Targeting the cancer cell proteasome and the TCA cycle by copper ionophores and proteasome inhibitors to overcome treatment resistance in solid tumors

• Applicant: Dr. Hannah Zillikens
• Subject Area: Dermatology
• Term: since 2025
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 562413011

Based on preliminary research suggesting that neocuproine, a copper chelator and ixazomib, a proteasome inhibitor alter mitochondrial activity and metabolism in cancer cells, we aim to investigate the following objectives. First, we will examine the behavioral response of solid tumor cells to drug synergy between ixazomib and neocuproine, including melanoma cell lines, breast cancer and non-small-cell lung cancer (NSCLC). Cell behavior will be assessed using proliferation assays and drug synergy analysis. To investigate the mode of cell death, we will conduct apoptosis assays using concentrations that show the highest synergy. Additionally, we will explore other forms of cell death, such as ferroptosis and cuproptosis, through protein analysis using Western blotting e.g. lipoylated proteins. Preliminary data indicate that proteasome inhibitors can disrupt mitochondrial complex I and II, particularly affecting SDHA and SDHB, subunits of succinate dehydrogenase (SDH). We plan to assess cancer cell dependency on mitochondrial function using rotenone (for complex I) and TTFA or malonate (for complex II) in both normoxic and hypoxic conditions, as hypoxia may shift cells toward glycolysis, reducing reliance on mitochondria. Comparing these conditions will help determine whether cancer cells depend more on mitochondrial activity or glycolysis for survival. Additionally, we will investigate the roles of SDHA and SDHB in cancer cell sensitivity to neocuproine and proteasome inhibitors. Using a doxycycline-inducible lentiviral shRNA system, we will temporally knock down SDHA and SDHB, impairing mitochondrial function and shifting MAPK-sensitive cells toward glycolysis. In contrast, MAPK-resistant cells are unable to adapt, making them more vulnerable to the loss of SDH, a target of neocuproine. This inducible system will help clarify the relationship between SDHA/SDHB loss and drug sensitivity. We will also assess the impact of SDHA/SDHB loss on pseudohypoxia by measuring HIF-1α and PGC-1α levels, which are elevated in a state of pseudohypoxia and cell stress. We will explore whether inhibiting HIF-1α restores sensitivity to drugs targeting oxidative metabolism, such as neocuproine and proteasome inhibitors. Since proteasome inhibitors can induce oxidative stress, we will measure ROS levels and examine their role in affecting drug response in return. To validate the effects of neocuproine and proteasome inhibitors ex vivo, we will use orthotopic slice cultures derived from tumor patients. This model closely mimics the tumors’ architecture, cellular heterogeneity, and interactions, making it ideal for personalized drug testing and biomarker validation. The aim is to bridge the gap between in vitro assays and clinical outcomes, potentially shape personalized therapy based on the unique molecular and metabolic profiles of patients' tumors.

• DFG Programme: WBP Fellowship
• International Connection: Switzerland
• Host: Professor Dr. Mitch Levesque