Granulin epithelin precursor promotes colorectal carcinogenesis by activating MARK/ERK pathway - PubMed (original) (raw)

doi: 10.1186/s12967-018-1530-7.

Yi Pan 1 2 3, Joanna Hung Man Tong 1 2 3, Ka Yee Tin 1 2, Wei Kang 1 2 3, Raymond Wai Ming Lung 1 2 3, Feng Wu 1 2 3, Hui Li 1 2 3, Simon Siu Man Ng 3 5, Tony Wing Chung Mak 5, Ka Fai To 6 7 8, Anthony Wing Hung Chan 9 10

Affiliations

Granulin epithelin precursor promotes colorectal carcinogenesis by activating MARK/ERK pathway

Yi Pan et al. J Transl Med. 2018.

Abstract

Background: Granulin epithelin precursor (GEP) is reported to function as a growth factor stimulating proliferation and migration, and conferring chemoresistance in many cancer types. However, the expression and functional roles of GEP in colorectal cancer (CRC) remain elusive. The aim of this study was thus to investigate the clinical significance of GEP in CRC and reveal the molecular mechanism of GEP in CRC initiation and progression.

Methods: The mRNA expression of GEP in CRC cell lines were detected by qRT-PCR. The GEP protein expression was validated by immunohistochemistry in tissue microarray (TMA) including 190 CRC patient samples. The clinicopathological correlation analysis were achieved by GEP expression on TMA. Functional roles of GEP were determined by MTT proliferation, monolayer colony formation, cell invasion and migration and in vivo studies through siRNA/shRNA mediated knockdown assays. The cancer signaling pathway identification was acquired by flow cytometry, western blot and luciferase activity assays.

Results: The mRNA expression of GEP in CRC was significantly higher than it in normal colon tissues. GEP protein was predominantly localized in the cytoplasm and most of the CRC cases demonstrated abundant GEP protein compared with non-tumorous tissues. GEP overexpression was associated with non-rectal location, advanced AJCC stage, regional lymph node and distant metastasis. By Kaplan-Meier survival analysis, GEP abundance served as a prognostic marker for worse survival in CRC patients. GEP knockdown exhibited anti-cancer effect such as inhibiting cell proliferation, monolayer colony formation, cell invasion and migration in DLD-1 and HCT 116 cells and decelerating xenograft formation in nude mice. siGEP also induced G1 cell cycle arrest and apoptosis. Luciferase activity assays further demonstrated GEP activation was involved in MAPK/ERK signaling pathway.

Conclusion: In summary, we compressively delineate the oncogenic role of GEP in colorectal tumorigenesis by activating MAPK/ERK signaling pathway. GEP might serve as a useful prognostic biomarker and therapeutic target for CRC.

Keywords: Colorectal cancer; GEP; MAPK/ERK pathway; Oncogene.

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Figures

Fig. 1

Fig. 1

The expression of GEP in CRC. a GEP mRNA expression was significantly higher in CRC cell lines (6/8) than normal colon. b GEP mRNA expression was significantly higher in colon cancer tissues than the normal colon in the Ki colon dataset of ONCOMINE. c Representative IHC images showing no expression in normal colon (H-score = 0), high expression in tumor cells (H-score > 150), and low expression in tumor cells (H-score ≤ 150) of GEP in human CRC samples. d GEP protein expression was higher in primary CRCs (51.6%), compared to normal colonic mucosa (4.3%). (*P < 0.05; **P < 0.01)

Fig. 2

Fig. 2

Clinicopathological and prognostic features of CRC patients with high GEP expression. a High GEP protein level was more frequently occurred in tumor located on colon than that on rectum (P < 0.05). b Higher GEP expression was also more commonly happened in patients with advanced AJCC stage (Stage III/IV, P < 0.01). c GEP upregulation was more involved in patients with lymphatic metastasis (P < 0.01). d, e GEP high expression correlated with both shorter disease-free survival (P < 0.01, d) and overall survival (P < 0.01, e). (*P < 0.05; **P < 0.01)

Fig. 3

Fig. 3

GEP exerts oncogenic function in CRC cells. a GEP showed decreased expression at the mRNA level by qRT-PCR in DLD-1 and HCT 116 cells. b A significantly decreased proliferation was observed in the siGEP treated group compared with siControl group in all 2 cell lines examined (P < 0.01). c GEP knockdown significantly reduced anchorage-dependent growth in CRC cell lines by Foci-Formation assay (P < 0.01). d, e Knockdown GEP expression by siRNA eliminated the ability of the and invasion (d) and migration (e) in both DLD-1 and HCT 116 cells compared to the control group. f Pictures of tumors isolated from nude mice at the end of investigation (Left); The tumor growth was monitored and calculated in the line chart (Middle) and histogram represented mean of the tumor weight (Right) from the shControl and shGEP groups. (*P < 0.05; **P < 0.01)

Fig. 4

Fig. 4

GEP knockdown results in G1 arrest and apoptosis in CRC. a Accumulation of G1 cells increased and S-phase cell percentage decreased in siGEP transfectants compared with siControls in DLD-1cell and HCT 116 cells lines. b The percentage of early apoptotic cells in siGEP treated cells was significantly increased compared to the siControl cells in these two cell lines. c Western blot of CyclinD1, activation of cleaved Caspase 3, activation of cleaved Caspase 8 and cleaved PARP expression after silencing GEP in DLD-1 and HCT 116 cells. d Relative luciferase reporter activity of cell cycle signaling shown in GEP suppressed DLD-1 and HCT 116 cells. e RNA expression of GEP was positively associated with CyclinD1 in TCGA database. (*P < 0.05; **P < 0.01)

Fig. 5

Fig. 5

GEP promotes carcinogenesis via MAPK/ERK pathway in CRC. a A serial of promoter-luciferase assays (p53, TGFβ, Cell cycle, Myc, Hypoxia, MAPK/ERK, NF-κB and Wnt) were performed to screen for GEP target signaling pathways in DLD-1 and HCT 116 cells with GEP knockdown cells. b Western blot showed significant suppression of phospho-MAPK/ERK in the siGEP-treated CRC tumors cells compared with control. c GEP was shown to upregulate the phosphorylation of MAPK/ERK, nucleus translocation and stimulate cell proliferation, cell survival and metastasis of CRC. (*P < 0.05; **P < 0.01)

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