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.2016 Mar 22:15:26.
doi: 10.1186/s12943-016-0510-x.

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

Affiliations

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

Danielle L Peacock Brooks et al. Mol Cancer..

Abstract

Background: Hypoxia-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.

Methods: We 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(neg or low) 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.

Results: We 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.

Conclusions: Our 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.

Keywords: Breast cancer; CD49f; Cancer stem cells (CSC); Hypoxia; Hypoxia-inducible factor (HIF); Invasion; Metastasis.

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Figures

Fig. 1
Fig. 1
Itga6 expression is down-regulated in response to deletion of HIF-1 in PyMT+ cultured cells and in tumors.a HIF-1 WT or KO cells were cultured at normoxia or hypoxia (6 h, 0.5 % O2) and the relative expression ofItga6 mRNA levels compared after normalization toInts3. The mean fold-change inItga6 levels was compared to KO cells cultured at hypoxia (set to 1.0) (*p < 0.01, ANOVA with Bonferroni post-test;n = 3 biological replicates).b Western blotting was performed for ITGA6 protein using whole cell extracts (WCE) prepared from near confluent HIF-1 WT and KO cells cultured at either normoxia (N) or hypoxia (H). Tata-binding protein (TBP) is shown as a loading control.c HIF-1 WT and KO adherent cells were exposed to normoxia or hypoxia (6 h, 0.5 % O2), fixed, immunostained with CD49f-FITC and counterstained with DAPI; the scale bar represents 20 μm.d The relative abundance ofItga6 mRNA after normalization toInts3 in the same set of HIF-1α WT and KO tumors as subjected to FACS analysis in panel (e) (p < 0.01, Student’st-test).e Representative histograms derived from FACS analysis of live, CD49-FITC stained cells isolated from HIF-1 WT (black histogram) and KO (blue histogram) tumors relative to the isotype antibody control (red histogram). To normalize the histogram height between samples, the y-axis shows the % Max (the number of cells in each bin divided by the number of cells in the bin that contains the largest number of cells). Data shown are representative of n ≥5 tumors/genotype. The percentage of tumor cells positive for CD49f-FITC was determined based on the live, singlet, Linneg parent population using FlowJo and the gating strategy presented in Additional file 4: Figure S4.f The mean ± SEM in the fold change of CD49f-FITC median fluorescence intensity (MFI) between WT and KO PyMT tumors; all data are expressed relative to each genotype’s corresponding isotype control MFI (p < 0.01, Student’st-test; n ≥5 tumors/genotype)
Fig. 2
Fig. 2
ITGA6/CD49f expression is reduced in response to loss of HIF activity in MDA-MB-231 cells and tumors.a Mean ± SEM of the expression ofITGA6 mRNA relative toPPIA, in adherent, cultured MDA-MB-231 cells (n = 3 independent biological experiments/genotype). mRNA levels were evaluated after 0, 6 or 24 h of hypoxic exposure. All data are expressed relative to shControl cells cultured at normoxia (set to 1.0); *p-value <0.05 by ANOVA.b Western blotting was performed for ITGA6 protein using whole cell extracts (WCE) prepared from near confluent MDA-MB-231 shControl or shHIF1A/shHIF2A cells cultured at either normoxia (N) or chronic hypoxia (H). Tata-binding protein (TBP) is shown as a loading control.c MDA-MB-231 shControl, shHIF1A, shHIF2A and shHIF1A/shHIF2A cells were harvested from normoxic cell culture at 90 % confluence, stained with CD49f-FITC in suspension, replated onto chamber well slides and stained with Hoechst 33342; scale bar represents 20 μM.d MDA-MB-231 shControl, shHIF1A, shHIF2A and shHIF1A/shHIF2A cells were harvested from normoxic or chronic hypoxic cell culture at 90 % confluence, stained with CD49f-FITC in suspension in flow buffer, and subjected to FACS analysis. Plots represent the % Max of stained cells (red histogram) versus the corresponding genotype’s isotype control (blue histogram); data presented are representative of 4 biological replicate experiments.e The average ± SEM in the fold change (FC) in the CD49f MFI among all four genotypes of cultured cells (n = 4 independent experiments/genotype; shControl set to 1.0;p<0.05.).f The average ± SEM in the fold change (FC) ofITGA6 mRNA levels expressed in tumors among all four genotypes (n = 4 tumors/genotype; shControl set to 1.0;p <0.05.); total RNA was prepared from homogenized whole tumors
Fig. 3
Fig. 3
ITGA6 is a direct HIF transcriptional target gene.a A schematic representation of putative HREs identified in the proximal promoter ofITGA6 that were assessed for HIF-1α and/or HIF-2α recruitment by ChIP assays.b MDA-MB-231 shControl and shHIF1A cells were cultured at hypoxia (0.5 % O2) for 6 h, and chromatin fragments were immunoprecipitated using HIF-1α or HIF-2α antibodies or anti-rabbit IgG (as the non-specific binding control). SYBR Green-based qRT-PCR was conducted on the purified, isolated DNA fragments to determine the site fold enrichment of HIFα recruitment relative to signal detected in the anti-rabbit IgG control per genotype (qRT-PCR values observed for the IgG control were set to 1.0 per genotype). As the positive control, qRT-PCR was also performed using primers flanking a previously validated, functional HRE site in the 3’EPO enhancer. Each panel shows the mean site fold enrichment ± SEM of technical replicates; data presented are representative of three replicate experiments.c Luciferase reporter assays were used to compare relative luciferase activity between MDA-MB-231 shControl or shHIF1A/shHIF2A cells transiently transfected with a wild type ITGA6 promoter linked to luciferase (ITGA6-Luc; white bars) or a HRE mutant promoter construct [ITGA6 (mutant)-Luc; grey bars] and then cultured at normoxia (Nor) or hypoxia (Hyp). In some cases, a stabilized version of murine HIF-1α was also co-transfected (+HIF1A). The mean ± standard deviation are shown;p <0.05 by one-way ANOVA followed by Bonferroni's multiple comparison test. The mutantITGA6 promoter contains three point mutations within the HIF consensus site
Fig. 4
Fig. 4
CD49f enriches for TIC activity in vitro and in vivo.a Tumors harvested from MMTV-PyMT+ females were harvest, digested, and stained with CD49f-FITC and CD24-PE antibodies prior to FACS sorting. CD49f+/CD24High and CD49fNeg/CD24Low sorted cell populations were plated in sphere culture conditions to assay for sphere formation efficiency (SFE), or were transplanted by limiting dilution transplantation into female FVB/Nj recipients.a Each sorted cell population was plated at a density of 30,000 cells/well in 6-well format and the grand mean SFE ± SEM determined (n ≥ 8 wells per genotype;n = 3 independent experiments). Thep-value was calculated by an unpaired Student’st-test.b Comparison of changes in mean tumor volume over time when 50, 100 or 200 CD49f+/CD24High or CD49fNeg/CD24Low sorted cells are transplanted into the cleared inguinal mammary fat pad to assay for TIC potential. The number of tumors that formed for each cohort is indicated in Table 1.c CD49f/CD24 expression was analyzed by FACS analysis of end-stage tumors derived from the transplantation of CD49f+/CD24High or CD49fNeg/CD24Low sorted cells; data are representative ofn ≥ 4 tumors/transplanted population.d Lung metastasis was evaluated in mice bearing tumors originating from CD49f+/CD24High or CD49fNeg/CD24Low cell populations. A subset of mice in the 100-cell and 200-cell inputs were housed until tumors grew to a volume of ~500 mm3 at which time the tumors and lungs were harvested. The mean total number of lung metastases ± SEM present in H&E-stained paraffin sections is shown in the box-and-whisker plot; *p <0.05 by Student’st-test; N.S. equals not significant
Fig. 5
Fig. 5
Manipulation of ITGA6 levels directly impacts invasion.a Non-sorted, cultured shControl or shHIF1A/shHIF2A MDA-MB-231 cells were evaluated for invasive potential using Boyden chamber assays. The grand mean of the invasion index (invasion corrected for random migration) ± SEM of 3 independent experiments is shown;p-values calculated by Student’st-test are indicated.b Knockdown ofITGA6 impairs invasion at normoxia and hypoxia. The grand mean of the invasion index (invasion corrected for random migration) ± SEM of 3 independent experiments is shown;p-values were calculated by Student’st-test.c Ectopic expression ofITGA6 in shHIF1A/shHIF2A cells partially rescues invasion at hypoxia. The grand mean of the invasion index (invasion corrected for random migration) ± SEM of 3 independent experiments is shown;p-values were calculated by Student’st-test.
Fig. 6
Fig. 6
Enriching for CD49f by cell sorting enhances cell invasion in MDA-MB-231 cells.a Mock-sorted, CD49fHigh or CD49fLow MDA-MB-231 cell populations were exposed for 24–48 h to hypoxia and invasion then compared (n = 3-4 replicate wells/population/experiment; data are representative of 3 independent sort experiments);p-values calculated by Student’st-test are shown. The grand mean invasion index ± SEM is expressed relative to Mock-sorted cells (cells stained with CD49f-FITC but not gated during sort; set to 1.0).b IF staining for CD49f-FITC and HIF-1α in cytospun CD49fHigh, CD49fLow or Mock sorted cells counterstained with DAPI; scale bar = 50 μm.c qRT-PCR analysis to compare expression levels of known HIF-dependent target genes in the CD49fHigh vs. CD49fLow cell populations; all values were first normalized for loading and were then normalized to Mock sorted cells (white bars; fold change, FC set to 1.0). The mean ± SEM is reported for three independent experiments
Fig. 7
Fig. 7
ITGA6 expression levels are prognostic of overall or recurrence-free survival of breast cancer patients.aITGA6 mRNA is differentially expressed in human breast cancers classified by the PAM50 subtype in the TCGA data set [30]; ***p < 0.001, ANOVA with Bonferroni post-test; (n = 93 basal, 56 HER2+, 228 Luminal A, 123 Luminal B and 7 normal-like tumors).bITGA6 mRNA levels also weakly positively correlate withHIF1A levels (p <0.05, correlation coefficientr2 = 0.0078; TCGA data), but not withHIF2A mRNA levels (p = 0.08).c-d Kaplan-Meier curves depict the probability of overall survival (c) or recurrence-free survival (d) based on relativeITGA6 mRNA expression from clinical data reported in [42].ITGA6 expression levels were stratified by the upper quartile or lowest quartiles (n = 37 tumors for “High” CD49f expression, andn = 45 tumors for “Low CD49f”) and significance determined by the log-rank test, *p < 0.05)
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