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.
Highlights
Cell growth regulator with RING-finger domain 1 (CGRRF1) is a growth suppressor and consists of a transmembrane domain and a RING-finger domain
We demonstrate that CGRRF1 suppresses the growth of breast cancer and the RING-finger domain is involved in its growth-inhibitory activity
We demonstrate that CGRRF1 suppresses growth of breast cancer and its E3 ligase activity is involved in CGRRF1-mediated growth suppression
Summary
CGRRF1 is a growth suppressor and consists of a transmembrane domain and a RING-finger domain. The expression of CGRRF1 is decreased in cancer tissues; the role of CGRRF1 in breast cancer and the mechanism(s) of its growth suppressor function remain to be elucidated. In endometrial cancer cell lines, overexpression of CGRRF1 suppresses cell proliferation and knockdown of CGRRF1 enhances cell growth. Several studies reported that the mRNA level of CGRRF1 is downregulated in tumor tissues including testicular germ cell tumor, endometrial tumor, and colorectal cancer [2, 4]. These findings suggest that CGRRF1 is a growth suppressor and might be involved in the development and/or progression of cancer.
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