Chaperone-mediated autophagy regulates proliferation by targeting RND3 in gastric cancer - PubMed (original) (raw)

doi: 10.1080/15548627.2015.1136770.

Jianjun Yang 1, Xing Fan 3, Sijun Hu 1, Fenli Zhou 1, Jiaqiang Dong 1, Song Zhang 1, Yulong Shang 1, Xiaoming Jiang 4, Hao Guo 1, Ning Chen 1, Xiao Xiao 1, Jianqiu Sheng 5, Kaichun Wu 1, Yongzhan Nie 1, Daiming Fan 1

Affiliations

Chaperone-mediated autophagy regulates proliferation by targeting RND3 in gastric cancer

Jinfeng Zhou et al. Autophagy. 2016.

Abstract

LAMP2A is the key protein of chaperone-mediated autophagy (CMA), downregulation of LAMP2A leads to CMA blockade. CMA activation has been implicated in cancer growth, but the exact mechanisms are unclear. Elevated expression of LAMP2A was found in 8 kinds of tumors (n=747), suggesting that LAMP2A may have an important role in cancer progression. Unsurprisingly, LAMP2A knockdown in gastric cancer (GC) cells hindered proliferation, accompanied with altered expression of cell cycle-related proteins and accumulation of RND3/RhoE. Interactomic and KEGG analysis revealed that RND3 was a putative CMA substrate. Further study demonstrated that RND3 silencing could partly rescue the proliferation arrest induced by LAMP2A knockdown; RND3 was increased upon lysosome inhibition via both chemicals and LAMP2A-shRNA; Furthermore, RND3 could interact with CMA components HSPA8 and LAMP2A, and be engulfed by isolated lysosomes. Thus, constant degradation of RND3 by CMA is required to sustain rapid proliferation of GC cells. At last, the clinical significance of LAMP2A was explored in 593 gastric noncancerous lesions and 173 GC tissues, the results revealed that LAMP2A is a promising biomarker for GC early warning and prognosis of female GC patients.

Keywords: CMA; LAMP2A; RND3; gastric cancer; proliferation.

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Figures

Figure 1.

Figure 1.

Identification of MG5 as a specific antibody against LAMP2A. (A) MG5 immunoblot in gastric cancer cell lines MKN45, BGC823, SGC7901 and AGS, the entire blot (from 10 to 250 kDa) is shown. ((B)and C) coimmunofluorescence of MG5 and LysoTracker Red (DND99) in BGC823 cells, GOLGB1 was used as a negative control, DAPI was used to stain the cell nucleus. Scale bar (in red) was 10 µm. (D) Immunoprecipitates of MG5 (MG5-IP) and control IgG (IgG-IP) form BGC823 cells were subject to western blot (WB) and Coomassie Blue staining. The bands in red rectangles of (D) were excised and prepared for mass spectrometry (MS). (E) Putative antigens for MG5 (in blue or red) were identified by MS and database matching. (F) MS peptide profile matching showed that LAMP2 is a candidate antigen of MG5. (G) WB assay of MG5-IP and IgG-IP using anti-LAMP2 (Santa Cruz Biotechnology). (H) 293T cells were transfected with LAMP2A, LAMP2B, LAMP2C or empty vector (control), and then were subjected to WB with either the MG5 or LAMP2 antibodies Anti-2L (Santa Cruz Biotechnology), ACTB was used as an internal loading control. (I) BGC823 cells were infected with lentivirus expressing LAMP2A, empty vector (Ctr), LAMP2A shRNA and control shRNA (NC), and were subjected to RT-PCR to confirm LAMP2A mRNA changes (I), then WB was performed to examine MG5 immunoreactivity (J), ACTB was used as an internal loading control.

Figure 2.

Figure 2.

CMA blockade impedes gastric cancer cell proliferation. (A, left) Immunostaining of LAMP2A using MG5 in 10 kinds of tumors and normal tissues. Abbreviations: GA, gastric adenocarcinoma; CA, colon adenocarcinoma; RA, rectal adenocarcinoma; PDC, pancreatic ductal carcinoma; LSCC-lung squamous cell carcinoma; LA, lung adenocarcinoma; BDC, breast ductal carcinoma; RCCC, renal clear cell carcinoma; the adjacent normal tissues were immunostained as controls. (Manifestation: X 200). (A, Right) LAMP2A positivity was examined by MG5 staining in 8 kinds of tumors and normal tissues (*, P < 0.05; **, P < 0.001; Chi-square test). ((B)and C) Four stable cell lines (BGC823-NC, BGC823-L2A−, AGS-NC, and AGS-L2A−) were established by infecting BGC823 with lentiviral vectors containing LAMP2A shRNA or control shRNA (NC), and LAMP2A expression was validated by WB, ACTB was used as an internal control. ((D)and E) Growth of the 4 stable cell lines were assayed by MTT (values are means ± SEM of 3 independent experiments; *, P < 0.05; t test). ((F)and G) Apoptosis of the 4 stable cell lines were assayed by FACS (values are means ± SEM of 3 independent experiments). (H) This panel shows the clones formed by BGC823-NC and BGC823-L2A− cell lines (left), and the bar diagram of colony formation numbers of the 2 cell lines (right), the values are means ± SEM of 3 independent experiments; *, P < 0.05; t test. (I) Expression of LAMP2A and cell cycle-related proteins were examined by WB, ACTB was used as an internal control (left). The densitometric analyses of the indicated proteins with respect to ACTB from 3 independent experiments are shown as bar diagram (right).

Figure 3.

Figure 3.

Screening for candidate substrates of CMA that can regulate proliferation, through an interactomic approach and KEGG analysis. (A) LAMP2A, TP53, GAPDG, PGK and RND3 levels were evaluated by WB in BGC823-NC, BGC823-L2A−, AGS-NC, and AGS-L2A− cell lines, ACTB was used as an internal control. (B) Expression correlation among LAMP2A, TP53 and RND3 based on IHC positivity in consecutive gastric cancer tissue slides, (n = 180, Spearman test). Scale bar was 100 µm. (C) HSPA8 and LAMP2A were immunoprecipitated from BGC823 cells and the IP efficacy was evaluated by WB. (D) Immunoprecipitates of (C) were subjected to SDS-PAGE and Coomassie Blue staining, and then the 2 lanes were excised and prepared for mass spectrum analysis, 624 proteins were selected as candidates for CMA substrates or cochaperones, and were subjected to KEGG analysis. The enriched KEGG pathways and related proteins are listed in (E).

Figure 4.

Figure 4.

RND3 mediates CMA blockade-induced proliferation arrest. The BGC823 cell line was transected with both RND3 or control plasmid (A), and the effect of RND3 overexpression on cell growth was assayed by MTT (B), additionally, the effect of RND3 overexpression on cell apoptosis was assayed by FACS (C). (D) Four stable cell lines (BGC823-NC, BGC823-L2A−, BGC823-L2A-/NC and BGC823-L2A− RND3− were established by infecting BGC823 with lentivirus expressing LAMP2A shRNA (L2A−), RND3 shRNA (RND3−), and control shRNA (NC), and the silencing efficacy was evaluated by WB. (E) Growth of the 4 cell lines was examined by MTT assay. (F) Apoptosis of the 4 cell lines was assayed by FACS. ((G)and H) Cell cycle distribution of the 4 cell lines was assayed by FACS. ((I)and J) Subcutaneous growth of the 4 cell lines in nude mice was evaluated by measuring the final volume at Day 10. The values are means ± SEM of 3 independent experiments (B, C, E, F, H, (J)*, P < 0.05; t test).

Figure 5.

Figure 5.

Identification of RND3 as a novel CMA substrate. (A) One “KFERQ”-like motif (QRIEL) was found in the amino sequence of RND3. ((B)and C) RND3 expression was examined in BGC823 cells after exposure to the lysosome inhibitor chloroquine (CQ, 50 µM), Baf, (10 nM), and the proteasome inhibitor MG132 (20 µM) for 12 h. ACTB was used as an internal control. (D) RND3 expression was examined by WB in BGC823-NC and BGC823-L2A−, ACTB was used as an internal control. (E) Interaction among LAMP2A, HSPA8 and RND3 was validated by reciprocal IP assay, LAMP1 was used as a negative control. (F) Interaction between HSPA8 and wild-type RND3 or the Q54A mutant was assayed by CoIP. (G) Coimmunofluorescence was performed to observe the colocalization of LAMP2A and RND3 in BGC823 cell upon inhibition of lysosome degradation by Baf, (10 nM, 12 h). Scale bar (in red) was 10 µm. (H) RND3 levels in whole cell lysates (WCL) and purified lysosomes (Lys) of BGC823 cells were assayed by WB following treatment with Baf (10 nM, 12 h); LAMP2A (lysosome marker) and ACTB (cytosol marker) were examined to verify the enrichment of lysosomes. ((I)and J) Binding and uptake assay was performed for RND3, with a bar diagram that shows percentage of RND3 with regard to the assay (* P < 0.05). GAPDH was used as a positive control. PI indicates protease inhibitors. ((K)and L) Competition assay with bona fide CMA substrate GAPDH for binding and uptake of RND3, bar diagram shows percentage of RND3 with regard to the assay (* P < 0.05). (M) Location of LAMP2A-positive lysosomes relative to nuclei in GES and BGC823 were assayed by IF, Red puncta indicate LAMP2A-positive lysosomes, Blue signal indicates cell nucleus. Scale bar (in red) was 10 µm. ((N)and O) HSPA8 and LAMP2A levels in isolated lys_o_somes of GES and BGC823 were examined by WB, bar diagram indicates densitometric analyses of the indicated proteins from 3 independent experiments. LAMP1 was used as an internal control for lysosomes (*, P < 0.05). (P) Location of RND3 in GES and BGC823 was assayed by IF; red signal indicates RND3, blue signal indicates cell nucleus. Scale bar (in red): 10 µm. (Q) Interaction between LAMP2A and RND3 in GES and BGC823 was assayed by CoIP. (R) Lysosomal binding and uptake assay was performed for endogenous RND3 in GES and BGC823, GAPDH was used as a positive control and PtdIns indicates protease inhibitors. (S) RND3 level in GES was examined by WB following LAMP2A knockdown. ACTB was used as an internal control.

Figure 6.

Figure 6.

LAMP2A expression in GC and noncancerous tissues with regard to prognosis and GC development. (A) Kaplan-Meier curves for postoperative survival of all the GC patients (left), of male GC patients (middle) and of female GC patients (right), according to LAMP2A positivity. The medium survival times of LAMP2A-negative and positive patients were compared by log-rank test, P < 0.05 was considered to be statistically significant. (B) Immunostaining of LAMP2A in gastric precancerous tissues and the corresponding GC tissues. Scale bar (in black): 200 µm. (X 200). (C) Correlation between LAMP2A positivity and GC development risk of gastric precancerosis was analyzed by unconditional logistic regression, P < 0.05 was considered to be statistically significant.

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This work was supported by the National Natural Science Foundation of China (81430072, 81120108005, 81372609, 81421003, 81272652, 81225003, 81227901), Project from Ministry of Science and Technology of China 2015BAI13B07, 2012AA02A504, 2012AA02A203-A03, 2012ZX09303011.

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