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TNFAIP8 Is a Novel Phosphoinositide-Binding Inhibitory Regulator of Rho Gtpases That

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TNFAIP8 Is a Novel Phosphoinositide-Binding Inhibitory Regulator of Rho Gtpases That bioRxiv preprint doi: https://doi.org/10.1101/2021.03.26.437243; this version posted March 26, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 TNFAIP8 is a novel phosphoinositide-binding inhibitory regulator of Rho GTPases that 2 promotes cancer cell migration 3 Mei Lin1,2,3, Honghong Sun1, Svetlana A. Fayngerts1, Peiwei Huangyang2, Youhai H. Chen1,2,3 4 1Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University 5 of Pennsylvania, Philadelphia, PA, USA. 6 2Cell and Molecular Biology Graduate Program, Perelman School of Medicine, University of 7 Pennsylvania, Philadelphia, PA, USA. 8 3Correspondence should be addressed to M.L. ([email protected]) or Y.H.C. 9 ([email protected]). 1 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.26.437243; this version posted March 26, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 10 SUMMARY 11 More than half of human tumors exhibit aberrantly dysregulated phosphoinositide 12 signaling, yet how this is controlled remains not fully understood. While somatic mutations 13 of PI3K, PTEN and Ras account for many cases of the hyperactivated lipid signals, other 14 mechanisms for these dysfunctions in cancer are also being discovered. We report here that 15 TNFAIP8 interacts with PtdIns(4,5)P2 and PtdIns(3,4,5)P3 and is likely to be hijacked by 16 cancer cells to facilitate directional migration during malignant transformation. TNFAIP8 17 maintains the quiescent cellular state by sequestering inactive Rho GTPases in the cytosolic 18 pool, which can be set free upon chemoattractant activation at the leading edge. 19 Consequently, loss of TNFAIP8 results in severe defects of chemotaxis and adhesion. Thus, 20 TNFAIP8, whose expression can be induced by inflammatory cytokines such as TNFa from 21 tumor microenvironment, represents a molecular bridge from inflammation to cancer by 22 linking NF-κB pathway to phosphoinositide signaling. Our study on the conserved 23 hydrophobic cavity structure will also advise in silico drug screening and development of 24 new TNFAIP8-based strategies to combat malignant human diseases. 25 INTRODUCTION 26 Cancer cell locomotion is an important form of eukaryotic cell migration. Failure to treat cancer 27 metastases from solid tumors in clinic has been responsible for the majority of patient deaths1. 28 The cascades for metastasis are complicated and encompass migration of cancerous cells away 29 from the primary site, intravasation into the circulation system, extravasation to secondary 30 tissues, and formation of distant metastatic tumors2. Cell migration is a pivotal step during these 31 metastatic processes. In order to move in one direction, the cells must first form a defined front 2 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.26.437243; this version posted March 26, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 32 and rear. This polarity is characterized by asymmetrical activation of proteins such as PI3Ks, 33 Rho GTPases, and actin regulatory proteins at the leading and trailing edges. Despite intensive 34 studies, how the shallow gradients of chemoattractants translate into axes of cell polarization 35 during chemotaxis is not well understood. Phosphatidylinositol (PtdIns) is a unique membrane 36 phospholipid that can be phosphorylated at the 3, 4 and 5 positions of the inositol ring to 37 generate seven phosphoinositides, among which PtdIns(4,5)P2 (PIP2) and PtdIns(3,4,5)P3 (PIP3) 3 38 are important lipid second messengers . PtdIns(4,5)P2 is the most abundant phosphoinositide and 39 binds to proteins important for actin polymerization, focal contacts formation, and cell adhesion. 40 PtdIns(3,4,5)P3 is highly enriched in the leading edge and controls cell migration by promoting 41 actin polymerization through both GTPase-dependent and independent mechanisms. In addition, 42 PI3K-mediated generation of PtdIns(3,4,5)P3 and the subsequent activation of Akt and its 43 downstream cascades (e.g., mTORC1) compose the signaling axis controlling cancer cell 44 survival and growth. Rho small GTPases are conformationally regulated by the binding of GTP 45 and GDP, and they are activated by guanine nucleotide exchange factors (GEFs) and inactivated 46 by GTPase activating proteins (GAPs). Moreover, Rho family can bind to guanine nucleotide 47 dissociation inhibitors (GDIs), which consist of RhoGDIα (ubiquitously expressed), RhoGDIβ 48 (hematopoietic tissue-specific expression), and RhoGDIγ (preferentially expressed in brain, 49 kidney, lung, pancreas, and testis)4. Of the at least 20 Rho family proteins identified so far in 50 human, Rac1/2, Cdc42, and RhoA/B have been the most widely studied for their importance in 51 cell migration. Rac and Cdc42 are mainly active at the leading edge, contributing to spatially 52 confined actin polymerization and protrusion via Arp2/3 and WASP/WAVE dependent 53 mechanisms, while Rho is involved in rear retraction5. It remains to be characterized how these 3 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.26.437243; this version posted March 26, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 54 molecular machineries are coordinated temporally and spatially to enable cells to sense 55 chemokine gradients, induce polarized morphology, and rapidly reorganize the cytoskeleton. 56 The TIPE (tumor necrosis factor-a-induced protein 8 (TNFAIP8)-like, or TNFAIP8L) 57 family of proteins have been implicated in inflammation and tumorigenesis. There are four 58 highly homologous mammalian TIPE family members: TNFAIP8, TIPE1 (TNFAIP8L1), TIPE2 59 (TNFAIP8L2), and TIPE3 (TNFAIP8L3). Existing evidences support that TNFAIP8 is an 60 oncogene, which is found to be frequently over-expressed in a wide range of human cancers and 61 promote tumor metastasis6–14. TIPE2 is almost exclusively expressed in hematopoietic cells and 62 is initially identified as one of the most highly upregulated genes in the spinal cord of 63 experimental autoimmune encephalomyelitis (EAE) mice15. TIPE2 is reported to be a negative 64 regulator of innate and adaptive immunity and essential for maintaining immune homeostasis16,17. 65 TIPE2 can direct myeloid cell polarization18 and play a redundant role with TNFAIP8 in 66 controlling murine lymphocyte migration during neural inflammation19. TIPE3 is preferentially 67 expressed in secretory epithelial tissues20, and its expression is also markedly upregulated in 68 cervical, colon, lung, and esophageal cancers21. The high-resolution crystal structures of murine 69 TNFAIP8 C165S mutant, human TIPE2, and human TIPE3 have been recently resolved21–23, and 70 they reveal a conserved TIPE homology (TH) domain composed of a large hydrophobic central 71 cavity. Although the outer surface is highly charged, the central cavity is predominantly 72 hydrophobic, implicating lipid binding capacity. In this report, we describe TNFAIP8 as a 73 phosphoinositide-binding negative regulator of Rho GTPases and examine its role in cancer cell 74 migration. 4 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.26.437243; this version posted March 26, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 75 RESULTS 76 Defective chemotaxis and adhesion in TNFAIP8-deficient differentiated HL-60 cells 77 TIPE proteins are newly discovered risk factors for inflammation and cancer (Supplementary 78 Fig. 1a). TNFAIP8, the founding member of this family, is originally identified as one of the 79 most differentially expressed genes in primary and matched metastatic head and neck squamous 80 cell carcinoma (HNSCC) from the same patient24,25. To investigate the role of TNFAIP8 in 81 directed cell migration, we used both loss- and gain-of-function approaches to manipulate 82 TNFAIP8 expression in HL-60 (Fig. 1a), a human promyelocytic leukemia cell line that can be 83 conveniently differentiated into neutrophil-like cells in vitro as a powerful model for eukaryotic 84 chemotaxis26. Human TNFAIP8 has six transcript variants (Variant 1 to 6) with different 85 transcription start sites/exons and can be translated to four protein isoforms (Isoform a to d). 86 Among these, Variants 3 to 6 were minimally expressed in normal or cancer tissues according to 87 recent RNA-seq analysis27, whereas Variant 2 was frequently over-expressed and Variant 1 was 88 commonly downregulated in multiple human malignancies. By targeting the shared exon with 89 CRISPR/Cas9, we validated the complete knockout of all TNFAIP8 isoforms in the established 90 HL-60 clonal cell lines (Supplementary Fig. 1b–d). To investigate the effects of TNFAIP8 91 deficiency in differentiated HL-60 (dHL-60) cells, we first tested chemoattractant EC50 and 92 verified that 10 nM fMLP was optimal for migration with an around eight-fold increase in cell 93 numbers through the Transwell filters (Supplementary Fig. 2a), while CXCL8 induced a two- 94 fold increase within a wide range of concentrations (Supplementary Fig. 2b). To demonstrate 95 whether TNFAIP8 was necessary and sufficient in regulating dHL-60 chemotaxis, TNFAIP8 96 expression was rescued by lentiviral titration and infection into knockout cells to the endogenous 97 level (Fig.
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