eRapport

Melanoma systems biology for therapeutic discovery

Prosjekt
Prosjektnummer
2014044
Ansvarlig person
Eivind Hovig
Institusjon
Oslo universitetssykehus HF
Prosjektkategori
Doktorgradsstipend
Helsekategori
Skin
Forskningsaktivitet
1. Underpinning
Rapporter
2020 - sluttrapport
This project utilized a systems biology approach to identify novel potential therapeutic targets in malignant melanoma. The project contained two published papers and one submitted manuscript. The PhD thesis based on this project has been successfully defended by the candidate and was accepted for the degree of PhD by the The Faculty of Medicine, University of Oslo, June 29th 2020. Project 1: The transcription factor MITF (Microphthalmia-Associated Transcription Factor) has been shown to play a role in the development of acquired vemurafenib drug resistance. In this subproject study, we discovered that under normal conditions (in melanoma cell lines), MITF inhibits growth factor receptor ERBB3 and its ligand NRG1. However, during vemurafenib treatment, MITF levels began to decrease, leading to an increase in ERBB3 and NRG1 expression. An up-regulation of ERBB3 and NRG1 expression contributed to sustained proliferation and survival of melanoma cell lines under vemurafenib treatment. This study was published in Oncotarget in 2016 doi: 10.18632/oncotarget.10422 Project 2: The role of MITF in melanoma development remains poorly understood. In a second subproject study, we attempted to gain insight into MITF regulation in wild type MC1R vs. MC1R mutants. MC1R mutants give rise to red hair, with a consequence for melanoma risk. To achieve insight, we analyzed the regulatory landscape of immortalized human melanocytes and gene modified variants by genome-wide MITF binding (Chip-Seq) and transcriptome (RNA-Seq). These data sets and corresponding histone modification sets were integrated by in silico regulatory landscape analysis to map regulatory variation between the two genotypes. We have here identified candidate factors involved in oncogenesis as potential targets of therapy. This paper was published in Cancers in 2020. https://doi.org/10.3390/cancers12071719 Project 3: The roles of AXL/GAS6 and ERBB3/NRG1 expression and signalling in melanoma have been associated with tumor growth, invasion and drug resistance. In this subproject, we show that these pathways are mainly controlled by the SOX10/MITF axis and that they interplay to sustain tumor growth. Melanoma cell lines can be divided into subgroups according to their expression levels of AXL and ERBB3. Our results show that all of these sub-groups utilize either the AXL/GAS6, ERBB3/NRG1 or both pathways for activation of the PI3K/AKT pathway. We demonstrate that evaluation of MITF/SOX10 and ERBB3/AXL levels in tumors is essential for finding the appropriate drug combination, and that AXL or ERBB3 inhibitors are useful in combination with MAPK inhibitors for overcoming MAPK resistance. A manuscript describing these findings has been submitted. Altogether, we have demonstrated potential therapeutic targets, and demonstrated that important pathways may be manipulated to modify signalling in these targets. We have developed a model system for induction of spontaneous transformation of melanocytes to cell able to form tumors in mice. We aim to develop this model further to derive insights into the epigenetic regulation of melanoma. This work has primarily been to build further underpinning for melanoma management, through better understanding of signalling mechanisms that are at play, and how the interconnect and may be manipulated.

NEI

2019
This project utilizes a systems biology approach to identify novel potential therapeutic targets in malignant melanoma. The project contains one published paper and two submitted manuscripts. The PhD thesis based on this project has been submitted for evaluation, and is expected to be defended this spring.Project 1 The transcription factor MITF (Microphthalmia-Associated Transcription Factor) has been shown to play a role in the development of acquired vemurafenib drug resistance. In this subproject study, we discovered that under normal conditions (in melanoma cell lines), MITF inhibits growth factor receptor ERBB3 and its ligand NRG1. However, during vemurafenib treatment, MITF levels begin to decrease, leading to an increase in ERBB3 and NRG1 expression. An up-regulation of ERBB3 and NRG1 expression contributed to sustained proliferation and survival of melanoma cell lines under vemurafenib treatment. This study was published in Oncotarget in 2016. Project 2 The role of MITF in melanoma development remains poorly understood. In a second subproject study, we attempted to gain insight into MITF regulation in wild type MC1R vs. MC1R mutants. MC1R mutants give rise to red hair, with a consequence for melanoma risk. To achieve insight, we analyzed the regulatory landscape of immortalized human melanocytes and gene modified variants by genome-wide MITF binding (Chip-Seq) and transcriptome (RNA-Seq). These data sets and corresponding histone modification sets were integrated by in silico regulatory landscape analysis to map regulatory variation between the two genotypes. We have here identified candidate factors involved in oncogenesis. A manuscript describing these findings has now been submitted. Project 3 The roles of AXL/GAS6 and ERBB3/NRG1 expression and signaling in melanoma have been associated with tumor growth, invasion and drug resistance. In this subproject, we show that these pathways are mainly controlled by the SOX10/MITF axis and that they interplay to sustain tumor growth. Melanoma cell lines can be divided into subgroups according to their expression levels of AXL and ERBB3. Our results show that all of these sub-groups utilize either the AXL/GAS6, ERBB3/NRG1 or both pathways for activation of the PI3K/AKT pathway. We demonstrate that evaluation of MITF/SOX10 and ERBB3/AXL levels in tumors is essential for finding the appropriate drug combination, and that AXL or ERBB3 inhibitors are useful in combination with MAPK inhibitors for overcoming MAPK resistance. A manuscript describing these findings has now been submitted.

NEI

2018
This project utilizes a systems biology approach to identify novel potential therapeutic targets in malignant melanoma. The project contains one published paper and two manuscripts be submitted by the end of 2019. Also, the PhD thesis based on this project is currently in development, and will be submitted before the end of this year.Project 1 The transcription factor MITF (Microphthalmia-Associated Transcription Factor) has been shown to play a role in the development of acquired vemurafenib drug resistance. In this subproject study, we discovered that under normal conditions (in melanoma cell lines) MITF inhibits growth factor receptor ERBB3 and its ligand NRG1. However, during vemurafenib treatment, MITF levels begin to decrease, leading to an increase in ERBB3 and NRG1 expression. An up-regulation of ERBB3 and NRG1 expression contributed to sustained proliferation and survival of melanoma cell lines under vemurafenib treatment. This study was published in Oncotarget in 2016. Project 2 The role of MITF in melanoma development remains poorly understood. In a second subproject study, we initiated and attempted to gain insight into MITF regulation in wild type MC1R vs. MC1R mutants. MC1R mutants give rise to red hair, with a consequence for melanoma risk. To achieve insight, we analyzed the regulatory landscape of immortalized human melanocytes and gene modified variants by genome-wide MITF binding (Chip-Seq) and transcriptome (RNA-Seq). These data sets and corresponding histone modification sets were integrated by in silico regulatory landscape analysis to map regulatory variation between the two genotypes. We have here identifying candidate factors involved in oncogenesis. The lab work included in article no. 2 is completed and a written article is currently under revision before submission. Project 3 The roles of AXL/GAS6 and ERBB3/NRG1 expression and signaling in melanoma have been associated with tumor growth, invasion and drug resistance. In this project, we show that these pathways are mainly controlled by the SOX10/MITF axis and that they interplay to sustain tumor growth. Melanoma cell lines can be divided in to sub- groups according to their expression levels of AXL and ERBB3. Our results show that all of these sub-groups utilize either the AXL/GAS6, ERBB3/NRG1 or both pathways for activation of the PI3K/AKT pathway. We demontrate that evaluation of MITF/SOX10 and ERBB3/AXL levels in tumors is essential for finding the appropriate drug combination and that AXL or ERBB3 inhibitors are useful in combination with MAPK inhibitors for overcoming MAPK resistance. A manuscript describing these findings is currently under progress.
2017
This project utilizes a systems biology approach to identify novel potential therapeutic targets in malignant melanoma. This entails modeling the complex signaling network involved in melanoma development. We here report on the progress of this project, which contains one published paper and two manuscripts in progress.The transcription factor MITF (Microphthalmia-Associated Transcription Factor) has been shown to play a role in the development of acquired vemurafenib drug resistance. In this subproject study, we discovered that under normal conditions (in melanoma cell lines) MITF inhibits growth factor receptor ERBB3 and its ligand NRG1. However, during vemurafenib treatment, MITF levels begin to decrease, leading to an increase in ERBB3 and NRG1 expression. An up-regulation of ERBB3 and NRG1 expression contributed to sustained proliferation and survival of melanoma cell lines under vemurafenib treatment. This study was published in Oncotarget in 2016. The role of MITF in melanoma development remains poorly understood. In a second subproject study, we initiated and attempted to gain insight into MITF regulation in wild type MC1R vs. MC1R mutants. MC1R mutants give rise to red hair, with a consequence for melanoma risk. To achieve insight, we analyzed the regulatory landscape of immortalized human melanocytes and gene modified variants by genome-wide MITF binding (Chip-Seq) and transcriptome (RNA-Seq). These data sets and corresponding histone modification sets were integrated by in silico regulatory landscape analysis to map regulatory variation between the two genotypes. We are now in the process of identifying candidate factors with possible causative links to increased oncogenesis. A manuscript is in preparation. A recent report suggests that MITF loss is involved in the development of vemurafenib resistance through up-regulation or activation of the receptor tyrosine kinase receptor (RTK) AXL. This has made AXL a target for melanoma therapy. We have investigated these reports closer and discovered that down-regulation of the AXL receptor in a panel of melanoma cell lines leads to an up-regulation of the RTK receptor ERBB3. We are now investigating the cross-talk between AXL and ERBB3 and their implications towards vemurafenib drug resistance in various melanoma cell lines harboring the BRAFV600E mutation. A manuscript describing our findings is currently in progress.
2016
This project utilizes a systems biology approach to identify novel potential therapeutic targets in malignant melanoma. This entails modeling the complex signaling network involved in melanoma development. We here report on the progress of this project, which already contains one published paper.The transcription factor MITF (Microphthalmia-Associated Transcription Factor) has been shown to play a role in the development of acquired drug resistance after long term vemurafenib treatment in many melanoma patients, through up-regulation of diverse growth factor receptors. In this project, we have uncovered a new signaling pathway under control of MITF that may play a role in vemurafenib resistance. Normally, MITF inhibits the growth factor receptor and its ligand, but after long term vemurafenib treatment, levels of MITF are significantly reduced. This leads to an increase in activation of both the growth factor receptor and its ligand, which in turn leads to increased cell growth and survival. This project has uncovered MITF to be involved in novel vemurafenib resistance not yet described in the literature. We have also shown that the inhibitory effect of MITF on the growth factor receptor and its ligand is a general mechanism and can be found in melanocytes, as well as in melanoma, and is independent of mutational status in the RAS- and RAF genes. It is important to emphasize that this is one of many signaling pathways that has shown to be under MITF control, and that a combination of growth factor receptor up-regulation most likely leads to resistance in patients after long-term vemurafenib treatment. Furthermore, it is also known that vemurafenib treatment can lead to an increase in MITF expression, and that the signaling pathways that lead to resistance in these tumors will be different from what we found in this project. Our findings may have implications for acquired drug resistance in melanoma. This work was published in Oncotarget in 2016. Researchers have generally been focusing on treatment strategies that will potentially reverse already acquired resistance. Consequently, as an extension of our earlier work, we are now testing possible treatment combinations to avoid acquired resistance. We are working with ten different melanoma cell lines harboring different levels of MITF, to investigate which growth factor receptor that is most important in resistance development, and which combination best fits with the genetic makeup in the form of active signaling mechanisms of the different cell lines. Since there are several growth factor receptors involved in resistance, we aim to uncover when combination strategies should be implemented, and to test the potential of alternating different treatment combinations to avoid or delay resistance in the panel of melanoma cell lines.
2015
Dette projected benytter systembiologiske angrepsvinkler for å identifisere nye angrepspunkter i ondartet føflekk-kreft. Dette innebærer modellering av de komplekse signalnettverk i melanom. Vi rapporterer her på første delprosjektet, som er definerte perturbasjoner for å predikere potensielle terapeutiske mål, med et fokus på MITF-proteinet.Transkripsjonsfaktoren MITF (Microphthalmia-Associated Transcription Factor) har vist seg å spille en rolle i utviklingen av resistens mot vemurafenib-behandling for en stor andel av melanompasientene (ondartet føflekk-kreft) via regulering av vekstfaktorreseptorer. Venurafenib er en av de nyutviklete legemidlene som har vist effekt i behandlingen av melanom. Vi har i dette prosjektet avdekket en ny MITF-styrt signalrute for utvikling av resistens mot vemurafenib. I normal tilstand hemmer MITF vekstfaktorreseptoren og dens ligand, men etter langvarig bruk av vemurafenib forsvinner MITF-uttrykket. Tap av MITF fører til at hemmingen av vekstfaktorreseptoren og dens ligand opphører, og signalveien aktiveres. Dette fører til økt cellvekst og overlevelse. Vi har med dette prosjektet vist at transkripsjonsfaktoren MITF spiller flere roller i utvikling av resistens mot vemurafenib enn hva som tidligere har vært kjent. Det viser seg at den nye MITF-styrte signalruten som har blitt avdekket er en generell mekansime som finnes både i melanocytter og i melanom, uavhengig av mutasjonsstatus i RAS- og RAF-genet. Det er viktig å presisere at dette kun er en av flere signalveier som har vist seg å være kontrollert av MITF, og at det er sannsynlig at det er en kombinajon av ulike vekstfaktorreseptorer som fører til resistensutvikling i pasienter etter langvarig vemurafenib-behandling. Videre er det også kjent at vemurafenib-behandling kan øke mengden av MITF-utrykk og at signalveiene som fører til resistensutvikling i disse kreftcellene/svulstene vil være anderledes enn hva vi har funnet i dette prosjektet. Imidlertid representerer det nye funnet en mulig ny angrepsvinkel for behandling av føflekk-kreft, og åpner også for mulighet for kombinert behandling med flere legemidler samtidig. Dette arbeidet er nå under sammenskriving.
2014
This project has recently started. The initial purpose is to elucidate the options for manipulation of the melanoma master switch MITF and other central signaling molecules of melanoma, utilizing molecular constructs and models of work mechanisms.Initially, we have developed functional mRNA molecules and lentiviral constructs of MITF and related factors, and are in the process of learning their effects in cell line systems. mRNA experiments We have started to optimize the delivery of MITF wild type and MITF dnR215del mRNA molecules into various melanoma cell line backgrounds. Our results so far have verified that our MITF wild type mRNA molecules are capable of inducing up-regulation of the tyrosinase gene (a downstream target gene of MITF) after MITF mRNA transfection. We are now in the process of exploring the novel targets of MITF in various stages of the melanoma progression by expression profiling. Furthermore, we have transfected melanoma cells with BRAF wild type and BRAF V600E, together with p16 wild type and p16 mutated, to investigate their function. Results so far have shown that BRAF V600E is able to activate ERK (phospho-ERK), showing that the functionality of the BRAF V600E mRNA is correct. p16 mRNA molecules has also been transfected and p16 protein was detected by Western blot, demonstrating successful translation of the given mRNA molecules. Further experiments are in progress to disclose their function in various mutational backrounds. Lentiviral construction We have transduced various melanoma cell lines with BRAFV600E and BRAF wild type to investigate possible induction of senescence. Our results so far suggest that BRAF V600E induce senescence in BRAF WT melanoma cell lines, but not in BRAF V600E cell lines. In addition we have stable constructs for MITF wild type, MITF dn, p16 wild type and p16 mutated, that have been verified by Western Blot and will be transduced in various melanoma cell line backgrounds.
Vitenskapelige artikler
Lavelle TJ, Alver TN, Heintz KM, Wernhoff P, Nygaard V, Nakken S, Øy GF, Bøe SL, Urbanucci A, Hovig E

Dysregulation of MITF Leads to Transformation in MC1R-Defective Melanocytes.

Cancers (Basel) 2020 Jun 28;12(7). Epub 2020 jun 28

PMID: 32605315 - Inngår i doktorgradsavhandlingen

Alver TN, Lavelle TJ, Longva AS, Øy GF, Hovig E, Bøe SL

MITF depletion elevates expression levels of ERBB3 receptor and its cognate ligand NRG1-beta in melanoma.

Oncotarget 2016 Aug 23;7(34):55128-55140.

PMID: 27391157 - Inngår i doktorgradsavhandlingen

Doktorgrader
Tine Norman Alver

Melanoma systems biology for therapeutic discovery

Disputert:
juni 2020
Hovedveileder:
Sigurd Bøe
Deltagere
  • Alfonso Urbanucci Prosjektdeltaker
  • Karl-Johan Malmberg Medveileder
  • Timothy J Lavelle Doktorgradsstipendiat (annen finansiering)
  • Sigurd Bøe Hovedveileder
  • Tine Norman Alver Doktorgradsstipendiat (annen finansiering)
  • Johannes Eivind Hovig Forskningsgruppeleder
  • Geir Frode Øy Prosjektdeltaker
  • Ane Sager Longva Prosjektdeltaker
  • Karl-Johan Malmberg Biveileder

eRapport er utarbeidet av Sølvi Lerfald og Reidar Thorstensen, Regionalt kompetansesenter for klinisk forskning, Helse Vest RHF, og videreutvikles av de fire RHF-ene i fellesskap, med støtte fra Helse Vest IKT

Alle henvendelser rettes til eRapport

Personvern  -  Informasjonskapsler