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

Catarina Macedo-Silva,Vera Miranda-Gonçalves,Ana Teresa Martins,Ana Lameirinhas,Isabel Bravo,Alexandre Pereira,Rui Henrique,João Lobo,Joana Lencart,Rita Guimarães,Carmen Jerónimo

doi:10.1038/s41419-020-03279-y

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.

Highlights

Esophageal cancer (EC) is the eighth most common cancer worldwide and the sixth most common cause of most patients are diagnosed with a loco-regional disease, surgery remains the cornerstone of curative-intent treatment, despite the high morbidity and mortality rates[6,7]
Hypoxia decreases RT response in Esophageal squamous cell carcinoma (ESCC) In both in vitro hypoxic conditions, Hypoxia-inducible factors (HIFs)-1α chemical induction with 50 μM CoCl2 or 0.5-1% of O2 levels, nuclear HIF-1α and cell membrane carbonic anhydrase IX (CAIX) expression were increased in all cell lines, a more impressive effect was observed in Kyse-30 and OE21 cells [which did not express these proteins in normoxia (21% O2 levels)] (Supplementary Fig. S1A, B)
To determine whether hypoxia might regulate double strand breaks (DSB) repair, γ-H2AX foci staining and DNA fragmentation were assessed through immunofluorescence (IF) and comet assay, respectively

Summary

Introduction

Esophageal cancer (EC) is the eighth most common cancer worldwide and the sixth most common cause of most patients are diagnosed with a loco-regional disease, surgery remains the cornerstone of curative-intent treatment, despite the high morbidity and mortality rates[6,7]. Radiotherapy (RT) is often used as the first-line treatment of EC, both as the main therapeutic strategy or in neoadjuvant context, combined with chemotherapy, entailing similar survival rates in advanced ESSC7,9. Hypoxia-inducible factors (HIFs) mediates tumor cells’ adaptation to hypoxic microenvironment. Is translocated to the nucleus to form the HIF–α/β complex, which binds to specific promoter regions in hypoxiaresponsive elements (HREs). Binding of HIF–α/β to HRE results in transcriptional upregulation of target genes typically involved in cell survival, cell metabolism, proliferation, and angiogenesis[13]. In EC, HIF-1α upregulation is considered a promising endogenous hypoxia biomarker[14] and HIF-1α-mediated upregulation of carbonic anhydrase IX (CAIX) associates with poor prognosis[11,15,16,17,18,19]

Methods

Results

Conclusion