Downregulation of ATG5-dependent macroautophagy by chaperone-mediated autophagy promotes breast cancer cell metastasis

Recent data have shown that the expression of lysosome-associated membrane protein type 2 A (LAMP2A), the key protein in the chaperone-mediated autophagy (CMA) pathway, is elevated in breast tumor tissues. However, the exact effects and mechanisms of CMA during breast cancer metastasis remain largely unknown. In this study, we found that the LAMP2A protein level was significantly elevated in human breast cancer tissues, particularly in metastatic carcinoma. The increased LAMP2A level was also positively correlated with the histologic grade of ductal breast cancer. High LAMP2A levels also predicted shorter overall survival of breast cancer patients. Downregulation of CMA activity by LAMP2A knockdown significantly inhibited the growth and metastasis of both MDA-MB-231 and MDA-MB-468 breast cancer cells in vivo and in vitro, while upregulation of CMA activity by LAMP2A overexpression had the opposite effect. Mechanistically, we found that elevated CMA activity mediated increased growth and metastasis of human breast cancer cells by downregulating the activity of autophagy-related gene 5 (ATG5)-dependent macroautophagy. Collectively, these results indicate that the anti-macroautophagic property is a key feature of CMA-mediated tumorigenesis and metastasis and may, in some contexts, serve as an attractive target for breast cancer therapies.


Lysosome isolation
Briefly, 200 mg of cell pellet (about 4×10 7 cells) was harvested by centrifuging and then suspended in 800 μl of lysosome enrichment buffer A added with protease inhibitors, and then the cell suspension was sonicated at 25 w of the power and 3 s one pulse for 3 bursts. 800 μl of lysosome enrichment buffer B with protease inhibitors was then added and mixed, centrifuged at 500 g for 10 min, the supernatant was collected and applied to a discontinuous density gradient (30%, 27%, 23%, 20% and 17% with Optiprep media). Then the samples were ultracentrifuged at 140000 g for 2 hours at 4°C, the top band of the gradient was carefully collected as the lysosomes. Then the lysosomes were mixed with 2-3 volumes of PBS and centrifuged at 18000 g for 30 min at 4°C. The lysosomal pellet integrity was verified by measuring the activity of β-hexosaminidase, a lysosomal enzyme, in the incubation medium 4 , and β-hexosaminidase ELISA kit was from ShangHai Qiaodu Biotechnology (QD30352).

Expression and purification of human GAPDH and HSC70
cDNAs of human GAPDH and HSC70 5 were cloned into the NdeI and XhoI sites of pet22b vector after codon partial tropism transformation. The PCR primers used for amplifying GAPDH and HSC70 cDNA were as follows: GAPDH forward: 5′-GTGACCATATGGGTAAAGTCAAAGTAGGT-3′, GAPDH reverse: 5′-GTGACCTCGAGTTCTTTGGACGCCATGTGC-3′, HSC70 forward: 5′-GACACCATATGAGCAAAGGCCCT-3′, HSC70 reverse: 5′-GTGTCCTCGAGGTCAACTTCTTCAATGGTCGGGCCGGAG-3′. Bacteria strains BL21 (DE3) carrying the pet22b-his-HSC70 and pet22b-his-GAPDH plasmid were induced expression by 0.3 mM of IPTG and incubated for an additional 12 h at 20 °C with 100 μg/ml ampicillin. Then, bacteria were collected by centrifugation (10000 g for 5 min) and washed by PBS, then the pellet was resuspended with 50 mM Tris-HCl, 2 mM EDTA, 100 mM NaCl (pH 8.0), and sonicated at 400 w of the power and 10 s one pulse for 40 bursts. After centrifuged at 10000 g for 30 min, the supernatant was collected and commonly purified by Nickel column.

Western blotting analysis
The cells were harvested and lysates were prepared with RIPA buffer (Beyotime, P0013B). Protein concentrations were determined with the BCA kit (Beyotime, P0011). Then, the proteins (30 µg/well) were subjected to electrophoresis on a 8-12% SDS-PAGE gel, transferred onto polyvinylidene fluoride membranes (Bio-Rad, 162-0177), blocked with 5% bovine serum albumin (BSA) and incubated with the corresponding primary antibodies at 4°C overnight. The membranes were washed and incubated with secondary antibodies and then detected by ECL kit (Millipore, WBKLS0500).

Immunofluorescence staining
Cells grown on sterile cover slips were stained with 25 nM LysoTracker red (Invitrogen, L12492), a lysosomal marker, for 30 min and washed with 1 × PBS followed by fixation with 4% paraformaldehyde for 15 min and then were blocked with goat serum for 30 minutes and incubated with the primary antibody rabbit IgG anti-LAMP2A (1:150) at 4°C overnight. The slides were then extensively washed with PBS and incubated with the fluorescent secondary antibody (Beyotime, P0179) for one hour. Finally, the slides were washed with PBS and visualized using a fluorescence microscope (Leica microsystems); the CMA activity was detected by investigating the distance between LAMP2A-positive lysosomes and the nucleus.

MTT assay
For cell growth, the MDA-MB-231 cells (1.5 × 10 3 /well), MDA-MB-468 cells (3 × 10 3 /well) and MCF10A cells (3 × 10 3 /well) were seeded in 96-well plates and cultured for 12 h for adhesion. The number of viable cells was determined using the MTT assay. The cell numbers were counted after an additional 0, 1, 2, 3, 4, 5, 6, and 7 days, respectively. For H2O2 treatment, the cells were treated with different concentrations (0, 0.05, 0.1 and 0.15 μΜ) of H2O2 for 24 h. Then, 20 μl of the MTT solution (5 mg/ml in PBS) was added to each well, and incubated for 4-6 h at 37°C. The solution was discarded and 200 μl DMSO was added to each well. After gently rocking for 10 min, the plates were detected at 490 nm.

Colony formation assay
The cells were seeded (300 cells/well) onto 6-well plates and allowed to form colonies for two weeks. Then, the colonies were fixed with 4% paraformaldehyde and stained with 1% crystal violet. After rinsing, the colonies were counted under a microscope.

Migration and invasion assay
For the migration assay, the cells were directly seeded in a transwell chamber (Millipore, PIEP12R48) with 8-µm pores in the membranes and cultured for 6 h in a 24-well plate. For the LAMP2A shRNA treatment, cells were seeded at 5 × 10 4 /well; for the LAMP2A overexpression and ATG5 siRNA treatment, the cells were seeded at 2 × 10 4 /well; for MCF10A, the cells were seeded at 6.5 × 10 4 /well. For the invasion assay, the upper chamber was coated with 40 µl extracellular matrix gel (Sigma, E0282) that was diluted 1:2 with cold DMEM medium and incubated 37°C for 30 min to form the ECM gel. The cells were seeded on the Matrigel and cultured for 48 h. The upper chamber was supplied with serum-free medium to allow the cells to migrate or invade towards the complete growth medium. Then, the cells in the upper chamber were wiped off and the cells on the lower surface were fixed and stained with 1% crystal violet before mounting on glass slides. Finally, the cells in five randomly chosen fields were counted under a microscope.

Transmission electron microscope
The cells (2 × 10 6 ) were seeded in 10-cm dishes and allowed to attach overnight. Then, the cells were collected and fixed in ice-cold 2.5% glutaraldehyde in 0.1 M PBS, rinsed with PBS, postfixed in 1% OsO4 with 0.1% potassium ferricyanide, washed again, dehydrated with graded alcohol, and embedded in Epon. Semithin sections (300 nm) were cut using a Reichart Ultracut (Leica). Then, the sections were counterstained with Reynold's lead citrate and examined under a JEOL 1210 transmission electron microscope.