7961 EMT Plasticity Induced by GRHL2 Overexpression Stimulates Dormancy and Enriches Stem Cell Characteristics in ER+ Breast Cancer Cells
Authors: Zheng Christy , Allen Kaelyn O , Liu Tianrui , Solodin Natalia M , Salem Kelley , Fowler Amy M , Vera Jessica , Tsourkas Philippos K , McIlwain Sean , Ozers Mary S , Alarid Elaine T 
Published: October 2024

Disclosure: C. Zheng: None. K.O. Allen: None. T. Liu: None. N.M. Solodin: None. K. Salem: None. A.M. Fowler: None. J. Vera: None. P.K. Tsourkas: None. S. McIlwain: None. M.S. Ozers, PhD: Owner/Co-Owner; Self; Co-Founder and CSO of Proteovista LLC. E.T. Alarid: None.

Metastasis is governed by an epithelial to mesenchymal transition (EMT) - the ability for an epithelial cell to adopt a mesenchymal phenotype to promote migration and tumor progression. Though EMT can be articulated as a distinctive transition between its binary states, a cell’s capacity to become plastic, and thus, express a hybrid EMT state with both epithelial and mesenchymal markers has re-defined the literature’s understanding of metastasis. To improve our comprehension of EMT, the nuclear transcription factor responsible for driving epithelial cell fate Grainyhead-like protein 2 (GRHL2) became the protein of interest as clinical evidence suggests high GRHL2 expression is indicative of poorer prognosis in breast cancer (BC). A tetracycline-inducible GRHL2 overexpression model in the estrogen receptor (ER) positive BC MCF7 cell line was developed to understand GRHL2’s role in EMT. Surprisingly, overexpression of the EMT suppressor GRHL2 induces EMT plasticity in ER-positive BC with increased E-cadherin and vimentin expression assessed by immunocytochemistry and mRNA expression. This GRHL2 overexpression model was used to further explore the dynamic nature of EMT plasticity. Gene expression analyses was performed to assess representative genes associated with a partial EMT in purified populations of GRHL2 overexpressing cells. Gene set enrichment analysis (GSEA) determined statistically significant hallmark pathways associated with GRHL2 overexpression. Isolated RNA of GRHL2 overexpressing cells revealed increased mRNA expression of p27 and NR2F1 via RT-qPCR, consistent with a dormancy phenotype. Interestingly, mammosphere and flow cytometry further expanded on the dynamic states of EMT plasticity by emphasizing on the enrichment of stem cell characteristics with GRHL2 overexpression. Extensive GRHL2 overexpression provides cells self-renewal capacity, as examined by mammosphere formation efficiency. Flow cytometry for stem cell markers CD44 and ALDH1 expression demonstrates the increased representation of stem cell markers in EMT plastic populations. To evaluate how GRHL2 overexpression influences tumor progression in vivo, immunocompromised mice were injected with GRHL2 overexpressing cells to examine tumor growth and the potential of cells to create micro-metastases using IVIS. Tumor growth responses differed between parental and GRHL2 overexpressing xenografts, with increased GRHL2 expression decreasing tumor growth. mRNA expression of harvested tumors mirrored in vitro expression of p27 and NR2F1. Together, these data indicates that GRHL2 overexpression induces EMT plasticity, in turn stimulating dormancy and stem-cell acquisition in vitro and in vivo, allowing hybrid cells the ability to adjust to its environment.

Presentation: 6/3/2024