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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

AKT regulates ERα protein expression.A. ERα levels determined by western blot are reduced in C4-HI tumors treated with PD98059 or LY294002 administrated as indicated in Figure 1. However, ERα levels are more variable between tumors and not regulated by either inhibitor in C4-HD tumor samples. Top. Two representative samples of a total of six are shown in the gel. Bottom. Quantification of ERα relative to β-actin (used as a loading control) levels. n = 6 for each treatment; **:p<0.01 vs. control. B. A similar pattern of regulation of ERα protein is observed in primary cells cultured on top of Matrigel and incubated for 48 hrs with 10 µM of either of the inhibitors. Top. Two representative samples of a total of six of each group are shown in two independent gels. Bottom. Quantificaton of ERα relative to β-actin in each cell type. n = 6 for each treatment; **p<0.01 vs. control. C. Representative confocal images showing ERα determined by immunofluoresence (green). Lower levels of the protein are seen in PD98059-treated and in LY294002-treated C4-HI cells growing on Matrigel, whereas similar levels of ERα are seen in C4-HD treated and un-treated cells. Nuclei were stained red with propidium iodide. Scale bar: 30 µm. D. Top. A representative sample for each treatment is shown in a western blot. Scp2, a mouse mammary cell line, transfected with a constitutively active form of AKT1, myristoylated AKT1-Δ4-129 (Scp2Akt), displays in the same gel AKT with a typical molecular weight of 59 kDa and the myristoylated deleted variant of AKT1 (p-myrAKT) with a molecular weight of 45 kDa. The antibody used to detect total AKT recognizes only wild type AKT. E-cadherin was used as a loading control for Scp2 cells. Scp2Akt cells exhibit higher levels of ERα than Scp2 cells transfected with the vector control (vc) or Scp2 control cells. In Scp2 and Scp2vc cells, but not in Scp2Akt cells, 2 and 5 µM LY294002 downregulate p-AKT and ERα levels, whereas total AKT levels remains invariable. Bottom. Quantification of ERα relative to E-cadherin levels. n = 4 for each treatment; a,b p<0.05 Scp2Akt vs. Scp2 and Scp2vc; *p<0.05 LY294002 vs. control.
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pone-0010786-g006: AKT regulates ERα protein expression.A. ERα levels determined by western blot are reduced in C4-HI tumors treated with PD98059 or LY294002 administrated as indicated in Figure 1. However, ERα levels are more variable between tumors and not regulated by either inhibitor in C4-HD tumor samples. Top. Two representative samples of a total of six are shown in the gel. Bottom. Quantification of ERα relative to β-actin (used as a loading control) levels. n = 6 for each treatment; **:p<0.01 vs. control. B. A similar pattern of regulation of ERα protein is observed in primary cells cultured on top of Matrigel and incubated for 48 hrs with 10 µM of either of the inhibitors. Top. Two representative samples of a total of six of each group are shown in two independent gels. Bottom. Quantificaton of ERα relative to β-actin in each cell type. n = 6 for each treatment; **p<0.01 vs. control. C. Representative confocal images showing ERα determined by immunofluoresence (green). Lower levels of the protein are seen in PD98059-treated and in LY294002-treated C4-HI cells growing on Matrigel, whereas similar levels of ERα are seen in C4-HD treated and un-treated cells. Nuclei were stained red with propidium iodide. Scale bar: 30 µm. D. Top. A representative sample for each treatment is shown in a western blot. Scp2, a mouse mammary cell line, transfected with a constitutively active form of AKT1, myristoylated AKT1-Δ4-129 (Scp2Akt), displays in the same gel AKT with a typical molecular weight of 59 kDa and the myristoylated deleted variant of AKT1 (p-myrAKT) with a molecular weight of 45 kDa. The antibody used to detect total AKT recognizes only wild type AKT. E-cadherin was used as a loading control for Scp2 cells. Scp2Akt cells exhibit higher levels of ERα than Scp2 cells transfected with the vector control (vc) or Scp2 control cells. In Scp2 and Scp2vc cells, but not in Scp2Akt cells, 2 and 5 µM LY294002 downregulate p-AKT and ERα levels, whereas total AKT levels remains invariable. Bottom. Quantification of ERα relative to E-cadherin levels. n = 4 for each treatment; a,b p<0.05 Scp2Akt vs. Scp2 and Scp2vc; *p<0.05 LY294002 vs. control.

Mentions: In order to find other mechanisms responsible for the difference in growth between C4-HD and C4-HI tumors, we investigated wether the PI3K/AKT and ERK1/2 pathways regulated the levels of ERα. Inhibition of either pathway significantly (p<0.01) reduced the expression levels of ERα in C4-HI tumors but not in C4-HD tumors as assessed by western blot (Figure 6A). This result, together with our finding that inhibition of p-ERK by PD98059 did not reduce tumor growth rate (Figure 1C), suggest that at least in C4-HI cells, cell proliferation and cell survival are not determined exclusively by ERα levels.


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)

AKT regulates ERα protein expression.A. ERα levels determined by western blot are reduced in C4-HI tumors treated with PD98059 or LY294002 administrated as indicated in Figure 1. However, ERα levels are more variable between tumors and not regulated by either inhibitor in C4-HD tumor samples. Top. Two representative samples of a total of six are shown in the gel. Bottom. Quantification of ERα relative to β-actin (used as a loading control) levels. n = 6 for each treatment; **:p<0.01 vs. control. B. A similar pattern of regulation of ERα protein is observed in primary cells cultured on top of Matrigel and incubated for 48 hrs with 10 µM of either of the inhibitors. Top. Two representative samples of a total of six of each group are shown in two independent gels. Bottom. Quantificaton of ERα relative to β-actin in each cell type. n = 6 for each treatment; **p<0.01 vs. control. C. Representative confocal images showing ERα determined by immunofluoresence (green). Lower levels of the protein are seen in PD98059-treated and in LY294002-treated C4-HI cells growing on Matrigel, whereas similar levels of ERα are seen in C4-HD treated and un-treated cells. Nuclei were stained red with propidium iodide. Scale bar: 30 µm. D. Top. A representative sample for each treatment is shown in a western blot. Scp2, a mouse mammary cell line, transfected with a constitutively active form of AKT1, myristoylated AKT1-Δ4-129 (Scp2Akt), displays in the same gel AKT with a typical molecular weight of 59 kDa and the myristoylated deleted variant of AKT1 (p-myrAKT) with a molecular weight of 45 kDa. The antibody used to detect total AKT recognizes only wild type AKT. E-cadherin was used as a loading control for Scp2 cells. Scp2Akt cells exhibit higher levels of ERα than Scp2 cells transfected with the vector control (vc) or Scp2 control cells. In Scp2 and Scp2vc cells, but not in Scp2Akt cells, 2 and 5 µM LY294002 downregulate p-AKT and ERα levels, whereas total AKT levels remains invariable. Bottom. Quantification of ERα relative to E-cadherin levels. n = 4 for each treatment; a,b p<0.05 Scp2Akt vs. Scp2 and Scp2vc; *p<0.05 LY294002 vs. control.
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getmorefigures.php?uid=PMC2877092&req=5

pone-0010786-g006: AKT regulates ERα protein expression.A. ERα levels determined by western blot are reduced in C4-HI tumors treated with PD98059 or LY294002 administrated as indicated in Figure 1. However, ERα levels are more variable between tumors and not regulated by either inhibitor in C4-HD tumor samples. Top. Two representative samples of a total of six are shown in the gel. Bottom. Quantification of ERα relative to β-actin (used as a loading control) levels. n = 6 for each treatment; **:p<0.01 vs. control. B. A similar pattern of regulation of ERα protein is observed in primary cells cultured on top of Matrigel and incubated for 48 hrs with 10 µM of either of the inhibitors. Top. Two representative samples of a total of six of each group are shown in two independent gels. Bottom. Quantificaton of ERα relative to β-actin in each cell type. n = 6 for each treatment; **p<0.01 vs. control. C. Representative confocal images showing ERα determined by immunofluoresence (green). Lower levels of the protein are seen in PD98059-treated and in LY294002-treated C4-HI cells growing on Matrigel, whereas similar levels of ERα are seen in C4-HD treated and un-treated cells. Nuclei were stained red with propidium iodide. Scale bar: 30 µm. D. Top. A representative sample for each treatment is shown in a western blot. Scp2, a mouse mammary cell line, transfected with a constitutively active form of AKT1, myristoylated AKT1-Δ4-129 (Scp2Akt), displays in the same gel AKT with a typical molecular weight of 59 kDa and the myristoylated deleted variant of AKT1 (p-myrAKT) with a molecular weight of 45 kDa. The antibody used to detect total AKT recognizes only wild type AKT. E-cadherin was used as a loading control for Scp2 cells. Scp2Akt cells exhibit higher levels of ERα than Scp2 cells transfected with the vector control (vc) or Scp2 control cells. In Scp2 and Scp2vc cells, but not in Scp2Akt cells, 2 and 5 µM LY294002 downregulate p-AKT and ERα levels, whereas total AKT levels remains invariable. Bottom. Quantification of ERα relative to E-cadherin levels. n = 4 for each treatment; a,b p<0.05 Scp2Akt vs. Scp2 and Scp2vc; *p<0.05 LY294002 vs. control.
Mentions: In order to find other mechanisms responsible for the difference in growth between C4-HD and C4-HI tumors, we investigated wether the PI3K/AKT and ERK1/2 pathways regulated the levels of ERα. Inhibition of either pathway significantly (p<0.01) reduced the expression levels of ERα in C4-HI tumors but not in C4-HD tumors as assessed by western blot (Figure 6A). This result, together with our finding that inhibition of p-ERK by PD98059 did not reduce tumor growth rate (Figure 1C), suggest that at least in C4-HI cells, cell proliferation and cell survival are not determined exclusively by ERα levels.

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