Melanoma systems biology for therapeutic discovery

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.

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.

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.