Acquired CYP19A1 amplification is an early specific mechanism of aromatase inhibitor resistance in ERα metastatic breast cancer

Abstract

Tumor evolution is shaped by many variables, potentially involving external selective pressures induced by therapies. After surgery, patients with estrogen receptor (ERα)-positive breast cancer are treated with adjuvant endocrine therapy, including selective estrogen receptor modulators (SERMs) and/or aromatase inhibitors (AIs). However, more than 20% of patients relapse within 10 years and eventually progress to incurable metastatic disease. Here we demonstrate that the choice of therapy has a fundamental influence on the genetic landscape of relapsed diseases. We found that 21.5% of AI-treated, relapsed patients had acquired CYP19A1 (encoding aromatase) amplification (CYP19A1^amp). Relapsed patients also developed numerous mutations targeting key breast cancer-associated genes, including ESR1 and CYP19A1. Notably, CYP19A1^amp cells also emerged in vitro, but only in AI-resistant models. CYP19A1 amplification caused increased aromatase activity and estrogen-independent ERα binding to target genes, resulting in CYP19A1^amp cells showing decreased sensitivity to AI treatment. These data suggest that AI treatment itself selects for acquired CYP19A1^amp and promotes local autocrine estrogen signaling in AI-resistant metastatic patients.

Figure 1. Clinical treatments shape cancer genetic evolution

A) Clinical discovery cohorts and sample design used in the study. CNA profiles for the CYP19A1 and ESR1 loci in the first relapse of patients treated with adjuvant Tamoxifen or AI mono-therapy B) Clinical discovery cohorts and sample design used in the study. CNA profiles for the CYP19A1 and ESR1 loci in the first relapse of patients treated with adjuvant Tamoxifen or AI mono-therapy. ESR1 data can be found in Supplementary figure 4 C) PDXs cohort. CNA profiles for the CYP19A1 and ESR1 loci in PDXs from patient treated with Tamoxifen or AI. ESR1 data can be found in Supplementary figure 4.

Figure 2. AI resistant metastases develop cluster amplification of the aromatase gene

A) Double-colour FISH analyses using 15 Alpha Satellite)/ CYP19A1 probes identify cluster amplification of the CYP19A1 locus B) Ratio of amplification obtained by computing CYP19A1/15α signals in 30 representative individual cancer cells from each validated tumor sample C) Breast Cancer mutations in Tamoxifen and AI treated metastatic samples D) Boxplots for individual patient/mutations in the two cohorts.

Figure 3. CYP19A1 amplification leads to increased aromatase activity

A) CNA profiles for the CYP19A1 and ESR1 loci in treatment naïve (MCF7) and estrogen deprived (LTED) cells B) DNA-FISH using CYP19A1-centered probes identifies widespread cluster amplification in LTED cells C-D) LTED clones uniquely upregulate aromatase mRNA and protein levels. Dot-blots represent mean and S.E.M. from 3 independent experiments. Western Blot have been cropped near the specific band. Full blot can be found in Supplementary Figure 11 E) Single-cell RNA-FISH highlight heterogeneity in aromatase expression. Asterisks denote a significant difference after Mann-Whitney test ****=P<10^-20 F) CYP19A1^amp cells transcriptionally activate estrogen-target genes in response to aromatizable androgens. AI treatment blocks transcriptional activation. Dot-blots represent mean and S.E.M. from 5 independent experiments. Asterisks denote a significant difference after two-way Anova or one-way ANOVA (bottom right panel) *=P<0.05.

Figure 4. CYP19A1^amp cells endogenously activate ERα and develop tolerance to AI

A) ChIP-seq heatmaps for ERα in treatment naïve (MCF7) and estrogen deprived (LTED) cells. Binding sites have been assigned to three clusters. The average profile of each cluster is reported in the central panels. Examples of ERα enrichment near important estrogen target genes are shown in the insets (right panels). B) MCF7 treatment with AI in the presence of estradiol. Dot-blots represent mean and S.E.M. from 3 independent experiments. C) LTED treatment with AI in the absence of estradiol. Dot-blots represent mean and S.D. from 3 independent experiments. Asterisks denote a significant difference after Student T-test *=P<0.05) D) CYP19A1 depleted LTED cells have increased sensitivity to AI. Dot-blots represent mean and S.D. from 3 independent experiments. Asterisks denote a significant difference after two-way ANOVA and Bonferroni post-test *,**,***,****= P<0.05, 0.01, 0.001 and 0.0001 E) CYP19A1 over-expressing cells have a growth advantage compared to WT in the absence of estradiol. Relative increase in growth rate is shown by plotting the ratio between the growth of CYP19A1 over-expressing cells to CYP19A1 WT cells under letrozole challenge. Dot-blots represent mean and S.E.M. from 3 independent experiments. Asterisks denote a significant difference after two-way ANOVA and Bonferroni post-test *=P<0.05 F) CYP19A1^amp LTED respond to low levels of irreversible steroidal AI. Dots represent mean and 95% C.I of 4 independent replicates. Asterisks denote a significant difference after two-way ANOVA and Bonferroni post-test ****= P<0.0001 G) Kaplan-Meier curve showing time to first relapse (TTF) for AI treated patients stratified retrospectively for CYP19A1 amplification. Dotted lines represent the 95% confidence intervals H) Working hypothesis for therapy-specific breast cancer progression. Genetic and epigenetic changes collaborate to increase tumor fitness by creating an estrogen-independent niche at metastatic sites treated with AI therapy.