Tumor-treating fields elicit a conditional vulnerability to ionizing radiation via the downregulation of BRCA1 signaling and reduced DNA double-strand break repair capacity in non-small cell lung cancer cell lines

Abstract

The use of tumor-treating fields (TTFields) has revolutionized the treatment of recurrent and newly diagnosed glioblastoma (GBM). TTFields are low-intensity, intermediate frequency, alternating electric fields that are applied to tumor regions and cells using non-invasive arrays. The predominant mechanism by which TTFields are thought to kill tumor cells is the disruption of mitosis. Using five non-small cell lung cancer (NSCLC) cell lines we found that there is a variable response in cell proliferation and cell killing between these NSCLC cell lines that was independent of p53 status. TTFields treatment increased the G2/M population, with a concomitant reduction in S-phase cells followed by the appearance of a sub-G1 population indicative of apoptosis. Temporal changes in gene expression during TTFields exposure was evaluated to identify molecular signaling changes underlying the differential TTFields response. The most differentially expressed genes were associated with the cell cycle and cell proliferation pathways. However, the expression of genes found within the BRCA1 DNA-damage response were significantly downregulated (Po0.05) during TTFields treatment. DNA double-strand break (DSB) repair foci increased when cells were exposed to TTFields as did the appearance of chromatid-type aberrations, suggesting an interphase mechanism responsible for cell death involving DNA repair. Exposing cells to TTFields immediately following ionizing radiation resulted in increased chromatid aberrations and a reduced capacity to repair DNA DSBs, which were likely responsible for at least a portion of the enhanced cell killing seen with the combination. These findings suggest that TTFields induce a state of 'BRCAness' leading to a conditional susceptibility resulting in enhanced sensitivity to ionizing radiation and provides a strong rationale for the use of TTFields as a combined modality therapy with radiation or other DNA-damaging agents. Lung cancer is the second most prevalent cancer and the leading cause of cancer-related death in the United States. 1 Non-small cell lung cancer (NSCLC) is the most prevalent type, accounting for~80% of new cases. 2,3 A plethora of treatment options exist including surgical resection, chemotherapy, radiation therapy and immunotherapy. 4,5 Fiveyear survival rates for patients with stage I and II NSCLC arẽ 50% and 30%, respectively. However, despite this myriad of options, 5-year survival rates for patients with late stage IIIA, IIIB and IV are 14%, 5% and 1%, respectively (www.cancer. net), highlighting the need for novel treatment modalities that can be utilized alone or in combination with conventional therapies to increase survival rates.The advent of Tumor-Treating Fields (TTFields), a novel physical treatment modality, has been effective for the treatment of solid, therapy-resistant primary and recurrent tumors. 6-9 TTFields electrodes are non-invasive and deliver a low-intensity (1-3 V/cm) intermediate frequency (100-300 kHz) alternating electric field across the tumor bed. 10 TTFie...

Tumor treating fields cause replication stress and interfere with DNA replication fork maintenance: Implications for cancer therapy

“…49 This suggests that stalled replication fork stabilization confers resistance to replication stress-inducing chemotherapeutics but because of the general suppression of a host of genes and proteins involved in replication fork maintenance and stability, checkpoint control and DNA repair pathways, TTFields exposure may overcome such resistance. Indeed the results herein provide a rationale for the additive to synergistic effects of TTFields seen in earlier preclinical reports 10,[12][13][14][15] with chemotherapeutic drugs such as paclitaxel, cisplatin, gemcitabine, doxorubicin, 5-FU, cyclophosphamide, DTIC, and Irinotecan which are known to primarily increase replication stress but not directly affect mitosis. [7][8][9][10][11] DNA replication is a mandatory and prerequisite step for mitosis.…”

“…Thus, using nucleotide analogs, newly synthesized DNA length was measured and confirmed that TTFields decreases the speed of DNA replication (Fig 1, BÀD), which suggests that TTFields induces replication stress. Although these results do not distinguish between co-directional and head-on collisions, it is clear that under TTFields treatment, these collisions are not resolved properly to facilitate the proper progression of replication given reduced expression of the FA pathway 15 and MCM genes (Fig 1, A) upon TTFields exposure. 15,26,27 Changes in DNA topology during collisions between replication and transcription machineries cause DNA-RNA hybrid structures called R-loops to form.…”

“…Although these results do not distinguish between co-directional and head-on collisions, it is clear that under TTFields treatment, these collisions are not resolved properly to facilitate the proper progression of replication given reduced expression of the FA pathway 15 and MCM genes (Fig 1, A) upon TTFields exposure. 15,26,27 Changes in DNA topology during collisions between replication and transcription machineries cause DNA-RNA hybrid structures called R-loops to form. R-loops have been implicated in a multitude of physiological roles in cells, such as regulation of gene expression, replication, Ig class switch recombination, DNA repair, and genomic instability.…”

“…TTFields treatment decreases replication fork speed and induces replication stress. An earlier study of DNA damage repair kinetics described higher levels of g-H2AX foci compared to co-localized g-H2AX/ 53BP1 foci and increased chromatid-type aberrations with TTFields exposure, 15 Phosphorylated g-H2AX has been described as an early sensor of DNA replication stress 17 but because phosphorylated g-H2AX can be generated by different DNA damage types it is not necessarily specific to replication stress. However, as seen in Fig 1, A, the expression of Mini-Chromosome Maintenance 10 (MCM10) and Mini-Chromosome Maintenance 6 (MCM6) genes, which are essential for replication initiation and DNA elongation processes, was decreased with time of exposure to TTFields supporting the notion of replication stress as a consequence of TTFields exposure (Fig 1, A).…”

“…In a previous study that was designed to determine whether there were differences in the response to TTFields in a series of nonÀsmall-cell lung cancers (NSCLC) of different genetic backgrounds and radiosensitivity, a genomics approach identified clear differences in gene expression patterns in NSCLC cells that were very sensitive to TTFields exposure compared to cells less responsive to TTFields exposure. A key finding was the decreased expression of the Fanconi's Anemia (FA) pathway genes, which play a key role in DNA double-strand break (DSB) repair 15 upon TTFields exposure. This downregulation of FA genes was accompanied by slower DNA repair postradiation and a greater number of residual (unrepaired) DNA lesions at 48 hours.…”