ITIH5 mediates epigenetic reprogramming of breast cancer cells

Abstract

BackgroundExtracellular 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.Methods 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.Results 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.ConclusionsOur 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-017-0610-2) contains supplementary material, which is available to authorized users.

ITIH5 induces a shift in TGF‐β superfamily signaling involving Endoglin and reduces risk for breast cancer metastasis and tumor death

“…(A) Heatmap illustrates an ITIH5‐associated signature of genes known being associated with metastasis ( P < 0.001). Comparison of gene expression profiles was performed using whole genome expression data of independent ITIH5‐expressing (ΔpBK‐ITIH5 clones) and ITIH5‐deficient MDA‐MB‐231 ΔpBK‐mock single‐cell clones including the MDA‐MB‐231 WT. The heatmap of significantly ( P ≤ 0.05) differential expressed (red: overexpressed; blue: suppressed) genes is shown.…”

“…As we recently demonstrated that ITIH5 mediates an epigenetic shift in breast cancer cells, we analyzed ENG promoter methylation in different MDA‐MB‐231 single cell clones by performing MSP (Supplementary Figure S2A). MDA‐MB‐231 WT and mock clones were found being completely methylated.…”

“…The breast cancer cell line MDA‐MB‐231 was obtained from the American Type Culture Collection (ATCC, Manassas, VA). MDA‐MB‐231 derived single‐cell clones with either ITIH5 overexpression (ITIH5 clones) or complete lack of ITIH5 expression (mock clones) were generated and characterized as described previously . The PCR‐based Venor® GeM Mycoplasma Detection Kit (Minerva Biolabs, Berlin, Germany) was used to avoid Mycoplasma infection.…”

“…For transient transfection, 3 × 10 5 cells per well were seeded in 6‐well dishes. The next day medium was exchanged and 1 h later cells were transfected using the Lipofectamine® 2000 reagent (Thermo Fisher, Waltham, MA) and the DNA vectors indicated (ΔpBK‐mock, ΔpBK‐ITIH5) at a ratio of 3:1 (μL reagent/μg DNA) for 24 h. The next day medium was exchanged to one including 0.5% FCS. After another 48 h, TGF‐β1 (1.0 ng/mL) was either added or not and cells were incubated for further 24 h. Thereafter, supernatants were collected and cells lysed in RIPA buffer for Western blot analysis as outlined below.…”

“…In vitro, ITIH5 overexpression has been reported to mediate suppression of tumor cell growth and colony formation, for example, in the RT112 bladder and the CaSki cervical cancer cell line . In aggressive MDA‐MB‐231 breast cancer cells ITIH5 expression induces epigenetic reprogramming and abrogates mesenchymal migration, a hallmark of metastatic tumor cells, involving the tumor suppressor gene “death associated protein kinase 1” ( DAPK1 ) . In addition, ITIH5 was recently identified as a metastasis suppressor gene in in pancreatic ductal carcinoma …”