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New cell culture model for aromatase inhibitor-resistant breast cancer shows sensitivity to fulvestrant treatment and cross-resistance between letrozole and exemestane.

Hole S, Pedersen AM, Hansen SK, Lundqvist J, Yde CW, Lykkesfeldt AE - Int. J. Oncol. (2015)

Bottom Line: Although many patients benefit from treatment, some will develop resistance, and models mimicking acquired resistance will be valuable tools to unravel the resistance mechanisms and to find new treatments and biomarkers.Letrozole, exemestane and tamoxifen were able to abrogate the testosterone stimulation but could not reduce growth to below the level in standard growth medium with AI, demonstrating cross-resistance between letrozole, exemestane and tamoxifen.These data show that ER is the main driver of growth of the AI-resistant cell lines and indicate ligand-independent activation of ER.

View Article: PubMed Central - PubMed

Affiliation: Breast Cancer Group, Cell Death and Metabolism, Danish Cancer Society Research Center, DK-2100 Copenhagen Ø, Denmark.

ABSTRACT
Aromatase inhibitor (AI) treatment is first-line systemic treatment for the majority of postmenopausal breast cancer patients with estrogen receptor (ER)-positive primary tumor. Although many patients benefit from treatment, some will develop resistance, and models mimicking acquired resistance will be valuable tools to unravel the resistance mechanisms and to find new treatments and biomarkers. Cell culture models for acquired resistance to the three clinically relevant AIs letrozole, anastrozole and exemestane were developed by selection and expansion of colonies of MCF-7 breast cancer cells surviving long-term AI treatment under conditions where endogenous aromatase-mediated conversion of androgen to estrogen was required for growth. Four cell lines resistant to each of the AIs were established and characterized. Maintenance of ER expression and function was a general finding, but ER loss was seen in one of twelve cell lines. HER receptor expression was increased, in particular EGFR expression in letrozole-resistant cell lines. The AI-resistant cell lines had acquired ability to grow without aromatase-mediated conversion of testosterone to estradiol, but upon withdrawal of AI treatment, testosterone induced minor growth stimulation. Letrozole, exemestane and tamoxifen were able to abrogate the testosterone stimulation but could not reduce growth to below the level in standard growth medium with AI, demonstrating cross-resistance between letrozole, exemestane and tamoxifen. In contrast, fulvestrant totally blocked growth of the AI resistant cell lines both after withdrawal of AI and with AI treatment. These data show that ER is the main driver of growth of the AI-resistant cell lines and indicate ligand-independent activation of ER. Fulvestrant is an efficient treatment option for these AI-resistant breast cancer cells, and the cell lines will be useful tools to disclose the underlying molecular mechanism for resistance to the different AIs.

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Effect of tamoxifen and fulvestrant on growth of MCF-7 and on AI-resistant cell lines in combination with AI. MCF-7 cells were grown in 10% NCS + 10−7 M testosterone alone or in combination with 10−6 M tamoxifen or 10−7 M fulvestrant. LetR-1, LetR-3, ExeR-1 and ExeR-3 were grown in their standard growth medium with the respective AI and in standard growth medium with the respective AI and with 10−6 M tamoxifen or 10−7 M fulvestrant for five days. Cell number was estimated by a colorimetric assay and expressed relative to the cell number in the control cultures (MCF-7 grown with 10% NCS + 10−7 M testosterone, LetR-1 and LetR-3 grown with 10% NCS + 10−7 M testosterone + 10−6 M letrozole, ExeR-1 and ExeR-3 grown with 10% NCS + 10−7 M testosterone + 10−7 M exemestane). Representative experiments of two independent experiments with four sample replicates are shown. Mean and SD are shown and the asterisks indicate statistically significant difference from the respective control cultures.
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f6-ijo-46-04-1481: Effect of tamoxifen and fulvestrant on growth of MCF-7 and on AI-resistant cell lines in combination with AI. MCF-7 cells were grown in 10% NCS + 10−7 M testosterone alone or in combination with 10−6 M tamoxifen or 10−7 M fulvestrant. LetR-1, LetR-3, ExeR-1 and ExeR-3 were grown in their standard growth medium with the respective AI and in standard growth medium with the respective AI and with 10−6 M tamoxifen or 10−7 M fulvestrant for five days. Cell number was estimated by a colorimetric assay and expressed relative to the cell number in the control cultures (MCF-7 grown with 10% NCS + 10−7 M testosterone, LetR-1 and LetR-3 grown with 10% NCS + 10−7 M testosterone + 10−6 M letrozole, ExeR-1 and ExeR-3 grown with 10% NCS + 10−7 M testosterone + 10−7 M exemestane). Representative experiments of two independent experiments with four sample replicates are shown. Mean and SD are shown and the asterisks indicate statistically significant difference from the respective control cultures.

Mentions: Tamoxifen is a weak estrogen antagonist and in medium with 10% NCS + 10−7 M testosterone, a dose-dependent growth inhibition of MCF-7 cells was seen. However, growth was not completely arrested after five days treatment with 10−6 M tamoxifen (Fig. 5A and B) as it is normally observed with MCF-7 cells grown in their standard medium with 1% FCS (41). Fulvestrant exerted complete growth arrest of MCF-7 cells at concentrations from 10−9 M (Fig. 5C and D). Dose-response growth experiments with antiestrogens were performed with AI-resistant cell lines withdrawn from AI treatment for one week (Fig. 5) and with cells grown with AI (Fig. 6). In AI-resistant cell lines grown with 10% NCS + 10−7 M testosterone and withdrawn from AI treatment for one week, tamoxifen induced a dose-dependent growth inhibition down to the level seen in 10% NCS (Fig. 5A and B). Fulvestrant also inhibited growth of AI-resistant cell lines in a dose-dependent manner, but in contrast to tamoxifen, fulvestrant reduced growth of the AI-resistant cell lines to below the level in NCS medium and down to the level seen in MCF-7 cells treated with 10−7 M fulvestrant (Fig. 5C and D). Tamoxifen treatment of AI-resistant cell lines grown in their standard growth medium with their respective AI had no effect or a stimulatory effect, whereas fulvestrant suppressed growth totally, as seen in MCF-7 cells (Fig. 6). To determine whether AI-resistant cell lines were completely growth arrested with fulvestrant, cultures with LetR-1 and ExeR-1 cells were treated with 10−7 M fulvestrant for one week, split and treated for further one week with fulvestrant. After a total of 14 days of treatment with 10−7 M fulvestrant, no viable LetR-1 or ExeR-1 cells could be detected.


New cell culture model for aromatase inhibitor-resistant breast cancer shows sensitivity to fulvestrant treatment and cross-resistance between letrozole and exemestane.

Hole S, Pedersen AM, Hansen SK, Lundqvist J, Yde CW, Lykkesfeldt AE - Int. J. Oncol. (2015)

Effect of tamoxifen and fulvestrant on growth of MCF-7 and on AI-resistant cell lines in combination with AI. MCF-7 cells were grown in 10% NCS + 10−7 M testosterone alone or in combination with 10−6 M tamoxifen or 10−7 M fulvestrant. LetR-1, LetR-3, ExeR-1 and ExeR-3 were grown in their standard growth medium with the respective AI and in standard growth medium with the respective AI and with 10−6 M tamoxifen or 10−7 M fulvestrant for five days. Cell number was estimated by a colorimetric assay and expressed relative to the cell number in the control cultures (MCF-7 grown with 10% NCS + 10−7 M testosterone, LetR-1 and LetR-3 grown with 10% NCS + 10−7 M testosterone + 10−6 M letrozole, ExeR-1 and ExeR-3 grown with 10% NCS + 10−7 M testosterone + 10−7 M exemestane). Representative experiments of two independent experiments with four sample replicates are shown. Mean and SD are shown and the asterisks indicate statistically significant difference from the respective control cultures.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4356498&req=5

f6-ijo-46-04-1481: Effect of tamoxifen and fulvestrant on growth of MCF-7 and on AI-resistant cell lines in combination with AI. MCF-7 cells were grown in 10% NCS + 10−7 M testosterone alone or in combination with 10−6 M tamoxifen or 10−7 M fulvestrant. LetR-1, LetR-3, ExeR-1 and ExeR-3 were grown in their standard growth medium with the respective AI and in standard growth medium with the respective AI and with 10−6 M tamoxifen or 10−7 M fulvestrant for five days. Cell number was estimated by a colorimetric assay and expressed relative to the cell number in the control cultures (MCF-7 grown with 10% NCS + 10−7 M testosterone, LetR-1 and LetR-3 grown with 10% NCS + 10−7 M testosterone + 10−6 M letrozole, ExeR-1 and ExeR-3 grown with 10% NCS + 10−7 M testosterone + 10−7 M exemestane). Representative experiments of two independent experiments with four sample replicates are shown. Mean and SD are shown and the asterisks indicate statistically significant difference from the respective control cultures.
Mentions: Tamoxifen is a weak estrogen antagonist and in medium with 10% NCS + 10−7 M testosterone, a dose-dependent growth inhibition of MCF-7 cells was seen. However, growth was not completely arrested after five days treatment with 10−6 M tamoxifen (Fig. 5A and B) as it is normally observed with MCF-7 cells grown in their standard medium with 1% FCS (41). Fulvestrant exerted complete growth arrest of MCF-7 cells at concentrations from 10−9 M (Fig. 5C and D). Dose-response growth experiments with antiestrogens were performed with AI-resistant cell lines withdrawn from AI treatment for one week (Fig. 5) and with cells grown with AI (Fig. 6). In AI-resistant cell lines grown with 10% NCS + 10−7 M testosterone and withdrawn from AI treatment for one week, tamoxifen induced a dose-dependent growth inhibition down to the level seen in 10% NCS (Fig. 5A and B). Fulvestrant also inhibited growth of AI-resistant cell lines in a dose-dependent manner, but in contrast to tamoxifen, fulvestrant reduced growth of the AI-resistant cell lines to below the level in NCS medium and down to the level seen in MCF-7 cells treated with 10−7 M fulvestrant (Fig. 5C and D). Tamoxifen treatment of AI-resistant cell lines grown in their standard growth medium with their respective AI had no effect or a stimulatory effect, whereas fulvestrant suppressed growth totally, as seen in MCF-7 cells (Fig. 6). To determine whether AI-resistant cell lines were completely growth arrested with fulvestrant, cultures with LetR-1 and ExeR-1 cells were treated with 10−7 M fulvestrant for one week, split and treated for further one week with fulvestrant. After a total of 14 days of treatment with 10−7 M fulvestrant, no viable LetR-1 or ExeR-1 cells could be detected.

Bottom Line: Although many patients benefit from treatment, some will develop resistance, and models mimicking acquired resistance will be valuable tools to unravel the resistance mechanisms and to find new treatments and biomarkers.Letrozole, exemestane and tamoxifen were able to abrogate the testosterone stimulation but could not reduce growth to below the level in standard growth medium with AI, demonstrating cross-resistance between letrozole, exemestane and tamoxifen.These data show that ER is the main driver of growth of the AI-resistant cell lines and indicate ligand-independent activation of ER.

View Article: PubMed Central - PubMed

Affiliation: Breast Cancer Group, Cell Death and Metabolism, Danish Cancer Society Research Center, DK-2100 Copenhagen Ø, Denmark.

ABSTRACT
Aromatase inhibitor (AI) treatment is first-line systemic treatment for the majority of postmenopausal breast cancer patients with estrogen receptor (ER)-positive primary tumor. Although many patients benefit from treatment, some will develop resistance, and models mimicking acquired resistance will be valuable tools to unravel the resistance mechanisms and to find new treatments and biomarkers. Cell culture models for acquired resistance to the three clinically relevant AIs letrozole, anastrozole and exemestane were developed by selection and expansion of colonies of MCF-7 breast cancer cells surviving long-term AI treatment under conditions where endogenous aromatase-mediated conversion of androgen to estrogen was required for growth. Four cell lines resistant to each of the AIs were established and characterized. Maintenance of ER expression and function was a general finding, but ER loss was seen in one of twelve cell lines. HER receptor expression was increased, in particular EGFR expression in letrozole-resistant cell lines. The AI-resistant cell lines had acquired ability to grow without aromatase-mediated conversion of testosterone to estradiol, but upon withdrawal of AI treatment, testosterone induced minor growth stimulation. Letrozole, exemestane and tamoxifen were able to abrogate the testosterone stimulation but could not reduce growth to below the level in standard growth medium with AI, demonstrating cross-resistance between letrozole, exemestane and tamoxifen. In contrast, fulvestrant totally blocked growth of the AI resistant cell lines both after withdrawal of AI and with AI treatment. These data show that ER is the main driver of growth of the AI-resistant cell lines and indicate ligand-independent activation of ER. Fulvestrant is an efficient treatment option for these AI-resistant breast cancer cells, and the cell lines will be useful tools to disclose the underlying molecular mechanism for resistance to the different AIs.

Show MeSH
Related in: MedlinePlus