CYLD-a potential mediator of site-specific melanoma metastasis

与柱状瘤病不同，黑色素瘤（一种高度侵袭性肿瘤）中去泛素酶CYLD的下调不是由CYLD基因突变引起的，而是由转录因子SNAIL 1的组成和高表达引起的。CYLD水平降低导致BCL-3/p50/p52依赖性NFκB激活，进而触发cyclinD1和N-cadherin等基因的表达。cyclinD1和N-cadherin水平升高促进黑色素瘤的增殖和侵袭。重要的是，在黑色素瘤患者中，我们可以将CYLD表达的丧失与Clark水平、肿瘤厚度、无进展和总生存期联系起来。在进一步的实验中，我们在黑色素瘤易发小鼠模型（Grm1-transgene）的背景下产生了一只cyd缺陷小鼠，Grm1-transgene是一种100%外显率自发发生黑色素瘤的模型。Grm1/Cyld+/+小鼠未出现真皮或局部转移，而Grm1/Cyld-/-小鼠出现多发局部皮肤转移并侵袭肌肉。在这个项目中，我们打算进一步利用这种小鼠模型，我们的目标是确定CYLD在有效的局部转移中的影响，并希望将我们的新知识也转移到远端部位特异性转移的事件中，特别是在肝脏，脑和淋巴结中。关于这一点，我们有兴趣发现肿瘤细胞的侵袭性途径（如淋巴途径），并希望区分对肿瘤细胞本身有影响的分子变化和改变微环境的cyld依赖因子（如肥大细胞）。我们对两种小鼠模型亚型的痣和原发肿瘤的组织样本进行了RNA-Seq分析。我们现在将表征表观遗传调控机制，这是相关的转移行为的差异。此外，我们将分析小鼠模型和小鼠细胞系的小鼠组织样本中的miRNA表达和变化，这些样本是我们在第一个资助期产生的。

Unlike in cylindromatosis, downregulation of the de-ubiquitinase CYLD in melanoma, a highly aggressive tumour, is not caused by mutations in the CYLD gene but rather by a constitutive and high expression of the transcription factor SNAIL 1. The reduced CYLD level leads to BCL-3/p50/p52-dependent NFκB activation, which in turn triggers expression of genes such as cyclinD1 and N-cadherin. Elevated levels of cyclinD1 and N-cadherin promote melanoma proliferation and invasion. Importantly, in melanoma patients we could link the loss of CYLD expression to Clark level, tumour thickness, and progression free and overall survival. In further experiments, we generated a Cyld-deficient mouse on the background of a melanoma-prone mouse model (Grm1-transgene), a model for spontaneously developing melanoma with 100% penetrance. Grm1/Cyld+/+ mice show no dermal or local invasion whereas Grm1/Cyld-/- mice present multiple local skin metastases and invasion into the muscle. In the project we intend to further exploit this mouse model and we aim to determine the impact of CYLD in efficient local metastasis and would like to transfer our new knowledge also to the event of distal site-specific metastasis, especially in liver, brain and lymph nodes. Regarding this we are interested to discover the invasive routes (e.g. lymphatic pathways), which are taken by the tumour cells and want to differentiate between molecular changes, which have an impact on the tumour cells themselves, and CYLD-dependent factors, which change the micro-environment (e.g. mast cells). We have performed RNA-Seq analysis of the tissue samples of nevi and primary tumours of both mice model subtypes. We will now characterize epigenetic regulation mechanisms, which are relevant for differences in metastatic behaviour. Furthermore, we will analyse miRNA expression and changes in murine tissue samples of the mouse model and murine cell lines, which we generated in the first funding period.