Using genetically encoded biosensors to investigate the cell and cilia cycles in melanoma

Brennan, Louise and Mort, Richard and Allinson, Sarah (2024) Using genetically encoded biosensors to investigate the cell and cilia cycles in melanoma. PhD thesis, Lancaster University.

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Abstract

Melanoma has innate phenotypic plasticity, allowing switching between heterogenous cell phenotypes in response to immune therapies, which contributes to resistance. These phenotypes closely resemble the differentiation trajectory in development, from the early embryo, through the neural crest and embryonic melanoblasts, to differentiated melanocytes. Primary cilia are protruding microtubule-based organelles present on differentiated melanocytes, their loss may be sufficient to drive cell cycle re-entry and metastatic progression. To investigate ciliary and cell cycle parameters in melanoma cells we developed a biosensor that allows discrimination of G1, S and G2/M cell cycle phases and the primary cilium by fusing the Fucci(CA) probes hCdt1-mCherry and hGeminin-mVenus with cilia localised Arl13b-RFP670. PiggyBac was used to integrate the construct into melanoblasts, melanocyte and melanoma lines and observe ciliated cells in all cell types. The newly developed Arl13b-Fucci(CA) biosensor allows for better cell and cilia cycle discrimination and our results suggest that ciliation status reflects melanoma differentiation status. The primary ciliome warrants further research for melanoma associated targets that could influence phenotype switching. Simultaneously, we screened cilia genes for association with melanoma using TCGA and melanoblast RNAseq data. We found a larger mutation frequency in cilia associated genes for melanoma compared to other cancers, including breast lung and ovarian. Cilia number and length increased with differentiation state of melanoma cell lines, based on MITF expression. Analysis of differentiation trajectory in TCGA melanoma data revealed a group of cilia genes specifically expressed in an undifferentiated subtype, associated with an invasive phenotype: The GLIS genes. Through genetically encoded biosensors, qPCR, RNAseq and shRNA knockdowns, we showed that knockdown of Glis genes resulted in a loss of cilia in melanoblasts. Further, we show that GLIS proteins are involved in sonic hedgehog signalling.