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GDNF-RET signaling in ER-positive breast cancers is a key determinant of response and resistance to aromatase inhibitors.

ABSTRACT: Most breast cancers at diagnosis are estrogen receptor-positive (ER(+)) and depend on estrogen for growth and survival. Blocking estrogen biosynthesis by aromatase inhibitors has therefore become a first-line endocrine therapy for postmenopausal women with ER(+) breast cancers. Despite providing substantial improvements in patient outcome, aromatase inhibitor resistance remains a major clinical challenge. The receptor tyrosine kinase, RET, and its coreceptor, GFR?1, are upregulated in a subset of ER(+) breast cancers, and the RET ligand, glial-derived neurotrophic factor (GDNF) is upregulated by inflammatory cytokines. Here, we report the findings of a multidisciplinary strategy to address the impact of GDNF-RET signaling in the response to aromatase inhibitor treatment. In breast cancer cells in two-dimensional and three-dimensional culture, GDNF-mediated RET signaling is enhanced in a model of aromatase inhibitor resistance. Furthermore, GDNF-RET signaling promoted the survival of aromatase inhibitor-resistant cells and elicited resistance in aromatase inhibitor-sensitive cells. Both these effects were selectively reverted by the RET kinase inhibitor, NVP-BBT594. Gene expression profiling in ER(+) cancers defined a proliferation-independent GDNF response signature that prognosed poor patient outcome and, more importantly, predicted poor response to aromatase inhibitor treatment with the development of resistance. We validated these findings by showing increased RET protein expression levels in an independent cohort of aromatase inhibitor-resistant patient specimens. Together, our results establish GDNF-RET signaling as a rational therapeutic target to combat or delay the onset of aromatase inhibitor resistance in breast cancer.

SUBMITTER: Morandi A

PROVIDER: S-EPMC3686594 | biostudies-literature | 2013 Jun

REPOSITORIES: biostudies-literature

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GDNF-RET signaling in ER-positive breast cancers is a key determinant of response and resistance to aromatase inhibitors.

Morandi Andrea A Martin Lesley-Ann LA Gao Qiong Q Pancholi Sunil S Mackay Alan A Robertson David D Zvelebil Marketa M Dowsett Mitch M Plaza-Menacho Ivan I Isacke Clare M CM

Cancer research 20130506 12

Most breast cancers at diagnosis are estrogen receptor-positive (ER(+)) and depend on estrogen for growth and survival. Blocking estrogen biosynthesis by aromatase inhibitors has therefore become a first-line endocrine therapy for postmenopausal women with ER(+) breast cancers. Despite providing substantial improvements in patient outcome, aromatase inhibitor resistance remains a major clinical challenge. The receptor tyrosine kinase, RET, and its coreceptor, GFRα1, are upregulated in a subset o ...[more]

PMID: 23650283

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Project description:BackgroundRearranged 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.MethodsTo 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.ResultsGDNF-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.ConclusionOutcomes 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.

Project description:BackgroundRearranged during transfection (RET) gene fusions are a validated target in non-small-cell lung cancer (NSCLC). RET-selective inhibitors selpercatinib (LOXO-292) and pralsetinib (BLU-667) recently demonstrated favorable antitumor activity and safety profiles in advanced RET fusion-positive NSCLC, and both have received approval by the US Food and Drug Administration for this indication. Insights into mechanisms of resistance to selective RET inhibitors remain limited.Patients and methodsThis study was performed at five institutions. Tissue and/or cell-free DNA was obtained from patients with RET fusion-positive NSCLC after treatment with selpercatinib or pralsetinib and assessed by next-generation sequencing (NGS) or MET FISH.ResultsWe analyzed a total of 23 post-treatment tissue and/or plasma biopsies from 18 RET fusion-positive patients who received an RET-selective inhibitor (selpercatinib, n = 10; pralsetinib, n = 7; pralsetinib followed by selpercatinib, n = 1, with biopsy after each inhibitor). Three cases had paired tissue and plasma samples, of which one also had two serial resistant tissue specimens. The median progression-free survival on RET inhibitors was 6.3 months [95% confidence interval 3.6-10.8 months]. Acquired RET mutations were identified in two cases (10%), both affecting the RET G810 residue in the kinase solvent front. Three resistant cases (15%) harbored acquired MET amplification without concurrent RET resistance mutations, and one specimen had acquired KRAS amplification. No other canonical driver alterations were identified by NGS. Among 16 resistant tumor specimens, none had evidence of squamous or small-cell histologic transformation.ConclusionsRET solvent front mutations are a recurrent mechanism of RET inhibitor resistance, although they occurred at a relatively low frequency. The majority of resistance to selective RET inhibition may be driven by RET-independent resistance such as acquired MET or KRAS amplification. Next-generation RET inhibitors with potency against RET resistance mutations and combination strategies are needed to effectively overcome resistance in these patients.

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GDNF-RET signaling in ER-positive breast cancers is a key determinant of response and resistance to aromatase inhibitors.

Publications

GDNF-RET signaling in ER-positive breast cancers is a key determinant of response and resistance to aromatase inhibitors.

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