Calmodulin-like protein 3 is an estrogen receptor alpha coregulator for gene expression and drug response in a SNP, estrogen, and SERM-dependent fashion

Sisi Qin,Richard M Weinshilboum,James N Ingle,D Lawrence Wickerham,Liewei Wang,Michiaki Kubo,Jia Yu,Mohan Liu

doi:10.1186/s13058-017-0890-x

Abstract

BackgroundWe previously performed a case–control genome-wide association study in women treated with selective estrogen receptor modulators (SERMs) for breast cancer prevention and identified single nucleotide polymorphisms (SNPs) in ZNF423 as potential biomarkers for response to SERM therapy. The ZNF423rs9940645 SNP, which is approximately 200 bp away from the estrogen response elements, resulted in the SNP, estrogen, and SERM-dependent regulation of ZNF423 expression and, “downstream”, that of BRCA1.MethodsElectrophoretic mobility shift assay–mass spectrometry was performed to identify proteins binding to the ZNF423 SNP and coordinating with estrogen receptor alpha (ERα). Clustered, regularly interspaced short palindromic repeats (CRISPR)/Cas9 genome editing was applied to generate ZR75-1 breast cancer cells with different ZNF423 SNP genotypes. Both cultured cells and mouse xenograft models with different ZNF423 SNP genotypes were used to study the cellular responses to SERMs and poly(ADP-ribose) polymerase (PARP) inhibitors.ResultsWe identified calmodulin-like protein 3 (CALML3) as a key sensor of this SNP and a coregulator of ERα, which contributes to differential gene transcription regulation in an estrogen and SERM-dependent fashion. Furthermore, using CRISPR/Cas9-engineered ZR75-1 breast cancer cells with different ZNF423 SNP genotypes, striking differences in cellular responses to SERMs and PARP inhibitors, alone or in combination, were observed not only in cells but also in a mouse xenograft model.ConclusionsOur results have demonstrated the mechanism by which the ZNF423 rs9940645 SNP might regulate gene expression and drug response as well as its potential role in achieving more highly individualized breast cancer therapy.

Highlights

We previously performed a case–control genome-wide association study in women treated with selective estrogen receptor modulators (SERMs) for breast cancer prevention and identified single nucleotide polymorphisms (SNPs) in ZNF423 as potential biomarkers for response to Selective estrogen receptor modulator (SERM) therapy
We demonstrated how the ZNF423 rs9940645 SNP that was not within an Estrogen response element (ERE) was able to affect the expression of ZNF423 and BRCA1 as well as treatment response as a result of the actions of calmodulin-like protein 3 (CALML3), which we identified as part of a complex bound to the ZNF423 SNP
As a first step to test this hypothesis, we designed DNA probes containing either the WT or variant rs9940645 SNP genotype sequences and performed electrophoretic mobility shift assays (EMSAs) using nuclear extracts from lymphoblastoid cell lines (LCLs) selected based on different ZNF423 SNP genotypes that had been treated with E2 alone or E2 plus 4-OH-TAM, the active metabolite of tamoxifen [8] (Fig. 1a)

Summary

Introduction

We previously performed a case–control genome-wide association study in women treated with selective estrogen receptor modulators (SERMs) for breast cancer prevention and identified single nucleotide polymorphisms (SNPs) in ZNF423 as potential biomarkers for response to SERM therapy. Our previous discovery genome-wide association study (GWAS) using samples from NSABP P-1 and P-2 subjects identified common single nucleotide polymorphisms (SNPs) in the ZNF423 gene as potential biomarkers for individualized SERM prevention therapy [8]. One of those SNPs, rs9940645 located approximately 200 bp distant from several estrogen response elements (EREs), resulted in SNP, estrogen and SERM-dependent regulation of ZNF423 expression and, “downstream”, that of BRCA1. It is possible that the rs9940645 SNP in the ZNF423 gene might be used as a biomarker to select patients for therapy with PARP inhibitors, either alone or in combination with SERMs, especially in patients who have low BRCA1 expression resulting from the effect of ZNF423 SNP genotypes in the presence of different drug treatments

Methods

Results

Discussion

Conclusion