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Histone deacetylase inhibitors restore toxic BH3 domain protein expression in anoikis-resistant mammary and brain cancer stem cells, thereby enhancing the response to anti-ERBB1/ERBB2 therapy.

Cruickshanks N, Hamed HA, Booth L, Tavallai S, Syed J, Sajithlal GB, Grant S, Poklepovic A, Dent P - Cancer Biol. Ther. (2013)

Bottom Line: AR mammary carcinoma cells had reduced expression of the toxic BH3 domain proteins BAX, BAK, NOXA, and PUMA.Knockdown of MCL-1 and BCL-XL caused necro-apoptosis in AR cells to a greater extent than in parental cells.These data argue that one mechanism to enhance the anti-tumor effect of chemotherapy could be HDACI pre-treatment.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurosurgery; Virginia Commonwealth University; Richmond, VA USA.

ABSTRACT
The present studies focused on defining the mechanisms by which anoikis-resistant (AR) mammary carcinoma cells can be reverted to a therapy-sensitive phenotype. AR mammary carcinoma cells had reduced expression of the toxic BH3 domain proteins BAX, BAK, NOXA, and PUMA. In AR cells expression of the protective BCL-2 family proteins BCL-XL and MCL-1 was increased. AR cells were resistant to cell killing by multiple anti-tumor cell therapies, including ERBB1/2 inhibitor + MCL-1 inhibitor treatment, and had a reduced autophagic flux response to these therapies, despite similarly exhibiting increased levels of LC3II processing. Knockdown of MCL-1 and BCL-XL caused necro-apoptosis in AR cells to a greater extent than in parental cells. Pre-treatment of anoikis-resistant cells with histone deacetylase inhibitors (HDACIs) for 24 h increased the levels of toxic BH3 domain proteins, reduced MCL-1 levels, and restored/re-sensitized the cell death response of AR tumor cells to multiple toxic therapies. In vivo, pre-treatment of AR breast tumors in the brain with valproate restored the chemo-sensitivity of the tumors and prolonged animal survival. These data argue that one mechanism to enhance the anti-tumor effect of chemotherapy could be HDACI pre-treatment.

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Figure 4. Regulation of lapatinib + obatoclax lethality by toxic BH3 domain proteins. (A) BT474 (wild type) cells were transfected with scrambled siRNA (siSCR, 20 nM) or siRNA molecules to knock down BAK, BAK, NOXA, and PUMA, as indicated. Thirty-six hours after transfection cells were treated with vehicle (VEH, DMSO) or with lapatinib (lap, 1 μM) and obatoclax (GX, 50 nM). Cells were isolated 12 h later and viability determined by trypan blue (± SEM, n = 3) #P < 0.05 less than corresponding value in siSCR cells; ##P < 0.05 less than corresponding single knock down cells. (B) BT474 (WT, wild type; AR, anoikis-resistant) cells were transfected with empty vector plasmid (CMV) or plasmids to express BAX, BAK, NOXA, and PUMA as indicated. Thirty-six hours after transfection cells were treated with vehicle (VEH, DMSO) or with lapatinib (lap, 1 μM) and obatoclax (GX, 50 nM). Cells were isolated 12 h later and viability determined by trypan blue (± SEM, n = 3) *P < 0.05 greater than corresponding value in CMV cells; **P < 0.05 greater than corresponding single expression cells. (C) BT474 (AR, anoikis-resistant) cells were transfected with scrambled siRNA (siSCR, 20 nM) or siRNA molecules to knock down MCL-1, c-FLIP-s, and BCL-XL, as indicated. Thirty-six hours after transfection cells were treated with vehicle (VEH, DMSO) or with lapatinib (lap, 1 μM) and obatoclax (GX, 50 nM). Cells were isolated 12 h later and viability determined by trypan blue (± SEM, n = 3) *P < 0.05 greater than corresponding value in siSCR cells; %P < 0.05 less than corresponding value in siMCL-1 cells. (D) SKBR3 (AR, anoikis-resistant) cells were transfected with scrambled siRNA (siSCR, 20 nM) or siRNA molecules to knock down MCL-1 and BCL-XL, as indicated. Thirty-six hours after transfection cells were treated with vehicle (VEH, DMSO) or with lapatinib (lap, 1 μM) and obatoclax (GX, 50 nM). Cells were isolated 12 h later and viability determined by trypan blue (± SEM, n = 3) *P < 0.05 greater than corresponding value in siSCR cells; %P < 0.05 less than corresponding value in siMCL-1 cells. (E) BT474 (AR, anoikis-resistant) cells were infected with empty vector adenovirus (CMV) or with adenoviruses to express dominant negative MEK1 or dominant negative AKT, as indicated. Thirty-six hours after infection cells were treated with vehicle (VEH, DMSO) or with lapatinib (lap, 1 μM) and obatoclax (GX, 50 nM). Cells were isolated 12 h later and viability determined by trypan blue (± SEM, n = 3) #P < 0.05 less than corresponding value in CMV cells.
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Figure 4: Figure 4. Regulation of lapatinib + obatoclax lethality by toxic BH3 domain proteins. (A) BT474 (wild type) cells were transfected with scrambled siRNA (siSCR, 20 nM) or siRNA molecules to knock down BAK, BAK, NOXA, and PUMA, as indicated. Thirty-six hours after transfection cells were treated with vehicle (VEH, DMSO) or with lapatinib (lap, 1 μM) and obatoclax (GX, 50 nM). Cells were isolated 12 h later and viability determined by trypan blue (± SEM, n = 3) #P < 0.05 less than corresponding value in siSCR cells; ##P < 0.05 less than corresponding single knock down cells. (B) BT474 (WT, wild type; AR, anoikis-resistant) cells were transfected with empty vector plasmid (CMV) or plasmids to express BAX, BAK, NOXA, and PUMA as indicated. Thirty-six hours after transfection cells were treated with vehicle (VEH, DMSO) or with lapatinib (lap, 1 μM) and obatoclax (GX, 50 nM). Cells were isolated 12 h later and viability determined by trypan blue (± SEM, n = 3) *P < 0.05 greater than corresponding value in CMV cells; **P < 0.05 greater than corresponding single expression cells. (C) BT474 (AR, anoikis-resistant) cells were transfected with scrambled siRNA (siSCR, 20 nM) or siRNA molecules to knock down MCL-1, c-FLIP-s, and BCL-XL, as indicated. Thirty-six hours after transfection cells were treated with vehicle (VEH, DMSO) or with lapatinib (lap, 1 μM) and obatoclax (GX, 50 nM). Cells were isolated 12 h later and viability determined by trypan blue (± SEM, n = 3) *P < 0.05 greater than corresponding value in siSCR cells; %P < 0.05 less than corresponding value in siMCL-1 cells. (D) SKBR3 (AR, anoikis-resistant) cells were transfected with scrambled siRNA (siSCR, 20 nM) or siRNA molecules to knock down MCL-1 and BCL-XL, as indicated. Thirty-six hours after transfection cells were treated with vehicle (VEH, DMSO) or with lapatinib (lap, 1 μM) and obatoclax (GX, 50 nM). Cells were isolated 12 h later and viability determined by trypan blue (± SEM, n = 3) *P < 0.05 greater than corresponding value in siSCR cells; %P < 0.05 less than corresponding value in siMCL-1 cells. (E) BT474 (AR, anoikis-resistant) cells were infected with empty vector adenovirus (CMV) or with adenoviruses to express dominant negative MEK1 or dominant negative AKT, as indicated. Thirty-six hours after infection cells were treated with vehicle (VEH, DMSO) or with lapatinib (lap, 1 μM) and obatoclax (GX, 50 nM). Cells were isolated 12 h later and viability determined by trypan blue (± SEM, n = 3) #P < 0.05 less than corresponding value in CMV cells.

Mentions: Based on our data in AR cells, we re-examined the roles of BH3 domain proteins in wild type parental cells. As previously noted knock down of BAX, BAK, NOXA, and PUMA suppressed lapatinib + obatoclax lethality in wild-type BT474 cells (Fig. 4A).17-19 Re-expression of toxic BH3 domain proteins BAX, BAK, NOXA, and PUMA restored lapatinib + obatoclax the responsiveness of AR cells; cells that have low expression of such proteins (Fig. 4B). Knockdown of MCL-1, BCL-XL, or c-FLIP-s also partially restored lapatinib + obatoclax responsiveness of AR cells (Figs. 4C and D). Expression of dominant negative MEK1 or of dominant negative AKT also partially restored lapatinib + obatoclax responsiveness of AR cells (Fig. 4E, cf. data in Fig. 2A).


Histone deacetylase inhibitors restore toxic BH3 domain protein expression in anoikis-resistant mammary and brain cancer stem cells, thereby enhancing the response to anti-ERBB1/ERBB2 therapy.

Cruickshanks N, Hamed HA, Booth L, Tavallai S, Syed J, Sajithlal GB, Grant S, Poklepovic A, Dent P - Cancer Biol. Ther. (2013)

Figure 4. Regulation of lapatinib + obatoclax lethality by toxic BH3 domain proteins. (A) BT474 (wild type) cells were transfected with scrambled siRNA (siSCR, 20 nM) or siRNA molecules to knock down BAK, BAK, NOXA, and PUMA, as indicated. Thirty-six hours after transfection cells were treated with vehicle (VEH, DMSO) or with lapatinib (lap, 1 μM) and obatoclax (GX, 50 nM). Cells were isolated 12 h later and viability determined by trypan blue (± SEM, n = 3) #P < 0.05 less than corresponding value in siSCR cells; ##P < 0.05 less than corresponding single knock down cells. (B) BT474 (WT, wild type; AR, anoikis-resistant) cells were transfected with empty vector plasmid (CMV) or plasmids to express BAX, BAK, NOXA, and PUMA as indicated. Thirty-six hours after transfection cells were treated with vehicle (VEH, DMSO) or with lapatinib (lap, 1 μM) and obatoclax (GX, 50 nM). Cells were isolated 12 h later and viability determined by trypan blue (± SEM, n = 3) *P < 0.05 greater than corresponding value in CMV cells; **P < 0.05 greater than corresponding single expression cells. (C) BT474 (AR, anoikis-resistant) cells were transfected with scrambled siRNA (siSCR, 20 nM) or siRNA molecules to knock down MCL-1, c-FLIP-s, and BCL-XL, as indicated. Thirty-six hours after transfection cells were treated with vehicle (VEH, DMSO) or with lapatinib (lap, 1 μM) and obatoclax (GX, 50 nM). Cells were isolated 12 h later and viability determined by trypan blue (± SEM, n = 3) *P < 0.05 greater than corresponding value in siSCR cells; %P < 0.05 less than corresponding value in siMCL-1 cells. (D) SKBR3 (AR, anoikis-resistant) cells were transfected with scrambled siRNA (siSCR, 20 nM) or siRNA molecules to knock down MCL-1 and BCL-XL, as indicated. Thirty-six hours after transfection cells were treated with vehicle (VEH, DMSO) or with lapatinib (lap, 1 μM) and obatoclax (GX, 50 nM). Cells were isolated 12 h later and viability determined by trypan blue (± SEM, n = 3) *P < 0.05 greater than corresponding value in siSCR cells; %P < 0.05 less than corresponding value in siMCL-1 cells. (E) BT474 (AR, anoikis-resistant) cells were infected with empty vector adenovirus (CMV) or with adenoviruses to express dominant negative MEK1 or dominant negative AKT, as indicated. Thirty-six hours after infection cells were treated with vehicle (VEH, DMSO) or with lapatinib (lap, 1 μM) and obatoclax (GX, 50 nM). Cells were isolated 12 h later and viability determined by trypan blue (± SEM, n = 3) #P < 0.05 less than corresponding value in CMV cells.
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Figure 4: Figure 4. Regulation of lapatinib + obatoclax lethality by toxic BH3 domain proteins. (A) BT474 (wild type) cells were transfected with scrambled siRNA (siSCR, 20 nM) or siRNA molecules to knock down BAK, BAK, NOXA, and PUMA, as indicated. Thirty-six hours after transfection cells were treated with vehicle (VEH, DMSO) or with lapatinib (lap, 1 μM) and obatoclax (GX, 50 nM). Cells were isolated 12 h later and viability determined by trypan blue (± SEM, n = 3) #P < 0.05 less than corresponding value in siSCR cells; ##P < 0.05 less than corresponding single knock down cells. (B) BT474 (WT, wild type; AR, anoikis-resistant) cells were transfected with empty vector plasmid (CMV) or plasmids to express BAX, BAK, NOXA, and PUMA as indicated. Thirty-six hours after transfection cells were treated with vehicle (VEH, DMSO) or with lapatinib (lap, 1 μM) and obatoclax (GX, 50 nM). Cells were isolated 12 h later and viability determined by trypan blue (± SEM, n = 3) *P < 0.05 greater than corresponding value in CMV cells; **P < 0.05 greater than corresponding single expression cells. (C) BT474 (AR, anoikis-resistant) cells were transfected with scrambled siRNA (siSCR, 20 nM) or siRNA molecules to knock down MCL-1, c-FLIP-s, and BCL-XL, as indicated. Thirty-six hours after transfection cells were treated with vehicle (VEH, DMSO) or with lapatinib (lap, 1 μM) and obatoclax (GX, 50 nM). Cells were isolated 12 h later and viability determined by trypan blue (± SEM, n = 3) *P < 0.05 greater than corresponding value in siSCR cells; %P < 0.05 less than corresponding value in siMCL-1 cells. (D) SKBR3 (AR, anoikis-resistant) cells were transfected with scrambled siRNA (siSCR, 20 nM) or siRNA molecules to knock down MCL-1 and BCL-XL, as indicated. Thirty-six hours after transfection cells were treated with vehicle (VEH, DMSO) or with lapatinib (lap, 1 μM) and obatoclax (GX, 50 nM). Cells were isolated 12 h later and viability determined by trypan blue (± SEM, n = 3) *P < 0.05 greater than corresponding value in siSCR cells; %P < 0.05 less than corresponding value in siMCL-1 cells. (E) BT474 (AR, anoikis-resistant) cells were infected with empty vector adenovirus (CMV) or with adenoviruses to express dominant negative MEK1 or dominant negative AKT, as indicated. Thirty-six hours after infection cells were treated with vehicle (VEH, DMSO) or with lapatinib (lap, 1 μM) and obatoclax (GX, 50 nM). Cells were isolated 12 h later and viability determined by trypan blue (± SEM, n = 3) #P < 0.05 less than corresponding value in CMV cells.
Mentions: Based on our data in AR cells, we re-examined the roles of BH3 domain proteins in wild type parental cells. As previously noted knock down of BAX, BAK, NOXA, and PUMA suppressed lapatinib + obatoclax lethality in wild-type BT474 cells (Fig. 4A).17-19 Re-expression of toxic BH3 domain proteins BAX, BAK, NOXA, and PUMA restored lapatinib + obatoclax the responsiveness of AR cells; cells that have low expression of such proteins (Fig. 4B). Knockdown of MCL-1, BCL-XL, or c-FLIP-s also partially restored lapatinib + obatoclax responsiveness of AR cells (Figs. 4C and D). Expression of dominant negative MEK1 or of dominant negative AKT also partially restored lapatinib + obatoclax responsiveness of AR cells (Fig. 4E, cf. data in Fig. 2A).

Bottom Line: AR mammary carcinoma cells had reduced expression of the toxic BH3 domain proteins BAX, BAK, NOXA, and PUMA.Knockdown of MCL-1 and BCL-XL caused necro-apoptosis in AR cells to a greater extent than in parental cells.These data argue that one mechanism to enhance the anti-tumor effect of chemotherapy could be HDACI pre-treatment.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurosurgery; Virginia Commonwealth University; Richmond, VA USA.

ABSTRACT
The present studies focused on defining the mechanisms by which anoikis-resistant (AR) mammary carcinoma cells can be reverted to a therapy-sensitive phenotype. AR mammary carcinoma cells had reduced expression of the toxic BH3 domain proteins BAX, BAK, NOXA, and PUMA. In AR cells expression of the protective BCL-2 family proteins BCL-XL and MCL-1 was increased. AR cells were resistant to cell killing by multiple anti-tumor cell therapies, including ERBB1/2 inhibitor + MCL-1 inhibitor treatment, and had a reduced autophagic flux response to these therapies, despite similarly exhibiting increased levels of LC3II processing. Knockdown of MCL-1 and BCL-XL caused necro-apoptosis in AR cells to a greater extent than in parental cells. Pre-treatment of anoikis-resistant cells with histone deacetylase inhibitors (HDACIs) for 24 h increased the levels of toxic BH3 domain proteins, reduced MCL-1 levels, and restored/re-sensitized the cell death response of AR tumor cells to multiple toxic therapies. In vivo, pre-treatment of AR breast tumors in the brain with valproate restored the chemo-sensitivity of the tumors and prolonged animal survival. These data argue that one mechanism to enhance the anti-tumor effect of chemotherapy could be HDACI pre-treatment.

Show MeSH
Related in: MedlinePlus