COUP-TFII in pancreatic adenocarcinoma: Clinical implication for patient survival and tumor progression
Corresponding Author
Simone Polvani
Department of Experimental and Clinical Biomedical Sciences, University of Florence, Firenze, Italy
Correspondence to: Simone Polvani, Department of Experimental and Clinical Biomedical Sciences, viale Pieraccini 6, 50139 Firenze, Italy, E-mail: [email protected]Search for more papers by this authorMirko Tarocchi
Department of Experimental and Clinical Biomedical Sciences, University of Florence, Firenze, Italy
Search for more papers by this authorSara Tempesti
Department of Experimental and Clinical Biomedical Sciences, University of Florence, Firenze, Italy
Search for more papers by this authorTommaso Mello
Department of Experimental and Clinical Biomedical Sciences, University of Florence, Firenze, Italy
Search for more papers by this authorElisabetta Ceni
Department of Experimental and Clinical Biomedical Sciences, University of Florence, Firenze, Italy
Search for more papers by this authorFrancesca Buccoliero
Department of Experimental and Clinical Biomedical Sciences, University of Florence, Firenze, Italy
Search for more papers by this authorMassimo D'Amico
Department of Experimental and Clinical Medicine, University of Florence, Firenze, Italy
Search for more papers by this authorVieri Boddi
Department of Public Health and Epidemiology, University of Florence, Firenze, Italy
Search for more papers by this authorMarco Farsi
Chirurgia Generale Oncologica, Azienda Ospedaliero Universitaria Careggi, Firenze, Italy
Search for more papers by this authorSilvia Nesi
Chirurgia Generale Oncologica, Azienda Ospedaliero Universitaria Careggi, Firenze, Italy
Search for more papers by this authorGabriella Nesi
Department of Translational Medicine and Surgery, University of Florence, Firenze, Italy
Search for more papers by this authorStefano Milani
Department of Experimental and Clinical Biomedical Sciences, University of Florence, Firenze, Italy
Search for more papers by this authorAndrea Galli
Department of Experimental and Clinical Biomedical Sciences, University of Florence, Firenze, Italy
Search for more papers by this authorCorresponding Author
Simone Polvani
Department of Experimental and Clinical Biomedical Sciences, University of Florence, Firenze, Italy
Correspondence to: Simone Polvani, Department of Experimental and Clinical Biomedical Sciences, viale Pieraccini 6, 50139 Firenze, Italy, E-mail: [email protected]Search for more papers by this authorMirko Tarocchi
Department of Experimental and Clinical Biomedical Sciences, University of Florence, Firenze, Italy
Search for more papers by this authorSara Tempesti
Department of Experimental and Clinical Biomedical Sciences, University of Florence, Firenze, Italy
Search for more papers by this authorTommaso Mello
Department of Experimental and Clinical Biomedical Sciences, University of Florence, Firenze, Italy
Search for more papers by this authorElisabetta Ceni
Department of Experimental and Clinical Biomedical Sciences, University of Florence, Firenze, Italy
Search for more papers by this authorFrancesca Buccoliero
Department of Experimental and Clinical Biomedical Sciences, University of Florence, Firenze, Italy
Search for more papers by this authorMassimo D'Amico
Department of Experimental and Clinical Medicine, University of Florence, Firenze, Italy
Search for more papers by this authorVieri Boddi
Department of Public Health and Epidemiology, University of Florence, Firenze, Italy
Search for more papers by this authorMarco Farsi
Chirurgia Generale Oncologica, Azienda Ospedaliero Universitaria Careggi, Firenze, Italy
Search for more papers by this authorSilvia Nesi
Chirurgia Generale Oncologica, Azienda Ospedaliero Universitaria Careggi, Firenze, Italy
Search for more papers by this authorGabriella Nesi
Department of Translational Medicine and Surgery, University of Florence, Firenze, Italy
Search for more papers by this authorStefano Milani
Department of Experimental and Clinical Biomedical Sciences, University of Florence, Firenze, Italy
Search for more papers by this authorAndrea Galli
Department of Experimental and Clinical Biomedical Sciences, University of Florence, Firenze, Italy
Search for more papers by this authorAbstract
Despite the accumulating knowledge of alterations in pancreatic cancer molecular pathways, no substantial improvements in the clinical prognosis have been made and this malignancy continues to be a leading cause of cancer death in the Western World. The orphan nuclear receptor COUP-TFII is a regulator of a wide range of biological processes and it may exert a pro-oncogenic role in cancer cells; interestingly, indirect evidences suggest that the receptor could be involved in pancreatic cancer. The aim of this study was to evaluate the expression of COUP-TFII in human pancreatic tumors and to unveil its role in the regulation of pancreatic tumor growth. We evaluated COUP-TFII expression by immunohistochemistry on primary samples. We analyzed the effect of the nuclear receptor silencing in human pancreatic cancer cells by means of shRNA expressing cell lines. We finally confirmed the in vitro results by in vivo experiments on nude mice. COUP-TFII is expressed in 69% of tested primary samples and correlates with the N1 and M1 status and clinical stage; Kaplan–Meier and Cox regression analysis show that it may be an independent prognostic factor of worst outcome. In vitro silencing of COUP-TFII reduces the cell growth and invasiveness and it strongly inhibits angiogenesis, an effect mediated by the regulation of VEGF-C. In nude mice, COUP-TFII silencing reduces tumor growth by 40%. Our results suggest that COUP-TFII might be an important regulator of the behavior of pancreatic adenocarcinoma, thus representing a possible new target for pancreatic cancer therapy.
Abstract
What's new?
The orphan nuclear receptor COUP-TFII influences many biological processes, and may play a role in pancreatic cancer. In this study, the authors discovered that COUP-TFII expression predicts poor outcome in pancreatic cancer. By silencing COUP-TFII in tumor cells, they were able to slow tumor growth and inhibit angiogenesis. The receptor may be an attractive target for therapy, they speculate, if a ligand can be identified that modulates its activity.
Supporting Information
Additional Supporting Information may be found in the online version of this article.
| Filename | Description |
|---|---|
| ijc28502-sup-0001-suppinfo01.pdf166.1 KB | Supplementary informations |
| ijc28502-sup-0002-suppfig1.tif9.5 MB | Supplementary Figure 1. COUP-TFII expression in primary samples. (A) Representative images of COUP-TFII (left) and cytokeratin 19 (right) staining in PDAC primary samples. (B) COUP-TFII staining in healthy pancreas showing a signal in Islets of Langherans (mostly cytoplasmic) and sparse acinar cells with low COUP-TFII expression. Original magnification 200x. |
| ijc28502-sup-0003-suppfig2.tif311.1 KB | Supplementary Figure 2. COUP-TFII mRNA expression in pancreatic cancer cell lines. (A) qPCR quantification of COUP-TFII mRNA expression levels 48h after plating in vitro in the indicated human pancreatic cancer cell lines compared to a pool of mRNA obtained from healthy pancreas. (B) COUP-TFI expression in the same samples as in (A). (C) Relative expression of COUP-TFI and COUP-TFII in the same samples as in (A). Data were normalized for GAPDH and in (C) COUP-TFI expression was further normalized to COUP-TFII. |
| ijc28502-sup-0004-suppfig3.tif284.3 KB | Supplementary Figure 3. COUP-TFII protein expression in pancreatic cancer cell lines. Representative Western Blot and densitometric analysis of COUP-TFII expression in vitro in PANC-1, MiaPaca2, PL-45, Su.86.86, BxPC3, Capan2, and hTERT-HPNE cell lines. COUP-TFII expression was normalized to hTERT-HPNE expression levels. Specifically, data were normalized to β-tubulin and COUP-TFII/β-tubulin ratio in hTERT-HPNE was set as 1. *p<0.05. |
| ijc28502-sup-0005-suppfig4.tif546.6 KB | Supplementary Figure 4. COUP-TFII silencing reduces proliferation in vitro. Pancreatic cancer cells were transiently transfected with pTER-shCOUP or pTER-NEG plasmids. 48h after transfection, the cells were collected and counted. WB for COUP-TFII (A) clearly confirmed that the NR was effectively down-regulated after transfection in all tested cell lines; however only in 4 (PANC-1, PL-45, MiaPaca2, BxPC3) out of 6 pancreatic cancer cells tested we demonstrated a reduced proliferation (B), suggesting that COUP-TFII role on proliferation may depend on the cross-talk with other factors. * p<0.05, ** p<.01. |
| ijc28502-sup-0006-suppfig5.tif1.4 MB | Supplementary Figure 5. Characterization of M2-shCOUP cells. (A) Representative qPCR evaluation of COUP-TFII and the closely homologue COUP-TFI mRNA at various time in vitro. In Dox treated cells, COUP-TFII is downregulated whereas expression of COUP-TFI is unaltered. RNA extraction and qPCR methods are described in Supplementary Information. (B) Representative WB and densitometric analysis of COUP-TFII protein. b-tubulin was used as internal loading control. (C) COUP-TFII expression as detected by IF with a specific mouse monoclonal antibody 48h after Dox addition to the medium. Dox induces a reduction in COUP-TFII expression without altering its cellular localization. Cyan is DAPI, Red is COUP-TFII. ** p<0.01. |
| ijc28502-sup-0007-suppfig6.tif1 MB | Supplementary Figure 6. Characterization of M2-shNEG cells. (A) Representative WB of COUP-TFII protein at various time after Dox treatment. b-tubulin was used as internal loading control. (B) COUP-TFII expression as detected by IF with a specific monoclonal antibody 48h after Dox addition to the medium. COUP-TFII expression and cellular localization is similar in treated and untreated M2-shNEG cells. |
| ijc28502-sup-0008-suppfig7.tif177.3 KB | Supplementary Figure 7. COUP-TFII role in cell death and cell cycle. (A) Percentage of death cells at 24 to 72 hours after induction of cell silencing in M2-shCOUP cells. Death cells were evaluated by Trypan Blue exclusion test; values represent the percentage of death cells respective to total number of cells in the same sample. Data refer to the results reported in Figure 2. (B) Caspase 3/7 activity at 48h after COUP-TFII silencing in M2-shCOUP cells. (C) Representative WB of E2F1 in M2-shCOUP cells 48h after silencing showing the reduced expression of E2F1. |
| ijc28502-sup-0009-suppfig8.tif135.8 KB | Supplementary Figure 8. Effect of COUP-TFII silencing on cell growth and invasion. (A) Anchorage-independent growth in PANC-shCOUP with and without Dox. The cells were treated for 48h with 1 mg/mL Dox then 2x104 cells were mixed with DMEM/10% FBS containing 0.3% agarose and layered over a solid base of 0.5% agarose in the same medium. Cells were cultured in the presence or absence of Dox for two weeks at 37°C then counted. Induction of RNAi significantly reduces colonies formation in PANC-shCOUP. (B) COUP-TFII specific downregulation in PANC-shCOUP cells reduces tumor cells invasion in a modified Boyden Chamber assay. * p<0.05, ** p<0.01. |
| ijc28502-sup-0010-suppfig9.tif321.9 KB | Supplementary Figure 9. COUP-TFII overexpression in Su.86.86 cells. To confirm the results obtained in silenced cells, a COUP-TFII expression plasmid was transiently transfected in the low expressing Su.86.86 cells. (A) Cell cycle analysis 48h after transfection clearly show the COUP-TFII involvement in cell cycle with an increase of cells in the G2/M and G1 phases. Right Panel Control M2-shCOUP, left panel Dox treated M2-shCOUP cells. (B) COUP-TFII up-regulation increases tumor cell invasion in a modified Boyden chamber assay. * p<0.05. |
| ijc28502-sup-0011-suppfig10.tif87 KB | Supplementary Figure 10. COUP-TFII regulates the expression of genes involved in stemness. qPCR evaluation of OCT-4, Sox2, and Nanog mRNA expression in M2-shCOUP cells 48h after Dox addition to the medium. * p<0.05, ** p<0.01. |
| ijc28502-sup-0012-suppfig11.tif3.9 MB | Supplementary Figure 11. RNAi induction has no effect on tumor growth. M2-shNEG tumor-bearing nude-Foxn1nu/nu mice were administered a Dox-containing chow for 4 weeks. Control mice received normal diet. (A) Growth curves of M2-shNEG xenografts in silenced and control groups. (B) Boxplot distribution of tumor masses at the end of the experiment and representative tumor masses from control and treated mice. (C) Representative IHC for PCNA, activated caspase 3 and CD34 from control and silenced mice. Original magnification 200x. (D) Quantitative analysis of the immunohistochemical results. |
| ijc28502-sup-0013-suppfig12.tif6.4 MB | Supplementary Figure 12. Expression of VEGF-C, in vitro and in vivo, and of Lyve-1 in M2-shCOUP xenograft tumors. (A) Expression of VEGF-C mRNA was evaluated by qPCR 48h after silencing in M2shCOUP and M2-shNEG cells in vitro. (B-C) Representative VEGF-C (B) and Lyve-1+ lymphatic vessels (C) IHC images of control (left panels) and Dox-treated (right panels) M2-shCOUP tumor-bearing mice, showing a reduction of VEGF-C and Lyve-1 staining in tumors where COUP-TFII was silenced. Original magnification 200x. (D) Lymphatic vessels quantification on Lyve-1 positive areas demonstrating a statistical significant reduction of lymphangiogenesis in COUP-TFII silenced tumors. * p<0.05. |
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