Discrete Adaptive Responses to MEK Inhibitor in Subpopulations of Triple-Negative Breast Cancer

Abstract

Daniel R. Goulet: Discrete Adaptive Responses to MEK Inhibitor in Subpopulations of Triple Negative Breast Cancer (Under the direction of Gary Johnson) Triple-negative breast cancer (TNBC) is characterized by genomic heterogeneity and high risk of relapse. Recent studies have demonstrated that breast cancers can contain a spectrum of epithelial and mesenchymal phenotypes. We identified epithelial (POS) and mesenchymal (NEG) subpopulations in a triple negative breast cancer cell line that have similar coding sequences, but unique enrichment of the epigenetic activators H3K27ac and BRD4. We show that localization of these epigenetic activators correlates with the expression of epithelial transcription factors TP63 and ELF3 in epithelial cells, and ZEB1 and TWIST1 in mesenchymal cells. Currently, there are no targeted therapies available for the treatment of triple negative breast cancer. However, overexpression of EGFR, KRAS, or BRAF occurs in approximately 30% of TNBC, suggesting that inhibiting the MAPK signaling pathway may be an effective therapy for TNBC. Acute treatment with the MEK inhibitor trametinib induced distinct transcriptional responses in epithelial and mesenchymal subpopulations. ChIP-Seq analysis of H3K27ac and BRD4 binding revealed epigenetic remodeling in response to trametinib treatment, especially at binding sites for AP-1 in NEG cells and epithelial-specific ETS transcription factors in POS cells. Chronic treatment with trametinib induced the emergence of trametinib resistant POS cells, but not NEG cells. In trametinib resistant POS cells, we observe increased expression of the Gi-coupled chemokine receptor CXCR7, and its cognate ligand adrenomedullin, as well as increased expression of KRAS. ChIP-Seq analysis revealed enrichment of H3K27ac and BRD4 at the CXCR promoter and enhancer, indicating epigenetic remodeling induces transcriptional activation. siRNA knockdown of CXCR7 and KRAS demonstrate these genes are essential for the proliferation of trametinib resistant cells, and reveals that epigenetic remodeling at the CXCR7 locus may be a mechanism for drug resistance. CCND1 Cyclin D1 CD49f Integrin alpha 6 CDH1 E-Cadherin CDK4 Cyclin-dependent kinase 4 CDK6 Cyclin-dependent kinase 6 CDKN2A Cyclin dependent kinase inhibitor 2A cDNA Complementary DNA CHD1 Chromodomain helicase binding protein 1 ChIP-Seq Chromatin Immunoprecipitation Sequencing xiv cm Centimeter CML Chronic Myelogenous Leukemia CpG Cytosine-Guanine dinucleotide CRISPR Clustered Regularly Interspaced Short Palindromic Repeats CTCF CCCTC-binding factor CTD C-terminal domain CXCR7 Chemokine receptor 7 DCLK1 Doublecortin-like kinase 1

Adaptive chromatin remodeling and transcriptional changes of the functional kinome in tumor cells in response to targeted kinase inhibition

“…This is consistent with studies showing that H3K27ac is associated with relaxed chromatin and enriched at enhancers along with BRD4, MED1, and the transcriptional coactivator p300 (29,71). Genes associated with epithelial or mesenchymal phenotypes were differentially expressed in POS and NEG cells, respectfully (69). For example, a subset of genes preferentially expressed in POS cells regulate cell-cell adhesion, a subset of genes expressed in NEG cells regulate binding to extracellular matrix.…”

“…The transcriptional response involves the expression of multiple RTKs in patients that in preclinical models are shown to contribute to overcoming growth inhibition and stimulate proliferation and survival. The dynamic transcriptional responses are seen in response to different kinase inhibitors in different tumor types (11,29,35,37,47,69). Transcriptional reprogramming associated with epigenetic remodeling is also observed with other, nonkinase inhibitors and chemotherapies (73)(74)(75)(76)(77).…”

“…Trametinib treatment for 24 h of POS and NEG SUM229PE cells showed by RNAseq that 15% and 10% of their transcriptomes were altered greater than twofold, respectfully (69). Kinome transcriptome analysis of the POS and NEG cells showed kinases upregulated and downregulated in response to trametinib.…”

“…SUM229PE cells maintain two populations in culture: one epithelial and basal-like (SUM229PE EpCAM+ referred to as POS) and the second mesenchymal and claudin low-like (SUM229PE EpCAMreferred to as NEG) (68). Exome sequencing showed that POS and NEG SUM229PE cells have no unique nucleotide or copy number alterations (69). In addition, genome-wide DNA methylation sequencing showed no significant difference between the two populations that would give a differential transcriptional response to MEK-ERK inhibition with trametinib treatment.…”

“…Jordan et al performed an siRNA screen in the SUM229PE NEG cells showing that SMARCD3/BAF60C, a regulatory subunit of the SWI/SNF chromatin remodeling complex, was necessary to maintain the mesenchymal phenotype (70). Loss of Smarcd3/Baf60c expression converted SUM229PE NEG cells to the epithelial EpCAM+ (POS) phenotype, consistent with the POS and NEG phenotypes being controlled by differing chromatin states (69). FAIRE (Formaldehyde-Assisted Isolation of Regulatory Elements) sequencing showed differences in open chromatin accessibility between POS and NEG cells that correlated with histone H3 lysine 27 acetylation (H3K27ac) measured by ChIPseq.…”