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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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Higher sensitivity of C4-HI tumors to inhibition of PI3K/AKT pathway is restored in 3D Matrigel.The 3D Matrigel culture system reproduces the in vivo differential sensitivity of primary cancer cells to PI3K and MEK inhibitors. Phase contrast microscopy showing the progression of C4-HD (top) and C4-HI (bottom) cultures in the presence of 10 µM PD98059, LY294002, or vehicle as control. Day 0 referred to the day of the isolation from the tumors. Primary cultures were maintained for 96 hrs on Matrigel, and the inhibitors were added into the culture medium for the last 48 hrs. During that time, under control conditions the clusters became bigger, due to proliferation and superposition of neighboring clusters. PD98059 causes a small reduction in the size of C4-HI clusters, but the effect of LY294002 is more dramatic on C4-HI than C4-HD cells. A higher number of structures with central lumen (indicated with red arrows) were noticeable in LY294002-treated C4-HI cells. Simultaneous treatment with the two inhibitors equally reduced the size of C4-HD and C4-HI clusters. Scale bar: 30 µm. The same progression was observed in three independent experiments.
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pone-0010786-g004: Higher sensitivity of C4-HI tumors to inhibition of PI3K/AKT pathway is restored in 3D Matrigel.The 3D Matrigel culture system reproduces the in vivo differential sensitivity of primary cancer cells to PI3K and MEK inhibitors. Phase contrast microscopy showing the progression of C4-HD (top) and C4-HI (bottom) cultures in the presence of 10 µM PD98059, LY294002, or vehicle as control. Day 0 referred to the day of the isolation from the tumors. Primary cultures were maintained for 96 hrs on Matrigel, and the inhibitors were added into the culture medium for the last 48 hrs. During that time, under control conditions the clusters became bigger, due to proliferation and superposition of neighboring clusters. PD98059 causes a small reduction in the size of C4-HI clusters, but the effect of LY294002 is more dramatic on C4-HI than C4-HD cells. A higher number of structures with central lumen (indicated with red arrows) were noticeable in LY294002-treated C4-HI cells. Simultaneous treatment with the two inhibitors equally reduced the size of C4-HD and C4-HI clusters. Scale bar: 30 µm. The same progression was observed in three independent experiments.

Mentions: We then explored the sensitivity of C4-HD and C4-HI cells growing for 96 hrs on Matrigel to PD98059 and LY294002 treatment. Analysis of phase contrast microscopy images revealed critical differences between the two cell types to kinase inhibitor treatment. Similar to what we found in vivo (Figure 1E), the PI3K inhibitor reduced cell survival (determined by cluster size) in C4-HI cells significantly more than in C4-HD cells (Figure 4). Furthermore, a small effect was observed using the MEK inhibitor in C4-HI cells. The simultaneous treatment with both inhibitors was remarkably effective both on C4-HD and C4-HI cells in reducing the size of the clusters. Moreover, treatment for 48 hrs with 10 µM LY294002 increased central lumen formation (indicated with red arrows in Figure 4) in C4-HI clusters. To evaluate if there is a selective effect of LY294002 in inducing cell death in C4-HI cells, we used the acridine orange/ethidium bromide (AO/EB) dye incorporation assay. By this technique, apoptotic cells are visualized by their red fluorescence whereas living cells fluoresce green. An analysis of phase contrast microscopy followed by confocal images from a fluorescence microscope of AO/EB staining demonstrated that C4-HD and C4-HI cell clusters were differentially sensitive to protein kinase inhibitors. After 48 hrs of LY294002 treatment, a significant increase (p<0.01) in the number of apoptotic C4-HI but not C4-HD cells was observed. In contrast, PD98059 did not significantly increase the percentage of C4-HI or C4-HD apoptotic cells (Figure 5A). Taken together, these data suggest that C4-HD clusters do not have lumen because of their failure to undergo cavitations via the apoptosis of centrally localized cells (Figure 5A).


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)

Higher sensitivity of C4-HI tumors to inhibition of PI3K/AKT pathway is restored in 3D Matrigel.The 3D Matrigel culture system reproduces the in vivo differential sensitivity of primary cancer cells to PI3K and MEK inhibitors. Phase contrast microscopy showing the progression of C4-HD (top) and C4-HI (bottom) cultures in the presence of 10 µM PD98059, LY294002, or vehicle as control. Day 0 referred to the day of the isolation from the tumors. Primary cultures were maintained for 96 hrs on Matrigel, and the inhibitors were added into the culture medium for the last 48 hrs. During that time, under control conditions the clusters became bigger, due to proliferation and superposition of neighboring clusters. PD98059 causes a small reduction in the size of C4-HI clusters, but the effect of LY294002 is more dramatic on C4-HI than C4-HD cells. A higher number of structures with central lumen (indicated with red arrows) were noticeable in LY294002-treated C4-HI cells. Simultaneous treatment with the two inhibitors equally reduced the size of C4-HD and C4-HI clusters. Scale bar: 30 µm. The same progression was observed in three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2877092&req=5

pone-0010786-g004: Higher sensitivity of C4-HI tumors to inhibition of PI3K/AKT pathway is restored in 3D Matrigel.The 3D Matrigel culture system reproduces the in vivo differential sensitivity of primary cancer cells to PI3K and MEK inhibitors. Phase contrast microscopy showing the progression of C4-HD (top) and C4-HI (bottom) cultures in the presence of 10 µM PD98059, LY294002, or vehicle as control. Day 0 referred to the day of the isolation from the tumors. Primary cultures were maintained for 96 hrs on Matrigel, and the inhibitors were added into the culture medium for the last 48 hrs. During that time, under control conditions the clusters became bigger, due to proliferation and superposition of neighboring clusters. PD98059 causes a small reduction in the size of C4-HI clusters, but the effect of LY294002 is more dramatic on C4-HI than C4-HD cells. A higher number of structures with central lumen (indicated with red arrows) were noticeable in LY294002-treated C4-HI cells. Simultaneous treatment with the two inhibitors equally reduced the size of C4-HD and C4-HI clusters. Scale bar: 30 µm. The same progression was observed in three independent experiments.
Mentions: We then explored the sensitivity of C4-HD and C4-HI cells growing for 96 hrs on Matrigel to PD98059 and LY294002 treatment. Analysis of phase contrast microscopy images revealed critical differences between the two cell types to kinase inhibitor treatment. Similar to what we found in vivo (Figure 1E), the PI3K inhibitor reduced cell survival (determined by cluster size) in C4-HI cells significantly more than in C4-HD cells (Figure 4). Furthermore, a small effect was observed using the MEK inhibitor in C4-HI cells. The simultaneous treatment with both inhibitors was remarkably effective both on C4-HD and C4-HI cells in reducing the size of the clusters. Moreover, treatment for 48 hrs with 10 µM LY294002 increased central lumen formation (indicated with red arrows in Figure 4) in C4-HI clusters. To evaluate if there is a selective effect of LY294002 in inducing cell death in C4-HI cells, we used the acridine orange/ethidium bromide (AO/EB) dye incorporation assay. By this technique, apoptotic cells are visualized by their red fluorescence whereas living cells fluoresce green. An analysis of phase contrast microscopy followed by confocal images from a fluorescence microscope of AO/EB staining demonstrated that C4-HD and C4-HI cell clusters were differentially sensitive to protein kinase inhibitors. After 48 hrs of LY294002 treatment, a significant increase (p<0.01) in the number of apoptotic C4-HI but not C4-HD cells was observed. In contrast, PD98059 did not significantly increase the percentage of C4-HI or C4-HD apoptotic cells (Figure 5A). Taken together, these data suggest that C4-HD clusters do not have lumen because of their failure to undergo cavitations via the apoptosis of centrally localized cells (Figure 5A).

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