Lrig1 is an estrogen-regulated growth suppressor and correlates with longer relapse-free survival in ERα-positive breast cancer

Abstract

Lrig1 is the founding member of the Lrig family and has been implicated in the negative regulation of several oncogenic receptor tyrosine kinases including ErbB2. Lrig1 is expressed at low levels in several cancer types but is overexpressed in some prostate and colorectal tumors. Given this heterogeneity, whether Lrig1 functions to suppress or promote tumor growth remains a critical question. Previously, we found that Lrig1 was poorly expressed in ErbB2-positive breast cancer, suggesting that Lrig1 has a growth-inhibitory role in this tumor type. However, breast cancer is a complex disease, with ErbB2-positive tumors accounting for just 25% of all breast cancers. To gain a better understanding of the role of Lrig1 in breast cancer, we examined its expression in estrogen receptor α (ERα)-positive disease which accounts for the majority of breast cancers. We find that Lrig1 is expressed at significantly higher levels in ERα-positive disease than in ERα-negative disease. Our study provides a molecular rationale for Lrig1 enrichment in ERα-positive disease by showing that Lrig1 is a target of ERα. Estrogen stimulates Lrig1 accumulation and disruption of this induction enhances estrogen-dependent tumor cell growth, suggesting that Lrig1 functions as an estrogen-regulated growth suppressor. In addition, we find that Lrig1 expression correlates with prolonged relapse-free survival in ERα-positive breast cancer, identifying Lrig1 as a new prognostic marker in this setting. Finally, we show that ErbB2 activation antagonizes ERα-driven Lrig1 expression, providing a mechanistic explanation for Lrig1 loss in ErbB2-positive breast cancer. This work provides strong evidence for a growth-inhibitory role for Lrig1 in breast cancer.

Figure 1

Lrig1 expression is associated with ERα status in human breast cancer. LRIG1 gene expression profiles and ERα phenotypic data of 363 breast carcinomas were obtained from two publicly available breast cancer microarray data sets. Correlation of Lrig1expression levels with ERα status of breast cancer specimens. Lrig1 is significantly overexpressed in the ER-positive tumors versus the ER-negative tumors using the t-test (p=0.0001). Left panel, Chin study. Right panel, Ivshina study.

Figure 2

Lrig1 is elevated in ER-positive tumors. (A) Western blot analysis of lysates from ER-negative and ER-positive tumors. Tissue lysates were blotted for Lrig1 and actin (loading control). Representative samples shown. (B) Densitometric analysis of ER-negative (n = 14) and ER-positive (n = 27) tumors. (C) qPCR analysis of Lrig1 transcript in ZR75-1 breast cancer cells. Hormone starved cells were treated with either vehicle control (VC) or E2 for 4 or 8 hours. (D) qPCR analysis of Lrig1 transcript in ZR75-1 breast cancer cells. Hormone starved cells were treated for 72 hours with either vehicle control (VC), 10 nM E2, 10 nM E2 plus 1 μM tamoxifen or 10 nM E2 plus 100 nM Fulvestrant. Columns, representative experiment performed in triplicate from at least three independent experiments bars, standard deviation.

Figure 3

Lrig1 accumulates following E2 stimulation. (A) Hormone starved ZR75-1 (left panel) or MCF7 (right panel) cells were treated with either vehicle control (VC) or 10 nM E2 for either 8 or 24 hours as indicated. Cell lysates were blotted for Lrig1 and actin (loading control). A representative blot is shown. (B) Schematic of the 4 Lrig1 enhancer elements (referred to as enh 1–4 in text) with approximate reference to the furthest downstream transcription start site (TSS) (sequence based on genome version hg18). Table lists size of ERα DNA binding region and ERE and Forkhead motif counts.

Figure 4

Chromatin Immunoprecipitation-qPCR analysis of the Lrig1 enhancer elements. Hormone starved ZR75-1 cells were treated with either vehicle control (VC) or 10 nM E2 for 30 minutes prior to cross-linking. Chromatin immunoprecipitation experiments were carried out using antibodies as indicated and subjected to qPCR with primers against the indicated regions. Enhancer # 1 of Xbp1 served as a positive control. Shown is the mean of three independent replicates with standard deviation. (A) Chromatin immunoprecipitation was performed with antibodies against p300 or ERα. Results are plotted as the fold enrichment of E2 treated cells over vehicle control treated cells. (B) Chromatin immunoprecipitation was performed with antibodies against H3K4me2. Results are plotted as fold enrichment over input DNA for both E2 and control treated cells. (C) Chromatin immunoprecipitation was performed with antibodies against FOXA1. Results are plotted as fold enrichment over input DNA for both E2 and control treated cells.

Figure 5

E2-mediated regulation of Lrig1 is FOXA1-dependent. (A) Representative western blot of FOXA1 knockdown efficiency. Hormone starved ZR75-1 cells were treated with either scramble control or FOXA1 siRNA. The following day the medium was changed to include either vehicle control (VC) or 10 nM E2 for an additional 24 hours. Cell lysates were collected at 48 hours post-transfection. (B) Hormone starved ZR75-1 cells were treated with either scramble control or FOXA1 siRNA 48 hours prior to treatment with 10 nM E2 or VC for 30 minutes followed by cross-linking. Chromatin immunoprecipitation was performed with antibodies against ERα and subjected to qPCR with primers against the indicated regions. Xbp1 enhancer 1 served as a positive control while the Tbx1 enhancer (FOXA1-independent) served as a negative control. Results are plotted as fold change in ERα occupancy, comparing scramble control and FOXA1 siRNA treatment. Shown is the mean of three independent replicates with standard deviation. Hormone starved ZR75-1 (C) and MCF7 (D) cells were treated with either scramble control or siRNA to FOXA1. Cells were then treated with either vehicle control (VC) or E2 for 8 hours before harvesting. Lrig1 transcript abundance was measured using Taqman real-time qPCR. Experiments were performed in triplicate and repeated at least three times with a representative experiment shown.

Figure 6

ErbB2 activation suppresses Lrig1 transcriptional output. All panels: ZR75-1 cells were transiently transfected with the pGL3-SV40 luciferase reporter vector (pGL3) or pGL3-SV40 containing enhancer elements 1–4. The β-galactosidase encoding pCMX vector was co-transfected and served as an internal control for transfection efficiency. 3X-ERE-Luc served as a positive control for E2 responsiveness. (A) Cells were hormone starved and treated with either vehicle control (VC) or 10 nM E2 for 18 hours. Cells were then assayed for firefly luciferase and β-galactosidase activity. Results shown are firefly luciferase activity normalized to β-galactosidase activity. Shown are representative experiments performed in triplicate with standard deviation. For (B), cells were treated as in (A) and in addition, cells were treated with VC or E2 plus 10 nM Nrg1β. For (C), cells were additionally transfected with either pcDNA3.1 vector control or NeuT expressing vector before treatment and assay as in (A).

Figure 7

Lrig1 suppresses growth of ER-positive breast cancer cells and correlates with prolonged relapse-free survival. Hormone starved T47D (A) and ZR75-1 (B) cells were treated with either scramble control or Lrig1 siRNA and then treated with E2 for 48 hours. Cell viability was measured using the MTT assay. The growth of E2 treated/scramble control cells was normalized to 1.0. (C) Representative western blot depicting efficiency of Lrig1 knockdown. (D) and (E) Association between Lrig1 expression and relapse-free survival (RFS) was determined. Patients were divided into groups based on Lrig1 expression levels. Two grouping systems were used: (D) tertiles; (E) cutoffs determined by mixed model (MM) clustering. Kaplan-Meier survival analysis was then performed for RFS with these expression groups as a factor. Significant survival differences between the groups were determined by log rank (Mantel-Cox) test (linear trend for factor levels). Events beyond 10 years were censored.