Regulation of alphaB-crystallin gene expression by the transcription factor Ets1 in breast cancer

Abstract

Recent studies indicate that the small heat shock protein alphaB-crystallin is expressed in poor prognosis basal-like breast tumors and likely contributes to their aggressive phenotype. However, the mechanisms underlying the deregulated expression of alphaB-crystallin in basal-like tumors are poorly understood. Using a bioinformatics approach, we identified a putative DNA binding motif in the human alphaB-crystallin promoter for the proto-oncogene Ets1, a member of the ETS transcription factor family that bind to DNA at palindromic ETS-binding sites (EBS). Here we demonstrate that ectopic expression of Ets1 activates the alphaB-crystallin promoter by an EBS-dependent mechanism and increases alphaB-crystallin protein levels, while silencing Ets1 reduces alphaB-crystallin promoter activity and protein levels. Chromatin immunoprecipitation analyses showed that endogenous Ets1 binds to the alphaB-crystallin promoter in basal-like breast cancer cells in vivo. Interrogation of publically available gene expression data revealed that Ets1 is expressed in human basal-like breast tumors and is associated with poor survival. Collectively, our results point to a previously unrecognized link between the oncogenic transcription factor Ets1 and alphaB-crystallin in basal-like breast cancer.

Figure 1. Schematic representation of the shared human αB-crystallin/HspB2 promoter and conservation of the putative ETS-binding site (EBS)

(A) Using the MOTIF-search platform, we performed a bioinformatics analysis of the shared human αB-crystallin/HspB2 promoter spanning the region between the start ATG for each gene. The search identified published (bold) transcriptional regulators of αB-crystallin, as well as additional putative (italics) regulators, including Ets1. (B) Alignment of the putative EBS in the human αB-crystallin promoter with the corresponding region from other species.

Figure 2. Transcriptional regulation of the human αB-crystallin promoter by Ets1

(A) Schematic representation of the reporter constructs used. The full-length WT human αB-crystallin promoter (−1081/+30) and 5’ truncations (−516/+30, and −356/+30) were subcloned into the pGL3-Basic luciferase vector (Basic). The mutant EBS (AAAAGATTTT) was generated by site-directed mutagenesis. A pGL3-MMP9 reporter was used as a positive control. (B) MCF-10A cells were transiently co-transfected with 700 ng of pcDNA3.1-Ets1 or vector, 100 ng of pGL3 firefly luciferase reporter, and 1 ng of control pRL-TK Renilla luciferase reporter. Firefly luciferase activity was normalized to Renilla luciferase activity and expressed as fold induction relative to the activity in empty vector-transfected cells. **p < 0.01 ***p < 0.001 versus pGL3-Basic. (C) MDA-MB-231 cells were transiently co-transfected with 300 ng pGL3 reporter, 3 ng of pRL-TK reporter, and 25 µM of siRNA targeting Ets1 or lamin A/C (control). Normalized firefly luciferase activity was expressed as fold induction relative to the activity in cells transfected with control lamin A/C siRNA. **p < 0.01 versus pGL3-Basic.

Figure 3. Ets1 binds to the αB-crystallin promoter in vitro and in vivo

(A) EMSA analysis of Ets1 binding to the putative EBS in the human αB-crystallin promoter in vitro. The human αB-crystallin promoter region containing the putative WT EBS or a mutant (Mut) EBS was duplexed with the corresponding antisense oligonucleotide and used as probes. Twenty fmol of biotinylated WT or mutant probe was added to nuclear extract in the absence or presence of 4 pmol of unbiotinylated WT probe. For antibody abrogation experiments, the binding reaction was preincubated with an Ets1 antibody or control IgG before adding the WT probe. Protein-DNA complexes were resolved by native-PAGE, transferred to a membrane, and detected by chemiluminescence. (B) ChIP analysis of endogenous Ets1 binding to the human αB-crystallin promoter in vivo. Input DNA-protein complexes or DNA-protein complexes immunoprecipitated with water, IgG or Ets1 antibody were PCR amplified using primers flanking the αB-crystallin promoter EBS.

Figure 4. Regulation of endogenous αB-crystallin protein levels by Ets1 in basal-like breast cancer cells

(A) MDA-MB-231 pools stably expressing pBABE vector or pBABE-Ets1 were created by retroviral transduction. Ets1, αB-crystallin and actin levels were determined by immunoblotting. (B) MDA-MB-231 cells were transiently transfected with 25 µM Ets1 siRNA or a control (C) lamin A/C siRNA. Ets1, αB-crystallin and actin levels were determined by immunoblotting 72 h later.

Figure 5. Ets1 is expressed in basal-like breast tumors and is associated with poor survival

Publically available gene expression datasets were interrogated by Oncomine analysis. (A) Ets1 expression as a function of ER-status in breast cancer [35]. (B) Ets1 expression in non-basal-like and basal-like breast tumors [36]. (C) Ets1 expression and five-year survival in breast cancer [35].