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Using the MCF10A/MCF10CA1a Breast Cancer Progression Cell Line Model to Investigate the Effect of Active, Mutant Forms of EGFR in Breast Cancer Development and Treatment Using Gefitinib.

Bessette DC, Tilch E, Seidens T, Quinn MC, Wiegmans AP, Shi W, Cocciardi S, McCart-Reed A, Saunus JM, Simpson PT, Grimmond SM, Lakhani SR, Khanna KK, Waddell N, Al-Ejeh F, Chenevix-Trench G - PLoS ONE (2015)

Bottom Line: The effects of EGFR over-expression and mutation on proliferation, migration, invasion, response to gefitinib, and tumour formation in vivo was investigated.Copy number analysis and whole exome sequencing of the MCF10A and MCF10CA1a cell lines were also performed.The EGFR-E746-A750 deletion increased MCF10CA1a cell migration and invasion, and greatly increased MCF10CA1a xenograft tumour formation and growth.

View Article: PubMed Central - PubMed

Affiliation: QIMR Berghofer Medical Research Institute, Brisbane, Queensland, Australia.

ABSTRACT

Background: Basal-like and triple negative breast cancer (TNBC) share common molecular features, poor prognosis and a propensity for metastasis to the brain. Amplification of epidermal growth factor receptor (EGFR) occurs in ~50% of basal-like breast cancer, and mutations in the epidermal growth factor receptor (EGFR) have been reported in up to ~ 10% of Asian TNBC patients. In non-small cell lung cancer several different mutations in the EGFR tyrosine kinase domain confer sensitivity to receptor tyrosine kinase inhibitors, but the tumourigenic potential of EGFR mutations in breast cells and their potential for targeted therapy is unknown.

Materials and methods: Constructs containing wild type, G719S or E746-A750 deletion mutant forms of EGFR were transfected into the MCF10A breast cells and their tumorigenic derivative, MCF10CA1a. The effects of EGFR over-expression and mutation on proliferation, migration, invasion, response to gefitinib, and tumour formation in vivo was investigated. Copy number analysis and whole exome sequencing of the MCF10A and MCF10CA1a cell lines were also performed.

Results: Mutant EGFR increased MCF10A and MCF10CA1a proliferation and MCF10A gefitinib sensitivity. The EGFR-E746-A750 deletion increased MCF10CA1a cell migration and invasion, and greatly increased MCF10CA1a xenograft tumour formation and growth. Compared to MCF10A cells, MCF10CA1a cells exhibited large regions of gain on chromosomes 3 and 9, deletion on chromosome 7, and mutations in many genes implicated in cancer.

Conclusions: Mutant EGFR enhances the oncogenic properties of MCF10A cell line, and increases sensitivity to gefitinib. Although the addition of EGFR E746-A750 renders the MCF10CA1a cells more tumourigenic in vivo it is not accompanied by increased gefitinib sensitivity, perhaps due to additional mutations, including the PIK3CA H1047R mutation, that the MCF10CA1a cell line has acquired. Screening TNBC/basal-like breast cancer for EGFR mutations may prove useful for directing therapy but, as in non-small cell lung cancer, accompanying mutations in PIK3CA may confer gefitinib resistance.

No MeSH data available.


Related in: MedlinePlus

Expression of the EGFR-GS and—DEL mutant proteins enhances MCF10A EGF-independent growth and sensitivity to gefitinib.A. Cells were cultured in the presence (+ EGF) or absence (- EGF) of EGF and their proliferation assessed using MTS assay by measuring absorbance at 490 nm over a period of 8 days. B. The doubling times of the cell lines were calculated from the rates of proliferation. As neither MCF10A nor MCF10A-WT cell proliferated in the absence of EGF, a doubling time could not be calculated. C. Cells were treated with varying concentrations of gefitinib in the absence of EGF for 72 hours and cell viability measured using the MTS assay. The IC50 was calculated and the differences compared using the unpaired t-Test (* p<0.05). D. Cells were treated with varying concentrations of gefitinib in the presence of EGF for 72 hours and cell viability measured using the MTS assay. The IC50 was calculated and the differences compared using the unpaired t-Test (**** p<0.0001). 10A: MCF10A-EV, WT: MCF10A-EGFR-WT, GS: MCF10A-EGFR-GS, DEL: MCF10A-EGFR-DEL. Data from duplicate experiments.
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pone.0125232.g001: Expression of the EGFR-GS and—DEL mutant proteins enhances MCF10A EGF-independent growth and sensitivity to gefitinib.A. Cells were cultured in the presence (+ EGF) or absence (- EGF) of EGF and their proliferation assessed using MTS assay by measuring absorbance at 490 nm over a period of 8 days. B. The doubling times of the cell lines were calculated from the rates of proliferation. As neither MCF10A nor MCF10A-WT cell proliferated in the absence of EGF, a doubling time could not be calculated. C. Cells were treated with varying concentrations of gefitinib in the absence of EGF for 72 hours and cell viability measured using the MTS assay. The IC50 was calculated and the differences compared using the unpaired t-Test (* p<0.05). D. Cells were treated with varying concentrations of gefitinib in the presence of EGF for 72 hours and cell viability measured using the MTS assay. The IC50 was calculated and the differences compared using the unpaired t-Test (**** p<0.0001). 10A: MCF10A-EV, WT: MCF10A-EGFR-WT, GS: MCF10A-EGFR-GS, DEL: MCF10A-EGFR-DEL. Data from duplicate experiments.

Mentions: As triple negative breast cancers (TNBC) have been found to harbour mutations in EGFR [28], we wished to determine how they affect a relatively normal breast cell line. We used MCF10A cells, a spontaneously immortalized, non-malignant cell line obtained from a patient with fibrocystic disease [32], and stably infected them with EGFR-wild type (WT), EGFR-G719S (GS) or EGFR-DEL (E746-A750) (DEL) retroviral constructs. All three constructs expressed EGFR well in vitro (S1 Fig). The proliferation of the MCF10A cell lines containing the EGFR constructs was monitored in the presence or absence of EGF (Fig 1A and 1B). Neither wild type EGFR, nor EGFR mutant expression, affected the proliferation of the MCF10A cells in the presence of EGF. However, in the absence of EGF both EGFR-GS and EGFR-DEL mutant expression resulted in increased proliferation of the MCF10A cells compared to the parental control, or the cells expressing wild type EGFR.


Using the MCF10A/MCF10CA1a Breast Cancer Progression Cell Line Model to Investigate the Effect of Active, Mutant Forms of EGFR in Breast Cancer Development and Treatment Using Gefitinib.

Bessette DC, Tilch E, Seidens T, Quinn MC, Wiegmans AP, Shi W, Cocciardi S, McCart-Reed A, Saunus JM, Simpson PT, Grimmond SM, Lakhani SR, Khanna KK, Waddell N, Al-Ejeh F, Chenevix-Trench G - PLoS ONE (2015)

Expression of the EGFR-GS and—DEL mutant proteins enhances MCF10A EGF-independent growth and sensitivity to gefitinib.A. Cells were cultured in the presence (+ EGF) or absence (- EGF) of EGF and their proliferation assessed using MTS assay by measuring absorbance at 490 nm over a period of 8 days. B. The doubling times of the cell lines were calculated from the rates of proliferation. As neither MCF10A nor MCF10A-WT cell proliferated in the absence of EGF, a doubling time could not be calculated. C. Cells were treated with varying concentrations of gefitinib in the absence of EGF for 72 hours and cell viability measured using the MTS assay. The IC50 was calculated and the differences compared using the unpaired t-Test (* p<0.05). D. Cells were treated with varying concentrations of gefitinib in the presence of EGF for 72 hours and cell viability measured using the MTS assay. The IC50 was calculated and the differences compared using the unpaired t-Test (**** p<0.0001). 10A: MCF10A-EV, WT: MCF10A-EGFR-WT, GS: MCF10A-EGFR-GS, DEL: MCF10A-EGFR-DEL. Data from duplicate experiments.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4430383&req=5

pone.0125232.g001: Expression of the EGFR-GS and—DEL mutant proteins enhances MCF10A EGF-independent growth and sensitivity to gefitinib.A. Cells were cultured in the presence (+ EGF) or absence (- EGF) of EGF and their proliferation assessed using MTS assay by measuring absorbance at 490 nm over a period of 8 days. B. The doubling times of the cell lines were calculated from the rates of proliferation. As neither MCF10A nor MCF10A-WT cell proliferated in the absence of EGF, a doubling time could not be calculated. C. Cells were treated with varying concentrations of gefitinib in the absence of EGF for 72 hours and cell viability measured using the MTS assay. The IC50 was calculated and the differences compared using the unpaired t-Test (* p<0.05). D. Cells were treated with varying concentrations of gefitinib in the presence of EGF for 72 hours and cell viability measured using the MTS assay. The IC50 was calculated and the differences compared using the unpaired t-Test (**** p<0.0001). 10A: MCF10A-EV, WT: MCF10A-EGFR-WT, GS: MCF10A-EGFR-GS, DEL: MCF10A-EGFR-DEL. Data from duplicate experiments.
Mentions: As triple negative breast cancers (TNBC) have been found to harbour mutations in EGFR [28], we wished to determine how they affect a relatively normal breast cell line. We used MCF10A cells, a spontaneously immortalized, non-malignant cell line obtained from a patient with fibrocystic disease [32], and stably infected them with EGFR-wild type (WT), EGFR-G719S (GS) or EGFR-DEL (E746-A750) (DEL) retroviral constructs. All three constructs expressed EGFR well in vitro (S1 Fig). The proliferation of the MCF10A cell lines containing the EGFR constructs was monitored in the presence or absence of EGF (Fig 1A and 1B). Neither wild type EGFR, nor EGFR mutant expression, affected the proliferation of the MCF10A cells in the presence of EGF. However, in the absence of EGF both EGFR-GS and EGFR-DEL mutant expression resulted in increased proliferation of the MCF10A cells compared to the parental control, or the cells expressing wild type EGFR.

Bottom Line: The effects of EGFR over-expression and mutation on proliferation, migration, invasion, response to gefitinib, and tumour formation in vivo was investigated.Copy number analysis and whole exome sequencing of the MCF10A and MCF10CA1a cell lines were also performed.The EGFR-E746-A750 deletion increased MCF10CA1a cell migration and invasion, and greatly increased MCF10CA1a xenograft tumour formation and growth.

View Article: PubMed Central - PubMed

Affiliation: QIMR Berghofer Medical Research Institute, Brisbane, Queensland, Australia.

ABSTRACT

Background: Basal-like and triple negative breast cancer (TNBC) share common molecular features, poor prognosis and a propensity for metastasis to the brain. Amplification of epidermal growth factor receptor (EGFR) occurs in ~50% of basal-like breast cancer, and mutations in the epidermal growth factor receptor (EGFR) have been reported in up to ~ 10% of Asian TNBC patients. In non-small cell lung cancer several different mutations in the EGFR tyrosine kinase domain confer sensitivity to receptor tyrosine kinase inhibitors, but the tumourigenic potential of EGFR mutations in breast cells and their potential for targeted therapy is unknown.

Materials and methods: Constructs containing wild type, G719S or E746-A750 deletion mutant forms of EGFR were transfected into the MCF10A breast cells and their tumorigenic derivative, MCF10CA1a. The effects of EGFR over-expression and mutation on proliferation, migration, invasion, response to gefitinib, and tumour formation in vivo was investigated. Copy number analysis and whole exome sequencing of the MCF10A and MCF10CA1a cell lines were also performed.

Results: Mutant EGFR increased MCF10A and MCF10CA1a proliferation and MCF10A gefitinib sensitivity. The EGFR-E746-A750 deletion increased MCF10CA1a cell migration and invasion, and greatly increased MCF10CA1a xenograft tumour formation and growth. Compared to MCF10A cells, MCF10CA1a cells exhibited large regions of gain on chromosomes 3 and 9, deletion on chromosome 7, and mutations in many genes implicated in cancer.

Conclusions: Mutant EGFR enhances the oncogenic properties of MCF10A cell line, and increases sensitivity to gefitinib. Although the addition of EGFR E746-A750 renders the MCF10CA1a cells more tumourigenic in vivo it is not accompanied by increased gefitinib sensitivity, perhaps due to additional mutations, including the PIK3CA H1047R mutation, that the MCF10CA1a cell line has acquired. Screening TNBC/basal-like breast cancer for EGFR mutations may prove useful for directing therapy but, as in non-small cell lung cancer, accompanying mutations in PIK3CA may confer gefitinib resistance.

No MeSH data available.


Related in: MedlinePlus