A 3D Organotypic Melanoma Spheroid Skin Model

Malignant transformation of melanocytes, the pigment cells of human skin, causes formation of melanoma, a highly aggressive cancer with increased ...

TU-Dresden

Induction of DNA damage by UVB and UVA radiation may generate mutations and genomic instability leading to carcinogenesis. Therefore, skin cells being repeatedly exposed to ultraviolet (UV) light have acquired multilayered protective mechanisms to avoid malignant transformation. Besides extensive DNA repair mechanisms, the damaged skin cells can be eliminated by induction of apoptosis, which is mediated through the action of tumor suppressor p53. In order to prevent the excessive loss of skin cells and to maintain the skin barrier function, apoptotic pathways are counteracted by anti-apoptotic signaling including the AKT/mTOR pathway. However, AKT/mTOR not only prevents cell death, but is also active in cell cycle transition and hyper-proliferation, thereby also counteracting p53. In turn, AKT/mTOR is tuned down by the negative regulators being controlled by the p53. This inhibition of AKT/mTOR, in combination with transactivation of damage-regulated autophagy modulators, guides the p53-mediated elimination of damaged cellular components by autophagic clearance. Alternatively, p53 irreversibly blocks cell cycle progression to prevent AKT/mTOR-driven proliferation, thereby inducing premature senescence. Conclusively, AKT/mTOR via an extensive cross talk with p53 influences the UV response in the skin with no black and white scenario deciding over death or survival. 

The role of AKT/mTOR pathway in stress response to UV-irradiation: implication in skin carcinogenesis by regulation of apoptosis, autophagy and senescence.

The transcription factor nuclear factor kappa-B (NFκB) is a key regulator of pro-inflammatory and pro-proliferative processes. Accordingly, uncontrolled NFκB activity may contribute to the development of severe diseases when the regulatory system is impaired. Since NFκB can be triggered by a huge variety of inflammatory, pro-and anti-apoptotic stimuli, its activation underlies a complex and tightly regulated signaling network that also includes multi-layered negative feedback mechanisms. Detailed understanding of this complex signaling network is mandatory to identify sensitive parameters that may serve as targets for therapeutic interventions. While many details about canonical and non-canonical NFκB activation have been investigated, less is known about cellular IκBα pools that may tune the cellular NFκB levels. IκBα has so far exclusively been described to exist in two different forms within the cell: stably bound to NFκB or, very transiently, as unbound protein. We created a detailed mathematical model to quantitatively capture and analyze the time-resolved network behavior. By iterative refinement with numerous biological experiments, we yielded a highly identifiable model with superior predictive power which led to the hypothesis of an NFκB-lacking IκBα complex that contains stabilizing IKK subunits. We provide evidence that other but canonical pathways exist that may affect the cellular IκBα status. This additional IκBα:IKKγ complex revealed may serve as storage for the inhibitor to antagonize undesired NFκB activation under physiological and pathophysiological conditions. 

Identification of new IκBα complexes by an iterative experimental and mathematical modeling approach.

In response to genotoxic stress, including UVB radiation, transcription factors NF-κB and p53 inevitably influence the cellular fate. Loss of p53 function has been attributed to malignant transformation and interferes with therapeutic interventions, whereas "gain of function" mutants even enhance tumor promotion. Constitutive NF-κB activation is linked to tumor maintenance and resistance against chemotherapy. The cross talk between p53 and NF-κB, however, is still under debate. Using the non-transformed keratinocyte cell line HaCaT, we shed light on the interplay between p53 and NF-κB by providing clear evidence that chronically activated NF-κB together with designated "gain of function" mutp53 promotes apoptosis via cooperative tumor necrosis factor (TNF) production in response to UVB+IL-1. Performing chromatin immunoprecipitation analysis we demonstrate that both transcription factors bind to the TNF promoter, whereas UVB-induced inhibition of Ser-Thr-phosphatase protein phosphatase 2A facilitates prolonged phosphorylation of NF-κB and the transcriptional cofactor cAMP response element-binding protein, both being required for extended TNF transcription. Thus, two major anti-apoptotic factors, NF-κB and mutp53, in concert may generate pro-apoptotic responses. As human skin is constantly exposed to UVB, causing IL-1 production as well, we hypothesize that the remarkable amount of hotspot p53 mutations within the epidermis (4%) may serve a protective function to eliminate precancerous cells at an early stage.

Anti-apoptotic NF-κB and "gain of function" mutp53 in concert act pro-apoptotic in response to UVB+IL-1 via enhanced TNF production.

Great advances have recently been made in treating patients with metastatic melanoma. However, existing therapies are less effective on cerebral than extracerebral metastases. This highlights the potential role of the brain environment on tumor progression and drug resistance and underlines the need for "brain-specific" therapies. We previously showed that the PI3K-AKT survival pathway is hyperactivated in brain but not extracerebral melanoma metastases and that astrocyte-conditioned medium activates AKT in melanoma cells in vitro We therefore tested the PI3K inhibitor buparlisib as an antitumor agent for melanoma brain metastases.

PI3K Pathway Inhibition Achieves Potent Antitumor Activity in Melanoma Brain Metastases In Vitro and In Vivo.

Tumor cell resistance to apoptosis, which is triggered by many anti-tumor therapies, remains a major clinical problem. Therefore, development of more efficient therapies is a priority to improve cancer prognosis. We have previously shown that a cell-permeable peptide derived from the p120 Ras GTPase-activating protein (RasGAP), called TAT-RasGAP317-326, bears anti-malignant activities in vitro and in vivo, such as inhibition of metastatic progression and tumor cell sensitization to cell death induced by various anti-cancer treatments. Recently, we discovered that this RasGAP-derived peptide possesses the ability to directly kill some cancer cells. TAT-RasGAP317-326 can cause cell death in a manner that can be either partially caspase-dependent or fully caspase-independent. Indeed, TAT-RasGAP317-326-induced toxicity was not or only partially prevented when apoptosis was inhibited. Moreover, blocking other forms of cell death, such as necroptosis, parthanatos, pyroptosis and autophagy did not hamper the killing activity of the peptide. The death induced by TAT-RasGAP317-326 can therefore proceed independently from these modes of death. Our finding has potentially interesting clinical relevance because activation of a death pathway that is distinct from apoptosis and necroptosis in tumor cells could lead to the generation of anti-cancer drugs that target pathways not yet considered for cancer treatment.

The TAT-RasGAP317-326 anti-cancer peptide can kill in a caspase-, apoptosis-, and necroptosis-independent manner.

 NRAS mutations in malignant melanoma are associated with aggressive disease requiring rapid antitumor intervention, but there is no approved targeted therapy for this subset of patients. In clinical trials, the MEK inhibitor (MEKi) binimetinib displayed modest antitumor activity, making combinations a requisite. In a previous study, the BRAF inhibitor (BRAFi) vemurafenib was shown to induce endoplasmic reticulum (ER) stress that together with inhibition of the RAF-MEK-ERK (MAPK) pathway amplified its proapoptotic activity in BRAF-mutant melanoma. The present study investigated whether this effect might extent to NRAS-mutant melanoma, in which MAPK activation would be expected. BRAFi increased pERK, but also significantly increased growth inhibition and apoptosis induced by the MEKi in monolayer, spheroids, organotypic, and patient-derived tissue slice cultures of NRAS-mutant melanoma. BRAFi such as encorafenib induced an ER stress response via the PERK pathway, as detected by phosphorylation of eIF2α and upregulation of the ER stress-related factors ATF4, CHOP, and NUPR1 and the proapoptotic protein PUMA. MEKi such as binimetinib induced the expression of the proapoptotic protein BIM and activation of the mitochondrial pathway of apoptosis, the latter of which was enhanced by combination with encorafenib. The increased apoptotic rates caused by the combination treatment were significantly reduced through siRNA knockdown of ATF4 and BIM, confirming its critical roles in this process. The data presented herein encourage further advanced  and clinical studies to evaluate MEKi in combination with ER stress inducing BRAFi as a strategy to treat rapidly progressing NRAS-mutant melanoma. .

Dagmar Kulms

Experimental Dermatology,
Medical Faculty,
Experimental Dermatology, Medical Faculty

TU-Dresden

A 3D Organotypic Melanoma Spheroid Skin Model

Dagmar Kulms

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Experimental Dermatology,Medical Faculty,Experimental Dermatology, Medical Faculty

TU-Dresden

A 3D Organotypic Melanoma Spheroid Skin Model

Experimental Dermatology,
Medical Faculty,
Experimental Dermatology, Medical Faculty