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RREB1 Integrates TGF-β and RAS Signals to Drive EMT
Developmental Cell
Spotlight RREB1 Integrates TGF-b and RAS Signals to Drive EMT Laurent Fattet1 and Jing Yang1,* 1Department of Pharmacology, Moores Cancer Center, University of California, San Diego, 3855 Health Sciences Drive, La Jolla, CA 92093, USA *Correspondence: [email protected] https://doi.org/10.1016/j.devcel.2020.01.020
TGF-b is long known to require Ras activation to induce EMT. In a recent issue of Nature, Massague´ and colleagues (Su et al., 2020) identify RAS-responsive element binding protein 1 (RREB1) as a critical integrator of TGF-b and Ras signals during both developmental and cancer EMT programs. TGF-b is a key inducer of EMT during various developmental and pathogenic EMT events. A large body of literature also show that the ability of TGF-b to induce EMT requires activation of Ras and its downstream MAPK (David et al., 2016; Horiguchi et al., 2009; Janda et al., 2002; Oft et al., 1996). However, how Ras and TGF-b signals are integrated into the EMT program is unknown. A recent study published in Nature by Su et al. (2020) identifies RAS-responsive element binding protein 1 (RREB1), a RAS transcriptional effector, as the key molecular integrator of Ras and TGF-b signals to induce EMT (Su et al., 2020). As TGF-b induces a lethal EMT in pancreatic adenocarcinoma (PDA) carrying KRAS oncogenic mutations (David et al., 2016), the authors sought to determine the common actors downstream of both pathways by analyzing the DNA binding motifs of TGF-b-activated transcription factors SMAD2/3 that also depend on oncogenic KRAS. ChIP-seq data, together with shRNA screening, identified zinc fingerscontaining protein RREB1, a poorly studied transcription factor previously involved in PDAC development (Kent et al., 2013; Thiagalingam et al., 1996), as a RAS-regulated SMAD cofactor. Further biochemical analyses demonstrated that ectopically expressed RREB1 interacts with and shares a similar genome-binding pattern as SMAD2/3, in a MAPK-dependent manner. Mutagenesis of RREB1 showed that phosphorylation of Ser-161 and Ser-970 by ERK mediates RREB1 effects during TGFb-induced EMT. Interestingly, knockout of RREB1 prevented the induction of EMT by TGF-b as shown by a decreased binding of SMAD2/3 to the promoters of
TGF-b-responsive genes SNAI1 and HAS2, and rescued expression of RREB1 could restore the induction of these genes by TGF-b. The authors next asked which EMT genes are regulated by RREB1 to coordinate the TGF-b and RAS signaling pathways. Gene ontology analysis of TGF-b response genes in an oncogenic RAS context highlighted genes involved in cell adhesion, migration, and EMT. A particular interest was given to a set of genes encoding extracellular matrix (ECM) components (laminins, collagens, proteases) or regulating ECM production and deposition by mesenchymal cells during fibrosis (such as IL-11, CTGF, WISP1, and PDGFB), as their expression is RREB1-dependent. Interestingly, depletion of EMT transcription factors SNAI1 or ZEB1 inhibited EMT but not the fibrogenic gene responses, suggesting that these two processes could be differentially controlled downstream of RREB1. In line with these results, the authors extended their findings to the breast tissue to show that TGF-b-induced EMT in normal mammary epithelial cells requires ERK-dependent activation of RREB1. To determine whether RREB1 plays a conserved role in mediating developmental EMT events, the authors performed RNA-seq analysis of WT and RREB1-KO embryonic stem (ES) cells upon induced differentiation in embryoid bodies. They found that RREB1 is required for proper differentiation as RREB1-null EBs failed to activate gene signatures associated with stem cell differentiation and EMT. Further analysis of the chromatin state by ATAC-seq suggested that SMAD2/3 and RREB1 accessibility to regulatory sequences of genes
regulating EMT might be context-dependent and vary for the induction of a cancer or a developmental EMT. The authors further showed through elegant in vivo analysis of chimeric embryos containing RREB1 / ES cells that the majority of mutated chimeras had failure in the gastrulation process. Interestingly, the authors analyzed the resulting embryos and found severe defects in EMT activation during gastrulation, as RREB1 depletion prevented the E- to N-cadherin switch observed in WT embryos and required for proper gastrulation. Intrigucells expressed ingly, RREB1 / Brachyury and Snai1 normally, with some chimeras displaying a coexpression of both E- and N-cadherin, or an aberrant expression of N-cadherin in the posterior epiblast, suggesting a more complex outcome than a total blockade of EMT. Consistent with this, previous studies in embryos lacking FGFR1 or FGF8 (and thus presumably defective in Ras activation) show that epiblasts succeeded in undergo EMT and gastrulate morphologically, but the resulting mesoderm cells failed to migrate (Sun et al., 1999; Yamaguchi et al., 1994). Together, these studies suggest that RREB1 may control a subset of the EMT program during gastrulation. This study by Su et al. (2020) identifies RREB1 as a key player that integrates both TGF-b and Ras signals to induce EMT during both gastrulation and pancreatic cancer development. Future studies are needed to understand how Ras signaling activates the cooperative binding of SMAD2/3 and RREB1 to their target DNA sequences. While the EMT program is thought to be largely conserved during development and cancer progression
Developmental Cell 52, February 10, 2020 ª 2020 Elsevier Inc. 259
Developmental Cell
Spotlight (Yang and Weinberg, 2008), comparison between the cancer and gastrulation EMT programs raises important questions on how RREB1 exerts some different roles during these two processes. Detailed analysis should aim to determine how the chromatin accessibility by the RREB1-SMAD complex differs in models of cancer and developmental EMTs, which could help to reveal these differential EMT transcription responses under developmental versus pathological contexts.
REFERENCES David, C.J., Huang, Y.H., Chen, M., Su, J., Zou, Y., Bardeesy, N., Iacobuzio-Donahue, C.A., and Massague´, J. (2016). TGF-b Tumor Suppression through a Lethal EMT. Cell 164, 1015–1030.
Horiguchi, K., Shirakihara, T., Nakano, A., Imamura, T., Miyazono, K., and Saitoh, M. (2009). Role of Ras signaling in the induction of snail by transforming growth factor-beta. J. Biol. Chem. 284, 245–253. Janda, E., Lehmann, K., Killisch, I., Jechlinger, M., €nert, Herzig, M., Downward, J., Beug, H., and Gru S. (2002). Ras and TGF[beta] cooperatively regulate epithelial cell plasticity and metastasis: dissection of Ras signaling pathways. J. Cell Biol. 156, 299–313. Kent, O.A., Fox-Talbot, K., and Halushka, M.K. (2013). RREB1 repressed miR-143/145 modulates KRAS signaling through downregulation of multiple targets. Oncogene 32, 2576–2585.
effector RREB1. Nature. https://doi.org/10.1038/ s41586-019-1897-5. Sun, X., Meyers, E.N., Lewandoski, M., and Martin, G.R. (1999). Targeted disruption of Fgf8 causes failure of cell migration in the gastrulating mouse embryo. Genes Dev. 13, 1834–1846. Thiagalingam, A., De Bustros, A., Borges, M., Jasti, R., Compton, D., Diamond, L., Mabry, M., Ball, D.W., Baylin, S.B., and Nelkin, B.D. (1996). RREB-1, a novel zinc finger protein, is involved in the differentiation response to Ras in human medullary thyroid carcinomas. Mol. Cell. Biol. 16, 5335–5345.
Oft, M., Peli, J., Rudaz, C., Schwarz, H., Beug, H., and Reichmann, E. (1996). TGF-beta1 and Ha-Ras collaborate in modulating the phenotypic plasticity and invasiveness of epithelial tumor cells. Genes Dev. 10, 2462–2477.
Yamaguchi, T.P., Harpal, K., Henkemeyer, M., and Rossant, J. (1994). fgfr-1 is required for embryonic growth and mesodermal patterning during mouse gastrulation. Genes Dev. 8, 3032–3044.
Su, J., Morgani, S.M., David, C.J., Wang, Q., Er, E.E., Huang, Y.-H., Basnet, H., Zou, Y., Shu, W., Soni, R.K., et al. (2020). TGF-b orchestrates fibrogenic and developmental EMTs via the RAS
Yang, J., and Weinberg, R.A. (2008). Epithelialmesenchymal transition: at the crossroads of development and tumor metastasis. Dev. Cell 14, 818–829.
260 Developmental Cell 52, February 10, 2020
Spotlight RREB1 Integrates TGF-b and RAS Signals to Drive EMT Laurent Fattet1 and Jing Yang1,* 1Department of Pharmacology, Moores Cancer Center, University of California, San Diego, 3855 Health Sciences Drive, La Jolla, CA 92093, USA *Correspondence: [email protected] https://doi.org/10.1016/j.devcel.2020.01.020
TGF-b is long known to require Ras activation to induce EMT. In a recent issue of Nature, Massague´ and colleagues (Su et al., 2020) identify RAS-responsive element binding protein 1 (RREB1) as a critical integrator of TGF-b and Ras signals during both developmental and cancer EMT programs. TGF-b is a key inducer of EMT during various developmental and pathogenic EMT events. A large body of literature also show that the ability of TGF-b to induce EMT requires activation of Ras and its downstream MAPK (David et al., 2016; Horiguchi et al., 2009; Janda et al., 2002; Oft et al., 1996). However, how Ras and TGF-b signals are integrated into the EMT program is unknown. A recent study published in Nature by Su et al. (2020) identifies RAS-responsive element binding protein 1 (RREB1), a RAS transcriptional effector, as the key molecular integrator of Ras and TGF-b signals to induce EMT (Su et al., 2020). As TGF-b induces a lethal EMT in pancreatic adenocarcinoma (PDA) carrying KRAS oncogenic mutations (David et al., 2016), the authors sought to determine the common actors downstream of both pathways by analyzing the DNA binding motifs of TGF-b-activated transcription factors SMAD2/3 that also depend on oncogenic KRAS. ChIP-seq data, together with shRNA screening, identified zinc fingerscontaining protein RREB1, a poorly studied transcription factor previously involved in PDAC development (Kent et al., 2013; Thiagalingam et al., 1996), as a RAS-regulated SMAD cofactor. Further biochemical analyses demonstrated that ectopically expressed RREB1 interacts with and shares a similar genome-binding pattern as SMAD2/3, in a MAPK-dependent manner. Mutagenesis of RREB1 showed that phosphorylation of Ser-161 and Ser-970 by ERK mediates RREB1 effects during TGFb-induced EMT. Interestingly, knockout of RREB1 prevented the induction of EMT by TGF-b as shown by a decreased binding of SMAD2/3 to the promoters of
TGF-b-responsive genes SNAI1 and HAS2, and rescued expression of RREB1 could restore the induction of these genes by TGF-b. The authors next asked which EMT genes are regulated by RREB1 to coordinate the TGF-b and RAS signaling pathways. Gene ontology analysis of TGF-b response genes in an oncogenic RAS context highlighted genes involved in cell adhesion, migration, and EMT. A particular interest was given to a set of genes encoding extracellular matrix (ECM) components (laminins, collagens, proteases) or regulating ECM production and deposition by mesenchymal cells during fibrosis (such as IL-11, CTGF, WISP1, and PDGFB), as their expression is RREB1-dependent. Interestingly, depletion of EMT transcription factors SNAI1 or ZEB1 inhibited EMT but not the fibrogenic gene responses, suggesting that these two processes could be differentially controlled downstream of RREB1. In line with these results, the authors extended their findings to the breast tissue to show that TGF-b-induced EMT in normal mammary epithelial cells requires ERK-dependent activation of RREB1. To determine whether RREB1 plays a conserved role in mediating developmental EMT events, the authors performed RNA-seq analysis of WT and RREB1-KO embryonic stem (ES) cells upon induced differentiation in embryoid bodies. They found that RREB1 is required for proper differentiation as RREB1-null EBs failed to activate gene signatures associated with stem cell differentiation and EMT. Further analysis of the chromatin state by ATAC-seq suggested that SMAD2/3 and RREB1 accessibility to regulatory sequences of genes
regulating EMT might be context-dependent and vary for the induction of a cancer or a developmental EMT. The authors further showed through elegant in vivo analysis of chimeric embryos containing RREB1 / ES cells that the majority of mutated chimeras had failure in the gastrulation process. Interestingly, the authors analyzed the resulting embryos and found severe defects in EMT activation during gastrulation, as RREB1 depletion prevented the E- to N-cadherin switch observed in WT embryos and required for proper gastrulation. Intrigucells expressed ingly, RREB1 / Brachyury and Snai1 normally, with some chimeras displaying a coexpression of both E- and N-cadherin, or an aberrant expression of N-cadherin in the posterior epiblast, suggesting a more complex outcome than a total blockade of EMT. Consistent with this, previous studies in embryos lacking FGFR1 or FGF8 (and thus presumably defective in Ras activation) show that epiblasts succeeded in undergo EMT and gastrulate morphologically, but the resulting mesoderm cells failed to migrate (Sun et al., 1999; Yamaguchi et al., 1994). Together, these studies suggest that RREB1 may control a subset of the EMT program during gastrulation. This study by Su et al. (2020) identifies RREB1 as a key player that integrates both TGF-b and Ras signals to induce EMT during both gastrulation and pancreatic cancer development. Future studies are needed to understand how Ras signaling activates the cooperative binding of SMAD2/3 and RREB1 to their target DNA sequences. While the EMT program is thought to be largely conserved during development and cancer progression
Developmental Cell 52, February 10, 2020 ª 2020 Elsevier Inc. 259
Developmental Cell
Spotlight (Yang and Weinberg, 2008), comparison between the cancer and gastrulation EMT programs raises important questions on how RREB1 exerts some different roles during these two processes. Detailed analysis should aim to determine how the chromatin accessibility by the RREB1-SMAD complex differs in models of cancer and developmental EMTs, which could help to reveal these differential EMT transcription responses under developmental versus pathological contexts.
REFERENCES David, C.J., Huang, Y.H., Chen, M., Su, J., Zou, Y., Bardeesy, N., Iacobuzio-Donahue, C.A., and Massague´, J. (2016). TGF-b Tumor Suppression through a Lethal EMT. Cell 164, 1015–1030.
Horiguchi, K., Shirakihara, T., Nakano, A., Imamura, T., Miyazono, K., and Saitoh, M. (2009). Role of Ras signaling in the induction of snail by transforming growth factor-beta. J. Biol. Chem. 284, 245–253. Janda, E., Lehmann, K., Killisch, I., Jechlinger, M., €nert, Herzig, M., Downward, J., Beug, H., and Gru S. (2002). Ras and TGF[beta] cooperatively regulate epithelial cell plasticity and metastasis: dissection of Ras signaling pathways. J. Cell Biol. 156, 299–313. Kent, O.A., Fox-Talbot, K., and Halushka, M.K. (2013). RREB1 repressed miR-143/145 modulates KRAS signaling through downregulation of multiple targets. Oncogene 32, 2576–2585.
effector RREB1. Nature. https://doi.org/10.1038/ s41586-019-1897-5. Sun, X., Meyers, E.N., Lewandoski, M., and Martin, G.R. (1999). Targeted disruption of Fgf8 causes failure of cell migration in the gastrulating mouse embryo. Genes Dev. 13, 1834–1846. Thiagalingam, A., De Bustros, A., Borges, M., Jasti, R., Compton, D., Diamond, L., Mabry, M., Ball, D.W., Baylin, S.B., and Nelkin, B.D. (1996). RREB-1, a novel zinc finger protein, is involved in the differentiation response to Ras in human medullary thyroid carcinomas. Mol. Cell. Biol. 16, 5335–5345.
Oft, M., Peli, J., Rudaz, C., Schwarz, H., Beug, H., and Reichmann, E. (1996). TGF-beta1 and Ha-Ras collaborate in modulating the phenotypic plasticity and invasiveness of epithelial tumor cells. Genes Dev. 10, 2462–2477.
Yamaguchi, T.P., Harpal, K., Henkemeyer, M., and Rossant, J. (1994). fgfr-1 is required for embryonic growth and mesodermal patterning during mouse gastrulation. Genes Dev. 8, 3032–3044.
Su, J., Morgani, S.M., David, C.J., Wang, Q., Er, E.E., Huang, Y.-H., Basnet, H., Zou, Y., Shu, W., Soni, R.K., et al. (2020). TGF-b orchestrates fibrogenic and developmental EMTs via the RAS
Yang, J., and Weinberg, R.A. (2008). Epithelialmesenchymal transition: at the crossroads of development and tumor metastasis. Dev. Cell 14, 818–829.
260 Developmental Cell 52, February 10, 2020