PKCα Modulates Epithelial-to-Mesenchymal Transition and Invasiveness of Breast Cancer Cells Through ZEB1

Abstract

ZEB1 is a master regulator of the Epithelial-to-Mesenchymal Transition (EMT) program. While extensive evidence confirmed the importance of ZEB1 as an EMT transcription factor that promotes tumor invasiveness and metastasis, little is known about its regulation. In this work, we screened for potential regulatory links between ZEB1 and multiple cellular kinases. Exploratory in silico analysis aided by phospho-substrate antibodies and ZEB1 deletion mutants led us to identify several potential phospho-sites for the family of PKC kinases in the N-terminus of ZEB1. The analysis of breast cancer cell lines panels with different degrees of aggressiveness, together with the evaluation of a battery of kinase inhibitors, allowed us to expose a robust correlation between ZEB1 and PKCα both at mRNA and protein levels. Subsequent validation experiments using siRNAs against PKCα revealed that its knockdown leads to a concomitant decrease in ZEB1 levels, while ZEB1 knockdown had no impact on PKCα levels. Remarkably, PKCα-mediated downregulation of ZEB1 recapitulates the inhibition of mesenchymal phenotypes, including inhibition in cell migration and invasiveness. These findings were extended to an in vivo model, by demonstrating that the stable knockdown of PKCα using lentiviral shRNAs markedly impaired the metastatic potential of MDA-MB-231 breast cancer cells. Taken together, our findings unveil an unforeseen regulatory pathway comprising PKCα and ZEB1 that promotes the activation of the EMT in breast cancer cells.

HERC1 Regulates Breast Cancer Cells Migration and Invasion

“…For the experimental metastasis assay, 1× 10 6 HERC1-silenced or control cells were resuspended in 200 μL of sterile 1 × PBS and injected in the lateral tail vein of male mice. Sixty days post injection, lungs were harvested, fixed in buffered formalin, and stored in 70% ethanol until DNA extraction and metastatic load quantification following experimental procedures as described previously [ 21 ].…”

High B3GALT5 expression confers poor clinical outcome and contributes to tumor progression and metastasis in breast cancer

“…Mechanistically, we found B3GALT5 could regulate cell proliferation, migration, and invasion through EMT activator ZEB1 in breast cancer cells as well as other properties of BCSCs, such as mammosphere formation, tumor initiation, and metastasis. ZEB1 is implicated as a prime element of a network of transcription factors that control cell proliferation, EMT, and cancer metastasis [ 27 , 28 ] in various cancers including breast cancer [ 29 , 30 ], pancreatic cancer [ 17 ], and lung cancer [ 31 ]. We found that overexpression of B3GALT5 altered cell morphology toward EMT and increased ZEB1 expression, whereas knockdown of B3GALT5 reduced EMT and cell proliferation as well as decreased ZEB1 expression.…”

USP19 modulates cancer cell migration and invasion and acts as a novel prognostic marker in patients with early breast cancer

“…Lungs were harvested 60 days post-injection, fixed in buffered formalin and then stored in 70% ethanol until use for DNA quantification (as described before ref. 36 ) or paraffin embedding, or insufflated with a 15% India Ink solution and counterstained with Fekete’s solution for macrometastasis exposure and imaging.…”