Novel target genes of ZEB1 and Snail1 in triple-negative human breast cancer.

Sammanfattning: Breast cancer is comprised of several subtypes that are different from one another and thedivergence leads to different outcomes of the disease. There are known prognostic factors andphenotypic distinction in different biological factors and expression patterns, such as theestrogen receptor (ER), progesterone receptor (PR), Ki67, HER2/neu expression (HER2). Ingeneral, there are three breast cancer subtypes with the most recurring subtype being luminalA, and the other two being luminal B and triple negative breast cancer. Triple negative breastcancer is a heterogeneous subtype which is defined by the lack of expression of ERα, PR andHER2. Triple negative breast cancers are also very aggressive and have the worst prognosiscompared to the other two ERα positive tumors. The luminal A subtype can develop into ametastatic cancer thanks to the so-called epithelial-mesenchymal transition (EMT), whichaffects a subpopulation of epithelial cancer cells. EMT is the name of a process that takesplace during the embryonic development, the wound healing and cancer metastasis, where theepithelial cells will transform into mesenchymal cells which have higher invasive andmigratory properties. EMT occurs when epithelial cells lose their apical-basal polarity andthen the adherens- and tight junctions are dissolved. The adherens junction dissolution can beobserved as a downregulation of CDH1 (E-cadherin), which is regularly measured in EMTstudies. Many signaling pathways are associated with the promotion and establishment ofEMT e.g. transforming growth factor β (TGFβ), Notch and Wnt signaling. Bioinformaticscreening was performed to look for mRNA expression levels of ZEB1 and Snail1 indifferent breast cancer cell lines. By using chromatin immunoprecipitation-sequencing (ChIPSeq)in the triple negative (ER-, PR- HER2-) Hs578T breast cancer cell line, a genome-widescreen for ZEB1 and Snail1 binding sites had been performed before the start of the project.The Hs578T cell line expresses many of the EMT transcription factors that are relevant forthe project. Since the signaling of TGFβ is crucial for these genes, manipulation of thissignaling pathway is needed to be able to analyse its importance for the function of thesegenes. To inhibit the activity of TGFβ, the small molecule GW6604 was used to inhibit theTGFβ type I receptor kinase (TβRI) and in that way inhibiting the signaling from thisreceptor. In addition, ZEB1 and Snail1 were knocked out by the use of the transfection andCRISPR/Cas9 knockout technique. By investigating mRNA and protein levels of chosengenes in both control Hs578T cells and ZEB1 and Snail1 knockout Hs578T cells, up or downregulation of some of these genes can be seen with stimulation with TGFβ. The knockout ofSnail1 but not of ZEB1 indicated that the loss of Snail1 generated breast cancer cells thatcould try to revert to epithelial at the phenotypic level.