The online version of this article (doi:10.1186/s12943-017-0610-2) contains supplementary material, which is available to authorized users.
Extracellular matrix (ECM) is known to maintain epithelial integrity. In carcinogenesis ECM degradation triggers metastasis by controlling migration and differentiation including cancer stem cell (CSC) characteristics. The ECM-modulator inter- α-trypsin inhibitor heavy chain family member five (ITIH5) was recently identified as tumor suppressor potentially involved in impairing breast cancer progression but molecular mechanisms underlying its function are still elusive.
ITIH5 expression was analyzed using the public TCGA portal. ITIH5-overexpressing single-cell clones were established based on T47D and MDA-MB-231 cell lines. Colony formation, growth, apoptosis, migration, matrix adhesion, traction force analyses and polarization of tumor cells were studied in vitro. Tumor-initiating characteristics were analyzed by generating a metastasis mouse model. To identify ITIH5-affected pathways we utilized genome wide gene expression and DNA methylation profiles. RNA-interference targeting the ITIH5-downstream regulated gene DAPK1 was used to confirm functional involvement.
ITIH5 loss was pronounced in breast cancer subtypes with unfavorable prognosis like basal-type tumors. Functionally, cell and colony formation was impaired after ITIH5 re-expression in both cell lines. In a metastasis mouse model, ITIH5 expressing MDA-MB-231 cells almost completely failed to initiate lung metastases. In these metastatic cells ITIH5 modulated cell-matrix adhesion dynamics and altered biomechanical cues. The profile of integrin receptors was shifted towards β1-integrin accompanied by decreased Rac1 and increased RhoA activity in ITIH5-expressing clones while cell polarization and single-cell migration was impaired. Instead ITIH5 expression triggered the formation of epithelial-like cell clusters that underwent an epigenetic reprogramming. 214 promoter regions potentially marked with either H3K4 and /or H3K27 methylation showed a hyper- or hypomethylated DNA configuration due to ITIH5 expression finally leading to re-expression of the tumor suppressor DAPK1. In turn, RNAi-mediated knockdown of DAPK1 in ITIH5-expressing MDA-MB-231 single-cell clones clearly restored cell motility.
Our results provide evidence that ITIH5 triggers a reprogramming of breast cancer cells with known stem CSC properties towards an epithelial-like phenotype through global epigenetic changes effecting known tumor suppressor genes like DAPK1. Therewith, ITIH5 may represent an ECM modulator in epithelial breast tissue mediating suppression of tumor initiating cancer cell characteristics which are thought being responsible for the metastasis of breast cancer.
Additional file 1: Cell plasticity of ITIH5-expressing MDA-MB-231 single-cell clones. This figure shows morphological characteristics of independent MDA-MB-231 single-cell clones using phase-contrast microscopy. (DOCX 109 kb)12943_2017_610_MOESM1_ESM.docx
Additional file 2: Overlap of hypo-and hypermethylated CpG sites with published gene sets (GSEA). This table summarizes an overlap of significantly hyper- and hypomethylated CpG sites between ΔpBK-ITIH5 and ΔpBK-mock clones with gene set data bases. (XLS 72 kb)12943_2017_610_MOESM2_ESM.xls
Additional file 3: CpG sites with potential H3 marks. This table lists CpG sites of the HumanMethylation450 BeadChip annotated with histone H3 modifications (H3K4Me3 and H3K27Me3). (XLSX 7866 kb)12943_2017_610_MOESM3_ESM.xlsx
Additional file 4: Identified promoter regions with potential H3 marks that are hyper- or hypomethylated due to ITIH5 expression. This table illustrates the 242 different promoter regions featuring a significant association with a potential H3 methylation status described for ES cells. (XLSX 23 kb)12943_2017_610_MOESM4_ESM.xlsx
Additional file 5: Primer sequences and reaction conditions for RT-PCR experiments. This table illustrates the sequences of all primers used in this study for RT-PCR analysis. (XLS 28 kb)12943_2017_610_MOESM5_ESM.xls
Additional file 6: Primer sequences and PCR conditions for MSP analyses. This table illustrates the sequences of all primers used in this study for MSP analysis. (XLSX 10 kb)12943_2017_610_MOESM6_ESM.xlsx
Additional file 7: Primer sequences for pyrosequencing. This table shows sequences of DAPK1 pyrosequencing. (XLSX 10 kb)12943_2017_610_MOESM7_ESM.xlsx
Additional file 8: Primary antibodies used in this study. This table lists all primary andtiboides used in this study. (XLS 26 kb)12943_2017_610_MOESM8_ESM.xls
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- ITIH5 mediates epigenetic reprogramming of breast cancer cells
Steffen K. Meurer
Antonio S. Sechi
- BioMed Central
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