Limits...
Autocrine regulation of cell proliferation by estrogen receptor-alpha in estrogen receptor-alpha-positive breast cancer cell lines.

Tan H, Zhong Y, Pan Z - BMC Cancer (2009)

Bottom Line: E2 stimulation of ICI182780 treated cells, however, promotes the expression and colocalization of ERalpha and Ki-67 as well as the cell cycle progressing through the S and G2/M phases.Inhibition of EGFR signaling does not inhibit the autocrine action of ERalpha.All of the three ERalpha-positive cell lines used in our study showed colocalization of ERalpha and Ki-67, indicating that these cell lines might be originated from primary tumor cells with autocrine regulation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Animal Science, Vermont Cancer Center, University of Vermont, Burlington, VT 05405, USA. htan1@uvm.edu

ABSTRACT

Background: Estrogen receptor-alpha (ERalpha) is essential for mammary gland development and is a major oncogene in breast cancer. Since ERalpha is not colocalized with the cell proliferation marker Ki-67 in the normal mammary glands and the majority of primary breast tumors, it is generally believed that paracrine regulation is involved in ERalpha mediated cell proliferation. In the paracrine model, ERalpha-positive cells don't proliferate but will release some paracrine growth factors to stimulate the neighboring cells to proliferate. In a subpopulation of cancer cells in some primary breast tumors, however, ERalpha does colocalize with the cell proliferation marker Ki-67, suggesting an autocrine regulation by ERalpha in some primary breast tumors.

Methods: Colocalization of ERalpha with Ki-67 in ERalpha-positive breast cancer cell lines (MCF-7, T47D, and ZR75-1) was evaluated by immunofluorescent staining. Cell cycle phase dependent expression of ERalpha was determined by co-immunofluorescent staining of ERalpha and the major cyclins (D, E, A, B), and by flow cytometry analysis of ERalphahigh cells. To further confirm the autocrine action of ERalpha, MCF-7 cells were growth arrested by ICI182780 treatment, followed by treatment with EGFR inhibitor, before estrogen stimulation and analyses for colocalization of Ki-67 and ERalpha and cell cycle progression.

Results: Colocalization of ERalpha with Ki-67 was present in all three ERalpha-positive breast cancer cell lines. Unlike that in the normal mammary glands and the majority of primary breast tumors, ERalpha is highly expressed throughout the cell cycle in MCF-7 cells. Without E2 stimulation, MCF-7 cells released from ICI182780 treatment remain at G1 phase. E2 stimulation of ICI182780 treated cells, however, promotes the expression and colocalization of ERalpha and Ki-67 as well as the cell cycle progressing through the S and G2/M phases. Inhibition of EGFR signaling does not inhibit the autocrine action of ERalpha.

Conclusion: Our data indicate that ERalpha can mediate estrogen-induced cell proliferation in an autocrine mode in ERalpha-positive breast cancer cell lines. All of the three ERalpha-positive cell lines used in our study showed colocalization of ERalpha and Ki-67, indicating that these cell lines might be originated from primary tumor cells with autocrine regulation.

Show MeSH

Related in: MedlinePlus

EGFR signaling is not required for the autocrine regulation of cell proliferation by ERα. A, MCF-7 cells treated with 10 nM ICI182780 for 72–96 hr, showing the down-regulation of ERα and Ki-67 by ICI182780 treatment. In A and B, ERα and Ki-67 were evaluated by immunofluorescent staining, nuclei were stained with DAPI. ERα-staining and Ki-67 staining were overlayed to show colocalization (Merge). Magnification, 400×. B, EGFR inhibitor Gefitinib does not inhibit E2 induced ERα and Ki-67 expression. MCF-7 cells were stimulated with 100 nM E2 in the presence of 10 nM ICI182780 and 20 μM EGFR inhibitor Gefitinib. MCF-7 cells were treated first with 10 nM ICI182780 for 72–96 hr, followed by adding the EGFR inhibitor Gefitinib to a final concentration of 20 μM for 2 hr, before adding estrogen to a final concentration of 100 nM for 16 hr. C, flow cytometry analysis of cell cycle progression, showing E2 stimulated cell cycle progression is not inhibited by Gefitinib. Upper panel, MCF-7 cells treated with ICI182780 for 72 hr. The cells were arrested at G1 phase. Lower panel, cell cycle progression was stimulated by 100 nM E2 in the presence of 10 nM ICI182780 and 20 μM EGFR inhibitor Gefitinib. MCF-7 cells were treated first with 10 nM ICI182780 for 72 hr, followed by treatment with the Gefitinib (20 μM) for 2 hr, before stimulation with 100 nM E2 for 36 hr. D, immunoblotting of phosphorylated ERK1/2, showing the inhibition of EGFR signaling by 20 μM Gefitinib. MCF-7 cells were serum starved for 24 hr, followed by treatment with 20 μM Gefitinib for 2 hr, before EGF stimulation for 5 min. The control group of cells was not treated with Gefitinib. Phosphorylation of ERK1/2 was used to assess the activation of the EGFR signaling.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2636826&req=5

Figure 6: EGFR signaling is not required for the autocrine regulation of cell proliferation by ERα. A, MCF-7 cells treated with 10 nM ICI182780 for 72–96 hr, showing the down-regulation of ERα and Ki-67 by ICI182780 treatment. In A and B, ERα and Ki-67 were evaluated by immunofluorescent staining, nuclei were stained with DAPI. ERα-staining and Ki-67 staining were overlayed to show colocalization (Merge). Magnification, 400×. B, EGFR inhibitor Gefitinib does not inhibit E2 induced ERα and Ki-67 expression. MCF-7 cells were stimulated with 100 nM E2 in the presence of 10 nM ICI182780 and 20 μM EGFR inhibitor Gefitinib. MCF-7 cells were treated first with 10 nM ICI182780 for 72–96 hr, followed by adding the EGFR inhibitor Gefitinib to a final concentration of 20 μM for 2 hr, before adding estrogen to a final concentration of 100 nM for 16 hr. C, flow cytometry analysis of cell cycle progression, showing E2 stimulated cell cycle progression is not inhibited by Gefitinib. Upper panel, MCF-7 cells treated with ICI182780 for 72 hr. The cells were arrested at G1 phase. Lower panel, cell cycle progression was stimulated by 100 nM E2 in the presence of 10 nM ICI182780 and 20 μM EGFR inhibitor Gefitinib. MCF-7 cells were treated first with 10 nM ICI182780 for 72 hr, followed by treatment with the Gefitinib (20 μM) for 2 hr, before stimulation with 100 nM E2 for 36 hr. D, immunoblotting of phosphorylated ERK1/2, showing the inhibition of EGFR signaling by 20 μM Gefitinib. MCF-7 cells were serum starved for 24 hr, followed by treatment with 20 μM Gefitinib for 2 hr, before EGF stimulation for 5 min. The control group of cells was not treated with Gefitinib. Phosphorylation of ERK1/2 was used to assess the activation of the EGFR signaling.

Mentions: In the paracrine regulation of cell proliferation by ERα, EGFR signaling is required [20]. Therefore we evaluated whether EGFR signaling is required for the autocrine regulation of cell proliferation by ERα in MCF-7 cells. EGFR inhibitor Gefitinib was used to block EGFR signaling, 20 μM of Gefitinib was chosen based on our preliminary experiments using a wide range concentrations of Gefitinib (1 μM – 50 μM) (Fig. 6D, and data not shown). We first evaluated whether Gefitinib treatment inhibits E2 stimulation induced expression and colocalization of ERα and Ki-67. MCF-7 cells were treated first with 10 nM ICI182780 for 72–96 hr, followed by adding the EGFR inhibitor Gefitinib to a final concentration of 20 μM for 2 hr, then by adding estrogen to a final concentration of 100 nM. In the presence of ICI182780 and Gefitinib, estrogen stimulation still induced the expression and colocalization of ERα and Ki-67 (Fig. 6B). Conceivably, Gefitinib treatment does not inhibit estrogen stimulation induced cell cycle progression (Fig. 6C lower panel). In the control groups of MCF-7 cells treated with ICI182780 for 72–96 hr, ERα and Ki-67 were down-regulated and the cells remained at G1 phase (Fig. 6A, C upper panel). In the growth curve studies and the EGF stimulation experiments, Gefitinib at 20 μM is sufficient to block EGFR signaling but not toxic to the cells (Fig. 6D, and data not shown). From these data, we concluded that EGFR signaling is not required for the autocrine action of ERα in mediating cell proliferation in MCF-7 cells.


Autocrine regulation of cell proliferation by estrogen receptor-alpha in estrogen receptor-alpha-positive breast cancer cell lines.

Tan H, Zhong Y, Pan Z - BMC Cancer (2009)

EGFR signaling is not required for the autocrine regulation of cell proliferation by ERα. A, MCF-7 cells treated with 10 nM ICI182780 for 72–96 hr, showing the down-regulation of ERα and Ki-67 by ICI182780 treatment. In A and B, ERα and Ki-67 were evaluated by immunofluorescent staining, nuclei were stained with DAPI. ERα-staining and Ki-67 staining were overlayed to show colocalization (Merge). Magnification, 400×. B, EGFR inhibitor Gefitinib does not inhibit E2 induced ERα and Ki-67 expression. MCF-7 cells were stimulated with 100 nM E2 in the presence of 10 nM ICI182780 and 20 μM EGFR inhibitor Gefitinib. MCF-7 cells were treated first with 10 nM ICI182780 for 72–96 hr, followed by adding the EGFR inhibitor Gefitinib to a final concentration of 20 μM for 2 hr, before adding estrogen to a final concentration of 100 nM for 16 hr. C, flow cytometry analysis of cell cycle progression, showing E2 stimulated cell cycle progression is not inhibited by Gefitinib. Upper panel, MCF-7 cells treated with ICI182780 for 72 hr. The cells were arrested at G1 phase. Lower panel, cell cycle progression was stimulated by 100 nM E2 in the presence of 10 nM ICI182780 and 20 μM EGFR inhibitor Gefitinib. MCF-7 cells were treated first with 10 nM ICI182780 for 72 hr, followed by treatment with the Gefitinib (20 μM) for 2 hr, before stimulation with 100 nM E2 for 36 hr. D, immunoblotting of phosphorylated ERK1/2, showing the inhibition of EGFR signaling by 20 μM Gefitinib. MCF-7 cells were serum starved for 24 hr, followed by treatment with 20 μM Gefitinib for 2 hr, before EGF stimulation for 5 min. The control group of cells was not treated with Gefitinib. Phosphorylation of ERK1/2 was used to assess the activation of the EGFR signaling.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2636826&req=5

Figure 6: EGFR signaling is not required for the autocrine regulation of cell proliferation by ERα. A, MCF-7 cells treated with 10 nM ICI182780 for 72–96 hr, showing the down-regulation of ERα and Ki-67 by ICI182780 treatment. In A and B, ERα and Ki-67 were evaluated by immunofluorescent staining, nuclei were stained with DAPI. ERα-staining and Ki-67 staining were overlayed to show colocalization (Merge). Magnification, 400×. B, EGFR inhibitor Gefitinib does not inhibit E2 induced ERα and Ki-67 expression. MCF-7 cells were stimulated with 100 nM E2 in the presence of 10 nM ICI182780 and 20 μM EGFR inhibitor Gefitinib. MCF-7 cells were treated first with 10 nM ICI182780 for 72–96 hr, followed by adding the EGFR inhibitor Gefitinib to a final concentration of 20 μM for 2 hr, before adding estrogen to a final concentration of 100 nM for 16 hr. C, flow cytometry analysis of cell cycle progression, showing E2 stimulated cell cycle progression is not inhibited by Gefitinib. Upper panel, MCF-7 cells treated with ICI182780 for 72 hr. The cells were arrested at G1 phase. Lower panel, cell cycle progression was stimulated by 100 nM E2 in the presence of 10 nM ICI182780 and 20 μM EGFR inhibitor Gefitinib. MCF-7 cells were treated first with 10 nM ICI182780 for 72 hr, followed by treatment with the Gefitinib (20 μM) for 2 hr, before stimulation with 100 nM E2 for 36 hr. D, immunoblotting of phosphorylated ERK1/2, showing the inhibition of EGFR signaling by 20 μM Gefitinib. MCF-7 cells were serum starved for 24 hr, followed by treatment with 20 μM Gefitinib for 2 hr, before EGF stimulation for 5 min. The control group of cells was not treated with Gefitinib. Phosphorylation of ERK1/2 was used to assess the activation of the EGFR signaling.
Mentions: In the paracrine regulation of cell proliferation by ERα, EGFR signaling is required [20]. Therefore we evaluated whether EGFR signaling is required for the autocrine regulation of cell proliferation by ERα in MCF-7 cells. EGFR inhibitor Gefitinib was used to block EGFR signaling, 20 μM of Gefitinib was chosen based on our preliminary experiments using a wide range concentrations of Gefitinib (1 μM – 50 μM) (Fig. 6D, and data not shown). We first evaluated whether Gefitinib treatment inhibits E2 stimulation induced expression and colocalization of ERα and Ki-67. MCF-7 cells were treated first with 10 nM ICI182780 for 72–96 hr, followed by adding the EGFR inhibitor Gefitinib to a final concentration of 20 μM for 2 hr, then by adding estrogen to a final concentration of 100 nM. In the presence of ICI182780 and Gefitinib, estrogen stimulation still induced the expression and colocalization of ERα and Ki-67 (Fig. 6B). Conceivably, Gefitinib treatment does not inhibit estrogen stimulation induced cell cycle progression (Fig. 6C lower panel). In the control groups of MCF-7 cells treated with ICI182780 for 72–96 hr, ERα and Ki-67 were down-regulated and the cells remained at G1 phase (Fig. 6A, C upper panel). In the growth curve studies and the EGF stimulation experiments, Gefitinib at 20 μM is sufficient to block EGFR signaling but not toxic to the cells (Fig. 6D, and data not shown). From these data, we concluded that EGFR signaling is not required for the autocrine action of ERα in mediating cell proliferation in MCF-7 cells.

Bottom Line: E2 stimulation of ICI182780 treated cells, however, promotes the expression and colocalization of ERalpha and Ki-67 as well as the cell cycle progressing through the S and G2/M phases.Inhibition of EGFR signaling does not inhibit the autocrine action of ERalpha.All of the three ERalpha-positive cell lines used in our study showed colocalization of ERalpha and Ki-67, indicating that these cell lines might be originated from primary tumor cells with autocrine regulation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Animal Science, Vermont Cancer Center, University of Vermont, Burlington, VT 05405, USA. htan1@uvm.edu

ABSTRACT

Background: Estrogen receptor-alpha (ERalpha) is essential for mammary gland development and is a major oncogene in breast cancer. Since ERalpha is not colocalized with the cell proliferation marker Ki-67 in the normal mammary glands and the majority of primary breast tumors, it is generally believed that paracrine regulation is involved in ERalpha mediated cell proliferation. In the paracrine model, ERalpha-positive cells don't proliferate but will release some paracrine growth factors to stimulate the neighboring cells to proliferate. In a subpopulation of cancer cells in some primary breast tumors, however, ERalpha does colocalize with the cell proliferation marker Ki-67, suggesting an autocrine regulation by ERalpha in some primary breast tumors.

Methods: Colocalization of ERalpha with Ki-67 in ERalpha-positive breast cancer cell lines (MCF-7, T47D, and ZR75-1) was evaluated by immunofluorescent staining. Cell cycle phase dependent expression of ERalpha was determined by co-immunofluorescent staining of ERalpha and the major cyclins (D, E, A, B), and by flow cytometry analysis of ERalphahigh cells. To further confirm the autocrine action of ERalpha, MCF-7 cells were growth arrested by ICI182780 treatment, followed by treatment with EGFR inhibitor, before estrogen stimulation and analyses for colocalization of Ki-67 and ERalpha and cell cycle progression.

Results: Colocalization of ERalpha with Ki-67 was present in all three ERalpha-positive breast cancer cell lines. Unlike that in the normal mammary glands and the majority of primary breast tumors, ERalpha is highly expressed throughout the cell cycle in MCF-7 cells. Without E2 stimulation, MCF-7 cells released from ICI182780 treatment remain at G1 phase. E2 stimulation of ICI182780 treated cells, however, promotes the expression and colocalization of ERalpha and Ki-67 as well as the cell cycle progressing through the S and G2/M phases. Inhibition of EGFR signaling does not inhibit the autocrine action of ERalpha.

Conclusion: Our data indicate that ERalpha can mediate estrogen-induced cell proliferation in an autocrine mode in ERalpha-positive breast cancer cell lines. All of the three ERalpha-positive cell lines used in our study showed colocalization of ERalpha and Ki-67, indicating that these cell lines might be originated from primary tumor cells with autocrine regulation.

Show MeSH
Related in: MedlinePlus