ARTICLE https://doi.org/10.1038/s41467-019-12832-z OPEN ZEB1/NuRD complex suppresses TBC1D2b to stimulate E-cadherin internalization and promote metastasis in lung cancer Roxsan Manshouri1,2, Etienne Coyaud3, Samrat T. Kundu1,2, David H. Peng1,2, Sabrina A. Stratton 4, Kendra Alton 4, Rakhee Bajaj 1,2, Jared J. Fradette1,2, Rosalba Minelli5, Michael D. Peoples5, Alessandro Carugo6, Fengju Chen7, Christopher Bristow6, Jeffrey J. Kovacs6, Michelle C. Barton 4, Tim Heffernan6, Chad J. Creighton 7, Brian Raught3 & Don L. Gibbons1,2* 1234567890():,; Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death worldwide, due in part to the propensity of lung cancer to metastasize. Aberrant epithelial-to- mesenchymal transition (EMT) is a proposed model for the initiation of metastasis. During EMT cell-cell adhesion is reduced allowing cells to dissociate and invade. Of the EMT- associated transcription factors, ZEB1 uniquely promotes NSCLC disease progression. Here we apply two independent screens, BioID and an Epigenome shRNA dropout screen, to define ZEB1 interactors that are critical to metastatic NSCLC. We identify the NuRD complex as a ZEB1 co-repressor and the Rab22 GTPase-activating protein TBC1D2b as a ZEB1/NuRD complex target. We find that TBC1D2b suppresses E-cadherin internalization, thus hindering cancer cell invasion and metastasis. 1 Department of Thoracic/Head and Neck Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA. 2 Department of Molecular and Cellular Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA. 3 Department of Medical Biophysics, Princess Margaret Cancer Centre, University of Toronto, Toronto, ON M5S 1A1, Canada. 4 Department of Epigenetics and Molecular Carcinogenesis, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA. 5 Department of Cancer Genomics, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA. 6 Institute for Applied Cancer Science, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA. 7 Department of Medicine and Dan L. Duncan Comprehensive Cancer Center, Baylor College of Medicine, Houston, TX, USA. *email: [email protected] NATURE COMMUNICATIONS | (2019) 10:5125 | https://doi.org/10.1038/s41467-019-12832-z | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-019-12832-z pithelial-to-mesenchymal transition (EMT) is the process to identify proteins in stable association with ZEB16,19; however, by which epithelial cells lose their apical-basal polarity and a high-confidence ZEB1 protein interactome has yet to be E 1,2 concomitantly acquire a migratory phenotype . Epithelial established. To elucidate ZEB1 transcriptional co-regulators we cells undergo EMT during normal embryonic development, applied the BioID screening method to identify ZEB1 interacting allowing cells to migrate and differentiate, however EMT also partners20,21. This technique harnesses an abortive E. coli biotin facilitates tumor progression. The metastatic cascade represents a ligase (BirA-R118, denoted BirA*) fused to a protein of interest. multi-step process and EMT empowers cancer cells to dis- BirA* can generate biotinoyl-AMP, but has lost the ability to seminate at the invasive fronts of tumors, intravasate, survive in interact with this intermediate. Highly reactive biotinoyl-AMP is the circulation, and extravasate into distant tissues. EMT is thus released into the vicinity of the bait protein, and reacts with activated by multiple transcription factors (e.g., TWIST, SNAIL, amine groups on nearby polypeptides. Biotinylated proteins can SLUG, ZEB1/2), however in lung adenocarcinoma, zinc finger E- then be isolated with streptavidin and identified using mass box binding homeobox 1 (ZEB1, δEF1, zfhx1a) expression is an spectrometry. In contrast to traditional AP-MS, this methodology early and pivotal event in pathogenesis; conferring metastatic allows for the elucidation of interactions that may be lost during properties, treatment resistance, and correlating with poor stringent lysis and washing (Fig. 1a). prognosis3–7. Hence, understanding how ZEB1 functions pro- A FlagBirA* tag was fused in-frame to either the N-terminus or vides promise for innovative therapeutic strategies to improve C-terminus of human ZEB1 and stably integrated into HEK293 lung cancer patient outcome, which remains the leading cause of Flp-In cells, under the control of a tetracycline-inducible cancer related death8. promoter. Two isogenic pools were generated for each bait ZEB1 orchestrates EMT through repression of epithelial genes protein, representing biological replicates. We performed RT- such as E-cadherin, a central component in adherens junctions, qPCR and immunoblot to validate ZEB1 upregulation and to and the microRNA-200 family. ZEB1 represses transcription of determine the effect on the expression of the known ZEB1 target target genes through the epigenetic regulation of promoter gene, E-cadherin. Upon tetracycline induction, exogenous chromatin architecture9. Densely arranged heterochromatin expression of ZEB1 consistently produced E-cadherin repression regions restrict the access of the transcription machinery thereby by both mRNA and protein expression (Fig. 1b, c and limiting the expression of the underlying gene10. ZEB1 enhances Supplementary Fig. 1a, b). To additionally assess the functionality heterochromatinization at target gene promoters by increasing of the FlagBirA*-tagged human ZEB1 proteins, we expressed the H3K27 deacetylation and tri-methylation9,11. Class I and II constructs in the murine 393P cell line (Supplementary Fig. 1c). HDAC inhibitors have been shown to have efficacy in restoring Both tagged ZEB1 constructs significantly upregulated the ZEB1 target gene expression, but the mechanism behind this migratory and invasive potential of the 393P cell line, confirming association remains incomplete11. ZEB1 can interact with the C- that the FlagBirA* tag did not hinder the biologic function of terminal binding protein (CtBP) corepressors to aid in the ZEB1 (Supplementary Fig. 1d). The expression of FlagBirA* recruitment of the CoREST complex in pancreatic tumors12–14, protein alone (denoted as –) had no effect on E-cadherin however subsequent work has proposed that ZEB1 represses expression levels or invasive potential in these assays. targets via CtBP-independent mechanisms in prostate cancer- Following validation of the biological activity of the N- suggesting that ZEB1 binding partners may be context specific15. terminal and C-terminal tagged ZEB1 proteins, cell pools were To expand the repertoire of treatments for metastatic NSCLC, incubated with tetracycline, biotin and the proteasome inhibitor we utilized two independent screening approaches to identify MG-132. Biotinylated proteins were isolated with streptavidin- ZEB1 interactors that are essential to cancer cell survival. Here we sepharose beads, washed, and subjected to trypsin proteolysis. describe an interaction with the Nucleosome Remodeling and The released peptides were identified using nanoflow liquid Deacetylase (NuRD) complex. The NuRD complex is one of four chromatography–electrospray ionization–tandem mass spectro- major chromatin remodeling complexes16,17. The catalytic core of metry (nLC–ESI–MS/MS). Using cells expressing the FlagBirA* NuRD complexes consists of the histone deacetylases (HDAC1/2) tag alone for comparison, the computational tool Significance and the chromodomain helicase DNA-binding proteins (CHD4/ Analysis of INTeractome22 was used to assign confidence scores Mi-2β and CHD3/Mi-2α), which also act as scaffolds for the other to individual protein-protein interactions with ZEB1. Proteins complex members. The current understanding of the biochemical confidently identified with a Max SAINT score >0.8, identified and structural features of NuRD components suggests that with >2 unique spectra in both analyses, and with at least combinatorial assembly of these factors confers functional spe- 2.5-fold greater peptide counts in the FlagBirA*-ZEB1 samples cificity to the NuRD complex. For instance, CHD3 and CHD4 are than in FlagBirA* samples, yielded a high-confidence list of 68 found in mutually exclusive NuRD complexes with non- ZEB1 interacting proteins (Table 1, extended list found in overlapping functions. Multiple biological functions are regu- Supplementary Data 1). Notably, BioID identified an association lated through NuRD chromatin modification, and alterations in between ZEB1 and several HDAC1 and HDAC2 containing co- NuRD complex activity have been implicated in a broad range of repressor complexes: Sin3, CoREST, and the NuRD complex. In human diseases, including cancer18. fact, all core members of the NuRD complex were identified as In this study, we find that ZEB1 recruits the NuRD complex in the top-ranking hits in the BioID screen. Several members of the NSCLC and link this association to the repression of known ZEB1 NuRD complex were previously identified as ZEB1 interactors target genes. Furthermore, we exploit the functional cooperation (Supplementary Fig. 1e). of ZEB1 and the NuRD complex to identify metastasis suppressors in NSCLC and establish the GTPase activating protein (GAP) TBC1D2b as a ZEB1/NuRD complex target gene. Mechanistically, Loss-of-function screen identifies vulnerabilities in NSCLC.To we find that aberrant suppression of TBC1D2b contributes to the ascertain the significance of these ZEB1 interactors as therapeutic