Elevated GRP78 expression is associated with poor prognosis in patients with pancreatic cancer

Effect of GRP78 expression on prognosis

We next examined the effect of GRP78 expression on prognosis using the Kaplan–Meier method and log-rank test; the univariate analysis showed that the poorer overall survival of patients was significantly associated with high GRP78 expression in the tumor tissues (p = 0.0077) (Fig. 1J), sex (p = 0.0075), histologic grade (p = 0.015), T stage (p = 0.0139), and peripheral nerve infiltration (PNI) (p = 0.0455) (Table 2). The Cox regression analysis showed that GRP78 expression as well as PNI, sex, tumor tissues, histologic grade, and T-stage are independent prognostic markers (p < 0.05) (Table 2). Additionally, the GRP78 expression in the tumor tissues was also an independent prognostic marker (p < 0.05) in many subgroups (female, G1-2, PNI-negative) of PDAC patients (Table 3; Fig. 2).

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Figure 2

Kaplan-Meier survival analysis of the PDAC patients after surgical resection based on the GRP78 expression in the tumor tissues in many subgroups.

(A) female patients (p = 0.0419); (B) G1-2 tumors (p = 0.0101); (C) patients with tumor size > 4 cm (p = 0.0354); (D) patients with carcinoma of the head of the pancreas (p = 0.0188); (E) PNI-negative tumors (p = 0.0198); (F) N1 tumors (p = 0.0468). G1, well differentiated; G2, moderately differentiated; PNI, perineural invasion; N, lymph node.

Expression of GRP78 in six PDAC cell lines

To investigate the effects of GRP78 on the behaviors of PDAC cells, we performed in vitro experiments to determine the proliferation and invasion of the cell lines. The expression of GRP78 in six PDAC cell lines, including AsPC-1, BxPC-3, Capan-1, MIA PaCa-2, PCT-3, and SU.86.86 cells, was evaluated by western blotting. As shown in Fig. 3, higher GRP78 expression was observed in the BxPC-3 and SU.86.86 cells. Additionally, relatively low GRP78 expression was observed in the Capan-1 cell line. Therefore, these three cell lines were chosen for additional in vitro experiments.

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Figure 3

Expression of GRP78 in six PDAC cell lines.

Cropped blots/gels were used in the figure and the gels had been run under the same experimental conditions; the full-length blots/gels are presented in Supplementary Figure S3B.

GRP78 promoted cell proliferation and regulated the cell cycle in PDAC cell lines

GRP78 was silenced using transient transfection of siRNAs in the BxPC-3 and SU.86.86 cell lines, which highly expressed GRP78. The expression of GRP78 was successfully suppressed at 48 hours after transfection and verified by western blotting (Fig. 4). The CCK8 assay showed that the proliferation of the two GRP78 knockdown cell lines was dramatically reduced compared to the negative control (Fig. 4A,B).

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Figure 4

Impact of GRP78 on the proliferation and cell cycle of the PDAC cell lines.

(A,B) The CCK8 assay showed that the proliferation capacity of the SU86.86 and BXPC-3 cells was decreased after GRP78 knockdown. (C,D) The percentage of SU.86.86 and BXPC-3 cells in G0/G1 phase was significantly increased after GRP78 knockdown. (E) The CCK8 assay showed that the proliferation capacity of the Capan-1 cells was increased after forced overexpression of GRP78. (F) After the forced overexpression of GRP78, the percentage of Capan-1 cells in G0/G1 phase was significantly decreased, while the percentage of cells in S phase was significantly increased. (G) Western blots for CyclinD1, CDK4, and CDK6 in the SU86.86, BXPC-3 and Capan-1 cells after knockdown or overexpression of GRP78 or the negative control. si-GRP78, siRNA-GRP78; OE-GRP78, overexpression of GRP78; NC, negative control. Cropped blots/gels were used in the figure and the gels had been run under the same experimental conditions; the full-length blots/gels are presented in Supplementary Figure S4.

We also evaluated the effect of GRP78 on the cell cycle. Compared to the control group, the downregulation of GRP78 in PDAC cells by transfecting siRNAs significantly decreased the percentage of cells in S phase and increased the percentage of cells in G0/G1 phase (Fig. 4C,D).

Additionally, the Capan-1 cell line was transfected with plasmids to overexpress GRP78 to confirm the effect of GRP78 on cell proliferation. Cell proliferation was significantly increased in the GRP78-overexpressing Capan-1 cell line compared to the negative control (Fig. 4E). Furthermore, the overexpression of GRP78 increased the percentage of cells in S phase in the cell cycle analysis (Fig. 4F).

Western blotting showed that the expression of cell cycle proteins, including CyclinD1, CDK4, and CDK6, was simultaneously suppressed in the GRP78 knockdown groups, while the expression levels of these proteins was upregulated in the GRP78-overexpressing group (Fig. 4G).

Tissue microarray construction and immunohistochemistry

The TMA was constructed with a manual tissue microarray (Beecher Instruments, Sun Prairie, WI, USA) using formalin-fixed paraffin-embedded blocks. Two cores of the tumor and adjacent nontumor tissue for each case were sampled from representative areas using a 1.5-mm punch.

Immunohistochemistry was performed to detect GRP78 expression. A rabbit GRP78 monoclonal antibody (ab108613, Abcam Biotech Company, Cambridge, UK) and an EnVision+ two-step staining kit (Dako, Glostrup, Denmark) were used for staining. Briefly, the 4-μm-thick formalin-fixed, paraffin-embedded tumor sections were mounted, deparaffinized in xylene, and rehydrated in a graded alcohol series. The slides were washed with phosphate-buffered saline (PBS), immersed in a 0.01 mol/L citrate buffer solution (pH 6.0), and heated in a microwave for 10 min for antigen retrieval. Peroxidase was quenched using 3% hydrogen peroxide for 10 min. After washing again with PBS, the slides were incubated at 4 °C overnight with the primary antibody (1:50). After washing with PBS four times, the horseradish peroxidase-conjugated secondary antibody was added for 30 min and diaminobenzidine was used as a chromogen. Finally, the slides were counterstained with hematoxylin (Sigma-Aldrich Co., LLC, Munich, Germany) to visualize the nuclei. Preimmune rabbit serum was used at the same dilution as the negative control.

Staining evaluation

A blinded staining evaluation was performed by two experienced pathologists who did not have access to the clinicopathological or follow-up data. Based on a previous study¹³, the frequencies of GRP78 staining were almost at the same level in the tissues; the staining intensity was used to evaluate the expression of GRP78. The proportion of stained cells was first classified using a scale of 0–3 as follows: 0 = negative, 1 = weakly positive, 2 = moderately positive and 3 = strongly positive. Finally, GRP78 expression was simply summarized as low (intensity 0 or 1) or high (intensity 2 or 3) as shown in Fig. 1.

Cell lines and culture conditions

The human PDAC cell lines AsPC-1, BxPC-3, Capan-1, MIA PaCa-2, PCT-3, and SU.86.86 were donated by the University of Heidelberg. The cells were cultured in HyClone RPMI-1640 medium (GE Healthcare Life Sciences, Logan, Utah, USA) supplemented with 10% HyClone fetal bovine serum (FBS, GE Healthcare Life Sciences, Logan, Utah, USA) in a humidified incubator with 5% CO₂ at 37 °C.

Small interfering RNA and transfection

The small interfering RNA for GRP78 (5′-CUAUGAAGCCCGUCCAGAAtt-3′) was purchased from Thermo Fisher Scientific Corporation (Waltham, MA, USA), and a nonsense sequence that is unrelated to siGRP78, 5′-UUCUCCGAACGUGUCACGUtt-3′, was used as a negative control. Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA) was used for transfection according to the manufacturer’s instructions. The SU.86.86 and BxPC-3 cells were plated in 6-well plates at a concentration of 5 × 10⁵ cells/well in 2 mL of medium supplemented with 10% FBS. After 18 h, when the confluence of cells reached approximately 80%, the cells were used for transient transfection. A mixture containing the 80 pM of the siGRP78 duplexes and the Lipofectamine 2000 reagent was added to each well for transfection. The cells were collected after 48 h.

Plasmids and transfection

The recombinant vectors encoding human GRP78 were constructed by PCR-based amplification and were then subcloned into the pcDNA3.1 expression vector (Thermo Fisher Scientific, Waltham, MA, USA). The vectors were transfected into the cells using Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturers’ protocol.

Western blot analysis

To obtain the whole-cell lysates, the transfected cells were digested with a 0.25% trypsin solution and lysed with RIPA buffer (Thermo Fisher Scientific, Waltham, MA, USA) containing a protease and phosphatase inhibitor cocktail (Sigma-Aldrich Co., LLC, St. Louis, MO, USA). The protein concentrations were measured with a BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA). The total proteins (50 μg) were first separated on 10% SDS-PAGE and then electrotransferred to a polyvinylidene difluoride membrane (PVDF, Millipore Corporation, Billerica, MA, USA). The PVDF membranes were blocked with 5% nonfat dry milk in TBS/Tween 0.5% (TBS-T) for 1 h, and then incubated overnight with primary antibodies at 4 °C. After washing with TBS-T three times for 5 min, the membranes were incubated in horseradish peroxidase–conjugated mouse or rabbit secondary antibodies (1:3,000; Golden Bridge Biological Technology, Beijing, China) in 5% (w/v) nonfat dry milk in TBST for 1 h at room temperature. The protein bands were detected using an enhanced chemiluminescence system (Merck & Co., Inc. Whitehouse Station, NJ, USA) and the intensities were quantified using Quantity One software. Equal protein sample loading was guaranteed by incubating the membranes with a β-actin antibody as the internal control standard. The anti-human-GRP78 primary antibody was used at a dilution of 1:1000 as described above. The anti-human-CyclinD1, anti-human-CDK4, anti-human-CDK6, anti-human-p-STAT3, anti-human-STAT3, anti-human-Smad4, anti-human-JAK2, anti-human-vimentin, anti-human-RhoA, and anti-human-ROCK1 primary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA) and used at a dilution of 1:500.

Cell proliferation analyses

Cell proliferation was detected using the CCK-8 kit (Dojindo Laboratories, Kumamoto, Japan). Briefly, the cells were seeded in flat-bottomed 96-well plates at 1000 cells/well with 100 μL medium, 10 μL of the CCK-8 solution was added to each well, and the plates were incubated for 3 h at 37 °C. The absorbance was measured at 450 nm using the SpectraMax 190 microplate reader (Molecular Devices, LLC, Sunnyvale, CA, USA) at time points from days 1 to 4. A wavelength of 630 nm was used as a reference.

Cell cycle assays

The cell cycle analysis was performed by fluorescence-activated cell sorting. The PDAC cells were harvested at 24 h after transfection. After washing with cold phosphate-buffered saline (PBS), the cells were resuspended in ice-cold PBS, and fixed in 70% ice-cold ethanol overnight at 4 °C. The cells were stained with a solution containing 0.2 mg/ml RNase A, 0.1% Triton X-100 and 20 μg/ml propidium iodide and analyzed using the Accuri C6 flow cytometer (Becton Dickinson, New Jersey USA).

Migration and invasion assays

The cell migration and invasion assays were performed using a double-chamber assay with a pore size of 8 μm (Corning Life Sciences, New York, NY, USA). The membranes for the invasion assay were coated with diluted Engelbreth-Holm-Swarm murine sarcoma extracellular matrix proteins (1:8, E1270, Sigma-Aldrich Co., LLC, St. Louis, MO, USA) and then incubated at 37 °C for 4 h. According to the manufacturer’s protocol, the cells (4 × 10⁴ cells per well) were added to the upper chambers containing serum-free media, and 600 μL of medium containing 10% FBS was added to the lower chamber. The cells for migration assays were carefully removed from the upper side of the membrane with a swab after incubating for 12 hours, and the cells for the invasion assays were removed after incubating for 24 hours. The migration and invasion activity was assessed by counting the number of cells on the lower side of the filter. The membranes were fixed with methanol and then stained with crystal violet. The number of cells in five randomly selected fields was counted under a light microscope at a magnification of 200×. All in vitro experiments were independently repeated in triplicate under identical conditions.

This work is supported by the grants from the National High Technology Research and Development Program (No. 2012AA02A212) and the National Natural Science Foundation of China (81272573).