In vitro evidence for senescent multinucleated melanocytes as a source for tumor-initiating cells.

Oncogenic signaling in melanocytes results in oncogene-induced senescence (OIS), a stable cell-cycle arrest frequently characterized by a bi- or multinuclear phenotype that is considered as a barrier to cancer progression. However, the long-sustained conviction that senescence is a truly irreversible process has recently been challenged. Still, it is not known whether cells driven into OIS can progress to cancer and thereby pose a potential threat. Here, we show that prolonged expression of the melanoma oncogene N-RAS(61K) in pigment cells overcomes OIS by triggering the emergence of tumor-initiating mononucleated stem-like cells from senescent cells. This progeny is dedifferentiated, highly proliferative, anoikis-resistant and induces fast growing, metastatic tumors. Our data describe that differentiated cells, which are driven into senescence by an oncogene, use this senescence state as trigger for tumor transformation, giving rise to highly aggressive tumor-initiating cells. These observations provide the first experimental in vitro evidence for the evasion of OIS on the cellular level and ensuing transformation.

Cystathionase mediates senescence evasion in melanocytes and melanoma cells.

The development of malignant melanoma is a highly complex process, which is still poorly understood. A majority of human melanomas are found to express a few oncogenic proteins, such as mutant RAS and BRAF variants. However, these oncogenes are also found in nevi, and it is now a well-accepted fact that their expression alone leads to senescence. This renders the understanding of senescence escape mechanisms an important point to understand tumor development. Here, we approached the question of senescence evasion by expressing the transcription factor v-myc myelocytomatosis viral oncogene homolog (c-MYC), which is known to act synergistically with many oncogenes, in melanocytes. We observed that MYC drives the evasion of reactive-oxygen stress-induced melanocyte senescence, caused by activated receptor tyrosine kinase signaling. Conversely, MIZ1, the growth suppressing interaction partner of MYC, is involved in mediating melanocyte senescence. Both, MYC overexpression and Miz1 knockdown led to a strong reduction of endogenous reactive-oxygen species (ROS), DNA damage and senescence. We identified the cystathionase (CTH) gene product as mediator of the ROS-related MYC and MIZ1 effects. Blocking CTH enzymatic activity in MYC-overexpressing and Miz1 knockdown cells increased intracellular stress and senescence. Importantly, pharmacological inhibition of CTH in human melanoma cells also reconstituted senescence in the majority of cell lines, and CTH knockdown reduced tumorigenic effects such as proliferation, H2O2 resistance and soft agar growth. Thus, we identified CTH as new MYC target gene with an important function in senescence evasion.

Oncogene activation in melanocytes links reactive oxygen to multinucleated phenotype and senescence.

Contrary to malignant melanoma, nevi are a benign form of melanocytic hyperproliferation. They are frequently observed as precursor lesions of melanoma, but they also feature biochemical markers of senescence. In particular, evidence for oncogene-induced melanocyte senescence as natural means to prevent tumorigenesis has been obtained in nevi with mutated B-Raf(V600E). Here, we demonstrate that strong oncogenic growth factor receptor signalling drives melanocytes into senescence, whereas weaker signals keep them in the proliferative state. Activation of oncogene-induced senescence also produces multinucleated giant cells, a long known histological feature of nevus cells. The protein levels of the senescence mediators, p53 and pRB, and their upstream activators do not correlate with senescence. However, strong oncogene signalling leads to pronounced reactive oxygen stress, and scavenging of reactive oxygen species (ROS) efficiently prevents the formation of multinucleated cells and senescence. Similarly, expression of oncogenic N-RAS results in ROS generation, DNA damage and the same multinuclear senescent phenotype. Hence, we identified oncogenic signalling-dependent ROS production as critical mediator of the melanocytic multinuclear phenotype and senescence, both of them being hallmarks of human nevus cells.