The Function of JNK and p38 in Melanoma Invasiveness

Das Melanom ist die tödlichste Art von Hautkrebs, verzeichnet den stärksten Anstieg unter bösartigen Hauterkrankungen, und ist ein sehr proliferativer Tumor mit einem hohen Risiko zur Metastasierung. Schon dünne Läsionen neigen dazu, in umliegendes Gewebe einzudringen, zum Wächter-Lymphknoten zu streuen und in entfernte Organe zu metastasieren vorzugsweise über das Lymphgefäßsystem. Während der mesenchymalen Transition eignen sich die Tumorzellen invasive Eigenschaften an, die die Metastasierung ermöglichen. Diese Veränderung von einer proliferativen zu einer invasiven Zelle ist ein bekanntes Phänomen während Melanomerkrankungen und wird durch definierte Marker bestimmt. Diese Heterogenität der Tumorzellen basiert auf der Aktivierung unterschiedlicher Signalwege, was deren Behandlung herausfordernd macht. Das Verständnis der molekularen Mechanismen, die diese Phänotyp Veränderung initiieren, ist essenziell für die Entwicklung neuer therapeutischer Strategien. Diese Doktorarbeit beabsichtigt, jene zugrundeliegenden Signalwege zu entschlüsseln, die für die Veränderung des Phänotyps verantwortlich sind. Zugleich sollen neue Marker für den proliferativen und den invasiven Phänotyp definiert werden. Vorliegende Ergebnisse tragen dazu bei, neue therapeutische Ansätze zur Melanomtherapie zu finden. Wir haben MITF und VEGF-C als Marker für den JNK, beziehungsweise für den p38 Signalweg identifiziert. Die Aktivierung von JNK führt zu erhöhter MITF Expression und daher zu Proliferation und schnellem Tumorwachstum, aber zu verminderter Invasion. Im Gegensatz dazu erhöht die Aktivierung von p38 die VEGF-C Expression, was die Hauptursache für Lymphangiogenese ist. Diese Zellen sind stark invasiv, haben aber langsamere Proliferationsraten. Nachdem wir die entgegengesetzten Rollen des JNK und p38 Signalweges in der Lymphangiogenese und die Rolle von p38 in der Tumorentwicklung identifiziert hatten, untersuchten wir die Funktion von p38/MK2 während der mesenchymalen Transition. Wir verwendeten 22 Melanomzelllinien und haben E-Cadherin Negativität als Marker für den invasiven Phänotyp identifiziert. Das Blockieren von p38 oder dessen downstream-target MK2 erhöht E-Cadherin und vermindert den Zusammenbruch der Endothelzell-Barriere, der durch Melanomzellen induziert wird. Darüber hinaus führt das Hemmen von MK2 zur verminderten Expression von PODXL und DEL-1 und verhindert die vaskuläre Streuung in vivo. Die Expression von PODXL und DEL-1 wird mit einer verschlechterten Prognose assoziiert und kann daher als Marker für eine geringere Überlebenswahrscheinlichkeit herangezogen werden, folglich sind MK2 downstream-targets potenziell für neue Therapien geeignet.Melanoma is the deadliest kind of skin cancer and the most increasing malignant skin disease. It is a highly proliferative tumor with a high risk to metastasize. Even thin lesions are prone to invade into the neighboring tissue, seed to the sentinel lymph node and metastasize to distant organs preferably via the lymphatic system. In order to metastasize, cancer cells have to gain invasive properties, this process is called mesenchymal transition. This switch from proliferative to invasive cells is a known phenomenon in melanoma and can be determined with defined markers. This heterogeneity of cells within the tumor is based on the activation of various pathways thus making it challenging to treat. The understanding of molecular mechanisms underlying this phenotypic switch is essential for the development of new therapeutic strategies. This thesis aims to decipher the underlying pathways responsible for the phenotypic switch and to identify novel markers for the proliferative and the invasive phenotypes. These findings contribute to clarify novel therapeutic targets for melanoma treatment. We identified MITF and VEGF-C as surrogate markers for the JNK and the p38 pathway, respectively. JNK activation leads to elevated MITF levels and, thus, to proliferation and fast tumor growth, but impaired invasive potential. In contrast, p38 activation increases VEGF-C expression which is the main driver of lymphangiogenesis. These cells are highly invasive but show decreased proliferation rates. Having identified the opposing roles of the JNK and the p38 signaling pathways in lymphangiogenesis and having discovered that p38 leads to tumor progression, we investigated p38/MK2 and its function in mesenchymal transition. Analyzing 22 patient-derived melanoma cell lines, we identified E-Cadherin negativity as marker for the invasive phenotype. Inhibition of p38 or its downstream target MK2 increases E-Cadherin expression and reduces melanoma-induced barrier disruption. Moreover, MK2 blocking results in PODXL and DEL-1 downregulation and inhibits vascular dissemination in vivo. PODXL and DEL-1 expression are associated with worse prognosis and can thus serve as markers for poor survival. Hence, MK2 downstream targets might be used as candidate therapeutics.Abweichender Titel laut Übersetzung der Verfasserin/des VerfassersArbeit an der Bibliothek noch nicht eingelangt - Daten nicht geprüftMedizinische Universität Wien, Diss., 2020(VLID)477084

Co-existence of BRAF and NRAS driver mutations in the same melanoma cells results in heterogeneity of targeted therapy resistance

Acquired chemotherapeutic resistance of cancer cells can result from a Darwinistic evolution process in which heterogeneity plays an important role. In order to understand the impact of genetic heterogeneity on acquired resistance and second line therapy selection in metastatic melanoma, we sequenced the exomes of 27 lesions which were collected from 3 metastatic melanoma patients treated with targeted or non-targeted inhibitors. Furthermore, we tested the impact of a second NRAS mutation in 7 BRAF inhibitor resistant early passage cell cultures on the selection of second line therapies.We observed a rapid monophyletic evolution of melanoma subpopulations in response to targeted therapy that was not observed in non-targeted therapy. We observed the acquisition of NRAS mutations in the BRAF mutated patient treated with a BRAF inhibitor in 1 of 5 of his post-resistant samples. In an additional cohort of 5 BRAF-inhibitor treated patients we detected 7 NRAS mutations in 18 post-resistant samples. No NRAS mutations were detected in pre-resistant samples. By sequencing 65 single cell clones we prove that NRAS mutations co-occur with BRAF mutations in single cells. The double mutated cells revealed a heterogeneous response to MEK, ERK, PI3K, AKT and multi RTK - inhibitors.We conclude that BRAF and NRAS co-mutations are not mutually exclusive. However, the sole finding of double mutated cells in a resistant tumor is not sufficient to determine follow-up therapy. In order to target the large pool of heterogeneous cells in a patient, we think combinational therapy targeting different pathways will be necessary

ROS Induction Targets Persister Cancer Cells with Low Metabolic Activity in NRAS-Mutated Melanoma

Clinical management of melanomas with NRAS mutations is challenging. Targeting MAPK signaling is only beneficial to a small subset of patients due to resistance that arises through genetic, transcriptional, and metabolic adaptation. Identification of targetable vulnerabilities in NRAS-mutated melanoma could help improve patient treatment. Here, we used multiomics analyses to reveal that NRAS-mutated melanoma cells adopt a mesenchymal phenotype with a quiescent metabolic program to resist cellular stress induced by MEK inhibition. The metabolic alterations elevated baseline reactive oxygen species (ROS) levels, leading these cells to become highly sensitive to ROS induction. In vivo xenograft experiments and single-cell RNA sequencing demonstrated that intratumor heterogeneity necessitates the combination of a ROS inducer and a MEK inhibitor to inhibit both tumor growth and metastasis. Ex vivo pharmacoscopy of 62 human metastatic melanomas confirmed that MEK inhibitor-resistant tumors significantly benefited from the combination therapy. Finally, oxidative stress response and translational suppression corresponded with ROS-inducer sensitivity in 486 cancer cell lines, independent of cancer type. These findings link transcriptional plasticity to a metabolic phenotype that can be inhibited by ROS inducers in melanoma and other cancers. Significance: Metabolic reprogramming in drug-resistant NRAS-mutated melanoma cells confers sensitivity to ROS induction, which suppresses tumor growth and metastasis in combination with MAPK pathway inhibitors

ROS induction as a strategy to target persister cancer cells with low metabolic activity in NRAS mutated melanoma

Metabolic reprogramming is an emerging hallmark of resistance to cancer therapy but may generate vulnerabilities that can be targeted with small molecules. Multi-omics analysis revealed that NRAS-mutated melanoma cells with a mesenchymal transcriptional profile adopt a quiescent metabolic program to resist cellular stress response induced by MEK-inhibitor resistance. However, as a result of elevated baseline ROS levels, these cells become highly sensitive to ROS induction. In vivo xenograft experiments and single-cell RNA sequencing demonstrated that intra-tumor heterogeneity requires the combination of a ROS-inducer and a MEK-inhibitor to target both tumor growth and metastasis. By ex vivo pharmacoscopy of 62 human metastatic melanomas, we found that MEK-inhibitor resistant tumors significantly benefitted from the combination therapy. Finally, we profiled 486 cancer cell lines and revealed that oxidative stress responses and translational suppression are biomarkers of ROS-inducer sensitivity, independent of cancer indication. These findings link transcriptional plasticity to a metabolic phenotype that can be inhibited by ROS-inducers in melanoma and other cancers. Statement of Significance Targeted-therapy resistance in cancer arises from genetic selection and both transcriptional and metabolic adaptation. We show that metabolic reprogramming sensitizes resistant cells to ROS-induction in combination with pathway inhibitors. Predictive biomarkers of metabolic sensitivity to ROS-inducing agents were identified in many cancer entities, highlighting the generalizability of this treatment approach