Knockdown of NR3C1 inhibits the proliferation and migration of clear cell renal cell carcinoma through activating endoplasmic reticulum stress–mitophagy

Abstract

Background Clear cell renal cell carcinoma (ccRCC) is closely associated with steroid hormones and their receptors affected by lipid metabolism. Recently, there has been growing interest in the carcinogenic role of NR3C1, the sole gene responsible for encoding glucocorticoid receptor. However, the specific role of NR3C1 in ccRCC remains unclear. The present study was thus developed to explore the underlying mechanism of NR3C1’s carcinogenic effects in ccRCC. Methods Expression of NR3C1 was verified by various tumor databases and assessed using RT-qPCR and western blot. Stable transfected cell lines of ccRCC with NR3C1 knockdown were constructed, and a range of in vitro and in vivo experiments were performed to examine the effects of NR3C1 on ccRCC proliferation and migration. Transcriptomics and lipidomics sequencing were then conducted on ACHN cells, which were divided into control and sh-NR3C1 group. Finally, the sequencing results were validated using transmission electron microscopy, mitochondrial membrane potential assay, immunofluorescence co-localization, cell immunofluorescent staining, and Western blot. The rescue experiments were designed to investigate the relationship between endoplasmic reticulum stress (ER stress) and mitophagy in ccRCC cells after NR3C1 knockdown, as well as the regulation of their intrinsic signaling pathways. Results The expression of NR3C1 in ccRCC cells and tissues was significantly elevated. The sh-NR3C1 group, which had lower levels of NR3C1, exhibited a lower proliferation and migration capacity of ccRCC than that of the control group (P < 0.05). Then, lipidomic and transcriptomic sequencing showed that lipid metabolism disorders, ER stress, and mitophagy genes were enriched in the sh-NR3C1 group. Finally, compared to the control group, ER stress and mitophagy were observed in the sh-NR3C1 group, while the expression of ATF6, CHOP, PINK1, and BNIP3 was also up-regulated (P < 0.05). Furthermore, Ceapin-A7, an inhibitor of ATF6, significantly down-regulated the expression of PINK1 and BNIP3 (P < 0.05), and significantly increased the proliferation and migration of ccRCC cells (P < 0.05). Conclusions This study confirms that knockdown of NR3C1 activates ER stress and induces mitophagy through the ATF6-PINK1/BNIP3 pathway, resulting in reduced proliferation and migration of ccRCC. These findings indicate potential novel targets for clinical treatment of ccRCC. Graphical Abstract

Hypoglycemic Activity of Rice Resistant-Starch Metabolites: A Mechanistic Network Pharmacology and In Vitro Approach

“…NR3C1 is a glucocorticoid receptor belonging to the nuclear receptor family and regulates the production of glucocorticoids in organisms [75]. Glucocorticoids have been associated with insulin resistance, glucose homeostasis, and diabetes risk [76].…”

Emerging evidence linking stress and glucocorticoid signaling with cancer phenotypes

“…While prior studies largely used high-dose or synthetic compounds to activate glucocorticoid signaling, less is known about how physiological levels of glucocorticoid activity influence cancer phenotypes. Addressing this question, a recent study showed that clear cell renal cell carcinoma (CCRCC) tissues are characterized by elevated expression of NR3C1 , the gene encoding the human glucocorticoid receptor (GR) in humans [ 2 ]. NR3C1 knockdown experiments further showed that reducing GR expression levels by 50% or more ameliorated cancer-related phenotypes, as shown by decreased cell proliferation and migration, and these effects were accompanied by reduced tumor size in cancer cell-injected mice [ 2 ].…”

“…Addressing this question, a recent study showed that clear cell renal cell carcinoma (CCRCC) tissues are characterized by elevated expression of NR3C1 , the gene encoding the human glucocorticoid receptor (GR) in humans [ 2 ]. NR3C1 knockdown experiments further showed that reducing GR expression levels by 50% or more ameliorated cancer-related phenotypes, as shown by decreased cell proliferation and migration, and these effects were accompanied by reduced tumor size in cancer cell-injected mice [ 2 ]. These findings align with another recent study that modeled chronic stress in cell culture through prolonged exposure of human fibroblasts to the endogenous human glucocorticoid cortisol at physiological levels reached in human tissues during in vivo stress [ 3 ].…”

“…Beyond epigenetic regulation, aberrant GR signaling likely also influences a wide range of biological processes implicated in cancer pathogenesis. For instance, downregulating the GR through NR3C1 knockdown was shown to activate endoplasmic reticulum stress and induce mitophagy in CCRCC cells, whereas directly inhibiting these processes counteracted the effect of GR downregulation on cell proliferation and migration [ 2 ]. Immune deregulation has also been recognized as a key link between stress and cancer, with potential pathogenic roles both within the tumor microenvironment and systemically.…”