RNF115 Deficiency Upregulates Autophagy and Inhibits Hepatocellular Carcinoma Growth

RNF115 Deficiency Upregulates Autophagy and Inhibits Hepatocellular Carcinoma Growth

Autophagy is an evolutionarily conserved lysosome-mediated degradation process in eukaryotic cells that plays a crucial role in maintaining cell homeostasis under normal conditions. This process involves the formation of a crescent-shaped phagophore, also known as the isolation membrane (IM), which expands and closes to form a double-membraned autophagosome. The autophagosome is then delivered to the late endosome/lysosomes for the degradation of sequestrated materials. Dysfunction in autophagy has been linked to various diseases, including tumors, neurodegenerative disorders, cardiovascular diseases, and autoimmune conditions.

Ring finger protein 115 (RNF115), also known as breast cancer-associated gene 2 (BCA2), is a RAB7 target protein. Studies have shown that RNF115 is overexpressed in invasive breast tumors, lung adenocarcinoma, and gastric cancer, with its levels negatively correlated with patient prognosis. Recent research has demonstrated that RNF115 negatively regulates phagosome maturation and the host response to bacterial infection. Additionally, RNF115 deficiency has been shown to alleviate acute liver injury in mice by promoting autophagy and inhibiting the inflammatory response. However, the role of RNF115 in hepatocellular carcinoma (HCC) remains unclear.

In this study, we investigated the function of RNF115 in HCC and its regulation of autophagy. We found that RNF115 acts as a negative regulator of basal autophagy in hepatoma cells. To explore this, we used HepG2 and HEK293T cell lines obtained from the American Type Culture Collection (Manassas, VA, USA) and BEL-7402 cells from the China Center for Type Culture Collection (Shanghai, China). All cell lines were authenticated using Short Tandem Repeat (STR) analysis and confirmed to be mycoplasma-free.

Animal experiments were conducted with approval from the Animal Ethics Committee of Peking University Health Sciences Center (LA2019203). We examined the physiological effects of RNF115 on autophagy regulation using RNF115-silenced hepatoma cells. Specifically, we monitored autophagic activity by assessing microtubule-associated protein 1 light chain 3 (MAP1LC3/LC3) lipidation and the degradation of the autophagy substrate SQSTM1/P62. RNF115 knockdown in HepG2 and BEL-7402 cells significantly increased endogenous LC3B-II accumulation under both basal and starved conditions compared to the control group. Additionally, RNF115-depleted BEL-7402 cells exhibited increased endogenous LC3B puncta. Simultaneously, RNF115 deficiency significantly decreased SQSTM1/P62 levels. Rapamycin (RAPA) treatment further facilitated SQSTM1 degradation in RNF115-silenced BEL-7402 cells, while bafilomycin A1 (Baf.A1) blocked this degradation, indicating that SQSTM1/P62 degradation involved the autophagy-lysosomal pathway. These results suggest that RNF115 negatively regulates autophagic activity in hepatoma cells.

The homotypic fusion and protein sorting (HOPS) complex is recruited to the lysosome membrane by binding to RAB7, promoting autophagosome maturation. We investigated whether RNF115-regulated autophagosome maturation was related to the HOPS complex. Co-immunoprecipitation (CO-IP) assays revealed that either FLAG-VPS39 or FLAG-VPS41 co-immunoprecipitated with overexpressed green fluorescent protein (GFP)-RNF115 and endogenous RNF115. RNF115 contains an N-terminal ubiquitin-binding zinc finger (BCA2 zinc finger [BZF]) domain, a protein kinase B (AKT) phosphorylation domain, and a C-terminal RING-H2 domain. We identified that GFP-VPS39 or -VPS41 co-precipitated the 101–200 amino acid region of RNF115, while the N- and C-terminals of RNF115 showed no interactions. Knocking down RNF115 increased the protein expression levels of VPS39 and VPS41, whereas overexpressing RNF115 reduced their expression. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) testing revealed that RNF115 knockdown increased VPS39 and VPS41 messenger RNA (mRNA) levels, while RNF115 overexpression decreased these levels. Further experiments showed that the decreased VPS39 in RNF115-overexpressing cells did not recover with MG132 treatment, whereas MG132 largely restored VPS41 expression, suggesting that RNF115 negatively regulates VPS39/VPS41 expression at both transcriptional and post-translational levels.

The HOPS complex and the GTPase RAB7 are critical for tethering autophagosomes with lysosomes, followed by fusion of the membrane bilayer. We demonstrated that RNF115 interacts with RAB7 and affects the RAB7–VPS39–VPS41 interaction. RNF115-depleted cells had significantly increased amounts of RAB7-precipitated VPS39/VPS41 compared to control cells, indicating that RNF115 negatively regulates the HOPS complex and the binding affinity of RAB7 with the HOPS complex, thereby decreasing autophagosome-lysosome fusion.

Autophagy related 14 (ATG14) is localized in the endoplasmic reticulum and is a component of the phosphatidylinositol 3-kinase catalytic subunit type 3 (PIK3C3) complex, which promotes autophagosome formation. ATG14 also binds to syntaxin 17 (STX17) and increases STX17–synaptosome associated protein 29 (SNAP29)–vesicle associated membrane protein 8 (VAMP8) complex assembly, promoting autophagosome-lysosome fusion. We found that RNF115 interacts with ATG14, and overexpressing RNF115 decreased GFP-ATG14 levels, while silencing RNF115 increased ATG14 protein expression. MG132 treatment of RNF115-overexpressing cells recovered the decreased ATG14, indicating that RNF115 downregulates ATG14 through the proteasome pathway. Additionally, RNF115 negatively regulated ATG14 transcript levels, suggesting that RNF115 negatively regulates ATG14 expression at both transcriptional and post-translational levels.

We further explored the function of RNF115 in HCC development by generating stable RNF115 knockdown HepG2 and BEL-7402 cell lines using HBLV-shRNF115 lentiviral vectors. The stable RNF115 knockdown cells exhibited decreased SQSTM1 levels and accumulated LC3B-II, indicating increased autophagic activity. Cell viability and colony formation assays demonstrated that knocking down RNF115 blocked hepatoma cell growth. Wound healing and Transwell assays showed that silencing RNF115 delayed hepatoma cell migration.

In vivo experiments using a nude mouse tumorigenicity assay revealed that knocking down RNF115 significantly inhibited BEL-7402 cell growth compared to the control group. We also generated Rnf115 knockout (KO) mice and administered diethylnitrosamine (DEN) to 14-day-old male Rnf115−/− mice and wild-type (WT) C57BL/6 mice. The Rnf115−/− mice developed fewer tumors than the WT mice, and hematoxylin–eosin (H&E) staining suggested that DEN-induced Rnf115+/+ livers contained more irregular tumor cell clusters than Rnf115−/− liver tissues. Immunofluorescence assays showed that DEN-induced Rnf115+/+ livers had a significantly higher fluorescence signal of SQSTM1 protein than Rnf115−/− liver tissues, indicating that decreased SQSTM1 accumulation in Rnf115−/− livers contributed to the inhibition of tumor progression.

In summary, our study demonstrates that RNF115 interacts with the HOPS complex and ATG14, negatively regulating their expression. Inactivating RNF115 promotes autophagic activity by enhancing RAB7–HOPS complex binding and ATG14 activities, which inhibits hepatoma cell growth both in vivo and in vitro. These findings provide an experimental foundation for HCC treatment by targeting RNF115.

doi.org/10.1097/CM9.0000000000003466

Was this helpful?

YesNo

0 / 0