Dependence receptor UNC5A restricts luminal to basal breast cancer plasticity and metastasis

Maria B. Padua, Poornima Bhat-Nakshatri, Manjushree Anjanappa, Mayuri S. Prasad, Yangyang Hao, Xi Rao, Sheng Liu, Jun Wan, Yunlong Liu, Kyle McElyea, Max Jacobsen, George Sandusky, Sandra Althouse, Susan Perkins, Harikrishna Nakshatri

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

Background: The majority of estrogen receptor-positive (ERα+) breast cancers respond to endocrine therapies. However, resistance to endocrine therapies is common in 30% of cases, which may be due to altered ERα signaling and/or enhanced plasticity of cancer cells leading to breast cancer subtype conversion. The mechanisms leading to enhanced plasticity of ERα-positive cancer cells are unknown. Methods: We used short hairpin (sh)RNA and/or the CRISPR/Cas9 system to knockdown the expression of the dependence receptor UNC5A in ERα+ MCF7 and T-47D cell lines. RNA-seq, quantitative reverse transcription polymerase chain reaction, chromatin immunoprecipitation, and Western blotting were used to measure the effect of UNC5A knockdown on basal and estradiol (E2)-regulated gene expression. Mammosphere assay, flow cytometry, and immunofluorescence were used to determine the role of UNC5A in restricting plasticity. Xenograft models were used to measure the effect of UNC5A knockdown on tumor growth and metastasis. Tissue microarray and immunohistochemistry were utilized to determine the prognostic value of UNC5A in breast cancer. Log-rank test, one-way, and two-way analysis of variance (ANOVA) were used for statistical analyses. Results: Knockdown of the E2-inducible UNC5A resulted in altered basal gene expression affecting plasma membrane integrity and ERα signaling, as evident from ligand-independent activity of ERα, altered turnover of phosphorylated ERα, unique E2-dependent expression of genes effecting histone demethylase activity, enhanced upregulation of E2-inducible genes such as BCL2, and E2-independent tumorigenesis accompanied by multiorgan metastases. UNC5A depletion led to the appearance of a luminal/basal hybrid phenotype supported by elevated expression of basal/stem cell-enriched ΔNp63, CD44, CD49f, epidermal growth factor receptor (EGFR), and the lymphatic vessel permeability factor NTN4, but lower expression of luminal/alveolar differentiation-associated ELF5 while maintaining functional ERα. In addition, UNC5A-depleted cells acquired bipotent luminal progenitor characteristics based on KRT14+/KRT19+ and CD49f+/EpCAM+ phenotype. Consistent with in vitro results, UNC5A expression negatively correlated with EGFR expression in breast tumors, and lower expression of UNC5A, particularly in ERα+/PR+/HER2- tumors, was associated with poor outcome. Conclusion: These studies reveal an unexpected role of the axon guidance receptor UNC5A in fine-tuning ERα and EGFR signaling and the luminal progenitor status of hormone-sensitive breast cancers. Furthermore, UNC5A knockdown cells provide an ideal model system to investigate metastasis of ERα+ breast cancers.

Original languageEnglish (US)
Article number35
JournalBreast Cancer Research
Volume20
Issue number1
DOIs
StatePublished - May 2 2018

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Breast Neoplasms
Neoplasm Metastasis
Epidermal Growth Factor Receptor
Gene Expression
Neoplasms
Clustered Regularly Interspaced Short Palindromic Repeats
Histone Demethylases
Phenotype
Lymphatic Vessels
Chromatin Immunoprecipitation
Heterografts
Estrogen Receptors
Small Interfering RNA
Reverse Transcription
Fluorescent Antibody Technique
Estradiol
Permeability
Analysis of Variance
Flow Cytometry
Carcinogenesis

Keywords

  • Breast cancer
  • Estradiol and metastasis
  • Estrogen receptor
  • Netrin-1
  • UNC5A

ASJC Scopus subject areas

  • Oncology
  • Cancer Research

Cite this

Dependence receptor UNC5A restricts luminal to basal breast cancer plasticity and metastasis. / Padua, Maria B.; Bhat-Nakshatri, Poornima; Anjanappa, Manjushree; Prasad, Mayuri S.; Hao, Yangyang; Rao, Xi; Liu, Sheng; Wan, Jun; Liu, Yunlong; McElyea, Kyle; Jacobsen, Max; Sandusky, George; Althouse, Sandra; Perkins, Susan; Nakshatri, Harikrishna.

In: Breast Cancer Research, Vol. 20, No. 1, 35, 02.05.2018.

Research output: Contribution to journalArticle

Padua, MB, Bhat-Nakshatri, P, Anjanappa, M, Prasad, MS, Hao, Y, Rao, X, Liu, S, Wan, J, Liu, Y, McElyea, K, Jacobsen, M, Sandusky, G, Althouse, S, Perkins, S & Nakshatri, H 2018, 'Dependence receptor UNC5A restricts luminal to basal breast cancer plasticity and metastasis', Breast Cancer Research, vol. 20, no. 1, 35. https://doi.org/10.1186/s13058-018-0963-5
Padua, Maria B. ; Bhat-Nakshatri, Poornima ; Anjanappa, Manjushree ; Prasad, Mayuri S. ; Hao, Yangyang ; Rao, Xi ; Liu, Sheng ; Wan, Jun ; Liu, Yunlong ; McElyea, Kyle ; Jacobsen, Max ; Sandusky, George ; Althouse, Sandra ; Perkins, Susan ; Nakshatri, Harikrishna. / Dependence receptor UNC5A restricts luminal to basal breast cancer plasticity and metastasis. In: Breast Cancer Research. 2018 ; Vol. 20, No. 1.
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abstract = "Background: The majority of estrogen receptor-positive (ERα+) breast cancers respond to endocrine therapies. However, resistance to endocrine therapies is common in 30{\%} of cases, which may be due to altered ERα signaling and/or enhanced plasticity of cancer cells leading to breast cancer subtype conversion. The mechanisms leading to enhanced plasticity of ERα-positive cancer cells are unknown. Methods: We used short hairpin (sh)RNA and/or the CRISPR/Cas9 system to knockdown the expression of the dependence receptor UNC5A in ERα+ MCF7 and T-47D cell lines. RNA-seq, quantitative reverse transcription polymerase chain reaction, chromatin immunoprecipitation, and Western blotting were used to measure the effect of UNC5A knockdown on basal and estradiol (E2)-regulated gene expression. Mammosphere assay, flow cytometry, and immunofluorescence were used to determine the role of UNC5A in restricting plasticity. Xenograft models were used to measure the effect of UNC5A knockdown on tumor growth and metastasis. Tissue microarray and immunohistochemistry were utilized to determine the prognostic value of UNC5A in breast cancer. Log-rank test, one-way, and two-way analysis of variance (ANOVA) were used for statistical analyses. Results: Knockdown of the E2-inducible UNC5A resulted in altered basal gene expression affecting plasma membrane integrity and ERα signaling, as evident from ligand-independent activity of ERα, altered turnover of phosphorylated ERα, unique E2-dependent expression of genes effecting histone demethylase activity, enhanced upregulation of E2-inducible genes such as BCL2, and E2-independent tumorigenesis accompanied by multiorgan metastases. UNC5A depletion led to the appearance of a luminal/basal hybrid phenotype supported by elevated expression of basal/stem cell-enriched ΔNp63, CD44, CD49f, epidermal growth factor receptor (EGFR), and the lymphatic vessel permeability factor NTN4, but lower expression of luminal/alveolar differentiation-associated ELF5 while maintaining functional ERα. In addition, UNC5A-depleted cells acquired bipotent luminal progenitor characteristics based on KRT14+/KRT19+ and CD49f+/EpCAM+ phenotype. Consistent with in vitro results, UNC5A expression negatively correlated with EGFR expression in breast tumors, and lower expression of UNC5A, particularly in ERα+/PR+/HER2- tumors, was associated with poor outcome. Conclusion: These studies reveal an unexpected role of the axon guidance receptor UNC5A in fine-tuning ERα and EGFR signaling and the luminal progenitor status of hormone-sensitive breast cancers. Furthermore, UNC5A knockdown cells provide an ideal model system to investigate metastasis of ERα+ breast cancers.",
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TY - JOUR

T1 - Dependence receptor UNC5A restricts luminal to basal breast cancer plasticity and metastasis

AU - Padua, Maria B.

AU - Bhat-Nakshatri, Poornima

AU - Anjanappa, Manjushree

AU - Prasad, Mayuri S.

AU - Hao, Yangyang

AU - Rao, Xi

AU - Liu, Sheng

AU - Wan, Jun

AU - Liu, Yunlong

AU - McElyea, Kyle

AU - Jacobsen, Max

AU - Sandusky, George

AU - Althouse, Sandra

AU - Perkins, Susan

AU - Nakshatri, Harikrishna

PY - 2018/5/2

Y1 - 2018/5/2

N2 - Background: The majority of estrogen receptor-positive (ERα+) breast cancers respond to endocrine therapies. However, resistance to endocrine therapies is common in 30% of cases, which may be due to altered ERα signaling and/or enhanced plasticity of cancer cells leading to breast cancer subtype conversion. The mechanisms leading to enhanced plasticity of ERα-positive cancer cells are unknown. Methods: We used short hairpin (sh)RNA and/or the CRISPR/Cas9 system to knockdown the expression of the dependence receptor UNC5A in ERα+ MCF7 and T-47D cell lines. RNA-seq, quantitative reverse transcription polymerase chain reaction, chromatin immunoprecipitation, and Western blotting were used to measure the effect of UNC5A knockdown on basal and estradiol (E2)-regulated gene expression. Mammosphere assay, flow cytometry, and immunofluorescence were used to determine the role of UNC5A in restricting plasticity. Xenograft models were used to measure the effect of UNC5A knockdown on tumor growth and metastasis. Tissue microarray and immunohistochemistry were utilized to determine the prognostic value of UNC5A in breast cancer. Log-rank test, one-way, and two-way analysis of variance (ANOVA) were used for statistical analyses. Results: Knockdown of the E2-inducible UNC5A resulted in altered basal gene expression affecting plasma membrane integrity and ERα signaling, as evident from ligand-independent activity of ERα, altered turnover of phosphorylated ERα, unique E2-dependent expression of genes effecting histone demethylase activity, enhanced upregulation of E2-inducible genes such as BCL2, and E2-independent tumorigenesis accompanied by multiorgan metastases. UNC5A depletion led to the appearance of a luminal/basal hybrid phenotype supported by elevated expression of basal/stem cell-enriched ΔNp63, CD44, CD49f, epidermal growth factor receptor (EGFR), and the lymphatic vessel permeability factor NTN4, but lower expression of luminal/alveolar differentiation-associated ELF5 while maintaining functional ERα. In addition, UNC5A-depleted cells acquired bipotent luminal progenitor characteristics based on KRT14+/KRT19+ and CD49f+/EpCAM+ phenotype. Consistent with in vitro results, UNC5A expression negatively correlated with EGFR expression in breast tumors, and lower expression of UNC5A, particularly in ERα+/PR+/HER2- tumors, was associated with poor outcome. Conclusion: These studies reveal an unexpected role of the axon guidance receptor UNC5A in fine-tuning ERα and EGFR signaling and the luminal progenitor status of hormone-sensitive breast cancers. Furthermore, UNC5A knockdown cells provide an ideal model system to investigate metastasis of ERα+ breast cancers.

AB - Background: The majority of estrogen receptor-positive (ERα+) breast cancers respond to endocrine therapies. However, resistance to endocrine therapies is common in 30% of cases, which may be due to altered ERα signaling and/or enhanced plasticity of cancer cells leading to breast cancer subtype conversion. The mechanisms leading to enhanced plasticity of ERα-positive cancer cells are unknown. Methods: We used short hairpin (sh)RNA and/or the CRISPR/Cas9 system to knockdown the expression of the dependence receptor UNC5A in ERα+ MCF7 and T-47D cell lines. RNA-seq, quantitative reverse transcription polymerase chain reaction, chromatin immunoprecipitation, and Western blotting were used to measure the effect of UNC5A knockdown on basal and estradiol (E2)-regulated gene expression. Mammosphere assay, flow cytometry, and immunofluorescence were used to determine the role of UNC5A in restricting plasticity. Xenograft models were used to measure the effect of UNC5A knockdown on tumor growth and metastasis. Tissue microarray and immunohistochemistry were utilized to determine the prognostic value of UNC5A in breast cancer. Log-rank test, one-way, and two-way analysis of variance (ANOVA) were used for statistical analyses. Results: Knockdown of the E2-inducible UNC5A resulted in altered basal gene expression affecting plasma membrane integrity and ERα signaling, as evident from ligand-independent activity of ERα, altered turnover of phosphorylated ERα, unique E2-dependent expression of genes effecting histone demethylase activity, enhanced upregulation of E2-inducible genes such as BCL2, and E2-independent tumorigenesis accompanied by multiorgan metastases. UNC5A depletion led to the appearance of a luminal/basal hybrid phenotype supported by elevated expression of basal/stem cell-enriched ΔNp63, CD44, CD49f, epidermal growth factor receptor (EGFR), and the lymphatic vessel permeability factor NTN4, but lower expression of luminal/alveolar differentiation-associated ELF5 while maintaining functional ERα. In addition, UNC5A-depleted cells acquired bipotent luminal progenitor characteristics based on KRT14+/KRT19+ and CD49f+/EpCAM+ phenotype. Consistent with in vitro results, UNC5A expression negatively correlated with EGFR expression in breast tumors, and lower expression of UNC5A, particularly in ERα+/PR+/HER2- tumors, was associated with poor outcome. Conclusion: These studies reveal an unexpected role of the axon guidance receptor UNC5A in fine-tuning ERα and EGFR signaling and the luminal progenitor status of hormone-sensitive breast cancers. Furthermore, UNC5A knockdown cells provide an ideal model system to investigate metastasis of ERα+ breast cancers.

KW - Breast cancer

KW - Estradiol and metastasis

KW - Estrogen receptor

KW - Netrin-1

KW - UNC5A

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