RNA-Seq Characterization of Melanoma Phenotype Switch in 3D Collagen After P38 MAPK Inhibitor Treatment
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biomolecules Communication RNA-seq Characterization of Melanoma Phenotype Switch in 3D Collagen after p38 MAPK Inhibitor Treatment Vladimír Cermˇ ák 1,2 , Aneta Škarková 1,2 , Ladislav Merta 1,2 , Veronika Kolomazníková 1,2, Veronika Palušová 3,4, Stjepan Uldrijan 3,4 , Daniel Rösel 1,2 and Jan Brábek 1,2,* 1 Department of Cell Biology, Charles University, Viniˇcná 7, 128 44 Prague, Czech Republic; [email protected] (V.C.);ˇ [email protected] (A.Š.); [email protected] (L.M.); [email protected] (V.K.); [email protected] (D.R.) 2 Biotechnology and Biomedicine Centre of the Academy of Sciences and Charles University (BIOCEV), Pr ˚umyslová 595, 252 42 Vestec u Prahy, Czech Republic 3 Department of Biology, Faculty of Medicine, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic; [email protected] (V.P.); [email protected] (S.U.) 4 International Clinical Research Center, St. Anne’s University Hospital, Pekaˇrská 53, 656 91 Brno, Czech Republic * Correspondence: [email protected]; Tel./Fax: +420-3258-73900 Abstract: Melanoma phenotype plasticity underlies tumour dissemination and resistance to therapy, yet its regulation is incompletely understood. In vivo switching between a more differentiated, proliferative phenotype and a dedifferentiated, invasive phenotype is directed by the tumour mi- croenvironment. We found that treatment of partially dedifferentiated, invasive A375M2 cells with two structurally unrelated p38 MAPK inhibitors, SB2021920 and BIRB796, induces a phenotype Citation: Cermák,ˇ V.; Škarková, A.; switch in 3D collagen, as documented by increased expression of melanocyte differentiation mark- Merta, L.; Kolomazníková, V.; ers and a loss of invasive phenotype markers. The phenotype is accompanied by morphological Palušová, V.; Uldrijan, S.; Rösel, D.; change corresponding to amoeboid–mesenchymal transition. We performed RNA sequencing with Brábek, J. RNA-seq Characterization an Illumina HiSeq platform to fully characterise transcriptome changes underlying the switch. Gene of Melanoma Phenotype Switch in 3D expression results obtained with RNA-seq were validated by comparing them with RT-qPCR. Tran- Collagen after p38 MAPK Inhibitor scriptomic data generated in the study will extend the present understanding of phenotype plasticity Treatment. Biomolecules 2021, 11, 449. in melanoma and its contribution to invasion and metastasis. https://doi.org/10.3390/biom11030449 Keywords: cancer; melanoma; metastasis; phenotype switch; amoeboid invasion Academic Editor: Masaaki Oyama Received: 27 January 2021 Accepted: 15 March 2021 1. Introduction Published: 17 March 2021 Despite significant progress in therapy, melanoma remains a life-threatening disease Publisher’s Note: MDPI stays neutral with a high risk of early metastasis. Understanding the biological processes underlying with regard to jurisdictional claims in melanoma metastasis is expected to generate new advances and therapeutic modalities published maps and institutional affil- in melanoma management [1]. Melanoma is a cancer arising from melanocytes, pigment- iations. producing cells located in the epidermis and a few other tissues. It shows unique biological features related to its developmental origin, namely specific differentiation plasticity and migratory behaviour. The central or master regulator of the melanocyte lineage is the transcription factor MITF [2,3]. MITF regulates expression of pigmentation-related genes, Copyright: © 2021 by the authors. and coordinates differentiation and proliferation in developing melanocytes. In addition, Licensee MDPI, Basel, Switzerland. it affects many other biological processes, including cell survival, invasion, senescence, This article is an open access article metabolism, autophagy, lysosomal biogenesis, and DNA damage repair [2]. MITF is reg- distributed under the terms and ulated at transcription and multiple post-transcriptional levels. Its transcription in the conditions of the Creative Commons melanocyte lineage is driven by SOX10, WNT/β-catenin and CREB, and is directly or indi- Attribution (CC BY) license (https:// rectly suppressed by NF-κB, JNK, hedgehog, TGFβ, activated RhoA, HIF1α, Brn2/POU3F2 creativecommons.org/licenses/by/ and ATF4 [2–6]. 4.0/). Biomolecules 2021, 11, 449. https://doi.org/10.3390/biom11030449 https://www.mdpi.com/journal/biomolecules Biomolecules 2021, 11, 449 2 of 13 A large-scale comparative study of published microarray data from melanoma cell Biomolecules 2021, 11, x FOR PEER REVIEW 2 of 14 lines identified two distinct gene expression profiles and associated phenotypes that were termed “invasive” and “proliferative” [7]. The different gene expression patterns corre- spond to low and high MITF activity and the related differentiation status of the cells. A large-scale comparative study of published microarray data from melanoma cell Thelines dedifferentiated,identified two distinct invasive gene expression phenotype profiles wasand associated originally phenotypes characterised that were by a sustained ac- tivationtermed “invasive” of TGF βandtarget “proliferative” genes, a[7]. shift The fromdifferent canonical gene expression to non-canonical patterns corre- WNT signalling, andspond a to low low expressionand high MITF of activity MITF-regulated and the related genes. differentiation Later, status it was of the found cells. The that it also involves sustaineddedifferentiated, NF-κ invasiveB, Yap/Taz-TEAD, phenotype was ER-stressoriginally characterised and Rho-ROCK by a sustained signalling activa- [5,7–11]. Melanoma dedifferentiationtion of TGFβ target genes, and a invasive shift from phenotypecanonical to non-canonical can be induced WNT signalling, or enhanced and a by inflammatory low expression of MITF-regulated genes. Later, it was found that it also involves sustained factorsNF-κB, Yap/Taz-TEAD, (IL1, TNFα), ER-stress TGFβ, glutamineand Rho-ROCK starvation, signalling [5,7–11] conditions. Melanoma of low dediffer- adhesion (e.g., growth inentiation melanospheres) and invasive phenotype and hypoxia can be [5 induced,12–18 ].or Itenhanced is associated by inflammatory with resistance factors to both BRAF inhibitors(IL1, TNFα), andTGFβ immunotherapy,, glutamine starvation, as conditions well as of with low adhesion enhanced (e.g., metastasisgrowth in mel- and poor progno- sisanospheres) [10,17– 21and]. hypoxia The underlying [5,12–18]. It is signallingassociated with pathways, resistance to their both BRAF cross-talks inhibitors and feed-forward and immunotherapy, as well as with enhanced metastasis and poor prognosis [10,17–21]. loopsThe underlying responsible signalling for, pathways, or, presumably, their cross-talks contributing and feed-forwa to,rd the loops preservation responsible of the invasive phenotypefor, or, presumably, in melanoma contributing cells to, and the pres exampleservation ofof transcriptsthe invasive phenotype specifically in mela- associated with either phenotype,noma cells and are examples summarised of transcripts in Figure specific1ally[8,11 associated,22–35]. with either phenotype, are summarised in Figure 1 [8,11,22–35]. Figure 1. Signalling circuitry of the invasive melanoma phenotype. Essential, broadly accepted Figureregulators 1. areSignalling marked by yellow circuitry colour. of Other the invasive regulators, melanoma relationships phenotype.and responses to Essential, stimuli broadly accepted regulatorswere compiled are from marked the literature by yellow related colour. to both Other invasive–proliferative regulators, relationships phenotype plasticity and responses and to stimuli were amoeboid invasion. Listed examples of genes whose high expression is characteristic of either phe- compilednotype were from assembled the literature from the pu relatedblished toliterature both invasive–proliferative and datasets. phenotype plasticity and amoeboid invasion. Listed examples of genes whose high expression is characteristic of either phenotype were assembledCancer fromcells, theincluding published melanoma, literature can indi andvidually datasets. invade a 3D environment in ei- ther a mesenchymal mode, which depends on integrin-mediated adhesion and extracel- lular proteaseCancer activity, cells, including or in an amoeboid melanoma, mode that can is individually independent of invade integrins a and 3D local- environment in either ised proteolysis, and is also characterised by largely dissociated microtubules and high aRhoA-ROCK-dependent mesenchymal mode, contractility which depends[36–38]. As onRhoA-ROCK integrin-mediated signalling is also adhesion required and extracellular proteasefor mesenchymal activity, invasion, or in anit is amoeboid an attractive mode target thatfor antimetastatic is independent therapy of integrins[39,40]. and localised proteolysis,A375M2 metastatic and ismelanoma also characterised cells have been by a largelypopular model dissociated for studying microtubules amoeboid and high RhoA- ROCK-dependentinvasion and its regulation, contractility for they show [36 prominent–38]. As blebbing RhoA-ROCK amoeboid signallingmorphology isin, also required for mesenchymalor on top of, soft invasion,collagen matrices, it is an good attractive long-term target viability for under antimetastatic such conditions, therapy and, [39,40]. A375M2 metastatic melanoma cells have been a popular model for studying amoeboid invasion and its regulation, for they show prominent blebbing amoeboid morphology in, or on top of, soft collagen matrices, good long-term