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.

Supplementary Information

   The online version contains supplementary material available at
   10.1038/s41598-025-85214-9.

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

   Subject terms: Radiotherapy, Cancer therapeutic resistance, Cancer
   genomics, Mechanisms of disease, Predictive markers

Introduction

   With approximately 900,000 cases per year, Head and Neck Squamous Cell
   Carcinoma (HNSCC) ranks as the 7th most frequent cancer globally^[34]1.
   It originates from the epithelial mucosa of the larynx, hypopharynx,
   oropharynx, nasopharynx, and oral cavity. The incidence is primarily
   linked to alcohol and tobacco consumption, as well as human
   papillomavirus (HPV) infection. First-line treatment for HNSCC often
   includes surgery and radiotherapy, with or without the addition of
   platinum-based chemotherapy^[35]2. Despite significant improvements in
   treatment, the 5-year overall survival rate remains below 50% for the
   HPV-negative subgroup, compared to 80% for the HPV-positive
   subgroup^[36]3,[37]4. The high recurrence rate after radiochemotherapy
   (RCTx) in HPV-negative patients underscores the need for better
   therapeutic strategies for this unfavorable group. High interpatient
   heterogeneity in the molecular characteristics of HNSCC tumors
   complicates further treatment optimization.

   As previously reviewed, amplification of the 11q13 locus is predictive
   for poor outcome in patients with HNSCC^[38]5. It occurs in about 30%
   of HPV-negative HNSCC patients (cBioPortal, HNSCC TCGA PanCancer Atlas
   database). The 11q13 locus encompasses several well-characterized
   genes, including: CCND1, ORAOV1, FGF19, FGF4, FGF3, ANO1, FADD, PPFIA1,
   and CTTN, all co-amplified in HNSCC tumors^[39]5. These genes are
   involved in crucial signaling pathways for tumor cell survival,
   proliferation, apoptosis, and cell migration^[40]5,[41]6, making them
   potential therapeutic targets for treatment optimization.

   Cyclin D1, encoded by CCND1, is a critical regulator of the cell cycle,
   contributing to uncontrolled cellular proliferation in cancer. Cyclin
   D1 is closely associated with the CDK4/6 complex, which phosphorylates
   the retinoblastoma protein (Rb). When phosphorylated, pRb detaches from
   the elongation factor E2F, allowing cell cycle progression and
   proliferation^[42]7. Although Cyclin D1 overexpression cannot be
   targeted directly, its deregulated functions in 11q13 amplified cases
   might be indirectly targeted by inhibiting the CDK4/6 complex.

   Another significant gene at the 11q13 locus is ANO1 (synonym: TMEM16A),
   which encodes Anoctamin-1 (referred to herein as Ano1), a transmembrane
   calcium-activated chloride channel involved in essential physiological
   functions of various tissues including salivary glands^[43]8 and airway
   epithelium^[44]9. One key feature of Ano1 is its ability to form a
   tight complex with the epidermal growth factor receptor (EGFR), thereby
   stabilizing EGFR expression^[45]10. Since EGFR is overexpressed in 90%
   of HNSCC cases^[46]11, and its overexpression is associated with poor
   prognosis^[47]12,[48]13, targeting Ano1 might represent a
   preferentially attractive therapeutic strategy for 11q13 amplified
   HNSCC^[49]14,[50]15.

   While targeting oncogenic drivers such as Cyclin D1 and Ano1 could be
   an attractive therapeutic strategy to improve outcomes in HPV-negative
   HNSCC, the high co-expression of these genes obscures their individual
   impact on radio and chemo-resistance. Analysis of patient outcome
   according to gene expression in the TCGA HNSCC dataset as well as a
   Charité patient cohort indicated a major role of Ano1 overexpression in
   poor prognosis. Using a previously established syngenic 11q13 amplified
   HNSCC cell line model of primary cisplatin (CDDP) resistance, we thus
   investigated in detail the role of CCND1 and ANO1 amplification in
   treatment resistance. Gene silencing by shRNA was combined with
   proteome analysis by mass spectrometry and functional phenotype
   analysis, including assessment of sensitivity to irradiation and CDDP.

Materials and methods

Cell lines

   Four HPV-ve cell lines were selected according to their 11q13
   amplification status: four single-cell derived subclones from the
   hypopharyngeal tumor cell line FaDu (ATCC^®HTB–43™, purchased from
   ATCC, Manassas, VA, USA) displaying sensitivity (clone 46, c46; clone
   54, c54) or primary resistance to CDDP (clone 5, c5; clone 78, c78)
   which had been established and comprehensively characterized in a
   previous study^[51]16 UM-SCC-22B, UT-SCC-9 and UT-SCC-15 (kindly
   provided by Prof. Dr. Kathrin Scheckenbach, University of Düsseldorf).
   Cells were maintained at 37 °C supplied with 5% CO[2] in a humidified
   atmosphere. Media composition with references of the reagents are