Poster Presentation 25th Lorne Cancer Conference 2013

Heterogeneity and plasticity in melanoma leads to treatment failure and increased invasiveness that can be blocked by targeting TSP-1 and/or SNAIL (#117)

Andreas Behren 1 , Aparna Jayachandran 1 , Matthew Anaka 1 , Christopher Hudson 1 , Sonja McKeown 2 , Jonathan Cebon 1
  1. Cancer Immunobiology Laboratory, Ludwig Institute for Cancer Research, Heidelberg, VIC, Australia
  2. University of Melbourne, Melbourne, VIC, Australia

We investigated emergent resistance to kinase inhibitors by assessing (i) cellular heterogeneity and (ii) plasticity; which can both yield resistant cells either stochastically or under the selective pressure of therapy. Cellular heterogeneity was evaluated in vitro with functional assays including the use of a membrane dye, CM-Dil, which identified a slow-cycling cell subpopulation within lines and xenografts. Cells were evaluated for migration in Boyden chamber assays and in vivo in the neural tube of the developing chick embryo. Chemoresistance was assessed following exposure to cytotoxic agents and the BRAF inhibitors PLX4720 and dabrafenib. Genome-wide gene expression was analyzed with Illumina HT12 arrays. Targets of interest were assessed by gene inhibition with siRNA. Label-retaining cells (LRC) were identified in multiple cell lines. These were more resistant to cytotoxic drugs and more invasive. Gene expression profiling identified a network of overexpressed genes related to epithelial-to-mesenchymal transition (EMT). Furthermore, resistance to BRAF inhibitors was associated with a phenotypic switch (which reduced differentiation and increased invasiveness) and with acquisition of the LRC gene signature. This was characterised by the transforming-growth factor beta (TGFb-activating enzyme Thrombospondin-1 (TSP-1), TGFb-induced (TGFBI) and a variety of other molecules which coding for extracellular proteins. Targeted knockdown of either SNAIL (a transcription factor that regulates EMT) or TSP-1 resulted in the inhibition of migration in vitro and in vivo. The LRC genotype links treatment failure, cellular plasticity, EMT and invasiveness. The plastic switch in vivo was blocked by inhibition of TSP-1 or SNAIL. These insights are informing the identification of targets for overcoming disease progression.