GDNF-RET signaling and EGR1 form a positive feedback loop that promotes tamoxifen resistance via cyclin D1
Abstract Background Rearranged during transfection (RET) tyrosine kinase signaling has been previously implicated in endocrine resistant breast cancer, however the mechanism by which this signaling cascade promotes resistance is currently not well described. We recently reported that glial cell-deri...
| Main Authors: | , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
BMC
2023-02-01
|
| Series: | BMC Cancer |
| Subjects: | |
| Online Access: | https://doi.org/10.1186/s12885-023-10559-1 |
| _version_ | 1811165868830228480 |
|---|---|
| author | Brooke A. Marks Ilissa M. Pipia Chinatsu Mukai Sachi Horibata Edward J. Rice Charles G. Danko Scott A. Coonrod |
| author_facet | Brooke A. Marks Ilissa M. Pipia Chinatsu Mukai Sachi Horibata Edward J. Rice Charles G. Danko Scott A. Coonrod |
| author_sort | Brooke A. Marks |
| collection | DOAJ |
| description | Abstract Background Rearranged during transfection (RET) tyrosine kinase signaling has been previously implicated in endocrine resistant breast cancer, however the mechanism by which this signaling cascade promotes resistance is currently not well described. We recently reported that glial cell-derived neurotrophic factor (GDNF)-RET signaling appears to promote a positive feedback loop with the transcription factor early growth response 1 (EGR1). Here we investigate the mechanism behind this feedback loop and test the hypothesis that GDNF-RET signaling forms a regulatory loop with EGR1 to upregulate cyclin D1 (CCND1) transcription, leading to cell cycle progression and tamoxifen resistance. Methods To gain a better understanding of the GDNF-RET-EGR1 resistance mechanism, we studied the GDNF-EGR1 positive feedback loop and the role of GDNF and EGR1 in endocrine resistance by modulating their transcription levels using CRISPR-dCAS9 in tamoxifen sensitive (TamS) and tamoxifen resistant (TamR) MCF-7 cells. Additionally, we performed kinetic studies using recombinant GDNF (rGDNF) treatment of TamS cells. Finally, we performed cell proliferation assays using rGDNF, tamoxifen (TAM), and Palbociclib treatments in TamS cells. Statistical significance for qPCR and chromatin immunoprecipitation (ChIP)-qPCR experiments were determined using a student’s paired t-test and statistical significance for the cell viability assay was a one-way ANOVA. Results GDNF-RET signaling formed a positive feedback loop with EGR1 and also downregulated estrogen receptor 1 (ESR1) transcription. Upregulation of GDNF and EGR1 promoted tamoxifen resistance in TamS cells and downregulation of GDNF promoted tamoxifen sensitivity in TamR cells. Additionally, we show that rGDNF treatment activated GDNF-RET signaling in TamS cells, leading to recruitment of phospho-ELK-1 to the EGR1 promoter, upregulation of EGR1 mRNA and protein, binding of EGR1 to the GDNF and CCND1 promoters, increased GDNF protein expression, and subsequent upregulation of CCND1 mRNA levels. We also show that inhibition of cyclin D1 with Palbociclib, in the presence of rGDNF, decreases cell proliferation and resensitizes cells to TAM. Conclusion Outcomes from these studies support the hypotheses that GDNF-RET signaling forms a positive feedback loop with the transcription factor EGR1, and that GDNF-RET-EGR1 signaling promotes endocrine resistance via signaling to cyclin D1. Inhibition of components of this signaling pathway could lead to therapeutic insights into the treatment of endocrine resistant breast cancer. |
| first_indexed | 2024-04-10T15:43:24Z |
| format | Article |
| id | doaj.art-a513dee986ef4a7189c12bc44866baf1 |
| institution | Directory Open Access Journal |
| issn | 1471-2407 |
| language | English |
| last_indexed | 2024-04-10T15:43:24Z |
| publishDate | 2023-02-01 |
| publisher | BMC |
| record_format | Article |
| series | BMC Cancer |
| spelling | doaj.art-a513dee986ef4a7189c12bc44866baf12023-02-12T12:14:55ZengBMCBMC Cancer1471-24072023-02-0123111410.1186/s12885-023-10559-1GDNF-RET signaling and EGR1 form a positive feedback loop that promotes tamoxifen resistance via cyclin D1Brooke A. Marks0Ilissa M. Pipia1Chinatsu Mukai2Sachi Horibata3Edward J. Rice4Charles G. Danko5Scott A. Coonrod6Department of Biomedical and Biological Sciences, College of Veterinary Medicine, Cornell UniversityBaker Institute for Animal Health, College of Veterinary Medicine, Cornell UniversityBaker Institute for Animal Health, College of Veterinary Medicine, Cornell UniversityDepartment of Biomedical and Biological Sciences, College of Veterinary Medicine, Cornell UniversityBaker Institute for Animal Health, College of Veterinary Medicine, Cornell UniversityDepartment of Biomedical and Biological Sciences, College of Veterinary Medicine, Cornell UniversityDepartment of Biomedical and Biological Sciences, College of Veterinary Medicine, Cornell UniversityAbstract Background Rearranged during transfection (RET) tyrosine kinase signaling has been previously implicated in endocrine resistant breast cancer, however the mechanism by which this signaling cascade promotes resistance is currently not well described. We recently reported that glial cell-derived neurotrophic factor (GDNF)-RET signaling appears to promote a positive feedback loop with the transcription factor early growth response 1 (EGR1). Here we investigate the mechanism behind this feedback loop and test the hypothesis that GDNF-RET signaling forms a regulatory loop with EGR1 to upregulate cyclin D1 (CCND1) transcription, leading to cell cycle progression and tamoxifen resistance. Methods To gain a better understanding of the GDNF-RET-EGR1 resistance mechanism, we studied the GDNF-EGR1 positive feedback loop and the role of GDNF and EGR1 in endocrine resistance by modulating their transcription levels using CRISPR-dCAS9 in tamoxifen sensitive (TamS) and tamoxifen resistant (TamR) MCF-7 cells. Additionally, we performed kinetic studies using recombinant GDNF (rGDNF) treatment of TamS cells. Finally, we performed cell proliferation assays using rGDNF, tamoxifen (TAM), and Palbociclib treatments in TamS cells. Statistical significance for qPCR and chromatin immunoprecipitation (ChIP)-qPCR experiments were determined using a student’s paired t-test and statistical significance for the cell viability assay was a one-way ANOVA. Results GDNF-RET signaling formed a positive feedback loop with EGR1 and also downregulated estrogen receptor 1 (ESR1) transcription. Upregulation of GDNF and EGR1 promoted tamoxifen resistance in TamS cells and downregulation of GDNF promoted tamoxifen sensitivity in TamR cells. Additionally, we show that rGDNF treatment activated GDNF-RET signaling in TamS cells, leading to recruitment of phospho-ELK-1 to the EGR1 promoter, upregulation of EGR1 mRNA and protein, binding of EGR1 to the GDNF and CCND1 promoters, increased GDNF protein expression, and subsequent upregulation of CCND1 mRNA levels. We also show that inhibition of cyclin D1 with Palbociclib, in the presence of rGDNF, decreases cell proliferation and resensitizes cells to TAM. Conclusion Outcomes from these studies support the hypotheses that GDNF-RET signaling forms a positive feedback loop with the transcription factor EGR1, and that GDNF-RET-EGR1 signaling promotes endocrine resistance via signaling to cyclin D1. Inhibition of components of this signaling pathway could lead to therapeutic insights into the treatment of endocrine resistant breast cancer.https://doi.org/10.1186/s12885-023-10559-1Breast cancerEstrogen receptor alphaTamoxifen resistanceGDNFRET signalingEGR1 |
| spellingShingle | Brooke A. Marks Ilissa M. Pipia Chinatsu Mukai Sachi Horibata Edward J. Rice Charles G. Danko Scott A. Coonrod GDNF-RET signaling and EGR1 form a positive feedback loop that promotes tamoxifen resistance via cyclin D1 BMC Cancer Breast cancer Estrogen receptor alpha Tamoxifen resistance GDNF RET signaling EGR1 |
| title | GDNF-RET signaling and EGR1 form a positive feedback loop that promotes tamoxifen resistance via cyclin D1 |
| title_full | GDNF-RET signaling and EGR1 form a positive feedback loop that promotes tamoxifen resistance via cyclin D1 |
| title_fullStr | GDNF-RET signaling and EGR1 form a positive feedback loop that promotes tamoxifen resistance via cyclin D1 |
| title_full_unstemmed | GDNF-RET signaling and EGR1 form a positive feedback loop that promotes tamoxifen resistance via cyclin D1 |
| title_short | GDNF-RET signaling and EGR1 form a positive feedback loop that promotes tamoxifen resistance via cyclin D1 |
| title_sort | gdnf ret signaling and egr1 form a positive feedback loop that promotes tamoxifen resistance via cyclin d1 |
| topic | Breast cancer Estrogen receptor alpha Tamoxifen resistance GDNF RET signaling EGR1 |
| url | https://doi.org/10.1186/s12885-023-10559-1 |
| work_keys_str_mv | AT brookeamarks gdnfretsignalingandegr1formapositivefeedbackloopthatpromotestamoxifenresistanceviacyclind1 AT ilissampipia gdnfretsignalingandegr1formapositivefeedbackloopthatpromotestamoxifenresistanceviacyclind1 AT chinatsumukai gdnfretsignalingandegr1formapositivefeedbackloopthatpromotestamoxifenresistanceviacyclind1 AT sachihoribata gdnfretsignalingandegr1formapositivefeedbackloopthatpromotestamoxifenresistanceviacyclind1 AT edwardjrice gdnfretsignalingandegr1formapositivefeedbackloopthatpromotestamoxifenresistanceviacyclind1 AT charlesgdanko gdnfretsignalingandegr1formapositivefeedbackloopthatpromotestamoxifenresistanceviacyclind1 AT scottacoonrod gdnfretsignalingandegr1formapositivefeedbackloopthatpromotestamoxifenresistanceviacyclind1 |