Eukaryotic initiation factor 4E-binding protein as an oncogene in breast cancer

Abstract

Background: Eukaryotic Initiation Factor 4E-Binding Protein (EIF4EBP1, 4EBP1) is overexpressed in many human cancers including breast cancer, yet the role of 4EBP1 in breast cancer remains understudied. Despite the known role of 4EBP1 as a negative regulator of cap-dependent protein translation, 4EBP1 is predicted to be an essential driving oncogene in many cancer cell lines in vitro, and can act as a driver of cancer cell proliferation. EIF4EBP1 is located within the 8p11-p12 genomic locus, which is frequently amplified in breast cancer and is known to predict poor prognosis and resistance to endocrine therapy.

Methods: Here we evaluated the effect of 4EBP1 targeting using shRNA knock-down of expression of 4EBP1, as well as response to the mTORC targeted drug everolimus in cell lines representing different breast cancer subtypes, including breast cancer cells with the 8p11-p12 amplicon, to better define a context and mechanism for oncogenic 4EBP1.

Results: Using a genome-scale shRNA screen on the SUM panel of breast cancer cell lines, we found 4EBP1 to be a strong hit in the 8p11 amplified SUM-44 cells, which have amplification and overexpression of 4EBP1. We then found that knock-down of 4EBP1 resulted in dramatic reductions in cell proliferation in 8p11 amplified breast cancer cells as well as in other luminal breast cancer cell lines, but had little or no effect on the proliferation of immortalized but non-tumorigenic human mammary epithelial cells. Kaplan-Meier analysis of EIF4EBP1 expression in breast cancer patients demonstrated that overexpression of this gene was associated with reduced relapse free patient survival across all breast tumor subtypes.

Conclusions: These results are consistent with an oncogenic role of 4EBP1 in luminal breast cancer and suggests a role for this protein in cell proliferation distinct from its more well-known role as a regulator of cap-dependent translation.

Keywords: 4E-BP1; 4EBP1; 8p11-p12; 8p11–12; 8p12–11; Amplification; Breast cancer; Chromosomal abnormality; Driver; EIF4EBP1; Estrogen receptor; Oncogene; PHAS-I.

Fig. 1

EIF4EBP1 is amplified in human breast cancer and correlates with reduced overall survival. (a) Amplification data from The Cancer Genome Atlas (TCGA) and (b) the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC). (c) Expression analysis using TCGA provisional data shows that high expression of A2 genes: BRF2, RAB11FIP1, EIF4EBP1, ASH2L, LSM1, BAG4, DDHD2, PLPP5, NSD3, FGFR1, TACC1, ADAM9, and ADAM32 correlates with reduced overall survival

Fig. 2

4EBP1 is highly expressed and phosphorylated in 8p11-p12 breast cancer cells. (a) Western blot of 4EBP1 and phospho-4EBP1 on residues Thr 37/37, Thr 70, and Ser 65 in SUM-44 (ER+), Cama-1 (ER+), and SUM-52 (ER-) cells with amplification of the 8p11-p12 genomic locus (8p11-12 CNA) as well as T47D (ER+), HCC1500 (ER+), MCF7 (ER+), and SUM-229 (ER-) cells without amplification of the 8p11-p12 genomic locus. Immortalized but non-tumorigenic breast epithelial cells are represented by MCF10A and H16N2 cells. (b) Cell proliferation was assessed in SUM-44, Cama-1, and SUM-52 cells in the presence or absence of 1 μM everolimus treatment for 72 h. Error bars represent standard deviation among replicates and p values are for the difference in cell proliferation in control versus treated cells. The p-value for the difference between the effect in SUM-52 cells and the other cell lines is <0.0001

Fig. 3

4EBP1 knockdown inhibits proliferation of ER+ 8p11-p12 breast cancer cells. (a) Western blot of 4EBP1, phospho-4EBP1 on residues Thr 37/46, and ERα in SUM-44 cells engineered with either control shRNA to lacZ or two individual shRNAs to EIF4EBP1 (4EBP sh_1 or sh_2). (b) Western blot of 4EBP1, phospho-4EBP1 on residues Thr 37/46, and ERα in Cama-1 cells engineered with either control shRNA to lacZ or two individual shRNAs to EIF4EBP1 (4EBP sh_1 or sh_2). (c) Cell proliferation was assessed in SUM-44 and (d) Cama-1 control and EIF4EBP1 knockdown cells on day 1 and day 4 in culture following selection in puromycin containing media. Error bars represent standard deviation among replicates and p-values represent the statistical comparison between each corresponding group

Fig. 4

4EBP1 knockdown does not affect proliferation of MCF10A and H16N2 non-transformed breast epithelial cells. (a) Western blot of 4EBP1 in MCF10A cells and (b) H16N2 cells engineered with either control shRNA to lacZ or two individual shRNAs to EIF4EBP1 (4EBP sh_1 or sh_2). (c) Cell proliferation was assessed in MCF10A and (d) H16N2 control and EIF4EBP1 knockdown cells on day 1 and day 4 in culture following selection in puromycin-containing medium. Error bars represent standard deviation among replicates and p-values represent significance between each corresponding group

Fig. 5

4EBP1 knockdown leads to G0/G1 arrest in ER+ 8p11-p12 breast cancer cells. (a) Cell cycle analysis of SUM-44 and (b) Cama-1 cells shows that 4EBP1 knockdown results in an accumulation of cells in G0/G1 with an associated decrease in cells in S-phase. (c) Western blot of cyclin D1 and p27 in SUM-44 and (d) Cama-1 cells engineered with either control shRNA to lacZ or two individual shRNAs to EIF4EBP1 (4EBP sh_1 or sh_2)

Fig. 6

4EBP1 knockdown inhibits proliferation of ER- 8p11-p12 breast cancer cells. (a) Western blot of 4EBP1, phospho-4EBP1 on residues Thr 37/46, ERα, cyclin D1, and p27 in SUM-52 cells engineered with either control shRNA to lacZ or two individual shRNAs to EIF4EBP1 (4EBP sh_1 or sh_2). (b) Cell proliferation was assessed in SUM-52 control and EIF4EBP1 knockdown cells on day 1 and day 4 in culture after selection in puromycin-containing medium. Error bars represent standard deviation among replicates and significance is the comparison between each corresponding group

Fig. 7

Kaplan-Meier analysis of breast cancer outcomes in patients with and without overexpression of 4EBP1. KM plotter analysis of EIF4EBP1 (probe ID: 221539_at) gene expression and overall survival in (a) ER+ populations (b) separated by luminal A (c) luminal B subtypes (d) all subtypes (no parameters selected) (e) post treatment with chemotherapy (f) tamoxifen or (g) endocrine therapy