Journal Article
. 2018 May; 20(1):35.
doi: 10.1186/s13058-018-0963-5.

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

Maria B Padua 1 Poornima Bhat-Nakshatri 1 Manjushree Anjanappa 1 Mayuri S Prasad 1 Yangyang Hao 2 Xi Rao 2 Sheng Liu 2 Jun Wan 2 Yunlong Liu 2 Kyle McElyea 3 Max Jacobsen 3 George Sandusky 3 Sandra Althouse 4 Susan Perkins 4 Harikrishna Nakshatri 5 
  • PMID: 29720215
  •     60 References
  •     4 citations


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 ∆Np63, 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.

Keywords: Breast cancer; Estradiol and metastasis; Estrogen receptor; Netrin-1; UNC5A.

Netrin-1 expression confers a selective advantage for tumor cell survival in metastatic breast cancer.
Julien Fitamant, Céline Guenebeaud, +6 authors, Patrick Mehlen.
Proc Natl Acad Sci U S A, 2008 Mar 21; 105(12). PMID: 18353983    Free PMC article.
Growth factor stimulation induces a distinct ER(alpha) cistrome underlying breast cancer endocrine resistance.
Mathieu Lupien, Clifford A Meyer, +8 authors, Myles Brown.
Genes Dev, 2010 Oct 05; 24(19). PMID: 20889718    Free PMC article.
Role of PRC2-associated factors in stem cells and disease.
Pedro Vizán, Malte Beringer, Cecilia Ballaré, Luciano Di Croce.
FEBS J, 2014 Oct 02; 282(9). PMID: 25271128
edgeR: a Bioconductor package for differential expression analysis of digital gene expression data.
Mark D Robinson, Davis J McCarthy, Gordon K Smyth.
Bioinformatics, 2009 Nov 17; 26(1). PMID: 19910308    Free PMC article.
Highly Cited.
Transcriptional activation of integrin beta6 during the epithelial-mesenchymal transition defines a novel prognostic indicator of aggressive colon carcinoma.
Richard C Bates, David I Bellovin, +6 authors, Arthur M Mercurio.
J Clin Invest, 2005 Jan 26; 115(2). PMID: 15668738    Free PMC article.
Highly Cited.
Cross-Stratification and Differential Risk by Breast Cancer Index and Recurrence Score in Women with Hormone Receptor-Positive Lymph Node-Negative Early-Stage Breast Cancer.
Ivana Sestak, Yi Zhang, +4 authors, Dennis Sgroi.
Clin Cancer Res, 2016 Jun 03; 22(20). PMID: 27252417
The netrin-1 receptors UNC5H are putative tumor suppressors controlling cell death commitment.
Karine Thiebault, Laetitia Mazelin, +6 authors, Patrick Mehlen.
Proc Natl Acad Sci U S A, 2003 Mar 26; 100(7). PMID: 12655055    Free PMC article.
Positive cross-regulatory loop ties GATA-3 to estrogen receptor alpha expression in breast cancer.
Jérôme Eeckhoute, Erika Krasnickas Keeton, +3 authors, Myles Brown.
Cancer Res, 2007 Jul 10; 67(13). PMID: 17616709
Highly Cited.
Evidence for a stem cell hierarchy in the adult human breast.
René Villadsen, Agla J Fridriksdottir, +5 authors, Ole W Petersen.
J Cell Biol, 2007 Apr 11; 177(1). PMID: 17420292    Free PMC article.
Highly Cited.
Luminal B breast cancer: molecular characterization, clinical management, and future perspectives.
Felipe Ades, Dimitrios Zardavas, +6 authors, Martine Piccart.
J Clin Oncol, 2014 Jul 23; 32(25). PMID: 25049332
Highly Cited. Review.
STAR: ultrafast universal RNA-seq aligner.
Alexander Dobin, Carrie A Davis, +6 authors, Thomas R Gingeras.
Bioinformatics, 2012 Oct 30; 29(1). PMID: 23104886    Free PMC article.
Highly Cited.
Estrogen-independent proliferation is present in estrogen-receptor HER2-positive primary breast cancer after neoadjuvant letrozole.
Matthew J Ellis, Yu Tao, +11 authors, J Michael Dixon.
J Clin Oncol, 2006 Jun 07; 24(19). PMID: 16754938
FOXA1 expression in breast cancer--correlation with luminal subtype A and survival.
Sunil Badve, Dmitry Turbin, +6 authors, Harikrishna Nakshatri.
Clin Cancer Res, 2007 Aug 03; 13(15 Pt 1). PMID: 17671124
The sox family of transcription factors: versatile regulators of stem and progenitor cell fate.
Abby Sarkar, Konrad Hochedlinger.
Cell Stem Cell, 2013 Jan 08; 12(1). PMID: 23290134    Free PMC article.
Highly Cited. Review.
H3K27 demethylation by JMJD3 at a poised enhancer of anti-apoptotic gene BCL2 determines ERα ligand dependency.
Amy Svotelis, Stéphanie Bianco, +4 authors, Nicolas Gévry.
EMBO J, 2011 Aug 16; 30(19). PMID: 21841772    Free PMC article.
Netrins: beyond the brain.
Vincenzo Cirulli, Mayra Yebra.
Nat Rev Mol Cell Biol, 2007 Mar 16; 8(4). PMID: 17356579
Highly Cited. Review.
AKT alters genome-wide estrogen receptor alpha binding and impacts estrogen signaling in breast cancer.
Poornima Bhat-Nakshatri, Guohua Wang, +7 authors, Harikrishna Nakshatri.
Mol Cell Biol, 2008 Oct 08; 28(24). PMID: 18838536    Free PMC article.
Identification of UNC5A as a novel transcriptional target of tumor suppressor p53 and a regulator of apoptosis.
Yuji Miyamoto, Manabu Futamura, +3 authors, Hiroufmi Arakawa.
Int J Oncol, 2010 Apr 08; 36(5). PMID: 20372800
Ethnicity-Dependent and -Independent Heterogeneity in Healthy Normal Breast Hierarchy Impacts Tumor Characterization.
Harikrishna Nakshatri, Manjushree Anjanappa, Poornima Bhat-Nakshatri.
Sci Rep, 2015 Aug 28; 5. PMID: 26311223    Free PMC article.
Intrinsic Subtypes and Gene Expression Profiles in Primary and Metastatic Breast Cancer.
Juan M Cejalvo, Eduardo Martínez de Dueñas, +24 authors, Aleix Prat.
Cancer Res, 2017 Mar 03; 77(9). PMID: 28249905    Free PMC article.
Highly Cited.
Molecular insights on basal-like breast cancer.
Mev Dominguez Valentin, Sabrina Daniela da Silva, +2 authors, Yves-Jean Bignon.
Breast Cancer Res Treat, 2012 Jan 12; 134(1). PMID: 22234518
Dependence receptors: from basic research to drug development.
Patrick Mehlen, Dale E Bredesen.
Sci Signal, 2011 Jan 27; 4(157). PMID: 21266712
The sesquiterpene lactone parthenolide in combination with docetaxel reduces metastasis and improves survival in a xenograft model of breast cancer.
Christopher J Sweeney, Sanjana Mehrotra, +8 authors, Harikrishna Nakshatri.
Mol Cancer Ther, 2005 Jun 16; 4(6). PMID: 15956258
Epidermal Growth Factor Receptor-Dependent Mutual Amplification between Netrin-1 and the Hepatitis C Virus.
Marie-Laure Plissonnier, Thomas Lahlali, +14 authors, Romain Parent.
PLoS Biol, 2016 Apr 01; 14(3). PMID: 27031829    Free PMC article.
Writing, erasing and reading histone lysine methylations.
Kwangbeom Hyun, Jongcheol Jeon, Kihyun Park, Jaehoon Kim.
Exp Mol Med, 2017 Apr 30; 49(4). PMID: 28450737    Free PMC article.
Highly Cited. Review.
SUMO1 negatively regulates the transcriptional activity of EVI1 and significantly increases its co-localization with EVI1 after treatment with arsenic trioxide.
Sneha Singh, Anjan Kumar Pradhan, Soumen Chakraborty.
Biochim Biophys Acta, 2013 Jun 19; 1833(10). PMID: 23770046
Netrin-1 and its receptors in tumorigenesis.
Hirofumi Arakawa.
Nat Rev Cancer, 2004 Dec 02; 4(12). PMID: 15573119
Mammary stem cells and the differentiation hierarchy: current status and perspectives.
Jane E Visvader, John Stingl.
Genes Dev, 2014 Jun 04; 28(11). PMID: 24888586    Free PMC article.
Highly Cited. Review.
Dual DNA methylation patterns in the CNS reveal developmentally poised chromatin and monoallelic expression of critical genes.
Jinhui Wang, Zuzana Valo, +3 authors, Judith Singer-Sam.
PLoS One, 2010 Nov 17; 5(11). PMID: 21079792    Free PMC article.
Biological and prognostic significance of stratified epithelial cytokeratins in infiltrating ductal breast carcinomas.
K Malzahn, M Mitze, M Thoenes, R Moll.
Virchows Arch, 1998 Sep 16; 433(2). PMID: 9737789
Differential expression analysis of multifactor RNA-Seq experiments with respect to biological variation.
Davis J McCarthy, Yunshun Chen, Gordon K Smyth.
Nucleic Acids Res, 2012 Jan 31; 40(10). PMID: 22287627    Free PMC article.
Highly Cited.
Maintaining epithelial stemness with p63.
Gerry Melino, Elisa Maria Memmi, Pier Giuseppe Pelicci, Francesca Bernassola.
Sci Signal, 2015 Jul 30; 8(387). PMID: 26221054
Highly Cited. Review.
ELF5 suppresses estrogen sensitivity and underpins the acquisition of antiestrogen resistance in luminal breast cancer.
Maria Kalyuga, David Gallego-Ortega, +27 authors, Christopher J Ormandy.
PLoS Biol, 2013 Jan 10; 10(12). PMID: 23300383    Free PMC article.
A gene expression signature identifying transient DNMT1 depletion as a causal factor of cancer-germline gene activation in melanoma.
Julie Cannuyer, Aurélie Van Tongelen, Axelle Loriot, Charles De Smet.
Clin Epigenetics, 2015 Oct 28; 7. PMID: 26504497    Free PMC article.
Phosphatidylinositol 3-kinase/AKT-mediated activation of estrogen receptor alpha: a new model for anti-estrogen resistance.
R A Campbell, P Bhat-Nakshatri, +3 authors, H Nakshatri.
J Biol Chem, 2001 Jan 15; 276(13). PMID: 11139588
Highly Cited.
Prognosis of hormone-dependent breast cancers: implications of the presence of dysfunctional transcriptional networks activated by insulin via the immune transcription factor T-bet.
Kasi McCune, Poornima Bhat-Nakshatri, +3 authors, Harikrishna Nakshatri.
Cancer Res, 2010 Jan 14; 70(2). PMID: 20068169    Free PMC article.
TFAP2C expression in breast cancer: correlation with overall survival beyond 10 years of initial diagnosis.
Susan M Perkins, Casey Bales, +5 authors, Harikrishna Nakshatri.
Breast Cancer Res Treat, 2015 Jul 15; 152(3). PMID: 26160249
Molecular Insights of Pathways Resulting from Two Common PIK3CA Mutations in Breast Cancer.
Poornima Bhat-Nakshatri, Chirayu P Goswami, +3 authors, Harikrishna Nakshatri.
Cancer Res, 2016 May 20; 76(13). PMID: 27197157
The cBio cancer genomics portal: an open platform for exploring multidimensional cancer genomics data.
Ethan Cerami, Jianjiong Gao, +12 authors, Nikolaus Schultz.
Cancer Discov, 2012 May 17; 2(5). PMID: 22588877    Free PMC article.
Highly Cited.
A new method for stranded whole transcriptome RNA-seq.
David F B Miller, Pearlly S Yan, +17 authors, Kenneth P Nephew.
Methods, 2013 Apr 06; 63(2). PMID: 23557989    Free PMC article.
featureCounts: an efficient general purpose program for assigning sequence reads to genomic features.
Yang Liao, Gordon K Smyth, Wei Shi.
Bioinformatics, 2013 Nov 15; 30(7). PMID: 24227677
Highly Cited.
KDM4B is a master regulator of the estrogen receptor signalling cascade.
Luke Gaughan, Jacqueline Stockley, +8 authors, Craig N Robson.
Nucleic Acids Res, 2013 Jun 01; 41(14). PMID: 23723241    Free PMC article.
Prospective identification of tumorigenic breast cancer cells.
Muhammad Al-Hajj, Max S Wicha, +2 authors, Michael F Clarke.
Proc Natl Acad Sci U S A, 2003 Mar 12; 100(7). PMID: 12629218    Free PMC article.
Highly Cited.
ChIP-Seq of ERalpha and RNA polymerase II defines genes differentially responding to ligands.
Willem-Jan Welboren, Marc A van Driel, +4 authors, Hendrik G Stunnenberg.
EMBO J, 2009 Apr 03; 28(10). PMID: 19339991    Free PMC article.
Highly Cited.
Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources.
Da Wei Huang, Brad T Sherman, Richard A Lempicki.
Nat Protoc, 2009 Jan 10; 4(1). PMID: 19131956
Highly Cited.
Endocrine-responsive breast cancer and strategies for combating resistance.
Simak Ali, R Charles Coombes.
Nat Rev Cancer, 2003 Mar 15; 2(2). PMID: 12635173
Highly Cited. Review.
Pioneer transcription factors: establishing competence for gene expression.
Kenneth S Zaret, Jason S Carroll.
Genes Dev, 2011 Nov 08; 25(21). PMID: 22056668    Free PMC article.
Highly Cited. Review.
UALCAN: A Portal for Facilitating Tumor Subgroup Gene Expression and Survival Analyses.
Darshan S Chandrashekar, Bhuwan Bashel, +4 authors, Sooryanarayana Varambally.
Neoplasia, 2017 Jul 22; 19(8). PMID: 28732212    Free PMC article.
Highly Cited.
Mechanisms of endocrine resistance in breast cancer.
C Kent Osborne, Rachel Schiff.
Annu Rev Med, 2010 Oct 05; 62. PMID: 20887199    Free PMC article.
Highly Cited. Review.
Stemness of the hybrid Epithelial/Mesenchymal State in Breast Cancer and Its Association with Poor Survival.
Anne Grosse-Wilde, Aymeric Fouquier d'Hérouël, +6 authors, Sui Huang.
PLoS One, 2015 May 29; 10(5). PMID: 26020648    Free PMC article.
Highly Cited.
Notch-Jagged signalling can give rise to clusters of cells exhibiting a hybrid epithelial/mesenchymal phenotype.
Marcelo Boareto, Mohit Kumar Jolly, +6 authors, Jose' N Onuchic.
J R Soc Interface, 2016 May 14; 13(118). PMID: 27170649    Free PMC article.
Netrin-4 induces lymphangiogenesis in vivo.
Frederic Larrieu-Lahargue, Alana L Welm, Kirk R Thomas, Dean Y Li.
Blood, 2010 Apr 22; 115(26). PMID: 20407033    Free PMC article.
Interleukin-1 alpha promotes tumor growth and cachexia in MCF-7 xenograft model of breast cancer.
Suresh Kumar, Hiromitsu Kishimoto, +4 authors, Harikrishna Nakshatri.
Am J Pathol, 2003 Nov 25; 163(6). PMID: 14633625    Free PMC article.
APOBEC3B-Mediated Cytidine Deamination Is Required for Estrogen Receptor Action in Breast Cancer.
Manikandan Periyasamy, Hetal Patel, +17 authors, Simak Ali.
Cell Rep, 2015 Sep 29; 13(1). PMID: 26411678    Free PMC article.
Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications.
T Sørlie, C M Perou, +14 authors, A L Børresen-Dale.
Proc Natl Acad Sci U S A, 2001 Sep 13; 98(19). PMID: 11553815    Free PMC article.
Highly Cited.
Progesterone receptor-cyclin D1 complexes induce cell cycle-dependent transcriptional programs in breast cancer cells.
Gwen E Dressing, Todd P Knutson, +5 authors, Carol A Lange.
Mol Endocrinol, 2014 Mar 13; 28(4). PMID: 24606123    Free PMC article.
Novel roles for Slits and netrins: axon guidance cues as anticancer targets?
Patrick Mehlen, Céline Delloye-Bourgeois, Alain Chédotal.
Nat Rev Cancer, 2011 Feb 18; 11(3). PMID: 21326323
Highly Cited. Review.
Identification of FDA-approved drugs targeting breast cancer stem cells along with biomarkers of sensitivity.
Poornima Bhat-Nakshatri, Chirayu P Goswami, +2 authors, Harikrishna Nakshatri.
Sci Rep, 2013 Aug 29; 3. PMID: 23982413    Free PMC article.
The DAVID Gene Functional Classification Tool: a novel biological module-centric algorithm to functionally analyze large gene lists.
Da Wei Huang, Brad T Sherman, +7 authors, Richard A Lempicki.
Genome Biol, 2007 Sep 06; 8(9). PMID: 17784955    Free PMC article.
Highly Cited.
Mechanisms of aromatase inhibitor resistance.
Cynthia X Ma, Tomás Reinert, Izabela Chmielewska, Matthew J Ellis.
Nat Rev Cancer, 2015 Apr 25; 15(5). PMID: 25907219
Highly Cited. Review.
Plasticity of Cancer Stem Cell: Origin and Role in Disease Progression and Therapy Resistance.
Plabon Kumar Das, Suja Pillai, +4 authors, Farhadul Islam.
Stem Cell Rev Rep, 2020 Jan 23; 16(2). PMID: 31965409
The Central Contributions of Breast Cancer Stem Cells in Developing Resistance to Endocrine Therapy in Estrogen Receptor (ER)-Positive Breast Cancer.
David Rodriguez, Marc Ramkairsingh, +3 authors, Damu Tang.
Cancers (Basel), 2019 Jul 25; 11(7). PMID: 31336602    Free PMC article.
CRISPR/Cas9: A powerful genome editing technique for the treatment of cancer cells with present challenges and future directions.
Abu Hazafa, Muhammad Mumtaz, +8 authors, Fatima Zafar.
Life Sci, 2020 Oct 09; 263. PMID: 33031826    Free PMC article.
UNC5A, an epigenetically silenced gene, functions as a tumor suppressor in non-small cell lung cancer.
Silu Ding, Hongwei Zhang, +2 authors, Guang Li.
Saudi J Biol Sci, 2020 Oct 27; 27(11). PMID: 33100860    Free PMC article.