The molecular determinants of development and chemoresistance in gestational trophoblastic disease (GTD)
Creators: name: Georgiou, Marina
Publisher: name: Imperial College London
Year: 2017

description: Gestational trophoblastic tumours may occur after either molar, normal or any other type of pregnancy. Due to their high vascularity, surgery is discouraged as a therapeutic option and patients are instead treated with chemotherapy. The chemotherapy regimen is selected through the International Federation of Gynaecology and Obstetrics (FIGO) scoring of the tumours to predict the probability of patients developing resistance to single drug therapy. The majority of patients (~95%) fall within the low-risk group and are treated with single-agent methotrexate (MTX), while high-risk patients receive combination chemotherapy, commonly consisting of etoposide, methotrexate, actinomycin-D alternating weekly with cyclophosphamide and vincristine (EMA/CO). Although MTX is curative as a monotherapy its effectiveness can be compromised by the development of resistance in &gt;33% of the cases. The MTX-resistant patients are then treated with EMA/CO, which is far more toxic. Thus, novel therapeutic strategies with reduced toxicity would be beneficial, especially for unresponsive patients. To identify novel pathways and druggable targets involved in MTX-resistance, stable isotopic labelling of amino acids in cell culture mass spectrometry (SILAC/MS) was used to compare the proteome and phosphoproteome of MTX-resistant and sensitive cells. Bioinformatics analysis of the proteomic screens identified cell cycle, DNA damage repair and metabolic processes as the major pathways involved in resistance. We show that MTX-resistant choriocarcinoma cells undergo cell cycle delay at the G1 phase in response to checkpoint activation downstream of DNA damage (DD). DD activates the ataxia telangiectasia and Rad3 related (ATR) pathway in MTX-resistant cells and stabilises p53, thereby enabling for a more efficient DNA damage repair, through non-homologous end joining (NHEJ). Here, we propose that extending G1 is central to the resistance phenotype, as it provides sufficient time for dihydrofolate reductase (DHFR) overexpression, the main target of MTX, and delays entry into S-phase where MTX exerts its action. This mechanism of resistance is further supported by the rewired cellular energetics of the resistant cells, which allow for slower proliferation, maintenance of energy metabolism and a beneficial reactive oxygen species (ROS) level. Moreover, MTX-resistant cells increase glutathione (GSH) levels and upregulate glutathione S-transferase Mu 1 (GSTM1) that functions in the detoxification of drugs, a phenotype that might directly contribute to MTX resistance. This work extended our understanding of the molecular determinants of MTX resistance in choriocarcinoma, which led to the therapeutic exploitation of both the delay at G1 and the activation of DNA damage repair downstream of the ATR pathway. Inhibition of cyclin dependent kinase 4 and 6 (CDK4/6) using small interference RNA or the clinically relevant inhibitor Palbociclib prevented cells from entering this beneficial G1 phase. Palbociclib, induced growth inhibition in in vitro and in vivo choriocarcinoma models, paving the way for its clinical use as a monotherapy for MTX-resistant choriocarcinoma patients. In addition, we suggest that ATR inhibition in combination with MTX treatment could also provide an alternative novel strategy to tackle MTX-resistance in the clinic. In short, the work carried out during my PhD, provided invaluable insight into the signalling and metabolic pathways associated with MTX resistance in choriocarcinoma and has the potential for translation into clinical studies.