Sandra Iden - A 6

Requirement of polarity networks in melanoma cell plasticity, therapy and drug resistance

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.  

Introduction 

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 and crucial for embryonic development. We recently identified Par3 as a rheostat of epidermal homeostasis by controlling stem cell maintenance and keratinocyte differentiation (Ali et al., 2016). 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, which integrates oncogenic Ras- and aPKC-mediated signalling (Iden et al., 2012). Furthermore, we demonstrated genetic interactions of Par3 and aPKCλ in different stages of skin tumorigenesis, with Par3/aPKCλ jointly fostering proliferative and 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 may be used to develop strategies for disease prevention and therapy.

Non-cell autonomous tumor suppression by polarity regulators 

Melanoma arises due to the malignant transformation of melanocytes, which are integrated in the basal layer of the epidermis. Intercellular crosstalk between melanocytes and keratinocytes is essential for the photoprotection of the skin but how the epidermal microenvironment controls melanomagenesis is largely unknown. Interestingly, we recently 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.

Epidermal polarity regulators control cyto-architecture & fate of other tissue-resident cells

To assess how epidermal Par3 counteracts melanoma we studied how melanocyte behaviour was affected by Par3 loss in the epithelial microenvironment. Epidermal Par3 inactivation resulted in melanocyte hyperplasia both in newborn and adult mice. 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). 

Expression of Par3 in the microenvironment inversely correlates with human melanoma progression 

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).

Melanoma-intrinsic functions of polarity regulators

Having identified Par3 as a strong extrinsic regulator of melanoma, we will now investigate putative intrinsic functions of polarity regulators in melanoma formation and progression. Analyses of large-scale human cancer genomic data sets disclosed a mutation rate of 8-12% for PARD3, PRKCI and PRKCZ in melanoma, frequently co-occurring with mutations in key tumor suppressors or melanoma oncogenes. We examined genetic alterations of a range of polarity regulators that in epithelial systems mostly act in concert. The results suggest that putative polarity networks in melanoma do not resemble those in carcinoma, as for instance mutations in PARD3 and PRKCI co-occur in melanoma, rather than following a mutually exclusive pattern. 

A major aim of this project is to understand intrinsic functions of single polarity regulators in melanoma, to dissect melanoma-specific polarity complexes, and unveil yet unknown polarity signalling networks that impart in disease progression.

Perspectives 

Our work revealed mechanistic insights in extrinsic roles of polarity regulators for melanocyte behaviour, melanoma formation and metastasis. Moreover, melanoma-intrinsic mutation profiles of polarity proteins deviate from those in epithelial cancers, suggesting novel functions and networks of conserved polarity proteins in melanoma. To dissect how melanoma cells utilize polarity signalling we generated a gRNA library to perform CRISPR/Cas9-based loss-of-function studies in melanocytes and melanoma cell lines. Relevant hits will be further tested for their significance in vivo, using a recently established metastatic melanoma model (Schlereth et al., 2015). Finally, we will ask how disturbed polarity gene function affects drug resistance mechanisms, an increasing problem in modern melanoma therapy.

Selected publications

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).

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)

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. 

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. 

Mescher M & Iden S (2015). Par proteins in tumor formation and progression. Cell Polarity 2. Role in development and disease. Springer, VIII, 145-166.


Dr. Sandra Iden

CECAD Cologne

Dr. Sandra Iden

Principal Investigator A 6

sandra.iden@uk-koeln.de

Work +49 221 478 89587

CECAD Research Center
Joseph-Stelzmann-Str. 26
50931 Cologne

http://iden.cecad-labs.uni-koeln.de/Home.84.0.html

Publications - Sandra Iden

Link to PubMed

Group Members

Sina Knapp (PhD student)
Annika Graband (PhD student)
Soriba Letzian (PhD student)
Martim Dias Gomes (PhD student)
Michael Saynisch (technician)
Peter Jeong (MD student)
Katherine Dodel (student assistant)
Susanne Bäß (student assistant)

Figure 1

CMMC Research Sandra Iden
Increased protrusion dynamics and motility of melanocytes co-cultured with Par3KO keratinocytes.

Figure 2

CMMC Research Sandra Iden
Par3 immunoreactivity in microenvironment of human melanoma decreases with disease progression.

Figure 3

Frequency of mutations in genes encoding key polarity regulators in human melanoma (cBioportal)