The Immunomodulatory Small Molecule Imiquimod Induces Apoptosis in Devil Facial Tumour Cell Lines

Abstract

The survival of the Tasmanian devil (Sarcophilus harrisii) is threatened by devil facial tumour disease (DFTD). This transmissible cancer is usually fatal, and no successful treatments have been developed. In human studies, the small immunomodulatory molecule imiquimod is a successful immunotherapy, activating anti-tumour immunity via stimulation of toll-like receptor-7 (TLR7) signaling pathways. In addition, imiquimod is a potent inducer of apoptosis in human tumour cell lines via TLR7 independent mechanisms. Here we investigate the potential of imiquimod as a DFTD therapy through analysis of treated DFTD cell lines and Tasmanian devil fibroblasts. WST-8 proliferation assays and annexin V apoptosis assays were performed to monitor apoptosis, and changes to the expression of pro- and anti-apoptotic genes were analysed using qRT-PCR. Our results show that DFTD cell lines, but not Tasmanian devil fibroblasts, are sensitive to imiquimod-induced apoptosis in a time and concentration dependent manner. Induction of apoptosis was accompanied by down-regulation of the anti-apoptotic BCL2 and BCLXL genes, and up-regulation of the pro-apoptotic BIM gene. Continuous imiquimod treatment was required for these effects to occur. These results demonstrate that imiquimod can deregulate DFTD cell growth and survival in direct and targeted manner. In vivo, this may increase DFTD vulnerability to imiquimod-induced TLR7-mediated immune responses. Our findings have improved the current knowledge of imiquimod action in tumour cells for application to both DFTD and human cancer therapy.

Transcriptome and proteome profiling reveals stress-induced expression signatures of imiquimod-treated Tasmanian devil facial tumor disease (DFTD) cells

“…Together these results indicate that imiquimod suppresses DFT1 cell cycle at the G1/S phase after 24 h of treatment. This finding supports previous analysis of imiquimod action in DFT1 cell lines, which demonstrated significant inhibition of cell proliferation after treatment [ 27 ].…”

“…To understand the molecular pathways regulated by imiquimod in DFT1, we analysed the transcriptome and proteome of untreated and imiquimod-treated cells using RNA-seq and nanoHPLC-MS, respectively. As we previously determined that the percentage of early apoptotic DFT1 cells increases after 48 h of imiquimod treatment, this time point was chosen for promeomics analysis [ 27 ]. The transcriptome of imiquimod-treated cells was generated at an earlier time point of 24 h to minimise the extent of RNA degradation for sequencing analysis.…”

“…An additional mechanism of stress-mediated apoptosis is activated through the calcium-dependent pro-apoptotic protein calpain after ER calcium depletion [ 60 , 61 ]. Previous analyses have shown that DFT1 cell lines undergo apoptosis after sustained treatment with imiquimod [ 27 ]. Direct measurement of pro- and anti-apoptotic factors in this previous study revealed down regulation of anti-apoptotic genes and up regulation of the pro-apoptotic gene BIM ( BCL2L11 ) at the mRNA level in response to imiquimod treatment.…”

“…Although the development of a prophylactic vaccine against DFTD is being investigated [ 25 , 26 ], there are currently no prophylactic or therapeutic options for the protection of wild Tasmanian devils from the disease. Our previous studies have found that cell lines established from DFTD tumors are sensitive to cell death induced by imiquimod [ 27 ]. Furthermore, TLR7 signalling is functional in the immune system of the Tasmanian devil, suggesting that imiquimod could be an effective DFTD immunotherapy [ 28 , 29 ].…”

“…As these analyses are challenged by a lack of Tasmanian devil specific reagents, the effects of imiquimod in DFTD have been explored in this study at the whole mRNA and protein level using RNA sequencing (RNA-seq) and proteomic mass spectrometry (MS). The DFT1 cell line C5065 was selected as a model line for this analysis, as previous investigations have demonstrated that the response of these cells to imiquimod treatment is representative of other DFTD cell lines [ 27 ]. This study will improve the current understanding of DFTD tumorigenesis and survival, and could reveal new targets for DFTD therapy.…”