Oral Presentation Australasian Society for Dermatological Research Annual Scientific Meeting 2017

A PI3K-AKT and Wnt/β-catenin pathway driven regulatory mechanism defines differentially cycling tumor cell sub-populations in a 3D melanoma model (#31)

Farzana Ahmed 1 , Crystal Tonnessen 2 , Loredana Spoerri 1 , Sheena Daignault 1 , Mathias Nierfeld 1 , Glen Boyle 3 , Mitchell Stark 4 , Helmut Schaider 4 , Michelle Hill 1 , Nikolas Haass 1 5 6
  1. University of Queensland Diamantina Institute, Woolloongabba, QLD, Australia
  2. Biochemistry, Tulane University, New Orleans, Louisiana, United states of America
  3. QIMR Berghofer Medical Research Institute, Brisbane, Queensland, Australia
  4. Faculty of Medicine, University of Queensland, Brisbane, Queensland, Australia
  5. Centenary Institute, Sydney, NSW, Australia
  6. 4Royal Prince Alfred Hospital, Sydney, NSW, Australia

Microenvironment-driven intra-tumoral dynamic heterogeneity is a leading cause of drug resistance acquisition in melanoma. It has been reported that slow cycling sub-populations within a tumour are more resistant to drug action compared to rapidly proliferating sub-populations. However, the molecular mechanism behind this phenomenon is still unknown. In this study, we aim to discover the molecular signatures of these two differentially cycling sub-populations and propose specific mechanism behind the microenvironment-driven emergence of these two sub-populations. We have generated 3D spheroids from fluorescent ubiquitination-based cell cycle indicator (FUCCI)-transduced melanoma cell lines. Confocal microscopy of spheroid sections revealed that there are two differentially cycling sub-populations present within the spheroid: a central G1-arrested (slow cycling) sub-population with low MITF expression and a peripheral rapidly proliferating sub-population with high MITF expression. To elucidate the molecular mechanism behind this phenomenon, we isolated cells from each sub-population by Hoechst dye diffusion and FACS and then confirmed their accurate separation by their respective MITF expression pattern. RNA seq analysis of cells isolated from these two sub-populations identified several specific molecules that showed significant differential expression pattern in these two sub-populations. Specially, melanocyte- and melanoma-specific isoforms of MITF (MITF-M, MITF-Mdel) showed more than 2 fold down-regulation in the G1 arrested central sub-population compared to cells in G1-phase of the cycling peripheral sub-population. Pathway enrichment analysis of the RNA seq data suggested that the PI3K-AKT pathway has been significantly altered in these two sub-populations. Based on these, we proposed a PI3K-AKT and Wnt/β-catenin pathway driven regulatory mechanism to explain the differential expression pattern of MITF in these differentially cycling tumor sub-populations. In addition, we will also investigate the downstream effectors of MITF to understand how differential expression of MITF and its activity in these two subpopulations regulate their segregation within spheroids.

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