Dysfunction of ubiquitin protein ligase MYCBP2 leads to cell resilience in human breast cancers

Abstract

Breast cancer is the most common type of cancer among women worldwide, and it is estimated that 294 000 new diagnoses and 37 000 deaths will occur each year in the United States alone by 2030. Large-scale genomic studies have identified a number of genetic loci with alterations in breast cancer. However, identification of the genes that are critical for tumorgenicity still remains a challenge. Here, we perform a comprehensive functional multi-omics analysis of somatic mutations in breast cancer and identify previously unknown key regulators of breast cancer tumorgenicity. We identify dysregulation of MYCBP2, an E3 ubiquitin ligase and an upstream regulator of mTOR signaling, is accompanied with decreased disease-free survival. We validate MYCBP2 as a key target through depletion siRNA using in vitro apoptosis assays in MCF10A, MCF7 and T47D cells. We demonstrate that MYCBP2 loss is associated with resistance to apoptosis from cisplatin-induced DNA damage and cell cycle changes, and that CHEK1 inhibition can modulate MYCBP2 activity and caspase cleavage. Furthermore, we show that MYCBP2 knockdown is associated with transcriptomic responses in TSC2 and in apoptosis genes and interleukins. Therefore, we show that MYCBP2 is an important genetic target that represents a key node regulating multiple molecular pathways in breast cancer corresponding with apparent drug resistance in our study.

Graphical Abstract

Figure 1.

Summary of evidence for MYCBP2 somatic mutational enrichment. (A) The analytic procedure for identifying MYCBP2 as a potential mutational driver. (B) Disease-free survival with MYCBP2 mutations.

Figure 2.

Gene co-expression analysis of MYCBP2 and related genes in the TCGA breast cancer samples. (A) MYCBP2 perturbation signature is enriched in the MYCBP2 centered BN constructed from the TCGA-BRCA samples. (B) BN subnetwork around MYCBP2. Pink and blue colors in the figure indicate downregulation and upregulation, respectively, of the gene in perturbed samples compared to baseline.

Figure 3.

Scatterplot matrix of gene co-expression between MYCBP2, ATM, ATR, CHEK1, MTOR, TSC1 and TSC2 among the TCGA-BRCA samples. Spearman correlation coefficients for gene pairs are given in the upper right corner of the plots as annotated. Colored points indicate that the underlying Spearman correlation is statistically significant (P-value <0.05), while gray points indicate that there is no correlation (P-value >0.05). The distribution of gene expression values for each gene is given on the diagonal by their kernel density estimation. (A) Gene co-expression by RNA-seq. (B) Protein co-expression by liquid chromatography–mass spectrometry, where protein expression was averaged across isoforms of each gene.

Figure 4.

MYCBP2 siRNA inhibition in MCF10A cells exposed to cisplatin. (A) Western blot and protein quantification of MYCBP2 and CHEK1 in NT cells, mock transfected cells and two different siRNAs against MYCBP2 (see the ‘Materials and Methods’ section) after 4 or 7 days of cisplatin exposure. (B) RT-qPCR quantification of MYCBP2 expression level after 4 days of cisplatin exposure. (C, D) Apoptosis assay by flow cytometry of NT, mock transfected and siMYCBP2-(5,6) groups after 4 days of cisplatin exposure. (E) Cell cycle assay by flow cytometry of NT, mock transfected and siMYCBP2-(5,6) groups of MCF10A cells after 4 days of cisplatin exposure.

Figure 5.

RNA-seq quantification of MYCBP2 knockdown signature on the transcriptome in MCF10A cells. (A) Proposed pathway for MYCBP2 from the previously published literature (6,12,29–31). (B) Gene expression in NT and siMYCBP2-6 exposed cells with and without cisplatin exposure of MYCBP2, proposed upstream genes, downstream genes and apoptosis-related genes.

Figure 6.

MYCBP2 shRNA knockdown in MCF7, MCF10A and T47D cell lines reduces caspase cleavage and is modulated by CHEK1 inhibition. Cell lines grown in DMSO-containing media, as described in the ‘Materials and Methods’ section, were exposed to no treatment, MYCBP2-targeting shRNA (pLV[shRNA]-EGFP:T2A:Puro-U6>hMYCBP2), rabusterib (4 μM solution) or a combination of both. Lines in panels (A)–(C) were exposed for 24 h to the mentioned conditions before western blot was performed; lines in panel (D) were exposed for 48 h.