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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

Overexpression of wild type or mutant EGFR increases the growth of MCF10CA1a mammary fat pad xeongrafts.Female BALB/c nude mice were injected in the mammary fat pad with 5x106 cells from the indicated cell line and tumour formation was monitored with bioluminescent imaging. CA: MCF10CA1a-EV, WT: MCF10CA1a -EGFR-WT, GS: MCF10CA1a -EGFR-GS, DEL: MCF10CA1a -EGFR-DEL A. Representative bioluminescent images of individual mice taken on day 14 and day 43. B. Plot of the increase in luciferase signal in each group of mice (*p<0.05, **p<0.01, ****p<0.0001, n = 5, vs CA control, One-Way ANOVA). C. Representative bioluminescent images of individual mice taken on day 49 post injection in an independent cohort of mice. D. Plot of the magnitude of luciferase signal in each group of mice at day 49 post injection (**p<0.01, n = 5).
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pone.0125232.g003: Overexpression of wild type or mutant EGFR increases the growth of MCF10CA1a mammary fat pad xeongrafts.Female BALB/c nude mice were injected in the mammary fat pad with 5x106 cells from the indicated cell line and tumour formation was monitored with bioluminescent imaging. CA: MCF10CA1a-EV, WT: MCF10CA1a -EGFR-WT, GS: MCF10CA1a -EGFR-GS, DEL: MCF10CA1a -EGFR-DEL A. Representative bioluminescent images of individual mice taken on day 14 and day 43. B. Plot of the increase in luciferase signal in each group of mice (*p<0.05, **p<0.01, ****p<0.0001, n = 5, vs CA control, One-Way ANOVA). C. Representative bioluminescent images of individual mice taken on day 49 post injection in an independent cohort of mice. D. Plot of the magnitude of luciferase signal in each group of mice at day 49 post injection (**p<0.01, n = 5).

Mentions: EGFR-DEL and EGFR-GS expression in MCF10A cells resulted in an increased proliferative ability in vitro and EGFR-DEL expression could induce spheroid formation in media lacking EGF. To determine whether these changes were reflected by increased tumorigenicity in vivo we examined the growth of mammary fat pad xenografts of the various MCF10A EGFR cell lines. The expression of the EGFR constructs in the MCF10A cells did not induce primary tumour formation up to 6 months post-injection (data not shown). As the MCF10A cell line is not malignant and the EGFR mutants were not sufficient to induce tumourigenesis, we decided to use the MCF10A derivative cell line, MCF10CA1a. MCF10CA1a is a fully malignant cell line that gained a spontaneous PIK3CA H1047R activating mutation [34] after in vivo passaging of MCF10AT cells, which were derived by transfecting active mutant HRAS G12V into MCF10A cells [33]. After generating a stable luciferase-expressing MCF10CA1a cell line, the cell line was engineered to express our EGFR constructs with an empty vector (EV) serving as a control (S1 Fig). The growth of the tumours induced by the MCF10CA1a cells was significantly increased by the expression of EGFR-DEL and to a lesser extent by EGFR-GS and EGFR-WT expression compared to the MCF10CA1a-EV cells (Fig 3).


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)

Overexpression of wild type or mutant EGFR increases the growth of MCF10CA1a mammary fat pad xeongrafts.Female BALB/c nude mice were injected in the mammary fat pad with 5x106 cells from the indicated cell line and tumour formation was monitored with bioluminescent imaging. CA: MCF10CA1a-EV, WT: MCF10CA1a -EGFR-WT, GS: MCF10CA1a -EGFR-GS, DEL: MCF10CA1a -EGFR-DEL A. Representative bioluminescent images of individual mice taken on day 14 and day 43. B. Plot of the increase in luciferase signal in each group of mice (*p<0.05, **p<0.01, ****p<0.0001, n = 5, vs CA control, One-Way ANOVA). C. Representative bioluminescent images of individual mice taken on day 49 post injection in an independent cohort of mice. D. Plot of the magnitude of luciferase signal in each group of mice at day 49 post injection (**p<0.01, n = 5).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4430383&req=5

pone.0125232.g003: Overexpression of wild type or mutant EGFR increases the growth of MCF10CA1a mammary fat pad xeongrafts.Female BALB/c nude mice were injected in the mammary fat pad with 5x106 cells from the indicated cell line and tumour formation was monitored with bioluminescent imaging. CA: MCF10CA1a-EV, WT: MCF10CA1a -EGFR-WT, GS: MCF10CA1a -EGFR-GS, DEL: MCF10CA1a -EGFR-DEL A. Representative bioluminescent images of individual mice taken on day 14 and day 43. B. Plot of the increase in luciferase signal in each group of mice (*p<0.05, **p<0.01, ****p<0.0001, n = 5, vs CA control, One-Way ANOVA). C. Representative bioluminescent images of individual mice taken on day 49 post injection in an independent cohort of mice. D. Plot of the magnitude of luciferase signal in each group of mice at day 49 post injection (**p<0.01, n = 5).
Mentions: EGFR-DEL and EGFR-GS expression in MCF10A cells resulted in an increased proliferative ability in vitro and EGFR-DEL expression could induce spheroid formation in media lacking EGF. To determine whether these changes were reflected by increased tumorigenicity in vivo we examined the growth of mammary fat pad xenografts of the various MCF10A EGFR cell lines. The expression of the EGFR constructs in the MCF10A cells did not induce primary tumour formation up to 6 months post-injection (data not shown). As the MCF10A cell line is not malignant and the EGFR mutants were not sufficient to induce tumourigenesis, we decided to use the MCF10A derivative cell line, MCF10CA1a. MCF10CA1a is a fully malignant cell line that gained a spontaneous PIK3CA H1047R activating mutation [34] after in vivo passaging of MCF10AT cells, which were derived by transfecting active mutant HRAS G12V into MCF10A cells [33]. After generating a stable luciferase-expressing MCF10CA1a cell line, the cell line was engineered to express our EGFR constructs with an empty vector (EV) serving as a control (S1 Fig). The growth of the tumours induced by the MCF10CA1a cells was significantly increased by the expression of EGFR-DEL and to a lesser extent by EGFR-GS and EGFR-WT expression compared to the MCF10CA1a-EV cells (Fig 3).

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