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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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Related in: MedlinePlus

Differential sensitivity to PI3K/AKT pathway regulation is lost in isolated cancer cells.A. Phase contrast microscopy of primary cultures isolated from C4-HD and C4-HI tumors showing flattened monolayers on tissue culture plastic. Scale bar: 30 µm. B. Top. Western blots showing p-AKT, total AKT, p-ERK1/2, ERK1/2 and β-actin in protein extracts from primary cells cultured for 96 hrs. Three representative samples of a total of six are shown in the gel. Bottom. Quantification of p-AKT/total AKT and p-ERK/total ERK levels, n = 6 for each cell type. C. Proliferation assay (3H-thymidine incorporation) in primary C4-HD (left) or C4-HI (right) cells incubated during the last 48 hrs with 0.01 µM MPA and 5, 10 and 20 µM PD98059 or LY294002. For the combined treatment, the inhibitor was used at a dose of 10 µM. 3H-thymidine was added in the last 18 hrs before harvesting the cells. MPA has a stimulatory effect while PD98059 and LY294002 have an inhibitory effect on MPA-treated and untreated cells. n = 8 for each treatment. ***:p<0.001; **:p<0.01; *: p<0.05 vs. control (untreated cells); different letters indicate significant differences between treatments in the presence of MPA with p<0.01. A representative experiment of a total of three is shown here. D. Left. Western blots showing that phosphorylation levels of AKT and ERK1/2 decrease in primary C4-HI cultures incubated for 48 hrs with 10 µM of specific inhibitors. β-actin levels in the blot serve as a loading control. Two representative samples of a total of six are shown (left). Right. Quantification of p-AKT/total AKT and p-ERK/total ERK levels. n = 6 for each group; *p<0.05; **p<0.01 vs. control.
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pone-0010786-g002: Differential sensitivity to PI3K/AKT pathway regulation is lost in isolated cancer cells.A. Phase contrast microscopy of primary cultures isolated from C4-HD and C4-HI tumors showing flattened monolayers on tissue culture plastic. Scale bar: 30 µm. B. Top. Western blots showing p-AKT, total AKT, p-ERK1/2, ERK1/2 and β-actin in protein extracts from primary cells cultured for 96 hrs. Three representative samples of a total of six are shown in the gel. Bottom. Quantification of p-AKT/total AKT and p-ERK/total ERK levels, n = 6 for each cell type. C. Proliferation assay (3H-thymidine incorporation) in primary C4-HD (left) or C4-HI (right) cells incubated during the last 48 hrs with 0.01 µM MPA and 5, 10 and 20 µM PD98059 or LY294002. For the combined treatment, the inhibitor was used at a dose of 10 µM. 3H-thymidine was added in the last 18 hrs before harvesting the cells. MPA has a stimulatory effect while PD98059 and LY294002 have an inhibitory effect on MPA-treated and untreated cells. n = 8 for each treatment. ***:p<0.001; **:p<0.01; *: p<0.05 vs. control (untreated cells); different letters indicate significant differences between treatments in the presence of MPA with p<0.01. A representative experiment of a total of three is shown here. D. Left. Western blots showing that phosphorylation levels of AKT and ERK1/2 decrease in primary C4-HI cultures incubated for 48 hrs with 10 µM of specific inhibitors. β-actin levels in the blot serve as a loading control. Two representative samples of a total of six are shown (left). Right. Quantification of p-AKT/total AKT and p-ERK/total ERK levels. n = 6 for each group; *p<0.05; **p<0.01 vs. control.

Mentions: In order to study the mechanisms that lead to the differential activation of AKT in C4-HI and C4-HD tumors, we isolated primary epithelial cells from the tumors and cultured them on plastic tissue culture plates. Under this two-dimensional (2D) condition, both C4-HD and C4-HI epithelial cells grow as clusters that adhere to the plastic (Figure 2A). In contrast to the results obtained with tumors growing in vivo, western blot analysis of epithelial cells isolated from C4-HD or C4-HI tumors that were placed on plastic for 96 hours show similar levels of p-AKT and p-ERK1/2 (Figure 2B). Furthermore, analysis of cell proliferation by 3H-thymidine uptake revealed that both cell types have a similar responsiveness to MPA or growth factors such as FGF-2 [41], and both display similar sensitivity to the inhibitors PD98059 and LY294002, as shown here (Figure 2C). In both cell types, inhibition of PI3K/AKT and MEK/ERK1/2 signaling interfered with the proliferative effect of 0.01 µM MPA (Figure 2C), suggesting that both pathways are involved in MPA-induced proliferation. Curiously, even though C4-HI tumor cells are MPA-independent in vivo, they are MPA-responsive in vitro.


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)

Differential sensitivity to PI3K/AKT pathway regulation is lost in isolated cancer cells.A. Phase contrast microscopy of primary cultures isolated from C4-HD and C4-HI tumors showing flattened monolayers on tissue culture plastic. Scale bar: 30 µm. B. Top. Western blots showing p-AKT, total AKT, p-ERK1/2, ERK1/2 and β-actin in protein extracts from primary cells cultured for 96 hrs. Three representative samples of a total of six are shown in the gel. Bottom. Quantification of p-AKT/total AKT and p-ERK/total ERK levels, n = 6 for each cell type. C. Proliferation assay (3H-thymidine incorporation) in primary C4-HD (left) or C4-HI (right) cells incubated during the last 48 hrs with 0.01 µM MPA and 5, 10 and 20 µM PD98059 or LY294002. For the combined treatment, the inhibitor was used at a dose of 10 µM. 3H-thymidine was added in the last 18 hrs before harvesting the cells. MPA has a stimulatory effect while PD98059 and LY294002 have an inhibitory effect on MPA-treated and untreated cells. n = 8 for each treatment. ***:p<0.001; **:p<0.01; *: p<0.05 vs. control (untreated cells); different letters indicate significant differences between treatments in the presence of MPA with p<0.01. A representative experiment of a total of three is shown here. D. Left. Western blots showing that phosphorylation levels of AKT and ERK1/2 decrease in primary C4-HI cultures incubated for 48 hrs with 10 µM of specific inhibitors. β-actin levels in the blot serve as a loading control. Two representative samples of a total of six are shown (left). Right. Quantification of p-AKT/total AKT and p-ERK/total ERK levels. n = 6 for each group; *p<0.05; **p<0.01 vs. control.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2877092&req=5

pone-0010786-g002: Differential sensitivity to PI3K/AKT pathway regulation is lost in isolated cancer cells.A. Phase contrast microscopy of primary cultures isolated from C4-HD and C4-HI tumors showing flattened monolayers on tissue culture plastic. Scale bar: 30 µm. B. Top. Western blots showing p-AKT, total AKT, p-ERK1/2, ERK1/2 and β-actin in protein extracts from primary cells cultured for 96 hrs. Three representative samples of a total of six are shown in the gel. Bottom. Quantification of p-AKT/total AKT and p-ERK/total ERK levels, n = 6 for each cell type. C. Proliferation assay (3H-thymidine incorporation) in primary C4-HD (left) or C4-HI (right) cells incubated during the last 48 hrs with 0.01 µM MPA and 5, 10 and 20 µM PD98059 or LY294002. For the combined treatment, the inhibitor was used at a dose of 10 µM. 3H-thymidine was added in the last 18 hrs before harvesting the cells. MPA has a stimulatory effect while PD98059 and LY294002 have an inhibitory effect on MPA-treated and untreated cells. n = 8 for each treatment. ***:p<0.001; **:p<0.01; *: p<0.05 vs. control (untreated cells); different letters indicate significant differences between treatments in the presence of MPA with p<0.01. A representative experiment of a total of three is shown here. D. Left. Western blots showing that phosphorylation levels of AKT and ERK1/2 decrease in primary C4-HI cultures incubated for 48 hrs with 10 µM of specific inhibitors. β-actin levels in the blot serve as a loading control. Two representative samples of a total of six are shown (left). Right. Quantification of p-AKT/total AKT and p-ERK/total ERK levels. n = 6 for each group; *p<0.05; **p<0.01 vs. control.
Mentions: In order to study the mechanisms that lead to the differential activation of AKT in C4-HI and C4-HD tumors, we isolated primary epithelial cells from the tumors and cultured them on plastic tissue culture plates. Under this two-dimensional (2D) condition, both C4-HD and C4-HI epithelial cells grow as clusters that adhere to the plastic (Figure 2A). In contrast to the results obtained with tumors growing in vivo, western blot analysis of epithelial cells isolated from C4-HD or C4-HI tumors that were placed on plastic for 96 hours show similar levels of p-AKT and p-ERK1/2 (Figure 2B). Furthermore, analysis of cell proliferation by 3H-thymidine uptake revealed that both cell types have a similar responsiveness to MPA or growth factors such as FGF-2 [41], and both display similar sensitivity to the inhibitors PD98059 and LY294002, as shown here (Figure 2C). In both cell types, inhibition of PI3K/AKT and MEK/ERK1/2 signaling interfered with the proliferative effect of 0.01 µM MPA (Figure 2C), suggesting that both pathways are involved in MPA-induced proliferation. Curiously, even though C4-HI tumor cells are MPA-independent in vivo, they are MPA-responsive in vitro.

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