Multi-Omics Analysis Identifies MGA as a Negative Regulator of the MYC Pathway in Lung Adenocarcinoma

Abstract

Genomic analysis of lung adenocarcinomas has revealed that the MGA gene, which encodes a heterodimeric partner of the MYC-interacting protein MAX, is significantly mutated or deleted in lung adenocarcinomas. Most of the mutations are loss of function for MGA, suggesting that MGA may act as a tumor suppressor. Here, we characterize both the molecular and cellular role of MGA in lung adenocarcinomas and illustrate its functional relevance in the MYC pathway. Although MGA and MYC interact with the same binding partner, MAX, and recognize the same E-box DNA motif, we show that the molecular function of MGA appears to be antagonistic to that of MYC. Using mass spectrometry-based affinity proteomics, we demonstrate that MGA interacts with a noncanonical PCGF6-PRC1 complex containing MAX and E2F6 that is involved in gene repression, while MYC is not part of this MGA complex, in agreement with previous studies describing the interactomes of E2F6 and PCGF6. Chromatin immunoprecipitation-sequencing and RNA sequencing assays show that MGA binds to and represses genes that are bound and activated by MYC. In addition, we show that, as opposed to the MYC oncoprotein, MGA acts as a negative regulator for cancer cell proliferation. Our study defines a novel MYC/MAX/MGA pathway, in which MYC and MGA play opposite roles in protein interaction, transcriptional regulation, and cellular proliferation. IMPLICATIONS: This study expands the range of key cancer-associated genes whose dysregulation is functionally equivalent to MYC activation and places MYC within a linear pathway analogous to cell-cycle or receptor tyrosine kinase/RAS/RAF pathways in lung adenocarcinomas.

Figure 1:. Genomic and proteomic analysis of MGA in lung adenocarcinomas

A. Mutation profile of MGA in lung adenocarcinomas. Missense (green dots) and truncating mutations (black dots) are highlighted. B. Immunoprecipitation-mass spectrometry (IP-Mass Spec) results in HEK293T cells (2 biological replicates). We identified proteins that are significantly enriched by the endogenous MGA antibody, relative to IgG control (adjusted P value < 0.05). The proteins that are part of a noncanonical PRC1 (ncPRC1) – the PCGF6-PRC1 complex – are highlighted by red dots. C. Immunoprecipitation (IP) of MGA and IgG, followed by immunoblotting of MGA-interacting proteins in HEK293T, A549 and NCI-H23 cells.

Figure 2:. ChIP-seq analyses reveal that MGA co-localizes with MYC and E2F6 in the genome.

A. Genomic distribution of MYC and MGA binding sites in A549 cells. B. Homer de novo motif analysis identified the top DNA motif enriched in MGA binding sites identified in A549 cells. C. ChIP-seq signal of MGA, MYC, MAX and E2F6 (A549 cells) at the 9,049 MGA binding sites (top to bottom ranked by MGA ChIP-seq signal ±2.5kb centered at each MGA binding site). D. Comparison of MGA ChIP-seq binding sites identified in A549 and NCI-H23 cells. E. ChIP-seq signal of MGA in A549 and NCI-H23 cells, MYC, MAX and E2F6 in A549 cells at the CDK4 and NME1 loci. The E-box DNA motif (CACGTG) positions are indicated.

Figure 3:. ChIP-seq/RNA sequencing analyses show that MGA binds to and represses MYC target genes.

A. Averaged ChIP-seq signal of MGA in A549 cells with and without MGA overexpression at the transcription start sites (TSS) of genes that are activated, repressed and unaffected by MGA (based on RNA sequencing results in A549 cells with and without MGA overexpression). B. ChIP-seq signal of MGA in A549 cells with and without MGA overexpression at the CDK4 locus. C. Gene set enrichment analysis of RNA sequencing results in A549 cells with and without MGA overexpression showed that MGA-repressed genes are enriched in MYC target genes identified by previous studies. D. Percentage of MGA-regulated genes that are activated by MYC (based on RNA sequencing results from A549 cells with and without siRNA-mediated MYC silencing). P values are derived from fisher exact tests. E. RT-qPCR assays in NCI-H23 cells validated that either silencing MYC or overexpressing MGA decreased the expression level of the MYC target genes CDK4, AURKA and NME1. Expression level is normalized to controls (siNC or Empty). Error bars: s.d.

Figure 4:. MGA acts as a repressor for cancer cell proliferation.

A. siRNA-mediated silencing of MYC (verified by immunoblots) decreased the proliferation of A549 cells. Cell number was counted five days post transfection and normalized to the negative control siRNA. P values are derived from t tests. Error bars: s.d., **P<0.01. B. Same experiment as A, but in NCI-H23 cells, Error bars: s.d., ***P<0.001. C. Schematic chart explaining the design of competitive cell proliferation assays. A549 and NCI-H23 cells were first transfected with empty-ZsGreen control or MGA-ZsGreen vector. The transfected cells were collected based on GFP signal and mixed with parental cells at 1:1 ratio. The percentage of GFP-positive cells was then counted over time. D. MGA overexpression (verified by immunoblots) decreased the proliferation of A549 cells. The percentage of GFP-positive cells was counted four and six days post seeding the cells and normalized to the empty-ZsGreen control. P values are derived from t tests. Error bars: s.d., *P<0.05; ***P<0.001. E. Same experiment as D, but in NCI-H23 cells, Error bars: s.d., **P<0.01; ***P<0.001.