CITED1 as a marker of favourable outcome in anti-endocrine treated, estrogen-receptor positive, lymph-node negative breast cancer

Abstract

Objective: To investigate CITED1 as a potential biomarker of anti-endocrine response and breast cancer recurrence, given its previously determined role in mediating estrogen-dependant transcription. The study is a continuation of earlier work establishing the role of CITED1 in mammary gland development.

Results: CITED1 mRNA is associated with estrogen-receptor positivity and selectively expressed in the GOBO dataset of cell lines and tumours representing the luminal-molecular subtype. In patients treated with tamoxifen, higher CITED1 correlated with better outcome, suggesting a role in anti-estrogen response. The effect was particularly evident in the subset of estrogen-receptor positive, lymph-node negative (ER+/LN-) patients although noticeable divergence of the groups was apparent only after five years. Tissue microarray (TMA) analysis further validated the association of CITED1 protein, by immunohistochemistry, with favourable outcome in ER+, tamoxifen-treated patients. Although we also found a favourable response to anti-endocrine treatment in a larger TCGA dataset, the tamoxifen-specific effect was not replicated. Finally, MCF7 cells overexpressing CITED1 showed selective amplification of AREG but not TGFα suggesting that maintenance of specific ERα-CITED1 mediated transcription is important for the long-term response to anti-endocrine therapy. These findings together confirm the proposed mechanism of action of CITED1 and support its potential use as a prognostic biomarker.

Keywords: Anti-endocrine; Aromatase inhibitor; Breast cancer; CITED1; ERα; Tamoxifen.

Fig. 1

CITED1 is expressed in cell lines of the ER+ luminal breast cancer subtype and correlates with tamoxifen response in vivo. A GOBO database of breast cancer cell lines showing relative expression of CITED1 mRNA ordered by tumour molecular subtypes representation. Luminal—orange, Basal A—light blue, Basal B—blue *Misclassified melanoma cell line, MDAMB435 [36]. B CITED1 protein expression is shown in breast cancer cell lines representing the different tumour molecular subtypes. β-actin is used as a loading control. C CITED1 expression in the various molecular subtypes represented in the GOBO breast tumour dataset. D CITED1 expression in ER+ and ER− tumours in the GOBO tumour dataset. E Survival analysis showing distant metastasis-free survival (DMFS) in patients with breast cancer treated with tamoxifen (TAM). Patients were classified into 3 groups according to expression: high CITED1—orange, medium CITED1—light blue, low CITED1—blue. F Survival analysis for the cohort of ER+/LN− patients treated with tamoxifen (TAM). The number of patients in each group at diagnosis is indicated as n

Fig. 2

Correlation of CITED1 protein (TMA) and gene expression (TGCA) with prognosis following anti-endocrine treatment. For the TMA analysis, patients were classified into 2 groups either as having low (negative/faint: blue line) or high (moderate/strong: orange line) CITED1 protein expression following IHC; A Relapse-free survival (RFS) following breast cancer diagnosis in the ER+ , TAM-treated cohort. B Breast cancer specific survival (BCSS) following diagnosis in the ER+ , TAM-treated cohort. The number of patients in each group at diagnosis is indicated in brackets. TCGA was used for validation of differential CITED1 gene expression (high expression: orange line, low expression: blue line) and its association with prognosis; C Relapse-free survival in all ER+/LN− TCGA breast tumours following any anti-endocrine (AE) treatment. D Relapse-free survival in the ER+/LN− breast tumours treated with aromatase inhibitors (AI). The number of patients in each group at diagnosis is indicated in brackets

Fig. 3

CITED1 overexpression alters expression of amphiregulin. A Characterization of CITED1 (28 kDa) and ERα (66KDa) protein expression in stable CITED1-overexpressing MCF7 cells compared to the empty vector (EV) control following passage (P) under selection (G418 antibiotic). β-tubulin (55 kDa) is used as a loading control. B CITED1 concentration, relative to a IPO8 control, in stable CITED1-overexpressing MCF7 cells (orange) compared to the empty vector control (blue) in response to estrogen stimulation (E2), tamoxifen (TAM) or simultaneous (E2/TAM) treatment. C, D Concentration of AREG and TGFα relative to a IPO8 control is shown in stable CITED1-overexpressing MCF7 cells (orange) compared to the stable empty vector control (blue). The response to estrogen stimulation (E2), tamoxifen (TAM) or simultaneous (E2/TAM) treatment is shown. E AREG protein expression in stable CITED1-overexpressing MCF7 cells compared to the empty vector (EV) control following passage under selection. The most intense band signal for AREG was just over the 25KDa marker which would likely correspond to the previously reported 26-28KDa cell surface form [30]. Total protein staining is used as a loading control. F AREG protein expression in stable CITED1-overexpressing MCF7 cells compared to the empty vector (EV) control in response to estrogen stimulation (E2), tamoxifen (TAM) or simultaneous (E2/TAM) treatment. Total protein staining is used as a loading control