Abstract

Background

   CXCR7 (ACKR3) has been well-supported as a promoter of growth and
   metastasis in hepatocellular carcinoma (HCC). Both CXCR7 and Hippo
   signaling play roles in organ development. We aimed to verify the
   involvement of Hippo-YAP signaling in CXCR7-regulated HCC
   proliferation, migration, and invasion.

Methods

   HCCLM3 cells were transfected with si-CXCR7, pcDNA-CXCR7, or related
   control RNA/empty vector. Cell proliferation was assessed using the
   Cell Counting Kit-8 (CCK-8), and mRNA and protein levels were measured
   via quantitative real-time PCR (qPCR) and Western blotting. Colony
   formation assays were conducted to evaluate proliferation capacity, and
   Transwell assays were used to assess invasion and migration.
   Transcriptome data from the TCGA-LIHC dataset were analyzed to
   investigate the potential effects of CXCR7 in HCC.

Results

   si-CXCR7 inhibited cell proliferation in HCCLM3 cells, while
   pcDNA-CXCR7 promoted it. Migration and invasion were suppressed by
   si-CXCR7 but enhanced by pcDNA-CXCR7. Patients with higher CXCR7
   expression in the TCGA-LIHC dataset had lower overall survival rates
   and increased gene transcription. The CXCR7-high expressing samples
   were characterized by the activation of several pathways, including
   PI3K-AKT signaling, calcium signaling, and the Hippo signaling pathway.
   si-CXCR7 reduced the relative protein levels of Gαq/11 and GαS while
   increasing phosphorylated LATS and phosphorylated YAP. Opposite trends
   in these proteins were observed with pcDNA-CXCR7. Finally, the
   inhibitory effects of si-CXCR7 on cell proliferation, migration, and
   invasion were reversed by the YAP inhibitor verteporfin.

Conclusion

   We suggest that CXCR7 promotes the growth and metastasis of HCC cells,
   at least in part, by inactivating the Hippo-YAP signaling pathway.

Supplementary Information

   The online version contains supplementary material available at
   10.1007/s12672-025-02324-6.

   Keywords: ACKR3, CXCL12, Liver cancer, Metastases, RDC1, TAZ

Highlights

     * CXCR7 regulates HCC progression possibly through the inactivation
       of the Hippo-YAP signaling pathway.
     * YAP inhibition reverses the oncogenic effects of CXCR7, including
       cell proliferation, migration, and invasion.
     * Transcriptome analysis from the TCGA-LIHC dataset links high CXCR7
       expression to PI3K-AKT signaling, the calcium signaling, MAPK, as
       well as Hippo signaling pathway.

Introduction

   Hepatocellular carcinoma (HCC) is one of the most common cancers
   worldwide [[32]1], accounting for over 80% of all liver cancers and
   ranking among the top causes of cancer-related deaths in many countries
   [[33]2]. The high mortality rate and extremely low 5-year overall
   survival rate of HCC are primarily due to the challenges in early
   detection [[34]3]. Therefore, exploring the complex regulation of
   metastasis in the tumor microenvironment (TME) is critical for
   developing effective therapeutic strategies [[35]4], particularly for
   HCC.

   CXC chemokine receptor (CXCR) signaling, mediated by CXCL12 (SDF1),
   CXCR4, and CXCR7, plays a pivotal role in organ development and normal
   liver regeneration [[36]5–[37]7], and is widely dysregulated in various
   cancers [[38]8]. In HCC, the co-expression of CXCL12 and CXCR4/CXCR7 is
   strongly associated with patient survival rates [[39]9]. CXCR7, also
   known as ACKR3 or RDC1, exhibits a much higher affinity for CXCL12 than
   CXCR4, underscoring its pivotal role in CXCL12-CXCR4/CXCR7 chemokine
   signaling within the TME [[40]10]. CXCR7 functions as a CXCL12
   scavenger receptor, predominantly expressed in liver endothelial cells,
   with its expression further upregulated in HCC [[41]11]. It regulates
   cell migration, particularly in endothelial cells, by establishing
   local CXCL12 gradients [[42]12]. Through β-arrestin-dependent or
   Gαi-dependent mechanisms, CXCR7 activates signaling pathways such as
   ERK/Akt, MAPK, and JAK/STAT. These activated pathways subsequently can
   promote processes like epithelial-to-mesenchymal transition (EMT),
   angiogenesis, migration, and tumor cell invasion [[43]13, [44]14].
   Notably, the downregulation of CXCR7 has been shown to inhibit the
   proliferation, invasion, and migration of HCC cells [[45]15, [46]16].

   Hippo signaling is a critical regulator of tissue homeostasis, organ
   development, regeneration, and cancer progression, with
   context-dependent effects [[47]17]. This evolutionarily conserved
   pathway mainly involves serine/threonine-protein kinases 4/3 (MST1/2),
   large tumor suppressor kinases (LATS) 1/2, yes-associated protein
   (YAP), and its paralog WW domain-containing transcription regulator
   protein 1 (WWTR1, also known as transcriptional co-activator with
   PDZ-binding motif [TAZ]) [[48]18]. In HCC, Hippo signaling restricts
   tumor progression by phosphorylating YAP/TAZ to destabilize them,
   thereby inhibiting the transcription of cancer-promoting genes
   [[49]19]. Specifically, activating Hippo signaling inhibits the
   migration and invasion of HCC cells, as demonstrated by the effects of
   Pien Tze Huang, Tubuloside B, and miR-100 [[50]16, [51]20, [52]21].

   The healthy liver has a strong regenerative capacity [[53]22], and both
   CXCR7 and Hippo signaling are involved in organ development and HCC
   progression. Interestingly, a novel feedback loop between Hippo
   signaling and CXCR7 has been identified in gastric cancer by Wang et
   al. CXCR7 can dephosphorylate YAP via the Gαq/11-ROCK-LATS axis,
   enhancing YAP’s nuclear localization, which in turn upregulates CXCR7
   transcription, thereby promoting gastric cancer progression [[54]23].
   However, whether CXCR7-mediated chemokine signaling interacts with
   Hippo signaling in HCC remains unknown. This study aims to verify the
   involvement of Hippo-YAP signaling in CXCR7-regulated HCC cell
   proliferation, migration, and invasion.

Materials and methods

Cell line information and cell transfection

   The hepatic carcinoma cell line HCCLM3, obtained from Shanghai FuHeng
   Biotechnology Co., Ltd. (Catalog #FH0096), was utilized in this study.
   This cell line exhibits high metastatic potential and adherent growth
   characteristics. Cultivation was carried out in DMEM culture medium
   with high glucose (Catalog #C3113-0500, VivaCell, Shanghai, China),
   supplemented with 10% fetal bovine serum (Catalog #04-001-1ACS,
   VivaCell) and 1% penicillin–streptomycin (Catalog #BL505A, Biosharp,
   Hefei, China).

   Based on the gene sequence of CXCR7 ([55]NM_020311.3), an
   overexpression sequence and a small interfering RNA (siRNA) targeting
   CXCR7 (5′-CGCUCUCCUUCAUUUACAUUU-3′) were designed and synthesized by
   Shanghai Genechem Co., Ltd. The coding sequence (CDS) of CXCR7 was
   cloned into the pcDNA3.1 vector (pCAMBIA1301, Catalog #HZB800277,
   HZbscience, Hangzhou, China) to generate the recombinant overexpression
   vector pcDNA-CXCR7. As corresponding controls, si-NC (sense:
   5′-ACGUGACACGUUCGGAGAAUU-3′ and antisense: 5′-UUCUCCGAACGUGUCACGU-3′)
   and an empty vector were used. HCCLM3 cells were resuspended and
   adjusted to a concentration of 8 × 10^5 cells/well before being seeded
   into a 6-well plate. For transfection, the overexpression plasmid or
   si-CXCR7 was mixed with PEI reagent (Catalog #23966-2,
   YEASEN, Shanghai, China) in Opti-MEM™ medium (Invitrogen, USA) and
   pre-incubated at room temperature for 20 min to form DNA-PEI complexes.
   The transfection complexes were then added to the cells in serum-free
   medium. After a 6-h incubation, the medium was replaced with complete
   growth medium, and the cells were further incubated at 37 °C for 24 h.
   Transfection efficiency was subsequently assessed.

CCK-8 activity assay and IC50 determination of verteporfin

   Cells in the logarithmic growth phase were seeded into a 96-well plate
   at a density of 3000 cells/well, with each group replicated three
   times, and incubated at 37 °C. At predetermined time points (0, 24, 48,
   72, and 96 h), CCK-8 solution (Catalog #C0037, Beyotime, Shanghai,
   China) was added, and the plates were returned to the incubator for an
   additional hour of incubation. Wells containing only the cell culture
   medium and CCK-8 solution, without cells, served as blank controls.
   Absorbance was measured at 450 nm, and the data were recorded.

   Verteporfin (VP; Catalog #HY-B0146, ECM, USA) was dissolved in DMSO and
   further diluted with PBS. HCCLM3 cells were seeded into a 96-well plate
   at a density of 5,000 cells/well, with each condition replicated in
   triplicate. Cells were then treated with varying concentrations of
   Verteporfin (0, 2, 4, 6, 10, 14 μM) and incubated at 37 °C for 72 h.
   The cytotoxicity of Verteporfin was evaluated using the CCK-8 assay,
   measuring absorbance at 450 nm to determine the IC50 value. A
   Verteporfin concentration of 16 μM (median for 16 and 18 μM) was
   selected for subsequent experiments according to its calculated IC50
   (about 17.90 μM).

Bioinformatics analysis of CXCR7

   Gene expression data specific to CXCR7 in LIHC cases, along with
   corresponding clinical data, were retrieved from the TCGA database (The
   Cancer Genome Atlas, [56]https://portal.gdc.cancer.gov). Patients were
   divided into high and low CXCR7 expression cohorts using the median
   expression level as a cutoff. Survival times and statuses were used to
   construct Kaplan–Meier survival curves, with a log-rank test assessing
   the statistical significance of survival differences between the two
   groups. Differential expression analysis was conducted using the
   Wilcoxon rank-sum test, applying criteria of |log2 Fold Change|> 1 and
   a p-value < 0.05 to identify significantly altered genes. The volcano
   plot of the differentially expressed genes (DEGs), including key DEGs,
   was visualized using the R package “ggplot2.” Enrichment analysis of
   Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG)
   pathway analysis and Gene Set Enrichment Analysis (GSEA) were conducted
   using the R packages clusterProfiler and org.Hs.eg.db. Genes highly
   correlated with CXCR7 were identified using GEPIA
   ([57]http://gepia.cancer-pku.cn/), while immune infiltration was
   assessed using Timer 2.0 ([58]http://timer.cistrome.org/).

Quantitative real-time PCR (qRT-PCR)

   Total RNA was extracted from cells using Trizol Reagent (Catalog
   #15596026, Invitrogen) and purified by phenol–chloroform extraction,
   following the manufacturer's instructions. The obtained RNA was
   converted into cDNA using a reverse transcription system (Catalog
   #R333-01, Vazyme, Nanjing, China). Quantitative PCR analysis of CXCR7
   was performed on a Bio-Rad CFX96 system using ChamQ Universal SYBR qPCR
   Master Mix (Catalog #R711-03, Vazyme) with the following primer
   sequences: GAPDH forward (F): 5′-GTTCGTCATGGGTGTGAACC-3′, reverse (R):
   5′-CATCCACAGTCTTCTGGGTG-3′; CXCR7 F: 5′-TGTCCTCACCATCCCAGTCT-3′, R:
   5′-CTGCCGAAGAGGTTGATGGA-3′. The 2^−ΔΔCt method was used to assess the
   relative expression levels of CXCR7 compared to the control group, with
   GAPDH as the internal reference.

Western blotting

   Antibodies used in this study include: CXCR7 (1:3000, 60 kDa, Catalog
   #60216-1-Ig, Proteintech, Shanghai, China), Gα q/11 (1:500, 41 kDa,
   Catalog #sc-365906, Santa Cruz, USA), Gα s (1:500, 49 kDa, Catalog
   #sc-135914, Santa Cruz), phosphorylated (p)-LATS (1:1000, 140 kDa,
   Catalog #8654S, CST, USA), LATS (1:1000, 140 kDa, Catalog #17049-1-AP,
   Proteintech), p-YAP (1:1000, 72 kDa, Catalog #13008T, CST), YAP
   (1:1000, 72 kDa, Catalog #abs134112, absin), and β-actin (1:150,000,
   42 kDa, Catalog #T0022, Affinity, USA). Protein concentration was
   determined (Catalog #P0010, Beyotime), and the samples were separated
   on a 12% SDS-PAGE gel, followed by semidry transfer. Membranes
   (0.22 µm) were blocked with a solution containing 5% non-fat milk and
   0.1% Tween. Membranes were then incubated with primary antibodies
   overnight at 4 °C. The following day, membranes were exposed to the
   corresponding secondary antibody at room temperature for one hour.
   Immunoblots were visualized using an enhanced chemiluminescence system
   (Catalog #G2014, Servicebio, Wuhan, China), and images were captured on
   GenoSens1800 autoradiography film (Shanghai Qinxiang Scientific
   Instrument Co., Ltd., China). β-actin was used for normalization.

Colony formation assay

   Cells were resuspended and adjusted to a concentration of 1 × 10^4
   cells/mL. Cells were then seeded at a density of 300 cells per well in
   a 6-well plate, with 2 mL of fresh culture medium added. Plates were
   incubated at 37 °C in a 5% CO[2] atmosphere. The colony size was
   periodically monitored, and the medium was refreshed regularly to
   maintain optimal growth conditions. After approximately two weeks,
   cells were fixed with tissue fixative (Catalog #P0099, Beyotime) for
   10 min to preserve colony morphology and then stained with a 0.2%
   crystal violet solution (Catalog #[59]C02121, Beyotime) for 15 min to
   visualize the cell clusters. Images of the colonies were captured for
   analysis.

Transwell assay

   Cells were resuspended and seeded into a 96-well plate at a density of
   5 × 10^4 cells onto the upper chamber of a Transwell insert, while the
   lower chamber was filled with culture medium supplemented with 10%
   serum. The Transwell plate was then incubated for 24 h. For the
   invasion assay, the membrane was precoated with Matrigel. Briefly,
   Matrigel (Catalog #111005, GENOM Bio, Hangzhou, China) was diluted with
   serum-free medium to a final concentration of 1 mg/mL. The diluted
   Matrigel was added to the center of the chamber and incubated at 37 °C
   for 4 h, followed by incubation at 4 °C overnight to allow for proper
   gelation. Migratory or invasive cells were fixed and stained using a
   0.1% crystal violet solution for 10 min. Images were captured under
   identical parameters.

Statistical analysis

   Protein gray scale analysis was performed using ImageJ software (1.48v,
   NIH, USA). Results are expressed as mean ± SD. The significance between
   two groups was evaluated using an unpaired two-tailed Student's t-test,
   while significance among multiple groups was assessed using one-way
   analysis of variance followed by Tukey's post hoc test. A
   p-value < 0.05 was considered significant.

Results

CXCR7 functions as an oncogene in HCC

   To explore the role and molecular mechanisms of CXCR7 in the biological
   characteristics of HCC, our study utilized transfection techniques to
   introduce both an overexpression plasmid (pcDNA-CXCR7) and siRNA
   targeting CXCR7 (si-CXCR7). Evidence from RT-qPCR and Western blotting
   confirmed the effectiveness of these transfections; si-CXCR7
   successfully induced a marked decrease in CXCR7 mRNA and protein
   expression (Fig. [60]1a, p < 0.01), while pcDNA-CXCR7 transfection led
   to a substantial increase in both (Fig. [61]1b, p < 0.01). Results from
   the CCK-8 assay demonstrated that HCCLM3 cells overexpressing CXCR7
   showed enhanced viability over time compared to the control, whereas
   suppression of CXCR7 resulted in a reduction in cellular viability
   (Fig. [62]1c, p < 0.01). During a two-week colony formation assay, we
   observed that the number of colonies formed in the si-CXCR7 group was
   notably lower than in the control group, whereas the pcDNA-CXCR7 group
   showed a significant increase in colony formation (Fig. [63]1d, e,
   p < 0.05). Moreover, Transwell assays revealed that inhibition of CXCR7
   resulted in a significant decrease in the number of migrating and
   invading cells, while overexpression of CXCR7 yielded the opposite
   outcomes (Fig. [64]1f–i). These findings suggest that high expression
   of CXCR7 in HCC is associated with enhanced cell activity,
   proliferation, migration, and invasion, indicating that CXCR7 plays a
   role in promoting cancer in HCC.

Fig. 1.

   [65]Fig. 1
   [66]Open in a new tab

   Chemokine C-X-C Motif Receptor 7 (CXCR7) promotes proliferation,
   migration, and invasion in hepatocellular carcinoma (HCC) cells. HCCLM3
   cells were transfected with non-targeting control siRNA (si-NC), CXCR7
   siRNA (si-CXCR7), pcDNA3.1 (Vector), or pcDNA3.1-CXCR7 (pcDNA-CXCR7). a
   RT-qPCR analysis of relative CXCR7 mRNA levels. b Western blot analysis
   of relative CXCR7 protein levels. c Cell viability was assessed using
   the CCK-8 assay. d, e Colony formation assays were conducted to assess
   cell proliferation. Transwell assays were used to evaluate cell
   migration f, g and invasion h, i. For A, B, E, and G: *p < 0.05,
   **p < 0.01; for C: **p < 0.01 versus si-NC, ##p < 0.01 versus Vector

CXCR7 correlates with the gene signature of the Hippo pathway in HCC

   The activation of the Hippo signaling pathway and its correlation with
   CXCR7 were evaluated through bioinformatics analysis. CXCR7 expression
   was significantly higher in HCC tissues compared to adjacent normal
   tissues; however, their mean expression levels were relatively similar
   (Fig. [67]2a). Across different disease stages, CXCR7 levels showed no
   significant variation (Fig. [68]2b). The top 70 genes predicted to be
   similar to CXCR7 demonstrated relatively low Pearson correlation
   coefficients (Fig. [69]2c). Furthermore, immune infiltration did not
   differ significantly between the top 10 samples with the highest and
   lowest CXCR7 expression (Fig. [70]2d). These findings suggest that
   CXCR7 signaling is broadly dysregulated in HCC. The activation of the
   Calcium signaling pathway, Chemokine signaling pathway, Hippo signaling
   pathway, MAPK signaling pathway, and PI3K-Akt signaling pathway was
   confirmed through GSEA ontology analysis (Fig. [71]2e). Additionally, a
   heatmap illustrating the expression of genes involved in the Hippo
   signaling pathway is presented in Fig. [72]2f. Patients with high CXCR7
   expression exhibited significantly worse prognoses compared to those
   with low expression (p = 0.016), as shown in Fig. [73]3a. The volcano
   plot revealed a total of 7152 upregulated DEGs, such as MUC5AC,
   KJRREL2, IGFN1, and MUCL3, alongside 1,112 downregulated genes
   (Fig. [74]3b). The number of upregulated genes was more than 6.4 times
   higher than the number of downregulated genes, suggesting a dominant
   effect of CXCR7 on gene upregulation. GO enrichment analysis of these
   DEGs showed that Biological Processes (BP) were primarily enriched in
   axon development, cell junctions, and external encapsulating structure
   organization. Cellular Component (CC) terms were enriched in
   collagen-containing extracellular matrix, cell-substrate junctions, and
   synaptic membranes (Fig. [75]3c). Molecular Function (MF) terms were
   enriched in channel activity, passive transmembrane transporter
   activity, and ion channel activity, highlighting the multifaceted
   impact of CXCR7 on cellular functions (Fig. [76]3c). Moreover, KEGG
   pathway enrichment analysis demonstrated significant enrichment in the
   PI3K-Akt signaling pathway, calcium signaling pathway, and MAPK
   signaling pathway, implicating the involvement of these critical
   signaling cascades in CXCR7-mediated effects (Fig. [77]3d).
   Interestingly, in our current research, we also found that the Hippo
   signaling pathway was significantly enriched.

Fig. 2.

   [78]Fig. 2
   [79]Open in a new tab

   Bioinformatics analysis of CXCR7 in HCC and its correlation with the
   Hippo signaling pathway. HCC samples were obtained from the TCGA-LIHC
   dataset. a Relative RNA expression of CXCR7 in HCC tissues compared to
   adjacent normal tissues. b Relative RNA expression of CXCR7 across
   different stages of HCC. c Predicted genes most closely related to
   CXCR7. d Immune infiltration in the top 10 samples with the highest and
   lowest CXCR7 expression. e GSEA ontology analysis of the pathways of
   interest. f Heatmap of Hippo signaling pathway genes arranged according
   to the relative RNA levels of CXCR7 for each sample *p < 0.05 by
   Pearson correlation for panels c & f, and by Two-way ANOVA for panel d.
   The “*” marked in red indicates upregulation, and marked in blue
   indicates downregulation in the ACKR3 high group

Fig. 3.

   [80]Fig. 3
   [81]Open in a new tab

   Bioinformatics analysis based on CXCR7 expression in patients with
   LIHC. Patients were divided into high-expression (N = 184) and
   low-expression (N = 184) groups based on the median expression level of
   the CXCR7 gene. a Kaplan–Meier survival curves were generated to
   compare the survival rates between the two groups. b A volcano plot was
   used to depict differential gene expression. GO enrichment analysis c
   and KEGG enrichment analysis d were performed on the differentially
   expressed genes

CXCR7 regulates Hippo-YAP signaling in HCC cells

   Several key components of the Hippo signaling pathway, including
   Gαq/11, Gαs, LATS, and YAP were evaluated with Western blotting. The
   result showed that suppression of CXCR7 levels led to a reduction in
   cellular Gαq/11 and Gαs protein levels, which in turn resulted in an
   elevation of LATS and YAP phosphorylation (Fig. [82]4a, b, p < 0.05).
   Conversely, the overexpression of CXCR7 caused an increase in
   intracellular Gαq/11 and Gαs protein levels, leading to a decrease in
   the phosphorylation levels of LATS and YAP (Fig. [83]4a, b, p < 0.05).
   This evidence suggests that CXCR7 is involved in regulating the
   Hippo-YAP signaling pathway, influencing cellular outcomes through
   alterations in protein expression and phosphorylation states.

Fig. 4.

   [84]Fig. 4
   [85]Open in a new tab

   CXCR7 overexpression inhibits Hippo-YAP signaling. HCCLM3 cells were
   transfected with si-NC, si-CXCR7, Vector, or pcDNA-CXCR7. a, b Western
   blot analysis was performed to measure the protein levels of Gαq/11,
   Gαs, p-LATS, LATS, p-YAP, and YAP, followed by densitometric analysis
   to quantify the immunoblot images. *p < 0.05, **p < 0.01

CXCR7 enhanced proliferation, migration and invasion by regulating Hippo-YAP
signaling in HCC cells

   Verteporfin, a photosensitizer and YAP inhibitor, was utilized to
   elucidate the interactions and functional significance of CXCR7
   modulation on the Hippo-YAP pathway. As shown in Fig. [86]5 (p < 0.05),
   following treatment with Verteporfin, HCCLM3 cells exhibited a marked
   enhancement in cell viability, accompanied by increased proliferation,
   migration, and invasion capabilities. Notably, in cells transfected
   with si-CXCR7 and subsequently treated with Verteporfin, the previously
   inhibited cell activity, proliferation, migration, and invasion were
   significantly reversed. These findings suggest a critical role for
   CXCR7 in modulating the effects of Verteporfin on cancer cell behavior,
   implicating its potential as a therapeutic target for interventions
   aimed at controlling tumor progression.

Fig. 5.

   [87]Fig. 5
   [88]Open in a new tab

   CXCR7 promotes proliferation, migration, and invasion by regulating
   Hippo-YAP signaling. HCCLM3 cells were transfected with si-NC,
   si-CXCR7, Vector, or pcDNA-CXCR7, followed by treatment with the YAP
   inhibitor Verteporfin (VP). a Cell viability was assessed using the
   CCK-8 assay. b, c Colony formation assays were conducted to assess cell
   proliferation. Transwell assays were used to evaluate cell migration
   (d, e) and invasion (f, g). For a: **p < 0.01 versus si-NC + PBS,
   ^##p < 0.01 versus si-CXCR7 + PBS; for c, e, and g: *p < 0.05,
   **p < 0.01

Discussion

   Angiogenesis is a universally significant therapeutic target that
   facilitates tumor growth, progression, metastasis, and chemoresistance
   [[89]24]. CXCR7, which is highly expressed in liver endothelial cells
   [[90]11], can be further upregulated under hypoxic conditions [[91]25],
   a key stimulus for inducing angiogenesis, normal liver regeneration,
   and HCC proliferation [[92]26, [93]27]. The promotive role of HCC
   derived CXCR7 on metastases, angiogenesis and tumor growth has been
   explored in earlier studies [[94]28, [95]29]. Several signaling
   pathways have been implicated in CXCR7-associated angiogenesis and
   tumor progression, including MEK/ERK, NF-κB, p38 MAPK and PI3K/AKT
   signaling pathways [[96]30–[97]33]. Additionally, CXCR7 contributes to
   HCC progression by functioning as a chemokine regulator, shaping the
   tumor microenvironment. Elevated CXCR7 expression promotes the
   recruitment of M2 macrophages, which facilitates immune escape in HCC
   [[98]34, [99]35]. In line with these findings, our study also
   demonstrated that silencing CXCR7 inhibited proliferation, colony
   formation, migration, and invasion of HCCLM3 cells, whereas increasing
   CXCR7 expression produced the opposite effects.

   Bioinformatics analysis was subsequently performed to gain a
   comprehensive understanding of CXCR7 in HCC using the TCGA-LIHC
   dataset. Our analysis revealed that CXCR7 expression was significantly
   dysregulated in HCC. The number of upregulated DEGs in samples with
   high CXCR7 expression was more than 6.4 times greater than in samples
   with low CXCR7 expression. This result suggests that CXCR7 has a broad
   impact on gene transcription in HCC, potentially activating or
   enhancing a wide range of signaling pathways. GO terms enriched include
   extracellular matrix organization, collagen-containing extracellular
   matrix, ion channel activity, gated channel activity, and extracellular
   matrix structural constituents. These enriched terms could support the
   roles of CXCR7 in cell migration, particularly through pathways related
   to extracellular matrix and cell adhesion [[100]8]. The most
   significantly enriched KEGG pathways are PI3K-AKT, calcium signaling,
   and MAPK signaling, which have been reported to be regulated by CXCR7
   to promote HCC progression, invasion, and migration [[101]30, [102]31].
   The Hippo signaling pathway was also significantly enriched by KEGG,
   which the activation was confirmed by GSEA ontology. Both Hippo
   signaling and CXCL12-CXCR7/CXCR4 are important in organ development,
   and CXCR7 can form a positive feedback loop with Hippo signaling (via
   YAP expression) to promote gastric cancer progression [[103]23]. The
   enrichment of the Hippo signaling pathway suggests that such a feedback
   loop may also be involved in tumor progression in HCC.

   The Hippo signaling pathway has been recognized as a critical regulator
   of organ size [[104]36]. When Hippo signaling is activated, the
   downstream effectors YAP/TAZ are phosphorylated (rendered inactive) and
   restricted to the cytoplasm; upon Hippo pathway inactivation, YAP/TAZ
   are dephosphorylated (activated) and transported into the nucleus,
   where they enhance the expression of target genes to increase cell
   proliferation and survival, enhance stemness, and promote tumor
   progression. Numerous studies have highlighted the key role of the
   Hippo signaling pathway in HCC [[105]18, [106]37]. Activation of Hippo
   signaling suppresses HCC progression by affecting multiple aspects,
   including proliferation and fate reprogramming, tumor microenvironment,
   and metabolic reprogramming [[107]18]. Similar to CXCR7, activation of
   YAP/TAZ in peritumoral tissues has been shown to exert suppressive
   effects on HCC progression [[108]38], which is contrary to what happens
   in HCC cells. Consistent with findings characterized by Wang et al. in
   gastric cancer [[109]23], we further demonstrated that Hippo signaling
   is regulated by CXCR7 in HCC cells. Specifically, Gαq/11 and Gαs
   protein levels were reduced, while p-LATS/LATS and p-YAP/YAP levels
   were increased with si-CXCR7 treatment; conversely, pcDNA-CXCR7 had the
   opposite effects on these proteins. Co-treatment with the YAP inhibitor
   verteporfin reversed the suppressive effects of si-CXCR7 on HCC cells,
   including cell viability, colony formation, migration, and invasion.
   Our results support the conclusion that Hippo signaling can be
   inhibited by CXCR7 in HCC cells, promoting proliferation, colony
   formation, migration, and invasion, as concluded in Fig. [110]6.

Fig. 6.

   Fig. 6
   [111]Open in a new tab

   Interaction of CXCR7 and Hippo signaling to promote HCC progression

   While our findings provide evidence for the interplay between CXCR7 and
   Hippo signaling in HCC, there are limitations. The in vitro tests were
   only carried out with HCCLM3 cells, it is not known whether the
   transfection can mimic physiological expression levels, and in vivo
   consequences of CXCR7 remain unexplored. CXCR7 has been reported to
   influence a wide range of signaling pathways, requiring further
   investigation to delineate its direct and indirect roles. Most
   importantly, both CXCR7 and Hippo signaling could have different
   implications in HCC and adjacent normal tissues, so their expression
   and interactions across cell types should be determined. Future studies
   should focus on validating these findings in vivo and exploring
   therapeutic strategies targeting the CXCR7-Hippo axis.

Conclusion

   Collectively, our findings indicate that two groups of morphogens,
   CXCL12-CXCR4/CXCR7 and Hippo signaling, may interact to promote HCC
   progression. CXCR7 inhibits Hippo signaling by activating LATS and YAP,
   which promotes the proliferation, colony formation, migration, and
   invasion of HCCLM3 cells.

Supplementary Information

   [112]Additional file 1 (JPG 2376 KB)^ (3.8MB, jpg)

Acknowledgements