Genetic alterations of KDM4 subfamily and therapeutic effect of novel demethylase inhibitor in breast cancer

Abstract

The histone lysine demethylase KDM4 subfamily, comprised of four members (A, B, C, and D), play critical roles in controlling transcription, chromatin architecture and cellular differentiation. We previously demonstrated that KDM4C is significantly amplified and overexpressed in aggressive basal-like breast cancers and functions as a transforming oncogene. However, information regarding the genomic and transcriptomic alterations of the KDM4 subfamily in different subtypes of breast cancer remains largely incomplete. Here, we conducted a meta-analysis of KDM4A, B, C and D in breast cancer and identified associations among recurrent copy number alterations, gene expression and breast cancer subtypes. We demonstrated that KDM4A and D are also significantly overexpressed in basal-like breast cancer, whereas KDM4B overexpression is more dominant in estrogen-receptor-positive, luminal breast cancer. Next, we investigated the therapeutic potential of a novel histone demethylase inhibitor, NCDM-32B, in breast cancer. The treatment of basal breast cancer cell lines with NCDM-32B resulted in the decrease of cell viability and anchorage independent growth in soft agar. Furthermore, we found that NCDM-32B impaired several critical pathways that drive cellular proliferation and transformation in breast cancer. Our findings demonstrate genetic amplification and overexpression of the KDM4 demethylases in different subtypes of breast cancer. Furthermore, histone methylation is reversible and KDM4 demethylases are druggable targets. Thus, KDM4 inhibitors may serve as a novel therapeutic approach for a subset of aggressive breast cancer.

Keywords: GASC1; KDM4; breast cancer; gene amplification; histone demethylase.

Figures

Figure 1

KDM4A-D copy number and expression levels in different subtypes breast cancer. A. High-level amplification and homozygous deletion of KDM4 subfamily members in TCGA breast cancer dataset (n=976). Data are displayed with the Oncoprint tool from cBioPortal. B. Frequencies of high-level amplification of KDM4A, B, C, and D in different subtypes of breast cancer. C. Expression levels of KDM4A, B, C, and D across five subtypes of breast cancer based on TCGA database. The differences in KDM4A, B, C, and D mRNA levels among breast cancer subtypes are statistically significant (P < 0.001).

Figure 2

KDM4 and H3 mark expression in breast cancer. A. mRNA expression levels of KDM4 demethylases in a panel of breast cancer cell lines were determined by qRT-PCR. mRNA expression levels in the MCF10A cells, an immortalized but nontumorigenic breast epithelial cell line, were arbitrarily set as 1. Relative expression levels in breast cancer cell lines are shown as fold changes compared with that of MCF10A cells. *The HCC1954 line belongs to the basal-like subtype even though it contains HER2 amplification. B. KDM4A, B, C, D and ER protein levels were analyzed by western blot in eight breast cancer cell lines and MCF10A line. Total H3 was used as the loading control. C. Global H3K4, K9, and K36 methylation levels in seven breast cancer cell lines and MCF10A line. Histone extracts were prepared from cells using the EpiQuik Total Histone Extraction Kit. Global histone methylation levels were detected by western blot assays with specific histone methylation antibodies. Total H3 level was used as the loading control.

Figure 3

Effects of inhibitor NCDM-32B on breast cancer growth and transformation. A. Chemical structure of the KDM4 inhibitor NCDM-32B. B. Western blot of H3K4me3/me2 and H3K9me3/me2 levels in HCC1954 cells with or without NCDM-32B (40 µM) treatment for 48 hours. C. Growth-inhibitory effect of NCDM-32B on HCC1954 and Colo824 breast cancer cells and non-tumorigenic MCF10A cells. D. The growth of Colo824 breast cancer cells in soft agar was also significantly inhibited by NCDM-32B treatment in a dose-dependent manner. Data are expressed as mean ± SD. *p

Figure 4

Gene expression changes with NCDM-32B inhibitor treatment in breast cancer. A. Ingenuity Pathway Analysis of the top pathways affected in differentially expressed genes in breast cancer after NCDM-32B treatment. The Y-axis is an inverse indication of P value or significance. B. qRT-PCR-validated mRNA microarray data in HCC1954 and Colo824 cells after NCDM-32B treatment.