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. 2021 Nov 3:11:758503.
doi: 10.3389/fonc.2021.758503. eCollection 2021.

Clinicopathological and Preclinical Patient-Derived Model Studies Define High Expression of NRN1 as a Diagnostic and Therapeutic Target for Clear Cell Renal Cell Carcinoma

Affiliations

Clinicopathological and Preclinical Patient-Derived Model Studies Define High Expression of NRN1 as a Diagnostic and Therapeutic Target for Clear Cell Renal Cell Carcinoma

Shuhei Kamada et al. Front Oncol. .

Abstract

Background: Acquired therapeutic resistance and metastasis/recurrence remain significant challenge in advance renal cell carcinoma (RCC), thus the establishment of patient-derived cancer models may provide a clue to assess the problem. We recently characterized that neuritogenesis-related protein neuritin 1 (NRN1) functions as an oncogene in testicular germ cell tumor. This study aims to elucidate the role of NRN1 in RCC.

Methods: NRN1 expression in clinical RCC specimens was analyzed based on immunohistochemistry. NRN1-associated genes in RCC were screened by the RNA-sequencing dataset from The Cancer Genome Atlas (TCGA). RCC patient-derived cancer cell (RCC-PDC) spheroid cultures were established and their viabilities were evaluated under the condition of gene silencing/overexpression. The therapeutic effect of NRN1-specific siRNA was evaluated in RCC-PDC xenograft models.

Results: NRN1 immunoreactivity was positively associated with shorter overall survival in RCC patients. In TCGA RCC RNA-sequencing dataset, C-X-C chemokine receptor type 4 (CXCR4), a prognostic and stemness-related factor in RCC, is a gene whose expression is substantially correlated with NRN1 expression. Gain- and loss-of-function studies in RCC-PDC spheroid cultures revealed that NRN1 significantly promotes cell viability along with the upregulation of CXCR4. The NRN1-specific siRNA injection significantly suppressed the proliferation of RCC-PDC-derived xenograft tumors, in which CXCR4 expression is significantly repressed.

Conclusion: NRN1 can be a potential diagnostic and therapeutic target in RCC as analyzed by preclinical patient-derived cancer models and clinicopathological studies.

Keywords: C-X-C chemokine receptor type 4; cancer stem-like cell; cancer stemness; neuritin 1; patient-derived cancer cell (PDC); patient-derived xenograft; renal cell carcinoma; spheroid.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial of financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
NRN1 is a poor prognostic factor for patients with RCC. (A) Representative immunohistochemistry (IHC) staining of low (left panel) and high (right panel) expression of NRN1 in RCC tissue sections. Scale bars, 100 µm. (B) Overall survival of 100 patients with clear cell RCC (ccRCC) of low or high NRN1 immunoreactivity was analyzed by Kaplan-Meier method. Statistical significance was evaluated by log-rank test. (C) Overall survival of RCC patients with low or high NRN1 mRNA levels were analyzed by Kaplan-Meier method. NRN1 expression data in TCGA RCC dataset were retrieved from The Human Protein Atlas (n = 845). High and low NRN1 expressions were determined at an expression cut off level as 6.6 fragments per kilobase of exon per million mapped fragments (FPKM) (expression level range: 0.1-204.2, expression median level: 2.9 FPKM, P = 3.4e-4). Statistical significance was evaluated by log-rank test.
Figure 2
Figure 2
Histological analysis of RCC primary tumors, and their patient-derived cancer models. Hematoxylin and eosin (HE) staining, NRN1 and CXCR4 immunohistochemistry of primary tumor specimens, corresponding patient-derived cancer cell (PDC) spheroid cultures, and their xenograft tumors. Star represents ccRCC tumor portion with eosinophilic cytoplasm. Scale bars, 50 µm.
Figure 3
Figure 3
NRN1 and CXCR4 promote RCC-PDC viability. (A–H) NRN1 silencing decreases whereas overexpression increases PDC viability. RCC-PDC1/2 spheroid cultures were transfected with NRN1 (siNRN1 #1 and #2) or control (siControl) siRNAs (A–D), and NRN1 or control expression vector (E–H). NRN1 mRNA levels were analyzed by qRT-PCR (A, C, E, G) (n = 3) and spheroid growth was estimated by cell viability assay based on ATP quantification in cell lysates (B, D, F, H) (n = 4). Data are shown as means ± SD. *P < 0.05 by two-sided Student’s t-test. (I–L) NRN1 silencing decreases whereas overexpression increases CXCR4 expression. RCC-PDC1/2 were transfected with siRNAs: siNRN1 #1, #2, or siControl (I, J), or expression vectors (NRN1 or control vector) (K, L). (M–P) CXCR4 silencing represses PDC viability. RCC-PDC1 and 2 were transfected with siRNAs targeting CXCR4 (siCXCR4 #1 and #2) or siControl. CXCR4 mRNA levels were analyzed by qRT-PCR (M, O) and spheroid growth was estimated by cell viability assay (N, P). Data are shown as means ± SD, n = 3. *P < 0.05 by two-sided Student’s t-test.
Figure 4
Figure 4
NRN1 silencing suppresses in vivo growth of RCC-PDC-derived xenograft tumors. (A) Representative images of xenograft tumor-bearing nude mice at time of sacrifice. (B) Volume of xenograft tumors derived from RCC-PDC1 cells treated with siControl (n = 5, red) or siNRN1 #1 (n = 5, blue). (C) Body weights of mice at time of sacrifice. (D) Representative images of hematoxylin and eosin (HE) staining and NRN1 and CXCR4 IHC staining in dissected xenograft tumors treated with siControl or siNRN1 #1. Scale bars, 50 μm. (E, F) NRN1 (E) and CXCR4 (F) levels in xenograft tumors analyzed by qRT-PCR. Data are shown as mean ± SD, n = 5; *P < 0.05, **P < 0.01 by two-sided Student’s t-test. (G) Schematic representation of oncogenic function of NRN1 and CXCR4 in RCC tumors.

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