KLF4 defines the efficacy of the epidermal growth factor receptor inhibitor, erlotinib, in triple-negative breast cancer cells by repressing the EGFR gene

Abstract

Background: Triple-negative breast cancer (TNBC) is characterized by high rates of recurrence and poor overall survival. This is due, in part, to a deficiency of targeted therapies, making it essential to identify therapeutically targetable driver pathways of this disease. While epidermal growth factor receptor (EGFR) is expressed in 60% of TNBCs and drives disease progression, attempts to inhibit EGFR in unselected TNBC patients have had a marginal impact on outcomes. Hence, we sought to identify the mechanisms that dictate EGFR expression and inhibitor response to provide a path for improving the utility of these drugs. In this regard, the majority of TNBCs express low levels of the transcription factor, Krüppel-like factor 4 (KLF4), while a small subset is associated with high expression. KLF4 and EGFR have also been reported to have opposing actions in TNBC. Thus, we tested whether KLF4 controls the expression of EGFR and cellular response to its pharmacological inhibition.

Methods: KLF4 was transiently overexpressed in MDA-MB-231 and MDA-MB-468 cells or silenced in MCF10A cells. Migration and invasion were assessed using modified Boyden chamber assays, and proliferation was measured by EdU incorporation. Candidate downstream targets of KLF4, including EGFR, were identified using reverse phase protein arrays of MDA-MB-231 cells following enforced KLF4 expression. The ability of KLF4 to suppress EGFR gene and protein expression and downstream signaling was assessed by RT-PCR and western blot, respectively. ChIP-PCR confirmed KLF4 binding to the EGFR promoter. Response to erlotinib in the context of KLF4 overexpression or silencing was assessed using cell number and dose-response curves.

Results: We report that KLF4 is a major determinant of EGFR expression and activity in TNBC cells. KLF4 represses transcription of the EGFR gene, leading to reduced levels of total EGFR, its activated/phosphorylated form (pEGFR), and its downstream signaling intermediates. Moreover, KLF4 suppression of EGFR is a necessary intermediary step for KLF4 to inhibit aggressive TNBC phenotypes. Most importantly, KLF4 dictates the sensitivity of TNBC cells to erlotinib, an FDA-approved inhibitor of EGFR.

Conclusions: KLF4 is a major regulator of the efficacy of EGFR inhibitors in TNBC cells that may underlie the variable effectiveness of such drugs in patients.

Keywords: Epidermal growth factor receptor (EGFR); Erlotinib; Krüppel-like factor 4 (KLF4); Triple-negative breast cancer (TNBC).

Fig. 1

KLF4 represses migration, invasion, and long-term cell growth of breast epithelial cells. a Western blot analysis and quantitation of KLF4 expression across eight different breast cell lines: MCF10A (10A), MCF-7, T47D, SKBR3, SUM159, HCC70, MDA-MB-468 (468), and MDA-MB-231 (231). b RT-qPCR analysis of KLF4 expression levels in MDA-MB-231 and MDA-MB-468 cell lines 3 days after infection with AdGFP (AdG) or AdKLF4 (AdK). c Western blot analysis and quantitation showing KLF4 expression in MDA-MB-231 and MDA-MB-468 following infection with AdGFP (G) or AdKLF4 (K) and compared to endogenous levels in MCF-10A cells. Two biological replicates were performed. Cells were infected with AdG or AdK for 2 days and then allowed to d migrate for 6 h or e invade into Matrigel for 24 h before they were stained and counted. f MDA-MB-231 and MDA-468 cells were infected with AdG or AdK and plated. After 24 h, they were counted using trypan blue exclusion. g RT-qPCR and h western blot analysis of the KLF4 expression in MCF10A cells 3 days post-infection with a non-targeting siRNA (siNS) or siRNA targeting KLF4 (siKLF4). Cells were infected with siNS or siKLF4 and allowed to i migrate for 6 h or j invade for 24 h before they were stained and counted. Cells were k, l infected with AdG or AdK or m transfected with siNS or siKLF4, and the cell number was counted at days 3–5 using trypan blue exclusion assay. Relative values were normalized versus the control, and error bars represent the standard deviation. Experiments were performed three independent times and in triplicate with *p < 0.05

Fig. 2

Identification of a KLF4-regulated protein signature reveals EGFR as a downstream target. a Western blot confirming the upregulation of KLF4 and its downstream target, E-cadherin, in MDA-MB-231 cells after AdGFP or AdKLF4 infection. b Reverse phase protein array heatmap showing differentially expressed genes after AdGFP or AdKLF4 infection in MDA-MB-231 cells. This analysis was completed as one experiment in duplicate. Proteins whose expression fold change was significantly different (p < 0.05) between AdKLF4 and AdGFP are shown. Red arrows indicate suppression of EGFR and phosphorylated EGFR by KLF4 overexpression while the green arrow identifies upregulated E-cadherin following KLF4 overexpression. c Western blot confirming EGFR repression in MDA-MB-231 cells infected with AdKLF4 compared to those infected with AdGFP. d Western blot analysis of EGFR expression across eight breast epithelial cell lines: MCF10A (10A), MCF7, T47D, SKBR3, HCC1143 (1143), SUM159, HCC70, MDA-MB-468 (468), and MDA-MB-231 (231). e Inverse correlation between KLF4 and EGFR protein in the eight breast epithelial cell lines, r² = 0.8542. *SKBR3 cell line

Fig. 3

KLF4 represses the EGFR signaling pathway. a Western blot analysis of KLF4, tEGFR, pEGFR (Y1068), tAKT, pAKT (S473), tERK1/2, and pERK1/2 (Y202/Y204) protein levels 3 days after AdGFP or AdKLF4 infection of MDA-MB-231 cells. b Graph depicting western blot quantification. All protein levels were normalized to total protein in the lane to control for loading, and phosphorylated proteins were normalized to their respective unphosphorylated proteins. AdKLF4 lanes were then expressed relative to AdGFP and are graphed with the horizontal bars indicating the median of the values of three replicate blots. Each dot represents the mean value from a different experiment, *p < 0.05. c, d Similar to a and b, but with lysates from infected MDA-MB-468 cells. e Western blot analysis of the same proteins in a, collected 3 days after transfection of MCF10A cells with siNS or siKLF4. f Quantitation and analysis of protein levels were completed as described in b. All western blots were completed in three independent experiments, each in triplicate, *p < 0.05

Fig. 4

KLF4 represses transcription of the EGFR gene. a RT-qPCR quantitation of EGFR mRNA levels following infection of MDA-MB-231 (231) and MDA-MB-468 (468) cells with AdGFP or AdKLF4. b RT-qPCR analysis of EGFR mRNA after silencing KLF4 in MCF10A cells. c Schematic of EGFR promoter-specific sites (1–6) used to detect KLF4 binding by ChIP-PCR. Three primer sets (A/B, C/D, and E/F) were used as indicated in the schematic and labels. Primers are listed in Table S1. d Gene-specific ChIP-PCR gel of MCF10A cells assessing the binding of KLF4 protein to the EGFR gene locus (hg19) where K1 and K2 are technical replicates for KLF4 immunoprecipitation. e Quantitation of d relative to the input. a, b Completed in three independent experiments in triplicate with *p < 0.05. d, e Completed in two independent experiments in duplicate

Fig. 5

Repression of EGFR is an obligatory intermediate step for KLF4 to inhibit aggressive breast cancer phenotypes. MCF10A cells were transfected with siNS, siKLF4, siEGFR, or siKLF4+siEGFR, and western blots were performed to confirm a KLF4 and b EGFR silencing. Cells were then allowed to c migrate for 6 h or d invade for 16 h before they were stained and counted. e MCF10A cells were transfected with siNS, siKLF4, siEGFR, or siKLF4+siEGFR, and cell number was counted at days 3–5 using trypan blue exclusion assay, *p < 0.05. f Apoptotic MCF10A cells were assessed using Hoechst stain 3 days after transfection. g EdU staining was used to quantify the number of proliferating MCF10A cells 3 days post-transfection. For all data, relative values were normalized versus the control, and error bars represent the standard deviation. Bars with distinct letters above them are significantly different from one another (p < 0.05). Experiments were performed three independent times in triplicate

Fig. 6

KLF4 expression dictates sensitivity to pharmacological inhibition of EGFR. a MDA-MB-231 and b MDA-MB-468 cells were infected with AdGFP or AdKLF4, and 2 days later, treated with 0–60 μM erlotinib for 3 days. The live cells were counted using trypan blue exclusion assay. Responses were normalized to effects observed with no drug for both AdGFP- and AdKLF4-infected cells. c MCF10A cells were transfected with siNS or siKLF4, and after 1 day, were treated with 0–60 μM erlotinib for 3 days. Cell number was counted, and the relative impact of the drug was normalized for siNS or siKLF4 in the absence of the drug. For each graph, nonlinear regression analysis was performed on IC₅₀ values of control versus experimental group, and each comparison resulted in statistical significance at p < 0.0001. Error bars represent the standard deviation. Experiments were performed three independent times in triplicate