Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2015 Apr 28;14:95.
doi: 10.1186/s12943-015-0356-7.

High Expression of Protein Phosphatase 4 Is Associated With the Aggressive Malignant Behavior of Colorectal Carcinoma

Affiliations
Free PMC article

High Expression of Protein Phosphatase 4 Is Associated With the Aggressive Malignant Behavior of Colorectal Carcinoma

Xinxiang Li et al. Mol Cancer. .
Free PMC article

Abstract

Background: Recent evidence suggests an important role of protein phosphatase 4 (PP4C) in the progression of several cancers, including breast cancer, lung cancer and pancreatic ductal adenocarcinoma. However, the contribution of PP4C to colorectal carcinoma (CRC) remains elusive.

Methods: The expression of PP4C in CRC tissues compared with matched non-tumor tissues and CRC cells was detected using quantitative RT-PCR, immunohistochemistry and western blotting assays. Through univariate and Kaplan-Meier analysis, we correlated the PP4C expression with clinicopathological features and patient survival. A series of experiments, including cell proliferation, lentiviral infection, cell invasion and MMP gelatinase activity assays, were performed to investigate the underlying mechanisms. Through further experiments, tumor growth and metastasis were evaluated in vivo using a xenogenous subcutaneously implant model and a tail vein metastasis model.

Results: In the present study, we found that PP4C expression is frequently increased in human CRC and that the upregulation of PP4C correlates with a more invasive tumor phenotype and poor prognosis. The ectopic expression of PP4C promoted CRC cell proliferation, migration and invasion in vitro and tumor growth and lung metastasis in vivo. Silencing the expression of PP4C resulted in the inhibition of cell proliferation and invasion. Further investigations showed that phosphorylated Akt (p-AKT) is required for the PP4C-mediated upregulation of MMP-2 and MMP-9, which promotes cell invasion.

Conclusions: Our data suggested a potential role of PP4C in tumor progression and provided novel insights into the mechanism of how this factor positively regulated cell proliferation and invasion in CRC cells.

Figures

Figure 1
Figure 1
PP4C was overexpressed in CRC tissues and cell lines. (A) The PP4C mRNA expression in CRC specimens and paired adjacent non-cancer colorectal tissues was determined by qRT-qPCR. (B) Representative images show PP4C expression in normal tissues adjacent to colorectal tissue (left) and colorectal cancer (right) (Original magnification, 100×). (C) Western blotting analysis of PP4C expression in five CRC cell lines. ** represents p < 0.01.
Figure 2
Figure 2
PP4C expression was determined in an immortalized human intestinal epithelial cell line (HIEC) and various CRC cell lines by real-time PCR analysis.
Figure 3
Figure 3
Increased PP4C expression was significantly associated with the overall survival (OS) of CRC patients. The data were analyzed using Kaplan-Meier survival analysis between patients with high PP4C expression and low PP4C expression according to the intensity scores.
Figure 4
Figure 4
PP4C mRNA expression were measured through real-time PCR in SW480 and HT29 cells transfected with PP4C.
Figure 5
Figure 5
PP4C regulated CRC cell proliferation and invasion. (A) Western blotting analysis of PP4C in SW480 and HT29 cells transfected with PP4C or a vector control. (B, C) Effect of PP4C overexpression on cell proliferation was evaluated by CCK-8 assay [(B) SW480 and (C) HT29]. (D) Representative micrographs of invasion assay of the indicated cells at 24 h. (E) Western blotting analysis of PP4C in SW620 and LOVO cells transfected with shPP4C or a negative control. (F, G) Proliferation rates of cell sublines detected by CCK-8 assay after PP4C knockdown [(F) SW480 and (G) HT29]. (H) Representative micrographs of the indicated cells grown for 24 h on Matrigel in the invasion assay (Original magnification, 100× for D and H). The data are shown as the means ± SD of three independent experiments. *P < 0.05, **P < 0.01.
Figure 6
Figure 6
Knockdown efficiency was shown after treatment of PP4C shRNA in SW620 and LOVO cell lines, which presented high endogenous PP4C expression.
Figure 7
Figure 7
MMP-2 and MMP-9 were essential for mediating PP4C-induced cell invasion. (A) The protein expression levels of MMP-2 and MMP-9 were detected by immunoblotting in SW480 and HT29 cells overexpressing PP4C. (B) Immunoblotting of MMP-2 and MMP-9 expression in SW620 and LOVO cells expressing lentiviral PP4C shRNA. (C, D) Conditioned medium from SW480 and HT29 cells overexpressing PP4C was collected and evaluated using the MMP gelatinase activity assay. (E, F) SW480 and HT29 cells overexpressing PP4C were pretreated with the MMP inhibitor GM6001 (25), a MMP-2 neutralizing antibody (Anti-MMP2) or a MMP-9 neutralizing antibody (Anti-MMP9) for 30 min, and the cell invasion was then determined using Matrigel invasion assay. *P < 0.05 and **P < 0.01 compared with the vector controls.
Figure 8
Figure 8
Involvement of the PI3K/AKT pathway in PP4C-induced MMP-2 and MMP-9 expression and activation and cell invasion. (A) Levels of p-AKT, T-AKT, MMP-2 and MMP-9 were evaluated in SW480 and HT29 cells after the indicated treatment. (B, C) The MMP gelatinase activity was determined after the indicated treatment. (D) Matrigel cell invasion was evaluated in SW480 and HT29 cells transfected with either vector control or PP4C followed by treatment with the PI3K inhibitor Ly294002 (25 μmol).
Figure 9
Figure 9
Effect of PP4C knockdown on AKT signaling molecules in CRC cells involved in MMP gelatinase activity. (A) Western blotting analysis of p-AKT and T-AKT expression was analyzed in PP4C-silenced SW620 and LOVO cells. (B, C) The MMP gelatinase activity was determined after PP4C silencing.
Figure 10
Figure 10
MK-2206 blocked PP4C-induced MMP-2 and MMP-9 expression and activation. (A) Levels of p-AKT, T-AKT, MMP-2 and MMP-9 were evaluated in SW480 and HT29 cells after the indicated treatment. (B, C) The MMP gelatinase activity was determined after the indicated treatment.
Figure 11
Figure 11
PP4C promoted CRC cell tumorigenicity and invasiveness of SW480 cells in vivo. (A) Representative photographs of tumors derived from SW480-PP4C or SW480-vector control cells in nude mice. The bar graphs showed the average tumor weight (gram). (B) At the end of the experiment, the expression of proteins of PP4C, MMP-2 and MMP-9 were determined in tissues from mice bearing SW480 xenograft tumors. (C) The number of tumor nodules and foci in the lungs was counted based on hematoxylin and eosin staining (Original magnification, 200 × for B). The values represent the means ± SD from 18 mice. **P < 0.01 (Student’s t test).

Similar articles

See all similar articles

Cited by 10 articles

See all "Cited by" articles

References

    1. Center MM, Jemal A, Smith RA, Ward E. Worldwide variations in colorectal cancer. CA Cancer J Clin. 2009;59(6):366–378. doi: 10.3322/caac.20038. - DOI - PubMed
    1. Ali R, Barnes I, Cairns BJ, Finlayson AE, Bhala N, Mallath M, et al. Incidence of gastrointestinal cancers by ethnic group in England, 2001–2007. Gut. 2013;62(12):1692–1703. doi: 10.1136/gutjnl-2012-303000. - DOI - PubMed
    1. Curley SA, Izzo F, Abdalla E, Vauthey JN. Surgical treatment of colorectal cancer metastasis. Cancer Metastasis Rev. 2004;23(1–2):165–182. doi: 10.1023/A:1025875332255. - DOI - PubMed
    1. Cunningham D, Atkin W, Lenz HJ, Lynch HT, Minsky B, Nordlinger B, et al. Colorectal cancer. Lancet. 2010;375(9719):1030–1047. doi: 10.1016/S0140-6736(10)60353-4. - DOI - PubMed
    1. Banky B, Raso-Barnett L, Barbai T, Timar J, Becsagh P, Raso E. Characteristics of CD44 alternative splice pattern in the course of human colorectal adenocarcinoma progression. Mol Cancer. 2012;11:83. doi: 10.1186/1476-4598-11-83. - DOI - PMC - PubMed

Publication types

MeSH terms

Feedback