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.

Keywords: Apoptosis; Cancer stem cells (CSCs); Chemotherapy; Cold-inducible RNA-binding protein (Cirbp); DNA damage; Hyperthermia; Nasopharyngeal carcinoma (NPC); Near-infrared (NIR) laser; Radiotherapy; Temperature-sensitive miRNA-377-3p (termed thermomiR-377-3p); Therapy resistance; Thermosensitivity.

Fig. 1

Hyperthermia significantly suppressed the proliferation and stemness of NPC cells. A, B CCK-8 assay A and colony formation assay B were performed in indicated NPC cells treated with hyperthermia at 42 °C or 44 °C for 30 min. C-E The xenograft subcutaneous tumor formation of hyperthermia-treated CNE2 cells in nude mice. CNE2 cells in vitro treated with hyperthermia at 42 °C or 44 °C for 30 min were injected subcutaneously into nude mice (n = 6). C Representative images of stripped xenograft tumors formed by CNE2 cells at the end of experiment. D The growth curve of tumor volumes within 20 days. E Tumor weight. F, G qRT-PCR F and Western blot G were employed to detect stemness-related gene expression in indicated NPC cells treated with hyperthermia at 42 °C for 30 min. H, I Flow cytometry analysis of the percentages of side population (SP) cells in indicated NPC cells treated with hyperthermia at 42 °C or 44 °C for 30 min. J, K Tumor sphere formation assay was used to detect the self-renewal ability of NPC cells treated with hyperthermia at 42 °C or 44 °C for 30 min. For tumor sphere formation assay, the indicated NPC cells (1 × 10³/well) treated with hyperthermia at 42 °C or 44 °C for 30 min were grown in serum-free DMEM-F12 supplemented with 10 μg/L bFGF, 20 μg/L EGF and 2% B27 in ultra-low adhesion plates. Two weeks later, spheres were counted by an inverted microscope, and images were acquired. Sphere size and density are shown in the left panels J, and the number of spheres is shown in the right panels K

Fig. 2

Combination treatment of hyperthermia and oridonin (Ori) significantly increased the killing effects on NPC cells and CSC-like population within NPC cells in vitro. A CCK-8 assay was performed in CNE2 and SUNE1 cells treated with oridonin at different concentrations for 24 h. B qRT-PCR was used to detect stemness-related gene expression in CNE2 and SUNE1 cells treated with different concentrations of oridonin (20, 40 and 60 μM) for 24 h. C, D Tumor sphere formation assay was performed in CNE2 and SUNE1 cells treated with oridonin at 20, 40 and 60 μM concentration for 24 h. E, F AnnexinV/PI apoptosis assay was performed in CNE2 and SUNE1 cells treated with the indicated concentrations of oridonin for 24 h. G qRT-PCR was used to detect apoptosis-related gene expression in CNE2 and SUNE1 cells treated with the indicated concentrations of oridonin for 24 h. H–L CCK-8 assay H, colony formation assay I, J and tumor sphere formation assay K, L were performed in CNE2 and SUNE1 cells treated with oridonin (20 μM) alone or combined treated with hyperthermia (42 °C for 30 min)

Fig. 3

Hyperthermia significantly enhanced the anti-tumor-killing activity of radiotherapy against radiation-resistant NPC cells and cancer stem-like cells. A Representative photographs of the morphology of CNE2 cells and radiation-resistant CNE2-8G cells. B Western blot was employed to detect stemness- and EMT-related gene expression in CNE2 and CNE2-8G cells. C CCK-8 assay was performed in CNE2 and CNE2-8G cells. D, E Tumor sphere formation assay was performed in CNE2 and CNE2-8G cells. F–H CCK-8 assay F, colony formation assay G and tumor sphere formation assay H were performed in CNE2 and CNE2-8G cells subjected to irradiation (IR) treatment at 0, 4 and 8Gy. I-K CCK-8 assay I colony formation assay J and tumor sphere formation assay K were performed in CNE2-8G cells treated by hyperthermia (42 °C for 30 min) and IR (4Gy) alone or combined. L qRT-PCR assay for detecting stemness-related gene expression in CNE2-8G cells treated by hyperthermia (42 °C for 30 min) and IR (4Gy) alone or combined

Fig. 4

RNAi-mediated silencing of endogenous Cirbp remarkably enhanced the tumor-killing effect of hyperthermia on NPC cells and cancer stem-like cells in vitro. A qRT-PCR assay for detecting the expression of Cirbp, Rbm3, miR-143 and miR-142-5p in the indicated NPC cells treated with or without hyperthermia at 42 °C for 30 min. B Western blot was employed to detect Cirbp expression in CNE2, SUNE1 and HONE1-EBV cells treated with or without hyperthermia at 42 °C for 30 min. C Class comparison and hierarchical clustering of differentially expressed hyperthermia-related genes between NPC cells treated with or without hyperthermia at 42 °C for 30 min. A cluster heat map for upregulated (red) and downregulated (blue) genes (see Tables S3 and S4) is shown. Other details as in Fig. S5. D Gene ontology (GO) analysis of up- and down-regulated genes (see Table S3) enriched in hyperthermia-associated biological processes, such as cellular response to heat, DNA damage and repair, cell cycle and cell death between NPC cells treated with or without hyperthermia at 42 °C for 30 min. E–H CCK-8 assay E, colony formation assay F, tumor sphere formation assay G and AnnexinV/PI apoptosis assay H were performed in shSCR- or shCirbp-expressing NPC cells treated with or without hyperthermia at 42 °C for 30 min. SCR: scrambled control shRNA. I Heatmap showing selected differentially expressed genes (see Table S6) related to cell death in shCirbp-expressing NPC cells. Right column lists the selected gene symbols. J GO and KEGG pathway analysis of up- and down-regulated genes (see Tables S6 and S7) related to cell survival and death in shSCR and shCirbp-expressing NPC cells

Fig. 5

Exogenous expression of Cirbp counteracted the tumor-killing effect of hyperthermia on NPC cells and cancer stem-like cells in vitro. A-D CCK-8 assay A, colony formation assay B, tumor sphere formation assay C and AnnexinV/PI apoptosis assay D were performed in Cirbp-expressing NPC cells treated with or without hyperthermia at 42 °C for 30 min

Fig. 6

ThermomiR-377-3p improved the sensitivity of NPC cells and cancer stem-like cells to hyperthermia in vitro by directly suppressing Cirbp expression. A-C qRT-PCR assay for detecting the expression of selected miRNAs of which Cirbp might be a potential target gene in the indicated NPC cells treated with or without hyperthermia at 40 °C and 42 °C for 30 min. ThermomiRs (i.e., miR-143 and miR-142-5p) were used as positive controls. D, E qRT-PCR assay for detecting the expression of miR-377-3p D and Cirbp E in NPC cells transiently transfected with miR-377-3p mimics or inhibitor. F Western blot was employed to detect Cirbp expression in NPC cells transiently transfected with miR-377-3p mimics or inhibitor. G Diagram of 3’-UTR-WT and 3’-UTR-MUT of Cirbp containing reporter constructs. H Luciferase reporter assays in HEK293T cells co-transfected with WT or MUT 3’-UTR and miRNAs as indicated. I, J Colony formation assay I and tumor sphere formation assay J were performed in miR-377-expressing NPC cells treated with or without hyperthermia at 42 °C for 30 min. K EdU assay was performed in NPC cells transiently transfected with miR-377-3p mimics and then treated with or without hyperthermia at 42 °C for 30 min. L Western blot was employed to detect Cirbp expression in miR-377- and Cirbp-expressing NPC cells. M, N Colony formation assay M and tumor sphere formation assay N were performed in miR-377- and Cirbp-expressing NPC cells treated with or without hyperthermia at 42 °C for 30 min

Fig. 7

Cirbp silencing-induced sensitization of subcutaneous tumor xenografts to hyperthermia by local thermal ablation with ICG and an NIR laser in vivo. A Schematic representation of the experimental design of hyperthermia treatment in nude mice harboring subcutaneous tumor xenografts formed by CNE2 or HONE1-EBV cells. B, E Representative images of stripped xenograft tumors formed by CNE2 B and HONE1-EBV E cells at the end of hyperthermia therapy (n = 3-4 mice/group). C, F The tumor growth curve (n = 3-4 mice/group). D, G Tumor weight (n = 3-4 mice/group). H Representative pictures of H&E staining of stripped xenograft tumors (showed in Fig. 7B, E)

Fig. 8

Ectopic expression of Cirbp counteracted the tumor-killing effect of hyperthermia on NPC cells and cancer stem-like cells in vivo. A Schematic representation of the experimental design of hyperthermia treatment in nude mice bearing subcutaneous tumor xenografts formed by CNE2 or SUNE1 cells. B, E Representative images of stripped xenograft tumors formed by CNE2 B and SUNE1 E cells at the end of hyperthermia therapy (n = 4 mice/group). C, F The tumor growth curve (n = 4 mice/group). D, G Tumor weight (n = 4 mice/group). H Representative pictures of H&E staining of stripped xenograft tumors (showed in Fig. 8B,E)

Fig. 9

Cirbp positively regulated the resistance of CSC-like cells to hyperthermia. A Western blot analysis of stemness-related gene expression in Cirbp-expressing and shCirbp-expressing NPC cells treated with or without hyperthermia at 42 °C for 30 min. B Western blot analysis of stemness-related gene expression in xenograft tumors (showed in Fig. 7B) formed by CNE2 cells. C Western blot analysis of stemness-related gene expression in xenograft tumors (showed in Fig. 8B) formed by CNE2 cells

Fig. 10

Cirbp silencing-induced inhibition of DNA damage repair. A, B Representative pictures of 53BP1 staining (red) A and quantification of the fraction of 53BP1⁺ cells B in shCirbp-expressing NPC cells treated with or without hyperthermia at 42 °C for 30 min. C, D Representative pictures of γ-H2AX staining(red) C and quantification of the fraction of γ-H2AX⁺ D in shCirbp-expressing NPC cells treated with or without hyperthermia at 42 °C for 30 min. E Western blot assay was used to detect Cirbp, p-ATM, p-ATR, p-Chk1, p-Chk2, p-BRCA1, p-p53 and γ-H2AX in indicated cells

Fig. 11

Ectopic expression of Cirbp counteracts the thermosensitivity of NPC cells by promoting DNA damage repair. A, B Representative pictures of 53BP1 staining(red) A and quantification of the fraction of 53BP1⁺ cells B in Cirbp-expressing NPC cells treated with or without hyperthermia at 42 °C for 30 min. C, D Representative pictures of γ-H2AX staining(red) C and quantification of the fraction of γ-H2AX⁺ D in Cirbp-expressing NPC cells treated with or without hyperthermia at 42 °C for 30 min. E Heatmap showing selected differentially expressed genes (see Table S10) involved in DNA damage and repair, and cell cycle in Cirbp-expressing CNE2 and SUNE1 cells. Right column lists the selected gene symbols. F GO and KEGG pathway analysis of up- and down-regulated genes (see Tables S11 and S12) involved in DNA damage and repair, and cell cycle in Cirbp-expressing CNE2 and SUNE1 cells. G Cirbp, p-ATM, p-ATR, p-Chk1, p-Chk2, p-BRCA1, p-p53 and γ-H2AX in indicated cells were determined by Western blot

Fig. 12

A proposed mechanism of Cirbp-mediated resistance and sensitization to hyperthermia