Abstract 1782: Reactive oxygen species (ROS) and mitochondrial DNA (mtDNA) damage in tumor hypoxia, poor radiotherapy response, and metastatic progression of rectal cancer
Authors: Bousquet Paula A. , Meltzer Sebastian , Sønstevold Linda , Esbensen Ying , Lyckander Lars G. , Dueland Svein , Flatmark Kjersti , Redalen Kathrine R. , Eide Lars , Ree Anne H. 
Published: July 2017

Background: Hypoxia is an important feature of the tumor microenvironment and correlates with radiotherapy resistance and metastatic progression. Additionally, as the malignant phenotype promotes aerobic glycolysis and contributes to the microenvironmental acidification, an underlying mechanism in radiation resistance and metastasis is likely linked to metabolic changes caused by mitochondrial reprogramming. We hypothesized that ROS and mtDNA damage might be indicators of poor therapy response and dissemination of rectal cancer.

Methods: Three colorectal cancer (CRC) cell lines (HCT116, HT29, LoVo) were cultured under hypoxia (0.2% O2) or normoxia (21% O2) for 24 hours. In a prospective rectal cancer trial, serum was sampled from patients at the time of diagnosis. Levels of ROS were determined by the ability to oxidize dihydrochlorofluoroscein into a fluorescent derivative. mtDNA damage was quantified by the ability of modification on template DNA to inhibit restriction enzyme cleavage. For the study patients, histologic tumor response to neoadjuvant radiotherapy was evaluated according to standard ypTN staging and tumor regression grade (TRG) scoring. Groups were compared using Student’s t-test. Survival differences were assessed by the log-rank test.

Results: Compared to normoxic conditions, hypoxia significantly reduced ROS levels in all CRC cell lines. Moreover, in serum samples, ROS levels were lower for T4 cases than for patients with T2-3 disease (p = 0.037), and accordingly, low circulating ROS was associated with adverse metastasis-free survival (MFS; p = 0.004) and overall survival (p &lt; 0.001). Hypoxic HCT116 and HT29 cell lines, but not the LoVo cells, showed significantly higher mtDNA damage than under normoxia. Given the indicated dependence of ROS and mtDNA damage to hypoxia, a low ratio of ROS to mtDNA damage turned out to be a promising indicator of hypoxic CRC cells. This ratio was lower in serum samples from patients who obtained poor histologic tumor response to radiotherapy (ypT3-4 versus ypT0-2 and poor versus good TRG; p = 0.044 for both). Accordingly, a high ratio of circulating ROS to mtDNA damage was associated with longer MFS (p = 0.021).

Conclusion: We showed that culturing of CRC cells under hypoxia caused decrease in ROS levels but increase in mtDNA damage. Interestingly, in serum samples from rectal cancer patients, low ROS levels were associated with T4 disease, representing a bulky and often hypoxic tumor, and thus adverse survival. Moreover, low ratio of ROS to mtDNA damage was correlated with poor histologic tumor response to neoadjuvant radiotherapy. These parameters may reflect an aggressive biological phenotype instigated by a hostile tumor microenvironment. The results are currently under validation in an independent patient cohort. Trial registration: NCT00278694

Citation Format: Paula A. Bousquet, Sebastian Meltzer, Linda Sønstevold, Ying Esbensen, Lars G. Lyckander, Svein Dueland, Kjersti Flatmark, Kathrine R. Redalen, Lars Eide, Anne H. Ree. Reactive oxygen species (ROS) and mitochondrial DNA (mtDNA) damage in tumor hypoxia, poor radiotherapy response, and metastatic progression of rectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1782. doi:10.1158/1538-7445.AM2017-1782