A Dkk1 Dependent Crosstalk Between Tgf Beta 2-Snail1 And Mirnas Contributes To Chemoresistance In Mesenchymal-Like Breast Cancer

The role of TGFβ in chemoresponse and progression of breast cancer (BC) remains unclarified, here we investigated the role of TGFβ2 in BC by taking into account BC heterogeneity. mRNA microarray identified TGFβ2 was upregulated in 5-Fu resistant MCF-7 BC cells (MCF-7/5-Fu) with EMT phenotype. TGFβ2 in mRNA and protein from MCF-7/5-Fu and triple-negative BC (TN-BC) cell lines MDA-MB-231, HCC38 and BT-549 with mesenchymal morphology was higher than luminal BC cell lines MCF-7, T47D, BT-474 and SKBR3. TGFβ signal was more active in mesenchymal BC cells than luminal BC cells charactered by p-smad2, p-smad3 and snial1 upregulated. TGFβ signal inhibition with TGFβ receptor (TGFβR) inhibitor SD208 or TGFβRI targeted shRNA reversed the mesenchymal phenotype accompanied with invasion inhibition, BC stem cell (BCSC) traits measured by CD44+CD24- subpopulation analysis and mammosphere assay, and resistance to 5-Fu and Paclitaxel, but played limited effect on MCF-7/5-Fu cells. As the same, TGFβ2 only partially induced EMT phenotype in MCF-7, T47D, BT474 and SKBR3 cells. miRNA microarray indentified downregulation of miR-141, miR-145 and miR-200a (Tβ2-miRNAs) in mesenchymal BC cells compared to lumial BC cells. Lentivirus system with two pre-miRNA units enhanced Tβ2-miRNAs expression accompanied with TGFβ2 downregulation and reversion of mesenchymal phenotype and drug resistance in mesenchymal BC cells. Luciferase activity assay also strongly suggested Tβ2-miRNAs directly target TGFβ2 synergistically. TGFβ signal inhibition and snail1 knockdown upregulated Tβ2-miRNAs in mesenchymal BC cells. ChIP assay showed snail1 bound to promoters of Tβ2-miRNAs, combined with promoter luciferase assay suggested snail1 transcriptionaly repress Tβ2-miRNAs expression in mesenchymal BC cells. However, DKK1 upregulated Tβ2-miRNAs expression in mesenchymal BC cells, but with limited effect in snail1 knockdown cells, suggesting DKK1 could reverse the repressive effect of snail1 on miRNAs expression. TGFβ2 upregulated Tβ2-miRNAs expression in MCF-7 and T47D cells with normal DKK1 expression, and snail1 konckdown abolished the effect of TGFβ2. In addition, using a tissue microarray of 93 BC biopsies containing 42 TN-BC, we found TGFβ2 and snail1 protein were present at higher levels in TN-BC than in luminal BC, and miRNAs measured by ISH and DKK1 protein were present at lower levels. Furthermore, the result of Tβ2-miRNAs detection in the serum samples from 76 BC patients including 34 TN-BC showed Tβ2-miRNAs abundance in serum samples from TN-BC was much lower. In conclusion, the role of TGFβ in BC is cell type dependent and our findings suggest TGFβ2 protein and Tβ2-miRNAs levels in serum will be valuable prognostic biomarkers and therapeutic strategies for mesenchymal BC. Note: This abstract was not presented at the meeting. Citation Format: Guopei Zheng, Xiaoting Jia, Zijuan Zhang, Zhijie Zhang, Chen Qu, Jiang Yin, Zhimin He. A DKK1 dependent crosstalk between TGFβ2-snail1 and miRNAs contributes to chemoresistance in mesenchymal-like breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4872. doi:10.1158/1538-7445.AM2014-4872