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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 8. Valproate pre-treatment of AR-BT474 cells growing in mouse brains enhances animal survival. (A) Intracerebral injection of WT-BT474 and AR-BT474 cells was performed over 10 min (1 × 106 cells, each). Seven days after tumor cell implantation, animals were segregated into treatment groups. For animal administration sodium valproate was dissolved in saline. Animals were treated with saline vehicle or sodium valproate to a final concentration of 100 mg/kg QD for 2 d. For animal administration, lapatinib and obatoclax were first dissolved in DMSO, and an equal volume of 50:50 Cremophor EL/ethanol (Sigma-Aldrich) was added. After mixing, a 1:10 dilution was made with sterile PBS. Animals were treated with vehicle (PBS/Cremophor EL/ethanol/DMSO), lapatinib, obatoclax, or a combination of lapatinib and obatoclax using oral gavage to a final concentration of 5 mg/kg QD body mass for obatoclax and 100 mg/kg BID for lapatinib for 3 d. *P < 0.05 greater survival than in AR-BT474 cells with vehicle; ¶P < 0.05 greater survival than vehicle treated WT-BT474 cells. Inset: H&E staining of tumors at the time of animal death. (B) WT-BT474 cells (1 × 106 cells, each animal) were injected into the fourth mammary fat pad. Fourteen days after tumor cell implantation tumors of ~100 mm3 had formed, and animals were segregated into treatment groups. For animal administration, lapatinib and flavopiridol were first dissolved in DMSO, and an equal volume of 50:50 Cremophor EL/ethanol (Sigma-Aldrich) was added. After mixing, a 1:10 dilution was made with sterile PBS. Animals were treated with vehicle (PBS/Cremophor EL/ethanol/DMSO), lapatinib, flavopiridol, or a combination of the drugs using oral gavage to a final concentration of 25 mg/kg QD body mass for flavopiridol and 50 mg/kg BID for lapatinib for 3 d. *P < 0.05 greater survival than in vehicle-treated cells.
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Figure 8: Figure 8. Valproate pre-treatment of AR-BT474 cells growing in mouse brains enhances animal survival. (A) Intracerebral injection of WT-BT474 and AR-BT474 cells was performed over 10 min (1 × 106 cells, each). Seven days after tumor cell implantation, animals were segregated into treatment groups. For animal administration sodium valproate was dissolved in saline. Animals were treated with saline vehicle or sodium valproate to a final concentration of 100 mg/kg QD for 2 d. For animal administration, lapatinib and obatoclax were first dissolved in DMSO, and an equal volume of 50:50 Cremophor EL/ethanol (Sigma-Aldrich) was added. After mixing, a 1:10 dilution was made with sterile PBS. Animals were treated with vehicle (PBS/Cremophor EL/ethanol/DMSO), lapatinib, obatoclax, or a combination of lapatinib and obatoclax using oral gavage to a final concentration of 5 mg/kg QD body mass for obatoclax and 100 mg/kg BID for lapatinib for 3 d. *P < 0.05 greater survival than in AR-BT474 cells with vehicle; ¶P < 0.05 greater survival than vehicle treated WT-BT474 cells. Inset: H&E staining of tumors at the time of animal death. (B) WT-BT474 cells (1 × 106 cells, each animal) were injected into the fourth mammary fat pad. Fourteen days after tumor cell implantation tumors of ~100 mm3 had formed, and animals were segregated into treatment groups. For animal administration, lapatinib and flavopiridol were first dissolved in DMSO, and an equal volume of 50:50 Cremophor EL/ethanol (Sigma-Aldrich) was added. After mixing, a 1:10 dilution was made with sterile PBS. Animals were treated with vehicle (PBS/Cremophor EL/ethanol/DMSO), lapatinib, flavopiridol, or a combination of the drugs using oral gavage to a final concentration of 25 mg/kg QD body mass for flavopiridol and 50 mg/kg BID for lapatinib for 3 d. *P < 0.05 greater survival than in vehicle-treated cells.

Mentions: We next examined the effect of a 24 h valproate pre-treatment of established AR-BT474 intra-cranial tumors on their response to a subsequent lapatinib + obatoclax treatment. AR-BT474 tumors grew more rapidly than WT-BT474 tumors, as judged by a more rapid animal nadir, in general agreement with our in vitro data examining these cells (Fig. 8A). Treatment of WT-BT474 tumors with lapatinib + obatoclax significantly prolonged animal survival compared with vehicle control and, to our surprise, treatment of AR-BT474 tumors with the drug combination also significantly promoted animal survival. Valproate treatment of AR-BT474 tumors caused a profound prolongation of survival, and treatment of AR-BT474 tumors with valproate followed by lapatinib + obatoclax also resulted in a significant enhancement in survival. In agreement with our in vitro data using CDK inhibitors wherein CDK9 inhibitors enhanced lapatinib toxicity; the CDK inhibitor flavopiridol also enhanced the anti-tumor effect of lapatinib in vivo (Fig. 8B).


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 8. Valproate pre-treatment of AR-BT474 cells growing in mouse brains enhances animal survival. (A) Intracerebral injection of WT-BT474 and AR-BT474 cells was performed over 10 min (1 × 106 cells, each). Seven days after tumor cell implantation, animals were segregated into treatment groups. For animal administration sodium valproate was dissolved in saline. Animals were treated with saline vehicle or sodium valproate to a final concentration of 100 mg/kg QD for 2 d. For animal administration, lapatinib and obatoclax were first dissolved in DMSO, and an equal volume of 50:50 Cremophor EL/ethanol (Sigma-Aldrich) was added. After mixing, a 1:10 dilution was made with sterile PBS. Animals were treated with vehicle (PBS/Cremophor EL/ethanol/DMSO), lapatinib, obatoclax, or a combination of lapatinib and obatoclax using oral gavage to a final concentration of 5 mg/kg QD body mass for obatoclax and 100 mg/kg BID for lapatinib for 3 d. *P < 0.05 greater survival than in AR-BT474 cells with vehicle; ¶P < 0.05 greater survival than vehicle treated WT-BT474 cells. Inset: H&E staining of tumors at the time of animal death. (B) WT-BT474 cells (1 × 106 cells, each animal) were injected into the fourth mammary fat pad. Fourteen days after tumor cell implantation tumors of ~100 mm3 had formed, and animals were segregated into treatment groups. For animal administration, lapatinib and flavopiridol were first dissolved in DMSO, and an equal volume of 50:50 Cremophor EL/ethanol (Sigma-Aldrich) was added. After mixing, a 1:10 dilution was made with sterile PBS. Animals were treated with vehicle (PBS/Cremophor EL/ethanol/DMSO), lapatinib, flavopiridol, or a combination of the drugs using oral gavage to a final concentration of 25 mg/kg QD body mass for flavopiridol and 50 mg/kg BID for lapatinib for 3 d. *P < 0.05 greater survival than in vehicle-treated cells.
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Figure 8: Figure 8. Valproate pre-treatment of AR-BT474 cells growing in mouse brains enhances animal survival. (A) Intracerebral injection of WT-BT474 and AR-BT474 cells was performed over 10 min (1 × 106 cells, each). Seven days after tumor cell implantation, animals were segregated into treatment groups. For animal administration sodium valproate was dissolved in saline. Animals were treated with saline vehicle or sodium valproate to a final concentration of 100 mg/kg QD for 2 d. For animal administration, lapatinib and obatoclax were first dissolved in DMSO, and an equal volume of 50:50 Cremophor EL/ethanol (Sigma-Aldrich) was added. After mixing, a 1:10 dilution was made with sterile PBS. Animals were treated with vehicle (PBS/Cremophor EL/ethanol/DMSO), lapatinib, obatoclax, or a combination of lapatinib and obatoclax using oral gavage to a final concentration of 5 mg/kg QD body mass for obatoclax and 100 mg/kg BID for lapatinib for 3 d. *P < 0.05 greater survival than in AR-BT474 cells with vehicle; ¶P < 0.05 greater survival than vehicle treated WT-BT474 cells. Inset: H&E staining of tumors at the time of animal death. (B) WT-BT474 cells (1 × 106 cells, each animal) were injected into the fourth mammary fat pad. Fourteen days after tumor cell implantation tumors of ~100 mm3 had formed, and animals were segregated into treatment groups. For animal administration, lapatinib and flavopiridol were first dissolved in DMSO, and an equal volume of 50:50 Cremophor EL/ethanol (Sigma-Aldrich) was added. After mixing, a 1:10 dilution was made with sterile PBS. Animals were treated with vehicle (PBS/Cremophor EL/ethanol/DMSO), lapatinib, flavopiridol, or a combination of the drugs using oral gavage to a final concentration of 25 mg/kg QD body mass for flavopiridol and 50 mg/kg BID for lapatinib for 3 d. *P < 0.05 greater survival than in vehicle-treated cells.
Mentions: We next examined the effect of a 24 h valproate pre-treatment of established AR-BT474 intra-cranial tumors on their response to a subsequent lapatinib + obatoclax treatment. AR-BT474 tumors grew more rapidly than WT-BT474 tumors, as judged by a more rapid animal nadir, in general agreement with our in vitro data examining these cells (Fig. 8A). Treatment of WT-BT474 tumors with lapatinib + obatoclax significantly prolonged animal survival compared with vehicle control and, to our surprise, treatment of AR-BT474 tumors with the drug combination also significantly promoted animal survival. Valproate treatment of AR-BT474 tumors caused a profound prolongation of survival, and treatment of AR-BT474 tumors with valproate followed by lapatinib + obatoclax also resulted in a significant enhancement in survival. In agreement with our in vitro data using CDK inhibitors wherein CDK9 inhibitors enhanced lapatinib toxicity; the CDK inhibitor flavopiridol also enhanced the anti-tumor effect of lapatinib in vivo (Fig. 8B).

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