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Head and neck squamous cell carcinoma (HNSCC) is the eight most common cancer in Europe. Patients with HNSCC receive primary radiochemotherapy (RCTx) and postoperative radiochemotherapy (PORT-C) as standard treatments. Patients show diverse responses due to tumor heterogeneity highlighting the importance of patient stratification. Tumor relapses can be attributed to cancer stem cells (CSCs). In HNSCC, CSCs are identified by several markers, including CD98hc and high aldehyde dehydrogenase (ALDH) activity contributed by ALDH1A1 and ALDH1A3 isoforms producing retinoic acid (RA) (1,2,3). CD98hc and CD98hc-related amino acid transporters (CD98hc-AATs) function as stress response and stemness regulators (4). Our previous study showed that CD98hc-associated signaling mechanisms play a critical role in HNSCC radioresistance. High CD98hc expression level is a prognostic marker of poor locoregional control in locally advanced HNSCC patients treated with PORT-C or primary RCTx (5,6). Our study aims to decipher the interplay between CD98hc-AATs and CSC phenotype. 

To analyze the relationship between CD98hc-AATs and ALDH isoforms, we treated HNSCC cell lines with all-trans retinoic acid (ATRA). We performed siRNA-mediated knockdown of ALDH1A1 or ALDH1A3 isoforms and RA receptors RARA and RXRA to investigate the influence of ALDH activity on CD98hc-AATs. The expression levels of CD98hc-AATs and ALDH isoforms were measured by RT-qPCR and Western blot. Sphere-forming capacity of HNSCC cell lines were investigated upon depletion of CD98hc-AATs. TCGA data mining was used to correlate RARA/RXRA expression with patient outcome.  

Our data show that ATRA treatment significantly increased CD98hc and LAT1 expression levels and resulted in HNSCC radiosensitization in a dose-dependent manner. Knockdown of ALDH1A1, ALDH1A3, RARA or RXRA lead to CD98hc and LAT1 downregulation. Expression levels of RARA/RXRA correlate with HNSCC radiosensitivity and patient outcomes. We identified putative RARA/RXRA binding sites on SLC3A2 and SLC7A5 promoters. Downregulation of ALDH1A1 and ALDH1A3 upon LAT1 knockdown suggests an interplay between CD98hc-AATs and ALDH isoforms. Additionally, knockdown of LAT2 and xCT affects size and numbers of spheres, respectively, in Cal33 cells.  

These results indicate that the expression of CD98hc-AATs are is associated with the regulation of HNSCC CSC properties, and are affected by ALDH activity. This interplay can be critical for tumor growth and radioresistance. Thus, evaluation of RARA and RXRA expression in samples of patients treated with PORT-C and RCTx will be performed to validate the clinical relevance of our findings.

References:

1. Kurth I, et al., Oncotarget. 2015.

2. Krause M, et al., Adv Drug Deliv Rev. 2017.

3. Peitzsch C, et al., Cancers (Basel). 2019.

4. Kahya U, et al., Cancers (Basel). 2021.

5. Linge A, et al., Clin Cancer Res. 2016.

6. Digomann D, et al., Clin Cancer Res. 2019.