The Role of Microenvironment in Development of Skin Cancer and Metastasis Arash Chitsazan BSc MSc A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2018 Faculty of Medicine Abstract The overarching aim of this thesis is to study the mechanisms by which giant congenital nevi form and to study if such mechanisms are targetable. Virtually nothing is known about genes conferring susceptibility to the development of giant congenital nevi, largely untreatable lesions which increase the risk of patients for development of neurocutaneous melanocytoma and malignant melanoma. To discover such genes, we utilized a nevus- prone transgenic mouse model together with the Collaborative Cross (CC), a genetic resource for discovery of genes for complex diseases. It not only allows gene discovery, but also a systems genetics approach that enabled us to determine how the gene functions in vivo to modify the nevogenesis. We also show that the identity of somatic oncogenic mutation (e.g. in NRAS) does not always define the histopathological subtype of nevus, as is generally thought. In sum, we have performed an unbiased genetic screen and discovered a novel gene (Cdon), a dependence receptor of sonic hedgehog (Shh) as the modifier gene for nevus exacerbation. We then used three different mouse models to validate it as the causal gene and determine its mechanism of action. We also show that Shh pathway components are active in the epidermis adjacent to human congenital nevi (Chapter 2 and 3). In summary this is the first time a gene has been successfully mapped and functionally validated using CC. In Chapter 4 we discuss the result from an unbiased genetic screen to map the modifier gene for development of sub-cutaneous (hypodermis) metastasis using 45 CC strains. Gja1 which encodes a gap junction subunit connexion 43 (Cx43) is the best candidate. Our gene expression and protein expression studies suggest that Gja1 could be a strong candidate for further study of sub-cutaneous metastasis. In Chapter 5 we went on to study the gene expression of normal skin, melanocytoma and malignant melanoma samples from our NRAS-Cdk4 mouse model to study the transformation and progression of melanocytic lesions. Dusp6 a negative regulator of MAPK pathway was highly upregulated with transformation and progression. Our protein expression study validated this result on murine and human nevi versus malignant melanoma samples. 2 Declaration by author This thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis. I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, financial support and any other original research work used or reported in my thesis. The content of my thesis is the result of work I have carried out since the commencement of my higher degree by research candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution. I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award. I acknowledge that an electronic copy of my thesis must be lodged with the University Library and, subject to the policy and procedures of The University of Queensland, the thesis be made available for research and study in accordance with the Copyright Act 1968 unless a period of embargo has been approved by the Dean of the Graduate School. I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material. Where appropriate I have obtained copyright permission from the copyright holder to reproduce material in this thesis and have sought permission from co-authors for any jointly authored works included in the thesis. 3 Publications during candidature Peer-reviewed papers: Chitsazan, A., Ferguson, B.,Villani, R., Handoko, HY., Mukhopadhyay, P., Gabrielli, B., Mooi, WJ., Soyer, HP., Lambie, D., Khosrotehrani, K., Morahan, G., and Walker, GJ. Keratinocyte Sonic Hedgehog Up-regulation Drives the Development of Giant Congenital Nevi via Paracrine Endothelin-1 Secretion. J Investigative Derma. Accepted for publication. Chitsazan, A., Ferguson, B., Ram, R., Mukhopadhyay, P., Handoko, H.Y., Gabrielli, B., Soyer, P.H., Morahan, G., and Walker, G.J. A mutation in the Cdon gene potentiates congenital nevus development mediated by NRAS(Q61K). Pigment Cell Melanoma Res. 2016;29(4):459-64 Young, A., Ngiow, S.F., Madore, J., Reinhardt, J., Landsberg, J., Chitsazan, A., Rautela, J., Bald, T., Barkauskas, D.S., Ahern, E., Huntington, ND., Schadendorf, D., Long, GV., Boyle, GM., Hölzel, M., Scolyer, RA., Smyth, MJ. Targeting Adenosine in BRAF-Mutant Melanoma Reduces Tumor Growth and Metastasis. Cancer Res. 2017;77, 4684-4696. Publications included in this thesis Chitsazan, A., Ferguson, B., Ram, R., Mukhopadhyay, P., Handoko, H.Y., Gabrielli, B., Soyer, P.H., Morahan, G., and Walker, G.J. A mutation in the Cdon gene potentiates congenital nevus development mediated by NRAS(Q61K). Pigment Cell Melanoma Res 2016;29(4):459-64.– incorporated as Chapter 2. 4 Conception and design (85%) Chitsazan A Analysis and interpretation (82.5%) Drafting and production (82.5%) Ferguson B Conception and design (2.5%) Ramesh R Analysis and interpretation (2.5%) Mukhopadhyay P Analysis and interpretation (2.5%) Handoko HY Conception and design (2.5%) Drafting and production Gabrielli B (2.5%) Soyer HP Analysis and interpretation (2.5%) Morahan G Drafting and production (5%) Walker GJ Conception and design (10%) Drafting and production (10%) Analysis and interpretation (10%) 5 Chitsazan, A., Ferguson, B.,Villani, R., Handoko, HY., Mukhopadhyay, P., Gabrielli, B., Mooi, WJ., Soyer, HP., Lambie, D., Khosrotehrani, K., Morahan, G., and Walker, GJ. Keratinocyte Sonic Hedgehog Up-regulation Drives the Development of Giant Congenital Nevi via Paracrine Endothelin-1 Secretion. J Investigative Derma. Accepted for publication. – incorporated as Chapter 3. Conception and design (85%) Chitsazan A Analysis and interpretation (83.5%) Drafting and production (80.5%) Ferguson B Conception and design (2.5%) Villani R Conception and design (2.5%) Handoko HY Conception and design (2.5%) Mukhopadhyay P Analysis and interpretation (2.5%) Gabrielli B Drafting and production (2%) Mooi WJ Drafting and production (2%) Soyer HP Analysis and interpretation (2%) Lambie D Analysis and interpretation (2%) Khosrotehrani K Drafting and production (2%) Morahan G Drafting and production (5%) Conception and Design (7.5%) Walker GJ Drafting and production (8.5%) Analysis and interpretation (10%) 6 Contributions by others to the thesis No contributions by others. Statement of parts of the thesis submitted to qualify for the award of another degree None. Research Involving Human or Animal Subjects Research involving human subjects: Approval number: 2011001201/HREC/11/QPAH/363 Approving committee: Human research ethics committee A & B Research involving animal subjects: Project number: P240 Approval number: A98004M Approving committee: QIMR Berghofer Medical Research Institute Animal Ethics Committee 7 Acknowledgements I owe a debt of gratitude to all people who have helped me during my candidacy. A huge thanks to Graeme for his meticulous mentorship and heart warming friendship. To Brian for his support, guidance and friendship. To Peter for his help and enthusiasm with diagnosis of murine melanocytic lesions. Big shut out to Walker lab, G floor staff, QIMRB histology and microscopy departments. Thanks to the UQDI and UQ for their financial support. Finally, a huge thanks to my family and friends for their love and support. 8 Financial support This project was funded by Melanoma Research Alliance, Washington DC., USA. Keywords collaborative cross, ednothelin-1, giant congenital nevus, melanocytoma, melanoma, cdon, shh, lde225, bosentan Australian and New Zealand Standard Research Classifications (ANZSRC) ANZSRC code: 111299, Oncology and Carcinogenesis not elsewhere classified, 80% ANZSRC code: 111202, Cancer Diagnosis, 10% ANZSRC code: 111205, Chemotherapy, 10% Fields of Research (FoR) Classification FoR code: 1112, Oncology and Carcinogenesis, 100% 9 List of Abbreviations used in the thesis α-Melanocyte-stimulating hormone α-MSH adrenocorticotropic hormone ADH basal cell carcinoma BCC collaborative cross CC congenital melanocytic nevi CMN dysplastic nevus syndrome DNS epithelial to mesenchymal transition EMT ednothelin-1 EDN1 endothelin receptor B EDNRB human keratin 1 HK1 hepatocyte growth factor HGF leukemia inhibitory factor LIF malignant melanoma MM non-melanoma skin cancers NMSC sonic hedgehog SHH stem cell factor SCF senescence-associated β-galactosidase SAβ-gal ultraviolet radiation UVR 10 Table of Contents 1.1 Skin ……………………………………………………………………………………......….19 Sonic hedgehog signaling in skin homeostasis ……………………………………….……..20 1.2 The melanocyte lineage in development ………………………………………………….22 1.3 Melanocyte growth in culture ……………………………………………………….………24 1.4 Non-cell autonomous effect of keratinocytes on melanocyte proliferation and differentiation ………………………………….…………………………………………..…..…24 1.5 Keratinocyte cytokines which