P1088 - Inhibition of oxidative phosphorylation in Medullary Thyroid Cancer cells affects mRNA levels of genes controlling cancer cell proliferation and apoptosis.

Objectives:
Most pediatric medullary thyroid cancers (MTC) result from dominantly inherited or de novo activating mutations in the Rearranged-during-transfection (RET) proto-oncogene.(1) Our group previously demonstrated that RET-positive MTC-derived cells have significantly increased mitochondrial respiratory chain molecules as compared to normal thyroid tissue. In this study, we examined how inhibition of oxidative phosphorylation affects expression of genes controlling response to oxidative stress and DNA damage in MTC-derived cells.

Methods:
Human MTC-derived TT cells (harboring C634W RET mutation) were grown in RPMI 1640 medium. Mitochondrial respiration was inhibited via silencing of cytochrome C oxidase subunit IV (COXIV) using either shRNA or treatment with potassium cyanide (KCN). Total RNA was extracted using the all prep DNA/RNA mini kit (Qiagen). The mRNA levels of genes involved in cellular response to oxidative stress and genes encoding molecules with established roles in cancer were examined using “Oxidative Stress” and “Human Cancer PathwayFinder” real-time PCR arrays (Qiagen).

Results:
In MTC-derived TT cells, the downregulation of COXIV expression by shRNA or treatment with KCN resulted in overexpression of genes controlling rapid oxidative stress (ROS) generating systems. The mRNA levels of NADPH oxidases (DUOX1, DUOX2) were increased, indicating oxidative cell damage. The mRNA level of Forkhead Box M1 (FOXM1), which is reported to protect cancer cells from the adverse effects of oxidative stress, was decreased in COXIV deficient cells. Analysis of known cancer genes showed that inhibition of oxidative phosphorylation in MTC cells was associated with significant downregulation of genes controlling angiogenesis (KDR, AMPGPT2) and genes regulating cell cycle progression (MKI67, MCM2, E2F4). At the same time, expression of genes involved in cell response to DNA damage (DDIT3, HMOX1) were increased after COXIV silencing. In addition, inhibition of mitochondrial respiration demonstrated an increase in expression of apoptotic markers PUMA and NOXA.

Conclusions:
While MTC accounts for only 4% of thyroid cancer, it represents more than 15% of thyroid cancer-related deaths.(2) Tyrosine kinase inhibitors are approved for metastatic MTC treatment, but only lead to increased survival rates rather than remission.(2) Our results suggest that targeting mitochondrial respiration may be an alternative treatment option in MTC patients. Our group has ongoing studies examining the metabolic response of TT cells to pharmacology agents that block oxidative phosphorylation.

End Note Citations:
1. Starenki D, Park JI. Pediatric Medullary Thyroid Carcinoma. J Pediatr Oncol. 2015;3(2):29-37.
2. Deshpande H, Roman S, Thumar J, Sosa JA. Vandetanib (ZD6474) in the Treatment of Medullary Thyroid Cancer. Clinical Medicine Insights: Oncology. 2011,5:213-221.