N=338) LUSC | (T=519, N=44) HNSC | a | |
| (T=426, N=88) OV | (T=523, N=100) KIRC | 2 EM | |
| (T=174, N=91) UCEC | . | (T=179, N=171) PAAD | A |
| (T=408, N=211) STAD | S A | ||
| (T=57, N=78) UCS | (T=118, N=339) TYHM |
LUAD
T (n=87) T (n=426)
PAAD
1.
SKCM
STAD
TGCT
00
0
N (n=28)
N (n=291)
N (n=349)
N (n=337)
N (n=44)
A
B
C
antibody staining of the tissues from the patients with non-small cell carcinoma (Fig. 3C).
Prognostic value of the NRP1 expression in different cancer types. The prognostic value of NRP1 was further explored with the median used as the cutoff value between the high and low expression groups. Low expression was significantly associ- ated with long OS time in adrenocortical carcinoma (ACC), brain lower grade glioma (LGG), stomach adenocarcinoma (STAD) and uveal melanoma (UVM) (Fig. 4A, C, E and G), as a P-value of <0.01 is being used for significance, implying that NRP1 may be an unfavorable marker. CESC, GBM and LUSC showed a trend but P-values were between 0.01 and 0.05 (Fig. 4B, F and G). However, low expression of NRP1 was significantly associated with a short OS time in KIRC only (Fig. 4I), implying that NRP1 may be favorable. The survival map of NRP1 expression among different cancer types is summarized in Fig. 4J.
Distribution and structure of NRP1 isoforms among cancer types. Different isoforms have different domains and roles that may be responsible for SARS-CoV-2 entry (43,44). NRP1 has 14 isoforms in different cancer types with differential expression levels (Fig. 5A). Expression levels of isoforms ENST00000374875.5 (NRP1-002) and ENST00000374867.6 (NRP1-202) were highest in most of the cancer types, followed by ENST00000395995.5 (NRP1-203), ENST00000413802.1 (NRP1-009) and ENST00000418675.5 (NRP1-008); others were very low or not detectable (Fig. 5A). Consistently, the isoform utilization for ENST00000374867.6 (NRP1-202) was highest, followed by ENST00000374875.5 (NRP1-002), across all 33 cancer types; others were very low (Fig. 5B). The genomic struc- tures of NRP1 isoforms from 33 different cancer types are shown in Fig. 5C. The isoforms NRP1-001, NRP1-202 and NRP1-203 have CUB, DUF3481, F5_F8_type_C and MAM domains. NRP1-001 and NRP1-202 are 923 amino acids long, and NRP1-203 is 906 amino acids long, but the others lack some or all functional domains (Fig. 5C). Homologs of the NRP1 gene are conserved in chimpanzee, Rhesus monkey, mouse, rat, dog, cow, chicken, zebrafish and frog, with functional domain and size in humans being the same as NRP1-001 and NRP1-202 in cancer (Fig. 5D). These results indicate that the isoform ENST00000374867.6 (NRP1-202)
might be involved in normal development in humans, in tumorigenesis and in SARS-CoV-2 entry in patients with different cancer types.
Mutations in NRP1 across cancer types. Mutation of NRP1 interaction sites, located in the PAN domain, was recently reported to reduce SARS-CoV-2 S-protein internalization (9). Mutations at any of the first three cysteines (C82A, C104A and C147A) of the NRP1 gene had significant negative impacts on SARS-CoV-2 S-protein binding. Thus, the present study aimed to determine which NRP1 mutations occur in pan-cancer tissues. In 32 cancer types from 10,953 patients, a total of 201 NRP1 mutations, including missense, truncating, splice and structural variation/fusion mutations, were found; skin cuta- neous melanoma showed the highest mutation frequency in 9.91% of 444 cases, followed by UCEC in 7.18% of 529 cases (Fig. 6A). The detailed NRP1 mutation landscapes appeared to be distributed across whole-gene regions, with missense mutations being dominant (Fig. 6B). However, there were no NRP1 mutations at the following three cysteines: C82, C104 and C147.
Next, the survival predictive value was analyzed, and the association with survival between groups with altered and unaltered NRP1 showed no significant difference, including with regard to overall survival (OS). The median OS time of the unaltered group was 79.46 months (95% CI, 73.68-84.20), while that of the altered group was shorter, at 65.33 months (95% CI, 48.76-107.18) (Fig. 6C).
Discussion
Cantuti-Castelvetri et al (6) and Daly et al (7) first found that NRP1 could act as a receptor to facilitate SARS-CoV-2 inva- sion into host cells (8). Recently, Lu et al (45) identified NRP1 as an entry receptor for Kaposi’s sarcoma-associated herpes- virus (KSHV), a double-stranded DNA virus, in mesenchymal stem cells. KSHV has been implicated in the pathogenesis of KS (46) and other malignancies, including multicentric Castleman’s disease (47), primary effusion lymphoma (48) and childhood osteosarcoma (49), highlighting NRP1 as a risk factor for viral entry in patients with cancer and viral-asso- ciated endemic cancer. However, NRP1 expression across cancer types and the potential roles of SARS-CoV-2 infection in patients with cancer are not clear.
A
Overall survival
B
Overall survival
C
1.0
Low NRP1 Group
1.0
Low NRP1 Group
1.0
Overall survival
High NRP1
Low NRP1 Group
Logrank p=0.0011
High NRP1 Group
High
HR(high)=3.9
Logrank p=0.016
Logrank p=0.00019
0.8
p(HR)=0.0024
0.8
HR(high)=1.8
0.8
HR(high)=1.8
n(high)=38
p(HR)=0.018
p(HR)=0.00023
n(high)=146
n(high)=192
Survival rate
0.6
n(low)=38
Survival rate
0.6
n(low)=146
Survival rate
0.6
n(low)=192
0.4
+
Q.
0.4
0.2
0.2
0.2
0.0
ACC
0.0
CESC
0.0
STAD
0
50
100
150
0
50
100
150
200
0
20
40
60
80
100
120
Months
Months
Months
D
Overall survival
m
Overall survival
F
1.0
Overall survival
1.0
LOW NRP1 Group
1.0
High NRP1 Group
Low
High NRP1 Group
Low NRP1 Group
Logrank p=0. 045
Logrank p=0.0019
High
0.8
HR(high)=66+08
0.8
0.8
p(HR)=1
HR(high)=4.3
Logrank p=0.037
n(high)]=68
p(HR)=0.0044
HR(high)=1.5
n(low) -68
n(high)=39
p(HR)=0.035
Survival rate
0.6
Survival rate
0.6
n(low)=39
Survival rate
0.6
n(high)=81
n(low)=81
4
0.4
0.4
0.2
TGC
0.2
0.2
0.0
T
0.0
UVM
0.0
GBM
0
50
100
150
200
250
0
20
40
60
80
0
20
40
Months
Months
Months
G
Overall survival
Overall survival
1.0
Overall survival
エ
1.0
1.0
Low NRP1 Group
Low NRP1 Group
High
High NRP1
Logrank p=9e-04
HR(high)=0.59
0.8
Logrank p=0.041
0.8
0
HR(high)=1.3
p(HR)=0.001
p(HR)=0.041
n(high)=258
Survival rate
0.6
n(high)=241
Survival rate
n(low)=258
Survival rate
0.6
n(low)=241
0.6
0.4
Low
+
0.4
High NRP1 Group
0.2
Logrank p=0.0086
HR(high)=1.6
0.2
0.2
p(HR)=0.0092
0.0
n(high)=257
LGG
LUSC
KIRC
n(low)=257
0.0
0.0
0
50
100
0
50
100
0
50
100
150
Months
Months
Months
J
Log10 (HR)
1.0
ENSG00000099250.17
☐
☐
☐
☐
☐
☐
☐
☐
☐
☐
☐
0.5
(NRP1)
0.0
ACC
BLCA
BRCA
CESC
CHOL
COAD
DLBC
ESCA
GBM
HNSC
KICH
KIRC
KIRP
LAML
LGG
LIHC
LUAD
LUSC
MESO
OV
PAAD
PCPG
PRAD
READ
SARC
SKCM
STAD
TGCT
THCA
THYM
UCEC
UCS
UVM
-0.5
-1.0
It is therefore important to investigate NRP1 expression across cancer types and the potential roles of SARS-CoV-2 in patients infected with cancer. In the current study, NRP1 mRNA and protein expression was found to be highest in the female reproductive tissues and the respiratory system, specifically in the nasopharynx, bronchus and fallopian tube, as well as in adipocytes, hepatic stellate cells, Sertoli cells,
endothelial cells and dendritic cells in the immune system. IHC showed that the NRP1 protein was mainly localized to the cytoplasm and membrane in the tissues of patients with non-small cell carcinoma, demonstrating its role in lung infection by SARS-CoV-2. The levels of NRP1 were signifi- cantly increased in DLBC, ESCA, GBM, HNSC, KIRC, PAAD, STAD and THYM and significantly decreased in
A
NRP1-010
1.2
0.0
NRP1-011
2.6
0.0
NRP1-006
3.1
0.0
NRP1-007
5.5
0.0 5.5
NRP1-008
0.0
+
A
2
1
NRP1-009
7.2
0.0
A
A
LIMARBAIRRADA
NRP1-203
4.4
0.0
5.
NRP1-002
7.6
0.0
.
NRP1-202
7.5
0.0
NRP1-005
3.1
0.0
NRP1-003
8.
0.0
NRP1-004
4.2
0.0
NRP1-201
3.7
0.0
NRP1-001
4.7
0.0
ACC
BLCA
BRCA
CESC
CHOL
COAD
DLBC
ESCA
GBM
HNSC
KICH
KIRC
KIRP
LAM
LLG
LIHC
LUAD
LUSC
MESO
OV
PAAD
PCPG
PRAD
READ
SARC
SKCM
STAD
TGCT
THCA
ΤΗΥΜ
UCEC
UCS
UVM
B
NRP1-010
NRP1-011
NRP1-006
NRP1-007
NRP1-008
NRP1-009
NRP1-203
NRP1-002
NRP1-202
NRP1-005
NRP1-003
NRP1-004
NRP1-201
NRP1-001
ACC
BLCA
BRCA
CESC
CHOL
COAD
DLBC
ESCA
GBM
HNSC
KICH
KIRC
KIRP
LAM
LLG
LIHC
LUAD
LUSC
MESO
OV -
PAAD
PCPG
PRAD
READ
SARC
SKCM
STAD
TGCT
THCA
THYM
UCEC
UCS
UVM
C
NRP1 (ENSG00000099250.17)
CUB
NRP1-001
923
DUF3481
NRP1-002
735
F5_F8_type_C
MAM
NRP1-003
644
NRP1-004
609
NRP1-005
704
NRP1-006
597
NRP1-007
100
NRP1-008
77
NRP1-009
105
NRP1-010
60
NRP1-011
170
NRP1-201
641
NRP1-202
923
NRP1-203
906
D
Genes
Proteins
Conserved domains
NRP1, H. sapiens
NP_003864.4
MAM (cd06263)
Neuropilin 1
923 aa
Meprin, A5 protein, and protein tyrosine phosphatase MU (MAM) domain. MAM is an extracellular domain
NRP1, P. troglodytes
XP_001143690.1
which mediates protein-protein interactions and is found in a diverse set of proteins, many of which are known to function in cell adhesion. Members …
Neuropilin 1
923 aa
NRP1, M. mulatta
XP_001087258.1
Neuropilin 1
923 aa
DUF3481 (pfam11980)
NRP1, C. lupus
XP_005617003.1
Domain of unknown function (DUF3481).
Neuropilin 1
926 aa
MAM (Smart00137)
NRP1, B. taurus
NP_001192589.1
Domain in meprin, A5, receptor protein tyrosine phosphatase mu (and others).
Neuropilin 1
923 aa
NrP1, M. musculus
NP_032763.2
MAM (pfam00629)
Neuropilin 1
923 aa
MAM domain.
NrP1, R. norvegicus
NP_659566.1
Neuropilin 1
CUB (cl00049)
922 aa
NRP1, G. gallus
CUB domain; extracellular domain; present in proteins mostly known to be involved in development; not found in prokaryotes, plants and yeast.
NP_990113.1
Neuropilin 1
914 aa
nrp1, X. tropicalis
NP_001093692.1
FA58C (cl19067)
Neuropilin 1
925 aa
Coagulation factor 5/8 C-terminal domain, discoidin domain; Cell surface-attached carbohydrate-binding domain, present in eukaryotes and assumed to have horizontally transferred to eubacterial genomes.
nrp1a, D. rerio
NP_852474.2
Neuropilin 1a
923 aa
A
10%
Alteration frequency
8%
Mutation
Structural variant
Amplification
Deep deletion
Multiple alterations
6%
4%
2%
Structural variant data + Mutation data
Skin cutaneous melanoma (TCGA, PanCancer Atlas)
Uterine corpus endometrial carcinoma (TCGA, PanCancer Atlas)
+ Stomach adenocarcinoma (TCGA, PanCancer Atlas)
+ Bladder urothelial carcinoma (TCGA, PanCancer Atlas)
+ + Mesothelioma (TCGA, PanCancer Atlas)
+ Ovarian serous cystadenocarcinoma (TCGA, PanCancer Atlas)
+ + Lung adenocarcinoma (TCGA, PanCancer Atlas)
+ + Esophageal adenocarcinoma (TCGA, PanCancer Atlas)
+ + Colorectal adenocarcinoma (TCGA, PanCancer Atlas)
+ + + Liver hepatocellular carcinoma (TCGA, PanCancer Atlas)
+ + + Cervical squamous cell carcinoma (TCGA, PanCancer Atlas)
+ + + Uterine carcinosarcoma (TCGA, PanCancer Atlas)
++ + Head and neck squamous cell carcinoma (TCGA, PanCancer Atlas)
+ + + Lung squamous cell carcinoma (TCGA, PanCancer Atlas)
+ + + Brain lower grade glioma (TCGA, PanCancer Atlas)
+ + + Breast invasive carcinoma (TCGA, PanCancer Atlas)
+ + + Prostate adenocarcinoma (TCGA, PanCancer Atlas)
+ + + Thymoma (TCGA, PanCancer Atlas)
+ + + Testicular germ cell tumors (TCGA, PanCancer Atlas)
+ + + Thyroid carcinoma (TCGA, PanCancer Atlas)
+ + + Pancreatic adenocarcinoma (TCGA, PanCancer Atlas)
+ + + Glioblastoma multiforme (TCGA, PanCancer Atlas)
+ + + Sarcoma (TCGA, PanCancer Atlas)
+ + + Kidney renal clear cell carcinoma (TCGA, PanCancer Atlas)
+ + + Kidney renal papillary cell carcinoma (TCGA, PanCancer Atlas)
+ + + Acute myeloid leukemia (TCGA, PanCancer Atlas)
+ + + Diffuse large B-cell lymphoma (TCGA, PanCancer Atlas)
+ + + Kidney chromophobe (TCGA, PanCancer Atlas)
+ + + Adrenocortical carcinoma (TCGA, PanCancer Atlas)
+ + + Cholangiocarcinoma (TCGA, PanCancer Atlas)
+ + + Pheochromocytoma and paraganglioma (TCGA, PanCancer Atlas)
+ + + Uveal melanoma (TCGA, PanCancer Atlas)
CNA data + + + + + + + + +
B
5
-| ENST00000265371
NRP1 Mutations
CCDS7177 | NRP1 HUMAN
A537V/T
Somatic mutation frequency 1.5%
1 Driver
201
VUS
0
Missense
158
Missense
0
0
Truncating
33 Truncating
CUB
CUB
F5_F8_type_C
F5_F8_type_C
MAM
DUF3481
0
Inframe
0 Inframe
0
200
400
600
800
923aa
0
Splice
7 Splice
1 SV/Fusion
3 SV/Fusion
View 3D Structure
C
X-Axis Max:
370 Months survival
100%
Logrank test P-value: 0.172
90%
Altered group
Unaltered group
Probability of overall survival
80%
70%
60%
50%
40%
30%
20%
10%
0%
0
20
40
60
80
100
1201
140
160 180 200 220 240 260 280 300 320 340 360
Overall survival (months)
Number of cases, total
Number of events
Median months overall (95% CI)
Altered group Unaltered group
235
91
65.33 (48.76-102.84) 79.46 (73.48-84.49)
10566
3420
CESC, KICH, LUSC, OV, UCEC and UCS, when compared with matched healthy tissues in different cancer types, indicating roles for viral invasion in most cancer types. Overall, low expression of ACC, LGG, STAD and UVM was significantly associated with longer OS time, but with shorter OS time in KIRC only, demonstrating that a poor prognosis was associated with high NRP1 expression in most cancer types. Notably, Morin et al (50) reported that NRP1 low expression is a biomarker of improved survival in patients with KIRC/renal cell carcinoma (RCC), thus confirming the bioinformatics results. In addition, NRP1 expression in KIRC/RCC patients might enhance infectivity or disease severity and the oncolytic properties of SARS-CoV-2 (51). The isoform ENST00000374867.6 (NRP1-202) is highly
expressed in most cancer types and thus might be involved in tumorigenesis and SARS-CoV-2 invasion in patients with different cancer types.
The limitations of the present study are based on the bioinformatics approach, and experimental validation of NRP1 expression may need to be performed in addition to IHC. Future studies will explore whether natural products, such as cordycepin and thymoquinone, would inhibit NRP1 expression and prevent susceptibility to SARS-CoV-2, as well as other viruses, such as EBV, KSHV, HTLV-1 and HTLV-2.
In conclusion, the present study highlights the significance of NRP1 expression, DNA mutation and prognostics in different cancer types and matched healthy tissue, and susceptibility to SARS-CoV-2 entry, and promotes the clinical potential and
practical implications of therapy for viral diseases, including COVID-19, and cancer by targeting NRP1.
Acknowledgements
The authors would like to thank Ms. Xiaoyan Liu from the Research Center for Preclinical Medicine, Southwest Medical University (Luzhou, China) for their technical help.
Funding
This study was supported by the Foundation of Science and Technology Department of Sichuan Province (grant nos. 2022NSFSC0737, 2023NSFSC0673 and 2022NSFSC1319), in part by the National Natural Science Foundation of China (grant nos. 81672887 and 82073263) and by the Primary Research and Development Plan of Hunan Province (grant no. 2020SK2071).
Availability of data and materials
The datasets used during the current study are available from the corresponding author on reasonable request.
Authors’ contributions
JiF, JH, LZ, CW and JC performed the experimental studies, data acquisition, data analysis and literature search. JuF collected and analyzed the data. JuF, DL and PZ designed and supervised the project. DL and JC confirm the authenticity of all the raw data. JuF wrote and edited the manuscript. All authors have read and approved the final manuscript.
Ethics approval and consent to participate
The study was approved by the Ethical Committee of Southwest Medical University (Luzhou, China) (approval no. 20221117-049). Written informed patient consent was obtained for participation.
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
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