PLK4 as a Key Regulator of Neuroblastoma Differentiation and a Promising Therapeutic Target

Abstract

Background: Neuroblastoma (NB) differentiation status critically influences prognosis and treatment response. Although differentiation therapy has shown clinical benefit, its efficacy remains limited. The molecular mechanisms driving NB differentiation are not fully understood. PLK4 has been linked to NB tumorigenesis, but its role in regulating differentiation remains unclear. Methods: We investigated the role of PLK4 in neuroblastoma differentiation by modulating its expression both in vitro and in vivo. Through comprehensive analyses employing Western blotting, co-immunoprecipitation, immunofluorescence and murine neuroblastoma models, we identified downstream signaling pathways involved in PLK4-mediated regulation of neuronal genes. Pharmacological inhibition of PLK4 further confirmed its functional relevance in promoting neuroblastoma differentiation. Results: PLK4 functions as a key regulator of neuroblastoma differentiation. Its depletion enhances neuronal maturation and sensitizes cells to 13-cis RA. Mechanistically, we identify a novel PLK4-CXCR4 signaling axis that governs neuroblastoma differentiation through PI3K/Akt-mediated modulation of cyclin D1 expression. The selective PLK4 inhibitor CFI-400945 exhibits dual anti-tumor activity by promoting terminal differentiation and suppressing proliferation. Conclusions: Our study identifies PLK4 as a potential molecular switch governing NB differentiation and a promising therapeutic target to overcome resistance to 13-cis RA.

Keywords: CXCR4; PLK4; cyclin D1; differentiation therapy; neuroblastoma.

Figure 1

The sensitivity of NB cell lines to 13-cis RA is correlated with the expression of PLK4. A. Neurite extension assessment by measuring neurite length to cell body diameter in neuroblastoma cells treated with 0.1% DMSO or 10 μM 13-cis RA for 5 days. B. Immunofluorescence staining showing β-III tubulin expression and neurite outgrowth in SH-SY5Y cells treated with DMSO or 13-cis RA. C. Western blotting analysis of PLK4, SYN, MYCN, PHOX2B and GAP43 expression in 5 NB cell lines. D. Quantification of relative protein expression was performed, and data represent mean ± SEM from 3-4 independent experiments. E. Relative expressions of PLK4 and differentiation-associated markers were measured by quantitative reverse transcription PCR (RT-PCR). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns: no significance.

Figure 2

PLK4 influences the differentiation of NB cells and PLK4-knockdown sensitizes them to the effects of 13-cis RA. A. PLK4 and differentiation-associated markers expression levels were assessed using Western blotting in NB cell lines (KD1: PLK4-knockdown1, KD2: PLK4-knockdown2, OE: PLK4 overexpression). B. Quantification of PLK4 and differentiation-associated markers expression was performed, and data represent mean ± SEM from 3-4 independent experiments. C & D. Expression levels of PLK4 and differentiation-associated markers were evaluated using RT-PCR in NB cell lines. E. Immunofluorescence of β-III Tubulin in SK-N-SH cells showing neurite outgrowths (red arrows) seen following PLK4-knockdown. F. Western blotting analysis of PLK4 and differentiation-associated markers protein levels in NB cell lines following PLK4-knockdown (the term KD as used in the text and figure pertains to KD2). G. Quantification of relative protein expression in 2G. Data were presented as mean ± SEM. n = 3-4. H. Rescue experiments assessing whether RA-induced differentiation is reversed by PLK4 overexpression in SK-N-SH. I. Quantification of above protein expression in 2H. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns: no significance.

Figure 3

The PI3K/Akt pathway plays a crucial role in PLK4-mediated differentiation. A. Results of a pathway analysis obtained from RNA sequencing (RNA-seq) in the PLK4-knockdown group (|Log2 fold change| > 1 and adjusted p value < 0.05). B. Western blotting analysis demonstrating the effect on different signaling pathway activation following PLK4-knockdown in SK-N-SH and SK-N-AS. C. Protein expression levels of various signaling pathways were quantified. Data represent mean ± SEM from 3-4 independent experiments. D. PLK4-overexpression NB cells were pretreated with LY294002. Western blotting analysis of the protein levels of p-Akt³⁰⁸, Akt, and differentiation-associated protein. E. Quantification of above protein expression in 3D. The mRNA (F) and protein (G) levels of PLK4, differentiation protein and Akt signaling pathway components assessed in murine tumor tissues by RT-PCR and Western blotting. H. IHC analysis of PLK4 and differentiation protein expressions in two groups. (KD1: PLK4-knockdown1, KD2: PLK4-knockdown2, OE: PLK4 overexpression). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns: no significance.

Figure 4

PLK4 mediates cell differentiation by interacting with CXCR4. A. Heatmap showing differentially expressed genes in PLK4-knockdown cells compared with the control cells. n = 3. B. Relative mRNA levels were of top differentially expressed genes were detected using RT-PCR. C. As shown in the left panel, Western blotting detected the expression of PLK4, CXCR4 and differentiation-associated markers after SH-SY5Y being treated with CFI-400945 or AMD-3100. The right panel corresponds to the quantification of relative protein expression. Data were presented as mean ± SEM. n = 3. D. Co-IP of endogenous as well as exogenous PLK4 interacts with CXCR4 in NB cells. E. PLK4-overexpression cells were treated with or without CCND1 siRNA, PLK4, CCND1 and differentiation-associated markers was examined. F. Quantification of relative protein expression in 3E. Data were presented as mean ± SEM. n = 3. Nuclear-cytoplasmic fractionation experiments (G) and Immunofluorescence (H-I) showed that PLK4 knockdown reduced the nuclear localization of cyclin D1. J. PLK4-overexpression cells were infected with LY294002 (5 μM, 48 h). The protein level of p-Akt and CCND1 were demonstrated by Western blotting. K. Quantification of relative protein expression in 3J (KD: PLK4-knockdown2, OE: PLK4 overexpression). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns: no significance.

Figure 5

PLK4 expression strongly correlates with the differentiation capacity and clinical outcome of NB. A. Quantification of IHC score (NB neuroblastoma; GNB ganglioneuroblastoma; GN ganglioneuroma). RT-PCR (B) and Western blotting (C) analysis of PLK4 expression in NB tissues with different differentiation status. D. Assessment of PLK4, PHOX2B, CXCR4, and cyclin D1 expression levels and their associations in the same tumor tissues. E. Intratumoral heterogeneity was confirmed through fluorescent multiplex immunohistochemistry. F. The Kaplan-Meier survival analysis of overall survival and progress free survival for NB patients in poor-differentiated subgroup. G. Correlation analysis between PLK4 expression and PHOX2B expression, differentiation status, CXCR4 expression and bone marrow metastasis. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 6

Inhibiting PLK4 can suppress the proliferation of NB cells and promote differentiation. A. IC50 values of CFI-400945 treatment in neuroblastoma cell lines for 72 h. IC50 was calculated in GraphPad Prism 8.0 and displayed respectively. B. Immunofluorescence of β-III Tubulin in SH-SY5Y cells treated with CFI-400945 or 0.1% DMSO for 5 days. C. Neurite extension assessment in neuroblastoma cells treated with CFI-400945 or 0.1% DMSO for 5 days. Treatment with CFI-400945 resulted in a significant increase in neurite outgrowth in NB cell lines, indicating differentiation of surviving cells. Data reported as mean ± SEM. D. Western blotting analysis of cells after treatment with CFI-400945 compared with the control cells. E. Quantification of relative protein expression in 6D. Data were presented as mean ± SEM. n = 3. F-G. Tumor development in SCID mice and tumor weights of tumors obtained from xenografts were assessed. H. Tumor volume in the all groups. I. The inhibition efficiency of PLK4, CXCR4 and the expression of differentiation-associated marker were examined by Western blotting. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns: no significance.

Figure 7

Working model.