Regulation of a Novel Splice Variant of Early Growth Response 4 (EGR4-S) by HER+ Signalling and HSF1 in Breast Cancer

Abstract

The zinc finger transcription factor EGR4 has previously been identified as having a critical role in the proliferation of small cell lung cancer. Here, we have identified a novel, shortened splice variant of this transcription factor (EGR4-S) that is regulated by Heat Shock Factor-1 (HSF1). Our findings demonstrate that the shortened variant (EGR4-S) is upregulated with high EGFR, HER2, and H-Ras^v12-expressing breast cell lines, and its expression is inhibited in response to HER pathway inhibitors. Protein and mRNA analyses of HER2+ human breast tumours indicated the novel EGR4-S splice variant to be preferentially expressed in tumour tissue and not detectable in patient-matched normal tissue. Knockdown of EGR4-S in the HER2-amplified breast cancer cell line SKBR3 reduced cell growth, suggesting that EGR4-S supports the growth of HER2+ tumour cells. In addition to chemical inhibitors of the HER2 pathway, EGR4-S expression was also found to be suppressed by chemical stressors and the overexpression of HSF1. Under these conditions, reduced EGR4-S levels were associated with the observed lower cell growth rate, but the augmentation of properties associated with higher metastatic potential. Taken together, these findings identify EGR4-S as a potential biomarker for HER2 pathway activation in human tumours that is regulated by HSF1.

Keywords: EGR4; HER2; HSF1; breast cancer; molecular stress.

Figure 1

Association of EGR4 with HSF1 and HER2/HER1 expression in breast cell lines. (A) EGR4 protein expression in non-transformed breast cells (mCherry) and oncogenically transformed breast cells (H-Ras^V12) with low (GFP Ctrl) and high HSF1 (HSF1 WT, HSF1 ΔRDT). (B) Protein expression in a panel of breast cancer cell lines of different cancer sub-types (Luminal, HER2+, Basal). (C) Merged light/chemiluminescent image showing molecular ladder (left and right) and EGR4 protein bands detected around ~51 kDa.

Figure 2

Structure of the EGR4 gene and splice variant expression in cells and tumour tissue. (A) Schematic diagram of the Human EGR4 gene, located on Chromosome 2, showing its 2 exon structure (top), the 3 possible mRNA transcripts (middle), and 3 hypothetical protein isoforms arising from these different transcripts (bottom). Based on: https://www.ensembl.info/known-bugs/ensembl-100/ (accessed on 15 August 2017). (B) RNAseq analysis for EGR4 exon expression in 56 different breast cancer cell lines. Reads were normalised to exon size by scaling to base-level coverage. (C) Example of Western blot results for HER2 and EGR4 expression in normal (N) and breast tumour (T) biopsies from HER2− (pt 1–4) and HER2+ (pt 5–9) patients.

Figure 3

Confirmation of the EGR4-S splice variant in cell lines and clinical samples. (A) Results from qPCR on three breast tumour patient biopsies showing the amplification cycle for a qPCR product (mean ± SEM) detected using primers binding in exon 1 vs. primers binding in exon 2 for the same tumour sample. (B) Analysis of TCGA BRCA RNA-seq dataset (n = 1085) showing distribution and median proportional change for the expression of EGR4 exon 1 and exon 2 mRNA in patients diagnosed with breast carcinoma (n = 1085). Expression of mRNA is separated according to breast cancer sub-type. (C) Representative punch biopsies from a HER2+ breast tumour at low magnification (left panels) and high magnification (right panels).

Figure 3

Confirmation of the EGR4-S splice variant in cell lines and clinical samples. (A) Results from qPCR on three breast tumour patient biopsies showing the amplification cycle for a qPCR product (mean ± SEM) detected using primers binding in exon 1 vs. primers binding in exon 2 for the same tumour sample. (B) Analysis of TCGA BRCA RNA-seq dataset (n = 1085) showing distribution and median proportional change for the expression of EGR4 exon 1 and exon 2 mRNA in patients diagnosed with breast carcinoma (n = 1085). Expression of mRNA is separated according to breast cancer sub-type. (C) Representative punch biopsies from a HER2+ breast tumour at low magnification (left panels) and high magnification (right panels).

Figure 4

Inverse association between HSF1 and EGR4-S expression. Western blots for EGR4-S and HSF1 expression in (A) Luminal, HER2+, and (B) Basal breast cancer cells, with overexpression or shRNA-knockdown of HSF1, respectively. (C) Western blots of HER2+ cells treated with varying concentrations of the stress-inducing compound, AUY922, for 24 h and (D) sulforaphane (SFN) for 24 h.

Figure 5

EGR4-S expression is regulated by HER- pathway targeted drug treatment. (A) Western blot analysis of HER2+ cell lysates treated for 24 h with increasing concentrations of TKI drugs (lapatinib, erlotinib) and (B) performed on indicated cell lysates treated over 24–48 h. (C) Western blot analysis of MDA-MB-468 breast cancer cell lysates treated with increasing concentrations of TKI drugs for 24 h (lapatinib, gefitinib, erlotinib).

Figure 6

EGR4-S expression is responsive to HER-targeted treatment and sensitive to altered HSF1 activity. (A) Lapatinib treatment of HER2+ cells, with/without elevated HSF1 and (B) with/without HSF1 shRNA knockdown. (C) EGR4-S expression in cells grown in increasing concentrations of lapatinib over the course of 7 weeks exhibits some reduction visible at 3 weeks. (D) Comparison of HER2 and EGR4-S protein expression in tumour (T) tissue and adjacent normal (N) tissue from eight different breast cancer patients (Pt 1–8) diagnosed with HER2+ tumours. Pt 11, 15, and 17 received pre-biopsy treatment (+PBT) to suppress the HER2 pathway.

Figure 7

Effect of altered EGR4 expression on cancer cell growth. (A) xCELLigence Assay for HER2+ cell viability with/without EGR4-S knockdown (EGR4-KD) over time. (B) A schematic of the proposed EGFR(HER1)/HER2 signalling pathway and its relationship with the downstream transcription factor, EGR4-S.