High Ano1 expression as key driver of resistance to radiation and cisplatin in HPV-negative head and neck squamous cell carcinoma

Abstract

Human papilloma virus-negative head and neck squamous cell carcinoma (HNSCC) frequently harbors 11q13 amplifications. Among the oncogenes at this locus, CCND1 and ANO1 are linked to poor prognosis; however, their individual roles in treatment resistance remain unclear. The impact of Cyclin D1 and Ano1 overexpression on survival was analyzed using the TCGA HNSCC dataset and a Charité cohort treated with cisplatin (CDDP)-based radiochemotherapy. High Ano1 expression was primarily associated with poor overall survival in both datasets. The effects of CCND1 and ANO1 knockdown (KD) on radio- and drug sensitivity, along with changes in global protein expression, cell viability, growth, and DNA repair, were studied in an 11q13-amplified HNSCC cell line model of primary cisplatin resistance. Unique pathway alterations- VEGF in CCND1 KD and the Rho GTPase cycle in ANO1 KD- were observed, along with shared changes like DNA damage and cell cycle dysregulation. Silencing CCND1 or ANO1 increased CDDP sensitivity, while only ANO1 silencing increased radiosensitivity. Copanlisib and afatinib were identified as promising candidates for combination therapy of 11q13-amplified HNSCC tumors. We demonstrated a predominant role for Ano1 in treatment resistance in Cyclin D1^highAno1^high HNSCC tumors and identified novel potential treatment combinations for this high-risk patient group.

Keywords: 11q13 amplification; Anoctamin-1; HNSCC; Mass spectrometry; Radiosensitivity; TMEM16A.

Fig. 1

High Ano1 expression in the absence of high cyclin D1 is associated with poor prognosis in HPV-negative HNSCC. (A, B) Patients (HPV- negative cases from the TCGA HNSCC PanCancer Atlas dataset; n = 415) were classified in four groups based on the expression levels of Ano1 and Cyclin D1. (A) Correlation between Cyclin D1 and Ano1 mRNA expression levels; (B) Kaplan Meier curves and Cox regression hazard ratios (+/- 95% confidence intervals) for overall survival according to Ano1 and Cyclin D1 expression levels; (C) Kaplan Meier curves of overall survival for locally advanced HNSCC patients treated with CDDP-based RCTx (Charité cohort, n = 34) according to Ano1 and Cyclin D1 expression levels are shown.

Fig. 2

Proteome analysis revealed common and unique protein expression changes uponCCND1orANO1silencing. (A, B) Volcano plots of fold changes upon CCND1(A) or ANO1(B) KD compared to LeGo, with significant upregulated proteins in red and downregulated proteins in blue; (C, D) Venn diagrams illustrating the number of common and unique proteins that were down- (C) or up-regulated (D) upon KD; (E-G) Pathway enrichment analysis referred to the Reactome dataset based on uniquely down- (blue) and upregulated proteins (red) upon CCND1 KD (E) and ANO1 KD (F), or common for both KD (G). Only the significant pathways (FDR < 0.05) are shown; (H) VEGF detection by ELISA upon CCND1 KD. Bars represent ± SEM from at least three independent experiments.

Fig. 3

Functional characterization of CCND1 and ANO1KD. (A)CCND1 and ANO1 KD significantly reduced the cell growth kinetic of FaDu compared to the control LeGo; (B) The plating efficiency is affected on ANO1 KD but not CCND1 KD, although the cell confluency is impaired for both KD (C, D); (E)CCND1 KD resulted in G0/G1 phase arrest and a simultaneous reduction of the S-phase fraction. Bars represent ± SEM from three independent experiments; (F) The knockdown of CCND1 and ANO1 significantly reduce the cell metabolic activity, measured by MTT assay.

Fig. 4

ANO1 silencing resensitizes cells to DNA damaging treatment (radiation and CDDP). (A,B) Dose response curves to CDDP treatment (A) and radiation (B) in parental cells (upper) and after gene silencing (lower panels). Bars represent ± SEM from at least three independent experiments; (C) Classification of cells according to their sensitivity to radiation based on their D0. Symbols for cell lines correspond to those in B; (D) Correlation between Ano1 mRNA expression and radiosensitivity (D0).

Fig. 5

Kinetics of DNA repair determined by the γH2AX foci assay. Bars represent the average number of γH2AX foci per nuclear area (µm) at 0 Gy and after irradiation with 4 Gy, measured at 2 h and 24 h post irradiation.

Fig. 6

CDK4/6 inhibition does not radiosensitize cells with high expression of Cyclin D1. (A) Western blot analysis revealed down-regulation of pRb and upregulation of Cyclin D1 by palbociclib. Blot lanes have been cropped from the same gel. Original blots are presented in Supplementary Image S1; (B-C) Pretreatment with palbociclib (0.5 µM for 6 h) radiosensitized cells upon CCND1 silencing (B) but not vector control cells (C). Bars represent ± SEM from at least three independent experiments.

Fig. 7

PI3K pathway inhibition with copanlisib leads to degradation of Cyclin D1 via the proteasome and sensitization to CDDP but not radiation. (A) Western blot representing downregulation of Cyclin D1 upon gene silencing or after treatment of 0.5 µM copanlisib for 24 h. This effect was reversed by treatment of the proteasome inhibitor MG132 (10 µM for 4 h). Samples were derived from the same experiment, and gels and blots were processed in parallel. Original blots are presented in Supplementary Image S2; (B) VEGF levels decreased upon copanlisib treatment (0.5 µM for 24 h); (C) Dose-response curves for copanlisib; (D-E) FaDu c5 and UM-22B, two HNSCC cell lines overexpressing Cyclin D1 were pretreated for 24 h with copanlisib before CDDP treatment (D) or irradiation with 4 Gy (E). Viability assay by MTT was done after 72 h (D) or 48 h (E). Bars represent ± SEM from at least three independent experiments.

Fig. 8

Additive inhibitory effect of copanlisib and afatinib in Cyclin D1^highAno1^highcells. (A) Dose-response curves of afatinib treatment in vector control (LeGo) and KD cells; (B) FaDu c5 or UM-SCC-22B cells were treated with 0.15 µM of copanlisib for 24 h before addition of afatinib for 48 h. Bars represent ± SEM from at least three independent experiments.