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

Granulin epithelin precursor promotes colorectal carcinogenesis by activating MARK/ERK pathway 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 clinicopatho- logical 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 com- pared 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 prolifera- tion, 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 dem- onstrated 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, Oncogene, MAPK/ERK pathway *Correspondence: kfto@cuhk.edu.hk; awh_chan@cuhk.edu.hk Department of Anatomical and Cellular Pathology, State Key Laboratory in Oncology in South China, Prince of Wales Hospital, The Chinese University of Hong Kong, 30-32 Ngan Shing Street, Shatin, NT, Hong Kong, SAR, China Full list of author information is available at the end of the article © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/ publi cdoma in/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Pan et al. J Transl Med (2018) 16:150 Page 2 of 12 follow-up. The last update of the database 31 December Background 2015. The study was approved by Committees for Clini - Colorectal cancer (CRC) is one of the major leading cal Research Ethics of Joint Chinese University of Hong causes of cancer-related deaths in the world [1]. Although Kong-New Territories East Cluster. the overall survival of CRC patients has improved, the prognosis of patients with metastasis or recurrence is still relatively poor [2]. Angiogenesis is one of the major hall- Cell lines and cell culture marks of cancer [3], facilitating tumor development, pro- Human CRC cell lines Caco2, DLD-1, HCT 116, HT-29, gression and metastasis [4, 5]. Thus, understanding the LoVo, LS 180, SW480 and SW620 were all obtained molecular mechanism and identifying of novel molecular American Type Culture Collection (ATCC, Manassas, biomarker in angiogenesis could benefit clinical manage - VA, USA). All cell lines were cultured in media according ment of CRC patients. to manufacturer’s instructions. Granulin epithelin precursor (GEP), also known as For transient transfection, DLD-1 or HCT 116 cells progranulin, acrogranin, proepithelin, and GP88/PC-cell were transfected using lipofectamine 2000 (Thermo derived growth factor, is a secreted glycoprotein com- Fisher Scientific, Waltham, MA, USA) by siGEP or posed of 7.5 repeats of cysteine-rich motif [6, 7]. Physi- siRNA control (Qiagen, Hilden, Germany) to interfere ologically, it is expressed in immune cells [8, 9], neurons GEP expression. Procedures of transfection were per- [10], epithelial cells [11] and chondrocytes [12], medi- formed according to the manufacturer’s instructions. ating wound healing, neurodegeneration and cartilage Cells stably expressing downregulated GEP mediated development [7, 10–12]. Pathologically, high expression by shRNA was established using retrovirus system. The levels of GEP are associated with poor prognosis in hepa- shRNA information for GEP knockdown can be found in tocellular carcinoma, ovarian cancer [13], bladder cancer Additional file 1. [14] and glioblastoma [15]. Functional studies reveals GEP acts as a growth factor to stimulate proliferation and Quantitative reverse transcriptase‑polymerase chain migration, and confer chemoresistance in many types of reaction (qRT‑PCR) cancers including breast cancer [16, 17], ovarian cancer Total RNA from cells was extracted by Trizol (Thermo [18, 19], liver cancer [20, 21] and bile duct cancer [22]. Fisher Scientific) according to the manufacturer’s As the expression and functional role of GEP in CRC instruction. cDNAs were synthesized using High-Capac- is unclear. In this study, the clinical significance and ity cDNA Reverse Transcription Kit (Thermo Fisher Sci - the function of GEP in CRC will be comprehensively entific) as protocol. Quantification was performed with revealed. the ABI 7500 Real Time PCR system (Applied Biosys- tems, Foster City, CA, USA). Primers and probes for GEP were GEP-forward (5′-CAA ATG GCC CAC AAC ACT Methods GA-3′), GEP-reverse (5′-CCC TGA GAC GGT AAA Patients and specimens GAT GCA-3′) and GEP-probe (5′-6FAM CCA CTG CTC Between 1999 and 2013, 190 patients undergoing resec- TGC CGG CCA CTC MGBNFQ-3′). Primer and probe tion of primary CRCs at Prince of Wales Hospital, the reagents for control 18s were ready-made reagents (Pre- Chinese University of Hong Kong were recruited in the Developed TaqMan Assay Reagents, Applied Biosys- present study. None of the patients received any neo- tems). All experiments were performed in a minimum of adjuvant therapy. The age of the patients ranged from 34 three replicates. to 92 years, with a median age of 67.4 years. There were 106 men and 84 women. Tumors were staged according Immunohistochemistry (IHC) to the pathological tumor-node-metastasis (pTNM) stag- Immunohistochemistry was performed on 5 µm sections ing system, 7th version. Distribution of the pTNM stages cut from tissue microarray blocks using Dako Envision and other clinicopathological features is listed in Table 1. Plus System (Dako, Carpinteria, CA, USA) following the The patients were regularly followed up according to the manufacturer’s instruction with modifications. Briefly, institutional practice. Disease free survival was defined as antigen retrieval was followed by endogenous peroxidase the period from the date of curative surgery of primary blocking and the following antibody in accordance with tumor to the date that the patient survived without any the manufacturer’s recommendations: GEP [23] (Clone: signs or symptoms of that cancer. Overall survival was A23, 1:800). The cytoplasmic expression of was assessed defined as the period from the date of curative surgery of by using histoscore (H-score) [24] for the staining inten- primary tumor to the date of cancer-related death or last sity and the actual percentage of stained cells in the Pan et al. J Transl Med (2018) 16:150 Page 3 of 12 Table 1 Clinicopathologic correlation of GEP expression in colorectal cancer (n = 190, significant P-value in italic format) GEP expression (n = 190) P‑ value All H‑score ≥ 150 H‑score < 150 98 (51.6%) 92 (48.4%) Male gender 106 (55.8%) 51 (52.0%) 55 (59.8%) 0.283 Age at operation (years, mean ± SD) 67.4 ± 12.1 66.7 ± 12.6 68.2 ± 11.6 0.386 Location 0.032 Right colon 51 (26.8%) 27 (29.0%) 24 (26.4%) Left colon 36 (18.9%) 23 (24.7%) 13 (14.3%) Rectum 88 (46.3%) 36 (38.7%) 52 (57.1%) Synchronous 9 (4.7%) 7 (7.5%) 2 (2.2%) Unknown 6 (3.2%) / / Size (cm, mean ± SD) 4.5 ± 1.7 4.6 ± 1.9 4.3 ± 1.6 0.257 Differentiation Well 2 (1.1%) 0 2 (2.2%) 0.052 Moderate 180 (94.7%) 92 (95.8%) 88 (97.8%) Poor 4 (2.1%) 4 (4.2%) 0 Others 4 (2.1%) / / AJCC stage < 0.01 I 17 (8.9%) 5 (5.1%) 12 (13.0%) II 57 (30.0%) 19 (19.4%) 38 (41.3%) III 56 (29.5%) 27 (27.6%) 29 (31.5%) IV 60 (31.6%) 47 (48.0%) 13 (14.1%) T stage 0.583 T1 5 (2.6%) 3 (3.1%) 2 (2.2%) T2 19 (10.0%) 8 (8.2%) 11 (12.0%) T3 122 (64.2%) 61 (62.2%) 61 (66.3%) T4 44 (23.2%) 26 (26.5%) 18 (19.8%) N stage < 0.01 N0 89 (46.8%) 35 (35.7%) 54 (58.7%) N1 61 (32.1%) 34 (34.7%) 27 (29.3%) N2 40 (21.1%) 29 (29.6%) 11 (12.0%) M stage < 0.01 M0 129 (67.9%) 50 (51.0%) 79 (85.9%) M1 61 (32.1%) 48 (49.0%) 13 (14.1%) Pre-ops CEA (ng/ml, mean ± SD) 142.0 ± 838.4 218.9 ± 1118.1 53.4 ± 256.5 0.194 cytoplasm by two of the investigators. The H-score was dilution). Other primary antibodies were from Cell Sign- obtained by the formula: 3 × percentage of strongly stain- aling (Danvers, MA, USA) commercially including Cyc- ing + 2 × percentage of moderately staining + percentage lin D1 (1:1000, #2926), cleaved PARP (Asp214) (1:1000, of weakly staining, giving a range of 0–300. The stain - #9541), phospho-MAPK/ERK (1:2000, #9106), MAPK/ ing was considered positive when there was moderate or ERK (1:1000, #9102), Caspase-8 (1:1000, #9746), Cleaved strong immunoreactivity over the cutoff point above 150. Caspase-8 (1:1000, #9748), Caspase-3 (1:1000, #9662), Cleaved Caspase-3 (1:1000, #9661). The secondary anti - Western blot analysis bodies were anti-Mouse IgG-HRP (1:15,000, 00049039, Equal total protein was separated by SDS-PAGE and Dako, Denmark) and anti-Rabbit IgG-HRP (1:5000, transferred to nitrocellulose membrane. GEP was 00028856, Dako). The western blot bands were quantified detected with a monoclonal anti-GEP antibody (1:5000 by ImageJ. Pan et al. J Transl Med (2018) 16:150 Page 4 of 12 Cell proliferation assay Flow cytometry analysis Cell proliferation assays were performed using Cells transfected with siGEP or siControl were harvested 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte-trazolium at 24, 48 or 72  h after transfection. Then live cells were bromide (MTT, Sigma-Aldrich, St. Louis, MO, USA) incubated in the mixture of propidium iodide (PI) and assay following the manufactures’ protocol. Briefly, annexin V-fluorescein isothiocyanate (Thermo Fisher CRC cells which have been transfected with siGEP or Scientific) for apoptosis study. Harvested cells were fixed siControl were plated in 96-well plate. Cell viability was and incubated with PI and RNase A at 4 °C for cell cycle measured at 24, 48, 72, 96, and 120 h. Finally, the optical analysis. Finally, cells were determined by FACS Cali- density was determined at 570 and 690  nm wavelength bur Flow Cytometer (Becton, Dickinson and Company, light absorption (Victor3, Perken Elmer, MA, USA). Franklin Lakes, NJ, USA) and CellQuest program. Luciferase reporter assay Monolayer colony formation assay The firefly luciferase construct was co-transfected with Anchorage-dependent growth was assessed by mon- Renilla luciferase vector (Qiagen) as a control into the olayer colony formation. 6-well plates were used at a den- siRNA treated cells. Dual Luciferase Reporter Assay 3 3 sity of 1 × 10 to 5 × 10 for transfected cells. Cells were System (Promega, Fitchburg, Wisconsin, USA) was incubated at 37 °C for 14–21 days until colonies can obvi- employed to check the luciferase activity after 48 h’ trans- ously be observed. Colonies were fixed with methanol fection. The results were expressed as the ratio of firefly for 5  min and stained with 0.5% crystal violet. Colonies luciferase activity to renilla luciferase activity. Experi- with cell numbers of more than 50 cells per colony were ments were repeated in triplicate. counted. The experiments were performed in duplicate wells in three independent experiments. Statistical analysis The statistical analysis was performed using IBM SPSS Statistics (Version 19.0, Armonk, NY, USA). The expres - Invasion and migration assay sion level of GEP in paired non-tumor and tumor tissues Cell invasion or migration activity were performed using was compared with paired Student’s t-test. Independent 24 well biocoat matrigel invasion chambers or sterilized Student’s t-test was used to compare the mean value of transwell insert chambers (Corning, Bedford, MA, USA). any two groups. The Pearson χ test was used to analyze Cells were harvested after transfected with siGEP or the association of target expression with clinicopatho- siControl for 24 h and re-plated with serum free medium logical parameters. Survival curves were drawn using in the upper chamber. The lower chamber was filled with the Kaplan–Meier method and compared by means of culture medium containing 10% FBS as the chemoat- the log-rank test. Univariable and multivariable Cox pro- tractant. After 48 h, the cells that moved to the lower sur- portional hazard regression models were used to analyze face of polycarbonate membrane were stained with 0.5% independent prognostic factors. P < 0.05 was considered crystal violet and counted at five random 200× fields. statistically significant. P < 0.01 was considered highly statistically significant. In vivo tumorigenic assays Results 1 × 10 transfected CRC cells in 100 μl PBS were injected GEP is overexpressed in CRC subcutaneously into the dorsal region of anaesthetized GEP mRNA expression was up-regulated in 6/8 CRC nude mice (5 mice/construct, control in left and treat- cell lines compared with the normal colon epithelium by ment in right). When tumor was formed, tumor diameter qRT-PCR (Fig. 1a). We analyzed GEP expression in cellu- was measured and documented every 3 days for 3 weeks. lar datasets available through ONCOMINE (http://www. At the end of investigation, mice were sacrificed, and oncom ine.org/), an online collection of microarrays. xenografts were collected for diameter check and weigh Using the Ki colon dataset [7], we observed that GEP valuation. The animal handling and all experimental expression was lower in normal colon compared to colon procedures were approved by the Department of Health cancer tissues (Fig.  1b). Using immunohistochemical of Hong Kong and the Animal Experimentation Ethics staining on 190 CRC and 70 normal colon samples, GEP Committee, The Chinese University of Hong Kong. Pan et al. J Transl Med (2018) 16:150 Page 5 of 12 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) protein was predominantly localized in the cytoplasm (P < 0.01), lymph node involvement (P < 0.01) and distant and most of the CRC cases demonstrated abundant GEP metastasis status (P < 0.01). Specifically, a high GEP pro - protein compared with non-tumorous tissues (Fig.  1c). tein level more frequently occurred in tumors located High expression (H-score ≥ 150) of GEP protein was on colon than that on rectum (P  <  0.05, Fig.  2a). High detected in 51.6% (98/190) of tumor tissues. Whereas, GEP expression was also more commonly happened in only 4.3% (3/70) of normal tissues were found high GEP patients with advanced AJCC stage (Stage III/IV, P < 0.01, expression (P < 0.01, Fig. 1d). Fig.  2b), including the presence of lymphatic (P < 0.01, Fig. 2c) and distant metastasis (P < 0.01, Table 1). High GEP expression was associated with shorter dis- High GEP expression correlates with poor survival in CRC ease-free survival and overall survival (P < 0.01, Fig.  2d, patients e). Other Clinicopathological parameters such as right- The correlation of GEP with clinicopathologic param - side colon, poor differentiation, tumor invades adjacent eters in CRC patients was summarized in Table  1. GEP organs or perforates the visceral peritoneum (T4), lymph expression varied significantly among CRC samples node involvement (N1/2), distant metastasis (M1), and with different tumor locations (P = 0.032), AJCC stages Pan et al. J Transl Med (2018) 16:150 Page 6 of 12 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) high CEA level before operation (> 10  ng/ml) also pre- the invasion (Fig.  3d) and migration (Fig.  3e) in both dicted worse disease-free survival and overall survival DLD-1 and HCT 116 cells compared to the control (Additional file  2). By multivariable Cox regression, com- group. These observations suggested that GEP had onco - pared with those classic prognostic parameters, high genic properties. GEP expression was not an independent prognosticator To determine the anti-tumor effect of shGEP in  vivo, for patients’ disease-free (Additional file  3) and overall DLD-1 cells stably expressing GEP shRNA was estab- survival (Additional file 4). lished using retrovirus system. shControl and shGEP were subcutaneously injected into the left and right GEP exerts an oncogenic function on CRC cells flanks of nude mice respectively. The shGEP group In DLD-1 and HCT 116 cell lines with endogenous high formed smaller tumor within 27  days (Fig.  3f ). In con- GEP expression, siRNA-mediated knockdown reduced trast to shControl injected mice, the mice injected GEP expression at mRNA (P < 0.01, Fig. 3a). siGEP treat- with shGEP exhibited greatly reduced mean tumor size ments significantly decreased cell proliferation (Fig.  3b), (P < 0.05) and mean tumor weight (P < 0.01) in xenograft anchorage-dependent growth (Fig.  3c), and abilities of model (Fig. 3f ). (See figure on next page.) 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) Pan et al. J Transl Med (2018) 16:150 Page 7 of 12 Pan et al. J Transl Med (2018) 16:150 Page 8 of 12 GEP knockdown results in G1 arrest and increased showed significant suppression of phosphop-MAPK/ERK apoptosis in CRC was observed in the siGEP treated CRC tumors (Fig. 5b), Since a growth inhibitory effect was involved in siGEP indicating that the MAPK/ERK signaling pathway was transfected cells, we further explored the molecular basis essential for anti-GEP-mediated growth inhibition in involved in siGEP-suppressed tumor cell growth. We CRC cells. GEP led to MAPK/ERK phosphorylation and analyzed the transfectants for cell cycle parameters and translocate into the nucleus, which stimulated cell prolif- apoptosis using flow cytometry. 24  h after transfection, eration, cell survival and metastasis of CRC (Fig. 5c). accumulation of G1 cells increased in siGEP transfect- ant compared with siControls (57.8% vs. 44.8% in DLD- Discussion 1; 52.6% vs. 36.2% in HCT 116 cells), while S-phase cell In this study, we characterized clinicopathological fea- percentage decreased after siGEP transfection (11.6% vs. tures of GEP expression and delineated its oncogenic 16.8% in DLD-1; 23.8% vs. 27.3% in HCT 116 cells) in function in colorectal carcinogenesis. these 2 cell lines (Fig. 4a). For apoptosis analysis, the per- GEP was overexpressed in CRC cell lines and patients’ centage of early apoptotic cells in siGEP treated cells was tumor samples. Overexpression of GEP in CRC was significantly increased compared to the siControl cells associated with nodal and distant metastasis and poorer in both DLD-1 (0.8%  ±  0.1% vs. 4.2% ± 0.2%, P < 0.01, clinical outcome. These findings concurred with previ - Fig.  4b) and HCT 116 cells (0.8% ± 0.1% vs. 5.5% ± 0.2%, ous observations showing GEP overexpression in differ - P < 0.01, Fig. 4b). ent human cancers including gliomas, renal, prostatic As proliferation-inhibition phenotypes were observed and hepatocellular carcinomas [25–28], and the correla- in siGEP groups, the associated cell cycle regulators tion with poor survival [29]. Moreover, GEP expression and apoptosis markers were analyzed by western blot. was also positively associated with the MSI/CIMP sub- Cyclin D1 was decreased in GEP knockdown cells, sup- types of CRC in the TCGA cohort and patients with this porting the G0/G1-phase cell cycle arrest (Fig.  4c). Acti- kind of subtype had a very poor survival rate after relapse vation of cleaved Caspase 3, 8 and cleaved PARP were [30, 31] (Additional file  5). More importantly, increased observed after silencing GEP expression (Fig. 4c), indicat- DNA copy number of GEP could be detected in tumor ing that GEP inhibited apoptosis via a caspase-dependent samples in TCGA and Kurashina colon statistics from pathway. Furthermore, luciferase reporter activity also ONCOMINE (Additional file  6), indicating a strong pos- showed GEP knockdown significantly suppressed cell sibility that overexpression of GEP was caused by copy cycle pathway in DLD-1and HCT 116 cells (Fig. 4d), con- number change from DNA level. firming the flow cytometry and western blot result for We further investigated the underlying mechanism of cell cycle analysis. Besides, GEP expression was positively upregulating GEP on CRC. A series of in vitro and in vivo associated with CyclinD1 expression from RNA level in experiments confirmed GEP as an oncogenic factor in TCGA database (Fig. 4e). CRC. Notably, a decreased GEP level by RNA interference decreased the cell proliferation rate by MTT and colony- GEP promotes carcinogenesis via MAPK/ERK pathway forming ability in the anchorage-dependent environment in CRCs and decelerating xenograft formation in nude mice by dis- To further gain insights into the downstream signal- turbing cell cycle and apoptosis process and targeting the ing pathways modulated by GEP in CRC tumorigene- MAPK/ERK pathway. Thus, GEP played a crucial role in sis, we examined the functional effect of GEP in several carcinogenesis of CRC. GEP expression has been reported important cancer pathways including p53, TGFβ, Myc, in other aggressive tumors [26, 29] with a promoting role Hypoxia, MAPK/ERK, NF-κB and Wnt by luciferase in cell growth regulation [32, 33], wound-healing process reporter activity assay. GEP knockdown significantly [34, 35], and murine development [36]. It has been previ- suppressed MAPK/ERK luciferase reporter activity in ously shown that GEP binds with TNFR1/2 and diminish DLD-1 and HCT 116 cells (Fig.  5a). Western blot results TNF-dependent activation of MAPK/ERK by altering the (See figure on next page.) 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) Pan et al. J Transl Med (2018) 16:150 Page 9 of 12 Pan et al. J Transl Med (2018) 16:150 Page 10 of 12 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) TNF/TNFR interaction [12]. Then it triggered activation of apoptosis is one of the major causative factors in tumo- the MAPK/ERK and PI3K/AKT/PKB signaling cascades as rigenesis and caspase activation has been considered well as focal adhesion kinase in the adhesion/motility path- a hallmark of apoptosis [40]. In this study, we demon- way in cell lines derived from adrenal, breast, cervical, and strated that knockdown of GEP induced cell apoptosis bladder cancer cells, as well as in modified mouse embryo with caspase activation, suggesting that GEP modulated fibroblasts [16, 37–39]. Therefore, control of CRC growth cell apoptosis via a caspase-dependent pathway. On the by GEP could be also mediated through these pathways. A other hand, p53 was well-documented to play an impor- decreased level of MAPK/ERK phosphorylated form was tant role in inducing cell apoptosis [41], and the inhibition observed in the GEP knockdown group from our study. of p53 may contribute to the anti-apoptotic effect of GEP. Here, we also provided evidence that GEP promoted cell We found that GEP also exhibited the effect of apoptosis migration and invasion in CRC cells in  vitro. A clinical both in p53 wild type and mutated CRC cells, indicating association evaluation showed that overexpression of GEP that GEP promoted tumorigenesis regardless of p53 muta- was associated significantly with advanced AJCC stage, tion status. In keeping with this, the p53 pathway was not suggesting that overexpression of GEP in CRC may facili- significantly affected when GEP knockdown by luciferase tate an invasive and metastatic phenotype. reporter activity assay for p53 pathway analysis. u Th s, GEP GEP enhanced tumor cell survival, which was partly knockdown activated cell apoptosis in a p53 independent attributable to its anti-apoptotic ability. Defective manner. Pan et al. J Transl Med (2018) 16:150 Page 11 of 12 Competing interests Conclusions The authors declare that they have no competing interests. Collectively, our findings demonstrated the oncogenic role of GEP in promoting tumorigenesis and metastasis Availability of data and materials The datasets during and/or analyzed during the current study available from in CRC. These findings suggest that GEP is a crucial fac - the corresponding author on reasonable request. tor in carcinogenesis of colon and rectum, and that GEP has the potential to serve as a prognostic marker and Consent for publication Not applicable. therapeutic target for CRC. Ethics approval and consent to participate Additional files The use of human samples was approved by Joint Chinese University of Hong Kong-New Territories East Cluster Clinical Research Ethics Committee, Hong Kong. Additional file 1. SuperArray SureSilencing shRNA plasmids mediated GEP knockdown. Additional file 2. Clinicopathological factors predicted prognosis for 190 Publisher’s Note CRC patients. Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. Additional file 3. Univariable and multivariable Cox regression of prog- nostic parameters for disease free survival in 190 patients with colorectal Received: 5 March 2018 Accepted: 28 May 2018 cancer. Additional file 4. Univariable and multivariable Cox regression of prognostic parameters for overall survival in 190 patients with colorectal cancer. References Additional file 5. GEP mRNA expression was higher in MSI/CIMP subtype 1. Torre LA, Bray F, Siegel RL, Ferlay J, Lortet-Tieulent J, Jemal A. Global than other subtypes in TCGA cohort. (*P < 0.05). cancer statistics, 2012. CA Cancer J Clin. 2015;65:87–108. Additional file 6. Copy number change of GEP in ONCOMINE colon tis- 2. Punt CJA, Tol J. OPINION More is less-combining targeted therapies in sue datasets. (*P < 0.05; P < 0.01). metastatic colorectal cancer. Nat Rev Clin Oncol. 2009;6:731–3. 3. 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The ability of the and invasion (d) and migration (e) in both DLD-1 and granulin–epithelin precursor/PC-cell-derived growth factor is a growth HCT 116 cells compared to the control group. f Pictures of tumors factor for epithelial ovarian cancer. Clin Cancer Res. 2003;9:44–51. isolated from nude mice at the end of investigation (Left); The tumor 30. Gao JJ, Aksoy BA, Dogrusoz U, Dresdner G, Gross B, Sumer SO, Sun YC, growth was monitored and calculated in the line chart (Middle) and Jacobsen A, Sinha R, Larsson E, et al. Integrative analysis of complex histogram represented mean of the tumor weight (Right) from the cancer genomics and clinical profiles using the cBioPortal. Sci Signaling. shControl and shGEP groups. (*P < 0.05; **P < 0.01) 2013;6:1. Ready to submit your research ? 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Granulin epithelin precursor promotes colorectal carcinogenesis by activating MARK/ERK pathway

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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 clinicopatho- logical 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 com- pared 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 prolifera- tion, 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 dem- onstrated 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, Oncogene, MAPK/ERK pathway *Correspondence: kfto@cuhk.edu.hk; awh_chan@cuhk.edu.hk Department of Anatomical and Cellular Pathology, State Key Laboratory in Oncology in South China, Prince of Wales Hospital, The Chinese University of Hong Kong, 30-32 Ngan Shing Street, Shatin, NT, Hong Kong, SAR, China Full list of author information is available at the end of the article © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/ publi cdoma in/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Pan et al. J Transl Med (2018) 16:150 Page 2 of 12 follow-up. The last update of the database 31 December Background 2015. The study was approved by Committees for Clini - Colorectal cancer (CRC) is one of the major leading cal Research Ethics of Joint Chinese University of Hong causes of cancer-related deaths in the world [1]. Although Kong-New Territories East Cluster. the overall survival of CRC patients has improved, the prognosis of patients with metastasis or recurrence is still relatively poor [2]. Angiogenesis is one of the major hall- Cell lines and cell culture marks of cancer [3], facilitating tumor development, pro- Human CRC cell lines Caco2, DLD-1, HCT 116, HT-29, gression and metastasis [4, 5]. Thus, understanding the LoVo, LS 180, SW480 and SW620 were all obtained molecular mechanism and identifying of novel molecular American Type Culture Collection (ATCC, Manassas, biomarker in angiogenesis could benefit clinical manage - VA, USA). All cell lines were cultured in media according ment of CRC patients. to manufacturer’s instructions. Granulin epithelin precursor (GEP), also known as For transient transfection, DLD-1 or HCT 116 cells progranulin, acrogranin, proepithelin, and GP88/PC-cell were transfected using lipofectamine 2000 (Thermo derived growth factor, is a secreted glycoprotein com- Fisher Scientific, Waltham, MA, USA) by siGEP or posed of 7.5 repeats of cysteine-rich motif [6, 7]. Physi- siRNA control (Qiagen, Hilden, Germany) to interfere ologically, it is expressed in immune cells [8, 9], neurons GEP expression. Procedures of transfection were per- [10], epithelial cells [11] and chondrocytes [12], medi- formed according to the manufacturer’s instructions. ating wound healing, neurodegeneration and cartilage Cells stably expressing downregulated GEP mediated development [7, 10–12]. Pathologically, high expression by shRNA was established using retrovirus system. The levels of GEP are associated with poor prognosis in hepa- shRNA information for GEP knockdown can be found in tocellular carcinoma, ovarian cancer [13], bladder cancer Additional file 1. [14] and glioblastoma [15]. Functional studies reveals GEP acts as a growth factor to stimulate proliferation and Quantitative reverse transcriptase‑polymerase chain migration, and confer chemoresistance in many types of reaction (qRT‑PCR) cancers including breast cancer [16, 17], ovarian cancer Total RNA from cells was extracted by Trizol (Thermo [18, 19], liver cancer [20, 21] and bile duct cancer [22]. Fisher Scientific) according to the manufacturer’s As the expression and functional role of GEP in CRC instruction. cDNAs were synthesized using High-Capac- is unclear. In this study, the clinical significance and ity cDNA Reverse Transcription Kit (Thermo Fisher Sci - the function of GEP in CRC will be comprehensively entific) as protocol. Quantification was performed with revealed. the ABI 7500 Real Time PCR system (Applied Biosys- tems, Foster City, CA, USA). Primers and probes for GEP were GEP-forward (5′-CAA ATG GCC CAC AAC ACT Methods GA-3′), GEP-reverse (5′-CCC TGA GAC GGT AAA Patients and specimens GAT GCA-3′) and GEP-probe (5′-6FAM CCA CTG CTC Between 1999 and 2013, 190 patients undergoing resec- TGC CGG CCA CTC MGBNFQ-3′). Primer and probe tion of primary CRCs at Prince of Wales Hospital, the reagents for control 18s were ready-made reagents (Pre- Chinese University of Hong Kong were recruited in the Developed TaqMan Assay Reagents, Applied Biosys- present study. None of the patients received any neo- tems). All experiments were performed in a minimum of adjuvant therapy. The age of the patients ranged from 34 three replicates. to 92 years, with a median age of 67.4 years. There were 106 men and 84 women. Tumors were staged according Immunohistochemistry (IHC) to the pathological tumor-node-metastasis (pTNM) stag- Immunohistochemistry was performed on 5 µm sections ing system, 7th version. Distribution of the pTNM stages cut from tissue microarray blocks using Dako Envision and other clinicopathological features is listed in Table 1. Plus System (Dako, Carpinteria, CA, USA) following the The patients were regularly followed up according to the manufacturer’s instruction with modifications. Briefly, institutional practice. Disease free survival was defined as antigen retrieval was followed by endogenous peroxidase the period from the date of curative surgery of primary blocking and the following antibody in accordance with tumor to the date that the patient survived without any the manufacturer’s recommendations: GEP [23] (Clone: signs or symptoms of that cancer. Overall survival was A23, 1:800). The cytoplasmic expression of was assessed defined as the period from the date of curative surgery of by using histoscore (H-score) [24] for the staining inten- primary tumor to the date of cancer-related death or last sity and the actual percentage of stained cells in the Pan et al. J Transl Med (2018) 16:150 Page 3 of 12 Table 1 Clinicopathologic correlation of GEP expression in colorectal cancer (n = 190, significant P-value in italic format) GEP expression (n = 190) P‑ value All H‑score ≥ 150 H‑score < 150 98 (51.6%) 92 (48.4%) Male gender 106 (55.8%) 51 (52.0%) 55 (59.8%) 0.283 Age at operation (years, mean ± SD) 67.4 ± 12.1 66.7 ± 12.6 68.2 ± 11.6 0.386 Location 0.032 Right colon 51 (26.8%) 27 (29.0%) 24 (26.4%) Left colon 36 (18.9%) 23 (24.7%) 13 (14.3%) Rectum 88 (46.3%) 36 (38.7%) 52 (57.1%) Synchronous 9 (4.7%) 7 (7.5%) 2 (2.2%) Unknown 6 (3.2%) / / Size (cm, mean ± SD) 4.5 ± 1.7 4.6 ± 1.9 4.3 ± 1.6 0.257 Differentiation Well 2 (1.1%) 0 2 (2.2%) 0.052 Moderate 180 (94.7%) 92 (95.8%) 88 (97.8%) Poor 4 (2.1%) 4 (4.2%) 0 Others 4 (2.1%) / / AJCC stage < 0.01 I 17 (8.9%) 5 (5.1%) 12 (13.0%) II 57 (30.0%) 19 (19.4%) 38 (41.3%) III 56 (29.5%) 27 (27.6%) 29 (31.5%) IV 60 (31.6%) 47 (48.0%) 13 (14.1%) T stage 0.583 T1 5 (2.6%) 3 (3.1%) 2 (2.2%) T2 19 (10.0%) 8 (8.2%) 11 (12.0%) T3 122 (64.2%) 61 (62.2%) 61 (66.3%) T4 44 (23.2%) 26 (26.5%) 18 (19.8%) N stage < 0.01 N0 89 (46.8%) 35 (35.7%) 54 (58.7%) N1 61 (32.1%) 34 (34.7%) 27 (29.3%) N2 40 (21.1%) 29 (29.6%) 11 (12.0%) M stage < 0.01 M0 129 (67.9%) 50 (51.0%) 79 (85.9%) M1 61 (32.1%) 48 (49.0%) 13 (14.1%) Pre-ops CEA (ng/ml, mean ± SD) 142.0 ± 838.4 218.9 ± 1118.1 53.4 ± 256.5 0.194 cytoplasm by two of the investigators. The H-score was dilution). Other primary antibodies were from Cell Sign- obtained by the formula: 3 × percentage of strongly stain- aling (Danvers, MA, USA) commercially including Cyc- ing + 2 × percentage of moderately staining + percentage lin D1 (1:1000, #2926), cleaved PARP (Asp214) (1:1000, of weakly staining, giving a range of 0–300. The stain - #9541), phospho-MAPK/ERK (1:2000, #9106), MAPK/ ing was considered positive when there was moderate or ERK (1:1000, #9102), Caspase-8 (1:1000, #9746), Cleaved strong immunoreactivity over the cutoff point above 150. Caspase-8 (1:1000, #9748), Caspase-3 (1:1000, #9662), Cleaved Caspase-3 (1:1000, #9661). The secondary anti - Western blot analysis bodies were anti-Mouse IgG-HRP (1:15,000, 00049039, Equal total protein was separated by SDS-PAGE and Dako, Denmark) and anti-Rabbit IgG-HRP (1:5000, transferred to nitrocellulose membrane. GEP was 00028856, Dako). The western blot bands were quantified detected with a monoclonal anti-GEP antibody (1:5000 by ImageJ. Pan et al. J Transl Med (2018) 16:150 Page 4 of 12 Cell proliferation assay Flow cytometry analysis Cell proliferation assays were performed using Cells transfected with siGEP or siControl were harvested 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte-trazolium at 24, 48 or 72  h after transfection. Then live cells were bromide (MTT, Sigma-Aldrich, St. Louis, MO, USA) incubated in the mixture of propidium iodide (PI) and assay following the manufactures’ protocol. Briefly, annexin V-fluorescein isothiocyanate (Thermo Fisher CRC cells which have been transfected with siGEP or Scientific) for apoptosis study. Harvested cells were fixed siControl were plated in 96-well plate. Cell viability was and incubated with PI and RNase A at 4 °C for cell cycle measured at 24, 48, 72, 96, and 120 h. Finally, the optical analysis. Finally, cells were determined by FACS Cali- density was determined at 570 and 690  nm wavelength bur Flow Cytometer (Becton, Dickinson and Company, light absorption (Victor3, Perken Elmer, MA, USA). Franklin Lakes, NJ, USA) and CellQuest program. Luciferase reporter assay Monolayer colony formation assay The firefly luciferase construct was co-transfected with Anchorage-dependent growth was assessed by mon- Renilla luciferase vector (Qiagen) as a control into the olayer colony formation. 6-well plates were used at a den- siRNA treated cells. Dual Luciferase Reporter Assay 3 3 sity of 1 × 10 to 5 × 10 for transfected cells. Cells were System (Promega, Fitchburg, Wisconsin, USA) was incubated at 37 °C for 14–21 days until colonies can obvi- employed to check the luciferase activity after 48 h’ trans- ously be observed. Colonies were fixed with methanol fection. The results were expressed as the ratio of firefly for 5  min and stained with 0.5% crystal violet. Colonies luciferase activity to renilla luciferase activity. Experi- with cell numbers of more than 50 cells per colony were ments were repeated in triplicate. counted. The experiments were performed in duplicate wells in three independent experiments. Statistical analysis The statistical analysis was performed using IBM SPSS Statistics (Version 19.0, Armonk, NY, USA). The expres - Invasion and migration assay sion level of GEP in paired non-tumor and tumor tissues Cell invasion or migration activity were performed using was compared with paired Student’s t-test. Independent 24 well biocoat matrigel invasion chambers or sterilized Student’s t-test was used to compare the mean value of transwell insert chambers (Corning, Bedford, MA, USA). any two groups. The Pearson χ test was used to analyze Cells were harvested after transfected with siGEP or the association of target expression with clinicopatho- siControl for 24 h and re-plated with serum free medium logical parameters. Survival curves were drawn using in the upper chamber. The lower chamber was filled with the Kaplan–Meier method and compared by means of culture medium containing 10% FBS as the chemoat- the log-rank test. Univariable and multivariable Cox pro- tractant. After 48 h, the cells that moved to the lower sur- portional hazard regression models were used to analyze face of polycarbonate membrane were stained with 0.5% independent prognostic factors. P < 0.05 was considered crystal violet and counted at five random 200× fields. statistically significant. P < 0.01 was considered highly statistically significant. In vivo tumorigenic assays Results 1 × 10 transfected CRC cells in 100 μl PBS were injected GEP is overexpressed in CRC subcutaneously into the dorsal region of anaesthetized GEP mRNA expression was up-regulated in 6/8 CRC nude mice (5 mice/construct, control in left and treat- cell lines compared with the normal colon epithelium by ment in right). When tumor was formed, tumor diameter qRT-PCR (Fig. 1a). We analyzed GEP expression in cellu- was measured and documented every 3 days for 3 weeks. lar datasets available through ONCOMINE (http://www. At the end of investigation, mice were sacrificed, and oncom ine.org/), an online collection of microarrays. xenografts were collected for diameter check and weigh Using the Ki colon dataset [7], we observed that GEP valuation. The animal handling and all experimental expression was lower in normal colon compared to colon procedures were approved by the Department of Health cancer tissues (Fig.  1b). Using immunohistochemical of Hong Kong and the Animal Experimentation Ethics staining on 190 CRC and 70 normal colon samples, GEP Committee, The Chinese University of Hong Kong. Pan et al. J Transl Med (2018) 16:150 Page 5 of 12 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) protein was predominantly localized in the cytoplasm (P < 0.01), lymph node involvement (P < 0.01) and distant and most of the CRC cases demonstrated abundant GEP metastasis status (P < 0.01). Specifically, a high GEP pro - protein compared with non-tumorous tissues (Fig.  1c). tein level more frequently occurred in tumors located High expression (H-score ≥ 150) of GEP protein was on colon than that on rectum (P  <  0.05, Fig.  2a). High detected in 51.6% (98/190) of tumor tissues. Whereas, GEP expression was also more commonly happened in only 4.3% (3/70) of normal tissues were found high GEP patients with advanced AJCC stage (Stage III/IV, P < 0.01, expression (P < 0.01, Fig. 1d). Fig.  2b), including the presence of lymphatic (P < 0.01, Fig. 2c) and distant metastasis (P < 0.01, Table 1). High GEP expression was associated with shorter dis- High GEP expression correlates with poor survival in CRC ease-free survival and overall survival (P < 0.01, Fig.  2d, patients e). Other Clinicopathological parameters such as right- The correlation of GEP with clinicopathologic param - side colon, poor differentiation, tumor invades adjacent eters in CRC patients was summarized in Table  1. GEP organs or perforates the visceral peritoneum (T4), lymph expression varied significantly among CRC samples node involvement (N1/2), distant metastasis (M1), and with different tumor locations (P = 0.032), AJCC stages Pan et al. J Transl Med (2018) 16:150 Page 6 of 12 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) high CEA level before operation (> 10  ng/ml) also pre- the invasion (Fig.  3d) and migration (Fig.  3e) in both dicted worse disease-free survival and overall survival DLD-1 and HCT 116 cells compared to the control (Additional file  2). By multivariable Cox regression, com- group. These observations suggested that GEP had onco - pared with those classic prognostic parameters, high genic properties. GEP expression was not an independent prognosticator To determine the anti-tumor effect of shGEP in  vivo, for patients’ disease-free (Additional file  3) and overall DLD-1 cells stably expressing GEP shRNA was estab- survival (Additional file 4). lished using retrovirus system. shControl and shGEP were subcutaneously injected into the left and right GEP exerts an oncogenic function on CRC cells flanks of nude mice respectively. The shGEP group In DLD-1 and HCT 116 cell lines with endogenous high formed smaller tumor within 27  days (Fig.  3f ). In con- GEP expression, siRNA-mediated knockdown reduced trast to shControl injected mice, the mice injected GEP expression at mRNA (P < 0.01, Fig. 3a). siGEP treat- with shGEP exhibited greatly reduced mean tumor size ments significantly decreased cell proliferation (Fig.  3b), (P < 0.05) and mean tumor weight (P < 0.01) in xenograft anchorage-dependent growth (Fig.  3c), and abilities of model (Fig. 3f ). (See figure on next page.) 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) Pan et al. J Transl Med (2018) 16:150 Page 7 of 12 Pan et al. J Transl Med (2018) 16:150 Page 8 of 12 GEP knockdown results in G1 arrest and increased showed significant suppression of phosphop-MAPK/ERK apoptosis in CRC was observed in the siGEP treated CRC tumors (Fig. 5b), Since a growth inhibitory effect was involved in siGEP indicating that the MAPK/ERK signaling pathway was transfected cells, we further explored the molecular basis essential for anti-GEP-mediated growth inhibition in involved in siGEP-suppressed tumor cell growth. We CRC cells. GEP led to MAPK/ERK phosphorylation and analyzed the transfectants for cell cycle parameters and translocate into the nucleus, which stimulated cell prolif- apoptosis using flow cytometry. 24  h after transfection, eration, cell survival and metastasis of CRC (Fig. 5c). accumulation of G1 cells increased in siGEP transfect- ant compared with siControls (57.8% vs. 44.8% in DLD- Discussion 1; 52.6% vs. 36.2% in HCT 116 cells), while S-phase cell In this study, we characterized clinicopathological fea- percentage decreased after siGEP transfection (11.6% vs. tures of GEP expression and delineated its oncogenic 16.8% in DLD-1; 23.8% vs. 27.3% in HCT 116 cells) in function in colorectal carcinogenesis. these 2 cell lines (Fig. 4a). For apoptosis analysis, the per- GEP was overexpressed in CRC cell lines and patients’ centage of early apoptotic cells in siGEP treated cells was tumor samples. Overexpression of GEP in CRC was significantly increased compared to the siControl cells associated with nodal and distant metastasis and poorer in both DLD-1 (0.8%  ±  0.1% vs. 4.2% ± 0.2%, P < 0.01, clinical outcome. These findings concurred with previ - Fig.  4b) and HCT 116 cells (0.8% ± 0.1% vs. 5.5% ± 0.2%, ous observations showing GEP overexpression in differ - P < 0.01, Fig. 4b). ent human cancers including gliomas, renal, prostatic As proliferation-inhibition phenotypes were observed and hepatocellular carcinomas [25–28], and the correla- in siGEP groups, the associated cell cycle regulators tion with poor survival [29]. Moreover, GEP expression and apoptosis markers were analyzed by western blot. was also positively associated with the MSI/CIMP sub- Cyclin D1 was decreased in GEP knockdown cells, sup- types of CRC in the TCGA cohort and patients with this porting the G0/G1-phase cell cycle arrest (Fig.  4c). Acti- kind of subtype had a very poor survival rate after relapse vation of cleaved Caspase 3, 8 and cleaved PARP were [30, 31] (Additional file  5). More importantly, increased observed after silencing GEP expression (Fig. 4c), indicat- DNA copy number of GEP could be detected in tumor ing that GEP inhibited apoptosis via a caspase-dependent samples in TCGA and Kurashina colon statistics from pathway. Furthermore, luciferase reporter activity also ONCOMINE (Additional file  6), indicating a strong pos- showed GEP knockdown significantly suppressed cell sibility that overexpression of GEP was caused by copy cycle pathway in DLD-1and HCT 116 cells (Fig. 4d), con- number change from DNA level. firming the flow cytometry and western blot result for We further investigated the underlying mechanism of cell cycle analysis. Besides, GEP expression was positively upregulating GEP on CRC. A series of in vitro and in vivo associated with CyclinD1 expression from RNA level in experiments confirmed GEP as an oncogenic factor in TCGA database (Fig. 4e). CRC. Notably, a decreased GEP level by RNA interference decreased the cell proliferation rate by MTT and colony- GEP promotes carcinogenesis via MAPK/ERK pathway forming ability in the anchorage-dependent environment in CRCs and decelerating xenograft formation in nude mice by dis- To further gain insights into the downstream signal- turbing cell cycle and apoptosis process and targeting the ing pathways modulated by GEP in CRC tumorigene- MAPK/ERK pathway. Thus, GEP played a crucial role in sis, we examined the functional effect of GEP in several carcinogenesis of CRC. GEP expression has been reported important cancer pathways including p53, TGFβ, Myc, in other aggressive tumors [26, 29] with a promoting role Hypoxia, MAPK/ERK, NF-κB and Wnt by luciferase in cell growth regulation [32, 33], wound-healing process reporter activity assay. GEP knockdown significantly [34, 35], and murine development [36]. It has been previ- suppressed MAPK/ERK luciferase reporter activity in ously shown that GEP binds with TNFR1/2 and diminish DLD-1 and HCT 116 cells (Fig.  5a). Western blot results TNF-dependent activation of MAPK/ERK by altering the (See figure on next page.) 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) Pan et al. J Transl Med (2018) 16:150 Page 9 of 12 Pan et al. J Transl Med (2018) 16:150 Page 10 of 12 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) TNF/TNFR interaction [12]. Then it triggered activation of apoptosis is one of the major causative factors in tumo- the MAPK/ERK and PI3K/AKT/PKB signaling cascades as rigenesis and caspase activation has been considered well as focal adhesion kinase in the adhesion/motility path- a hallmark of apoptosis [40]. In this study, we demon- way in cell lines derived from adrenal, breast, cervical, and strated that knockdown of GEP induced cell apoptosis bladder cancer cells, as well as in modified mouse embryo with caspase activation, suggesting that GEP modulated fibroblasts [16, 37–39]. Therefore, control of CRC growth cell apoptosis via a caspase-dependent pathway. On the by GEP could be also mediated through these pathways. A other hand, p53 was well-documented to play an impor- decreased level of MAPK/ERK phosphorylated form was tant role in inducing cell apoptosis [41], and the inhibition observed in the GEP knockdown group from our study. of p53 may contribute to the anti-apoptotic effect of GEP. Here, we also provided evidence that GEP promoted cell We found that GEP also exhibited the effect of apoptosis migration and invasion in CRC cells in  vitro. A clinical both in p53 wild type and mutated CRC cells, indicating association evaluation showed that overexpression of GEP that GEP promoted tumorigenesis regardless of p53 muta- was associated significantly with advanced AJCC stage, tion status. In keeping with this, the p53 pathway was not suggesting that overexpression of GEP in CRC may facili- significantly affected when GEP knockdown by luciferase tate an invasive and metastatic phenotype. reporter activity assay for p53 pathway analysis. u Th s, GEP GEP enhanced tumor cell survival, which was partly knockdown activated cell apoptosis in a p53 independent attributable to its anti-apoptotic ability. Defective manner. Pan et al. J Transl Med (2018) 16:150 Page 11 of 12 Competing interests Conclusions The authors declare that they have no competing interests. Collectively, our findings demonstrated the oncogenic role of GEP in promoting tumorigenesis and metastasis Availability of data and materials The datasets during and/or analyzed during the current study available from in CRC. These findings suggest that GEP is a crucial fac - the corresponding author on reasonable request. tor in carcinogenesis of colon and rectum, and that GEP has the potential to serve as a prognostic marker and Consent for publication Not applicable. therapeutic target for CRC. Ethics approval and consent to participate Additional files The use of human samples was approved by Joint Chinese University of Hong Kong-New Territories East Cluster Clinical Research Ethics Committee, Hong Kong. Additional file 1. SuperArray SureSilencing shRNA plasmids mediated GEP knockdown. Additional file 2. Clinicopathological factors predicted prognosis for 190 Publisher’s Note CRC patients. Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. Additional file 3. Univariable and multivariable Cox regression of prog- nostic parameters for disease free survival in 190 patients with colorectal Received: 5 March 2018 Accepted: 28 May 2018 cancer. Additional file 4. Univariable and multivariable Cox regression of prognostic parameters for overall survival in 190 patients with colorectal cancer. References Additional file 5. GEP mRNA expression was higher in MSI/CIMP subtype 1. Torre LA, Bray F, Siegel RL, Ferlay J, Lortet-Tieulent J, Jemal A. Global than other subtypes in TCGA cohort. (*P < 0.05). cancer statistics, 2012. CA Cancer J Clin. 2015;65:87–108. Additional file 6. Copy number change of GEP in ONCOMINE colon tis- 2. Punt CJA, Tol J. OPINION More is less-combining targeted therapies in sue datasets. (*P < 0.05; P < 0.01). metastatic colorectal cancer. Nat Rev Clin Oncol. 2009;6:731–3. 3. 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Choose BMC and benefit from: fast, convenient online submission thorough peer review by experienced researchers in your field rapid publication on acceptance support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year At BMC, research is always in progress. Learn more biomedcentral.com/submissions

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Journal of Translational MedicineSpringer Journals

Published: Jun 4, 2018

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