RREB1: A Critical Transcription Factor, Integrates TGF-β and RAS Signals to Drive Cancer Metastasis via Regulation of Enhancers

Abstract

A recent research article published by Lee et al. [1] in Cell revealed that transforming growth factor β (TGF-β) and rat sarcoma viral oncogene homolog (RAS) signaling, together trigger expression of epithelial-to-mesenchymal transition (EMT) and fibrogenic factors enhancing cancer metastasis through a precise and complex system. The authors elucidated that RAS-responsive element-binding protein 1 (RREB1)-mediated TGF-β-dependent fibrogenesis, and EMT come together to form a program to regulate cancer metastasis (Figure 1). This study enhances our understanding of the crosstalk between RAS and TGF-β in cancer metastasis, providing a potential therapeutic target.

RREB1, comprising 15 zinc finger (ZF) domains, is a critical transcription factor downstream of the RAS/mitogen-activated protein kinase (MAPK) signaling cascade, which plays a significant role in integration of RAS and TGF-β signaling pathways. TGF-β-activated small mother against decapentaplegic (SMAD) transcription factors are recruited by MAPK-activated RREB1 to Snail family transcriptional repressor (SNAIL). The recruitment of SMADs to SNAIL increases expression of SNAIL and triggers induction of developmental and fibrogenic EMT in carcinoma cells [2, 3]. Furthermore, RREB1-eukaryotic translation elongation factor 1A1 (eEF1A1)-3′ UTR axis enhances the translation of mitochondrial respiratory complex proteins encoded in nucleus and offers a novel therapeutic target for combating leukemia stem cells (LSCs) [4].

Cancer metastasis is the primary cause of patient mortality. During cancer metastasis, EMT is a crucial process in which epithelial cells lose their typical characteristics and acquire traits of mesenchymal cells, enhancing cell migration, invasion of surrounding tissues, and resistance to treatments. Su et al. [2] and Fontana et al. [5] revealed that the synergy between the TGF-β and RAS pathways trigger the EMT in fibrogenesis. Additionally, they identified RREB1, a RAS transcriptional effector, as an important cofactor of SMAD inducing EMT-transcription factors' (TFs) expression. Furthermore, in human acute myeloid leukemia (AML), a short variant of RREB1, known as RREB1S (1368 bp), enhances translation of nuclear-encoded mitochondrial genes mediated through its interaction with the translational factor eEF1A1, to maintain the characteristics of LSCs [4]. However, the subset of TGF-β mediated EMT-TFs regulated by RREB1 and the specific mechanism of RREB1-integrated RAS and TGF-β signaling transduction in cancer metastasis remains unknown.

Activated mutant-Kirsten RAS (KRAS) is a key driver mutation in lung adenocarcinoma (LUAD) accounting for one of the most common genetic subsets of human LUAD [1]. Single cell RNA-sequencing and immunofluorescence findings of metastasis samples isolated from KRAS-mutated patients with LUAD suggested that EMT-TFs and fibrogenic signatures are upregulated in metastases. To determine how EMT and fibrogenesis mediate LUAD metastasis, authors constructed EMT and fibrogenic-related gene knockout models, both in vivo and in vitro. The assay for transposase-accessible chromatin using sequencing (ATAC-seq) indicated that RREB1-dependent chromatin accessibility related to RAS signaling, TGF-β signaling, and EMT are induced by addition of TGF-β. Did RREB1 combine with the genome in response to TGF-β? Chromatin immunoprecipitation and sequencing indicated that the RREB1 could interact with the chromatin without recognizing the RRE motif to prime them for the TGF-β response. And the cleavage under targets and release using nuclease (CUT&RUN) assays showed that the histone H3K4me1is enriched near RRE-free RREB1-binding regions of EMT-TFs and fibrogenic-TFs, but not in regions marked by H3K4me3, which is a marker for active promoters. Indeed, these enhancers can transition to an active form by TGF-β, to gain the histone modification H3K27ac (acetylation of histone H3 at lysine 27), a hallmark of active enhancers. For investigating the role of TGF-β in LUAD metastasis, the authors employed CRISPR knockout screening to identify cofactors that regulate EMT and fibrogenesis, deliberately excluding the intersection with pancreatic ductal adenocarcinoma (PDAC). This method identifies LUAD-specific cofactors without confounding influences from PDAC, for its aggressiveness, in which, activation of both EMT and fibrogenesis can lead to death. Furthermore, authors demonstrated that SMAD could combine with DExH-Box Helicase 9 (DHX9) and INO80 Complex ATPase Subunit (INO80) as cofactors alongside RREB1. The functions of these two complexes were elucidated: SMAD3-DHX9 recruits CREB-binding protein (CBP), a coactivator with intrinsic acetyltransferase activity, to activate RREB1-primed enhancers. And TGF-β treatment induces structural modifications in SMAD4, particularly in the α-helix/loop extension and C-terminal tail, facilitating the recruitment of INO80. INO80, a chromatin remodeling ATPase recruited by SMAD4, removes the repressive histone variant H2A.Z to activate RREB1-primed enhancers. Finally, using domain mapping, the authors demonstrated that the zinc-finger 1–5 domain of RREB1(ZF1-5) could interact with H4K20acK16ac, reducing RREB1-mediated pulmonary metastasis. Collectively, the authors elucidated a specific mechanism of MAPK-TGF-β mediated EMT and fibrogenesis during cancer metastasis outgrowth. RAS-induced RREB1 primes enhancers, which are subsequently activated through the interaction of SMAD3-DHX9 and SMAD4-INO80 with RREB1. This activation enhances the transcriptional activities of EMT transcription factors and fibrogenic factors, ultimately promoting pulmonary metastasis (Figure 1).

In 2020, the research team led by Joan Massagué published TGF-beta orchestrates fibrogenic and developmental EMTs via the RAS effector RREB1 in Nature. They clarified the transcription factor RREB1, activated by the MAPK signaling pathway, recruited SMAD transcriptional complexes and regulated the expression of SNAIL, a critical driver of EMT. The interplay between RREB1 and SMAD enhanced chromatin accessibility, promoting the expression of additional EMT-related genes. In pancreatic adenocarcinoma, this interaction stimulated myofibroblast activation, intratumoral fibrosis, and tumor progression [2]. However, the underlying molecular mechanisms driving these processes required further investigation. The same research team not only reproduced the findings of their former paper in vivo and in vitro, but also identified that EMT and fibrogenic genes driven by TGF-β can fuel metastasis published in Cell. Simultaneously, the results suggested that the SMAD complex recruited by RREB1-H4K20acK16ac cleared H2A.Z, thereby enhancing chromatin accessibility [1]. This study is the first to show how SMAD4-recruited INO80 regulates RREB1-primed enhancers to activate gene transcription, providing new insights into SMAD-mediated regulation and potential therapies for LUAD metastasis. However, it focuses only on pulmonary metastasis, neglecting other sites like bones, liver, or brain. While highlighting RREB1's role in EMT and fibrosis, the mechanisms and specific TGF-β ligands or receptors involved remain unclear, warranting further investigation.

Furthermore, the competitive inhibition of RREB1 by its ZF1-5 domain results in reduced lung metastasis and fibrosis, underscoring its pivotal role in these pathological processes. Additionally, the expression of the short RREB1 variant (RREB1S) has been shown to confer resistance to venetoclax in AML cells, correlating with poorer patient prognosis. These findings suggest that RREB1 may serve as a therapeutic target across various diseases. Future research focusing on RREB1 protein structure, posttranslational modifications, and associated molecular mechanisms could provide valuable insights for the development of more effective treatments.

In summary, RAS-activated RREB1 primes enhancers of fibrogenic EMT genes, which are subsequently activated through the recruitment of chromatin remodeling complexes by the TGF-β/SMAD signaling pathways in LUAD metastasis. RAS and TGF-β synergistically enhance the expression of fibrogenic EMT genes, thereby promoting carcinoma metastasis. Furthermore, research has first identified the role of SMAD4 in recruiting INO80 to facilitate aberrant nucleosome elimination under TGF-β treatment. The study additionally offers a therapeutic strategy to inhibit LUAD metastasis by targeting RREB1, which effectively suppresses the expression of fibrogenic EMT genes. The study expands our understanding of the crosstalk between various signaling pathways. Moving forward, the mechanism underlying the regulation of fibrogenic EMT genes by RREB1 should be further investigated in other diseases. Moreover, it is likely that additional cross-talks between distinct signaling pathways remain to be uncovered.

Zhe Wang wrote the manuscript and prepared the figure. Feng Xie provided valuable discussion. Fangfang Zhou approved the final version of the manuscript. All authors have read and approved the final manuscript.

The authors have nothing to report.

The authors declare no conflicts of interest.