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Newick, Kheng

Cell and Molecular Biology Program

2013

Ph. D.

Mesothelioma, a rare tumor associated with asbestos exposure, responds very poorly to standard therapies, creating a need for new therapeutic approaches. FOXM1, a redoxresponsive transcription factor, has emerged as a therapeutic target in a variety of human cancers. FOXM1, which is expressed exclusively in proliferating cells, controls the expression of mitotic genes. Studies showed that FOXM1 is stimulated by reactive oxygen species (ROS), and inhibited by antioxidant treatments, indicating that it plays a critical role in redox homeostasis. This is supported by the observation that FOXM1 is required for adaptation to oncogene-induced oxidative stress. In this capacity, FOXM1 regulates the expression of pro-survival factors and antioxidant enzymes, including the mitochondrial antioxidant enzyme peroxiredoxin 3 (PRX3). PRX3, a peroxidase responsible for metabolism of up to 90% of mitochondrial hydrogen peroxide, has been shown to protect cells from apoptosis induced by certain chemotherapeutic drugs. Due to its essential roles in cell cycle progression and resistance to oxidative stress, the loss of FOXM1 generally leads to tumor cell death.
Here, FOXM1 expression was examined in human malignant mesothelioma (MM) tumors and MM cells in culture. Immunostaining showed that FOXMI is widely expressed in all MM, and studies with several human MM tumor cell lines indicate that the expression of oncogenic isoforms of FOXM1 cells is mitogen-independent. Studies with the antioxidant N-acetyl-L-cysteine showed that the activity of thiostrepton (TS), a thiazole antibiotic that inhibits FOXM1 expression, is redox-dependent in MM cells. Experiments with recombinant PRX3 indicated that TS most likely adducts reactive cysteine residues in PRX3, thereby inactivating its peroxidase activity. Immunoblotting of cell extracts showed TS produces a modified form of PRX3 that migrates at the apparent molecular weight of crosslinked PRX3 homodimers.
The abundance of this modified species was markedly increased by coincidental treatment with gentian violet (GV), a triphenylmethane that inhibits the expression of thioredoxin 2 (TRX2), the mitochondrial oxidoreductase that regenerates PRX3. Disruption of the mitochondrial antioxidant adaptive response by TS and GV generates severe mitochondrial oxidative stress, and these two compounds show synergistic effects on MM cell death. Our studies also show that MM cells produce more mitochondrial ROS than primary or immortalized human mesothelial cells, suggesting that the increased sensitivity 6f MM tumor cells to TS and GV may be linked to differences in mitochondrial redox homeostasis.
To test the efficacy of candidate compounds on MM tumor growth, human MM cells were injected into severe combined immunodeficient (SCID) mice, either subcutaneously (SQ) or intraperitoneally (IP) prior to treatment with TS, GV, or a combination of both drugs. In the SQ model, TS-treated mice showed a statistically significant reduction in tumor volume. In the IP model, TS showed no statistically significant effect on tumor progression. However, treatment with GV, or a combination of TS and GV, produced statistically significant reductions in tumor volume over the same treatment course. Expression of FOXM1 and PRX3 in xenoplant tumors was variable, and did not correlate with effects on tumor progression, suggesting that TS and GV act through other redox-responsive targets in addition to FOXM1 and PRX3. Overall, our results show that perturbing mitochondrial redox homeostasis provides a novel approach for targeting MM tumor progression.