Pro-Oncogenic Transcription Factors BACH1 and Nrf2 Associate with Cytoplasmic Biomolecular Condensates of GFP-MxA in Oral Cancer Cells

Phase-separated biomolecular condensates in the cytoplasm and nucleus are now recognized to contribute to carcinogenesis through aberrant signaling by assorted transcription factors and fusion oncoproteins. Oral cancer, the sixth most prevalent malignancy worldwide, frequently occurs in a U-shaped “high-risk” zone (floor of mouth, side of tongue, and anterior fauces) reflecting the path of liquid transit through the mouth. We previously reported that environmental stresses of saliva-like hypotonicity and beverage-like temperature changes triggered cycles of disassembly/reassembly of biomolecular condensates of GFP-tagged human myxovirus resistance protein (MxA; alias Mx1) in oral cancer cells. In the present study we identified some of the constituents of GFP-MxA cytoplasmic condensates in oral cells. GFP-MxA condensates were isolated from interferon (IFN)-λ1-treated GFP-MxA expressing OECM1 human oral cancer cells using magnetic bead-based immunoisolation. Unbiased peptide identification confirmed presence of MxA/Mx1 peptides; however, the strongest intensity was for the BACH1 transcription factor family. Immunofluorescence analyses confirmed the association of BACH1 and the family member Nrf2 with cytoplasmic human GFP-MxA condensates. Moreover, GFP-BACH1 and GFP-Nrf2 colocalized with cytoplasmic human HA-MxA condensates in transiently transfected OECM1 cells. Western blot assays confirmed presence of BACH1 and Nrf2 proteins in complexes isolated using anti-MxA pAb. In as much as BACH1 and Nrf2 regulate oxidative stress response genes, it was remarkable that immunofluorescence assays revealed the presence of heme oxygenase 1 (HO1) – a downstream redox regulator - in GFP-MxA condensates. In terms of aberrant function, in live cells, the Nrf2 transcription factor underwent rapid disassembly and reassembly cycles driven by saliva-like hypotonicity. The data highlight the unexpected intersections in oral cells between MxA condensates and BACH1, Nrf2 and HO1 – proteins well known to be involved in pathways regulating cellular responses to environmental and oxidative stresses, antiviral defense, oral epithelial dysplasia, and cancer progression and metastases.