Abstract
Epigenetic deregulation, especially mutagenesis or the abnormal expression of epigenetic regulatory factors (ERFs), plays an important role in malignant tumorigenesis. To screen natural inhibitors of breast cancer metastasis, we adopted small interfering RNAs (siRNAs) to transiently knock down 591 ERF-coding genes in luminal breast cancer MCF-7 cells and found that depletion of AF9 significantly promoted MCF-7 cell invasion and migration. A mouse model of metastasis further confirmed the suppressive role of AF9 in breast cancer metastasis. RNA profiling revealed enrichment of AF9 targets genes in the epithelial-mesenchymal transition (EMT). Mechanistically, tandem mass spectrometry showed that AF9 interacts with Snail, which hampers Snail transcriptional activity in basal-like breast cancer (BLBC) cells. AF9 reconstitutes an activated state on the promoter of Snail, which is a master regulator of EMT, and derepresses genes by recruiting CBP or GCN5. Additionally, microRNA-5694 (miR-5694) targeted and degraded AF9 messenger RNA (mRNA) in BLBC cells, further enhancing cell invasion and migration. Notably, AF9 and miR-5694 expression in BLBC clinical samples correlated inversely. Hence, miR-5694 mediates downregulation of AF9 and provides metastatic advantages in BLBC. Restoring expression of the metastasis suppressor AF9 is a possible therapeutic strategy against metastatic breast cancer.
Highlights
Malignant breast cancer can metastasize to lymph nodes and multiple distant organs, such as the lungs, bones, and brain.[1]
To identify which epigenetic regulatory factor (ERF) contributes to suppressing luminal cancer cell mobility and invasive growth, we adopted small interfering RNA (siRNA) to knock down 591 genes[30] encoding ERFs in MCF-7 cells and performed wound healing assays to examine tumor cell migration using an IncuCyte high-throughput screening system (Figure 1A)
CDH1 and BRMS1 were used as positive controls when their loss drove wound healing (Figure 1B), and AF9 was depleted in the MCF-7 cells through transient transfection with 4 independent siRNAs in this screening system (Figure 1C)
Summary
Malignant breast cancer can metastasize to lymph nodes and multiple distant organs, such as the lungs, bones, and brain.[1] Metastasis is the multistep process in which tumor cells detach from primary sites and disseminate to other sites. The prevailing view is that metastatic capacity is a late, acquired event during tumorigenesis and that it is difficult to reverse this transition once metastasis occurs.[2] it is important to explore the early regulators initiating cancer metastasis. Molecular Therapy Vol 29 No 3 March 2021 a 2020 The Author(s).
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