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Nigam J, Kazmi HR, Khare L, Srivastava M, Chandra A. Heterodimerization of cholecystokinin 1 and cholecystokinin 2 receptors in gallbladder cancer: a new mechanism for carcinogenesis. J Cancer Res Clin Oncol 2023; 149:7069-7078. [PMID: 36871090 DOI: 10.1007/s00432-023-04653-x] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/09/2022] [Accepted: 02/14/2023] [Indexed: 03/06/2023]
Abstract
PURPOSE Cholecystokinin is present in abundance in gallbladder tissue and mediates function through two structurally related receptors, CCK1R and CCK2R. Heterodimerization of these receptors is known to impact cell growth in vitro. However, the significance of these heterodimers in gallbladder carcinogenesis is relatively unknown. METHODS Therefore, we evaluated the expression and the dimerization status of CCK1 and CCK2 receptors in human gallbladder carcinoma cell line (GBC-SD) and resected gallbladder tissue from normal (n = 10), cholelithiasis (n = 25) and gallbladder cancer (n = 25) by immunofluorescence/immunohistochemistry and western blot. The dimerization status of CCK1R and CCK2R was evaluated by co-immunoprecipitation. To understand the effect of heterodimerization of these receptors on growth-related signaling pathways, the expression of p-AKT, rictor, raptor and p-ERK was evaluated by western blot. RESULTS We demonstrated the expression and heterodimerization of CCK1 and CCK2 receptor in GBC-SD gall bladder carcinoma cell line. Knockdown of CCK1R and CCK2R in the cell line led to significant reduction in p-AKT (P = 0.005; P = 0.0001) and rictor (P < 0.001; P < 0.001) levels. In tissue samples, significantly higher expression of CCK1R and CCK2R was observed in gallbladder cancer when compared to other groups both by immunohistochemistry (P = 0.008 and P = 0.013) and western blot (P = 0.009 and P = 0.003). An increase in heterodimer formation of CCK1R with CCK2R was observed in gallbladder cancer when compared to normal and cholelithiasis tissues. No significant difference in the expression of p-AKT and p-ERK was observed between the three groups. CONCLUSION Our results provide the first evidence of heterodimerization of CCK1R and CCK2R in gallbladder tissue, and its association with development of gallbladder cancer. This finding has potential clinical and therapeutic significance.
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Affiliation(s)
- Jaya Nigam
- Department of Surgical Gastroenterology, King George's Medical University, Lucknow, India
| | - Hasan Raza Kazmi
- Fels Cancer Institute for Personalized Medicine, Temple University, Lewis Katz School of Medicine, Philadelphia, PA, USA
| | | | - Meenu Srivastava
- Department of Surgical Gastroenterology, King George's Medical University, Lucknow, India
| | - Abhijit Chandra
- Department of Surgical Gastroenterology, King George's Medical University, Lucknow, India.
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Structural insights into human brain-gut peptide cholecystokinin receptors. Cell Discov 2022; 8:55. [PMID: 35672283 PMCID: PMC9174195 DOI: 10.1038/s41421-022-00420-3] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/21/2021] [Accepted: 05/11/2022] [Indexed: 11/26/2022] Open
Abstract
The intestinal hormone and neuromodulator cholecystokinin (CCK) receptors CCK1R and CCK2R act as a signaling hub in brain–gut axis, mediating digestion, emotion, and memory regulation. CCK receptors exhibit distinct preferences for ligands in different posttranslational modification (PTM) states. CCK1R couples to Gs and Gq, whereas CCK2R primarily couples to Gq. Here we report the cryo-electron microscopy (cryo-EM) structures of CCK1R–Gs signaling complexes liganded either by sulfated cholecystokinin octapeptide (CCK-8) or a CCK1R-selective small-molecule SR146131, and CCK2R–Gq complexes stabilized by either sulfated CCK-8 or a CCK2R-selective ligand gastrin-17. Our structures reveal a location-conserved yet charge-distinct pocket discriminating the effects of ligand PTM states on receptor subtype preference, the unique pocket topology underlying selectivity of SR146131 and gastrin-17, the conformational changes in receptor activation, and key residues contributing to G protein subtype specificity, providing multiple structural templates for drug design targeting the brain–gut axis.
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Ma Y, Weng J, Wang N, Zhang Y, Minato N, Su L. A novel nuclear localization region in SIPA1 determines protein nuclear distribution and epirubicin-sensitivity of breast cancer cells. Int J Biol Macromol 2021; 180:718-728. [PMID: 33753200 DOI: 10.1016/j.ijbiomac.2021.03.101] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/30/2020] [Revised: 02/26/2021] [Accepted: 03/17/2021] [Indexed: 01/03/2023]
Abstract
Signal-induced proliferation-associated protein 1 (SIPA1) is highly expressed and mainly located in the nucleus in some breast cancer cell lines and clinical tumor tissues. Previous study revealed that nuclear localization of SIPA1 is functionally involved in breast cancer metastasis in the lymphatic gland. In the current study, we identified a non-typical region (140-179aa) of SIPA1 as a novel nuclear localization region (NLR) which is crucial for translocating the proteins into the nucleus in HEK293 cells and breast cancer cells. This region contained one basic amino acid, His160, and had no common features of typical nuclear localization signals. In addition, overexpressing SIPA1 without NLR could suppress breast cancer cell proliferation but could not promote cell migration in MCF7 cells. Furthermore, we found that a high expression of SIPA1 upregulated the expression of ABCB1, encoding multi-drug resistance protein MDR1, and promoted the resistance to epirubicin in breast cancer cells, while this effect was largely abolished in the cells with the expression of NLR-deleted SIPA1. This study overall, identified a nuclear localization-dependent region determining the nuclear distribution of SIPA1 and its regulation on epirubicin-sensitivity in breast cancer cells, which could be a potential drug target to facilitate the development of breast cancer chemotherapy.
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Affiliation(s)
- Ying Ma
- Key Laboratory of Molecular Biophysics of Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, China
| | - Jun Weng
- Key Laboratory of Molecular Biophysics of Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, China
| | - Ning Wang
- Key Laboratory of Molecular Biophysics of Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, China
| | - Yilei Zhang
- Department of Experimental Radiation Oncology, University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA
| | - Nagahiro Minato
- Department of Immunology and Cell Biology, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan
| | - Li Su
- Key Laboratory of Molecular Biophysics of Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, China.
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Wang N, Weng J, Xia J, Zhu Y, Chen Q, Hu D, Zhang X, Sun R, Feng J, Minato N, Gong Y, Su L. SIPA1 enhances SMAD2/3 expression to maintain stem cell features in breast cancer cells. Stem Cell Res 2020; 49:102099. [PMID: 33296812 DOI: 10.1016/j.scr.2020.102099] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/12/2020] [Revised: 09/30/2020] [Accepted: 11/18/2020] [Indexed: 02/06/2023] Open
Abstract
SIPA1, a GTPase activating protein that negatively regulates Ras-related protein (Rap), is a potential modulator of tumor metastasis and recurrence. In this study, we first showed that SIPA1 facilitated the stemness features of breast cancer cells, such as of tumorsphere formation capability and the expression of stemness marker CD44. In addition, SIPA1 promoted the expression of four stemness-associated transcription factors through increasing the expression of SMAD2 and SMAD3 in vitro and in vivo. The stemness features were abolished by blocking the phosphorylation of SMAD3 with its specific inhibitor SIS3. Furthermore, SIPA1 decreased the breast cancer cell sensitivity to chemotherapy drugs. This effect was, however, competitively reversed by blocking the SMAD3 phosphorylation by SIS3 treatment in breast cancer cells. Taken together, SIPA1 promotes and sustains the stemness of breast cancer cells and their resistance to chemotherapy by increasing the expression of SMAD2 and SMAD3, and blocking SMAD3 phosphorylation could suppress the cancer cell stemness and increase the sensitivity to chemotherapy in breast cancer cells expressing a high level of SIPA1.
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Affiliation(s)
- Ning Wang
- Key Laboratory of Molecular Biophysics of Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, China
| | - Jun Weng
- Key Laboratory of Molecular Biophysics of Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, China
| | - Jing Xia
- Key Laboratory of Molecular Biophysics of Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, China
| | - Yangjin Zhu
- Key Laboratory of Molecular Biophysics of Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, China
| | - Qiongrong Chen
- Department of Pathology, Zhongnan Hospital of Wuhan University, Wuhan University, Wuhan 430071, China
| | - Die Hu
- Key Laboratory of Molecular Biophysics of Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, China
| | - Xue Zhang
- Department of Breast Surgery, Renmin Hospital of Wuhan University, Wuhan University, Wuhan 430060, China
| | - Rui Sun
- Department of Oncology, Puai Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430034, China
| | - Jueping Feng
- Department of Oncology, Puai Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430034, China
| | - Nagahiro Minato
- Department of Immunology and Cell Biology, Graduate School of Medicine, Kyoto University, Yoshida, Sakyo-ku, Kyoto 606-8501, Japan
| | - Yiping Gong
- Department of Breast Surgery, Renmin Hospital of Wuhan University, Wuhan University, Wuhan 430060, China; Department of Breast Surgery, Hubei Cancer Hospital, Wuhan 430079, China.
| | - Li Su
- Key Laboratory of Molecular Biophysics of Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, China.
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Zeng Q, Ou L, Wang W, Guo DY. Gastrin, Cholecystokinin, Signaling, and Biological Activities in Cellular Processes. Front Endocrinol (Lausanne) 2020; 11:112. [PMID: 32210918 PMCID: PMC7067705 DOI: 10.3389/fendo.2020.00112] [Citation(s) in RCA: 65] [Impact Index Per Article: 13.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/05/2020] [Accepted: 02/20/2020] [Indexed: 12/24/2022] Open
Abstract
The structurally-related peptides, gastrin and cholecystokinin (CCK), were originally discovered as humoral stimulants of gastric acid secretion and pancreatic enzyme release, respectively. With the aid of methodological advances in biochemistry, immunochemistry, and molecular biology in the past several decades, our concept of gastrin and CCK as simple gastrointestinal hormones has changed considerably. Extensive in vitro and in vivo studies have shown that gastrin and CCK play important roles in several cellular processes including maintenance of gastric mucosa and pancreatic islet integrity, neurogenesis, and neoplastic transformation. Indeed, gastrin and CCK, as well as their receptors, are expressed in a variety of tumor cell lines, animal models, and human samples, and might contribute to certain carcinogenesis. In this review, we will briefly introduce the gastrin and CCK system and highlight the effects of gastrin and CCK in the regulation of cell proliferation and apoptosis in both normal and abnormal conditions. The potential imaging and therapeutic use of these peptides and their derivatives are also summarized.
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Affiliation(s)
- Qiang Zeng
- Health Management Institute, People's Liberation Army General Hospital, Beijing, China
| | - Lei Ou
- Health Management Institute, People's Liberation Army General Hospital, Beijing, China
| | - Wei Wang
- Department of Clinical Laboratory, Xiamen Huli Guoyu Clinic, Co., Ltd., Xiamen, China
- *Correspondence: Wei Wang
| | - Dong-Yu Guo
- Department of Clinical Laboratory, Xiamen Huli Guoyu Clinic, Co., Ltd., Xiamen, China
- Dong-Yu Guo
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Gastrin stimulates pancreatic cancer cell directional migration by activating the Gα12/13-RhoA-ROCK signaling pathway. Exp Mol Med 2018; 50:1-14. [PMID: 29717112 PMCID: PMC5938061 DOI: 10.1038/s12276-018-0081-6] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/11/2017] [Revised: 02/02/2018] [Accepted: 02/05/2018] [Indexed: 12/24/2022] Open
Abstract
The mechanism by which gastrin promotes pancreatic cancer cell metastasis is unclear. The process of directing polarized cancer cells toward the extracellular matrix is principally required for invasion and distant metastasis; however, whether gastrin can induce this process and its underlying mechanism remain to be elucidated. In this study, we found that gastrin-induced phosphorylation of paxillin at tyrosine 31/118 and RhoA activation as well as promoted the metastasis of PANC-1 cancer cells. Depletion of Gα12 and Gα13 inhibited the phosphorylation of paxillin and downstream activation of GTP-RhoA, blocked the formation and aggregation of focal adhesions and facilitated polarization of actin filaments induced by gastrin. Suppression of RhoA and ROCK also exhibited identical results. Selective inhibition of the CCKBR–Gα12/13–RhoA–ROCK signaling pathway blocked the reoriented localization of the Golgi apparatus at the leading edge of migrated cancer cells. YM022 and Y-27632 significantly suppressed hepatic metastasis of orthotic pancreatic tumors induced by gastrin in vivo. Collectively, we demonstrate that gastrin promotes Golgi reorientation and directional polarization of pancreatic cancer cells by activation of paxillin via the CCKBR–Gα12/13–RhoA–ROCK signal pathway. A hormone found in high levels in pancreatic cancer sufferers helps the disease spread by co-ordinating cellular migration. Pancreatic cancer is one of the most deadly forms of cancer, being highly aggressive and likely to metastasize. Honggang Yu at Renmin Hospital of Wuhan University and scientists across China have demonstrated that gastrin, a hormone expressed at higher levels in patients with pancreatic cancer, helps to co-ordinate directional cell migration and ensure the disease spreads effectively. By activating two key molecules via a specific signalling pathway, gastrin ensures the correct orientation of the Golgi apparatus, a cellular organelle tasked with packaging proteins for transportation. This in turn activates directional migration of the cancer cells. The results explain why gastrin is over-expressed in both tumors and blood in cancer patients, and may inform future therapies.
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Kowalski-Chauvel A, Teissier G, Toulas C, Cohen-Jonathan-Moyal E, Seva C. By modulating α2β1 integrin signalling, gastrin increases adhesion oF AGS-GR gastric cancer cells. Exp Cell Res 2018; 362:498-503. [PMID: 29253536 DOI: 10.1016/j.yexcr.2017.12.014] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/09/2017] [Revised: 12/13/2017] [Accepted: 12/14/2017] [Indexed: 01/01/2023]
Abstract
Peritoneal metastasis is a major cause of recurrence of gastric cancer and integrins are key molecules involved in gastric cancer cells attachment to the peritoneum. The peptide hormone, gastrin, initially identified for its role in gastric acid secretion is also a growth factor for gastric mucosa. Gastrin has also been shown to contribute to gastric cancers progression. Here, we provide the first evidence that gastrin increases the adhesion of gastric cancer cells. Gastrin treatment induces the expression of α2 integrin subunit through a mechanism that involves the ERK pathway. We also observed in response to gastrin an increase in the amount of α2 integrin associated with β1subunit. In addition, gastrin-stimulated cell adhesion was blocked with an anti-α2β1 integrin neutralizing antibody. We also show that gastrin activates the integrin pathway via the phosphorylation of β1 integrin by a Src family kinase. This mechanism may contribute to the enhancement of cell adhesion observed in response to gastrin since we found an inhibition of gastrin-mediated cell adhesion when cells were treated with a Src inhibitor. By regulating one of the key step of the metastatic process gastrin might contribute to increase the aggressive behaviour of human gastric tumours.
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Affiliation(s)
- Aline Kowalski-Chauvel
- INSERM UMR.1037-Cancer Research Center of Toulouse (CRCT)/University Paul Sabatier Toulouse III, team 11, Oncopole 2 Avenue Hubert Curien, CS 53717, 31037 Toulouse, France
| | - Guy Teissier
- INSERM UMR.1037-Cancer Research Center of Toulouse (CRCT)/University Paul Sabatier Toulouse III, team 11, Oncopole 2 Avenue Hubert Curien, CS 53717, 31037 Toulouse, France
| | - Christine Toulas
- INSERM UMR.1037-Cancer Research Center of Toulouse (CRCT)/University Paul Sabatier Toulouse III, team 11, Oncopole 2 Avenue Hubert Curien, CS 53717, 31037 Toulouse, France; IUCT-oncopole Toulouse, France
| | - Elizabeth Cohen-Jonathan-Moyal
- INSERM UMR.1037-Cancer Research Center of Toulouse (CRCT)/University Paul Sabatier Toulouse III, team 11, Oncopole 2 Avenue Hubert Curien, CS 53717, 31037 Toulouse, France; IUCT-oncopole Toulouse, France
| | - Catherine Seva
- INSERM UMR.1037-Cancer Research Center of Toulouse (CRCT)/University Paul Sabatier Toulouse III, team 11, Oncopole 2 Avenue Hubert Curien, CS 53717, 31037 Toulouse, France.
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Eibl G, Rozengurt E. KRAS, YAP, and obesity in pancreatic cancer: A signaling network with multiple loops. Semin Cancer Biol 2017; 54:50-62. [PMID: 29079305 DOI: 10.1016/j.semcancer.2017.10.007] [Citation(s) in RCA: 40] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/02/2017] [Accepted: 10/22/2017] [Indexed: 02/08/2023]
Abstract
Pancreatic ductal adenocarcinoma (PDAC) continues to be a lethal disease with no efficacious treatment modalities. The incidence of PDAC is expected to increase, at least partially because of the obesity epidemic. Increased efforts to prevent or intercept this disease are clearly needed. Mutations in KRAS are initiating events in pancreatic carcinogenesis supported by genetically engineered mouse models of the disease. However, oncogenic KRAS is not entirely sufficient for the development of fully invasive PDAC. Additional genetic mutations and/or environmental, nutritional, and metabolic stressors, e.g. inflammation and obesity, are required for efficient PDAC formation with activation of KRAS downstream effectors. Multiple factors "upstream" of KRAS associated with obesity, including insulin resistance, inflammation, changes in gut microbiota and GI peptides, can enhance/modulate downstream signals. Multiple signaling networks and feedback loops "downstream" of KRAS have been described that respond to obesogenic diets. We propose that KRAS mutations potentiate a signaling network that is promoted by environmental factors. Specifically, we envisage that KRAS mutations increase the intensity and duration of the growth-promoting signaling network. As the transcriptional activator YAP plays a critical role in the network, we conclude that the rationale for targeting the network (at different points), e.g. with FDA approved drugs such as statins and metformin, is therefore compelling.
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Affiliation(s)
- Guido Eibl
- Departments of Surgery, David Geffen School of Medicine at UCLA, Los Angeles, CA, United States; CURE: Digestive Diseases Research Center, University of California at Los Angeles, Los Angeles, CA, United States.
| | - Enrique Rozengurt
- Departments of Medicine, David Geffen School of Medicine at UCLA, Los Angeles, CA, United States; CURE: Digestive Diseases Research Center, University of California at Los Angeles, Los Angeles, CA, United States
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Bi JJ, Li J, Cheng BF, Yang HJ, Ding QQ, Wang RF, Chen SJ, Feng ZW. NCAM affects directional lamellipodia formation of BMSCs via β1 integrin signal-mediated cofilin activity. Mol Cell Biochem 2017; 435:175-183. [PMID: 28536952 DOI: 10.1007/s11010-017-3066-1] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/18/2017] [Accepted: 05/05/2017] [Indexed: 12/20/2022]
Abstract
The neural cell adhesion molecule (NCAM), a key member of the immunoglobulin-like CAM family, was reported to regulate the migration of bone marrow-derived mesenchymal stem cells (BMSCs). However, the detailed cellular behaviors including lamellipodia formation in the initial step of directional migration remain largely unknown. In the present study, we reported that NCAM affects the lamellipodia formation of BMSCs. Using BMSCs from Ncam knockout mice we found that Ncam deficiency significantly impaired the migration and the directional lamellipodia formation of BMSCs. Further studies revealed that Ncam knockout decreased the activity of cofilin, an actin-cleaving protein, which was involved in directional protrusions. To explore the molecular mechanisms involved, we examined protein tyrosine phosphorylation levels in Ncam knockout BMSCs by phosphotyrosine peptide array analyses, and found that the tyrosine phosphorylation level of β1 integrin, a protein upstream of cofilin, was greatly upregulated in Ncam-deficient BMSCs. Notably, by blocking the function of β1 integrin with RGD peptide or ROCK inhibitor, the cofilin activity and directional lamellipodia formation of Ncam knockout BMSCs could be rescued. Finally, we found that the effect of NCAM on tyrosine phosphorylation of β1 integrin was independent of the fibroblast growth factor receptor. These results indicated that NCAM regulates directional lamellipodia formation of BMSCs through β1 integrin signal-mediated cofilin activity.
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Affiliation(s)
- Jia-Jia Bi
- School of Life Sciences and Technology, Xinxiang Medical University, 601 Jinsui Road, Xinxiang, 453003, Henan, China
| | - Jing Li
- School of Life Sciences and Technology, Xinxiang Medical University, 601 Jinsui Road, Xinxiang, 453003, Henan, China
| | - Bin-Feng Cheng
- School of Life Sciences and Technology, Xinxiang Medical University, 601 Jinsui Road, Xinxiang, 453003, Henan, China
| | - Hai-Jie Yang
- School of Life Sciences and Technology, Xinxiang Medical University, 601 Jinsui Road, Xinxiang, 453003, Henan, China
| | - Qiong-Qiong Ding
- School of Life Sciences and Technology, Xinxiang Medical University, 601 Jinsui Road, Xinxiang, 453003, Henan, China
| | - Rui-Fei Wang
- College of Life Sciences, Henan Normal University, Xinxiang, 453007, China
| | - Su-Juan Chen
- School of Life Sciences and Technology, Xinxiang Medical University, 601 Jinsui Road, Xinxiang, 453003, Henan, China.
| | - Zhi-Wei Feng
- School of Basic Medical Sciences, Xinxiang Medical University, 601 Jinsui Road, Xinxiang, 453003, Henan, China.
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Nuclear SIPA1 activates integrin β1 promoter and promotes invasion of breast cancer cells. Oncogene 2014; 34:1451-62. [DOI: 10.1038/onc.2014.36] [Citation(s) in RCA: 35] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/10/2013] [Revised: 12/31/2013] [Accepted: 01/06/2014] [Indexed: 12/14/2022]
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Zhang R, Li M, Zang W, Chen X, Wang Y, Li P, Du Y, Zhao G, Li L. MiR-148a regulates the growth and apoptosis in pancreatic cancer by targeting CCKBR and Bcl-2. Tumour Biol 2013; 35:837-44. [PMID: 23975374 DOI: 10.1007/s13277-013-1115-2] [Citation(s) in RCA: 47] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/22/2013] [Accepted: 08/13/2013] [Indexed: 01/01/2023] Open
Abstract
Our previous studies have revealed that miR-148a is downregulated in pancreatic cancer. Bioinformatics analysis has shown cholecystokinin-B receptor (CCKBR) and B cell lymphoma (Bcl-2) to be potential targets of miR-148a. But the pathophysiologic role of miR-148a and its relevance to the growth and development of pancreatic cancer are yet to be investigated. The purpose of this study is to elucidate the molecular mechanisms where miR-148a acts as a tumor suppressor in pancreatic cancer. Our results showed significant downregulation of miR-148a in 28 pancreatic cancer tissue samples and five pancreatic cancer cell lines, compared with their non-tumor counterparts by qRT-PCR. MiR-148a was found to not only inhibit the proliferation of pancreatic cancer cells (PANC-1 and AsPC-1) in vitro by MTT assay and colony formation assay, but also to promote cells apoptosis in vitro by Annexin V-FITC apoptosis detection and caspase activity assay. Using western blot and luciferase activity assay, CCKBR and Bcl-2 were identified as targets of miR-148a. Moreover, we also found that the expression of Bcl-2 lacking in 3'UTR could abrogate the pro-apoptosis function of miR-148a. These findings suggest the importance of miR-148a's targeting of CCKBR and Bcl-2 in the regulation of pancreatic cancer growth and apoptosis.
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Affiliation(s)
- Rui Zhang
- Department of Emergency, The First Affiliated Hospital of Zhengzhou University, No. 1 Jianshe Road, Zhengzhou, Henan, 450052, China
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Dvorshchenko KO. Stress-responsive systems in rat pancreas upon long-term gastric hypochlorhydria and administration of multiprobiotic “Symbiter. UKRAINIAN BIOCHEMICAL JOURNAL 2013. [DOI: 10.15407/ubj85.02.068] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/09/2023] Open
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Matters GL, Clawson GA. A Speculative Role for Stromal Gastrin Signaling in Development and Dissemination of Pancreatic Ductal Adenocarcinoma. ACTA ACUST UNITED AC 2013; Suppl 4:003. [PMID: 25346875 PMCID: PMC4208305 DOI: 10.4172/2165-7092.s4-003] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
The peptide growth factor gastrin and its receptor, the G-protein coupled cholecystokinin receptor type B (CCKBR), play an integral role in the growth and progression of pancreatic ductal adenocarcinoma (PDAC). Gastrin immunoreactivity is found in the fetal pancreas but its expression is not detected in normal pancreas after birth, except when it is re-expressed in malignant lesions.
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Affiliation(s)
- Gail L Matters
- Department of Biochemistry and Molecular Biology, Hershey Medical Center, Pennsylvania State University, Hershey, PA, USA
| | - Gary A Clawson
- Department of Biochemistry and Molecular Biology, Hershey Medical Center, Pennsylvania State University, Hershey, PA, USA ; Gittlen Cancer Research Foundation and Departments of Pathology, Biochemistry and Molecular Biology, USA
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Gigoux V, Fourmy D. Acting on Hormone Receptors with Minimal Side Effect on Cell Proliferation: A Timely Challenge Illustrated with GLP-1R and GPER. Front Endocrinol (Lausanne) 2013; 4:50. [PMID: 23641235 PMCID: PMC3638125 DOI: 10.3389/fendo.2013.00050] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/11/2012] [Accepted: 04/10/2013] [Indexed: 12/18/2022] Open
Abstract
G protein-coupled receptors (GPCRs) constitute a large family of receptors that sense molecules outside the cell and activate inside signal transduction pathways and cellular responses. GPCR are involved in a wide variety of physiological processes, including in the neuroendocrine system. GPCR are also involved in many diseases and are the target of 30% of marketed medicinal drugs. Whereas the majority of the GPCR-targeting drugs have proved their therapeutic benefit, some of them were associated with undesired effects. We develop two examples of used drugs whose therapeutic benefits are tarnished by carcinogenesis risks. The chronic administration of glucagon-like peptide-1 (GLP-1) analogs widely used to treat type-2 diabetes was associated with an increased risk of pancreatic or thyroid cancers. The long-term treatment with the estrogen antagonist tamoxifen, developed to target breast cancer overexpressing estrogen receptors ER, presents agonist activity on the G protein-coupled estrogen receptor which is associated with an increased incidence of endometrial cancer and breast cancer resistance to hormonotherapy. We point out and discuss the need of pharmacological studies to understand and overcome the undesired effects associated with the chronic administration of GPCR ligands. In fact, biological effects triggered by GPCR often result from the activation of multiple intracellular signaling pathways. Deciphering which signaling networks are engaged following GPCR activation appears to be primordial to unveil their contribution in the physiological and physiopathological processes. The development of biased agonists to elucidate the role of the different signaling mechanisms mediated by GPCR activation will allow the generation of new therapeutic agents with improved efficacy and reduced side effects. In this regard, the identification of GLP-1R biased ligands promoting insulin secretion without inducing pro-tumoral effects would offer therapeutic benefit.
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Affiliation(s)
- Véronique Gigoux
- Université de Toulouse, Université Paul SabatierToulouse, France
- *Correspondence: Véronique Gigoux, CHU Rangueil – INSERM, Université de Toulouse, Université Paul Sabatier, EA4552, 1 Avenue Jean Poulhès, BP 84225, 31432 Toulouse Cedex 4, France. e-mail:
| | - Daniel Fourmy
- Université de Toulouse, Université Paul SabatierToulouse, France
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15
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Transcriptome analysis during human trophectoderm specification suggests new roles of metabolic and epigenetic genes. PLoS One 2012; 7:e39306. [PMID: 22761758 PMCID: PMC3382239 DOI: 10.1371/journal.pone.0039306] [Citation(s) in RCA: 37] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/08/2011] [Accepted: 05/18/2012] [Indexed: 11/28/2022] Open
Abstract
In humans, successful pregnancy depends on a cascade of dynamic events during early embryonic development. Unfortunately, molecular data on these critical events is scarce. To improve our understanding of the molecular mechanisms that govern the specification/development of the trophoblast cell lineage, the transcriptome of human trophectoderm (TE) cells from day 5 blastocysts was compared to that of single day 3 embryos from our in vitro fertilization program by using Human Genome U133 Plus 2.0 microarrays. Some of the microarray data were validated by quantitative RT-PCR. The TE molecular signature included 2,196 transcripts, among which were genes already known to be TE-specific (GATA2, GATA3 and GCM1) but also genes involved in trophoblast invasion (MUC15), chromatin remodeling (specifically the DNA methyltransferase DNMT3L) and steroid metabolism (HSD3B1, HSD17B1 and FDX1). In day 3 human embryos 1,714 transcripts were specifically up-regulated. Besides stemness genes such as NANOG and DPPA2, this signature included genes belonging to the NLR family (NALP4, 5, 9, 11 and 13), Ret finger protein-like family (RFPL1, 2 and 3), Melanoma Antigen family (MAGEA1, 2, 3, 5, 6 and 12) and previously unreported transcripts, such as MBD3L2 and ZSCAN4. This study provides a comprehensive outlook of the genes that are expressed during the initial embryo-trophectoderm transition in humans. Further understanding of the biological functions of the key genes involved in steroidogenesis and epigenetic regulation of transcription that are up-regulated in TE cells may clarify their contribution to TE specification and might also provide new biomarkers for the selection of viable and competent blastocysts.
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Fino KK, Matters GL, McGovern CO, Gilius EL, Smith JP. Downregulation of the CCK-B receptor in pancreatic cancer cells blocks proliferation and promotes apoptosis. Am J Physiol Gastrointest Liver Physiol 2012; 302:G1244-52. [PMID: 22442157 PMCID: PMC3378167 DOI: 10.1152/ajpgi.00460.2011] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/08/2023]
Abstract
Gastrin stimulates the growth of pancreatic cancer cells through the activation of the cholecystokinin-B receptor (CCK-BR), which has been found to be overexpressed in pancreatic cancer. In this study, we proposed that the CCK-BR drives growth of pancreatic cancer; hence, interruption of CCK-BR activity could potentially be an ideal target for cancer therapeutics. The effect of CCK-BR downregulation in the human pancreatic adenocarcinoma cells was examined by utilizing specific CCK-BR-targeted RNA interference reagents. The CCK-BR receptor expression was both transiently and stably downregulated by transfection with selective CCK-BR small-interfering RNA or short-hairpin RNA, respectively, and the effects on cell growth and apoptosis were assessed. CCK-BR downregulation resulted in reduced cancer cell proliferation, decreased DNA synthesis, and cell cycle arrest as demonstrated by an inhibition of G(1) to S phase progression. Furthermore, CCK-BR downregulation increased caspase-3 activity, TUNEL-positive cells, and decreased X-linked inhibitor of apoptosis protein expression, suggesting apoptotic activity. Pancreatic cancer cell mobility was decreased when the CCK-BR was downregulated, as assessed by a migration assay. These results show the importance of the CCK-BR in regulation of growth and apoptosis in pancreatic cancer. Strategies to decrease the CCK-BR expression and activity may be beneficial for the development of new methods to improve the treatment for patients with pancreatic cancer.
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Affiliation(s)
| | - Gail L. Matters
- Departments of 1Medicine and ,2Biochemistry and Molecular Biology, College of Medicine, Pennsylvania State University, Hershey, Pennsylvania
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Cayrol C, Bertrand C, Kowalski-Chauvel A, Daulhac L, Cohen-Jonathan-Moyal E, Ferrand A, Seva C. α V integrin: A new gastrin target in human pancreatic cancer cells. World J Gastroenterol 2011; 17:4488-95. [PMID: 22110279 PMCID: PMC3218139 DOI: 10.3748/wjg.v17.i40.4488] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/30/2010] [Revised: 02/12/2011] [Accepted: 02/19/2011] [Indexed: 02/06/2023] Open
Abstract
AIM: To analyse αV integrin expression induced by gastrin in pancreatic cancer models.
METHODS: αV integrin mRNA expression in human pancreatic cancer cells was analysed using a “cancer genes” array and confirmed by real-time reverse transcription-polymerase chain reaction (PCR). Western blotting and semi-quantitative immunohistochemistry were used to examine protein levels in human pancreatic cancer cell lines and pancreatic tissues, respectively. The role of αV integrin on gastrin-induced cell adhesion was examined using blocking anti-αV integrin monoclonal antibodies. Adherent cells were quantified by staining with crystal violet.
RESULTS: Using a “cancer genes” array we identified αV integrin as a new gastrin target gene in human pancreatic cancer cells. A quantitative real-time PCR approach was used to confirm αV integrin gene expression. We also demonstrate that Src family kinases and the PI 3-kinase, two signalling pathways specifically activated by the CCK-2 receptor (CCK2R), are involved in gastrin-mediated αV integrin expression. In contrast, inhibition of the ERK pathway was without any effect on αV integrin expression induced by gastrin. Our results also show that gastrin modulates cell adhesion viaαV integrins. Indeed, in vitro adhesion assays performed on fibronectin show that gastrin significantly increases adhesion of pancreatic cancer cells. The use of blocking anti-αV integrin monoclonal antibodies completely reversed the increase in cell-substrate adhesion induced by gastrin. In addition, we showed in vivo that the targeted CCK2R expression in the pancreas of Elas-CCK2 mice, leads to the overexpression of αV integrin. This process may contribute to pancreatic tumour development observed in these transgenic animals.
CONCLUSION: αV integrin is a new gastrin target in pancreatic cancer models and contributes to gastrin effects on cell adhesion.
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Berna MJ, Seiz O, Nast JF, Benten D, Bläker M, Koch J, Lohse AW, Pace A. CCK1 and CCK2 receptors are expressed on pancreatic stellate cells and induce collagen production. J Biol Chem 2010; 285:38905-14. [PMID: 20843811 DOI: 10.1074/jbc.m110.125534] [Citation(s) in RCA: 61] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022] Open
Abstract
The gastrointestinal hormone cholecystokinin (CCK) can induce acute pancreatitis in rodents through its action on acinar cells. Treatment with CCK, in combination with other agents, represents the most commonly used model to induce experimental chronic pancreatitis. Pancreatic stellate cells (PSC) are responsible for pancreatic fibrosis and therefore play a predominant role in the genesis of chronic pancreatitis. However, it is not known whether PSC express CCK receptors. Using real time PCR techniques, we demonstrate that CCK1 and CCK2 receptors are expressed on rat PSC. Interestingly both CCK and gastrin significantly induced type I collagen synthesis. Moreover, both inhibit proliferation. These effects are comparable with TGF-β-stimulated PSC. Furthermore, the natural agonists CCK and gastrin induce activation of pro-fibrogenic pathways Akt, ERK, and Src. Using specific CCK1 and CCK2 receptor (CCK2R) inhibitors, we found that Akt activation is mainly mediated by CCK2R. Akt activation by CCK and gastrin could be inhibited by the PI3K inhibitor wortmannin. Activation of ERK and the downstream target Elk-1 could be inhibited by the MEK inhibitor U0126. These data suggest that CCK and gastrin have direct activating effects on PSC, are able to induce collagen synthesis in these cells, and therefore appear to be important regulators of pancreatic fibrogenesis. Furthermore, similar to TGF-β, both CCK and gastrin inhibit proliferation in PSC.
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Affiliation(s)
- Marc J Berna
- Universitätsklinikum Eppendorf, Medizinische Klinik I, 20246 Hamburg, Germany
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Zhou J, Zhang ZX, Li DC. Effects of lorglumide on growth and invasion of human pancreatic cancer cell line Mia PaCa-2 in vitro through the cholecystokinin-cholecystokinin-1 receptor pathway. Curr Ther Res Clin Exp 2010; 71:239-51. [PMID: 24688146 DOI: 10.1016/j.curtheres.2010.08.005] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 06/21/2010] [Indexed: 12/29/2022] Open
Abstract
BACKGROUND Cholecystokinin (CCK) has been found to be a growth stimulant through its special receptor pathway, especially for gastrointestinal malignancies. Although the CCK-1 receptor has been shown to be highly expressed in resected human pancreatic cancer samples, its role is less clear. OBJECTIVE The aim of this in vitro study was to investigate the CCK-1 receptor expression and the function of the CCK-CCK-1 receptor pathway in the human pancreatic adenocarcinoma cell line, Mia PaCa-2. METHODS The expression of the CCK-1 receptor in Mia PaCa-2 cells was detected by reverse-transcriptase polymerase chain reaction and flow cytometry. CCK-1 receptor agonist CCK-8S (the major transmitter form of CCK) and antagonist lorglumide were cultured respectively with Mia PaCa-2. Three groups were created for this study: CCK-8S group (Mia PaCa-2 cells treated with CCK-8S), lorglumide group (Mia PaCa-2 cells treated with lorglumide), and the control group (Mia PaCa-2 cells alone). Investigators were blinded to group designation. 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and flow cytometry were used to detect the cell growth, cell cycle, and apoptosis. Apoptosis index rate was measured by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling. Cell invasion ability was observed by invasion assay. Expression of matrix metalloproteinase-2 (MMP-2) was measured by Western blotting. RESULTS Mia PaCa-2 cells were found to express the CCK-1 receptor. Compared with the control group (70.2% [1.5%]), CCK-8S was associated with significant mean (SD) cell proliferation (85.1% [1.7%]; P = 0.039), and the ratio in the S stage of the cell cycle increased significantly (50.5% [1.7%] vs 42.2% [1.4%]; P = 0.021). CCK-8S was also associated with increased Mia PaCa-2 cell invasion ability (123.8 [1.7] vs 102.1 [5.8]; P = 0.005 vs control). Compared with the control group, lorglumide was associated with significantly inhibited cell growth (52.1% [1.8%]; P = 0.002) and cell invasion (77.6% [1.2%]; P = 0.003). Lorglumide also induced G0/G1 cell cycle arrest and apoptosis (27.1% [3-5%] vs 3-7% [0.6%]; P = 0.003 vs control). The change of invasion ability appeared to be mediated by MMP-2 expression, which was upregulated by CCK-8S and downregulated by lorglumide. CONCLUSION The findings of this in vitro study suggest that CCK may exert a trophic action on the Mia PaCa-2 cell line, while lorglumide inhibited the cell growth and invasion.
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Affiliation(s)
- Jin Zhou
- General Surgery Department, The First Affiliated Hospital of Soochow University, Suzhou, China
| | - Zi-Xiang Zhang
- General Surgery Department, The First Affiliated Hospital of Soochow University, Suzhou, China
| | - De-Chun Li
- General Surgery Department, The First Affiliated Hospital of Soochow University, Suzhou, China
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Ashurst HL, Varro A, Dimaline R. Regulation of mammalian gastrin/CCK receptor (CCK2R) expression in vitro and in vivo. Exp Physiol 2007; 93:223-36. [PMID: 17933865 PMCID: PMC2253704 DOI: 10.1113/expphysiol.2007.040683] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022]
Abstract
The gastrin/CCK receptor (CCK2R) mediates the physiological functions of gastrin in the stomach, including stimulation of acid secretion and cellular proliferation and migration, but little is known about the factors that regulate its expression. We identified endogenous CCK2R expression in several cell lines and used luciferase promoter-reporter constructs to define the minimal promoter required for transcription in human gastric adenocarcinoma, AGS, and rat gastric mucosa, RGM1, cells. Consensus binding sites for SP1, C/EBP and GATA were essential for activity. Following serum withdrawal from RGM1 and AR42J cells, endogenous CCK2R mRNA abundance and the activity of a CCK2R promoter-reporter construct were significantly elevated. Transcription of CCK2R was also increased in AGS-G(R) and RGM1 cells by gastrin through mechanisms partly dependent upon protein kinase C (PKC) and mitogen/extracellular signal-regulated kinase (MEK). Gastrin significantly increased endogenous CCK2R expression in RGM1 cells, and CCK2R protein expression was elevated in the stomach of hypergastrinaemic animals. In mice with cryoulcers in the acid-secreting mucosa, CCK2R expression increased progressively in the regenerating mucosa adjacent to the ulcer repair margin, evident at 6 days postinjury and maximal at 13 days. De novo expression of CCK2R was observed in the submucosa beneath the repairing ulcer crater 6-9 days postinjury. Many of the cells in mucosa and submucosa that expressed CCK2R in response to cryoinjury were identified as myofibroblasts, since they coexpressed vimentin and smooth muscle alpha-actin but not desmin. The data suggest that increased CCK2R expression might influence the outcome of epithelial inflammation or injury and that the response may be mediated in part by myofibroblasts.
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Affiliation(s)
- H Louise Ashurst
- Physiological Laboratory, School of Biomedical Sciences, University of Liverpool, Crown Street, Liverpool L69 3BX, UK
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Grabowska AM, Watson SA. Role of gastrin peptides in carcinogenesis. Cancer Lett 2007; 257:1-15. [PMID: 17698287 DOI: 10.1016/j.canlet.2007.06.017] [Citation(s) in RCA: 45] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/12/2007] [Revised: 06/25/2007] [Accepted: 06/28/2007] [Indexed: 01/22/2023]
Abstract
Gastrin gene expression is upregulated in a number of pre-malignant conditions and established cancer through a variety of mechanisms. Depending on the tissue where it is expressed and the level of expression, differential processing of the polypeptide product leads to the production of different biologically active peptides. In turn, acting through the classical CCK-2R receptor, CCK-2R isoforms and alternative receptors, these peptides trigger signalling pathways which influence the expression of downstream genes that affect cell survival, angiogenesis and invasion. Here we review this network of events, highlighting the importance of cellular context for interpreting the role of gastrin peptides and a possible role for gastrin in supporting the early stage of carcinogenesis.
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Affiliation(s)
- Anna M Grabowska
- Division of Pre-Clinical Oncology, D Floor, West Block, Queen's Medical Centre, University Hospital, Nottingham NG7 2UH, UK.
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