Nrf2 Modulates the Hybrid Epithelial/Mesenchymal Phenotype and Notch Signaling During Collective Cancer Migration.

Hybrid epithelial/mesenchymal cells (E/M) are key players in aggressive cancer metastasis. It remains a challenge to understand how these cell states, which are mostly non-existent in healthy tissue, become stable phenotypes participating in collective cancer migration. The transcription factor Nrf2, which is associated with tumor progression and resistance to therapy, appears to be central to this process. Here, using a combination of immunocytochemistry, single cell biosensors, and computational modeling, we show that Nrf2 functions as a phenotypic stability factor for hybrid E/M cells by inhibiting a complete epithelial-mesenchymal transition (EMT) during collective cancer migration. We also demonstrate that Nrf2 and EMT signaling are spatially coordinated near the leading edge. In particular, computational analysis of an Nrf2-EMT-Notch network and experimental modulation of Nrf2 by pharmacological treatment or CRISPR/Cas9 gene editing reveal that Nrf2 stabilizes a hybrid E/M phenotype which is maximally observed in the interior region immediately behind the leading edge. We further demonstrate that the Nrf2-EMT-Notch network enhances Dll4 and Jagged1 expression at the leading edge, which correlates with the formation of leader cells and protruding tips. Altogether, our results provide direct evidence that Nrf2 acts as a phenotypic stability factor in restricting complete EMT and plays an important role in coordinating collective cancer migration.

Optimizing locked nucleic acid modification in double-stranded biosensors for live single cell analysis.

Double-stranded (ds) biosensors are homogeneous oligonucleotide probes for detection of nucleic acid sequences in biochemical assays and live cell imaging. Locked nucleic acid (LNA) modification can be incorporated in the biosensors to enhance the binding affinity, specificity, and resistance to nuclease degradation. However, LNA monomers in the quencher sequence can also prevent the target-fluorophore probe binding, which reduces the signal of the dsLNA biosensor. This study investigates the influence of LNA modification on dsLNA biosensors by altering the position and amount of LNA monomers present in the quencher sequence. We characterize the fluorophore-quencher interaction, target detection, and specificity of the biosensor in free solution and evaluate the performance of the dsLNA biosensor in 2D monolayers and 3D spheroids. The data indicate that a large amount of LNA monomers in the quencher sequence can enhance the specificity of the biosensor, but prevents effective target binding. Together, our results provide guidelines for improving the performance of dsLNA biosensors in nucleic acid detection and gene expression analysis in live cells.

Decoding leader cells in collective cancer invasion.

Collective cancer invasion with leader-follower organization is increasingly recognized as a predominant mechanism in the metastatic cascade. Leader cells support cancer invasion by creating invasion tracks, sensing environmental cues and coordinating with follower cells biochemically and biomechanically. With the latest developments in experimental and computational models and analysis techniques, the range of specific traits and features of leader cells reported in the literature is rapidly expanding. Yet, despite their importance, there is no consensus on how leader cells arise or their essential characteristics. In this Perspective, we propose a framework for defining the essential aspects of leader cells and provide a unifying perspective on the varying cellular and molecular programmes that are adopted by each leader cell subtype to accomplish their functions. This Perspective can lead to more effective strategies to interdict a major contributor to metastatic capability.

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

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'.