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Responsiveness to PI3K and MEK inhibitors in breast cancer. Use of a 3D culture system to study pathways related to hormone independence in mice.

Polo ML, Arnoni MV, Riggio M, Wargon V, Lanari C, Novaro V - PLoS ONE (2010)

Bottom Line: A three-dimensional (3D) culture system, by maintaining differential cellular organization that is typical of each tumor variant, may allow for the maintenance of particular hormone responses and thus be appropriate for the study of the effects of specific inhibitors of signaling pathways associated with disease progression.Induction of cell death by anti-hormones such as ICI182780 and ZK230211 was more effective in MPA-dependent tumors with lower AKT activity.Inhibition of MEK with PD98059 did not affect tumor growth in any tested variant.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Hormonal Carcinogenesis, Institute of Experimental Biology and Medicine (IBYME)-National Council for Scientific and Technical Research (CONICET), Buenos Aires, Argentina.

ABSTRACT

Background: A significant proportion of breast cancer patients face failure of endocrine therapy due to the acquisition of endocrine resistance. We have explored mechanisms involved in such disease progression by using a mouse breast cancer model that is induced by medroxyprogesterone acetate (MPA). These tumors transit through different stages of hormone sensitivity. However, when cells from tumor variants were seeded on plastic, all were stimulated by progestins and inhibited by antiprogestins such as RU486. Furthermore, cells from a RU486-resistant tumor variant recovered antiprogestin sensitivity.

Hypothesis: A three-dimensional (3D) culture system, by maintaining differential cellular organization that is typical of each tumor variant, may allow for the maintenance of particular hormone responses and thus be appropriate for the study of the effects of specific inhibitors of signaling pathways associated with disease progression.

Method: We compared the behavior of tumors growing in vivo and cancer cells ex vivo (in 3D Matrigel). In this system, we evaluated the effects of kinase inhibitors and hormone antagonists on tumor growth.

Principal findings: LY294002, a PI3K/AKT pathway inhibitor, decreased both tumor growth in vivo and cell survival in Matrigel in MPA-independent tumors with higher AKT activity. Induction of cell death by anti-hormones such as ICI182780 and ZK230211 was more effective in MPA-dependent tumors with lower AKT activity. Inhibition of MEK with PD98059 did not affect tumor growth in any tested variant. Finally, while Matrigel reproduced differential responsiveness of MPA-dependent and -independent breast cancer cells, it was not sufficient to preserve antiprogestin resistance of RU486-resistant tumors.

Conclusion: We demonstrated that the PI3K/AKT pathway is relevant for MPA-independent tumor growth. Three-dimensional cultures were useful to test the effects of kinase inhibitors on breast cancer growth and highlight the need for in vivo models to validate experimental tools used for selective therapeutic targeting.

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Loss of endocrine resistance in isolated C4-HIR tumor cells is not prevented by 3D Matrigel.Isolated C4-HI and C4-HIR tumor cells are sensitive to cell death in response to RU486 when cultured on plastic, and the 3D Matrigel culture system is not sufficient to revert this phenotype. A. C4-HI and C4-HIR tumors were transplanted in syngeneic mice and measured every 2 days (length and width). RU486 was inoculated s.c. at a dose of 12 mg/kg/day once the tumors were 60 mm2. C4-HI tumors (left) regress following treatment with antiprogestin, while C4-HIR tumors (right) are unresponsive. B. Fluorescent confocal images of AO/EB staining show similar number of apoptotic cells (red cells) after 48 hrs of treatment with 0.01 µM MPA and a higher number of apoptotic cells with 0.01 µM RU486 treatment. Right. Quantification of the percentage of apoptotic C4-HI (upper) and C4-HIR (lower) cells per cluster, of three independent experiments with ten clusters in each. Data corresponds to the mean +/− SEM; ***:p<0.001 vs. control. Scale bar: 100 µm.
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pone-0010786-g008: Loss of endocrine resistance in isolated C4-HIR tumor cells is not prevented by 3D Matrigel.Isolated C4-HI and C4-HIR tumor cells are sensitive to cell death in response to RU486 when cultured on plastic, and the 3D Matrigel culture system is not sufficient to revert this phenotype. A. C4-HI and C4-HIR tumors were transplanted in syngeneic mice and measured every 2 days (length and width). RU486 was inoculated s.c. at a dose of 12 mg/kg/day once the tumors were 60 mm2. C4-HI tumors (left) regress following treatment with antiprogestin, while C4-HIR tumors (right) are unresponsive. B. Fluorescent confocal images of AO/EB staining show similar number of apoptotic cells (red cells) after 48 hrs of treatment with 0.01 µM MPA and a higher number of apoptotic cells with 0.01 µM RU486 treatment. Right. Quantification of the percentage of apoptotic C4-HI (upper) and C4-HIR (lower) cells per cluster, of three independent experiments with ten clusters in each. Data corresponds to the mean +/− SEM; ***:p<0.001 vs. control. Scale bar: 100 µm.

Mentions: Finally, we evaluated whether endocrine resistance of C4-HIR tumors can be reproduced in culture using Matrigel as a substratum. As previously reported [7] and reproduced here (Figure 8A), C4-HI tumors regress after antiprogestin treatment (ZK230211 or RU486). This is in contrast to C4-HIR tumors, which continue growing following the same treatment. However, when primary cells were isolated from each tumor and placed on plastic, both cell types were sensitive to RU486 (unpublished data). Furthermore, this loss of endocrine resistance of C4-HIR tumor cells could not be prevented by culturing the cells on Matrigel. After 48 hrs of 0.01 µM RU486 treatment, both C4-HI and C4-HIR tumor cells were equally sensitive to the antiprogestin, showing similar increase (p<0.001) in the percentages of apoptotic cells (visualized by red fluorescence) when assayed by AO/EB dye uptake (Figure 8B). Under the same conditions, it was noticeable that treatment with 0.01 µM MPA for 48 hrs did not significantly affect basal cell death in both C4-HI and C4-HIR cultures (Figure 8B). It is important to mention that C4-HIR cells remained more disorganized than C4-HI cells on Matrigel (Figure 8B, photographs).


Responsiveness to PI3K and MEK inhibitors in breast cancer. Use of a 3D culture system to study pathways related to hormone independence in mice.

Polo ML, Arnoni MV, Riggio M, Wargon V, Lanari C, Novaro V - PLoS ONE (2010)

Loss of endocrine resistance in isolated C4-HIR tumor cells is not prevented by 3D Matrigel.Isolated C4-HI and C4-HIR tumor cells are sensitive to cell death in response to RU486 when cultured on plastic, and the 3D Matrigel culture system is not sufficient to revert this phenotype. A. C4-HI and C4-HIR tumors were transplanted in syngeneic mice and measured every 2 days (length and width). RU486 was inoculated s.c. at a dose of 12 mg/kg/day once the tumors were 60 mm2. C4-HI tumors (left) regress following treatment with antiprogestin, while C4-HIR tumors (right) are unresponsive. B. Fluorescent confocal images of AO/EB staining show similar number of apoptotic cells (red cells) after 48 hrs of treatment with 0.01 µM MPA and a higher number of apoptotic cells with 0.01 µM RU486 treatment. Right. Quantification of the percentage of apoptotic C4-HI (upper) and C4-HIR (lower) cells per cluster, of three independent experiments with ten clusters in each. Data corresponds to the mean +/− SEM; ***:p<0.001 vs. control. Scale bar: 100 µm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2877092&req=5

pone-0010786-g008: Loss of endocrine resistance in isolated C4-HIR tumor cells is not prevented by 3D Matrigel.Isolated C4-HI and C4-HIR tumor cells are sensitive to cell death in response to RU486 when cultured on plastic, and the 3D Matrigel culture system is not sufficient to revert this phenotype. A. C4-HI and C4-HIR tumors were transplanted in syngeneic mice and measured every 2 days (length and width). RU486 was inoculated s.c. at a dose of 12 mg/kg/day once the tumors were 60 mm2. C4-HI tumors (left) regress following treatment with antiprogestin, while C4-HIR tumors (right) are unresponsive. B. Fluorescent confocal images of AO/EB staining show similar number of apoptotic cells (red cells) after 48 hrs of treatment with 0.01 µM MPA and a higher number of apoptotic cells with 0.01 µM RU486 treatment. Right. Quantification of the percentage of apoptotic C4-HI (upper) and C4-HIR (lower) cells per cluster, of three independent experiments with ten clusters in each. Data corresponds to the mean +/− SEM; ***:p<0.001 vs. control. Scale bar: 100 µm.
Mentions: Finally, we evaluated whether endocrine resistance of C4-HIR tumors can be reproduced in culture using Matrigel as a substratum. As previously reported [7] and reproduced here (Figure 8A), C4-HI tumors regress after antiprogestin treatment (ZK230211 or RU486). This is in contrast to C4-HIR tumors, which continue growing following the same treatment. However, when primary cells were isolated from each tumor and placed on plastic, both cell types were sensitive to RU486 (unpublished data). Furthermore, this loss of endocrine resistance of C4-HIR tumor cells could not be prevented by culturing the cells on Matrigel. After 48 hrs of 0.01 µM RU486 treatment, both C4-HI and C4-HIR tumor cells were equally sensitive to the antiprogestin, showing similar increase (p<0.001) in the percentages of apoptotic cells (visualized by red fluorescence) when assayed by AO/EB dye uptake (Figure 8B). Under the same conditions, it was noticeable that treatment with 0.01 µM MPA for 48 hrs did not significantly affect basal cell death in both C4-HI and C4-HIR cultures (Figure 8B). It is important to mention that C4-HIR cells remained more disorganized than C4-HI cells on Matrigel (Figure 8B, photographs).

Bottom Line: A three-dimensional (3D) culture system, by maintaining differential cellular organization that is typical of each tumor variant, may allow for the maintenance of particular hormone responses and thus be appropriate for the study of the effects of specific inhibitors of signaling pathways associated with disease progression.Induction of cell death by anti-hormones such as ICI182780 and ZK230211 was more effective in MPA-dependent tumors with lower AKT activity.Inhibition of MEK with PD98059 did not affect tumor growth in any tested variant.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Hormonal Carcinogenesis, Institute of Experimental Biology and Medicine (IBYME)-National Council for Scientific and Technical Research (CONICET), Buenos Aires, Argentina.

ABSTRACT

Background: A significant proportion of breast cancer patients face failure of endocrine therapy due to the acquisition of endocrine resistance. We have explored mechanisms involved in such disease progression by using a mouse breast cancer model that is induced by medroxyprogesterone acetate (MPA). These tumors transit through different stages of hormone sensitivity. However, when cells from tumor variants were seeded on plastic, all were stimulated by progestins and inhibited by antiprogestins such as RU486. Furthermore, cells from a RU486-resistant tumor variant recovered antiprogestin sensitivity.

Hypothesis: A three-dimensional (3D) culture system, by maintaining differential cellular organization that is typical of each tumor variant, may allow for the maintenance of particular hormone responses and thus be appropriate for the study of the effects of specific inhibitors of signaling pathways associated with disease progression.

Method: We compared the behavior of tumors growing in vivo and cancer cells ex vivo (in 3D Matrigel). In this system, we evaluated the effects of kinase inhibitors and hormone antagonists on tumor growth.

Principal findings: LY294002, a PI3K/AKT pathway inhibitor, decreased both tumor growth in vivo and cell survival in Matrigel in MPA-independent tumors with higher AKT activity. Induction of cell death by anti-hormones such as ICI182780 and ZK230211 was more effective in MPA-dependent tumors with lower AKT activity. Inhibition of MEK with PD98059 did not affect tumor growth in any tested variant. Finally, while Matrigel reproduced differential responsiveness of MPA-dependent and -independent breast cancer cells, it was not sufficient to preserve antiprogestin resistance of RU486-resistant tumors.

Conclusion: We demonstrated that the PI3K/AKT pathway is relevant for MPA-independent tumor growth. Three-dimensional cultures were useful to test the effects of kinase inhibitors on breast cancer growth and highlight the need for in vivo models to validate experimental tools used for selective therapeutic targeting.

Show MeSH
Related in: MedlinePlus