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. 2022 Dec 15;46(1):133–143. doi: 10.1007/s13402-022-00739-9

Targeting ARHGEF12 promotes neuroblastoma differentiation, MYCN degradation, and reduces tumorigenicity

Yi Yang 1,#, Siqi Wang 2,#, Jiaoyang Cai 2,#, Jianwei Liang 2,#, Yingwen Zhang 1, Yangyang Xie 2, Fei Luo 3, Jingyan Tang 2, Yijin Gao 2,✉, Shuhong Shen 2,✉, Haizhong Feng 3,✉, Yanxin Li 1,✉
PMCID: PMC12974740  PMID: 36520365

Abstract

Purpose

Neuroblastoma arises from developmental block of embryonic neural crest cells and is one of the most common and deadly pediatric tumors. However, the mechanism underlying this block is still unclear. Here, we show that targeting Rho guanine nucleotide exchange factor 12 (ARHGEF12, also named LARG) promotes MYCN degradation and neuroblastoma differentiation, leading to reduced neuroblastoma malignancy.

Methods

The neuroblastoma TARGET dataset was downloaded to assess ARHGEF12 expression. Cell differentiation, proliferation, colony formation and cell migration analyses were performed to investigate the effects of ARHGEF12 knockdown on neuroblastoma cells. Western blotting and immunohistochemistry were employed to determine protein expression. Animal xenograft models were used to investigate antitumor effects after ARHGEF12 knockdown or treatment with the ARHGEF12 inhibitor Y16 in vivo.

Results

We found that the expression level of ARHGEF12 was higher in neuroblastoma than in better-differentiated ganglioneuroblastoma. Knockdown of ARHGEF12 promoted neuroblastoma differentiation, decreased stemness-related gene expression, and increased differentiation-related gene expression. ARHGEF12 knockdown reduced tumor growth, and the resulting tumors showed bigger tumor cells compared to those in control neuroblastoma xenografts. In addition, it was found that ARHGEF12 knockdown promoted MYCN ubiquitination and degradation in MYCN-amplified tumors through RhoA/ROCK/GSK3β signaling. Targeting ARHGEF12 with the small molecular inhibitor Y16 induced cell differentiation and attenuated neuroblastoma tumorigenicity.

Conclusion

Our findings provide new insight into the mechanism by which ARHGEF12 regulates neuroblastoma tumorigenicity and suggest a translatable therapeutic approach by targeting ARHGEF12 with a small molecular inhibitor.

Supplementary Information

The online version contains supplementary material available at 10.1007/s13402-022-00739-9.

Keywords: Neuroblastoma, ARHGEF12/LARG, RhoA, Cell differentiation, Y16

Introduction

Neuroblastoma is the most common malignancy in infants and the most common extracranial solid tumor in children [1]. Although many efforts have been made to improve its outcome, it still accounts for ~ 10% of cancer-related deaths among the pediatric population [2]. Neuroblastoma originates from poorly differentiated neural crest progenitors, and aberrant or blocked differentiation is a central aspect of neuroblastoma genesis [3]. It is well-recognized that a higher tumor differentiation state is related to a more favorable clinical stage and a better clinical outcome [4]. However, the mechanisms underlying neuroblastoma differentiation induction are still elusive.

RhoA has been shown to be a neural differentiation inhibitor [5, 6], and aberrant activation of RhoA enhances neuroblastoma tumorigenicity [7, 8]. Rho guanine nucleotide exchange factor 12 (ARHGEF12), also known as LARG, is known to act as a RhoA-GEF and to regulate RhoA activity [9]. ARHGEF12 has been shown to regulate cell morphology and cell invasion [10, 11], and the mechanical response to forces on integrins [12, 13], mesenchymal stem cell stemness [14], smooth muscle cell differentiation [15], and the differentiation of neural stem cells into oligodendrocytes [16]. ARHGEF12 has also been found to function as a tumor suppressor in human breast and colorectal cancer [17]. By contrast, it has also been reported that ARHGEF12 can promote breast cancer cell proliferation [18]. Y16, an ARHGEF12 small-molecule inhibitor [18–20], has been reported to inhibit RhoA activity and suppress breast cancer cell sphere formation [19, 20]. Recently, we found that ARHGEF12 may regulate erythropoiesis and be involved in erythroid regeneration after chemotherapy in acute lymphoblastic leukemia patients [21]. As yet, however, the function of ARHGEF12 in neuroblastoma is still unknown.

Here, we found that the expression level of ARHGEF12 mRNA is higher in neuroblastoma than in better-differentiated ganglioneuroblastoma (GNB). ARHGEF12 knockdown promoted neuroblastoma cell differentiation and reduced its tumor growth. In addition, we found that ARHGEF12 regulates MYCN ubiquitination and stability through RhoA/ROCK/GSK3β signaling. Targeting ARHGEF12 with the small molecule inhibitor Y16 enhanced neuroblastoma differentiation and reduced its malignancy.

Materials and methods

Cell lines

HEK293T (RRID: CVCL_0063), SK-N-BE(2) (RRID: CVCL_0528), CHP-134 (RRID: CVCL_1124), SK-N-SH (RRID: CVCL_0531) and SH-SY5Y (RRID: CVCL_0019) were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Gibco) and antibiotics. The cells were cultured in a humidified atmosphere at 37 °C with 5% CO2. All cell lines were authenticated using SNP profiling within the last three years and were tested negative for mycoplasma contamination using a PCR-based method.

Plasmids and transfection

ARHGEF12 shRNAs were generated as previously described [22]. ARHGEF12 shRNAs were co-transfected with packaging plasmids, pMD2.G and psPAX2, into HEK293T cells using Hieff Trans™ Liposomal Transfection Reagent (Yeason Biotechnology Co. Ltd) as previously described [22]. Viruses were concentrated by centrifugation and added to the culture medium supplemented with 5 μg/ml polybrene. After 48 h, infected neuroblastoma cells were selected with puromycin and validated by Western blotting.

Subcutaneous xenograft assays

All animal experiments were approved by the Shanghai Jiao Tong University Institutional Animal Care and Use Committee (IACUC). NOD/SCID female mice aged 6–8 weeks (SLAC, Shanghai, China) were randomly divided into 5–6 per group. To assess the effects of ARHGEF12 knockdown, SK-N-BE(2) cells (5 × 106) with or without ARHGEF12 shRNAs (Table S1) were injected subcutaneously into the mice. To assess the efficacy of Y16 in neuroblastoma treatment, intraperitoneal injection with vehicle or Y16 (30 mg/kg) daily was initiated when the tumors reached an average size of 150–250 mm3. The tumor volumes were measured every other day. Finally, the mice were sacrificed and photographed.

RNA extraction and quantitative real-time PCR (qRT-PCR)

Total RNA was extracted from cell lines and tumor tissues using an EZ-press RNA Purification Kit (B0004DP; EZB) or Trizol (Thermo Fisher Scientific), respectively, according to the manufacturer's instructions. Reverse transcription was performed using a Hifair II 1st Strand cDNA Synthesis SuperMix for qPCR (gDNA digester plus; Yeasen Biotechnology). Quantitative real-time PCR was performed on a CFX Connect Real-Time PCR System (Bio-Rad Laboratories) using a SYBR Green master mix (Yeasen Biotechnology). All target gene-expression levels were normalized to β-actin. The primers used are listed in Table S2.

Immunofluorescence assay

Cells were fixed with 4% polyformaldehyde and permeabilized with 0.1% Triton X-100 as previously described [23]. After being blocked with 10% goat serum in PBS, cells were stained with F-actin (1:500; ab130935, Abcam). This primary antibody was visualized by goat anti-mouse IgG bound to Alexa 647 (1:500; ab150115, Abcam). Nuclear staining was performed with DAPI, and fluorescence images were obtained using a Zeiss inverted LSM confocal microscope (Carl Zeiss).

Hematoxylin–Eosin (H&E) staining and immunohistochemistry (IHC)

Formalin-fixed paraffin-embedded sections were stained with hematoxylin–eosin (HE) or immunostained with anti-Ki-67 (1:100, GT209429, Gene Tech) or anti-MYCN (1:200, #84,406, Cell Signaling Technology, CST) antibodies using a standard immunohistochemistry protocol. MYCN and Ki-67 positive cells were counted in three random fields per slide.

Western blotting

Western blotting (WB) was performed as previously described [24] using the following antibodies: anti-MYCN (1:1000, #84,406), anti-GSK3β (1:1000, #12,456), anti-GSK3β(ser9) (1:1000, #5558) and anti-β-actin (1:1000, #3700) from Cell Signaling Technology (CST), anti-ARHGEF12 (1:200, sc-166318), anti-ROCK1 (1:200, sc-17794) anti-ROCK2 (1:200, sc-398519) from Santa Cruz, and anti-GAPDH (1:50,000, No. 60004–1-Ig) from Proteintech.

Cell growth assay

Cells were seeded at a density of 5 × 104 per well in 12-well formats with at least three replicates per condition. Cell numbers were calculated using an automated cell counter (Bio-Rad Laboratories).

Cell cycle analysis

Cell cycle analysis was performed as previously described [22]. Cells were fixed in 70% cold ethanol and stained with propidium iodide (PI)-phycoerythrin (PE) after being treated with RNase A. Data were acquired using flow cytometry.

Colony-forming assay

Cells were seeded at a density of 1500–2000 per well in 6-well formats with at least three replicates per condition. After 10–15 days, cells were fixed with 4% paraformaldehyde and stained with 0.05% crystal violet. Cell colonies were imaged and counted for quantification.

Scratch wound healing cell migration assay

Cells were cultured in FBS-free DMEM for 12 h after they reached a confluent state in 6-well formats. Next, sterile 10 μl pipette tips were used to make scratches. After being rinsed with 1 × PBS, the cells were further cultured in DMEM without FBS. Images of the scratches were captured at 0 and 24 h. Cells migrated to the wound area were quantified by the relative percentage of wound area covered by migrated cells to the original wound area. Assays were repeated three times for each cell line.

RhoA activity assay

RhoA activity was assessed using a RhoA G-LISA activation assay kit (Cytoskeleton, BK-124) according to the manufacturer's instructions. Cells were harvested and lysed using the provided lysis buffer. Next, 10 µg cell lysates were subjected to a G-LISA™ assay. Absorbance was read at 490 nm wavelength.

Cycloheximide chase experiments

Cells cultured in DMEM with 10% FBS (Gibco) were treated with cycloheximide (CHX, 50 μg/ml) and harvested after 0, 15, 30, 45 and 60 min, respectively. Treated cells were lysed and analyzed by Western blotting using anti-MYCN (1:1000, #84,406, CST) and anti-GAPDH (1:50,000, No.60004–1-Ig, Proteintech) antibodies. MYCN protein expression was quantified using Image-Pro Plus 6.0 software.

Co-immunoprecipitation assay

Co-immunoprecipitation was performed as previously described [23]. Cells were harvested and lysed in IP lysis buffer (20 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 2 mM Na3VO4, 5 mM NaF, 1% Triton X-100, and protease inhibitor cocktail) at 4 °C for 30 min. Equal amounts of cell lysates were immunoprecipitated using specific antibodies and protein A-agarose beads (Invitrogen). Proteins were lysed in SDS lysis buffer after which standard Western blotting was performed using anti-MYCN (1:1000, #84,406), anti-ubiquitin (1:1000, #3936) and anti-β-actin (1:1000, #3700) antibodies from CST.

Data availability

The data supporting the findings of this study are available within the article and its supporting information files and/or available from the corresponding author upon reasonable request.

Statistical analysis

Data were processed using GraphPad Prism 8.0. Statistical differences between groups were assessed using paired or unpaired student's t-test or one-way ANOVA. Statistical details are described in the figures. Sample sizes were selected based on previous experience.

Results

ARHGEF12 knockdown promotes neuroblastoma cell differentiation

Since ARHGEF12 has been reported to regulate neural stem cell differentiation [16], we hypothesized that ARHGEF12 might also mediate neuroblastoma cell differentiation. To test this hypothesis, we first downloaded the clinical neuroblastoma TARGET dataset and found that ARHGEF12 mRNA levels were higher in neuroblastoma than in better-differentiated ganglioneuroblastoma (GNB) (Fig. 1a). Next, we evaluated ARHGEF12 protein expression in four neuroblastoma cell lines and found that ARHGEF12 was highly expressed in SK-N-BE(2) and SH-SY5Y cell lines (Fig. 1b). After knockdown ARHGEF12 with two different shRNAs in SK-N-BE(2) and SH-SY5Y cells (Fig. 1c), we found that this knockdown (KD) increased the sizes of cells in adherent culture or when digested with pancreatin (Fig. 1d and f). In addition, we found that inhibition of ARHGEF12 caused longer neurite-like protrusions and remarkable actin stress fiber changes (Fig. 1e and g). Finally, to further assess the effects of ARHGEF12 KD on neuroblastoma differentiation, we analyzed the expression of stemness-related genes and neuronal differentiation markers by qRT-PCR. We found that compared with the control, ARHGEF12 KD decreased the expression of SOX2 and OCT4 and increased the expression of NSE, SYP, NDRG1, and MAPT (Fig. 1h). These data indicate that ARHGEF12 KD promotes neuroblastoma differentiation.

Fig. 1.

Fig. 1

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ARHGEF12 knockdown promotes neuroblastoma differentiation. (a) Expression levels of ARHGEF12 mRNA in ganglioneuroblastoma and neuroblastoma specimens in the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) database. (b) Western blotting analysis of ARHGEF12 expression in four neuroblastoma cell lines. (c) Western blotting validation of ARHGEF12 knockdown (KD) in SK-N-BE(2) and SH-SY5Y cells treated with a control shRNA (shC) or two different ARHGEF12 shRNAs (shAR#1 and shAR#2). (d and f) Representative bright-field pictures of SK-N-BE(2) (d) and SH-SY5Y (f) cells after ARHGEF12 KD. Upper panel, digested by pancreatin, lower panel, adherent cultured. Scale bar, 50 µm. (e and g) Representative immunostaining pictures of SK-N-BE(2) (e) and SH-SY5Y (g) cells after ARHGEF12 KD. Red: F-actin, Blue: DAPI. Scale bar, 10 µm. (h) Effects of ARHGEF12 KD on the expression of stemness-related genes SOX2 and OCT4, and neuronal differentiation markers NSE, SYP, NDRG1 and MAPT in SK-N-BE(2) and SH-SY5Y cells. In a and h, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, by two-tailed t-test or one-way ANOVA

ARHGEF12 knockdown decreases neuroblastoma tumorigenicity

To investigate the functions of ARHGEF12 in neuroblastoma, we analyzed the effects of ARHGEF12 KD in vitro. We found that ARHGEF12 KD decreased cell proliferation (Fig. S1a), cell cycle progression (Fig. S1b), cell migration (Fig. S1c and S1d) and colony formation (Fig. S1e and S1f) in SK-N-BE(2) and SH-SY5Y cells compared with the corresponding controls.

We further assessed the role of ARHGEF12 using a SK-N-BE(2) xenograft model. We found that compared with the control, ARHGEF12 KD decreased tumor growth (Fig. 2a) and endpoint tumor weight without affecting overall mice body weight (Fig. 2b to d). More interestingly, we found that ARHGEF12 KD reduced tumor cell proliferation (Fig. 2e and f) and increased tumor cell sizes (Fig. 2e). Our data indicate that ARHGEF12 KD reduces neuroblastoma tumorigenicity.

Fig. 2.

Fig. 2

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ARHGEF12 knockdown decreases neuroblastoma tumorigenicity. (a) In vivo effects of ARHGEF12 KD on tumor growth. SK-N-BE(2) cells were xenografted into SCID mice. Tumor growth was monitored every other day. (b) Representative images of animals with SK-N-BE(2) tumors. (c) Representative images of SK-N-BE(2) tumors at the endpoint. (d) Quantification of tumor weights in c. (e) Representative images of H&E and Ki-67 staining. Scale bar, 50 µm. (f) Quantification of Ki-67 positive cells in e. In a, d and f, * p < 0.05, *** p < 0.001, by two-tailed t-test

ARHGEF12 knockdown promotes MYCN ubiquitination and degradation

MYCN is a core oncogene in neuroblastoma and high MYCN expression has been correlated with a poor prognosis [25]. We found in a cohort of clinical neuroblastoma tissues that the ARHGEF12 protein levels were positively associated with MYCN protein expression (Fig. 3a). ARHGEF12 KD markedly decreased MYCN protein levels (Fig. 3b) but not MYCN mRNA levels (Fig. 3c). This result was validated in SK-N-BE(2) xenograft tumors (Fig. 3d to g). These data indicate that ARHGEF12 may be related to MYCN protein stability.

Fig. 3.

Fig. 3

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ARHGEF12 knockdown promotes MYCN ubiquitination and degradation. (a) Western blotting analysis of ARHGEF12 and MYCN expression in 10 neuroblastoma specimens with or without MYCN amplification. (b and c) Effects of ARHGEF12 KD on protein (b) and mRNA (c) expression of MYCN in SK-N-BE(2) and SH-SY5Y cells. (d and e) Effects of ARHGEF12 KD on protein (d) and mRNA (e) expression of MYCN in SK-N-BE(2) xenograft tumors. (f) Representative images of MYCN staining in d. Scale bar, 50 µm. (g) Quantification of positive MYCN cells in f. (h) Western blotting analysis of MYCN in SK-N-BE(2) cells after being treated with MG132 (10 μM) or vehicle for 6 h. (i) Western blotting analysis of MYCN in SK-N-BE(2) cells after being treated with cycloheximide (CHX) for up to 60 min. (j) Quantification of MYCN levels in i. (k) Western blotting analysis of ubiquitination in SK-N-BE(2) cells after being immunoprecipitated with MYCN antibody. In c, e, g and j, * p < 0.05, ** p < 0.01, **** p < 0.0001, by two-tailed t-test or one-way ANOVA

To test this possibility, we treated SK-N-BE(2) cells with or without MG132, a proteasome inhibitor. We found that MG132 significantly inhibited ARHGEF12 KD-decreased MYCN expression (Fig. 3h). Next, we performed cycloheximide chase experiments and found that ARHGEF12 KD decreased MYCN stability (Fig. 3i and j). In addition, we found that ARHGEF12 KD increased MYCN ubiquitination (Fig. 3k). Taken together, these data suggest that ARHGEF12 knockdown promotes MYCN ubiquitination and degradation.

ARHGEF12 knockdown promotes neuroblastoma differentiation and decreases MYCN protein stability through the RhoA/ROCK/GSK3β pathway

ARHGEF12 is known as an activator of the RhoA/ROCK axis [9], which regulates cell stemness and differentiation [5, 6]. Thus, we hypothesized that ARHGEF12 might regulate neuroblastoma differentiation through RhoA/ROCK signaling. To test this hypothesis, we assessed the effects of ARHGEF12 KD on RhoA activity. Using a G-LISA RhoA activation assay kit, we found that ARHGEF12 KD markedly decreased RhoA activity in SK-N-BE(2) and SH-SY5Y cells (Fig. 4a). Besides, we found that ARHGEF12 KD reduced the expression of ROCK1 and ROCK2 in both cell lines. These data indicate that ARHGEF12/RhoA/ROCK axis-mediated neuroblastoma differentiation is not dependent on MYCN amplification.

Fig. 4.

Fig. 4

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ARHGEF12 knockdown promotes neuroblastoma differentiation and decreases MYCN protein stability through RhoA/ROCK/GSK3β pathway. (a) Effects of ARHGEF12 KD on RhoA activity in SK-N-BE(2) and SH-SY5Y cells. RhoA activity was analyzed using a G-LISA RhoA activation assay kit. *** p < 0.001, **** p < 0.0001, by one-way ANOVA. (b) Western blotting analysis of ROCK1/2 expression after ARHGEF12 KD in SK-N-BE(2) and SH-SY5Y cells. (c) Wnt3A rescue of p-GSK3β(Ser9) inhibited by ARHGEF12 KD. SK-N-BE(2) cells were treated with or without Wnt3A (50 ng/ml) for 1 h. (d) A working model of ARHGEF12-regulated neuroblastoma differentiation and MYCN degradation through RhoA/ROCK/GSK3β signaling. Targeting ARHGEF12 with the small molecular inhibitor Y16 delays neuroblastoma xenograft tumor growth

Accumulating data indicate that GSK3β may regulate the phosphorylation and stability of MYCN protein [26, 27], and that ROCK2 inhibition may decrease GSK3β(Ser9) phosphorylation and increase MYCN phosphorylation, resulting in enhanced MYCN degradation [7]. Thus, ARHGEF12 may regulate MYCN protein stability through a RhoA/ROCK-mediated GSK3β pathway. We found that ARHGEF12 KD markedly decreased GSK3β(Ser9) phosphorylation and MYCN expression (Fig. 4c). To further substantiate this result, we treated ARHGEF12-KD SK-N-BE(2) cells with Wnt3A, which may induce the phosphorylation of GSK-3β(Ser9) [28]. We found that Wnt3A treatment rescued GSK-3β(Ser9) phosphorylation inhibited by ARHGEF12 KD and decreased ARHGEF12 KD-induced MYCN degradation (Fig. 4c). Taken together, these results indicate that ARHGEF12 knockdown promotes neuroblastoma cell differentiation and reduces MYCN protein stability through the RhoA/ROCK/GSK3β pathway (Fig. 4d).

Targeting ARHGEF12 with the small molecule inhibitor Y16 reduces neuroblastoma tumorigenicity

Y16 is a small molecule inhibitor that may inhibit ARHGEF12-RhoA complex formation and RhoA activity [19, 20]. We found that treatment with Y16 caused a time-dependent and dose-dependent inactivation of the RhoA/ROCK axis and downregulation of MYCN protein levels (Fig. 5a and b). In addition, we found that Y16 slowed down cell proliferation (Fig. S2a) and cell migration (Fig. S2b and 2c).

Fig. 5.

Fig. 5

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Targeting ARHGEF12 with the small molecular inhibitor Y16 reduces neuroblastoma tumorigenicity. (a) Western blotting analysis of the effects of Y16 treatment on the expression of ROCK1/2, GSK3β, MYCN and pGSK3β(Ser9). Cells were treated with 50 μM Y16 for 0 h, 24 h, 48 h or 72 h, respectively. (b) Western blot analysis of the effects of Y16 treatment on the expression of ROCK1/2, GSK3β, MYCN and pGSK3β(Ser9). Cells were treated with 0 μM, 10 μM, 30 μM, 50 μM, 70 μM or 100 μM Y16 for 72 h, respectively. (c) Effects of Y16 treatment on tumor growth. SK-N-BE(2) cells were xenografted into SCID mice. When tumors reached a palpable size, the mice were treated with vehicle or Y16 (30 mg/kg) daily by intraperitoneal injection for 21 days. Tumor growth was monitored every other day. (d) Representative images of tumors after Y16 treatment. (e) Quantification of tumor weights in d. (f) Representative images of MYCN and Ki-67 staining in tumor sections from d. Scale bar, 50 µm. (g) Quantification of cell numbers with a positive signal in f. In c, e and g, * p < 0.05, *** p < 0.001, by two-tailed t-test

Next, the SK-N-BE(2) subcutaneous xenograft model was employed to assess Y16 antitumor activity in vivo. Mice with palpable tumors detected 1 to 2 weeks after inoculation were treated with 30 mg/kg Y16 intraperitoneally (i.p.) every day for a maximum of 3 weeks. We found that treatment with Y16 caused a significant reduction in tumor growth rate and final tumor weight (Fig. 5c to e). In contrast to the control group, MYCN protein expression in the Y16 treated group was lower (Fig. 5f and g). The reduction in cell proliferation rate in the experimental group was confirmed by a lower positive intensity and narrower positive distribution of Ki-67 staining (Fig. 5f and g). These results suggest that targeting ARHGEF12 may be a promising therapeutic approach for neuroblastoma, and that Y16 may have the potential for neuroblastoma treatment.

Discussion

Accumulating data indicate that inducing cell differentiation is a key to treating neuroblastoma. In this study, we show that ARHGEF12 may act as a new factor inhibiting neuroblastoma differentiation. Inhibition of ARHGEF12 by a pharmacological inhibitor or shRNA depletion markedly promoted neuroblastoma differentiation and reduced neuroblastoma tumorigenicity.

Previous studies have shown that ARHGEF12 can regulate cell differentiation [14–16, 21], but its exact functions are still not clear. It has been found, for example, that ARHGEF12 promotes erythropoiesis in acute lymphoblastic leukemia patients [21], and that depletion of ARHGEF12 significantly inhibited RhoA activation and increased the expression of differentiation markers in smooth muscle cells [15]. In addition, it has been reported that ARHGEF12 KD accelerated mesenchymal stem cell adipogenesis and reduced basal RhoA activity [14]. In this study, we show that ARHGEF12 KD promotes neuroblastoma differentiation through RhoA/ROCK signaling.

Although ARHGEF12 is known as an essential factor in development [14–16, 21], its functions in cancer are still elusive. A KMT2A-ARHGEF12 gene fusion has been found in high-grade B-cell lymphoma with a poor prognosis [29–31] and ARHGEF12 has been reported to act as a tumor suppressor in human breast and colorectal cancer [17]. In contrast, Gurrapu et al. [18] reported that ARHGEF12 inhibitor Y16 treatment may inhibit breast cancer cell proliferation. Here, we show that ARHGEF12 functions as an oncogene in neuroblastoma and that depletion of ARHGEF12 inhibits in vitro tumor cell proliferation, migration and colony formation, and in vivo tumor growth.

We also found that ARHGEF12 regulates MYCN stability in neuroblastoma. Previously, it has been reported that PI3K blockade may activate GSK3β, leading to phosphorylation and degradation of MYCN, while phosphorylation-defective MYCN mutants are stabilized [26]. ROCK2 is a downstream effector of ARHGEF12/RhoA [9]. ROCK2 inhibition has been found to decrease GSK3β(Ser9) phosphorylation and increase MYCN phosphorylation, resulting in enhanced MYCN degradation [7]. However, it is still unknown whether ARHGEF12 regulates MYCN stability. Here, we show that genetic inhibition of ARHGEF12 promotes MYCN ubiquitination and degradation through RhoA-mediated ROCK/GSK3β signaling, suggesting that targeting ARHGEF12 may be a potential way to treat neuroblastoma.

Differentiation-induction may be a good choice for treating cancer. Retinoic acid (RA) has, for example, been used as an adjuvant to differentiate or eliminate remaining tumor cells after intensive multimodal therapy [32, 33]. SNAI2, a transcriptional repressor, has been reported to interfere with neuronal cell differentiation, and it was found that loss of SNAI2 could reduce the metastatic spread of neuroblastoma cells and sensitize them to RA therapy [34]. Treatment with Parnate, a SNAI2 inhibitor, could mimic phenotypes resulting from SANI2 deduction in various tumor cells [35, 36]. Here, we show that inhibition of ARHGEF12 by the pharmacological inhibitor Y16 or by shRNA depletion markedly promotes neuroblastoma differentiation and reduces neuroblastoma tumorigenicity.

Taken together, our findings provide new insights into the mode of action of ARHGEF12 and suggest that targeting ARHGEF12 may be a potential therapeutic strategy for neuroblastoma.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 1646 KB) (1.6MB, docx)

Abbreviations

GEFs

Guanine nucleotide exchange factors

NB

Neuroblastoma

GNB

Ganglioneuroblastoma

RA

Retinoic acid

ARHGEF12

Rho guanine nucleotide exchange factor 12

DMEM

Dulbecco's modified Eagle medium

FBS

Fetal bovine serum

PBS

Phosphate-buffered saline

PI

Propidium Iodide

PE

Phycoerythrin

CHX

Cycloheximide

ARHGEF12 KD

ARHGEF12 knockdown

H&E

Hematoxylin-eosin

co-IP

co-immunoprecipitation

Author contribution

Y.L., H.F., S.S. and J.G. designed the experiments. Y.Y., S.W., J.C., J.L. and F.L. performed the experiments. Y.Y., S.W., J.C., Y.W.Z., Y.Y.X. and J.Y.T. analyzed and interpreted the data. Y.Y., Y.L. and H.F. wrote the paper. Y.L. supervised the project.

Funding

This work was supported in part by the National Key R&D Program of China (2018YFC1313000/2018YFC1313005) to Y.G. and Y. L; the National Natural Science Foundation of China (81972341 and 32271007) to Y.L.; the Shanghai Municipal Commission of Science and Technology (201409002700 to Y. L., 17411950400 to J. T.); Program of Shanghai Academic/Technology Research Leader (21XD1403100) to H.F.; the Shanghai Jiao Tong University Medical Engineering Cross Fund (No. YG2017MS32); and the Pudong New Area Science & Technology Development Fund (PKJ2018-Y47) to Y. L.; the State Key Laboratory of Oncogenes and Related Genes (KF2115) to F.L.

Data availability

All relevant data and material are available from the corresponding author upon reasonable request.

Code availability

Not applicable.

Declarations

Ethics statement

The study was conducted according to the Ethical Principles of Measures for Ethical Review of Biomedical Research Involving Human Beings and the Declaration of Helsinki.

Consent to participate

All patients provided written informed consent prior to participating in any study-specific procedures.

Consent for publication

All authors read and approved the final manuscript.

Conflict of interest

The authors declare no conflicts of interest.

Footnotes

The original version of this article was revised: In Fig. 5f of this article the representative MYCN IHC images of tumor from vehicle-treated mice presented the wrong samples. Fig. 5f has been corrected in the original article. The authors declare that this correction does not affect the description, interpretation, or the original conclusions of the manuscript.

Publisher's note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Yi Yang, Siqi Wang, Jiaoyang Cai, and Jianwei Liang contributed equally to this work

Change history

10/9/2023

The original version of this article was revised: In Fig. 5f of this article the representative MYCN IHC images of tumor from vehicle-treated mice presented the wrong samples. Fig. 5f has been corrected in the original article. The authors declare that this correction does not affect the description, interpretation, or the original conclusions of the manuscript.

Change history

10/16/2023

A Correction to this paper has been published: 10.1007/s13402-023-00890-x

Contributor Information

Yijin Gao, Email: gaoyijin@scmc.com.cn.

Shuhong Shen, Email: shenshuhong@scmc.com.cn.

Haizhong Feng, Email: fenghaizhong@sjtu.edu.cn.

Yanxin Li, Email: liyanxin@scmc.com.cn.

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Associated Data

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Supplementary Materials

Supplementary file1 (DOCX 1646 KB) (1.6MB, docx)

Data Availability Statement

The data supporting the findings of this study are available within the article and its supporting information files and/or available from the corresponding author upon reasonable request.

All relevant data and material are available from the corresponding author upon reasonable request.

Not applicable.

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