A novel CREB5/TOP1MT axis confers cisplatin resistance through inhibiting mitochondrial apoptosis in head and neck squamous cell carcinoma

Abstract

Background: Cisplatin resistance is one of the main causes of treatment failure and death in head and neck squamous cell carcinoma (HNSCC). A more comprehensive understanding of the cisplatin resistance mechanism and the development of effective treatment strategies are urgent.

Methods: RNA sequencing, RT-PCR, and immunoblotting were used to identify differentially expressed genes associated with cisplatin resistance. Gain- and loss-of-function experiments were performed to detect the effect of CREB5 on cisplatin resistance and mitochondrial apoptosis in HNSCC. Chromatin immunoprecipitation (ChIP) assay, dual-luciferase reporter assay, and immunoblotting experiments were performed to explore the underlying mechanisms of CREB5.

Results: CREB5 was significantly upregulated in cisplatin-resistant HNSCC (CR-HNSCC) patients, which was correlated with poor prognosis. CREB5 overexpression strikingly facilitated the cisplatin resistance of HNSCC cells in vitro and in vivo, while CREB5 knockdown enhanced cisplatin sensitivity in CR-HNSCC cells. Interestingly, the activation of AKT signaling induced by cisplatin promoted nucleus translocation of CREB5 in CR-HNSCC cells. Furthermore, CREB5 transcriptionally activated TOP1MT expression depending on the canonical motif. Moreover, CREB5 silencing could trigger mitochondrial apoptosis and overcome cisplatin resistance in CR-HNSCC cells, which could be reversed by TOP1MT overexpression. Additionally, double-targeting of CREB5 and TOP1MT could combat cisplatin resistance of HNSCC in vivo.

Conclusions: Our findings reveal a novel CREB5/TOP1MT axis conferring cisplatin resistance in HNSCC, which provides a new basis to develop effective strategies for overcoming cisplatin resistance.

Keywords: CREB5; Cisplatin resistance; HNSCC; Mitochondrial apoptosis; TOP1MT.

Fig. 1

CREB5 is upregulated in cisplatin-resistant HNSCC cells and promotes cisplatin resistance. A MTT assay was used to analyze IC50_cisplatin in parent and cisplatin-resistant cells. B Venn diagram shows the expression of 241 genes upregulated simultaneously in HN4/DDP and HN30/DDP cells; the log FC (fold change) value is greater than 2. C Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of 241 genes upregulated in HN4/DDP and HN30/DDP cells compared to control cells. D, E Immunoblotting was used to detect the correlation of AKT phosphorylation level with cisplatin-stimulated concentration and time. F List of genes in PI3K-Akt signaling pathway. G RT-PCR analysis of the gene expression contained in the PI3K-Akt signaling pathway in parent and cisplatin-resistant cells. H Immunoblotting analysis of CREB5 expression in parent and cisplatin-resistant cells. I Immunoblotting was used to detect the correlation of CREB5 expression with cisplatin-stimulated concentration. J Immunoblotting analysis of phosphor-AKT (Ser 473) and nuclear CREB5 expression in parent cells and untreated or MK2206-treated (10 μM for 24 h) cisplatin-resistant cells. K RT-PCR analysis of CREB5 expression in 40 CR-HNSCC samples and 32 non-CR-HNSCC tissues. L Kaplan-Meier analysis of the correlation between CREB5 expression and overall survival. M, N RT-PCR and immunoblotting were used to detect the overexpression or knockout efficiency of CREB5. O, P MTT assay was used to analyze the correlation of IC50_cisplatin with CREB5 expression in parent and cisplatin-resistant cells. Q The tumor volume and weight in nude mice subcutaneously inoculated with un-transfected or CREB5-transfected HN30 cells at the end of the experiment are shown. R The tumor volume of control or CREB5-transfected HN30 cells was calculated every 3–4 days; cisplatin was injected intraperitoneally every 6 days for a total of four times. S Immunoblotting and RT-PCR analysis of CREB5 expression in subcutaneous tumor tissues of nude mice. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001

Fig. 2

CREB5 regulates mitochondrial apoptosis in parent and cisplatin-resistant cells. A, B MTT assay was used to analyze the relationship between CREB5 expression and proliferation in parent and cisplatin-resistant cells. C, D The relationship between CREB5 expression and the ability to form colonies was analyzed in parent and cisplatin-resistant cells. E Flow cytometry was used to analyze the effect of CREB5 overexpression on the basal and cisplatin-induced (5 μM for 48 h) apoptosis percentage in HN4 and HN30 cells. F Flow cytometry was used to analyze the effect of CREB5 knockdown on the basal and cisplatin-induced (20 μM for 48 h) apoptosis percentage in HN4/DDP and HN30/DDP cells. G The flow chart of the mitochondrial apoptotic pathway. H Immunoblotting was used to analyze the effect of CREB5 knockdown or overexpression on the mitochondrial apoptosis pathway in cisplatin-resistant and parent cells, respectively. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001

Fig. 3

CREB5 promotes TOP1MT transcription in HNSCC. A RT-PCR was used to detect the effect of CREB5 on the expression of mitochondrial-related gene in HN4 and HN30 cells. B Immunoblotting was used to detect the effect of CREB5 on TOP1MT expression in HN4 and HN30 cells. C, D RT-PCR and immunoblotting were used to detect the effect of CREB5 knockdown on TOP1MT expression in HN4/DDP and HN30/DDP cells. E RT-PCR analysis of correlation of CREB5 expression with TOP1MT expression in HNSCC patients. F, G Immunoblotting was used to analyze the effect of TOP1MT on the mitochondrial apoptosis pathway in parent and cisplatin-resistant cells. H Schematic representation of TOP1MT promoter amplified by eight ChIP PCR primers. ChIP-PCR analysis of anti-CREB5- or IgG-immunoprecipitated TOP1MT promoter fragments extracted from HNSCC cells stably transfected with CREB5. I The luciferase deletion mutation vector of the predicted CREB5 target sequence (canonical motif: TGACG, non-canonical motif: TGGCG) in the TOP1MT gene. J Relative TOP1MT reporter activity in HN4 and HN30 cells co-transfected with CREB5 and luciferase reporter. K The effect of cisplatin resistance on TOP1MT reporter luciferase activity in HN4 and HN30 cells. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001

Fig. 4

CREB5 regulates mitochondrial activity through TOP1MT. A–D Real-time OCR measurement of HN4 and HN30 cells treated as indicated. Cells were incubated with 1 μM oligomycin (O), 0.5 μM luoro-carbonyl cyanide phenylhydrazone (F), and 1 μM antimycin (A) for the indicated times. E, F Mito-Tracker Red CMXRos was used to detect the effect of CREB5 and TOP1MT expression on mitochondrial activity in parent and cisplatin-resistant cells. G, H Relative red fluorescence (Mito-Tracker Red CMXRos) values are shown. I, J The effect of CREB5 and TOP1MT expression on ATP levels was analyzed in HN4, HN30, HN4/DDP, and HN30/DDP cells. K Immunoblotting was used to detect the expression levels of CREB5, TOP1MT, Bcl-2, Bcl-xL, Bax, cytochrome c, caspase 3, cleaved caspase 3, caspase 7, cleaved caspase 7, PARP, cleaved PARP, and GAPDH in HN4 and HN30 CREB5 cells with TOP1MT knockdown. L Immunoblotting was used to detect the expression levels of CREB5, TOP1MT, Bcl-2, Bcl-xL, Bax, cytochrome c, caspase 3, cleaved caspase 3, caspase 7, cleaved caspase 7, PARP, cleaved PARP, and GAPDH in HN4/DDP and HN30/DDP siCREB5 cells with TOP1MT overexpression. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001

Fig. 5

CREB5 inhibits apoptosis and promotes resistance to cisplatin by TOP1MT in HNSCC cells. A Flow cytometry analysis of cisplatin-induced (5 μM) apoptosis percentage in HN4 CREB5 cells with TOP1MT knockdown. B Percent cell survival was detected by MTT when TOP1MT was knocked down in HN4 CREB5 cells. IC50_cisplatin values are shown on the right. C Flow cytometry analysis of cisplatin-induced (5 μM) apoptosis percentage in HN30 CREB5 cells with TOP1MT knockdown. D Percent cell survival was detected by MTT when TOP1MT was knocked down in HN30 CREB5 cells. IC50_cisplatin values are shown on the right. E Flow cytometry analysis of cisplatin-induced (20 μM) apoptosis percentage in HN4/DDP siCREB5 cells with TOP1MT overexpression. F Percent cell survival was detected by MTT when TOP1MT was knocked down in HN4/DDP siCREB5 cells. IC50_cisplatin values are shown on the right. G Flow cytometry analysis of cisplatin-induced (20 μM) apoptosis percentage in HN30/DDP siCREB5 cells with TOP1MT overexpression. H Percent cell survival was detected by MTT when TOP1MT was overexpressed in HN30/DDP siCREB5 cells. IC50_cisplatin values are shown on the right. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001

Fig. 6

Double-targeting of CREB5 and TOP1MT has a potent antitumor effect in cisplatin-resistant HNSCC in vivo. A The tumor weight in nude mice subcutaneously inoculated with HN30/DDP cells treated with cholesterol-modified siCREB5 or/and siTOP1MT (5 μM) at the end of the experiment are shown. B The tumor volume of HN30/DDP cells treated with cholesterol-modified siCREB5 or/and siTOP1M (5 μM) was calculated every 3–4 days, and cisplatin was injected intraperitoneally every 6 days for a total of four times. C Ki67, TOP1MT, and CREB5 in subcutaneous tumor tissue of nude mice were detected and analyzed using IHC. D, E RT-PCR and immunoblotting analysis showed the expression of TOP1MT, CREB5, and GAPDH in the subcutaneous tumor tissue of nude mice. F Sample information of PDX model. G, H Image of mice and tumor after siCREB5 and siTOP1MT administration for 3 weeks. I, J Nude mice were used to construct the PDX model. The treatment group was given siCREB5 and siTOP1MT (5 μM) every day. The tumor volume and weight were recorded every week, and the mice were killed after 3 weeks. *p < 0.05; **p < 0.01; ***p < 0.001 ****p < 0.0001

Fig. 7

A positive correlation between CREB5 and TOP1MT expression was observed in HNSCC tissues. A CREB5 and TOP1MT expression was detected using IHC in normal oral tissues. B Representative images of CREB5 and TOP1MT expression in HNSCC tissues. C The expression scores of CREB5 and TOP1MT were analyzed in normal and HNSCC tissues. D IHC analysis of the correlation between CREB5 and TOP1MT expression in the HNSCC tissue microarray. E–H The correlations between the expression scores of TOP1MT and CREB5 and gender, grade, TNM stage, and age were analyzed in normal and HNSCC tissues. I The proposed model illustrates the regulatory role of TOP1MT and CREB5 in promoting cisplatin resistance in HNSCC. *p < 0.05; **p < 0.01; ***p < 0.001