Limits...
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.

Show MeSH

Related in: MedlinePlus

Cell death induced by LY294002 in C4-HI cancer cells involves intrinsic BAX/mitochondrial/caspase-9 apoptotic pathway.The 3D Matrigel culture system of primary C4-HI cancer cells reproduces the increased pro-apoptotic effect of the PI3K inhibitor observed in vivo. A. Top. Phase contrast microscopy showing a representative C4-HD (left) and C4-HI (right) cell cluster cultured for 96 hrs on Matrigel and treated for the last 48 hrs with 10 µM PD98059, LY294002, or vehicle as control. Confocal images from a fluorescence microscope of acridine orange/ethidium bromide (AO/EB) staining was used to discriminate live from apoptotic cells. AO fluoresces green in live cells and EB fluoresces orange/red when intercalated with DNA in dead cells. Most C4-HI cell clusters on Matrigel exhibit a central lumen. In contrast, no C4-HD cell clusters possess a central lumen. A higher number of apoptotic cells in and around the central lumen of LY294002-treated C4-HI cells was also noted. Scale bar: 30 µm. Bottom. Quantification of the percentage of apoptotic cells per cluster, of four independent experiments with ten clusters in each. Data corresponds to the mean +/− SEM. LY294002 induces cell death in C4-HI cells; **:p<0.01 vs. control. B. Confocal images showing higher BAX and activated caspase-9 staining, and lower Bcl-XL staining in C4-HI cells treated with 10 µM LY294002. Nuclei were stained red with propidium iodide. Scale bar: 30 µm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2877092&req=5

pone-0010786-g005: Cell death induced by LY294002 in C4-HI cancer cells involves intrinsic BAX/mitochondrial/caspase-9 apoptotic pathway.The 3D Matrigel culture system of primary C4-HI cancer cells reproduces the increased pro-apoptotic effect of the PI3K inhibitor observed in vivo. A. Top. Phase contrast microscopy showing a representative C4-HD (left) and C4-HI (right) cell cluster cultured for 96 hrs on Matrigel and treated for the last 48 hrs with 10 µM PD98059, LY294002, or vehicle as control. Confocal images from a fluorescence microscope of acridine orange/ethidium bromide (AO/EB) staining was used to discriminate live from apoptotic cells. AO fluoresces green in live cells and EB fluoresces orange/red when intercalated with DNA in dead cells. Most C4-HI cell clusters on Matrigel exhibit a central lumen. In contrast, no C4-HD cell clusters possess a central lumen. A higher number of apoptotic cells in and around the central lumen of LY294002-treated C4-HI cells was also noted. Scale bar: 30 µm. Bottom. Quantification of the percentage of apoptotic cells per cluster, of four independent experiments with ten clusters in each. Data corresponds to the mean +/− SEM. LY294002 induces cell death in C4-HI cells; **:p<0.01 vs. control. B. Confocal images showing higher BAX and activated caspase-9 staining, and lower Bcl-XL staining in C4-HI cells treated with 10 µM LY294002. Nuclei were stained red with propidium iodide. Scale bar: 30 µm.

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)

Cell death induced by LY294002 in C4-HI cancer cells involves intrinsic BAX/mitochondrial/caspase-9 apoptotic pathway.The 3D Matrigel culture system of primary C4-HI cancer cells reproduces the increased pro-apoptotic effect of the PI3K inhibitor observed in vivo. A. Top. Phase contrast microscopy showing a representative C4-HD (left) and C4-HI (right) cell cluster cultured for 96 hrs on Matrigel and treated for the last 48 hrs with 10 µM PD98059, LY294002, or vehicle as control. Confocal images from a fluorescence microscope of acridine orange/ethidium bromide (AO/EB) staining was used to discriminate live from apoptotic cells. AO fluoresces green in live cells and EB fluoresces orange/red when intercalated with DNA in dead cells. Most C4-HI cell clusters on Matrigel exhibit a central lumen. In contrast, no C4-HD cell clusters possess a central lumen. A higher number of apoptotic cells in and around the central lumen of LY294002-treated C4-HI cells was also noted. Scale bar: 30 µm. Bottom. Quantification of the percentage of apoptotic cells per cluster, of four independent experiments with ten clusters in each. Data corresponds to the mean +/− SEM. LY294002 induces cell death in C4-HI cells; **:p<0.01 vs. control. B. Confocal images showing higher BAX and activated caspase-9 staining, and lower Bcl-XL staining in C4-HI cells treated with 10 µM LY294002. Nuclei were stained red with propidium iodide. Scale bar: 30 µm.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0010786-g005: Cell death induced by LY294002 in C4-HI cancer cells involves intrinsic BAX/mitochondrial/caspase-9 apoptotic pathway.The 3D Matrigel culture system of primary C4-HI cancer cells reproduces the increased pro-apoptotic effect of the PI3K inhibitor observed in vivo. A. Top. Phase contrast microscopy showing a representative C4-HD (left) and C4-HI (right) cell cluster cultured for 96 hrs on Matrigel and treated for the last 48 hrs with 10 µM PD98059, LY294002, or vehicle as control. Confocal images from a fluorescence microscope of acridine orange/ethidium bromide (AO/EB) staining was used to discriminate live from apoptotic cells. AO fluoresces green in live cells and EB fluoresces orange/red when intercalated with DNA in dead cells. Most C4-HI cell clusters on Matrigel exhibit a central lumen. In contrast, no C4-HD cell clusters possess a central lumen. A higher number of apoptotic cells in and around the central lumen of LY294002-treated C4-HI cells was also noted. Scale bar: 30 µm. Bottom. Quantification of the percentage of apoptotic cells per cluster, of four independent experiments with ten clusters in each. Data corresponds to the mean +/− SEM. LY294002 induces cell death in C4-HI cells; **:p<0.01 vs. control. B. Confocal images showing higher BAX and activated caspase-9 staining, and lower Bcl-XL staining in C4-HI cells treated with 10 µM LY294002. Nuclei were stained red with propidium iodide. Scale bar: 30 µm.
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