MOB1A regulates glucose deprivation-induced autophagy via IL6-STAT3 pathway in gallbladder carcinoma

Abstract

MOB kinase activator 1A (MOB1A) plays an important role in many diseases and cancers. Here, we observed that MOB1A was substantially overexpressed in gallbladder carcinoma (GBC) tissues compared with nontumor tissues. The high expression of MOB1A was closely associated with poor survival in patients with GBC at advanced TNM stages. Furthermore, our study indicated that MOB1A promoted autophagy by activating the IL6/STAT3 signaling pathway and regulating the chemosensitivity to gemcitabine under glucose deprivation conditions both in vitro and in vivo. In conclusion, these findings suggested that MOB1A is critical for the development of GBC via the MOB1A-IL6/STAT3-autophagy axis.

Keywords: IL6-STAT3 pathways; MOB1A; autophagy; gallbladder carcinoma; glucose-deprivation.

Figure 1

The expression and clinical significance of MOB1A in GBC tissues. (A) Part of the cluster analysis of miRNA expression profiles of GBC tissues and nontumor tissues from our previous microarray results. (B, C) Ten pairs of fresh GBC tissues and corresponding adjacent nontumor tissues were used for qRT-PCR analysis (B) and western blotting (C) to compare the expression levels of MOB1A. (D) Distribution of IHC results in 45 GBC tissues. (E) Sections used for the analysis of the expression levels of MOB1A in GBC tumor and cholecystitis tissues. Representative images of cholecystitis and GBC tissues are shown for weak (+), moderate (++), and strong (+++) staining. (F) Kaplan-Meier overall survival curve of patients with GBC based on different levels of MOB1A. Tests of significance were two-sided; *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 2

MOB1A promotes the survival of cancer cells in a glucose-deprivation microenvironment. (A) Protein expression of MOB1A in GBC cells lines, including NOZ, GCB-SD, OCUG, and SGC996, as well as in normal gallbladder cells, HGEPC. (A, B) Indicated GBC cells were transfected with lentiviruses to knockdown (A) or upregulate (B) the expression of MOB1A. Western blotting was performed to verify the stable cell lines. (C, D) Representative image (C) and expression levels of MOB1A and Ki-67 detected in a GBC subcutaneous xenograft model by IHC (D) after scarification (scale bar, 100 μm). (E) The tumor size and weight formed in nude mice injected with either scramble or shMOB1A-NOZ expressing cells, were measured. (F, G) Flow cytometry analysis (F) was used to detect cell apoptosis (G) of GBC-SD, and NOZ scramble, or shMOB1A cells under nutritional-deprived conditions (**P < 0.01).

Figure 3

MOB1A promotes autophagy and protects cancer cells from apoptosis in glucose-deprivation conditions. (A) Indicated GBC cells were incubated with or without glucose for 24 h and then collected for western blotting to detect the level of LC3, SQSTM1, and BECN1. (B, C) Cells were treated with glucose-free medium for 24 h and then collected to detect the level of LC3, SQSTM1, and BECN1, following downregulation (B) or upregulation (C) of MOB1A. (D, E) The level of LC3 in GBC-SD cells was visualized by immunofluorescent staining (D). GBC-SD cells expressing scramble or shMOB1A, cultured in glucose-free medium for 24 h, and analyzed for LC3-expressing puncta (E) (**P < 0.01). (F) Following incubation with glucose-free medium for 24 h indicated MOB1A-overexpressing GBC cells prevented cell apoptosis induced by the glucose deprivation conditions. Western blotting was performed to detect the levels of cleaved-PARP and cleaved-caspase 3. (G) Following treatment with HCQ (10 μM) to block autophagy and incubated in glucose-free medium for 24 h, the inhibition effect of MOB1A on apoptosis was abolished. The levels of cleaved-PARP and cleaved-caspase 3 were detected by western blotting. (H, I) Following incubation in glucose-free medium for 24 h, MOB1A-overexpressing GBC cells exhibited increased cell viability; when treated with HCQ (10 μM), no difference was observed between the EV and the MOB1A group. Flow cytometry analysis (H) was used to detect cell viability (I). **P < 0.01; ***P < 0.001.

Figure 5

MOB1A promotes autophagy under glucose-deprivation conditions via the IL6-STAT3 pathway. (A, B) Indicated GBC cells either downregulated (A) or upregulated (B) for the expression of MOB1A were subjected to western blotting to detect the expression level of LC3, IL6, and p-STAT3. (C) Indicated MOB1A-overexpressing GBC cells were transfected with anti-IL6 silencing RNA (IL6 siRNA) or control RNA. (D) The expression level of LC3 was detected by western blotting in MOB1A-downregulated GBC-SD cells following treatment with or without rhIL-6. (E) IHC was used to detect the expression of LC3-II, p-STAT3, cleave-cas3, and IL6 in the scramble and shMOB1A groups in vivo.

Figure 4

MOB1A regulates the chemosensitivity to gemcitabine via autophagy in GBC cells. (A, B) Indicated MOB1A-overexpressing GBC cells exhibited resistance to GEM, as assessed by a cell viability assay following treatment with GEM for 48 h at indicated doses (B); shMOB1A cells showed a corresponding tendency (A). (C) Indicated GBC cells either expressing scramble or shMOB1A assessed by apoptotic assay for flow cytometry after treatment with GEM for 48 h. (D) Following treatment with or without HCQ to block autophagy, MOB1A-overexpressing SGC996 cells were subjected to flow cytometry to detect their apoptotic level. (E-G) Overexpression of MOB1A prevented the growth inhibition of the xenograft and apoptosis induced by intraperitoneal injection (i.p.) of GEM in SGC996 MOB1A-overexpressing cell xenografts, as evaluated by representative images (E), tumor growth volume (F), and Ki-67 (upper) and TUNEL (down) staining in xenograft tissues after scarification (G). *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 6

Schematic of the role of MOB1A in the survival of GBC cells under glucose-deprivation conditions. MOB1A promoted autophagy via the IL6/STAT3 signaling pathway to attenuate cell apoptosis, regulate the chemoresistance to GEM, and then help cells survive in glucose deprivation conditions.