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. Here, 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 destabilised 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'.

NFATc acts as a non-canonical phenotypic stability factor for a hybrid epithelial/mesenchymal phenotype

“…Additionally, NFATc1 knockdown reduced the migration potential of H1975 cells as observed in scratch and trans-well migration assays. These findings indicate that the hybrid E/M cells may exhibit greater migratory and invasive potential when compared to mesenchymal cells ( Fig 2B-C), reminiscent of our previous observations comparing hybrid E/M cells with mesenchymal ones (17). Overall, these experimental results provide a proof-of-principle validation of our model predictions that NFATc can stabilize a hybrid E/M phenotype.…”

“…Even though NFATc extended the range of SNAIL values enabling a hybrid E/M state, the hybrid E/M state always co-existed with epithelial and/or mesenchymal states ({E, H, M} and {H, M} phases in Fig 1B); no monostable regime ({H}) for a hybrid E/M state was seen in the case of NFATc, as observed with GRHL2, OVOL1/2, ΔNP63α, NUMB and NRF2 (17)(18)(19)(20)(21)34). Similarly, compared to the other PSFs, the presence of NFATc does not increase the absolute value of MRT for a hybrid E/M phenotype as compared to the case without NFATc .…”

“…Overexpression of ZEB promotes EMT and silences the micro-RNAs which act as a safeguard for maintaining an epithelial phenotype (13). Recent studies have indicated the involvement of phenotypic stability factors (PSF) such as GRHL2, OVOL2, NUMB and NRF2 that can maintain the cells in a hybrid E/M phenotype(s) and prevent the cells from undergoing a complete EMT (17)(18)(19)(20)(21)(22). Knockdown of these PSFs usually drove hybrid E/M cells towards a completely mesenchymal phenotype, as observed in H1975 non-small cell lung cancer (NSCLC) cells which can maintain a hybrid E/M phenotype stably over multiple passages in vitro (20).…”

A biophysical model of Epithelial-Mesenchymal Transition uncovers the frequency and size distribution of Circulating Tumor Cell clusters across cancer types

“…Indeed, a high expression of 'phenotypic stability factors' that can stabilize hybrid epithelial/mesenchymal phenotype(s) and increase the mean residence times in these phenotypes by preventing them from undergoing a complete EMT (i.e. reduce &T<UVOWO "#$ effectively) have been correlated with higher aggressiveness and worse patient survival in multiple cancer datasets (26)(27)(28)(29), corroborating the evidence of clusters being the primary 'bad actors' of metastasis.…”

“…7). Thus, counterintuitive to the long-standing paradigm, mechanisms that inhibit a complete EMT and instead stabilize hybrid E/M state(s) might bear a higher metastatic potential (27,28,59). Future investigations should therefore focus on identifying the molecular mechanisms that drive a progression to a mesenchymal state or maintain a partial EMT instead.…”