Poster Presentation 28th Lorne Cancer Conference 2016

MITF regulates EMT and drives subcompartment-specific distribution of differentially cycling melanoma cells (#104)

Farzana Ahmed 1 , Crystal Tonnessen 1 , Loredana Spoerri 1 , Sheena Daignault 1 , Kimberley Beaumont 2 3 , Wolfgang Weninger 2 3 4 , Nikolas Haass 1 2 3
  1. University of Queensland Diamantina Institute, Woolloongabba, QLD, Australia
  2. Centenary Institute, Sydney, NSW, Australia
  3. University of Sydney, Sydney, NSW, Australia
  4. 4Royal Prince Alfred Hospital, Sydney, NSW, Australia

Melanoma drug resistance may be due, in part, to dynamic heterogeneity. Cancer cells within a tumor exhibit various phenotypes in response to environmental stress. This results in populations with different proliferative and invasive capabilities and drug sensitivities. Understanding the molecular signature of dynamic heterogeneity is crucial to design effective therapies.

Our 3D spheroid model generated from fluorescent ubiquitination-based cell cycle indicator (FUCCI) transduced melanoma cell line allows for real-time cell cycle analysis in response to environmental stress, e.g.  hypoxia and nutrient deprivation. We have previously shown that melanoma spheroids (but also xenografts in vivo), segregate into differentially cycling subpopulations – namely G1 arrested cells in the center and cycling cells in the periphery. Moreover, we have demonstrated that this phenomenon is dependent on the expression levels of Microphthalmia-associated transcription factor (MITF).

Here we set out to decipher the role that MITF plays in this phenomenon to more detail. Using confocal microscopy of sectioned spheroids we found that expression of MITF, SLUG and N-Cadherin co-localized with the proliferating sub compartment, while E-Cadherin co-localized with the G1-arrested center. Vimentin was highly expressed only at the very periphery of the spheroid and in the cells invading into the collagen matrix. Furthermore, ectopic MITF overexpression resulted in a random distribution of MITF throughout the spheroid and also increased SLUG and Vimentin expression in a similar random pattern. We then isolated cells from the different sub compartments by Hoechst dye diffusion and FACS and isolated RNA. Real time PCR confirmed the above described findings at the transcriptional level.

This novel technique of isolating these two subpopulations will allow us to perform transcriptome analysis of them and to interrogate the resulting datasets for a better understanding of the drivers of dynamic tumour heterogeneity and eventually cancer drug sensitivity and resistance.