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RGC-32 induces transition of pancreatic cancer to epithelial mesenchyme in vivo
Pancreatology xxx (2018) 1e5
Contents lists available at ScienceDirect
Pancreatology journal homepage: www.elsevier.com/locate/pan
RGC-32 induces transition of pancreatic cancer to epithelial mesenchyme in vivo Liang Zhu a, *, 1, Ying Ding b, 1 a b
Department of Gastroenterology, The First Affiliated Hospital of Nanchang University, Nanchang, 330006, Jiangxi, China Department of Plastic and Cosmetic Surgery, The Second Affiliated Hospital of Nanchang University, Nanchang, 330006, Jiangxi, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 14 March 2018 Received in revised form 15 May 2018 Accepted 17 May 2018 Available online xxx
Objectives: This study was undertaken to investigate the induction of transition of pancreatic cancer to epithelial mesenchyme by RGC-32. Methods: Primary human pancreatic cancer cell line BXPC-3 was transfected with lentivirus overexpressing the response gene to complement-32 gene (RGC-32) and used to induce tumor in mice. The tumor sizes were measured and the expression of cytokeratin, e-cadherin and vimentin at mRNA using real time PCR and at protein levels by Western blot. Results: Compared with the control, mice inoculated with the cells transfected with empty vector had similar tumor size while those inoculated with the cells transfected with RGC-32 expressing virus had significantly greater tumor size. HE staining showed that tumors were formed in all treatments. Molecular analyses showed that there was no difference in the expression of the cytokeratin, e-cadherin and vimentin genes at mRNA and protein levels between control and empty vector groups. However, mice derived from cells transfected with RGC-32 expressing virus had reduced cytokeratin and e-cadherin expression and increased vimentin expression. Conclusions: These data suggest that RGC-32 promotes the proliferation of pancreatic cancer and induces the epithelialemesenchymal transition (EMT). It would be a future direction of research to investigate the regulatory mechanism of signal molecules downstream RGC-32 on EMT-related transcription factors and deliberate the role of RGC-32 in tumorigenicity. As a result, RGC-32 may become a new therapeutic target for cancers. © 2018 IAP and EPC. Published by Elsevier B.V. All rights reserved.
Keywords: RGC-32 Pancreatic cancer BXPC-3 Epithelial mesenchymal transition
1. Introduction Pancreatic cancer is a highly malignant and early metastatic digestive tract tumor with very poor prognosis. It is difficult to be diagnosed early. Most of the diagnosed patients already have local invasion and/or distant metastasis and have lose the best chance of treatment. As a consequence, the five year survival rate is less than 5% [1]. Therefore, a better understanding of the mechanism underlying the invasion and metastasis of the cancer is of great significance for controlling its progression and prolonging the survival time of pancreatic cancer patients. Epithelial mesenchymal transition (EMT) refers to a process by
* Corresponding author. Department of Gastroenterology, The First Affiliated Hospital of Nanchang University, 17 Yongwaizhen Street, Nanchang, Jiangxi 330006, China. E-mail address: [email protected] (L. Zhu). 1 These authors contributed equally to this work.
which epithelial cells lose their cell polarity and cell-cell adhesion, and gain migratory and invasive properties to become mesenchymal stem cells under certain physiological and pathological circumstances [2]. At the same time, the phenotype of the cells also changes, resulting in the gradual disappearance of epithelial phenotype markers such as E-cadherin and cytokeratins and the enhancement of mesenchymal markers such as vimentin and Ncadherin [3]. More and more studies have shown that EMT plays an important role in the progression of tumor. Tumor cells enhance their migration and invasion through EMT to become malignant [4]. Therefore, EMT has been considered as an important pathological process that promotes the invasion and metastasis of tumor [2]. The response gene to complement-32 (RGC-32) was first cloned in 1998 as response gene induced by complement activation [5], which is involved in the regulation of cell cycle and differentiation [6]. It is found that RGC-32 is expressed in a variety of normal tissues including the pancreas [7]. A number of studies have reported the role of RGC-32 in tumor [8] and found that RGC-32 is
https://doi.org/10.1016/j.pan.2018.05.480 1424-3903/© 2018 IAP and EPC. Published by Elsevier B.V. All rights reserved.
Please cite this article in press as: Zhu L, Ding Y, RGC-32 induces transition of pancreatic cancer to epithelial mesenchyme in vivo, Pancreatology (2018), https://doi.org/10.1016/j.pan.2018.05.480
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abnormally expressed in human cancers but differently in different organs [6,9e11], suggesting that the expression may be organ- and tissue-specific. However, little is known about the role of RGC-32 in pancreatic cancer. In our preliminary experiments, we have found that the expression of RGC-32 is significantly more deregulated in pancreatic cancer tissue than in chronic pancreatitis and normal pancreatic tissues, and is related to the tumor, node, metastasis (TNM) staging (P < 0.05), the pancreatic cancer positive rate and Ecadherin abnormal expression rate (P < 0.05) [12]. Furthermore, we have shown that RGC-32 has impact on hypoxia-induced EMT, and the possible link between HIF-1 and EMT may have therapeutic potential for pancreatic cancer [12]. Therefore, we hypothesized that RGC-32 may be one of metastasis-promoting genes in pancreatic cancer and may be involved in the invasion and metastasis of pancreatic cancer via EMT process. To better understand the role of the RGC-32 gene in pancreatic cancer, we investigated the expression of genes in the TGF-a and HIF-a pathways and the induction and mechanism of EMT in pancreatic cancer. The results would provide new insights into molecular targets for gene therapy of pancreatic cancer.
Once the solid tumors grew to 1 cm3 in size, they were isolated and cut into pieces of 1 mm3 in size and grafted subcutaneously to neck back of the nude mice to obtain xenotransplanted tumor models. Six animals were used in each treatment. The volumes (V) of tumor were measured every 3 days based on the length, width and height (V ¼ 0.5 x L x W x H). At the end of experiments, mice were scarified by cervical dislocation and tumors were isolated for subsequent analysis. 2.5. Immunohistochemistry
Human pancreatic cancer cell line BXPC-3 (cat no. BNCC338477) was purchased from Yingniurui Biotech, Wuxi, China.
10 mm transverse sections of tumor tissue were incubated with antibodies against E-cadherin (rabbit anti- E-cadherin, cat no. ab40772, 1:1000, Abcam, USA), vimentin (rabbit anti-vimentin, cat no. ab92574 1:500, Abcam) and cytokeratin (rabbit anticytokeratin, cat no. ab181597, 1:200, Abcam) for 2 h at room temperature followed by incubation with appropriate secondary antibody (goat anti-mouse IgG, cat. no. ZB-2305, ZSGB-Bio, Beijing) at 37 C for 2 h, and mounted with ProLong antifade reagent (Invitrogen). Each antibody series was performed on all tissue sections simultaneously to control for variations in processing and allow quantitative comparisons of staining intensity. The slides were reacted to DAB (diaminobenzidine) chromogenic solution at room temperature for 3 min before microscopy study. Images for a single antibody series were acquired at the same fluorescence intensity to keep imaging conditions constant and allow quantitative comparisons of staining intensity.
2.2. Reagents and instruments
2.6. Real-time quantitative PCR for mRNA expression
DMEM high glucose medium (lot no. KGM12800S-500) was purchased from Sijiqing Biologicals, Hangzhou, China; DMSO (lot no. 302A0325) was purchased from Amresco, USA; 0.25% trypsin (containing EDTA, lot no. 20160818) was obtained from KGI Biologicals, Jiangsu, China; fetal bovine serum (lot no. 1552680) was purchased from BI Technologies, USA; Transwell was obtained from FALCON, USA; 2xGoldStar Taq MasterMix and 2xULtraSYBR Mixture were obtained from Keyene, Beijing, China; Goldview I nucleic acid stain (cat no. G8140) was purchased from Solarbio, USA; ultrasensitive chemiluminescence imaging system (ChemiDocXRSþ) and quantitative PCR instrument (CFX Connect) were purchased from BioRad, USA.
Total RNA was isolated from the pancreatic cancer cells using the Trizol reagent according to the supplier's instruction (Invitrogen, USA) and reversely transcripted into cDNA using the RNA reverse transcription kit (Applied Biosystems by Life Technologies, Carlsbad, California, USA). RT-qPCR was performed using TaqMan RNA Assays (Applied Biosystems) in a total volume of 50 ml containing 4 ml of cDNA, 25 ml of TaqMan Gene Expression Master Mix and 1 ml of each fluorescence probe. The cycling conditions were 95 C for 30 s followed by 40 cycles, each one consisting of 95 C for 30s, 95 C for 5s, 60 C for 30s. Samples were run in five replicate and the mean value was calculated for each case. Primer sequences for RGC-32 were forward: 50 -TCAACCTTCTACCAGGCCACTC-3'; reverse: 50 -GCAAGCAGGTAAACAAAGTCAG-3'. GAPDH (forward: 50 AGGTCGGTGTGAACGGATTTG-30 , reverse: 50 -GGGGTCGTTGATGGCAACA-30 ) was used as internal control. The data were managed according to previously described protocol [13].
2. Materials and methods 2.1. Cell line
2.3. Construction of Ad-RGC-32 Wild type RGC-32 cDNA was inserted to KpnI and NotI digested adenovirus shuttle vector pAdTrack-CMV to obtain pAdTrack-CMVRGC-32. The plasmid was co-transformed with adenovirus backbone plasmid pAdEasy-1 into Escherichia coli DH5a cells to generate recombinant vector Ad-RGC-32 harboring the RGC-32 gene via homologous recombination in E. coli cells. The adenovirus vector was transfected into the human embryonic kidney 293 (HEK293, Sigma, USA) cells to produce recombinant adenovirus Ad-RGC-32. The virus particles were examined by fluorescence microscopy and tittered for TCID50 (tissue culture infectious dose 50%/mL). The negative control (empty vector) was produced similarly using pAdTrack-CMV and pAdEasy-1.
2.7. Western blot analysis Cells were washed twice with cold PBS and lysed with RIPA buffer that contains protease and phosphotase inhibitors cocktail (Roche, UK). The supernatants were collected after centrifugation at 12000 rpm for 20 min. 50mg/lane protein was applied to polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a PVDF membrane, and then detected by the proper primary and secondary antibodies before visualization with a chemiluminescence kit. The intensity of blot signals was quantitated using ImageQuant TL analysis software (General Electric, UK).
2.4. Tumor graft model 2.8. H & E staining BxPC-3 cells were transfected with lentivirus expressing RGC-32 or harboring empty vector and grown to a confluence of 80%e90% at 37 C in 5% CO2 in DMEM medium and the cell suspensions (3 106 tumor cells/mouse) were injected subcutaneously into the anterior neck of 4 week-old nude mice (Slack Inc, Hunan, China).
HE staining was preformed as previously described [14]. Briefly, paraffin sections were dewaxed, dehydrated, washed in PBS and dropped with hematoxylin to stain for 5 min. The stained slides were counterstained with eosin for 2 min after washed with PBS
Please cite this article in press as: Zhu L, Ding Y, RGC-32 induces transition of pancreatic cancer to epithelial mesenchyme in vivo, Pancreatology (2018), https://doi.org/10.1016/j.pan.2018.05.480
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and mounted with neutral balata before microscopy study. 2.9. Statistical analysis All data were expressed as means ± standard error of the mean (SEM) obtained from at least three independent experiments. Means were compared using the student's t-test or one-way ANOVA with the LSD test using SPSS 20 software. A p-value 0.05 was considered statistically significant. 3. Results Fig. 1. Morphology and weight of tumors in mice after grafted with pancreatic cancer cells. The cells were either un-transfected (control) or transfected with empty vector (negative vector) or RGC-32 expression virus (RGC-32). Upper pane: tumor morphology; low pane: tumor weight. * denotes P < 0.05 vs control.
3.1. RGC-32 increased the tumor size As shown in Fig. 1, the volumes of tumor derived from cells transfected with virus expressing RGC-32 were significantly greater
Fig. 2. Expression of EMT-related genes in tumors in mice after grafted with pancreatic cancer cells. The cells were either un-transfected (control) or transfected with empty vector (negative vector) or RGC-32 expression virus (RGC-32). A. mRNA level; B. left panel: representative Western blot, right pane: relative protein level. * denotes P < 0.05 vs control.
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those of control or transfected with empty vector (p < 0.05), while the tumor volumes were similar between control and empty vector groups. 3.2. RGC-32 regulated the expression of EMT-related genes qRT-PCR analysis showed that the mRNA levels of cytokeratin and E-cadherin were significantly down-regulated, and the mRNA level of vimentin was significantly up-regulated in the tumor derived from the cells overexpressing RGC-32 (Fig. 2A, p < 0.05), as compared with control and empty-vector groups. In the latter two groups, the expression levels of these genes were similar (Fig. 2A). Similar results were obtained at the protein levels (Fig. 2B). These data suggest that the overexpression of RGC-32 may result in increased expression of EMT-related genes in pancreatic cancer. 3.3. HE staining and immunohistochemistry results HE staining showed that there were tumor tissues in all mice (Fig. 3A). Immunohistochemically, there were significantly more vimentin-positive cells and less cytokeratin- and E-cadherin-positive cells in the tumors derived from the cells overexpressing RGC32 (Fig. 3B), than in control and empty vector groups. For the latter two groups, no difference was observed in the expression level of these genes (Fig. 3B). These results further demonstrate that the overexpression of RGC-32 may result in increased expression of EMT-related genes in pancreatic cancer. 3.4. Discussion In recent years, the incidence of pancreatic ductal adenocarcinomas (PDAC) has being increasing obviously in China. It now accounts for 2.2% of the total malignant tumors. However, due to the difficulty of early diagnosis and high degree of malignancy, the 5
Fig. 3. HE and immunohistochemistry assays of tumor tissues in mice after grafted with pancreatic cancer cells. The cells were either un-transfected (control) or transfected with empty vector (negative vector) or RGC-32 expression virus (RGC-32). A: HE staining; B: percentage of positive cells. * denotes P < 0.05 vs control.
year survival rate is still less than 5%, and has not been improved in the past 40 years. Therefore, it is urgent to study the pathogenesis, prevention and treatment of the cancer. The RGC-32 gene, also known as C13orf15 and response gene to complement 32, is an important complement response gene. It is located in the human chromosome 13q14.11 [15] and widely expressed in many tissues, regulating cell cycle and immune response, promoting cell proliferation and differentiation [8,16]. Recent studies show that it is highly expressed in malignant tumors, such as colorectal cancer, pancreatic cancer, and may be involved in the regulation of tumor proliferation and invasion as oncogene [17,18]. In this study, we show that the overexpression of the RGC-32 gene significantly increases the tumor volume, demonstrating that RGC-32 could promote tumor growth. This is consistent with the previous studies [17,18]. EMT has been shown to be an important pathological process that promotes tumor invasion and metastasis. Tumor cells can enhance their mobility and invasiveness through EMT process, thus becoming malignant [19]. A number of signaling pathways are involved in the EMT in PDAC, among which the most important is the TGF-b signaling pathway. Several studies have shown that TGFb-induced EMT is one of the important factors that promote the invasion and metastasis of PDAC [20]. In 41.4% of PDAC tissues, the expression of TGF-b was increased, and the serum level of TGF- b was found to be negatively correlated with the prognosis of PDAC patients [21]. Zhu et al. showed that the positive rates of cell expressing RGC32 and E-cadherin were very higher in pancreatic cancer with lymph node metastasis or at high TNM stage [22]. E-cadherin is not only closely related to the progression of pancreatic cancer, but also plays an important role in the development of esophageal squamous cell carcinoma and gastric adenocarcinoma [23]. Using the pancreatic cancer cell line BXPC-3 with homozygous deletion of Smad4, it was shown that sustained stimulation of TGF-b induces EMT in BXPC-3 cells and up-regulates the expression of RGC-32 [12]. RGC-32 not only mediates the TGF- induced EMT process via at least ERK-MAPK and p38-MAPK signaling pathways, but also induces EMT independently, which is not dependent on the Smad signaling pathway [24]. In addition, RGC-32 was shown to be involved in the regulation of TGF-b-induced cell migration, and inhibit the proliferation but not apoptosis of BXPC-3 cells [25]. These results further confirm that RGC-32 enhances the transformation of the metastatic phenotype of pancreatic cancer. Furthermore, a number of recent studies have shown that RGC-32 not only plays an important role in the EMT process in tumor cells, but also in normal cells [26]. For example, Kurahara et al. found that RGC-32 mediates TGF-b-induced EMT process in human proximal tubular epithelial cells (HPTC) [26]. Our study shows that the overexpression of RGC-32 resulted in significant downregulation of cytokeratin and epithelial marker protein E-cadherin and significant up-regulation of mesenchymal marker protein vimentin, suggesting that RGC-32 promotes tumor metastasis. So far, the role of RGC-32 in cancer has not been fully elucidated and could be very complicated. For example, in colorectal and pancreatic cancer, RGC-32 is an oncogene while in glioma, it appears to be antiandrogenic [9]. Therefore, further studies are needed to further identify and deliberate the signal transduction pathways and mechanism by which RGC-32 participates in oncogenesis. It is likely that RGC-32 is one of the key molecules mediating the dual roles of TGF-b. Exploring the regulatory mechanism of signaling molecules downstream RGC-32 on EMT-related transcription factors may become a hot research topic. In addition, further investigations are needed to understand whether RGC-32 participates in more oncogenesis processes and its roles in these
Please cite this article in press as: Zhu L, Ding Y, RGC-32 induces transition of pancreatic cancer to epithelial mesenchyme in vivo, Pancreatology (2018), https://doi.org/10.1016/j.pan.2018.05.480
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processes. The elucidation would result in the identification of new therapeutic target for the cancer. Declaration of conflict of interest None. Acknowledgement This work was supported by the Natural Science Foundation of Jiangxi Province, China (The grant number is 20171BAB215044), National Natural Science Foundation of China (The grant number is 81302152) and Key Project of Educational Commission of Jiangxi Province of China (The grant number is GJJ170006). References [1] Hidalgo M. Pancreatic cancer. N Engl J Med 2010;362:1605e17. [2] Polyak K, Weinberg RA. Transitions between epithelial and mesenchymal states: acquisition of malignant and stem cell traits. Nat Rev Canc 2009;9: 265e73. [3] Thiery JP, Acloque H, Huang RY, Nieto MA. Epithelial-mesenchymal transitions in development and disease. Cell 2009;139:871e90. [4] Thiery JP. Epithelial-mesenchymal transitions in tumour progression. Nat Rev Canc 2002;2:442e54. [5] Badea TC, Niculescu FI, Soane L, Shin ML, Rus H. Molecular cloning and characterization of rgc-32, a novel gene induced by complement activation in oligodendrocytes. J Biol Chem 1998;273:26977e81. [6] Saigusa K, Imoto I, Tanikawa C, Aoyagi M, Ohno K, Nakamura Y, et al. Rgc32, a novel p53-inducible gene, is located on centrosomes during mitosis and results in g2/m arrest. Oncogene 2007;26:1110e21. [7] Badea T, Niculescu F, Soane L, Fosbrink M, Sorana H, Rus V, et al. Rgc-32 increases p34cdc2 kinase activity and entry of aortic smooth muscle cells into sphase. J Biol Chem 2002;277:502e8. [8] Vlaicu SI, Tegla CA, Cudrici CD, Danoff J, Madani H, Sugarman A, et al. Role of c5b-9 complement complex and response gene to complement-32 (rgc-32) in cancer. Immunol Res 2013;56:109e21. [9] Fosbrink M, Cudrici C, Niculescu F, Badea TC, David S, Shamsuddin A, et al. Overexpression of rgc-32 in colon cancer and other tumors. Exp Mol Pathol 2005;78:116e22. [10] Kang Y, Siegel PM, Shu W, Drobnjak M, Kakonen SM, Cordon-Cardo C, et al. A multigenic program mediating breast cancer metastasis to bone. Canc Cell
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2003;3:537e49. [11] Chandran UR, Ma C, Dhir R, Bisceglia M, Lyons-Weiler M, Liang W, et al. Gene expression profiles of prostate cancer reveal involvement of multiple molecular pathways in the metastatic process. BMC Canc 2007;7:64. [12] Zhu L, Zhao Q. Hypoxia-inducible factor 1alpha participates in hypoxiainduced epithelial-mesenchymal transition via response gene to complement 32. Exp Ther Med 2017;14:1825e31. [13] Livak KJ, Schmittgen TD. Analysis of relative gene expression data using realtime quantitative pcr and the 2(-delta delta c(t)) method. Methods 2001;25: 402e8. [14] Fischer AH, Jacobson KA, Rose J, Zeller R. Hematoxylin and eosin staining of tissue and cell sections. Cold Spring Harb. Protoc. 2008;2008. pdb prot4986. [15] Vlaicu SI, Cudrici C, Ito T, Fosbrink M, Tegla CA, Rus V, et al. Role of response gene to complement 32 in diseases. Arch Immunol Ther Exp 2008;56:115e22. [16] Wang QJ, Song BF, Zhang YH, Ma YY, Shao QQ, Liu J, et al. Expression of rgc32 in human normal and preeclamptic placentas and its role in trophoblast cell invasion and migration. Placenta 2015;36:350e6. [17] Lu Y, Hu XB. C5a stimulates the proliferation of breast cancer cells via aktdependent rgc-32 gene activation. Oncol Rep 2014;32:2817e23. [18] Vlaicu SI, Tegla CA, Cudrici CD, Fosbrink M, Nguyen V, Azimzadeh P, et al. Epigenetic modifications induced by rgc-32 in colon cancer. Exp Mol Pathol 2010;88:67e76. [19] Vaquero J, Guedj N, Claperon A. Nguyen Ho-Bouldoires TH, Paradis V, Fouassier L: epithelial-mesenchymal transition in cholangiocarcinoma: from clinical evidence to regulatory networks. J Hepatol 2017;66:424e41. [20] Pang MF, Georgoudaki AM, Lambut L, Johansson J, Tabor V, Hagikura K, et al. Tgf-beta1-induced emt promotes targeted migration of breast cancer cells through the lymphatic system by the activation of ccr7/ccl21-mediated chemotaxis. Oncogene 2016;35:748e60. [21] Kabashima A, Higuchi H, Takaishi H, Matsuzaki Y, Suzuki S, Izumiya M, et al. Side population of pancreatic cancer cells predominates in tgf-beta-mediated epithelial to mesenchymal transition and invasion. Int J Canc 2009;124: 2771e9. [22] Carson RC, Juszczak M, Davenport A, Burns A. Is maximum conservative management an equivalent treatment option to dialysis for elderly patients with significant comorbid disease? Clin J Am Soc Nephrol 2009;4:1611e9. [23] Davison SN, Holley JL. Ethical issues in the care of vulnerable chronic kidney disease patients: the elderly, cognitively impaired, and those from different cultural backgrounds. Adv Chron Kidney Dis 2008;15:177e85. [24] He SCW, Tang C. Effects of erk inhibitor azd6244 on the growth of multidrugresistance human gastric carcinoma cell lines. Gastroenterology 2010;138. S503. [25] Tegla C, Cudrici C, Boodhoo D. Rgc-32 regulates tgf- b extracellular matrix expression in reactive astrocytes. J Immunol 2014;192(1 supplement):52. [26] Kagawa-Singer M, Blackhall LJ. Negotiating cross-cultural issues at the end of life: "You got to go where he lives". J Am Med Assoc 2001;286:2993e3001.
Please cite this article in press as: Zhu L, Ding Y, RGC-32 induces transition of pancreatic cancer to epithelial mesenchyme in vivo, Pancreatology (2018), https://doi.org/10.1016/j.pan.2018.05.480
Contents lists available at ScienceDirect
Pancreatology journal homepage: www.elsevier.com/locate/pan
RGC-32 induces transition of pancreatic cancer to epithelial mesenchyme in vivo Liang Zhu a, *, 1, Ying Ding b, 1 a b
Department of Gastroenterology, The First Affiliated Hospital of Nanchang University, Nanchang, 330006, Jiangxi, China Department of Plastic and Cosmetic Surgery, The Second Affiliated Hospital of Nanchang University, Nanchang, 330006, Jiangxi, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 14 March 2018 Received in revised form 15 May 2018 Accepted 17 May 2018 Available online xxx
Objectives: This study was undertaken to investigate the induction of transition of pancreatic cancer to epithelial mesenchyme by RGC-32. Methods: Primary human pancreatic cancer cell line BXPC-3 was transfected with lentivirus overexpressing the response gene to complement-32 gene (RGC-32) and used to induce tumor in mice. The tumor sizes were measured and the expression of cytokeratin, e-cadherin and vimentin at mRNA using real time PCR and at protein levels by Western blot. Results: Compared with the control, mice inoculated with the cells transfected with empty vector had similar tumor size while those inoculated with the cells transfected with RGC-32 expressing virus had significantly greater tumor size. HE staining showed that tumors were formed in all treatments. Molecular analyses showed that there was no difference in the expression of the cytokeratin, e-cadherin and vimentin genes at mRNA and protein levels between control and empty vector groups. However, mice derived from cells transfected with RGC-32 expressing virus had reduced cytokeratin and e-cadherin expression and increased vimentin expression. Conclusions: These data suggest that RGC-32 promotes the proliferation of pancreatic cancer and induces the epithelialemesenchymal transition (EMT). It would be a future direction of research to investigate the regulatory mechanism of signal molecules downstream RGC-32 on EMT-related transcription factors and deliberate the role of RGC-32 in tumorigenicity. As a result, RGC-32 may become a new therapeutic target for cancers. © 2018 IAP and EPC. Published by Elsevier B.V. All rights reserved.
Keywords: RGC-32 Pancreatic cancer BXPC-3 Epithelial mesenchymal transition
1. Introduction Pancreatic cancer is a highly malignant and early metastatic digestive tract tumor with very poor prognosis. It is difficult to be diagnosed early. Most of the diagnosed patients already have local invasion and/or distant metastasis and have lose the best chance of treatment. As a consequence, the five year survival rate is less than 5% [1]. Therefore, a better understanding of the mechanism underlying the invasion and metastasis of the cancer is of great significance for controlling its progression and prolonging the survival time of pancreatic cancer patients. Epithelial mesenchymal transition (EMT) refers to a process by
* Corresponding author. Department of Gastroenterology, The First Affiliated Hospital of Nanchang University, 17 Yongwaizhen Street, Nanchang, Jiangxi 330006, China. E-mail address: [email protected] (L. Zhu). 1 These authors contributed equally to this work.
which epithelial cells lose their cell polarity and cell-cell adhesion, and gain migratory and invasive properties to become mesenchymal stem cells under certain physiological and pathological circumstances [2]. At the same time, the phenotype of the cells also changes, resulting in the gradual disappearance of epithelial phenotype markers such as E-cadherin and cytokeratins and the enhancement of mesenchymal markers such as vimentin and Ncadherin [3]. More and more studies have shown that EMT plays an important role in the progression of tumor. Tumor cells enhance their migration and invasion through EMT to become malignant [4]. Therefore, EMT has been considered as an important pathological process that promotes the invasion and metastasis of tumor [2]. The response gene to complement-32 (RGC-32) was first cloned in 1998 as response gene induced by complement activation [5], which is involved in the regulation of cell cycle and differentiation [6]. It is found that RGC-32 is expressed in a variety of normal tissues including the pancreas [7]. A number of studies have reported the role of RGC-32 in tumor [8] and found that RGC-32 is
https://doi.org/10.1016/j.pan.2018.05.480 1424-3903/© 2018 IAP and EPC. Published by Elsevier B.V. All rights reserved.
Please cite this article in press as: Zhu L, Ding Y, RGC-32 induces transition of pancreatic cancer to epithelial mesenchyme in vivo, Pancreatology (2018), https://doi.org/10.1016/j.pan.2018.05.480
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abnormally expressed in human cancers but differently in different organs [6,9e11], suggesting that the expression may be organ- and tissue-specific. However, little is known about the role of RGC-32 in pancreatic cancer. In our preliminary experiments, we have found that the expression of RGC-32 is significantly more deregulated in pancreatic cancer tissue than in chronic pancreatitis and normal pancreatic tissues, and is related to the tumor, node, metastasis (TNM) staging (P < 0.05), the pancreatic cancer positive rate and Ecadherin abnormal expression rate (P < 0.05) [12]. Furthermore, we have shown that RGC-32 has impact on hypoxia-induced EMT, and the possible link between HIF-1 and EMT may have therapeutic potential for pancreatic cancer [12]. Therefore, we hypothesized that RGC-32 may be one of metastasis-promoting genes in pancreatic cancer and may be involved in the invasion and metastasis of pancreatic cancer via EMT process. To better understand the role of the RGC-32 gene in pancreatic cancer, we investigated the expression of genes in the TGF-a and HIF-a pathways and the induction and mechanism of EMT in pancreatic cancer. The results would provide new insights into molecular targets for gene therapy of pancreatic cancer.
Once the solid tumors grew to 1 cm3 in size, they were isolated and cut into pieces of 1 mm3 in size and grafted subcutaneously to neck back of the nude mice to obtain xenotransplanted tumor models. Six animals were used in each treatment. The volumes (V) of tumor were measured every 3 days based on the length, width and height (V ¼ 0.5 x L x W x H). At the end of experiments, mice were scarified by cervical dislocation and tumors were isolated for subsequent analysis. 2.5. Immunohistochemistry
Human pancreatic cancer cell line BXPC-3 (cat no. BNCC338477) was purchased from Yingniurui Biotech, Wuxi, China.
10 mm transverse sections of tumor tissue were incubated with antibodies against E-cadherin (rabbit anti- E-cadherin, cat no. ab40772, 1:1000, Abcam, USA), vimentin (rabbit anti-vimentin, cat no. ab92574 1:500, Abcam) and cytokeratin (rabbit anticytokeratin, cat no. ab181597, 1:200, Abcam) for 2 h at room temperature followed by incubation with appropriate secondary antibody (goat anti-mouse IgG, cat. no. ZB-2305, ZSGB-Bio, Beijing) at 37 C for 2 h, and mounted with ProLong antifade reagent (Invitrogen). Each antibody series was performed on all tissue sections simultaneously to control for variations in processing and allow quantitative comparisons of staining intensity. The slides were reacted to DAB (diaminobenzidine) chromogenic solution at room temperature for 3 min before microscopy study. Images for a single antibody series were acquired at the same fluorescence intensity to keep imaging conditions constant and allow quantitative comparisons of staining intensity.
2.2. Reagents and instruments
2.6. Real-time quantitative PCR for mRNA expression
DMEM high glucose medium (lot no. KGM12800S-500) was purchased from Sijiqing Biologicals, Hangzhou, China; DMSO (lot no. 302A0325) was purchased from Amresco, USA; 0.25% trypsin (containing EDTA, lot no. 20160818) was obtained from KGI Biologicals, Jiangsu, China; fetal bovine serum (lot no. 1552680) was purchased from BI Technologies, USA; Transwell was obtained from FALCON, USA; 2xGoldStar Taq MasterMix and 2xULtraSYBR Mixture were obtained from Keyene, Beijing, China; Goldview I nucleic acid stain (cat no. G8140) was purchased from Solarbio, USA; ultrasensitive chemiluminescence imaging system (ChemiDocXRSþ) and quantitative PCR instrument (CFX Connect) were purchased from BioRad, USA.
Total RNA was isolated from the pancreatic cancer cells using the Trizol reagent according to the supplier's instruction (Invitrogen, USA) and reversely transcripted into cDNA using the RNA reverse transcription kit (Applied Biosystems by Life Technologies, Carlsbad, California, USA). RT-qPCR was performed using TaqMan RNA Assays (Applied Biosystems) in a total volume of 50 ml containing 4 ml of cDNA, 25 ml of TaqMan Gene Expression Master Mix and 1 ml of each fluorescence probe. The cycling conditions were 95 C for 30 s followed by 40 cycles, each one consisting of 95 C for 30s, 95 C for 5s, 60 C for 30s. Samples were run in five replicate and the mean value was calculated for each case. Primer sequences for RGC-32 were forward: 50 -TCAACCTTCTACCAGGCCACTC-3'; reverse: 50 -GCAAGCAGGTAAACAAAGTCAG-3'. GAPDH (forward: 50 AGGTCGGTGTGAACGGATTTG-30 , reverse: 50 -GGGGTCGTTGATGGCAACA-30 ) was used as internal control. The data were managed according to previously described protocol [13].
2. Materials and methods 2.1. Cell line
2.3. Construction of Ad-RGC-32 Wild type RGC-32 cDNA was inserted to KpnI and NotI digested adenovirus shuttle vector pAdTrack-CMV to obtain pAdTrack-CMVRGC-32. The plasmid was co-transformed with adenovirus backbone plasmid pAdEasy-1 into Escherichia coli DH5a cells to generate recombinant vector Ad-RGC-32 harboring the RGC-32 gene via homologous recombination in E. coli cells. The adenovirus vector was transfected into the human embryonic kidney 293 (HEK293, Sigma, USA) cells to produce recombinant adenovirus Ad-RGC-32. The virus particles were examined by fluorescence microscopy and tittered for TCID50 (tissue culture infectious dose 50%/mL). The negative control (empty vector) was produced similarly using pAdTrack-CMV and pAdEasy-1.
2.7. Western blot analysis Cells were washed twice with cold PBS and lysed with RIPA buffer that contains protease and phosphotase inhibitors cocktail (Roche, UK). The supernatants were collected after centrifugation at 12000 rpm for 20 min. 50mg/lane protein was applied to polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a PVDF membrane, and then detected by the proper primary and secondary antibodies before visualization with a chemiluminescence kit. The intensity of blot signals was quantitated using ImageQuant TL analysis software (General Electric, UK).
2.4. Tumor graft model 2.8. H & E staining BxPC-3 cells were transfected with lentivirus expressing RGC-32 or harboring empty vector and grown to a confluence of 80%e90% at 37 C in 5% CO2 in DMEM medium and the cell suspensions (3 106 tumor cells/mouse) were injected subcutaneously into the anterior neck of 4 week-old nude mice (Slack Inc, Hunan, China).
HE staining was preformed as previously described [14]. Briefly, paraffin sections were dewaxed, dehydrated, washed in PBS and dropped with hematoxylin to stain for 5 min. The stained slides were counterstained with eosin for 2 min after washed with PBS
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and mounted with neutral balata before microscopy study. 2.9. Statistical analysis All data were expressed as means ± standard error of the mean (SEM) obtained from at least three independent experiments. Means were compared using the student's t-test or one-way ANOVA with the LSD test using SPSS 20 software. A p-value 0.05 was considered statistically significant. 3. Results Fig. 1. Morphology and weight of tumors in mice after grafted with pancreatic cancer cells. The cells were either un-transfected (control) or transfected with empty vector (negative vector) or RGC-32 expression virus (RGC-32). Upper pane: tumor morphology; low pane: tumor weight. * denotes P < 0.05 vs control.
3.1. RGC-32 increased the tumor size As shown in Fig. 1, the volumes of tumor derived from cells transfected with virus expressing RGC-32 were significantly greater
Fig. 2. Expression of EMT-related genes in tumors in mice after grafted with pancreatic cancer cells. The cells were either un-transfected (control) or transfected with empty vector (negative vector) or RGC-32 expression virus (RGC-32). A. mRNA level; B. left panel: representative Western blot, right pane: relative protein level. * denotes P < 0.05 vs control.
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those of control or transfected with empty vector (p < 0.05), while the tumor volumes were similar between control and empty vector groups. 3.2. RGC-32 regulated the expression of EMT-related genes qRT-PCR analysis showed that the mRNA levels of cytokeratin and E-cadherin were significantly down-regulated, and the mRNA level of vimentin was significantly up-regulated in the tumor derived from the cells overexpressing RGC-32 (Fig. 2A, p < 0.05), as compared with control and empty-vector groups. In the latter two groups, the expression levels of these genes were similar (Fig. 2A). Similar results were obtained at the protein levels (Fig. 2B). These data suggest that the overexpression of RGC-32 may result in increased expression of EMT-related genes in pancreatic cancer. 3.3. HE staining and immunohistochemistry results HE staining showed that there were tumor tissues in all mice (Fig. 3A). Immunohistochemically, there were significantly more vimentin-positive cells and less cytokeratin- and E-cadherin-positive cells in the tumors derived from the cells overexpressing RGC32 (Fig. 3B), than in control and empty vector groups. For the latter two groups, no difference was observed in the expression level of these genes (Fig. 3B). These results further demonstrate that the overexpression of RGC-32 may result in increased expression of EMT-related genes in pancreatic cancer. 3.4. Discussion In recent years, the incidence of pancreatic ductal adenocarcinomas (PDAC) has being increasing obviously in China. It now accounts for 2.2% of the total malignant tumors. However, due to the difficulty of early diagnosis and high degree of malignancy, the 5
Fig. 3. HE and immunohistochemistry assays of tumor tissues in mice after grafted with pancreatic cancer cells. The cells were either un-transfected (control) or transfected with empty vector (negative vector) or RGC-32 expression virus (RGC-32). A: HE staining; B: percentage of positive cells. * denotes P < 0.05 vs control.
year survival rate is still less than 5%, and has not been improved in the past 40 years. Therefore, it is urgent to study the pathogenesis, prevention and treatment of the cancer. The RGC-32 gene, also known as C13orf15 and response gene to complement 32, is an important complement response gene. It is located in the human chromosome 13q14.11 [15] and widely expressed in many tissues, regulating cell cycle and immune response, promoting cell proliferation and differentiation [8,16]. Recent studies show that it is highly expressed in malignant tumors, such as colorectal cancer, pancreatic cancer, and may be involved in the regulation of tumor proliferation and invasion as oncogene [17,18]. In this study, we show that the overexpression of the RGC-32 gene significantly increases the tumor volume, demonstrating that RGC-32 could promote tumor growth. This is consistent with the previous studies [17,18]. EMT has been shown to be an important pathological process that promotes tumor invasion and metastasis. Tumor cells can enhance their mobility and invasiveness through EMT process, thus becoming malignant [19]. A number of signaling pathways are involved in the EMT in PDAC, among which the most important is the TGF-b signaling pathway. Several studies have shown that TGFb-induced EMT is one of the important factors that promote the invasion and metastasis of PDAC [20]. In 41.4% of PDAC tissues, the expression of TGF-b was increased, and the serum level of TGF- b was found to be negatively correlated with the prognosis of PDAC patients [21]. Zhu et al. showed that the positive rates of cell expressing RGC32 and E-cadherin were very higher in pancreatic cancer with lymph node metastasis or at high TNM stage [22]. E-cadherin is not only closely related to the progression of pancreatic cancer, but also plays an important role in the development of esophageal squamous cell carcinoma and gastric adenocarcinoma [23]. Using the pancreatic cancer cell line BXPC-3 with homozygous deletion of Smad4, it was shown that sustained stimulation of TGF-b induces EMT in BXPC-3 cells and up-regulates the expression of RGC-32 [12]. RGC-32 not only mediates the TGF- induced EMT process via at least ERK-MAPK and p38-MAPK signaling pathways, but also induces EMT independently, which is not dependent on the Smad signaling pathway [24]. In addition, RGC-32 was shown to be involved in the regulation of TGF-b-induced cell migration, and inhibit the proliferation but not apoptosis of BXPC-3 cells [25]. These results further confirm that RGC-32 enhances the transformation of the metastatic phenotype of pancreatic cancer. Furthermore, a number of recent studies have shown that RGC-32 not only plays an important role in the EMT process in tumor cells, but also in normal cells [26]. For example, Kurahara et al. found that RGC-32 mediates TGF-b-induced EMT process in human proximal tubular epithelial cells (HPTC) [26]. Our study shows that the overexpression of RGC-32 resulted in significant downregulation of cytokeratin and epithelial marker protein E-cadherin and significant up-regulation of mesenchymal marker protein vimentin, suggesting that RGC-32 promotes tumor metastasis. So far, the role of RGC-32 in cancer has not been fully elucidated and could be very complicated. For example, in colorectal and pancreatic cancer, RGC-32 is an oncogene while in glioma, it appears to be antiandrogenic [9]. Therefore, further studies are needed to further identify and deliberate the signal transduction pathways and mechanism by which RGC-32 participates in oncogenesis. It is likely that RGC-32 is one of the key molecules mediating the dual roles of TGF-b. Exploring the regulatory mechanism of signaling molecules downstream RGC-32 on EMT-related transcription factors may become a hot research topic. In addition, further investigations are needed to understand whether RGC-32 participates in more oncogenesis processes and its roles in these
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processes. The elucidation would result in the identification of new therapeutic target for the cancer. Declaration of conflict of interest None. Acknowledgement This work was supported by the Natural Science Foundation of Jiangxi Province, China (The grant number is 20171BAB215044), National Natural Science Foundation of China (The grant number is 81302152) and Key Project of Educational Commission of Jiangxi Province of China (The grant number is GJJ170006). References [1] Hidalgo M. Pancreatic cancer. N Engl J Med 2010;362:1605e17. [2] Polyak K, Weinberg RA. Transitions between epithelial and mesenchymal states: acquisition of malignant and stem cell traits. Nat Rev Canc 2009;9: 265e73. [3] Thiery JP, Acloque H, Huang RY, Nieto MA. Epithelial-mesenchymal transitions in development and disease. Cell 2009;139:871e90. [4] Thiery JP. Epithelial-mesenchymal transitions in tumour progression. Nat Rev Canc 2002;2:442e54. [5] Badea TC, Niculescu FI, Soane L, Shin ML, Rus H. Molecular cloning and characterization of rgc-32, a novel gene induced by complement activation in oligodendrocytes. J Biol Chem 1998;273:26977e81. [6] Saigusa K, Imoto I, Tanikawa C, Aoyagi M, Ohno K, Nakamura Y, et al. Rgc32, a novel p53-inducible gene, is located on centrosomes during mitosis and results in g2/m arrest. Oncogene 2007;26:1110e21. [7] Badea T, Niculescu F, Soane L, Fosbrink M, Sorana H, Rus V, et al. Rgc-32 increases p34cdc2 kinase activity and entry of aortic smooth muscle cells into sphase. J Biol Chem 2002;277:502e8. [8] Vlaicu SI, Tegla CA, Cudrici CD, Danoff J, Madani H, Sugarman A, et al. Role of c5b-9 complement complex and response gene to complement-32 (rgc-32) in cancer. Immunol Res 2013;56:109e21. [9] Fosbrink M, Cudrici C, Niculescu F, Badea TC, David S, Shamsuddin A, et al. Overexpression of rgc-32 in colon cancer and other tumors. Exp Mol Pathol 2005;78:116e22. [10] Kang Y, Siegel PM, Shu W, Drobnjak M, Kakonen SM, Cordon-Cardo C, et al. A multigenic program mediating breast cancer metastasis to bone. Canc Cell
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