The establishment and maintenance of cellular polarity is crucial for tissue homeostasis, and its loss is a hallmark of carcinogenesis. This project aims to identify the role of conserved polarity proteins, master regulators of cytoarchitecture, in the initiation and progression of melanoma skin cancer. An ultimate goal is to translate the knowledge gained in this project into strategies for improved diagnosis and treatment of human melanoma.
A major goal of our research is to unveil molecular connections between the regulation of cell polarity and carcinogenesis. Conserved polarity proteins couple cell shape to control of growth, migration and differentiation. The Par3-atypical PKC-Par6 polarity complex is a key regulatory module of cellular asymmetry. We recently identified Par3 as a rheostat of epidermal homeostasis by controlling stem cell maintenance and keratinocyte differentiation (Ali et al., 2016). We unravelled underlying mechanochemical signalling networks, with Par3 coupling genome integrity and epidermal fate through shaping keratinocyte mechanics (Dias Gomes et al., 2019). Moreover, dysregulation of polarity proteins is a frequent event associated with tumor formation or progression (Mescher & Iden, 2015). In epidermal cancers, we found tumor-type dependent pro-oncongenic and tumor-suppressive functions of Par3 (Iden et al., 2012). Furthermore, we demonstrated genetic interactions of Par3 and aPKCλ in different stages of skin tumorigenesis, with Par3/aPKCλ jointly fostering tumor-promoting inflammatory signals (Vorhagen, Kleefisch et al., 2018).
Malignant melanoma is the most aggressive form of skin cancer with a rising incidence and mortality in the western world. The major risk factors are UV exposure and genetic predisposition. In this project, we ask how polarity protein signalling affects the formation and progression of metastatic melanoma. We combine mouse disease models with cell-based assays and analyses of human cancers to elucidate whether and how targeting the polarity machinery can be used to develop strategies for disease prevention and therapy.
Melanoma arises due to the malignant transformation of melanocytes located in the epidermis. Intercellular crosstalk between melanocytes and keratinocytes is essential for photoprotection of the skin. How the epidermal micro-environment controls melanoma outgrowth, however, is largely unknown. Interestingly, we identified epidermal Par3 as a non-cell autonomous suppressor of melanoma formation and metastasis. In an autochthonous malignant melanoma mouse model (HGF;Cdk4R24C) combined with epidermal loss of Par3, melanoma multiplicity as well as distant metastasis to the lung was significantly increased (Mescher, Jeong et al., 2017), revealing a previously unrecognized anti-tumorigenic function of polarity regulators in the tumor microenvironment.
To assess how epidermal Par3 counteracts melanoma we studied melanocyte behaviour upon Par3 loss in the epithelial microenvironment. Epidermal Par3 inactivation resulted in melanocyte hyperplasia. In vitro co-culture assays revealed a phenotypic switch of melanocytes accompanied by increased motility when co-cultured with Par3-deficient keratinocytes. We further demonstrated that melanocyte dedifferentiation and hyperproliferation as consequence of epidermal Par3 loss is driven by altered P-cadherin-dependent heterologous keratinocyte-melanocyte interactions. Collectively, these studies revealed that Par3 loss in the epidermis fosters a permissive niche for melanocyte transformation, invasion and metastasis (Mescher, Jeong et al., 2017).
To examine the significance of our findings for human disease we analysed tissues from melanoma patients at different disease stages. In line with our mouse data, Par3 expression in the area surrounding primary tumors inversely correlated with melanoma progression. In contrast, P-cadherin expression, which in mice was elevated following Par3 loss, increased with progressing melanoma grade (Mescher, Jeong et al., 2017).
Having identified Par3 as a strong extrinsic regulator of melanoma, we investigated putative intrinsic functions of polarity regulators in melanoma formation and progression. CRISPR-Cas9 mediated polarity protein inactivation was used to analyse growth behaviour in human melanoma cell lines. Interestingly, Par3 knock-out (BFP labelled) melanoma cells were overgrowing control melanocytes in the course of 7 days. In summary, these data support the hypothesis that the polarity protein Par3, intrinsically and extrinsically, prevents melanoma outgrowth and progression, identifying it as a relevant clinical target for melanoma treatment.
Our work revealed mechanistic insights in extrinsic roles of polarity regulators for melanocyte behaviour, melanoma formation and metastasis. Further, we provided first insights in melanoma-intrinsic functions of polarity proteins, which will be further followed by in vivo studies using a recently established metastatic melanoma model (Schlereth et al., 2015). The goal of these experiments is testing the relevance of CRISPR/Cas9-mediated deletion of polarity proteins and its consequence for primary tumor growth and metastasis in vivo. To understand how disturbed polarity gene function affects drug resistance mechanisms, we will treat polarity mutant melanoma cells with clinically used BRAF or MEK inhibitors and analyse the resulting drug sensitivity, respectively. Our long-term goal is to understand how tumors utilize polarity networks and to translate this knowledge into clinical practice for prevention and therapy of human cancers.
1. Dias Gomes M*, Letzian S*, Saynisch M, Iden S (2019). Polarity signaling ensures epidermal homeostasis by coupling cellular mechanics and genomic integrity. Nat Commun, in press. doi : 10.1038/s41467-019-11325-3 (*shared)
2. Vorhagen S*, Kleefisch D*, Persa OD, Graband A, Schwickert A, Saynisch M, Leitges M, Niessen CM# & Iden S# (2018). Shared and independent functions of aPKCλ and Par3 in skin tumorigenesis. Oncogene, doi:10.1038/s41388-018-0313-1 (shared *first, #corresp. authors).
3. Mescher M*, Jeong P*, Knapp SK, Rübsam M, Saynisch M, Kranen M, Landsberg J, Schlaak M, Mauch C, Tüting T, Niessen CM, & Iden S (2017). The epidermal polarity protein Par3 is a non-cell autonomous suppressor of malignant melanoma. J Exp Med, 214, 339-358 (*shared)
4. Ali NJA, Dias Gomes M, Bauer R, Brodesser S, Niemann C, & Iden S (2016). Essential role of polarity protein Par3 for epidermal homeostasis through regulation of barrier function, keratinocyte differentiation and stem cell maintenance. J Invest Dermat, 136, 2406-2416.
5. Schlereth SL, Iden S, Mescher M, Ksander BR, Bosch JJ, Cursiefen C, Heindl LM (2015). A novel model of metastatic conjunctival melanoma in immune-competent mice. Invest Ophthalmol Vis Sci, 56, 5965-73.
6. Mescher M & Iden S (2015). Par proteins in tumor formation and progression. Cell Polarity 2. Springer, VIII, 145-166.
Dias Gomes, M., Letzian, S., Saynisch, M., and Iden, S. (2019). Polarity signaling ensures epidermal homeostasis by coupling cellular mechanics and genomic integrity. Nat Commun 10, 3362.
Vorhagen S, Kleefisch D, Persa OD, Graband A, Schwickert A, Saynisch M, Leitges M, Niessen CM, and Iden S (2018). Shared and independent functions of aPKClambda and Par3 in skin tumorigenesis. Oncogene10.1038/s41388-018-0313-1.
Mescher M, Jeong P, Knapp SK, Rubsam M, Saynisch M, Kranen M, Landsberg J, Schlaak M, Mauch C, Tuting T, Niessen CM, and Iden S (2017). The epidermal polarity protein Par3 is a non-cell autonomous suppressor of malignant melanoma. J Exp Med 214, 339-58.
Rahn E, Thier K, Petermann P, Rubsam M, Staeheli P, Iden S, Niessen CM, and Knebel-Morsdorf D (2017). Epithelial Barriers in Murine Skin during Herpes Simplex Virus 1 Infection: The Role of Tight Junction Formation. J Invest Dermatol 137, 884-93.
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Tissue-scale control of epidermal immunity by polarity networks in homeostasis and disease
Sina Knapp (PhD student)
Annika Graband (PhD student)
Soriba Letzian (PhD student)
Martim Dias Gomes (PhD student)
Michael Saynisch (technician)
Peter Jeong (Medical student)
Katherine Dodel (student assistant)
Susanne Bäß (student assistant)
Theresa Schneider (student assistant)