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. Statistical significance for qPCR and chromatin immunoprecipitation (ChIP)-qPCR was determined using a student’s t-test.
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 p-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.
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.
Competing Interest Statement
The authors have declared no competing interest.
Abbreviations
- BC
- Breast cancer
- ERα
- estrogen receptor alpha
- GDNF
- Glial cell-derived neurotrophic factor
- RET
- rearranged during transfection
- EGR1
- early growth response 1
- MAPK
- mitogen-activated protein kinase
- ERK 1/2
- extracellular signal-regulated kinases 1 and 2
- CRISPR
- Clustered Regularly Interspaced Short Palindromic Repeats
- dCAS9
- Endonuclear deficient CRISPR-associated protein 9
- ELK-1
- Ets Like-1
- TF
- Transcription factor
- TAM
- Tamoxifen
- TamS
- Tamoxifen sensitive
- TamR
- Tamoxifen resistant
- HER2
- human epidermal growth factor receptor 2
- EGFR
- Epidermal growth factor receptor