CXCR4 promotes gefitinib resistance of Huh7 cells by activating the c‐Met signaling pathway

C‐X‐C chemokine receptor type 4 (CXCR4) expression is associated with poor prognosis of hepatocellular carcinoma (HCC). The aim of this study was to explore the biological role of CXCR4 in gefitinib resistance of HCC. Compared with a normal, non‐gefitinib‐resistant, human HCC cell line (Huh7), CXCR4 mRNA and protein were highly expressed in gefitinib‐resistant Huh7 cells (Huh7‐R). Cell proliferation was decreased, and apoptosis was enhanced in Huh7 cells in the presence of gefitinib. These influences conferred by gefitinib treatment on proliferation and apoptosis of Huh7 cells were abolished by CXCR4 overexpression. CXCR4 knockdown reduced the proliferation ability of HuH‐7R cells after gefitinib treatment. Importantly, CXCR4 overexpression had no influence on caveolin 1 (Cav‐1) expression; similarly, Cav‐1 silencing did not cause a substantive change in CXCR4 expression. However, CXCR4 activated Cav‐1, c‐Met, and Raf‐1 in Huh7 cells, whereas Cav‐1 silencing repressed the expression of Raf‐1 and phosphorylated c‐Met in Huh7 cells. CXCR4 overexpression promoted proliferation and repressed apoptosis in gefitinib‐treated Huh7 cells, which was partly rescued by PHA‐665752 (a c‐Met inhibitor) treatment or c‐Met deficiency. Finally, we constructed a tumor xenograft model to determine the influence of CXCR4 overexpression on tumor growth of HCC. CXCR4 overexpression accelerated tumor growth of HCC, which was abrogated by c‐Met deficiency. These findings demonstrate that CXCR4 overexpression activates c‐Met via the Cav‐1 signaling pathway, thereby promoting gefitinib resistance of Huh7 cells. Thus, this study highlights novel insights into the mechanism of gefitinib resistance of HCC and CXCR4 may become a potential target for HCC treatment.

C-X-C chemokine receptor type 4 (CXCR4) expression is associated with poor prognosis of hepatocellular carcinoma (HCC). The aim of this study was to explore the biological role of CXCR4 in gefitinib resistance of HCC. Compared with a normal, non-gefitinib-resistant, human HCC cell line (Huh7), CXCR4 mRNA and protein were highly expressed in gefitinib-resistant Huh7 cells (Huh7-R). Cell proliferation was decreased, and apoptosis was enhanced in Huh7 cells in the presence of gefitinib. These influences conferred by gefitinib treatment on proliferation and apoptosis of Huh7 cells were abolished by CXCR4 overexpression. CXCR4 knockdown reduced the proliferation ability of HuH-7R cells after gefitinib treatment. Importantly, CXCR4 overexpression had no influence on caveolin 1 (Cav-1) expression; similarly, Cav-1 silencing did not cause a substantive change in CXCR4 expression. However, CXCR4 activated Cav-1, c-Met, and Raf-1 in Huh7 cells, whereas Cav-1 silencing repressed the expression of Raf-1 and phosphorylated c-Met in Huh7 cells. CXCR4 overexpression promoted proliferation and repressed apoptosis in gefitinibtreated Huh7 cells, which was partly rescued by PHA-665752 (a c-Met inhibitor) treatment or c-Met deficiency. Finally, we constructed a tumor xenograft model to determine the influence of CXCR4 overexpression on tumor growth of HCC. CXCR4 overexpression accelerated tumor growth of HCC, which was abrogated by c-Met deficiency. These findings demonstrate that CXCR4 overexpression activates c-Met via the Cav-1 signaling pathway, thereby promoting gefitinib resistance of Huh7 cells. Thus, this study highlights novel insights into the mechanism of gefitinib resistance of HCC and CXCR4 may become a potential target for HCC treatment.
Hepatocellular carcinoma (HCC) is a highly heterogeneous malignant tumor with a high morbidity and mortality [1]. HCC is insidious and develops rapidly. Many patients have progressed to the middle and late stages of HCC when they are discovered. Therefore, patients with HCC often lose the opportunity to undergo radical surgery. So far, although scholars chemotherapy, radiotherapy, biological therapy, and minimally invasive treatment. In recent years, molecular targeted therapy has become a research focus [3].
Epidermal growth factor receptor (EGFR) belongs to a member of the receptor tyrosine kinase superfamily, which is expressed in various malignant tumors. The ligand forms a complex with EGFR to activate tyrosine kinases and signal transduction pathways and thus regulates cell proliferation, invasion, metastasis and antioxidation [4]. EGFR tyrosine kinase inhibitor (TKI) drugs, such as gefitinib and erlotinib, have been used in clinical practice [5]. However, some patients are not sensitive to EGFR-TKIs or develop drug resistance during treatment, which directly affects the clinical efficacy of EGFR-TKIs [6]. In addition to the partial secondary mutations of EGFR, many molecules located in the downstream signaling pathways or subactivation pathways of EGFR are also involved in the formation of drug resistance, such as the c-Met signaling pathway [7,8]. Amplification or up-regulation of c-Met may participate in the mechanisms of EGFR-TKI resistance. c-Met is a transmembrane receptor for hepatocyte growth factor (HGF) with autonomous phosphorylation activity, and it is a member of the receptor tyrosine kinases superfamily [9]. The combination of HGF and c-Met promotes autophosphorylation of tyrosine residues in the cytoplasm, which in turn activates the protein tyrosine kinase domain in the cytoplasm. The activated protein tyrosine kinase causes autophosphorylation of tyrosine at the carboxyl terminus of c-Met. Subsequently, various substrate proteins in the cytoplasm are rapidly phosphorylated. The activated signaling pathways avoid the damage of TKI to cells, promote proliferation of cancer cells, and ultimately lead to the resistance of patients with cancer to EGFR-TKIs [10]. However, the molecular mechanism of EGFR-TKIs in regulating c-Met expression in HCC is still unclear.
C-X-C chemokine receptor type 4 (CXCR4) is highly expressed in HCC cells, and the up-regulation of CXCR4 is closely associated with the poor prognosis of HCC . This study has suggested that CXCR4 may be involved in the regulation of EGFR-TKIs resistance in HCC. A previous study has reported that CXCL12/ CXCR4 induces c-Met activation by activating the caveolin 1 (Cav-1) signaling pathway [11]. Cav-1 and c-Met are colocalized in HCC cells, and the reciprocal activating crosstalk between c-Met and Cav-1 promotes invasive phenotype in HCC by promoting the oncogenic signaling of c-Met [12]. Overexpression of Cav-1 initiates carcinogenesis in liver cirrhosis and subsequently contributes to the progression of HCC [13]. Cav-1 accelerates invasion and metastasis of HCC, which attributes to up-regulate the expression of Pofut1 [14]. Moreover, a previous study has confirmed that Cav-1 inhibition enhances the sensitivity to gefitinib in lung adenocarcinoma cells. Thus, we speculated whether CXCR4 can enhance EGFR-TKI (gefitinib) resistance in HCC by activating the Cav-1/c-Met signaling pathway. This work conducted research on this hypothesis. Gefitinib-resistant Huh7 cells (Huh7-R cells) were induced by treating with a gradual gradient of gefitinib concentration. Cells were initially treated with 10 nM gefitinib, until the cells could grow stably in the DMEM containing 10 lM gefitinib. Two weeks before the experiment, Huh7-R cells were cultured in DMEM without gefitinib for further use. Huh7-R cells were treated with 10 lM gefitinib for 48 h.

Tumor xenograft experiments
Male BALB/c nude mice, 4-6 weeks old (weighing 15-20 g), were purchased from Vital River Laboratory Animal Technology (Beijing, China). These mice were housed under specified pathogen-free conditions with an optimal temperature (22-24°C) and humidity (40-60%) and a 12-h light/dark cycle. Mice had ad libitum access to food and water. A tumor xenograft mouse model was constructed by subcutaneous injection of the transfected Huh7 cells (1 9 10 6 cells/200 lL) through the right armpit. The mice were randomly divided into four groups (n = 4): (a) normal group, mice were subcutaneously injected with Huh7 cells; (b) LV-NC group, mice were subcutaneously injected with LV-NC-transfected Huh7 cells; (c) LV-CXCR4 group, mice were subcutaneously injected with LV-CXCR4-transfected Huh7 cells; and (d) LV-CXCR4 + LV-sh c-Met group, mice were subcutaneously injected with LV-CXCR4transfected Huh7 cells and LV-sh c-Met-transfected Huh7 cells. When the tumor volume was 100 mm 3 , mice received 45 mgÁkg À1 gefitinib continuously by gavage. Ten days after inoculation, the tumor volume of HCC was measured every 5 days. The long and short diameters of the tumor tissues were measured using a vernier caliper; tumor volume (mm 3 ) = (length/width 2 )/2. After 30 days, the mice were anesthetized by 5% isoflurane inhalation and then euthanized by cervical dislocation. The tumor tissues were stripped from the mice. The tumors were collected and weighed. All protocols were carried out following the Guide for the Care and Use of Laboratory Animals (National Institutes of Health), with the approval of the Ethics Committee of First Affiliated Hospital of Harbin Medical University.

Gene expression
Total RNA from Huh7 and Huh7-R cells was extracted using TRIzol reagent (Cat: 15596026; Invitrogen, Carlsbad, CA, USA). The concentration and integrity of RNA was detected using NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and 1.5% agarose gel electrophoresis. Total RNA was reverse transcribed using PrimeScript TM RT reagent Kit (Perfect Real Time; Cat: RR047A; Takara, Tokyo, Japan) to generate complementary DNA. The relative quantification of genes was assessed by performing quantitative real-time PCR (qRT-PCR) applying TB GreenÒ Premix Ex Taq TM II (Tli RNa-seH Plus; Cat: RR820A; Takara). The data were analyzed using 2 ÀΔΔCT method.

Cell proliferation
Cell proliferation was assessed applying Cell Counting Kit-8 (CCK-8) assay. Huh7 or Huh7-R cells were seeded into a 96-well plate and cultured at 37°C for 48 h. Then, cells (100 lL) were incubated with 10 lL CCK-8 reagent (Cat: C0037; Beyotime, Shanghai, China) at 37°C for 1 h. The absorbance of each well at 450 nm was measured by a microplate reader (Thermo Fisher Scientific).

Statistical analysis
Each assay was performed three times. All data are reported as mean AE standard deviation. SPSS 22.0 statistical software (IBM, Armonk, NY, USA) was used for statistical analysis. Two-tailed Student's t test and one-way ANOVA were used to analyze the statistical difference. A P value <0.05 was considered as a significant difference.

CXCR4 overexpression promoted gefitinib resistance of Huh7 cells
To explore the biological role of CXCR4 in gefitinib resistance of HCC, we compared the expression of CXCR4 between Huh7 and Huh7-R cells. qRT-PCR and WB data showed that CXCR4 mRNA and protein were highly expressed in Huh7-R cells with respect to Huh7 cells (Fig. 1A-C). We also found that the expression of p-c-Met was significantly increased in Huh7-R cells as compared with Huh7 cells. However, there was no change in EGFR and c-Met expression between Huh7 and Huh7-R cells (Fig. S1A-C). Then, we overexpressed CXCR4 in Huh7 cells and assessed the impact of CXCR4 overexpression on cell proliferation and apoptosis in gefitinib-treated Huh7 cells by CCK-8 assay and flow cytometry. Cell proliferation of Huh7 cells was severely reduced by gefitinib treatment, which was effectively rescued by CXCR4 up-regulation (Fig. 1D). Moreover, apoptosis of Huh7 cells was notably enhanced in the presence of gefitinib. CXCR4 overexpression partly repressed gefitinib-induced apoptosis in Huh7 cells (Fig. 1E,F). Furthermore, we silenced CXCR4 in HuH-7R cells and measured the influence of CXCR4 knockdown on the gefitinib resistance of HuH-7R cells. Gefitinib treatment had no effect on proliferation ability of HuH-7R cells. CXCR4 deficiency reduced the proliferation ability of HuH-7R cells after gefitinib treatment (Fig. S1D). Thus, these data suggested that CXCR4 overexpression promoted gefitinib resistance of Huh7 cells, and CXCR4 knockdown reversed gefitinib resistance of HuH-7R cells.

CXCR4 overexpression promoted gefitinib resistance of Huh7 cells by activating the c-Met signaling pathway
We further investigated the mechanism of action of CXCR4 in regulating gefitinib resistance of Huh7 cells. WB was performed to examine the impact of CXCR4 overexpression on the expression of Cav-1 and c-Met signaling pathway-related proteins in the Huh7 cells. The total expressions of Cav-1 and c-Met were not affected by CXCR4 overexpression in Huh7 cells. However, the expressions of p-Cav-1, p-c-Met and Raf-1 were increased in Huh7 cells after transfection of the CXCR4 overexpression vector ( Fig. 2A,B). Similarly, gefitinib treatment also had no effect on the expression of Cav-1 and c-Met in Huh7 cells. Gefitinib treatment caused a down-regulation of p-Cav-1, p-c-Met and Raf-1 in Huh7 cells, which was effectively abolished by CXCR4 overexpression (Fig. 2C-F). Furthermore, we determined the influence of Cav-1 knockdown on the Cav-1 and c-Met signaling pathway by WB analysis, showing that the expression of c-Met in Huh7 cells had no changes in the presence of pcDNA3.1-CXCR4 or si-Cav-1. The expressions of pc-Met and Raf-1 were enhanced by CXCR4 upregulation in Huh7 cells. Cav-1 deficiency caused a decrease in the expression of p-c-Met and Raf-1 in Huh7 cells. However, the influence conferred by CXCR4 up-regulation was partly rescued by Cav-1 silencing (Fig. 3A-C). In addition, CXCR4 overexpression enhanced the expression of CXCR4, and Cav-1 deficiency reduced Cav-1 expression in Huh7 cells. Importantly, CXCR4 overexpression had no influence on Cav-1 expression; similarly, Cav-1 silencing did not cause a substantive change in CXCR4 expression. CXCR4 overexpression caused an up-regulation of p-Cav-1, and Cav-1 silencing led to a down-regulation of p-Cav-1 in Huh7 cells. Inhibition of Cav-1-mediated down-regulation of p-Cav-1 was rescued by CXCR4 overexpression (Fig. 3D-G). Next, Huh7 cells were treated with PHA-665752 to inhibit c-Met activity. The results obtained from CCK-8 assay and flow cytometry showed that CXCR4 overexpression enhanced proliferation and inhibited apoptosis of Huh7 cells, which was effectively rescued by PHA-665752 treatment (Fig. 4A-C). We also found that the expressions of p-c-Met and Raf-1 were significantly increased by CXCR4 overexpression in the gefitinib-treated Huh7 cells. PHA-665752 treatment reversed CXCR4 overexpression-mediated promotion of p-c-Met and Raf-1 expression (Fig. S2A-C). Furthermore, CXCR4 overexpression caused an increase in proliferation and led to a decrease in apoptosis of Huh7 cells. Knockdown of c-Met effectively rescued the influence of CXCR4 overexpression on proliferation and apoptosis of Huh7 cells (Fig. 4D-F). In addition, CXCR4 overexpression enhanced p-c-Met and Raf-1 expression in gefitinib-treated Huh7 cells. CXCR4 overexpression-mediated promotion of Raf-1 expression was repressed by c-Met knockdown (Fig. 2D-F).
Taken together, these findings demonstrated that CXCR4 overexpression activated c-Met via the Cav-1 signaling pathway, thereby promoting gefitinib resistance of Huh7 cells.  combined with c-Met knockdown to verify the impact of CXCR4 on tumor growth in HCC. LV-CXCR4transfected Huh7 cells of inoculation significantly enhanced the weight and volume of tumor in mice. However, the weight and volume of tumor in mice were notably decreased after inoculation of LV-CXCR4 + LV-sh c-Met-transfected Huh7 cells (Fig. 5A-C). Thus, in vivo results confirmed that CXCR4 overexpression promoted tumor growth of HCC in mice by activating the c-Met signaling pathway.

Discussion
Gefitinib is one of the important new molecular alternative drugs developed in recent years. Gefitinib is a kind of EGFR-TKI drug that has a synergistic effect on chemotherapy drugs [15,16]. The inhibitory effect of gefitinib on tumors may be related to block the cell cycle, promote cell apoptosis and inhibit the secretion of vascular endothelial growth factor and angiopoiesis. In addition, gefitinib also can be used to inhibit the growth of HCC cells and tumor growth in mice [17,18]. Moreover, a previous study has reported that CXCR4 is associated with gefitinib resistance in cancer [19]. Gefitinib promotes migration and epithelial-to-mesenchymal transition of lung adenocarcinoma cells harboring EGFR mutation by enhancing CXCR4 expression. However, the occurrence of gefitinib resistance in patients with HCC is the main reason for failure of tumor treatment. In our study, we investigated the molecular mechanism of CXCR4 in gefitinib resistance in HCC. We found that CXCR4 and p-c-Met were highly expressed in Huh7-R cells. CXCR4 overexpression promoted proliferation and inhibited apoptosis of Huh7 cells in the presence of gefitinib. CXCR4 knockdown reversed gefitinib resistance of HuH-7R cells. These data suggested that CXCR4 and c-Met were associated with the occurrence of gefitinib resistance in HCC.
CXCR4 has been reported to be associated with the progression of HCC. CXCR4 participates in promoting migration and invasion of HCC cells. Cordycepin, a traditional Chinese medicine extract, causes downregulation of CXCR4, thereby inhibiting the migration and invasion of human HCC cells [20]. Benedicto et al. [21] have confirmed that the CXCL12/CXCR4 signaling pathway plays a crucial role in promoting liver metastasis. AMD3100 inhibits CXCR4 activity and blocks the CXCL12/CXCR4 axis, thereby inhibiting the contribution of both stromal and cancer cells to liver metastasis. Celastrol inhibits proliferation and migration of HCC cells by inhibiting the CXCR4 signaling pathway to impede the progression of HCC [22]. Our work revealed that gefitinib inhibited Cav-1 and c-Met signaling pathways, which were rescued by CXCR4 overexpression. Moreover, CXCR4 overexpression had no influence on Cav-1 expression; similarly, Cav1 silencing did not cause a substantive change in CXCR4 expression. However, CXCR4 up-regulation activated Cav-1 and c-Met signaling pathways by promoting the expression of p-Cav-1 and p-c-Met, which was abolished by Cav-1 silencing. Thus, CXCR4 may promote the activation of c-Met through the Cav-1 signaling pathway. In addition, PHA-665752 blocked c-Met activity, repressed proliferation and enhanced apoptosis of Huh7 cells. The effect of CXCR4 overexpression on proliferation and apoptosis of Huh7 cells was abolished by c-Met deficiency. Thus, these findings demonstrated that CXCR4 overexpression activated c-Met via the Cav-1 signaling pathway, thereby promoting gefitinib resistance of Huh7 cells.
Many studies have confirmed the role of c-Met in the procession of HCC. P-Rex1 promotes proliferation and migration of HCC cells and enhances xenograft tumor growth in vivo by activating the HGF/c-Met signaling pathway [23]. The HGF/c-Met signaling pathway participates in the liver metastasis of colorectal cancer in mice [24]. Consistently, we verified the effect of CXCR4 on gefitinib resistance in HCC in vivo. CXCR4 overexpression significantly promoted tumor growth of HCC in mice. However, c-Met deficiency notably suppressed tumor growth of HCC. Thus, CXCR4 overexpression promotes tumor growth of HCC by activating the c-Met signaling pathway. Of course, this article also has some shortcomings. This article studied only the impact of CXCR4 on the tolerance of Huh7 cells to gefitinib, and whether CXCR4 had the same effect on other HCC cell lines still needed further study. We will conduct research on this topic in future work.
Previous study has confirmed that the positive crosstalk between CXCR4 and EGFR increases the migration ability in gastric cancer [25]. In lung cancers, c-Met amplification induces gefitinib resistance by regulating the phosphorylation of the epidermal growth factor receptor (ERBB3)/phosphatidylinositol 3-kinase (PI3K) axis [26]. CXCR4 promotes the phosphorylation of c-Met and then induces epithelial-tomesenchymal transition and accelerates migration in gastric cancer cells [11]. Consistently, this work also confirmed the carcinogenic effects of CXCR4 in HCC. CXCR4 overexpression accelerated the invasive phenotype in HCC through the Cav-1/c-Met signaling pathway and thus promoted gefitinib resistance of HCC.
In conclusion, our work demonstrates the mechanism of action of CXCR4 in gefitinib resistance of HCC. CXCR4 overexpression activates c-Met via the Cav-1 signaling pathway, thereby promoting gefitinib resistance of HCC. Thus, this study highlights novel insights into the mechanism of gefitinib resistance of HCC, and CXCR4 may be a potential target for HCC treatment.