1 Somatic mutational landscapes of adherens junctions and their 2 functional consequences in cutaneous melanoma development 3 4 Praveen Kumar Korla,1 Chih-Chieh Chen,2 Daniel Esguerra Gracilla,1 Ming-Tsung Lai,3 Chih- 5 Mei Chen,4 Huan Yuan Chen,5 Tritium Hwang,1 Shih-Yin Chen,4,6,* Jim Jinn-Chyuan Sheu1,4, 6-9,* 6 1Institute of Biomedical Sciences, National Sun Yat-sen University, Kaohsiung 80242, Taiwan; 7 2Institute of Medical Science and Technology, National Sun Yat-sen University, Kaohsiung 80424, 8 Taiwan; 3Department of Pathology, Taichung Hospital, Ministry of Health and Welfare, Taichung 9 40343, Taiwan; 4Genetics Center, China Medical University Hospital, Taichung 40447, Taiwan; 10 5Institute of Biomedical Sciences, Academia Sinica, Taipei 11574, Taiwan; 6School of Chinese 11 Medicine, China Medical University, Taichung 40402, Taiwan; 7Department of Health and 12 Nutrition Biotechnology, Asia University, Taichung 41354, Taiwan; 8Department of 13 Biotechnology, Kaohsiung Medical University, Kaohsiung 80708, Taiwan; 9Institute of 14 Biopharmaceutical Sciences, National Sun Yat-sen University, Kaohsiung 80242, Taiwan 15 16 PKK, CCC and DEG contributed equally to this study. 17 *Correspondence to: Dr. Shih-Yin Chen ([email protected]) at Genetics Center, China 18 Medical University Hospital, Taichung, 40447, TAIWAN; or Dr. Jim Jinn-Chyuan Sheu 19 ([email protected]) at Institute of Biomedical Sciences, National Sun Yat-sen 20 University, Kaohsiung City 80424, TAIWAN. 21 22 Running title: mutational landscape of cadherins in melanoma 1 23 Abstract 24 Cell-cell interaction in skin homeostasis is tightly controlled by adherens junctions (AJs). 25 Alterations in such regulation lead to melanoma development. However, mutations in AJs and 26 their functional consequences are still largely unknown. 27 Methods: Cadherin mutations in skin cutaneous melanoma were identified using sequencing data 28 from TCGA dataset, followed by cross-validation with data from non-TCGA cohorts. Mutations 29 with significant occurrence were subjected to structural prediction using MODELLER and 30 functional protein simulation using GROMACS software. Neo-antigen prediction was carried out 31 using NetMHCpan tool. Cell-based fluorescence reporter assay was used to validate β-catenin 32 activity in the presence of cadherin mutations. Clinical significance was analyzed using datasets 33 from TCGA and other non-TCGA cohorts. Targeted gene exon sequencing and 34 immunofluorescence staining on melanoma tissues were performed to confirm the in silico 35 findings. 36 Results: Highly frequent mutations in type-II classical cadherins were found in melanoma with 37 one unique recurrent mutation (S524L) in the fifth domain of CDH6, which potentially destabilizes 38 Ca2+-binding and cell-cell contacts. Mutational co-occurrence and physical dynamics analyses 39 placed CDH6 at the center of the top-four mutated cadherins (core CDHs; all type-II), suggesting 40 altered heterophilic interactions in melanoma development. Mutations in the intracellular domains 41 significantly disturbed CDH6/β-catenin complex formation, resulting in β-catenin translocation 42 into cytosol or nucleus and dysregulation of canonical Wnt/β-catenin signaling. Although 43 mutations in core CDH genes correlated with advanced cancer stages and lymph node invasion, 44 the overall and disease-free survival times in those patients were longer in patients with wild-type. 45 Peptide/MHC-I binding affinity predictions confirmed overall increased neo-antigen potentials of 2 46 mutated cadherins, which associated with T-lymphocyte infiltration and better clinical outcomes 47 after immunotherapy. 48 Conclusion: Changes in cell-cell communications by somatic mutations in AJ cadherins function 49 as one of mechanisms to trigger melanoma development. Certain mutations in AJs may serve as 50 potential neo-antigens which conversely benefit patients for longer survival times. 51 52 Keywords: adherens junctions (AJ), cadherin-6 (CDH6), somatic mutation, melanoma, neo- 53 antigen 54 55 Graphical Abstract 56 57 Heterophilic interactions between melanocyte and keratinocyte maintain skin homeostasis. UV- 58 induced CDH mutations cause contact control loss from keratinocyte and melanomagenesis. 59 During metastasis, mutation-derived Neo-Ags boost T-lymphocytes and activate anti-cancer 60 immunity. 3 61 Introduction 62 Cutaneous melanoma is the most dangerous form of skin cancer with high invasive and metastatic 63 potentials, resulting in more than 55,000 deaths per year worldwide [1] . Even though the cure rate 64 for patients with localized tumors is high, the overall five-year survival rates could be very low 65 (<20%) for those in whom the spread has occurred, indicating critical roles of cell-cell and cell- 66 matrix crosstalk in disease development. In normal epidermis, melanocytes or their stem cells form 67 multiple contacts with keratinocytes, which in turn establish regulatory networks in paracrine 68 manners to fine-tune melanocyte growth and epidermal homeostasis [2, 3]. During 69 melanomagenesis, melanoma cells alter such regulatory contacts via extracellular matrix 70 remodeling and then promote dermal invasion by activating inflammation-dependent cell 71 migration/translocation [4]. These findings suggest that loss of the control by keratinocytes enables 72 melanocyte undergoing uncontrolled cell growth, leading to cell transformation and melanoma 73 development. 74 Adherens junctions (AJs) are cadherin-based dynamic structures which link the physical 75 contact from the extracellular matrix to cytoskeleton networks via complex formation between 76 cadherins and catenins (, and p120-catenin). Dissociation of -catenin by growth factor 77 stimulation leads to loss of cadherin-mediated cell-cell contact and increase of -catenin levels in 78 cytoplasm and nucleus, which subsequently promotes cell proliferation via activating TCF/Wnt 79 signaling [5, 6]. On the other hand, AJs also regulate stress-sensing and force-transmission through 80 signaling cascades, known as mechanotransduction [7, 8]. Clustering of core cadherin-catenin 81 complexes can further strengthen cell-cell adhesion, allowing cadherins to nucleate signaling hubs 82 and establish the regulatory Hippo network [9, 10]. Breakdown of Hippo network by up-regulating 83 expression and nuclear translocation of YAP/TAZ, the nuclear mechanosensor/transducer, was 4 84 found during melanoma progression, resulting in invasion, metastasis and drug resistance [11-14]. 85 Accordingly, AJ-mediated mechanical and cytoskeletal signaling are important for melanoma to 86 create 3D microenvironment for further progression. 87 Recent studies by using lineage-tracing approach or global gene expression analyses 88 suggested that skin cutaneous melanoma cells may originate from adult melanocyte stem cells and 89 show feature properties of multipotent progenitors [2, 4]. Within the specialized anatomical 90 microenvironments, both hemophilic (via type-I cadherins) and heterophilic (via type-II cadherins) 91 cell-cell interactions are required for melanocyte stem cells to develop new cell partnerships with 92 stroma and/or endothelial cells. For examples, cadherin switching from N-cadherin (CDH2) to K- 93 cadherin (CDH6) was found critical for neural crest cell formation, emigration and detachment 94 from the neural tube to be melanocyte [15, 16]. Furthermore, down-regulation of key junction 95 proteins for communication between melanocytes and keratinocytes frequently occur during 96 melanoma development, including E-cadherin (CDH1) and P-cadherin (CDH3) [2, 17, 18]. 97 Alterations in such interacting network enable melanocyte stem cells to proliferate and migrate out 98 of the niche after activation or transformation by extrinsic stimuli like UV exposure [2, 4]. 99 With advanced whole genome sequencing and other integrative technologies, a set of major 100 driver mutations in cutaneous melanoma, e.g. BRAF, RAS and NF1, have been discovered which 101 contribute to activation of mitogen-activated protein kinase and phosphoinositol kinase signaling 102 [19, 20]. Notably, immune signatures with higher lymphocyte infiltration have been correlated 103 with improved survival outcomes but are irrespective of the genomic subtypes in patients [19, 21]. 104 These data indicate the need to further explore certain genes that determine microenvironment 105 remodeling and lymphocyte recruitment through cell-cell/cell-matrix interactions. In this study, 5 106 we aimed to study somatic mutational landscapes of key AJ genes in melanoma and characterize 107 functional impacts of those mutations with molecular and clinical meanings. 6 108 Materials and Methods 109 Data collection and functional relevance with mutations in classical cadherin genes 110 Somatic mutation, gene expression and clinical information data of individual cadherins in skin 111 cutaneous melanoma were collected from cBioPortal databank (https://www.cbioportal.org/) Data 112 from TCGA cohort (TCGA, PanCancer Atlas; n = 448) were utilized as the studying cohort, and 113 the resultant data were validated by using data from non-TCGA cohort (n = 396). The functional 114 interactome among classical cadherins were analyzed by using STRING protein-protein 115 interactome database (http://string-db.org/). Oncoprinter and Mutation mapper tools in cBioportal 116 database were performed to analyze mutation types, hotspot mutation sites, and the conserved 117 mutation sites in cadherin and CTNNB1