ABSTRACT
Background Breast tumors overexpressing human epidermal growth factor receptor (HER2) confer intrinsic resistance to endocrine therapy (ET), and patients with HER2/ estrogen receptor-positive (HER2+/HR+) breast cancer (BCa) are less responsive to ET than HER2−/ER+. However, real-world evidence reveals that a large subset of HER2+/ER+ patients receive ET as monotherapy, positioning this treatment pattern as a clinical challenge. In the present study, we developed and characterized two distinct in vitro models of ET-resistant (ETR) HER2+/ER+ BCa to identify possible therapeutic vulnerabilities.
Methods To mimic ETR to aromatase inhibitors (AI), we developed two long-term estrogen-deprived (LTED) cell lines from BT-474 (BT474) and MDA-MB-361 (MM361). Growth assays, PAM50 molecular subtyping, genomic and transcriptomic analyses, followed by validation and functional studies, were used to identify targetable differences between ET-responsive parental and ETR-LTED HER2+/ER+ cells.
Results Compared to their parental cells, MM361 LTEDs grew faster, lost ER, and increased HER2 expression, whereas BT474 LTEDs grew slower and maintained ER and HER2 expression. Both LTED variants had reduced responsiveness to fulvestrant. Whole-genome sequencing of the more aggressive MM361 LTED model system identified exonic mutations in genes encoding transcription factors and chromatin modifiers. Single-cell RNA sequencing demonstrated a shift towards non-luminal phenotypes, and revealed metabolic remodeling of MM361 LTEDs, with upregulated lipid metabolism and antioxidant genes associated with ferroptosis, including GPX4. Combining the GPX4 inhibitor RSL3 with anti-HER2 agents induced significant cell death in both the MM361 and BT474 LTEDs.
Conclusions The BT474 and MM361 AI-resistant models capture distinct phenotypes of HER2+/ER+ BCa and identify altered lipid metabolism and ferroptosis remodeling as vulnerabilities of this type of ETR BCa.
Competing Interest Statement
SB, HS, LJ, ST, DM, MB, MP, and MDM have nothing to declare. RAB consults for AstraZeneca LP and Boehringer Ingelheim, and is the Chief Scientific Officer and Managing Member of Onco-Mind, LLC, which owns patents related to cancer precision medicine. RBR is an Associate Editor for the Journal of the Endocrine Society.
Footnotes
ABBREVIATIONS
- 4-HNE
- 4-hydroxynonenal
- BCa
- Breast cancer
- BT474
- BT-474
- CSS
- Charcoal-stripped bovine serum
- DEG
- Differential expressed gene
- DMFS
- Distant metastasis-free survival
- E2
- 17β-estradiol
- EMT
- epithelial-mesenchymal transition
- ER
- Estrogen receptor
- ET
- Endocrine therapy
- ETR
- Endocrine therapy-resistant
- FDR
- False discovery rate
- FSP1
- Ferroptosis suppressor protein 1
- GSEA
- Gene set enrichment assay
- HER2
- human epidermal growth factor receptor 2
- HR
- Hormone receptor
- ICI
- fulvestrant
- IMEM
- Improved minimum essential medium
- LTED
- Long-term estrogen deprivation
- MDA
- Malondialdehyde
- MM361
- MDA-MB-361
- MSigDB
- Molecular signature database
- OS
- Overall survival
- PFS
- Progression-free survival
- qRT-PCR
- Real-time PCR
- RFS
- Relapse-free survival
- SBS
- Single base substitution
- scRNAseq
- Single-cell RNA sequencing
- SERD
- Selective estrogen receptor degrader
- TR
- Transcriptional regulator
- TP
- Trastuzumab + pertuzumab
- WGS
- whole genome sequencing