ARHGEF15 overexpression worsens the prognosis in patients with pancreatic ductal adenocarcinoma through enhancing the motility and proliferative activity of the cancer cells

Tumor samples from a total of 39 patients with pancreatic ductal adenocarcinoma (PDAC) were collected between 2002 and 2010, and subjected to molecular profiling using DNA microarrays. Details of the clinical characteristics of the patients are presented in Table 1. Among the PDAC patients, there were 27 male and 12 female patients, respectively, with an overall median age of 64.5 years (range, 42 to 78 years). The median overall survival (OS) was 17.5 months (range, 1.7 to 62.8 months), and the pathological stage ranged from I to IVb. No statistically significant relationship was found between the OS and any of the patient characteristics, including the gender, age, tumor histology, tumor size, tumor location or the pathologic stage (Table 1, p > 0.05, log rank test).

To identify the molecular mechanism underlying the exceptionally poor prognosis of PDAC, we performed a global gene expression analysis using the Affymetrix X3P GeneChip microarray, and carried out an analysis of the prognosis in relation to the tumor gene expression profiles of the 39 PDAC patients enrolled in the study. The samples were divided into two groups: the poor prognosis group, in which the OS was shorter than the median OS, and the better prognosis group, in which the OS was longer than the median OS. We found that the expressions of 46 genes were significantly correlated with the OS in the PDAC patients (Additional file 1: Table S1, p < 0.0001). Of the 46 genes, 32 were overexpressed in the worse prognosis group, whereas 14 were overexpressed in the better prognosis group (Fig. 1a). The top five most differentially expressed genes between the poor prognosis group and better prognosis group were HNF1B, ARHGEF15, SEPT6, MATR3 and PMSE4. The gene showing the most significant differential expression was HNF1B (Additional file 2: Figure S1, p < 0.00001), known as a transcription factor, whose higher expression level in the PDAC specimens was correlated with a worse survival of the patients [17]. On the other hand, the relationships between the other 4 genes and the prognosis of the patients with PDAC have not been reported to date.

To verify the differential expressions of the genes identified by microarray analysis, we performed independent real-time RT-PCR analyses for ARHGEF15, SEPT6, MATR3, and PMSE4. In the RNA samples obtained from the 39 formalin-fixed tumors that were analyzed, reliable data were obtained for 35 samples, as judged from the expression of ACTB, the internal control gene. Real-time RT-PCR analysis showed higher expression levels of the ARHGEF15, SEPT6 and MATR3 genes among the four genes in the worse prognosis group as compared to the better prognosis group. The results were analyzed by constructing Kaplan-Meier plots and compared by the log-rank test (Fig. 1b). In particular, the patient with higher expression levels of ARHGEF15 showed a significantly shorter OS as compared to the group showing lower ARHGE15 expression levels (median OS 14.0 months in the high ARHGEF15 expression group vs. 26.2 months in the low ARHGEF15 expression group; p < 0.01, log rank test). These data indicated that ARHGEF15 upregulation might enhance the aggressiveness of PDAC cells, resulting in a worse prognosis of the patients.

Since ARHGEF15 is a specific GEF for the Rho-family proteins that is involved in multiple cancer signaling pathways, we further investigated the molecular role of ARHGEF15 in the development of PDAC using pancreatic cancer cell lines. We initially assessed the ARHGEF15 expression levels in 11 pancreatic cancer cell lines by real-time RT-PCR analysis. As shown in Fig. 2a and b, varied expression levels were observed in the cell lines, with a maximum difference in the ARHGEF15 expression level of approximately 86-fold among the pancreatic cancer cell line panel. While the AsPC-1 and MIAPaCa-2 cells showed lower ARHGEF15 mRNA expression levels, the Hs766T cells showed the highest expression levels. ARHGEF15 protein expression levels in the three cell lines were also examined by Western blotting, which revealed expression patterns consistent with the mRNA expression patterns (Fig. 2c).

Since higher ARHGEF15 expression levels were associated with a worse prognosis, we attempted to investigate the biological effects of overexpression or knockdown of ARHGEF15 on the cellular phenotype using pancreatic cancer cell lines. First, we examined the effect of gene silencing of ARHGEF15 by RNA interference in the Hs766T cells, which showed the highest endogenous expression levels of ARHGEF15 among the pancreatic cell line panel, as shown in Fig. 2a. We confirmed by real-time RT-PCR that small-interfering (si) RNAs for ARHGEF15 (siARHGEF15s) suppressed the ARHGEF15 mRNA expression level by more than 80 % as compared to the control cells (Fig. 3a). ARHGEF15 protein expression was also examined by Western blotting, which revealed expression patterns consistent with the mRNA expression patterns. Given the fact that the Rho-family proteins are direct downstream effectors of ARHGEF15, the amounts of activated RhoA, Rac1 and Cdc42 were determined in the ARHGEF15-silenced cells; pull-down assay was conducted to measure the amounts of GTP-bound activated RhoA, Rac1 and Cdc42. Lysates of HeLa cells treated with GTP as positive controls showed increased amounts of the activated forms of the Rho-family proteins, while the lysates of the cells treated with GDP as negative controls showed decreased amounts of the activated forms of the Rho-family proteins, confirming the specificity of the assay (Fig. 3b). ARHGEF15 gene silencing in Hs766T cells reduced the amounts of activated GTP-RhoA, -Rac1 and -Cdc42 at 72 h (Fig. 3c and Additional file 3: Figure S2a). Then, we investigated the effect of ARHGEF15 overexpression on the amounts of the activated forms of the Rho-family proteins. Both AsPC-1 and MIAPaCa-2 cells, which showed low endogenous expression levels of ARHGEF15, were transiently transfected with an expression vector encoding ARHGEF15. Detection of the Halo tag attached to the ARHGEF15 protein confirmed that more than 50 % of the cells overexpressed the exogenous ARHGEF15 (Fig. 3d and e). Pull-down assay for active Rho-family proteins showed that the levels of GTP-bound active RhoA, Rac1 and Cdc42 were significantly increased in the AsPC-1 cells that showed ARHGEF15 overexpression (Fig. 3f). ARHGEF15 overexpression in MIAPaCa-2 cells was also associated with a tendency towards increase in the amounts of active Rho-family proteins. Collectively, the results indicate that both gene silencing and overexpression of ARHGEF15 modulate the degree of activation of the Rho-family proteins.

Rho-family GTPases are critical intracellular signaling molecules that regulate cytoskeleton organization, gene expression, cell cycle progression and cell motility [3]. Therefore, we examined whether activation of the Rho-family proteins by ARHGEF15 affected the aggressiveness of the pancreatic cancer. The effect of the ARHGEF15 expression level on the cellular motility was investigated using the transwell and wound healing assays. As shown in Fig. 4a, gene silencing of ARHGEF15 caused a notable reduction in the number of Hs766T cells, as compared to the control cells, migrating through the holes in the filters of the transwell chambers. In addition, we assessed the effect of ARHGEF15 gene silencing on the migration of cells into the scratch wound area at 24 h after generation of the wound in the presence of the cells. While almost complete closure of the entire wound area was observed in the control cells, the wound closure area decreased in the cells treated with siARHGEF15 (Fig. 4b). The retarding effect on wound healing and migration was confirmed again by using two different siRNA sequences for ARHGEF15. In addition, we assessed the effect of ARHGEF15 overexpression on the motility of the AsPC-1 and MIAPaCa-2 cells by the transwell and wound healing assays. ARHGEF15 overexpression in both of these cell lines was associated with a significant increase in the number of migrating cells in both the transwell (Fig. 5a and b) and wound healing (Fig. 5c) assays, consistent with the phenotypes of ARHGEF15 gene silencing.

In addition to promoting cell motility, the Rho-family proteins are also critical intracellular signaling molecules that contribute to cell growth through associating with various proteins. We next examined whether modulation of ARHGEF15 expression affected the proliferation of pancreatic cancer cell lines, using Cell Counting Kit-8, a colorimetric modified MTT assay kit. First, we examined the effect of suppression of ARHGEF15 on the growth rate of Hs766T cells which were demonstrated to show high endogenous ARHGEF15 expression levels. As shown in Fig. 6a, the Hs766T cells treated with siARHGEF15s showed a 44.7 % and 36.7 % decrease of the cell proliferative activity at 72 h as compared to the controls. The decreased cell proliferation was confirmed by an independent time-course assay using a different siRNA for ARHGEF15 (Additional file 3: Figure S2a). Next, we assessed the effect of ARHGEF15 overexpression on the growth rate of the AsPC-1 and MIAPaCa-2 cells, which revealed an approximately 60 % increase in the proliferative activity of the AsPC-1 cells, and approximately 30 % increase in the proliferative activity of the MIAPaCa-2 cells at 72 h (Fig. 6b). The time-course study of ARHGEF15 overexpression also confirmed the effect of ARHGEF15 overexpression of enhancing the proliferative activity of the pancreatic cells (Additional file 3: Figure S2b). The results of the upregulation and downregulation experiments led us to infer that ARHGEF15 overexpression in the tumor contributes to the aggressiveness of PDAC.

Finally, we assessed the effect of a RhoA inhibitor (CCG-1423) and RhoA gene silencing on the cellular phenotypes to obtain evidence in support of our findings. In accordance with the observation found in association with ARHGEF15 depletion, both the RhoA inhibitor and RhoA gene silencing reduced the proliferative activity, migration and invasiveness of the cancer cells (Fig. 7a–d).

All FFPE blocks were sectioned into 4-μm-thick sections with a Leica SM2010R microtome (Leica Microsystems K.K., Tokyo, Japan) using an RNase-free technique, and mounted on Superfrost slides. Two slides were prepared for each block: one stained with hematoxylin and eosin, and the other used for the subsequent RNA extraction. The tumor areas of the tissue sections were macro-dissected and RNA from these areas was isolated, linearly amplified and hybridized to the Affymetrix GeneChip Human X3P Array (Affymetrix, Santa Clara, CA, USA) using labeling methods, in accordance with the manufacturer’s instructions for the Arcturus Paradise PLUS Reagent System (Life Technologies, Grand Island, NY, USA) and GeneChip 3’ IVT Express Reagent kit (Affimetrix). Affymetrix array CEL files were processed by the RMA algorithm [37] to obtain probe set-level gene expression data, using the Expression Console software (Affymetrix). Hierarchical clustering of the microarray data was performed using the MATLAB software (The MathWorks, Natick, MA, USA). The R survival package was used for the survival analysis.

The human pancreatic cancer cell lines AsPC-1, MIAPaCa-2, CFPAC-1, CAPAN-1, CAPAN-2, SU.86.86., BxPC-3, CoLo587, Panc-1 and Hs766T were purchased from American Type Culture Collection (ATCC, Rockville, MD, USA), while the KP-4 cell line was purchased from the Japanese Collection of Research Bioresources Cell Bank (JCRB Cell Bank, Osaka, Japan). Cells were cultured in the recommended media supplemented with fetal bovine serum and the required reagents. All cells were incubated at 37 °C in a humidified atmosphere containing 5 % CO₂. The Halo-tagged ARHGEF15 expression vector pFN21A was purchased from Promega (Madison, WI, USA). The small-interfering RNAs (siRNA) against ARHGEF15 (ARHGEF15HSS117853 and ARHGEF15HSS117854) and RhoA (RHOAVHS40471 and RHOAHSS100655) were purchased from Life Technologies. CCG-1423, a RhoA-specific inhibitor, was purchased from Sigma-Aldrich (St. Louis, MO, USA).

The cell viability was quantified by a colorimetric modified MTT assay using Cell Counting Kit-8 (Dojindo, Kumamoto, Japan), in accordance with the manufacturer’s instructions. Cells were seeded on to four wells of 96-well plates at a density of 5 × 10² cells/well. The cell viability assay was performed 72 h after the seeding. Ten μL of Cell Counting Kit-8 was then added to each well. After incubation at 37 °C for 4 h, the absorption at 570 nm was measured using a microplate reader. The proliferative activities of the cells transfected with siRNA or plasmid were also quantified at 72 h after the transfection by a colorimetric assay.

Cell migration assays were performed using transwell chambers (24-well, 8-μm pore size; Corning, Corning, NY, USA) and cell invasiveness assays were conducted using BD Falcon Cell culture inserts coated with BD matrigel matrix (24-well, 8-μm pore size; BD Bioscience, San Jose, CA, USA). About 1 × 10⁵ cells in DMEM containing 0.5 % serum were loaded into the upper chambers. The lower chambers were filled with the same medium supplemented with 10 % FBS. The plates were incubated at 37 °C for 12 h. Cells that did not migrate through the pores were removed with a cotton swab. Cells on the lower side of the insert filter were fixed with 10 % formalin and stained with hematoxylin and eosin. The number of cells on the underside of the filter was counted.

Scratch wound-healing assays were performed in 24-well tissue culture plates (Corning). At 24 h after the cells were seeded (by which time, the cell confluence usually reached 90–100 %), scratches were made using the tip of a 200-μL pipette. The wells were then washed twice with the medium and cultured for an additional 24 or 48 h, followed by assessment of the wound area.

Cells were grown in 10-cm dishes, starved in serum-free medium for 24 h and then stimulated with 10 % fetal bovine serum for 2 h. Then, the cells were lysed in a buffer containing 20 mM Tris–HCl, pH 7.6, 100 mM NaCl, 1 % Triton-X-100, 10 mM MgCl₂, 2 mM NaF, and protease inhibitors. The lysates were clarified and the protein concentrations were determined by the bicinchoninic acid protein assay (Thermo Fisher Scientific, Waltham, MA, USA) and normalized. Cell lysates of HeLa cell lines were treated with GTP or GDP and used as positive or negative controls to assess the validity of the pull-down assay [38, 39]. The amounts of GTP-bound RhoA, Cdc42 and Rac1 in equal amounts of total protein were enriched and extracted using their affinity for the downstream effector proteins. The pull-downs (active RhoA, Cdc42 or Rac1) and cell extracts (total RhoA, Cdc42 or Rac1) were analyzed by SDS-PAGE followed by Western blotting with a RhoA-, Cdc42- or Rac1-specific antibody, respectively. Proteins were separated by 4–15 % SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted on to polyvinylidene difluoride membranes. After blocking, the membranes were probed with primary antibodies against RhoA, Cdc42 and Rac1. Subsequently, after incubation with horseradish peroxidase-conjugated secondary antibodies, the antigen-antibody complexes were visualized using enhanced chemiluminescence (Thermo Fisher Scientific). Images were captured using an image analyzer (LAS 3000; Fuji Film, Tokyo, Japan).

Pancreatic cancer cells were lysed using a radioimmunoprecipitation assay buffer (Thermo Fisher Scientific) containing protease inhibitors and phosphatase inhibitors. The lysates were clarified and the protein concentrations were determined by the bicinchoninic acid protein assay (Thermo Fisher Scientific) and normalized. Proteins were separated by 4–15 % SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted on to polyvinylidene difluoride membranes. After blocking in 3%FBS in wash buffer (0.2 M Tris-HCl, pH 7.6, 1.5 M NaCl, 0.1 % Tween 20), the membranes were incubated with the specific primary antibodies against ARHGEF15 (Santa Cruz Biotechnology, Dallas, TX, USA) diluted in blocking solution, at the appropriate dilutions. To ensure equal loading, the membranes were probed with anti-β-Actin antibody (Sigma-Aldrich). The membranes were then washed five times, incubated at room temperature for 1 h with a secondary antibody diluted in 5 % non-fat milk in wash buffer. After an additional five washes, the proteins were detected using ECL Western Blotting Detection Reagent, in accordance with the manufacturer’s protocol.

Cells were transfected with stealth RNA-mediated interference (RNAi; Life Technologies) for ARHGEF15, RhoA or stealth RNAi negative control (Life Technologies) using Lipofectamine RNAiMAX (Life Technologies), in accordance with the manufacturer’s protocol. The Halo-tagged ARHGEF15 expression vector pFN21A was transfected using the Viafect transfection kit (Promega), in accordance with the manufacturer’s protocol. All the experiments, both those involving downregulation of ARHGEF15/RhoA, and those with overexpression of ARHGEF15 were carried out using transient assays.

Total RNA was isolated from the cells using the RNeasy Mini kit (Qiagen, Venlo, Netherlands). For the clinical PDAC analysis, total RNA was extracted from the tumor area using the Arcturus Paradise PLUS Whole Transcript Reverse Transcription Kit (Life Technologies), in accordance with the manufacturer’s protocol. The cDNAs were synthesized using the Vilo Reverse Transcription kit (Life Technologies). Reactions were carried out using the TaqMan Gene Expression Master Mix (Life Technologies) in an ABI Prism 7900 platform (Life Technologies), in accordance with the manufacturer’s protocol. ACTB was used to normalize the gene expressions (ΔCt) and 2^−ΔΔCt to calculate the mRNA expression levels. The following primers and probe sets were used to analyze the respective genes: Hs01060665_g1 for ACTB, Hs00209087_m1 for ARHGEF15, Hs00938813_m1 for SEPT6, Hs00251579_m1 for MATR3 and Hs01056041_m1 for PMSE4 (Life Technologies). If the Ct value of ACTB in real-time RT-PCR was less than 35, the data were used to calculate the gene expression level. The relative quantification of ARHGEF15 was performed using the comparative cycle threshold method.

Statistical analysis to determine the significances of differences in the proliferative activity and motility of the cells was conducted using a two-tailed Student’s t-test. P-values of <0.05 were considered as denoting statistical significance. Error bars represent standard deviations.