Mevastatin blockade of autolysosome maturation stimulates LBH589-induced cell death in triple-negative breast cancer cells

Abstract

Histone deacetylase inhibitors (HDACi) are promising anti-cancer agents, and combining a HDACi with other agents is an attractive therapeutic strategy in solid tumors. We report here that mevastatin increases HDACi LBH589-induced cell death in triple-negative breast cancer (TNBC) cells. Combination treatment inhibited autophagic flux by preventing Vps34/Beclin 1 complex formation and downregulating prenylated Rab7, an active form of the small GTPase necessary for autophagosome-lysosome fusion. This means that co-treatment with mevastatin and LBH589 activated LKB1/AMPK signaling and subsequently inhibited mTOR. Co-treatment also led to cell cycle arrest in G2/M phase and induced corresponding expression changes of proteins regulating the cell cycle. Co-treatment also increased apoptosis both in vitro and in vivo, and reduced tumor volumes in xenografted mice. Our results indicate that disruption of autophagosome-lysosome fusion likely underlies mevastatin-LBH589 synergistic anticancer effects. This study confirms the synergistic efficacy of, and demonstrates a potential therapeutic role for mevastatin plus LBH589 in targeting aggressive TNBC, and presents a novel therapeutic strategy for further clinical study. Further screening for novel autophagy modulators could be an efficient approach to enhance HDACi-induced cell death in solid tumors.

Keywords: HDAC inhibitor; LBH589; autophagy; breast cancer; mevastatin.

Figure 1. Mevastatin enhances LBH589-induced autophagy and cell death in TNBC cells

Screening for suitable partners acting in synergy with LBH589 in TNBC cells (A) With or without LBH589 (25 nM), endogenous LC3B and p62/SQSTM1 levels were detected by Western blotting in mevastatin-treated MDA-MB-231 (0, 0.5, 1, 2 μM) (B) and MDA-MB-468 cells (0, 4, 8, 16 μM) (C) for 24 h. Synergistic cell death induction by mevastatin and LBH589 for 24 h in MDA-MB-231 (D) and MDA-MB-468 cells (E) followed by FACS analysis. Mevastatin enhanced LBH589-induced apoptosis-related proteins dose-dependently in MDA-MB-231 (F) and MDA-MB-468 cells (G) as shown by Western blotting.

Figure 2. Mevastatin enhances LBH589-induced G2-M arrest in TNBC cells

Cell cycle distribution after treatment with or without LBH589 (25 nM) plus mevastatin (0, 0.5, 1, 2 μM in MDA-MB-231 cells (A); 0, 4, 8, 16 μM in MDA-MB-468 cells (B) for 24 h followed by FACS analysis. Mevastatin regulates cell cycle-related protein expression more efficiently in the presence of LBH589, in a dose-dependent manner. Western blotting of cells treated with or without LBH589 (25 nM) plus mevastatin (0, 0.5, 1, 2 μM in MDA-MB-231 cells (C) 0, 4, 8, 16 μM in MDA-MB-468 cells (D) for 24 h.

Figure 3. Effect of mevastatin on LBH589-induced cell death

MDA-MB-231 and MDA-MB-468 cells were treated with indicated agents for 24 h. Prior to treatment, z-VAD (0, 5, 10, 20 μM) or IETD (20 μM) was added for 2 h to MDA-MB-231 (A) and MDA-MB-468 cells (B), and apoptosis was assessed via FACS analysis. Cleaved PARP, and caspases 8 and 3 were analyzed via Western blotting with MDA-MB-231 (C and E) and MDA-MB-468 cells (D and F).

Figure 4. Mevastatin and LBH589 activate AMPK and inhibit mTOR in TNBC cell lines

AMPK involvement in the synergistic effects of mevastatin and LBH589 on MDA-MB-231 (A) and MDA-MB-468 (B) cells for 24 h. Cell lysates were immunoblotted with indicated antibodies. Co-treatment effect on ERK does not depend on Ras in MDA-MB-231 (C) and MDA-MB-468 (D) cells after 24 h. Cell lysates were analyzed by Western blotting for phosphorylated or total MEK, phosphorylated or total ERK, with GAPDH as an internal control. MDA-MB-231 (E) and MDA-MB-468 cells (F) were treated with mevastatin plus LBH589 for 6, 12, 24 and 36 h. Lysates were immunoblotted with the indicated antibodies.

Figure 5. The mevalonate pathway is involved in mevastatin enhancement of LBH-induced TNBC cell death

Mevalonate and compound C rescued mevastatin-induced proliferation inhibition in MDA-MB-231 and MDA-MB-468 cells. Prior to mevastatin and/or LBH589 (25 nM) treatment for 48 h, MDA-MB-231 (A) and MDA-MB-468 (B) cells were incubated with or without mevalonate (250 μM) for 6 h. Alternatively, prior to treatment, MDA-MB-231 (C) and MDA-MB-468 (D) cells were incubated with or without compound C (1, 2 μM) for 2 h. Mevalonate and compound C blocked LKB1/AMPK signaling in MDA-MB-231 (E) and MDA-MB-468 (F) cells treated with indicated agents for 24 h. Cell lysates were immunoblotted with indicated antibodies. Mevalonate and Compound C reduced cleavage of apoptosis-related proteins in MDA-MB-231 (G) and MDA-MB-468 (H) cells. Cleavage of PARP, and caspases 8 and 3 was analyzed by Western blotting. Data are representative of at least three experiments. Student's t-test. **P < 0.01; ***P < 0.001.

Figure 6. Mevastatin blockade of mevalonate signaling abrogates LBH589-induced autophagy maturation in TNBC cells

Prior to mevastatin plus LBH589 (25 nM) treatment for 24 h, MDA-MB-231 (A) and MDA-MB-468 (B) cells were incubated with or without z-VAD (20 μM) for 2 h. Cell lysates were analyzed by Western blotting for Vps34, Beclin 1, LC3B, p62/SQSTM1, and NBR1, with GAPDH as a loading control. Relative p62/SQSTM1 expression (compared with GAPDH) was analyzed by qPCR. Student's t-test. *P < 0.05; **P < 0.01 (C). MDA-MB-231 cells were treated with mevastatin (0, 0.5, 1, 2 μM) and/or LBH589 (25 nM) for 24 h. Mevastatin reduced LBH589-dependent expression of Vps34 and Beclin 1 in a dose-dependent manner (D). MDA-MB-231 cells were treated with mevastatin (0, 0.5, 1, 2 μM) for 24 h and probed for endogenous Vps34 and Beclin 1. Mevastatin abrogated Vps34-Beclin 1 complex formation dose-dependently in the presence of LBH589 (E). MDA-MB-231 cells were treated with mevastatin (0, 0.5, 1, 2 μM) plus 25 nM LBH589. Immunoblot analyses were performed on immuoprecipitates as indicated. tfLC3 stable MDA-MB-231 cells were analyzed by confocal microscopy (F). MDA-MB-231_tfLC3 cells were treated with mevastatin (2 μM) and/or LBH589 (25 nM) for 24 h. Representative fluorescence images are shown. MDA-MB-231 cells transfected with flag-Rab7 (or pcDNA3.1) were treated with mevastatin (2 μM) and/or LBH589 (25 nM) for 48 h, and cell proliferation was measured. Student's t-test. **P < 0.01; ***P < 0.001 (G). Mevastatin with or without LBH589 (25 nM) reduced prenylated Rab7 in a dose-dependent manner (H). MDA-MB-231 cells were treated with mevastatin (0, 0.5, 1, 2 μM) and/or LBH589 (25 nM) for 24 hours. Cell lysates were immunoblotted for unprenylated and prenylated Rab7. Prior to treatment with mevastatin (2 μM) and LBH589 (25 nM) for 24 h, MDA-MB-231 cells were incubated with or without mevalonate (250 μM) for 6 h, or with or without compound C (2 μM) for 2 h (I). Cell lysates were immunoblotted for LC3B, p62/SQSTM1, and unprenylated and prenylated Rab7, with GAPDH as a loading control.

Figure 7. Mevastatin plus LBH589 enhances TNBC cell death in vivo in MDA-MB-231 cell xenografted mice

Photographs of subcutaneous tumors resulting from indicated treatments in xenografted nude mice (A). Tumor-bearing mice were treated with vehicle control, mevastatin (10 mg/kg orally daily), LBH589 (0.5 mg/kg intraperitoneal injection daily), or combination as indicated (B). Mouse body weight curves following treatments (C). Detection of DNA strand breaks by TUNEL assay in MDA-MB-231 tumors after treatment with vehicle, mevastatin (10 mg/Kg orally daily), LBH589 (0.5 mg/kg intraperitoneal injection daily), or combination (D) Green fluorescence indicates TUNEL positive cells. Tumors were stained with DAPI to identify nuclei. Quantitative evaluation of TUNEL positive cells treated with indicated agents (E). Five images were analyzed in each group; bars over columns represent means ± SD; Student's t-test. **P < 0.01; ***P < 0.001. Immunoblotting analysis of xenograft tumor tissues (F and G).

Figure 8. Schematic diagram of the antitumor activities of mevastatin and LBH589 in TNBC

This schematic diagram shows that mevastatin increased HDACi LBH589-induced cell death in TNBC cells. Combination treatment inhibited autophagic flux by preventing Vps34/Beclin 1 complex formation and the maturation of autophagosome-lysosome via down regulated prenylated Rab7 as well as the activity of P70S6K by activated LKB1/AMPK signaling, resulting in Caspase8 dependent cell death.