SUMMARY The first epithelial to mesenchymal transition (EMT) occurs in trophoblast stem (TS) cells during implantation. Inactivation of the serine/threonine kinase MAP3K4 in TS cells (TSKI4 cells) induces an intermediate state of EMT, where cells retain stemness, lose epithelial markers, and gain mesenchymal characteristics. Investigation of relationships between MAP3K4 activity, stemness, and EMT in TS cells may reveal key regulators of EMT. Here, we show that MAP3K4 activity controls EMT through the ubiquitination and degradation of HDAC6. Loss of MAP3K4 activity in TSKI4 cells results in elevated HDAC6 expression and the deacetylation of cytoplasmic and nuclear targets. In the nucleus, HDAC6 deacetylates the promoters of tight junction genes, promoting the dissolution of tight junctions. Importantly, HDAC6 knockdown in TSKI4 cells restores epithelial features including cell-cell adhesion and barrier formation. These data define an epigenetic role for HDAC6 in regulating gene expression during transitions between epithelial and mesenchymal phenotypes.

SUMMARY O-GalNAc glycosylation is initiated in the Golgi by glycosyltransferases called GALNTs. Proteomic screens identified >600 O-GalNAc-modified proteins, but the biological relevance of these modifications has been difficult to determine. We have discovered a conserved function for GALNT3 in trophoblast stem (TS) cells, blastocyst trophectoderm, and human mammary epithelial cells (HMECs). The loss of GALNT3 expression in these systems reduces O-GalNAc glycosylation and induces epithelial-mesenchymal transition. Furthermore, Galnt3 expression is reduced in aggressive, mesenchymal claudin-low breast cancer cells. We show that GALNT3 expression controls the O-GalNAc glycosylation of multiple proteins, including E-cadherin in both TS cells and HMECs. The loss of GALNT3 results in the intracellular retention of E-cadherin in the Golgi. Significantly, re-expression of GALNT3 in TS cells increases O-GalNAc glycosylation and restores the epithelial state. Together, these data demonstrate the critical biological role of GALNT3 O-GalNAc glycosylation to promote the epithelial phenotype in TS cells, blastocyst trophectoderm, and HMECs.

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Cytochrome P4501B1 (CYP1B1) is elevated in breast cancer. Studies indicate a relationship between CYP1B1 and aggressive cancer phenotypes. Here, we report on in vitro studies in triple-negative breast cancer cell lines, where knockdown (KD) of CYP1B1 was used to determine the influence of its expression on invasive cell phenotypes. CYP1B1 KD in MDA-MB-231 cells resulted in the loss of mesenchymal morphology, altered expression of epithelial–mesenchymal genes, and increased claudin (CLDN) RNA and protein. CYP1B1 KD cells had increased cell-to-cell contact and paracellular barrier function, a reduced rate of cell proliferation, abrogation of migratory and invasive activity, and diminished spheroid formation. Analysis of clinical breast cancer tumor samples revealed an association between tumors exhibiting higher CYP1B1 RNA levels and diminished overall and disease-free survival. Tumor expression of CYP1B1 was inversely associated with CLDN7 expression, and CYP1B1HI/CLDN7LOW identified patients with lower median survival. Cells with CYP1B1 KD had an enhanced chemosensitivity to paclitaxel, 5-fluorouracil, and cisplatin. Our findings that CYP1B1 KD can increase chemosensitivity points to therapeutic targeting of this enzyme. CYP1B1 inhibitors in combination with chemotherapeutic drugs may provide a novel targeted and effective approach to adjuvant or neoadjuvant therapy against certain forms of highly metastatic breast cancer.

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