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. 2014 Nov 6;5(11):e1510.
doi: 10.1038/cddis.2014.468.

Bortezomib enhances cancer cell death by blocking the autophagic flux through stimulating ERK phosphorylation

C Kao  1 , A Chao  2 , C-L Tsai  2 , W-C Chuang  1 , W-P Huang  3 , G-C Chen  4 , C-Y Lin  2 , T-H Wang  5 , H-S Wang  6 , C-H Lai  2
Affiliations

Bortezomib enhances cancer cell death by blocking the autophagic flux through stimulating ERK phosphorylation

C Kao et al. Cell Death Dis. .

Abstract

The antitumor activity of an inhibitor of 26S proteasome bortezomib (Velcade) has been observed in various malignancies, including colon cancer, prostate cancer, breast cancer, and ovarian cancer. Bortezomib has been proposed to stimulate autophagy, but scientific observations did not always support this. Interactions between ERK activity and autophagy are complex and not completely clear. Autophagy proteins have recently been shown to regulate the functions of ERK, and ERK activation has been found to induce autophagy. On the other hand, sustained activation of ERK has also been shown to inhibit the maturation step of the autophagy process. In this study, we sought to identify the mechanism of autophagy regulation in cancer cells treated with bortezomib. Our results indicate that bortezomib blocked the autophagic flux without inhibiting the fusion of the autophagosome and lysosome. In ovarian cancer, as well as endometrial cancer and hepatocellular carcinoma cells, bortezomib inhibited protein degradation in lysosomes by suppressing cathepsins, which requires the participation of ERK phosphorylation, but not JNK or p38. Our findings that ERK phosphorylation reduced cathepsins further explain how ERK phosphorylation inhibits the autophagic flux. In conclusion, bortezomib may induce ERK phosphorylation to suppress cathepsin B and inhibit the catalytic process of autophagy in ovarian cancer and other solid tumors. The inhibition of cisplatin-induced autophagy by bortezomib can enhance chemotherapy efficacy in ovarian cancer. As we also found that bortezomib blocks the autophagic flux in other cancers, the synergistic cytotoxic effect of bortezomib by abolishing chemotherapy-related autophagy may help us develop strategies of combination therapies for multiple cancers.

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Figures

Figure 1
Figure 1
Bortezomib (BTZ) induces autophagy. (a) Immunofluorescent puncta formation in TOV112D cells transfected with GFP-LC3 in the presence or absence of 0.05 μM BTZ for 24 h. Results shown are mean±standard error for three independent experiments. Scale bar represents 30 μm. (b) Transmission electron microscopy showed that 24 h of BTZ treatment induced the formation of initial autophagic vacuoles (indicated with arrowhead) and degrading autophagic vacuoles (indicated with arrow). Of note, many degrading autophagic vacuoles had double membranes. Scale bar = 100 nm. (c) BTZ induced autophagy in a time-dependent manner. After treatment with 0.05 μM BTZ for 0, 1, 2, 4, 8, 16, and 24 h, TOV112D cells were analyzed for immunofluorescent GFP-LC3 puncta formation. Results shown are mean±standard error for three independent experiments. Scale bar represents 30 μm. (d) After treatment with designated concentrations of BTZ for 24 h, ovarian cancer cells (TOV112D, TOV21G, OV90, and ES2) were analyzed with western blots for the intensity changes in LC3-I and LC3-II, as indicated. CTR, control
Figure 2
Figure 2
Bortezomib (BTZ) specifically initiates autophagy but blocks the degradation of p62. (a) Left panel: A time-dependent increase of immunofluorescent GFP-LC3 puncta formation was shown in TOV112D cells treated with 0.05 μM BTZ, but not in those with 0.05 μM MG132. Scale bar represents 30 μm. Right panel: The numbers of puncta in every cell were counted and averaged. The fold change of puncta was normalized by the puncta number in the cells at 0 h. Results shown are mean±standard error for three independent experiments. (b) Ovarian cancer cell lines (TOV112D, TOV21G, OV90, and ES2) were treated with designated concentrations of BTZ or MG132 for 24 h, and the intensity changes in LC3-II and p62 were analyzed with western blots. (c) TOV112D cells were treated with either 0.05 μM BTZ or 0.05 μM MG132 in presence of 100 μM cycloheximide (CHX) for 1, 2, and 4 h, and the changes in p62 levels were analyzed with western blots
Figure 3
Figure 3
Bortezomib (BTZ) inhibits lysosome functions after the fusion of autophagosome and lysosome. (a) Ovarian cancer cells (TOV112D, TOV21G, and OV90) were treated with designated concentrations of BTZ for designated hours, and the level changes in LC3-I and p62 were analyzed with western blots. Treatment with 30 μM chloroquine (CQ) in TOV112D cells for the same time periods was used to show the effects of fusion disruption between autophagosome and lysosome. (b) After being transfected with GFP-LC3 for 72 h, TOV112D cells were treated with 0.05 μM BTZ, 5 μM rapamycin (RAPA), or 30 μM chloroquine (CQ) for 24 h, and analyzed for LAMP-2 levels with immunofluorescent microscopy. In the cells treated with rapamycin or BTZ, the merged yellow signals of GFP-LC3 (green) and LAMP-2 (red) indicated the co-localization of autophagosome (marked by GFP-LC3) and lysosome (marked by LAMP-2). Chloroquine blocked the fusion of autophagosome and lysosome, thus minimized yellow signals. Scale bar represents 30 μm. CTR, control
Figure 4
Figure 4
Bortezomib (BTZ) suppresses the catalytic process of autophagy in lysosome. (a) TOV112D cells were treated with 0.05 μM BTZ, 5 μM rapamycin (RAPA), or 30 μM CQ for 24 h, and subjected to lysosomal analysis with 300 nM Lysotracker for 16 h. Rapamycin-treated cells were shown as the positive control of autophagy, showing the merged yellow signals of autophagosome (green fluorescence) and functional lysosome (red Lysotracker signals). Even in the cells treated with rapamycin, co-treatment with chloroquine blocked the fusion of autophagosome and lysosome and diminished lysosomal function, shown by the absence of red Lysotracker signal. (b) TOV112D cells were transiently transfected with GFP-RFP-LC3 vector for 72 h, and subsequently treated with 0.05 μM BTZ, 5 μM rapamycin (RAPA), or 30 μM CQ for 24 h. Using the GFP-RFP-LC3 expression vector, autophagosomes (GFP) and autolysomes (RFP) are shown with green and red signals, respectively. Thus, the fluorescence changes can be used as an indicator for the autophagic flux. Scale bar represents 30 μm. CTR, control
Figure 5
Figure 5
Bortezomib (BTZ) induces the phosphorylation of ERK, reduces the level of cathepsin B (CTSB), and suppresses the catalytic process in autophagic lysosomes. (a) TOV112D cells were treated with 0.05 μM BTZ for 24 h. CTSB protein and mRNA levels were measured by immunoblotting and real-time quantitative PCR, respectively. Results shown are mean±standard error for three independent experiments. (b) The effects of the overexpression of CTSB for 72 h on the TOV112D cells treated with 0.05 μM BTZ for 24 h were analyzed by measuring the p62 protein level and (c) using the MTT assays. Forced expression of C9-CTSB was detected by anti-C9 antibody with western blot analysis. Results shown are the mean±standard error for three independent experiments. (d) The effects induced by the treatment with 0.05 μM BTZ and 30 μM PD98059 for 24 h were analyzed in TOV112D cells using immunoblotting for the levels of ERK phosphorylation, CTSB, and p62. (e) The effect of forced expression of a constitutively active p-ERK (Y204D) for 72 h in TOV112D cells were analyzed by measuring the level of CTSB. Results shown are mean ± standard error for three independent experiments. (f) TOV112D cells were transfected with a C9-CTSB expression vector and treated with 0.05 μM BTZ. After lysosomal labeling with 300 nM Lysotracker (red) for 16 h, the colocalization of CTSB and functional lysosomes was shown by yellow merged signals of anti-C9 (green) into Lysotracker (red). Scale bar represents 30 μm
Figure 6
Figure 6
Bortezomib (BTZ) blocks cisplatin (CDDP)-induced autophagy and enhances cytotoxicity in mice. (a) Treatment with 5 μM CDDP caused a time-dependent autophagy of TOV112D, shown by increasing levels of LC3-II and decreasing levels of p62. (b) Puncta formation (arrow) in TOV112D cells treated with 0.05 μM BTZ, 5 μM CDDP, or the combination of both for 24 h. Scale bar represents 30 μm. (c) Treatment with 0.05 μM BTZ inhibited CDDP-induced autophagy, shown by the increased level of p62 and LC3-II. (d) CPPD-induced autophagy was characterized with increased levels of cathepsin B, which was inhibited by the co-treatment with BTZ. (e) Ten millions of MOSEC/LUC cells were intraperitoneally injected into C57BL/6 mice. Subsequently, the mice were intraperitoneally injected with 100 μL HBSS (vehicle alone), 20 μg/mL BTZ per mouse, 100 μL CDDP (80 μM) per mouse, or both twice a week. The mice were then analyzed with the IVIS 200 in vivo imaging system on a weekly basis. (f) Protein levels of p-ERK, cathepsin B (CTSB), and p62 from representative tumors from the aforementioned studied mice were analyzed with immunohistochemistry. Protein levels were shown as different intensities of brown color. Cell nuclei were counterstained with hematoxylin. Scale bar represents 20 μm. CTR, control
Figure 7
Figure 7
ERK phosphorylation induced by bortezomib inhibits the catalytic process of autophagy and enhances cisplatin-induced cytotoxicity of cancer cells. (a) Upon the treatment with cisplatin, cancer cells often use autophagy as a form of self-rescue. Increased levels of cathepsin B (CTSB) are required to complete the catabolic process of autophagy. (b) Treatment with bortezomib induces phosphorylation of ERK, which decreases CTSB levels and blocks the catabolic process of autophagy. By adding bortezomib to cisplatin-based chemotherapy, self-rescuing autophagy of cancer cells is suppressed, resulting in an enhanced anticancer efficacy
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