ThermomiR-377-3p-induced suppression of Cirbp expression is required for effective elimination of cancer cells and cancer stem-like cells by hyperthermia

Abstract

Background In recent years, the development of adjunctive therapeutic hyperthermia for cancer therapy has received considerable attention. However, the mechanisms underlying hyperthermia resistance are still poorly understood. In this study, we investigated the roles of cold‑inducible RNA binding protein (Cirbp) in regulating hyperthermia resistance and underlying mechanisms in nasopharyngeal carcinoma (NPC). Methods CCK-8 assay, colony formation assay, tumor sphere formation assay, qRT-PCR, Western blot were employed to examine the effects of hyperthermia (HT), HT + oridonin(Ori) or HT + radiotherapy (RT) on the proliferation and stemness of NPC cells. RNA sequencing was applied to gain differentially expressed genes upon hyperthermia. Gain-of-function and loss-of-function experiments were used to evaluate the effects of RNAi-mediated Cirbp silencing or Cirbp overexpression on the sensitivity or resistance of NPC cells and cancer stem-like cells to hyperthermia by CCK-8 assay, colony formation assay, tumorsphere formation assay and apoptosis assay, and in subcutaneous xenograft animal model. miRNA transient transfection and luciferase reporter assay were used to demonstrate that Cirbp is a direct target of miR-377-3p. The phosphorylation levels of key members in ATM-Chk2 and ATR-Chk1 pathways were detected by Western blot. Results Our results firstly revealed that hyperthermia significantly attenuated the stemness of NPC cells, while combination treatment of hyperthermia and oridonin dramatically increased the killing effect on NPC cells and cancer stem cell (CSC)‑like population. Moreover, hyperthermia substantially improved the sensitivity of radiation‑resistant NPC cells and CSC‑like cells to radiotherapy. Hyperthermia noticeably suppressed Cirbp expression in NPC cells and xenograft tumor tissues. Furthermore, Cirbp inhibition remarkably boosted anti‑tumor‑killing activity of hyperthermia against NPC cells and CSC‑like cells, whereas ectopic expression of Cirbp compromised tumor‑killing effect of hyperthermia on these cells, indicating that Cirbp overexpression induces hyperthermia resistance. ThermomiR-377-3p improved the sensitivity of NPC cells and CSC‑like cells to hyperthermia in vitro by directly suppressing Cirbp expression. More importantly, our results displayed the significantly boosted sensitization of tumor xenografts to hyperthermia by Cirbp silencing in vivo, but ectopic expression of Cirbp almost completely counteracted hyperthermia-mediated tumor cell-killing effect against tumor xenografts in vivo. Mechanistically, Cirbp silencing-induced inhibition of DNA damage repair by inactivating ATM-Chk2 and ATR-Chk1 pathways, decrease in stemness and increase in cell death contributed to hyperthermic sensitization; conversely, Cirbp overexpression-induced promotion of DNA damage repair, increase in stemness and decrease in cell apoptosis contributed to hyperthermia resistance. Conclusion Taken together, these findings reveal a previously unrecognized role for Cirbp in positively regulating hyperthermia resistance and suggest that thermomiR-377-3p and its target gene Cirbp represent promising targets for therapeutic hyperthermia.

Global trends in research of nasopharyngeal carcinoma: a bibliometric and visualization analysis

“…It is one of the key mechanisms for NPC cells to resist anti-tumor measures, leading to metastasis and invasion ( 59 ). Multiple studies have confirmed that TGF-β ( 60 ), NF-κB ( 61 ), Wnt ( 62 ), Akt ( 63 ), Notch ( 64 ), and other signaling pathways are involved in EMT in NPC, while EBV infection ( 65 ), abnormal gene expression ( 66 ), hypoxia ( 67 ), and abnormal expression of non-coding RNA ( 68 ) are involved in regulating these signaling pathways. Stanniocalcin-2 promotes cell EMT by activating the ITGB2/FAK/SOX6 signaling pathway in NPC ( 69 ).…”

“…In terms of drug resistance, tumor repopulating cells (TRCs) with stem cell-like cancer cell characteristics resist chemotherapy and radiotherapy by reducing the sensitivity to iron death ( 114 ). Based on such an ecological theory, researchers can consider using ecological therapies to combat NPC (by altering the surrounding environment to eliminate specific targets), such as destroying tumor habitats through hyperthermia ( 68 ) and reducing drug resistance in NPC cells through adaptive therapies ( 115 ). Therefore, it is helpful for researchers to develop more effective anti-cancer strategies to examine NPC in a new system.…”