CGRRF1, a growth suppressor, regulates EGFR ubiquitination in breast cancer

Abstract

BackgroundCGRRF1 is a growth suppressor and consists of a transmembrane domain and a RING-finger domain. It functions as a RING domain E3 ubiquitin ligase involved in endoplasmic reticulum-associated degradation. The expression of CGRRF1 is decreased in cancer tissues; however, the role of CGRRF1 in breast cancer and the mechanism(s) of its growth suppressor function remain to be elucidated.MethodsTo investigate whether CGRRF1 inhibits the growth of breast cancer, we performed MTT assays and a xenograft experiment. Tumors harvested from mice were further analyzed by reverse phase protein array (RPPA) analysis to identify potential substrate(s) of CGRRF1. Co-immunoprecipitation assay was used to verify the interaction between CGRRF1 and its substrate, followed by in vivo ubiquitination assays. Western blot, subcellular fractionation, and reverse transcription quantitative polymerase chain reaction (qRT-PCR) were performed to understand the mechanism of CGRRF1 action in breast cancer. Publicly available breast cancer datasets were analyzed to examine the association between CGRRF1 and breast cancer.ResultsWe show that CGRRF1 inhibits the growth of breast cancer in vitro and in vivo, and the RING-finger domain is important for its growth-inhibitory activity. To elucidate the mechanism of CGRRF1, we identified EGFR as a new substrate of CGRRF1. CGRRF1 ubiquitinates EGFR through K48-linked ubiquitination, which leads to proteasome degradation. In addition to regulating the stability of EGFR, knockout of CGRRF1 enhances AKT phosphorylation after EGF stimulation. By analyzing the breast cancer database, we found that patients with low CGRRF1 expression have shorter survival. As compared to normal breast tissues, the mRNA levels of CGRRF1 are lower in breast carcinomas, especially in HER2-positive and basal-like breast cancers. We further noticed that CGRRF1 promoter methylation is increased in breast cancer as compared to that in normal breast tissue, suggesting that CGRRF1 is epigenetically modified in breast cancer. Treatment of 5-azactidine and panobinostat restored CGRRF1 expression, supporting that the promoter of CGRRF1 is epigenetically modified in breast cancer. Since 5-azactidine and panobinostat can increase CGRRF1 expression, they might be potential therapies for breast cancer treatment.ConclusionWe demonstrated a tumor-suppressive function of CGRRF1 in breast cancer and identified EGFR as its target.

Shared and Distinctive Neighborhoods of Emerin and Lamin B Receptor Revealed by Proximity Labeling and Quantitative Proteomics

“…The fifth (Lsg1) is a non-membrane protein with large intranuclear and cytoplasmic pools that is implicated in nuclear export and cytoplasmic maturation of the large ribosomal subunit . The TM proteins selected for this analysis included Cgrrf1 , and Rnf185, ,, ubiquitin E3 ligases implicated in ER-associated degradation (ERAD). These were enriched very highly with LBR-TbID (∼13–18-fold), and to a lesser degree by Emd-TbID (∼4–6-fold).…”

Shared and distinctive neighborhoods of emerin and LBR revealed by proximity labeling and quantitative proteomics

“…The fifth (Lsg1) is a non-membrane protein with large intranuclear and cytoplasmic pools that is implicated in nuclear export and cytoplasmic maturation of the large ribosomal subunit (Lo et al, 2010). The TM proteins selected for this analysis included Cgrrf1 (Glaeser et al, 2018; Lee et al, 2019) and Rnf185 (El Khouri et al, 2013; Glaeser et al, 2018; van de Weijer et al, 2020), ubiquitin E3 ligases implicated in ER-associated degradation (ERAD). These were enriched very highly with LBR-TbID (∼13-18-fold), and to a lesser degree by Emd-TbID (∼4-6-fold).…”

LncSEA: a platform for long non-coding RNA related sets and enrichment analysis

“…The identification of novel drug targets and the development of new candidate drugs are of great significance for the targeted treatment of cancer. In the results section of the ‘Drug’ category, we found these lncRNAs were significantly enriched to the anti-breast cancer drugs Topotecan and Panobinostat ( 72 , 73 ). Interestingly, the drug, TKI258, that ranked third most significant in the enrichment analysis was recently reported to lead to the suppression of downstream signaling by RAS-RAF-MAPK and PI3K-AKT molecules, which are involved in cell proliferation, cell survival, and tumor invasion ( 74 ).…”