CXCL12/CXCR7/β-arrestin1 biased signal promotes epithelial-to-mesenchymal transition of colorectal cancer by repressing miRNAs through YAP1 nuclear translocation

CXCR7 overexpression promotes EMT and upregulates the expression of stem marker DCLK1

CXCR7 is highly expressed in many cancers and has predominantly pro-metastatic roles in cancer [24]. EMT is a process by which polarized epithelial cells are transformed into mesenchymal cells with the properties of increased motility and invasion. It is characterized by decreasing of E-cadherin and increasing of Vimentin expression. To explore whether CXCR7 contributes to EMT, we performed RNA-sequencing in CXCR7-overexpressing HCT116 cells and control cells. Among all the differentially expressed genes, Vimentin and ZEB1 were significantly increased and the intestinal stem cell marker DCLK1 was also markedly enhanced when CXCR7 was overexpressed (Fig. 1A). The most significantly upregulated genes were listed in Additional file 2: Table S1. To further confirm the association of CXCR7 on the regulation of EMT and DCLK1, HCT116, HT29 and SW620 cells were infected by lentivirus expressing CXCR7 (LV-CXCR7) and siRNA targeting CXCR7 (siCXCR7). Firstly, we examined the basal levels of CXCR7 expression in these cells (Additional file 1: Fig. S1A, B), the results showed that HCT116 and HT29 cells have moderate expression levels of CXCR7 while SW620 cells have a higher expression level of CXCR7 than the other cells. HCT116 cells have the epithelial phenotype with high expression of E-cadherin but minimal level of mesenchymal marker Vimentin expression at protein levels. In contrast, SW620 cells have the high metastatic capacity to lymph node with high expression of Vimentin but trace amount of E-cadherin expression at protein levels. HT29 cells are more similar to HCT116 cells in phenotype and gene expression. Therefore, we initially selected the three cell lines for manipulation of CXCR7 expression. The results indicated that we can successfully overexpress and knockdown of CXCR7 in these cells (Fig. 1B, C). Particularly, there was no significant difference between parental cells and vector control or scramble siRNA in CXCR7 expression (Fig. 1C). Interestingly, although there was genetic heterogeneity among the CRC cells, they displayed a consistent EMT phenotype when CXCR7 was overexpressed. Notably, Vimentin was prominently upregulated with concurrent downregulation of E-cadherin in CXCR7-overexpressing cells compared with vector control. In addition, EMT-related transcriptional factors ZEB1 and SNAI1 were also found to be upregulated when CXCR7 was overexpressed. Transwell assay indicated that the invasive capacity was potentially enhanced in CXCR7-overexpressing CRC cells compared with that of control cells (Additional file 1: Fig. S2). In contrast, these EMT-related proteins were downregulated when the cells were transfected with CXCR7 siRNA (Fig. 1B). RT-qPCR analysis further confirmed the regulation of E-cadherin and Vimentin at mRNA level by overexpressing or knockdown of CXCR7 (Fig. 1C, D). In parallel, as a stem marker associated with metastasis, DCLK1 had similar changes in line with EMT progression, suggesting overexpression of CXCR7 contributed to CRC metastasis by upregulating DCLK1 expression.

Fig. 1figure 1

CXCR7 overexpression promotes EMT and upregulates the expression of DCLK1. A RNA-sequencing was performed in HCT116 cells infected with lentiviral expressed CXCR7 (LV-CXCR7) and control. Hierarchical clustering analysis of differentially expressing mRNAs between HCT116Control and HCT116LV−CXCR7. B Western blot analysis of the expression levels of CXCR7, DCLK1, E-cadherin, Vimentin, N-cadherin, ZEB1 and SANI1 in HCT116, SW620 and HT29 cells infected with LV-CXCR7 and control lentivirus or transfected with siCXCR7 and siNC. Statistical analysis was performed compared with control group normalized to β-actin. Bars are means ± SD, *P < 0.05, **P < 0.01, ***P < 0.001, n = 3. C, D RT-qPCR was performed to determine the mRNA levels of CXCR7, E-cadherin, Vimentin and DCLK1 in HCT116, HT29 and SW620 cells overexpressing or knockdown of CXCR7. Statistical analysis was performed compared with control group. Bars are means ± SD, *P < 0.05, **P < 0.01, ***P < 0.001, n = 3. E The correlation of CXCR7 (ACKR3) with Vimentin and DCLK1 was determined in CRC tissues by Gene Expression Profiling Interactive Analysis (GEPIA) online tools. F The overall survival analysis of gastrointestinal cancer patients (gastric adenocarcinoma plus colorectal adenocarcinoma) divided by expression of Vimentin and DCLK1 using GEPIA. The patients were divided with high and low gene expression levels using the median cutoff and log-rank P value was shown

To determine the clinical relevance of CXCR7 expression with EMT, we analyzed the correlation of the expression of CXCR7 (ACKR3) with EMT markers and DCLK1 in human CRC tissues by Gene Expression Profiling Interactive Analysis (GEPIA) (http://GEPIA.cancer-pku.cn/index.html) using TCGA datasets. Notably, we found a robust and statistically significant association between Vimentin, DCLK1 and CXCR7 (R = 0.64 and 0.63 respectively, P < 0.001) (Fig. 1E). We also found a significant positive correlation between CXCR7 and EMT-related transcriptional factors (ZEB1 and SNAI1) respectively (Additional file 1: Fig. S3). These TCGA datasets analysis strongly support the association of the expression of CXCR7 with EMT. Furthermore, we evaluated the expression of Vimentin and DCLK1 as prognostic gene signature by using gastrointestinal cancer datasets, we found that high Vimentin and DCLK1 mRNA expression significantly correlated with a worse overall survival in gastrointestinal cancer (Fig. 1F). Collectively, these findings suggest that overexpression of CXCR7 promotes EMT and upregulates the expression of DCLK1, which is possibly associated with poor clinical outcome.

CXCR7 overexpression contributes to EMT by repressing miR-124-3p and miR-188-5p

In order to investigate the mechanism that CXCR7 signal activation contributes to CRC progression and EMT, miRNA sequencing was performed in above HCT116 cells (HCT116Control and HCT116LV−CXCR7), and the significantly upregulated and downregulated miRNAs were listed in Additional file 2: Table S2. Among all the differentially expressed miRNAs, miR-124-3p and miR-188-5p were significantly downregulated in HCT116LV−CXCR7 compared with HCT116Control, indicating that these miRNAs were downregulated by CXCR7 activation (Fig. 2A). To verify the results, we determined the expression of these miRNAs in CRC cells in response to activation of the CXCL12/CXCR7 axis. As a result, RT-qPCR analysis showed that miR-124-3p and miR-188-5p were significantly downregulated by overexpression of CXCR7, which was further suppressed in response to CXCL12 stimulation. In contrast, knockdown of CXCR7 markedly elevated these miRNAs in HCT116 and HT29 cells (Fig. 2B). CXCL12 is known as the common ligand for activation of CXCR4 and CXCR7, to elucidate whether activation of CXCR7 represses the expression of miRNAs through biased signaling, we used AMD3100, the specific inhibitor of CXCR4, to exclude the effect of activation of CXCL12/CXCR4 axis. The results showed that CXCL12/CXCR7 produced a similar effect on the downregulation of miR-124-3p and miR-188-5p with or without the treatment of AMD3100. Specifically, the biased signal of CXCL12/CXCR7 profoundly suppressed miR-124-3p and miR-188-5p. (Fig. 2B).

Fig. 2figure 2

CXCR7 biased signal activation contributes to EMT by repressing miR-124-3p and miR-188-5p. A RNA-sequencing was performed in HCT116 cells infected with CXCR7 and control lentivirus. Hierarchical clustering analysis of differentially expressing miRNAs between HCT116Control and HCT116LV−CXCR7 cells. B RT-qPCR analysis of miR-124-3p and miR-188-5p levels in HCT116 and HT29 cells infected with CXCR7 lentivirus or siRNA-CXCR7 and Controls with or without CXCL12 (100 ng/ml) in the presence of AMD3100 (2 μM) stimulation for 48 h. Statistical analysis was performed compared with vector control or siNC groups. *P < 0.05, **P < 0.01, n = 3 C, D RT-qPCR and Western blot analysis of DCLK1and Vimentin at mRNA and protein levels in CRC cells transfected with miR-124-3p and miR-188-5p mimics (124m, 188m) or inhibitors (124i, 188i) and their respective negative control (NCm, NCi). β-actin was used as loading control. Statistical analysis was performed compared with control group. Bars are means ± SD, *P < 0.05, **P < 0.01, ***P < 0.001, n = 3. E, F HCT116 cells were co-transfected with DCLK1 and Vimentin luciferase constructs and negative control (NC), miR-188-5p and miR-124-3p mimics respectively. The comparison of luciferase activity of wild-type (WT) and mutant (MUT) DCLK1-3′UTR or Vimentin-3′UTR constructs was performed 36 h after transfection. Con081 luciferase plasmid was used as the vector control. Data was normalized to Renilla activity. Bars are means ± SD; *P < 0.05, **P < 0.01, n = 3

To gain an insight into the molecular mechanism of these miRNAs on CRC progression and EMT, two mRNA target-predicting algorithms (MiRDB and Targetscan) were utilized to identify the potential downstream targets of miR-124-3p and miR-188-5p. Vimentin and DCLK1 were predicted to be the potential target gene of miR-124-3p and miR-188-5p respectively. To verify the hypothesis, HCT116, HT29 and SW620 cells were transfected with miR-124-3p and miR-188-5p mimics and inhibitors respectively, and the results showed that these miRNAs were significantly enhanced or suppressed in these CRC cells (Fig. S4A, B). As expected, the expression of Vimentin was significantly suppressed by miR-124-3p mimics, while substantially enhanced by miR-124-3p inhibitors. Similarly, the expression of DCLK1 was robustly reduced by miR-188-5p mimics and increased by miR-188-5p inhibitors (Fig. 2C, D). Of note, we also observed the regulation of DCLK1 by miR-124-3p, possibly due to the indirect regulatory effect of miR-124-3p on DCLK1 gene expression since miR-124-3p has multiple target genes and exhibits tumor suppressive effect in colorectal cancer [25]. Furthermore, luciferase reporter assay was performed to confirm the direct binding of miR-124-3p with Vimentin 3′-UTR and miR-188-5p with DCLK1 3′-UTR respectively. The predicted binding sites of the miRNAs with wild type and mutant 3′-UTR luciferase reporter constructs are shown in Fig. 2E. In accordance with these results, the binding was abolished by mutation of the binding sites of these miRNAs on Vimentin 3′-UTR and DCLK1 3′-UTR, suggesting that these miRNAs could directly bind to Vimentin 3′-UTR and DCLK1 3′-UTR and regulate its expression at the post-transcriptional level (Fig. 2F). Taken together, these results establish that CXCR7 biased signal activation contributes to CRC progression and EMT by repressing miR-124-3p and miR-188-5p that targeting Vimentin and DCLK1.

YAP1 manipulates CXCR7 biased activation-induced EMT by repressing miR-124-3p and miR-188-5p

EMT is a highly dynamic and reversible process, conferring cancer cells with the plasticity for distant dissemination and invasive capacity. YAP1/TEAD transcriptional activation has emerged as the important regulator of EMT by cooperation with ZEB1 and AP-1 [26]. Based on this, we hypothesize that YAP1 promotes EMT via the regulation of miRNAs in CRC cells.

Knockdown of YAP1 with two different siRNAs led to a pronounced decrease of DCLK1 and Vimentin at protein levels in CRC cells (Fig. 3A, B). As a transcriptional coactivator, YAP1 translocates into nucleus to exert the regulatory effects with TEAD that contains an N-terminal DNA-binding domain. To further investigate whether nuclear YAP1 orchestrates the expression of Vimentin and DCLK1 by regulating miRNAs expression, we transfected CRC cells with a construct expressing the constitutive active form of YAP1 (YAP-5SA), resistant to LATS-mediated phosphorylation, which directly leads to YAP1 nuclear translocation. Intriguingly, enforced expression of YAP-5SA rescued, to a large extent, the marked downregulation of DCLK1 and Vimentin caused by YAP1 knockdown (Fig. 3C, D). Moreover, overexpression of Flag-YAP-5SA significantly enhanced the expression of DCLK1 and Vimentin compared with vector control in both HCT116 and SW620 cells (Additional file 1: Fig. S5). Interestingly, RT-qPCR analysis further confirmed the downregulation of mRNA levels of DCLK1 and mesenchymal marker Vimentin upon YAP1 knockdown. Of relevance, the expression levels of miR-124-3p and miR-188-5p were strongly enhanced by YAP1 silencing (Fig. 3E, F).

Fig. 3figure 3

YAP1 promotes EMT and upregulates DCLK1 by repressing miR-124-3p and miR-188-5p. A, B Western blot analysis of the expression levels of YAP1, DCLK1 or Vimentin in HCT116 and SW620 cells transfected with two different YAP1 siRNAs. β-actin was used as loading control. Statistical analysis was performed compared with siNC group. C, D Western blot analysis of YAP1, DCLK1 or Vimentin in HCT116 and SW620 cells transfected with YAP1 siRNA and then rescued with transfection of Flag-YAP-5SA plasmid. Flag tag was used to indicate the overexpression of YAP-5SA. E, F RT-qPCR analysis of DCLK1 and Vimentin at mRNA levels and concurrent expression levels of miR-124-3p and miR-188-5p in HCT116 and SW620 cells transfected with YAP1 siRNAs. Statistical analysis was performed compared with siNC group. G RT-qPCR analysis of expression levels of miR-124-3p and miR-188-5p in HCT116 cells transfected with YAP1 siRNA and Flag-YAP-5SA plasmid compared with siNC and vector control respectively. H, I Western blot analysis of YAP1, DCLK1 or Vimentin in CXCR7-overexpressing HCT116 and SW620 cells transfected with YAP1 siRNA and siNC. The vector control CRC cells were transfected with siNC as controls. Bars are means ± SD; *P < 0.05, **P < 0.01, ***P < 0.001 (n = 3)

More importantly, enforced expression of YAP-5SA resulted in a drastic reduction of miR-124-3p and miR-188-5p and it could also attenuate the prominent elevation of these miRNAs in YAP1 knockdown cells (Fig. 3G). These results suggested that YAP-5SA rescued the suppression of EMT markers caused by silencing of YAP1 through the repression of miR-124-3p and miR-188-5p. To further prove that YAP1 is involved in the regulation of CXCR7-induced EMT, CRC cells overexpressing CXCR7 were transfected with siYAP1, as a result, knockdown of YAP1 significantly suppressed the upregulation of Vimentin and DCLK1 induced by overexpression of CXCR7 (Fig. 3H, I). Taken together, these findings reveal that nuclear YAP1 critically promoted CXCR7-induced EMT by repressing miR-124-3p and miR-188-5p in CRC cells.

CXCR7/β-arr1-mediated biased signal induces YAP1 nuclear translocation in CRC cells

Since nuclear YAP1 plays a crucial role in regulation of EMT by repression of miR-124-3p and miR-188-5p, next, we explored whether CXCL12/CXCR7 biased activation promotes YAP1 nuclear translocation. Notably, CXCL12 induced the reduction of YAP1 in the cytoplasm paralleled with YAP1 nuclear accumulation, which was potentiated by overexpression of CXCR7 (Fig. 4A). The CXCL12-induced YAP1 nuclear accumulation was also confirmed by immunofluorescence analysis in HCT116 and HT29 cells, showing the time-dependent nuclear translocation of YAP1 and reached plateau after 60 min stimulation by CXCL12 (Fig. 4B, C and Additional file 1: Fig. S6).

Fig. 4figure 4

CXCR7/β-arr1-mediated biased signal induces YAP1 nuclear translocation in CRC cells. A Western blot analysis of YAP1 expression in cytoplasmic and nuclear extracts of HCT116Control and HCT116LV−CXCR7 cells treated with or without CXCL12 (100 ng/ml) for 60 min. GAPDH and Lamin B1 were used as cytoplasmic and nuclear loading control, respectively. B, C YAP1 localization evaluated by immunofluorescence (IF) in HCT116 and HT29 cells treated with or without CXCL12 (100 ng/ml). YAP1 was labeled with Alexa Fluor® 488 donkey anti-rabbit secondary antibodies, nuclei were visualized with DAPI, shown in blue. Scale bars, 50 µm. D Analysis of endogenous YAP1-β-arr1 interaction in HCT116 cells by Co-immunoprecipitation (Co-IP). Normal rabbit IgG antibodies were used as control. E IF staining was performed to determine the colocalization of YAP1 (red) and β-arrestin1 (green) or β-arrestin2 (green) in HCT116 cells treated with CXCL12 (100 ng/ml) in the presence of AMD3100 (2 μM). DAPI was used for nuclear staining. Scale bars, 50 µm. F Western blot analysis of YAP1 and β-arrestin1 expression in cytoplasmic and nuclear extracts of HCT116 and HT29 cells treated with or without CXCL12 (100 ng/ml) in the presence of AMD3100 (2 μM). GAPDH and Histone H3 were used as cytoplasmic and nuclear loading control, respectively. G Western blot analysis of β-arrestin1 expression in HCT116 cells transfected with β-arrestin1 siRNA. β-actin was used as loading control. H Western blot analysis of YAP1 expression in cytoplasmic and nuclear extracts of HCT116 cells transfected with β-arrestin1 siRNA or siNC and treated with or without CXCL12 (100 ng/ml) plus AMD3100 (2 μM). GAPDH and Histone H3 were used as cytoplasmic and nuclear loading control, respectively. Bars are means ± SD; *P < 0.05, **P < 0.01, ***P < 0.001, NS stands for no significance (n = 3)

β-arr1, previously known as a cytosolic regulator and scaffold of GPCR signaling, has recently been revealed to translocate to the nucleus mediating receptor endocytosis and signal transduction. Therefore, it is likely that β-arr1 shuttles between the cytoplasm and the nucleus mediating CXCL12/CXCR7 biased signal transduction. To assess whether β-arr1 could functionally contribute to YAP1 activity regulation consequently to CXCL12/CXCR7 axis activation, we performed co-immunoprecipitation analysis in whole cell lysates derived from HCT116 cells, and found that endogenous YAP1 could physically interact with β-arr1 (Fig. 4D). Here we ask whether the interaction between β-arr1 and YAP1 facilitates the nuclear translocation of YAP1 with the concurrent nuclear shuttling of β-arr1 or not? Immunofluorescence analysis indicated that although CXCL12 stimulation at early stage (for 90 min) led to substantial YAP1 nuclear translocation, β-arr1 and β-arr2 were predominantly located in the cytoplasm and not involved in this process (Fig. 4E). However, it was true that we observed the nuclear translocation of β-arr1 at later stage (> 6 h) of CXCL12 stimulation (Additional file 1: Fig. S7). Furthermore, nuclear-cytoplasmic fractions indicated that CXCL12 induced dramatic reduction of YAP1 in the cytoplasm accompanied by the significant increase of nuclear YAP1. Concurrently, AMD3100, as a CXCR4 antagonist, was used to rule out any effects of CXCL12/CXCR4 signaling activation (Fig. 4F). These results indicated that CXCL12/CXCR7 biased signal activation substantially promoted nuclear translocation of YAP1. Noticeably, β-arr1 did not exhibit a simultaneous nuclear translocation at early stage of CXCL12 stimulation in CRC cells (Fig. 4F). Then we ask whether β-arr1 is required for YAP1 nuclear translocation upon CXCL12/CXCR7 biased signal activation? As shown in Fig. 4G, H, knockdown of β-arr1 interfered with the nuclear translocation of YAP1 in response to CXCL12 stimulation.

These results reveal that CXCL12/CXCR7 biased signal activation promotes YAP1 nuclear translocation by recruiting β-arr1 in the cytoplasm, which could not be abrogated by AMD3100 pretreatment. Overall, these findings establish that CXCR7 activation by CXCL12 induces YAP1 nuclear enrichment in CRC cells and prove the critical role of β-arr1 in transducing CXCL12/CXCR7-dependent YAP1 cytoplasmic-nuclear shuttling.

YAP1 inhibits miR-124-3p and miR-188-5p expression by recruiting YY1 to the promoters

Nuclear YAP1 functions as a potent transcriptional cofactor by binding with TEAD1. YAP1/TEAD1 complex interacts with other transcriptional factors to regulate the expression of target genes [27]. Generally, YAP1/TEAD complex activates the oncogenic downstream genes to trigger carcinogenesis. To further explore the inhibitory effects of nuclear YAP1 on the expression of miR-124-3p and miR-188-5p, TransmiR v2.0 database (http://www.cuilab.cn/transmir) and interface of mirTrans (http://mcube.nju.edu.cn/jwang/lab/soft/mirtrans/) were used to predict the transcriptional factor binding sites at the promoters of miR-124-3p and miR-188-5p. As a result, Yin Yang 1 (YY1) was predicted to be the potentially common transcriptional factor that could suppress the expression of these miRNAs (Additional file 1: Fig. S8).

We hypothesized that YAP1 functions as a transcriptional repressor by interacting with YY1, transcriptionally repressing the expression of miR-124-3p and miR-188-5p, which promotes EMT and metastasis. To prove this hypothesis, HCT116 and SW620 cells were used to confirm whether YY1 is involved in the regulation of miR-124-3p and miR-188-5p expression. As shown in Fig. 5A, miR-124-3p and miR-188-5p were robustly upregulated by YY1 silencing. Expectedly, knockdown of YY1 led to remarkably downregulation of mesenchymal marker Vimentin in SW620 cells. Consistently, the expression of DCLK1 was also profoundly impaired with YY1 depletion in both HCT116 and SW620 cells (Fig. 5B, C). These results indicated that YY1 was implicated with the regulation of EMT. To further assess the specificity of YY1 in promoting EMT, we transfected CRC cells with a construct expressing YY1 with HA-tag which led to YY1 overexpression. Remarkably, YY1 silencing led to a marked reduction of the expression of Vimentin and DCLK1, which could be rescued by enforced expression of HA-YY1 at protein levels (Fig. 5D). In particular, overexpression of YY1 could enhance the expression of DCLK1 and Vimentin in CRC cells (Additional file 1: Fig. S9). In order to prove that YY1 is required for promoting EMT by repressing the expression levels of miR-124-3p and miR-188-5p, we performed RT-qPCR analysis and found that YY1 silencing significantly enhanced the levels of miR-124-3p and miR-188-5p, which can be strongly attenuated by enforced expression of HA-YY1 (Fig. 5E).

Fig. 5figure 5

YAP1 inhibits miR-124-3p and miR-188-5p expression by recruiting YY1 to the promoters. A, B RT-qPCR analysis of the mRNA levels of DCLK1, Vimentin and concurrent expression levels of miR-124-3p and miR-188-5p in HCT116 and SW620 cells transfected with YY1 siRNAs. C Western blot analysis of the expression of YY1, DCLK1 or Vimentin in HCT116 and SW620 cells transfected with two different YY1 siRNAs. β-actin was used as an internal control. D Western blot analysis of YY1, DCLK1 or Vimentin in HCT116 and SW620 cells transfected with YY1 siRNA and then rescued with HA-YY1 plasmid. E RT-qPCR analysis of miR-124-3p and miR-188-5p levels in HCT116 cells transfected with YY1 siRNA and HA-YY1 plasmid compared with siNC and vector control respectively. Bars are means ± SD; *P < 0.05, **P < 0.01, ***P < 0.001, n = 3. F Analysis of the interaction of endogenous YAP1 with YY1 and transfected Flag-YAP-5SA with HA-YY1 by Co-IP. IP was performed using anti-YAP1 or anti-Flag antibodies and normal rabbit IgG antibodies. G IF staining was performed to determine the colocalization of YAP1 (red) and YY1 (green) in HCT116 and HT29 cells treated with CXCL12 (100 ng/ml) in the presence of AMD3100 (2 μM). DAPI was used for nuclear staining. Scale bars, 50 µm. (H) HCT116 cells were co-transfected with miR-124-3p or miR-188-5p promoter luciferase construct (miR-124 or miR-188 promoter) together with HA-YY1 plasmid. pGL3-basic plasmid was used as the vector control. The comparison of luciferase activities of promoter constructs normalized to Renilla activity was indicated. Bars are means ± SD; *P < 0.05, **P < 0.01 (n = 3)

In order to address our hypothesis that YAP1 inhibits the expression of miR-124-3p and miR-188-5p by recruiting YY1, Co-IP assay was performed in HCT116 cells. We found that endogenous YAP1 could physically interact with YY1. Furthermore, when HCT116 cells were transfected with Flag-YAP-5SA and HA-YY1, YAP1 was found to interact with HA-YY1 in the nucleus by exogenously overexpression (Fig. 5F). Consistently, immunofluorescence analysis further showed the nuclear colocalization of YAP1 and YY1 in CRC cells upon CXCL12 stimulation with or without the treatment of AMD3100 (Fig. 5G). Moreover, luciferase reporter assay showed that the promoter activities of miR-124-3p and miR-188-5p were robustly enhanced compared with control vector of pGL3-basic, which was significantly impaired upon YY1 overexpression (Fig. 5H). Taken together, these results unveil that YAP1 inhibited miR-124-3p and miR-188-5p expression by recruiting YY1 to the promoters, therefore, they cooperated to regulate EMT plasticity and metastasis of CRC cells.

YAP1 inhibitor suppresses CXCL12/CXCR7-induced EMT and tumor metastasis

In light of the crucial role of YAP1 in promoting EMT by repressing miR-124-3p and miR-188-5p via interacting with YY1, we wonder if YAP1 inhibitor Verteporfin could blunt CXCL12/CXCR7-induced EMT and distant metastasis. As shown in Fig. 6A, CXCL12/CXCR7 biased activation strongly upregulated the expression of DCLK1 and Vimentin, which was attenuated by Verteporfin. We further examined the effects of Verteporfin on distant metastasis when nude mice were injected with HCT116LV−CXCR7 cells via tail veins. The results indicated that CXCR7 overexpression facilitated more distant liver metastasis as shown by the larger metastatic nodules, which was greatly impaired by Verteporfin. The representative Hematoxylin and Eosin (HE) staining of metastatic nodules in livers were illustrated in Fig. 6B. However, there was no significant difference in lung metastasis among these groups.

Fig. 6figure 6

YAP1 inhibitor suppresses CXCR7 induced tumor progression and metastasis. A Western blot analysis of the expression of DCLK1 or Vimentin normalized to β-actin in HCT116 and SW620 cells treated with CXCL12 (100 ng/ml) in the presence of AMD3100 (2 μM) and Verteporfin (3 µM) for 48 h. B Representative images of livers and lungs and H&E-stained sections of liver metastatic nodules in nude mice inoculated with HCT116LV−CXCR7 and HCT116Control cells via tail veins with or without the treatment of Verteporfin (10 mg/kg, n = 3 per group). The arrows point out the visible metastatic nodules of liver. C Representative images of colons from AOM/DSS-treated WT, Villin-CXCR7 mice and Villin-CXCR7 mice treated with Verteporfin (10 mg/kg). Average size of colon polyps was analyzed in different groups (n = 5). D RT-qPCR analysis of expression levels of miR-124-3p and miR-188-5p in colon cancer tissues from above-mentioned C57BL/6 mice. E Representative IHC staining of Vimentin in AOM/DSS-induced colon adenocarcinoma tissues from wild type C57BL/6 mice and Villin-CXCR7 mice administered with Verteporfin (10 mg/kg) or vehicle control via intraperitoneal injection daily. F Western blot analysis of DCLK1 and Vimentin expression in colon cancer tissues from these mice. GAPDH was used as loading control and statistical analysis was performed. *P < 0.05, **P < 0.01, ***P < 0.001

To investigate the role of YAP1 in colitis-associated carcinogenesis and progression upon CXCL12/CXCR7 biased activation in vivo, wild type (WT) and Villin-CXCR7 transgenic mice (Villin-CXCR7) were treated with AOM and DSS for 3 cycles as described in methods. We found that AOM/DSS treatment seriously aggravated colonic inflammation and tumor burden in Villin-CXCR7 mice compared with WT mice, as indicated by the larger size of colonic adenocarcinoma. Importantly, Verteporfin, a YAP1 inhibitor which disrupts YAP-TEAD interactions, led to a significant suppression of the colonic adenocarcinomas (Fig. 6C). To investigate whether YAP1 inhibitor hinders EMT process in CRC by regulation of miR-124-3p and miR-188-5p in vivo, we performed RT-qPCR analysis and found that pharmacological inactivation of YAP1 with Verteporfin reversed the repression of miRNAs in AOM/DSS induced Villin-CXCR7 mice (Fig. 6D). Further IHC and Western blot analysis showed that Vimentin and DCLK1 were highly expressed in colonic adenocarcinoma of Villin-CXCR7 mice, which was abrogated by Verteporfin (Fig. 6E, F). These results suggested that YAP1 inhibitor significantly suppressed distant metastasis of HCT116LV−CXCR7 tumor xenografts in nude mice and AOM/DSS-induced colonic adenocarcinoma in Villin-CXCR7 transgenic mice, highlighting the therapeutic potential of targeting YAP1 in the control of CRC progression and metastasis.

CXCL12/CXCR7/β-arr1-induced YAP1 nuclear translocation is associated with EMT and metastasis in human CRC tissues

To explore the clinical relevance of CXCR7 activation with expression of nuclear YAP1 and EMT markers in colorectal cancer patients, we collected 22 pairs of human CRC specimens and adjacent normal colonic tissues for immunohistochemistry, Western blot and RT-qPCR analysis. As shown in Fig. 7A, CXCR7 was highly expressed in human CRC tissues compared with normal colon tissues, particularly with higher expression in metastatic CRC tissues than non-metastatic counterparts. YAP1 was remarkably overexpressed in nuclei in CRC tissues whereas predominantly expressed in cytoplasm in adjacent normal colon tissues. More importantly, there was also a higher expression of nuclear YAP1 in metastatic CRC tissues than non-metastatic counterparts. Similarly, the expression of Vimentin was higher in CRC particularly metastatic CRC tissues compared with adjacent normal colon tissues (Fig. 7A). Further protein analysis exhibited prominently high expression of YAP1 and DCLK1 in CRC tissues compared with adjacent normal tissues (Fig. 7B). To explore the potential link between YAP1, Vimentin, DCLK1 and upstream CXCR7, we used public accessible online tool GEPIA (http://GEPIA.cancer-pku.cn/index.html). It revealed a significant positive correlation of the expression of YAP1 and Vimentin (R = 0.35, P < 0.001) as well as other EMT-related transcriptional factors (Additional file 1: Fig. S10). In addition, the expression of YAP1 and DCLK1 also displayed a potent correlation (R = 0.42, P < 0.001) (Fig. 7C). Notably, the expression level of CXCR7 was strongly correlated with that of YAP1 (R = 0.43, P < 0.001) in CRC tissues. The expression level of miR-124-3p was significantly reduced in human CRC tissues and was negatively correlated with the expression of Vimentin at mRNA levels (Pearson R = -0.3386, P < 0.05) (Fig. 7D, E), highlighting that miR-124-3p functions as a tumor suppressive miRNA in CRC tissues. Taken together, these data suggest that CXCL12/CXCR7/β-arr1 biased activation triggers YAP1 nuclear translocation, which contributes to EMT and CRC metastasis by repressing miR-124-3p and miR-188-5p in clinical CRC specimens (Fig. 8).

Fig. 7figure 7

YAP1 nuclear translocation is associated with EMT and metastasis in human CRC tissues. A Representative immunohistochemistry images of the expression of CXCR7, YAP1 and Vimentin in paired normal colonic tissues, CRC tissues and matched liver metastatic CRC tissues from CRC patients. Scale bar = 100, 50, 20 μm. B Western blot analysis of YAP1 and DCLK1 expression in 22 pairs of human CRC tissues and adjacent normal tissues. β-actin was used as an internal control. Statistical analysis was performed to compare the gene expression in colorectal cancer tissues (T) compared with adjacent normal colonic tissues (N). *P < 0.05. C The correlations of YAP1 with DCLK1 and Vimentin as well as association of CXCR7 with YAP1 were determined in CRC tissues by GEPIA. D RT-qPCR analysis of miR-124-3p levels in 22 pairs of human CRC tissues and adjacent normal tissues. E The correlation of miR-124-3p and Vimentin mRNA level was performed by Pearson correlation analysis (n = 40). *P < 0.05, **P < 0.01

Fig. 8figure 8

Schematic depiction of the working model for CXCL12/CXCR7/β-arrestin1 biased signal promoting EMT through YAP1 nuclear translocation

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