ITGA6 is directly regulated by hypoxia-inducible factors and enriches for cancer stem cell activity and invasion in metastatic breast cancer modelsReportar como inadecuado

ITGA6 is directly regulated by hypoxia-inducible factors and enriches for cancer stem cell activity and invasion in metastatic breast cancer models - Descarga este documento en PDF. Documentación en PDF para descargar gratis. Disponible también para leer online.

Molecular Cancer

, 15:26

First Online: 22 March 2016Received: 10 July 2015Accepted: 11 March 2016DOI: 10.1186-s12943-016-0510-x

Cite this article as: Brooks, D.L.P., Schwab, L.P., Krutilina, R. et al. Mol Cancer 2016 15: 26. doi:10.1186-s12943-016-0510-x


BackgroundHypoxia-inducible factors HIFs are well-established mediators of tumor growth, the epithelial to mesenchymal transition EMT and metastasis. In several types of solid tumors, including breast cancers, the HIFs play a critical role in maintaining cancer stem cell CSC activity. Thus, we hypothesized that HIFs may also regulate transcription of markers of breast CSC activity. One approach to enrich for breast cells with stem-like phenotypes is FACS sorting, in which sub-populations of live cells are gated based on the expression of cell surface antigens, including various integrin subunits. Integrin alpha 6 ITGA6; CD49f is routinely used in combination with other integrin subunits to enrich for breast stem cells by FACS. Integrins not only mediate interactions with the extracellular matrix ECM, but also drive intracellular signaling events that communicate from the tumor microenvironment to inside of the tumor cell to alter phenotypes including migration and invasion.

MethodsWe used two models of metastatic breast cancer MBC, polyoma middle T MMTV-PyMT and MDA-MB-231 cells, to compare the expression of ITGA6 in wild type and knockout KO or knockdown cells. Chromatin immunoprecipitation ChIP and luciferase reporter assays verified that ITGA6 is a direct HIF transcriptional target. We also used FACS sorting to enrich for CD49f  cells to compare tumorsphere formation, tumor initiating cell activity, invasion and HIF activity relative to CD49f cells. Knockdown of ITGA6 significantly reduced invasion, whereas re-expression of ITGA6 in the context of HIF knockdown partially rescued invasion. A search of public databases also revealed that ITGA6 expression is an independent prognostic factor of survival in breast cancer patients.

ResultsWe report that ITGA6 is a HIF-dependent target gene and that high ITGA6 expression enhances invasion and tumor-initiating cell activities in models of MBC. Moreover, cells that express high levels of ITGA6 are enriched for HIF-1α expression and the expression of HIF-dependent target genes.

ConclusionsOur data suggest that HIF-dependent regulation of ITGA6 is one mechanism by which sorting for CD49f  cells enhances CSC and metastatic phenotypes in breast cancers. Our results are particularly relevant to basal-like breast cancers which express higher levels of the HIFα subunits, core HIF-dependent target genes and ITGA6 relative to other molecular subtypes.

KeywordsHypoxia Hypoxia-inducible factor HIF Breast cancer CD49f Cancer stem cells CSC Invasion Metastasis AbbreviationsCAR9carbonic anhydrase 9

ChIPchromatin immunoprecipitation

CSCcancer stem cells

ECMextracellular matrix

ELDAextreme limiting dilution analysis

EpCAMepithelial cell adhesion molecule


ERestrogen receptor

HIFhypoxia-inducible factor

HREhypoxia response element

Ints3integrator complex subunit 3

ITGA6; CD49fintegrin alpha 6


Linhematopoietic lineage


MBCmetastatic breast cancer

MMTVmouse mammary tumor virus

OSoverall survival

PHD3prolyl hydroxylase 3

PPIAcyclophilin A

PyMTpolyoma virus middle T

RFSrecurrence free survival


SFEsphere formation efficiency

TCGAThe Cancer Genome Atlas

TICtumor initiating cell

WISP2Wnt-inducible signaling protein 2

Electronic supplementary materialThe online version of this article doi:10.1186-s12943-016-0510-x contains supplementary material, which is available to authorized users.

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Autor: Danielle L. Peacock Brooks - Luciana P. Schwab - Raisa Krutilina - Deanna N. Parke - Aarti Sethuraman - David Hoogewijs


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