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. 2015 Jan 27;14(1):12.
doi: 10.1186/s12943-014-0276-y.

SPOCK1 as a potential cancer prognostic marker promotes the proliferation and metastasis of gallbladder cancer cells by activating the PI3K/AKT pathway

Yi-Jun Shu  1   2 , Hao Weng  3   4 , Yuan-Yuan Ye  5   6 , Yun-Ping Hu  7   8 , Run-Fa Bao  9   10 , Yang Cao  11   12 , Xu-An Wang  13   14 , Fei Zhang  15   16 , Shan-Shan Xiang  17   18 , Huai-Feng Li  19   20 , Xiang-Song Wu  21   22 , Mao-Lan Li  23   24 , Lin Jiang  25   26 , Wei Lu  27   28 , Bao-San Han  29 , Zhi-Gang Jie  30 , Ying-Bin Liu  31   32
Affiliations

SPOCK1 as a potential cancer prognostic marker promotes the proliferation and metastasis of gallbladder cancer cells by activating the PI3K/AKT pathway

Yi-Jun Shu et al. Mol Cancer. .

Abstract

Background: Gallbladder cancer (GBC) is a leading cause of cancer-related death worldwide, and its prognosis remains poor, with 5-year survival of approximately 5%. In this study, we analyzed the involvement of a novel proteoglycan, Sparc/osteonectin, cwcv, and kazal-like domains proteoglycan 1 (SPOCK1), in the tumor progression and prognosis of human GBC.

Methods: SPOCK1 expression levels were measured in fresh samples and stored specimens of GBC and adjacent nontumor tissues. The effect of SPOCK1 on cell growth, DNA replication, migration and invasion were explored by Cell Counting Kit-8, colony formation, EdU retention assay, wound healing, and transwell migration assays, flow cytometric analysis, western blotting, and in vivo tumorigenesis and metastasis in nude mice.

Results: SPOCK1 mRNA and protein levels were increased in human GBC tissues compared with those in nontumor tissues. Immunohistochemical analysis indicated that SPOCK1 levels were increased in tumors that became metastatic, compared with those that did not, which was significantly associated with histological differentiation and patients with shorter overall survival periods. Knockdown of SPOCK1 expression by lentivirus-mediated shRNA transduction resulted in significant inhibition of GBC cell growth, colony formation, DNA replication, and invasion in vitro. The knockdown cells also formed smaller xenografted tumors than control GBC cells in nude mice. Overexpression of SPOCK1 had the opposite effects. In addition, SPOCK1 promoted cancer cell migration and epithelial-mesenchymal transition by regulating the expression of relevant genes. We found that activation of the PI3K/Akt pathway was involved in the oncogenic functions of SPOCK1 in GBC.

Conclusions: SPOCK1 activates PI3K/Akt signaling to block apoptosis and promote proliferation and metastasis by GBC cells in vitro and in vivo. Levels of SPOCK1 increase with the progression of human GBC. SPOCK1 acts as an oncogene and may be a prognostic factor or therapeutic target for patients with GBC.

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Figures

Figure 1
Figure 1
Clinical significance of SPOCK1 in human GBC. (A) Scatterplots of the relative expression of SPOCK1 in GBC tissues and their matched nontumor counterparts. SPOCK1 expression was calculated and is expressed as the SPOCK1/GADPH expression ratio (2-ΔCT). (B) Comparison of the SPOCK1 expression level between GBC tissues and their corresponding nontumor tissues. (C) Representative image of GBC staining with an anti-SPOCK1 antibody. (a, b) Weak expression of SPOCK1 in well and moderately differentiated GBC tissues; (c) strong expression of SPOCK1 in poorly differentiated GBC tissues (scale bar, 100 μm). (D) Scatterplots of the average staining scores of SPOCK1 expression in patients with or without metastasis. (E) Kaplan-Meier overall survival curve of GBC patients correlated with SPOCK1 expression.
Figure 2
Figure 2
Expression of SPOCK1 in GBC cell lines. (A and B) mRNA and protein expression of SPOCK1 in tumor and nontumor tissues, and GBC cell lines GBC-SD, NOZ, SGC-996, OCUG, and EH-GB-1. (C) Immunofluorescence images of SPOCK1 (red) and DAPI (blue) staining in GBC-SD and NOZ cells (×200). (D) Western blot analysis of SPOCK1 expression in SPOCK1-depleting GBC-SD and NOZ cells and SPOCK1 overexpressing SGC-996 cells. GADPH was used as the loading control.
Figure 3
Figure 3
Effect of SPOCK1 overexpression and silencing on the growth of GBC cells in vitro . (A) The cells growth rates were determined by CCK-8 proliferation assays at various time points (*P < 0.05, **P < 0.01, and ***P < 0.001). (B) Representative images of colony formation induced by Lv-shNC, Lv-shSPOCK1, MOCK-SGC-996, and SPOCK1-SGC-996 cells. The numbers of colonies were calculated and are depicted in the bar chart (*P < 0.05, **P < 0.01, and ***P < 0.001). (C) Knockdown of SPOCK1 expression inhibited DNA replication in GBC-SD and NOZ cells compared to control as determined by the EdU incorporation assay. Elevated expression of SPOCK1 increased DNA replication in SGC-996 cells.
Figure 4
Figure 4
Effect of SPOCK1 overexpression and silencing on the growth of GBC cells in vivo . (A and C) Representative examples of tumors formed in nude mice injected with the indicated cells. (B and D) Tumor growth curves are summarized in the line chart. A statistical plot of average tumor weights in the subcutaneous xenograft model (*P < 0.05, **P < 0.01, and ***P < 0.001).
Figure 5
Figure 5
SPOCK1 promotes tumor invasion and metastasis in vitro by inducing EMT. (A) Wound closure was delayed in Lv-shSPOCK1-transduced cells compared with that in CTRL and Lv-shNC cells at 48 h in both GBC-SD and NOZ cells. Overexpression of SPOCK1 in SGC-996 cells had the opposite effects (*P < 0.05, **P < 0.01, and ***P < 0.001). (B) Matrigel invasion assay of CTRL, Lv-shNC, Lv-shSPOCK1, MOCK, and SPOCK1 transfectants cells. The number of invaded cells was calculated and is depicted in the bar chart. (*P < 0.05, **P < 0.01, and ***P < 0.001). (C and D) The protein expression of Snail, vimentin, N-cadherin and E-cadherin in the indicated cells was examined by western blotting. The protein expression of vementin and E-cadherin was examined by immunofluorescence analysis. The red signal represents staining for E-cadherin or vimentin. Nuclei were counterstained with DAPI.
Figure 6
Figure 6
SPOCK1 promotes tumor invasion and metastasis in vivo by inducing EMT. (A) An in vivo metastasis assay was performed to evaluate the effect of Lv-shSPOCK1 cells on tumor metastasis. Mice that received SPOCK1-depleted NOZ cells exhibited little ascites at 4 weeks after implantation. (B) The tumor incidence rate during the 4-week observation period. (C) Immunohistochemical staining of SPOCK1, E-cadherin, and vimentin in tumor tissues of the peritoneal metastasis model.
Figure 7
Figure 7
SPOCK1 exerts an anti-apoptotic effect via the PI3K/Akt pathway. (A) Apoptosis was determined by flow cytometry. Cells stained with annexin-V-APC were considered as apoptotic. The apoptotic index was defined as the percentage of apoptotic cells. (B) Apoptotic changes in the nuclear morphology of GBC-SD and NOZ cells as indicated by Hoechst 44322 staining (blue). The apoptotic index, defined as the percentage of apoptotic cells, was calculated and is summarized in the bar chart (*P < 0.05, **P < 0.01, and ***P < 0.001). (C) The levels of phosphorylated PI3K (Tyr607), total PI3K, phosphorylated Akt (Ser473), total Akt, Bax, Bcl-2, caspase-9, caspase-3, and PARP were detected in CTRL, Lv-shNC, and Lv-shSPOCK1 cells by western blot analysis. GADPH was used as the loading control.
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References

    1. Wu XS, Shi LB, Li ML, Ding Q, Weng H, Wu WG, et al. Evaluation of two inflammation-based prognostic scores in patients with resectable gallbladder carcinoma. Ann Surg Oncol. 2014;21:449–457. doi: 10.1245/s10434-013-3292-z. - DOI - PubMed
    1. Li M, Zhang S, Wang Z, Zhang B, Wu X, Weng H, Ding Q, Tan Z, Zhang N, Mu J, et al. Prognostic significance of nemo-like kinase (NLK) expression in patients with gallbladder cancer. Tumour Biol. 2013;34:3995–4000. doi: 10.1007/s13277-013-0988-4. - DOI - PubMed
    1. Li M, Zhang Z, Li X, Ye J, Wu X, Tan Z, Liu C, Shen B, Wang XA, Wu W, et al. Whole-exome and targeted gene sequencing of gallbladder carcinoma identifies recurrent mutations in the ErbB pathway. Nat Genet. 2014;46:872–876. doi: 10.1038/ng.3030. - DOI - PubMed
    1. Tan Z, Li M, Wu W, Zhang L, Ding Q, Wu X, et al. NLK is a key regulator of proliferation and migration in gallbladder carcinoma cells. Mol Cell Biochem. 2012;369:27–33. doi: 10.1007/s11010-012-1365-0. - DOI - PubMed
    1. Dong P, Zhang Y, Gu J, Wu W, Li M, Yang J, et al. Wogonin, an active ingredient of Chinese herb medicine Scutellaria baicalensis, inhibits the mobility and invasion of human gallbladder carcinoma GBC-SD cells by inducing the expression of maspin. J Ethnopharmacol. 2011;137:1373–1380. doi: 10.1016/j.jep.2011.08.005. - DOI - PubMed

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