JmjC-KDMs KDM3A and KDM6B modulate radioresistance under hypoxic conditions in esophageal squamous cell carcinoma

Abstract

Esophageal squamous cell carcinoma (ESCC), the most frequent esophageal cancer (EC) subtype, entails dismal prognosis. Hypoxia, a common feature of advanced ESCC, is involved in resistance to radiotherapy (RT). RT response in hypoxia might be modulated through epigenetic mechanisms, constituting novel targets to improve patient outcome. Post-translational methylation in histone can be partially modulated by histone lysine demethylases (KDMs), which specifically removes methyl groups in certain lysine residues. KDMs deregulation was associated with tumor aggressiveness and therapy failure. Thus, we sought to unveil the role of Jumonji C domain histone lysine demethylases (JmjC-KDMs) in ESCC radioresistance acquisition. The effectiveness of RT upon ESCC cells under hypoxic conditions was assessed by colony formation assay. KDM3A/KDM6B expression, and respective H3K9me2 and H3K27me3 target marks, were evaluated by RT-qPCR, Western blot, and immunofluorescence. Effect of JmjC-KDM inhibitor IOX1, as well as KDM3A knockdown, in in vitro functional cell behavior and RT response was assessed in ESCC under hypoxic conditions. In vivo effect of combined IOX1 and ionizing radiation treatment was evaluated in ESCC cells using CAM assay. KDM3A, KDM6B, HIF-1α, and CAIX immunoexpression was assessed in primary ESCC and normal esophagus. Herein, we found that hypoxia promoted ESCC radioresistance through increased KDM3A/KDM6B expression, enhancing cell survival and migration and decreasing DNA damage and apoptosis, in vitro. Exposure to IOX1 reverted these features, increasing ESCC radiosensitivity and decreasing ESCC microtumors size, in vivo. KDM3A was upregulated in ESCC tissues compared to the normal esophagus, associating and colocalizing with hypoxic markers (HIF-1α and CAIX). Therefore, KDM3A upregulation in ESCC cell lines and primary tumors associated with hypoxia, playing a critical role in EC aggressiveness and radioresistance. KDM3A targeting, concomitant with conventional RT, constitutes a promising strategy to improve ESCC patients’ survival.

The component of the m6A writer complex VIRMA is implicated in aggressive tumor phenotype, DNA damage response and cisplatin resistance in germ cell tumors

“…Additionally, this was further demonstrated in vivo, with reduced tumor size and angiogenesis, evaluated both macroscopically and histologically. We again highlight the versatility of the CAM assay for assessing tumor properties and response to drugs in vivo [61], also recently demonstrated for GCTs as well [62,63]. Interestingly, VIRMA knockdown resulted in significant protein expression decrease of all members of the methyltransferase complex (METTL3, the only component with catalytic activity; METTL14, which is relevant for substrate recognition, specificity and activity; and WTAP, which stabilizes the complex).…”

Hypoxia in Solid Tumors: How Low Oxygenation Impacts the “Six Rs” of Radiotherapy

“…Thus, hypoxia can prime cancer cells for DSBs repair initiation (79). In this sense, it has been recently demonstrated that hypoxia (1% O 2 ) is associated with a higher ɣH2AX foci decay rate and enhanced RAD51 recruitment in irradiated HNSCC cells, suggesting a higher efficacy of HRR under hypoxia in this model (75,80).…”

“…This potentially primes hypoxic cells for postirradiation DSBs repair initiation. It has also been shown that hypoxia can modify the epigenetic landscape (global loss of H3K27me3 and H3K9me2) by modulating epigenetic enzyme expressions, such as KDM3A and KDM6B and finally promotes DDR activity and radioresistance in HNSCC (80). Moreover, via HIFs, hypoxia can also increase KDM4B expression participating in HRR orchestration (93)(94)(95).…”

Hydralazine and Enzalutamide: Synergistic Partners against Prostate Cancer

“…Cell viability was evaluated by MTT before the first treatment and 24 h after the final treatment. The CompuSyn software [19] was used to calculate the drug combination index (CI), under the assumption that a value lower than 1 indicates synergistic effects, a value of 1 denotes an additive effect, and a value higher than 1 implies antagonistic effects.…”

“…PCa cells (2 × 10 5 for all cell lines) were seeded in flasks and the following day submitted to treatment with the combination of hydralazine and enzalutamide at the concentrations previously calculated. Daily treatments were performed for 72 h. DNA damage was assessed using the protocol described elsewhere [19]. Representative pictures were taken on a microscope (model IX51, Olympus, Tokyo, Japan) and cells were analyzed using the ImageJ software OpenComet Plugin, which measured the tail moment: tail % DNA × means of head × tail distance.…”