NRF2 activates a partial epithelial-mesenchymal transition and is maximally present in a hybrid epithelial/mesenchymal phenotype

Abstract

The epithelial-mesenchymal transition (EMT) is a key process implicated in cancer metastasis and therapy resistance. Recent studies have emphasized that cells can undergo partial EMT to attain a hybrid epithelial/mesenchymal (E/M) phenotype – a cornerstone of tumour aggressiveness and poor prognosis. These cells can have enhanced tumour-initiation potential as compared to purely epithelial or mesenchymal ones and can integrate the properties of cell-cell adhesion and motility that facilitates collective cell migration leading to clusters of circulating tumour cells (CTCs) – the prevalent mode of metastasis. Thus, identifying the molecular players that can enable cells to maintain a hybrid E/M phenotype is crucial to curb the metastatic load. Using an integrated computational-experimental approach, we show that the transcription factor NRF2 can prevent a complete EMT and instead stabilize a hybrid E/M phenotype. Knockdown of NRF2 in hybrid E/M non-small cell lung cancer cells H1975 and bladder cancer cells RT4 destabilized a hybrid E/M phenotype and compromised the ability to collectively migrate to close a wound in vitro. Notably, while NRF2 knockout simultaneously downregulated E-cadherin and ZEB-1, overexpression of NRF2 enriched for a hybrid E/M phenotype by simultaneously upregulating both E-cadherin and ZEB-1 in individual RT4 cells. Further, we predict that NRF2 is maximally expressed in hybrid E/M phenotype(s) and demonstrate that this biphasic dynamic arises from the interconnections among NRF2 and the EMT regulatory circuit. Finally, clinical records from multiple datasets suggest a correlation between a hybrid E/M phenotype, high levels of NRF2 and its targets and poor survival, further strengthening the emerging notion that hybrid E/M phenotype(s) may occupy the ‘metastatic sweet spot’.

Unresolved Complexity in the Gene Regulatory Network Underlying EMT

“…We are also beginning to decipher the requirement of different TFs for EMT initiation, maintenance, and progression (Figure 3). For example, the TF NRF2 delays the transition of a cell to a full mesenchymal phenotype; it maintains the hybrid epithelial/mesenchymal (partial/intermediate EMT) state (106). Similarly, research from our lab has shown that JNK-induced TFs and subsequent signaling are not required for EMT initiation, but was essential for progression of phenotypic hallmarks of EMT (91).…”

Cancer Stem Cells and Epithelial-to-Mesenchymal Transition in Cancer Metastasis

“…Application of MAGIC to TGFβ-treated HMLE suggested that 80% of cells showed one or more hybrid E/M phenotypes, and identified many genes whose expression peaked during these hybrid phenotypes while maintained low levels in both epithelial and mesenchymal states (van Dijk et al 2018). Identification of those markers unique to hybrid E/M phenotypes (Bocci et al 2019c) may reveal novel MetSC markers. Thus, computational analysis of static/dynamic single-cell expression data can possibly discover novel regulatory networks for EMP/CSC and pinpoint potential therapeutic vulnerabilities (Fig.…”

Decoupling of Nrf2 Expression Promotes Mesenchymal State Maintenance in Non-Small Cell Lung Cancer

“…This is consistent with the attenuated proliferation, increased migration, and increased senescent behaviors of mesenchymal-like tumor cells. Analysis of breast cancer patient samples for high Nrf2 expression has correlated with a significant decrease in overall survival ( p = 0.024) [64]. Here in NSCLC adenocarcinomas we observed a trend for poor overall survival with increased Nrf2 target gene expression ( p = 0.15), which is correlated with a proliferative, epithelial phenotype.…”

“…For example, in fibrotic lung disease, Nrf2 expression decreased the EMT promoting transcription factor Snail [63]. In contrast, in NSCLC H1975 and bladder RT4 cells Nrf2 knockdown had mixed impacts on epithelial and mesenchymal markers, suggesting the generation of an intermediate ‘hybrid’ EMT state [64]. Here in mutant KRAS and mutant EGFR NSCLC models we find Nrf2 expression promotes a more epithelial cell state while antagonizing the mesenchymal state.…”