The putative tumor activator ARHGEF3 promotes nasopharyngeal carcinoma cell pathogenesis by inhibiting cellular apoptosis

Nasopharyngeal carcinoma (NPC) is one of the most prevalent forms of highly invasive malignancy in Southern China and Southeast Asia. The pathogenesis of NPC is a multistep process driven by the acquisition of numerous genetic abnormalities. We investigated the potential oncogenic role of the Rho-guanine nucleotide exchange factor 3 gene, ARHGEF3, in NPC pathogenesis. Expression levels of ARHGEF3 were frequently up-regulated in NPC cell lines and tissues. In a large cohort of clinical NPC tissues high expression of ARHGEF3 was positively associated with an increased T status, distant metastasis, and a more advanced clinical stage (P < 0.05). Survival analysis revealed that ARHGEF3 expression was a significant and independent prognosis factor for NPC patients. In NPC cell lines, knockdown of ARHGEF3 was sufficient to inhibit cell growth, motility, and invasion in vitro, whereas ectopic overexpression of ARHGEF3 substantially enhanced NPC cells tumorigenesis and metastasis in vivo. Depletion of ARHGEF3 in NPC cells dramatically promoted caspase-3 induced apoptosis and an anti-apoptosis factor, BIRC8, was identified as a critical downstream target of the ARHGEF3. Our findings suggest that increased expression of ARHGEF3 plays a critical oncogenic role in NPC pathogenesis by preventing cell apoptosis through the up-regulation of BIRC8, and ARHGEF3 might be employed as a novel prognostic marker and effective therapeutic target for human NPC.


INTRODUCTION
Nasopharyngeal carcinoma (NPC) is a unique head and neck cancer which is highly prevalent in Southern China and throughout Southeast Asia [1], with an incidence of 25-30 cases per 100,000 persons annually [2].NPC pathogenesis is a multistep process driven by an accumulation of genetic alterations, including the loss of tumor suppressor genes and activation of oncogenes [3]. Malignant cell proliferation and apoptosis inhibition are the main factors influencing the development and progression of NPC and lead to the poor overall survival of NPC patients [4,5]. It is essential to elucidate the molecular mechanisms underlying tumorigenesis and invasiveness in NPC in order to identify novel therapeutic targets and develop new modalities of treatment.
It has been reported that ARHGEF3, a Rhoguanine nucleotide exchange factor (GEF) upregulated in acute myeloid leukemia (AML), modulates AML differentiation through activation of RhoA and pathways directly controlled by small GTPase family proteins [6]. The human gene ARHGEF3 is located at chromosome 3p13-21 and encodes a polypeptide of 526 amino acids with homology to neuroepithelial transforming gene 1 (NET1) [7][8][9]. ARHGEF3 belongs to the family of Rho-GEFs which specifically activates two members of the Rho-GTPase family, RHOA and RHOB, and accelerates Rho-GTPase activity by conversion of GTP to GDP [10,11]. Mutations in some members of the GEF family, such as DOCK2, DOCK8 and ARHGEF6, are associated with invasiveness and metastasis of human malignancies [12][13][14][15]. However, the potential roles and biological mechanisms of the GEF family gene ARHGEF3 in human cancers have not been studied. To investigate if abnormalities in ARHGEF3 are involved in NPC pathogenesis, we examined ARHGEF3 protein levels in a series of carcinomatous and non-neoplastic human nasopharyngeal cells and tissues, assessed the clinicopathologic/prognostic significance of ARHGEF3 expression in our NPC cohort, and investigated the mechanisms underlying the oncogenic and tumorigenic role of ARHGEF3 in NPC.
We found that high expression of ARHGEF3 in NPCs is important in the acquisition of an aggressive phenotype. Silencing ARHGEF3 in NPC cells was sufficient to inhibit cell growth, migration, and invasion in vitro, while overexpression of ARHGEF3 supported the tumorigenic and metastatic capacities of NPC cells in vivo. Further, we demonstrated that depletion of ARHGEF3 in NPC cells promoted caspase3-induced apoptosis. We also identified the anti-apoptosis factor BIRC8 as a critical downstream target of ARHGEF3. Collectively, our results provide an explanation for the malignant nature of NPC involving ARHGEF3 overexpression and the underlying mechanism that links ARHGEF3 to BIRC8 in NPC cell apoptosis.

Analysis of ARHGEF3 protein levels in NPC cells and nasopharyngeal tissues
We analyzed endogenous ARHGEF3 protein levels in 8 human nasopharyngeal cell lines by Western blotting and found that ARHGEF3 was overexpressed in 5 NPC cell lines (CNE2, SUNE1, 5-8F, 6-10B and C666), while the other 2 NPC lines (CNE1 and HONE1) and the immortalized normal nasopharyngeal cell line NP69 exhibited low ARHGEF3 protein levels ( Figure 1A, left). At the same time, we found that ARHGEF3 protein expression was higher in 8 primary NPC tissues, compared with adjacent non-neoplastic nasopharyngeal tissues. But there were no difference between the tumor and adjacent tissues in 1 case. (Figure 1A, right).

IHC staining of ARHGEF3 expression in NPC tissues and its correlation with NPC patients' pathological features and survival
Using IHC staining, we observed high expression of ARHGEF3 ( Figure 1B, left) in 111 of 192 (57.8%) primary NPC tissues (Table 1). 17 cases of NPC were not informative due to unrepresentative samples or lost samples. We used the whole NPC tissue slides of these cases to improve the limitation of TMA technology in our study. Correlation analysis demonstrated that high expression of ARHGEF3 was positively associated with an increased T status, distant metastasis, and/ or a more advanced clinical stage of NPCs (P< 0.05, Table 1). Kaplan-Meier survival curves showed that the mean disease-free survival time in NPC patients with high expression of ARHGEF3 was significantly shorter than in patients with low expression of ARHGEF3 (P=0.001, long-rank test, Figure 1C, Table 2). Multivariate Cox proportional hazards regres sion analysis demonstrated that high expression of ARHGEF3 was a significant and independent prognostic factor for poor survival of NPC patients (relative risk: 1.709, confidence interval: 1.002-2.913, P=0.049, Table 2).

Knockdown of ARHGEF3 suppresses NPC cell growth, migration, and invasion in vitro
The above observations prompted us to explore the biological function of ARHGEF3 in NPC tumorigenesis and progression. The capacity for colony formation was evaluated in two NPC cell lines (CNE2 and SUNE1) that were transfected with siARHGEF3 or control siNC. The efficiency of ARHGEF3 knockdown by siARHGEF3 was examined by Western blotting (Figure 2A). Both ARHGEF3-silenced CNE2 and SUNE1 cells had fewer and smaller colonies than that siNC-transfected cells ( Figure 2B), indicating that depletion of ARHGEF3 inhibits growth in NPC cells. Next, the effect of ARHGEF3 levels on NPC cell migration and invasion capacities were characterized by the wound-healing and Matrigel invasion assays, respectively. Knockdown of ARHGEF3 in both CNE2 and SUNE1 cells caused a dramatic suppression of cell migration and invasion abilities as compared to control cells ( Figure 2C and 2D).

Upregulated expression of ARHGEF3 supports the tumorigenic and metastatic capacities of NPC cells in vivo
To investigate whether levels of ARHGEF3 influence the tumorigenic function of NPC cells in vivo, we first constructed a HONE1-ARHGEF3 cell line which stably overexpressed ARHGEF3 ( Figure 2E, right, upper) . Next, HONE1-ARHGEF3 cells were transplanted into the backs of BALB/C-nu athymic nude mice, while HONE1-vector cells were used as a negative control (n = 5 mice per group). Thirty days after cell injection mice were sacrificed and the size and the weight of the subcutaneous tumors were examined. Tumors developed from HONE1-ARHGEF3 cells were significantly larger and heavier (P=0.02) than those arising from control cells ( Figure 2E). To investigate if increased expression of ARHGEF3 in NPC cells is causative in an in vivo experimental metastasis model, we injected HONE1-ARHGEF3 or control HONE1-vector cells into the tail vein of BALB/C-nu athymic nude mice (n = 5 mice per group). Eight weeks after injection, mice were killed and metastatic tumor nodules formed in lung and liver were examined. We did not detect tumor nodule formation in the livers of all mice examined, but overexpression of ARHGEF3 significantly increased metastasis in lung (P<0.01, Figure 2F).

Expression level of ARHGEF3 influences the apoptosis of NPC cells in vitro
To further study the effect of ARHGEF3 on NPC cell apoptosis, we transfected CNE2 and SUNE1 cells with either siARHGEF3 or siBIRC8 for 48h and found that knockdown of ARHGEF3 and BIRC8 increased apoptosis in both CNE2 and SUNE1 cells compared with control cells. Then in rescue experiment, three days after the transfection of siARHGEF3, cells were transfected with pcDNA3.1(+)-BIRC8. Finally, the apoptotic assay was performed with flow cytometry. Ectopic expression of BIRC8 in CNE2 and SUNE1 cells with the knockdown of ARHGEF3 reversed the pro-apoptotic function of siARHGEF3 ( Figure 3A). We also determined that the levels of active, cleaved caspase-3 were substantially increased in siARHGEF3-CNE2 and siARHGEF3-SUNE1 cells when compared to matched control cells ( Figure 3B). These data suggest that the attenuation of ARHGEF3 expression promotes NPC cell apoptosis.

ARHGEF3 regulates apoptosis-related gene expressions in NPC cells
In an effort to determine the potential downstream targets of ARHGEF3 that are involved in the promotion of NPC cell apoptosis, we compared mRNA expression profiles of siARHGEF3-CNE2 cells with those of control siNC-CNE2 cells using a Human Tumor Apoptosis RT 2 Profiler™ PCR Array containing 84 apoptosis-related genes. We identified a total of 6 downregulated genes (NAIP, BIRC2, BIRC3, XIAP, BIRC6, and BIRC8) and 1 upregulated gene (FASLG) in siARHGEF3-transfected CNE2 cells, which showed more than a twofold change in mRNA levels compared to control siNC-CNE2 cells ( Figure 3C and Table 3). Downregulation of XIAP and BIRC8 (ILP-2) was further validated by Western blotting assay in CNE2 and SUNE1 cells after ARHGEF3 knockdown ( Figure 3D). Further, we found a significant positive correlation between the expression of ARHGEF3 and BIRC8 in our large cohort of NPC tissues (P=0.015, Table 4). There was no significant difference in XIAP expression between the ARHGEF3 high-expressing and low-expressing groups (P = 0.321, Table 4).

DISCUSSION
ARHGEF3 is a key activator of Rho-GTPases including, in particular RhoA, Rac1, and CDC42, which function as molecular switches in a variety of cellular signaling pathways [16,17]. Increasing evidence suggests that Rho-GTPases are frequently deregulated during tumor progression, which promotes malignant phenotypes in cancer cells and is correlated with poor patient prognosis [18][19][20]. However, the In the present study, we demonstrated that the majority of human NPC cell lines and tissues expressed high levels of endogenous ARHGEF3 protein compared to control non-neoplastic nasopharyngeal cells and tissues. These findings suggest that upregulated expression of ARHGEF3 provides a selective advantage in NPC pathogenic processes.
Our analyses also found that high ARHGEF3 expression in our NPC cohorts was positively correlated with tumor T status, distant metastasis, and advanced clinical stage, suggesting that high expression of ARHGEF3 facilitates a malignant phenotype in NPC. Further, we observed that expression of ARHGEF3 was a strong and independent prognostic predictor for NPC patients. These findings underscore a potentially important role of ARHGEF3 in the development and progression of NPC, and suggest that examination of ARHGEF3 expression by IHC could be used as an additional tool in identifying those NPC patients at increased risk of tumor growth and/or metastasis.
Our investigation of the mechanisms through which ARHGEF3 regulates NPC cell malignancy demonstrated that knockdown of ARHGEF3 in CNE2 and SUNE1 NPC cells dramatically repressed cell growth, migration, and invasion in vitro. In contrast, ARHGEF3 overexpression in HONE1 NPC cells induced cell tumorigenicity in vivo. Moreover, in a tail vein injection mouse model of cancer metastasis ectopic overexpression of ARHGEF3 in HONE1 cells led to a significant increase in the number of metastatic lung lesions. These data support our emerging view that increased ARHGEF3 expression is a critical molecular event in the process of NPC pathogenesis.
It has been reported that cancer cells exhibit deficiencies in the induction of apoptosis, resulting in accelerated tumor development and reduced responsiveness to anti-cancer therapies [21][22][23]. Conversely, increased apoptosis in cancer cells may provide therapeutic benefits, especially in apoptosis-defective cancers [24,25]. In the current study, we found that silencing ARHGEF3 in NPC cells could induce apoptosis, as measured by an increased percentage of annexin V positive cells and increased cleavage of caspases3. Gene expression profiling also revealed that silencing of ARHGEF3 resulted in downregulation of a number of genes, most notably BIRC8. Further, we did observe a significant positive correlation between expression of ARHGEF3 and BIRC8 in our large cohort of NPC clinical tissues.BIRC8 (ILP-2) belongs to the inhibitors of apoptosis protein (IAP) family, which are apoptosis inhibitors that may protect against apoptotic stimuli and suppress apoptosis [26]. It has been reported that BIRC8 exerts its effects by association with an inhibition of specific caspases [27]. In addition, studies have documented that BIRC8 (ILP-2) is a tumor biomarker and promotes cancer progression [28,29]. These results suggest that ARHGEF3 regulates cell apoptosis via control of BIRC8 expression, which is in turn involved in attenuation of caspases3-induced apoptosis in the pathogenesis of NPC.

Figure 3: Knockdown of ARHGEF3 promotes cellular apoptosis and regulates apoptosis-associated gene expression in NPC cells. A. A representative image showing that knockdown of ARHGEF3 and BIRC8 in CNE2 and SUNE1 cells significantly increases
cell apoptosis compared to control cells (A). At first, CNE2 and SUNE1 cells were transfected with siARHGEF3 or siBIRC8 alone. Then in rescue experiment, three days after the transfection of siARHGEF3, cells were transfected with pcDNA3.1(+)-BIRC8. Finally, the apoptotic assay was performed with flow cytometry. The knockdown of ARHGEF3 and BIRC8 in CNE2 and SUNE1 cells significantly increases cell apoptosis compared to control cells. Ectopic expression of BIRC8 in CNE2 and SUNE1 cells with the knockdown of ARHGEF3 reversed the pro-apoptotic function of siARHGEF3 (left). Data represent the mean ± SD of at least 3 independent experiments. **P<0.01, ***P<0.001 by Student's t test (right). B. Western blot showing that siARHGEF3 in CNE2 and SUNE1 cells increased levels of the active cleaved form of caspase-3 compared with siNC treatment. C. A total of six down-regulated genes (BIRC2, BIRC3, BIRC6, BIRC8, NAIP and XIAP) and one up-regulated gene (FASLG) showed more than a two-fold mRNA differential expression in siARHGEF3-CNE2 cells. D. Knockdown of ARHGEF3 by siARHGEF3 down-regulated protein levels of BIRC8 and XIAP in both CNE2 and SUNE1 cells. Our study demonstrates, for the first time, the protein expression dynamics of ARHGEF3 in a large cohort of clinical NPC tissues. High expression of ARHGEF3 may be important in tumorigenesis and acquisition of a poor prognostic phenotype of human NPC. Importantly, our functional and mechanistic studies suggest an important oncogenic role for ARHGEF3 in the suppression of NPC cell apoptosis by regulating BIRC8 expression and caspases3-induced apoptosis, activities that might be responsible for the development and progression of human NPCs.

Western blotting assay
Equal amounts of whole cell and tissue lysates were resolved by SDS-polyacrylamide gel electrophoresis and electrotransferred on a polyvinylidenedifluoride membrane

Immunohistochemistry (IHC) staining
IHC studies were performed using a standard streptavidin biotin-peroxidase complex method. For antigen retrieval, TMA slides were microwave treated in 10 mM citrate buffer (pH 6.0) for 10 min. The TMA slides were incubated with anti-ARHGEF3 (1:100 dilution; Abgent Laboratories, San Diego, CA), in a moist chamber overnight at 4ºC. A negative control was obtained by replacing the primary antibody with a normal rabbit IgG.
A semi-quantitative scoring criterion for IHC of ARHGEF3 was used, in which both staining intensity and positive areas were recorded. A staining index (values 0 to 12) obtained as the intensity of positive staining (negative = 0, weak = 1, moderate = 2, or strong = 3 scores) and the proportion of immunopositive cells of interest (< 25% = 1, 25 to 50% = 2, 51% to 75% = 3, > 75% = 4 scores) were calculated. Because the staining index of expression of ARHGEF3 in all 50 non-neoplastic nasopharyngeal mucosa tissues was no more than 4, we designated staining index scores of 0-4 as "low" expression of ARHGEF3 ( Figure 1B right) and staining index scores of 6-12 was as "high" expression of ARHGEF3 (Figure 1B left).The cutoff scores for determining "high" level expression of XIAP and BIRC8 (ILP-2) were determined at staining index scores of >4 and >6, respectively.

Small interfering RNA (siRNA)
CNE2 and SUNE1 cells were cultured in six-well plates. The cells were transfected with anti-ARHGEF3 siRNA or anti-control siRNA (Ambion, Austin, Texas) using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. The gene silencing effect was measured by Western blotting 48 h post-transfection.

Colony formation assay
Five hundred infected cells were placed in a fresh six-well plate and maintained in RPMI1640 containing 10%FBS for 10 days. Colonies were fixed with methanol and stained with 0.1% Giemsa in 20% methanol for 15 minutes.

Wound-healing and matrigel invasion assays
Cell migration was assessed by measuring the movement of cells into a scraped cellular area created by 200μL pipette tip.The spread of wound closure was observed after 48h and photographed under a microscope. We measured the fraction of cell coverage across the line to calculate migration rate.
For invasion assays, 1×10 4 cells were added to a Matrigel invasion chamber (BD Biosciences, Becton Dickson Labware, Franklin Lakes, NJ) present in the insert of a 24-well culture plate. FBS was added to the lower chamber as a chemoattractant. After 48h, the noninvading cells were gently removed with a moist cotton swab. Invasive cells located on the lower side of the chamber were fixed with paraformaldehyde and then stained with crystal violet, air dried, and photographed. For colorimetric assays, the samples were treated with 150μL 10% acetic acid and absorbance was measured with a spectrophotometer at 560nm.

In vivo tumorigenesis and metastasis assays
For the in vivo assays of subcutaneous tumorigenesis of NPC cells, 1×10 6 mixed populations of HONE1-ARHGEF3 cells stably overexpressing ARHGEF3 or the control HONE1-vector were injected subcutaneously into the backs of 4-week-old male BALB/C-nu athymic nude mice. At day 30, the mice were sacrificed, the tumors were removed, and tumor weight was calculated.
To evaluate the metastasis potential of NPC cells in vivo, five 4-week-old male BALB/C-nu athymic nude mice in each experimental group were injected with HONE1-ARHGEF3 and HONE1-vector cells, separately. Briefly, each mouse received 2×10 5 cells via tail vein injection. Eight weeks after cell injection mice were killed and the lungs and the liver were removed from each mouse and fixed with phosphate-buffered neutral formalin. Consecutive tissue sections were made for each block of the tissues, which were then stained with haematoxylineosin. Finally, the slides of the lungs and the liver were carefully examined under a microscope. All experimental procedures involving animals were are accordant with the Guidelines for the Care and Use of Laboratory Animals (NIH publications Nos. 80-23, revised 1996).

Apoptosis assay
To assess the rate of apoptosis, transfected cells were harvested and washed twice with cold PBS, and the Annexin V-PI Kit (Nanjing Keygen, Nanjing, China) was used according to the manufacturer's guidelines. The detection was performed with a FACS Calibur using Cell Quest software (BDIS, San Jose, CA).

Real-time PCR gene array
RNA was extracted from siARHGEF3-CNE2 and siNC-CNE2 cells using Trizol (Invitrogen, Carlsbad, CA) and was cleaned them using the RNeasy MinElute cleanup kit (Qiagen, Valencia, CA). Messenger RNA expression levels were quantified with a Human Tumor Apoptosis RT 2 Profiler PCR array (Super Array Bioscience, Frederick, MD). SiARHGEF3-CNE2 cells were compared with control siNC-CNE2 cells using a Human Apoptosis RT 2 Profiler PCR array containing 84 key genes involved in programmed cell death.

Statistical analysis
Statistical analysis was performed using the SPSS software package (SPSS Standard version18.0, SPSS Inc). Differences between variables were assessed by the Chi-square test or Fisher's exact test. For survival analysis, we analyzed all patients with NPC by Kaplan-Meier analysis. A log rank test was used to compare different survival curves. Multivariate survival analysis was performed on all parameters that were found to be significant in univariate analysis using the Cox regression model. Data derived from cell line experiments are presented as mean ±SD and assessed by the two-tailed Student's t test. P values of < 0.05 were considered statistically significant.