Selective inhibition of HDAC6 promotes bladder cancer radiosensitization and mitigates the radiation-induced CXCL1 signalling

British Journal of Cancer ARTICLE www.nature.com/bjc OPEN Translational Therapeutics Selective inhibition of HDAC6 promotes bladder cancer radiosensitization and mitigates the radiation-induced CXCL1 signalling Yu-Chieh Tsai1,2, Tzu-Yin Wang1, Chia-Lang Hsu 2,3,4, Wei-Chou Lin5, Jyun-Yu Chen1, Jia-Hua Li6, Yeong-Shiau Pu ✉ Ann-Lii Cheng 8, Jason Chia-Hsien Cheng 1,2 and Sheng-Fang Su 2,9 7 , © The Author(s) 2023 1234567890();,: BACKGROUND: Although trimodality therapy resecting tumours followed by chemoradiotherapy is emerged for muscle-invasive bladder cancer (MIBC), chemotherapy produces toxicities. Histone deacetylase inhibitors have been identified as an effective strategy to enhance cancer radiotherapy (RT). METHODS: We examined the role of HDAC6 and specific inhibition of HDAC6 on BC radiosensitivity by performing transcriptomic analysis and mechanism study. RESULTS: HDAC6 knockdown or HDAC6 inhibitor (HDAC6i) tubacin exerted a radiosensitizing effect, including decreased clonogenic survival, increased H3K9ac and α-tubulin acetylation, and accumulated γH2AX, which are similar to the effect of panobinostat, a pan-HDACi, on irradiated BC cells. Transcriptomics of shHDAC6-transduced T24 under irradiation showed that shHDAC6 counteracted RT-induced mRNA expression of CXCL1, SERPINE1, SDC1 and SDC2, which are linked to cell migration, angiogenesis and metastasis. Moreover, tubacin significantly suppressed RT-induced CXCL1 and radiation-enhanced invasion/ migration, whereas panobinostat elevated RT-induced CXCL1 expression and invasion/migration abilities. This phenotype was significantly abrogated by anti-CXCL1 antibody, indicating the key regulator of CXCL1 contributing to BC malignancy. Immunohistochemical evaluation of tumours from urothelial carcinoma patients supported the correlation between high CXCL1 expression and reduced survival. CONCLUSION: Unlike pan-HDACi, the selective HDAC6i can enhance BC radiosensitization and effectively inhibit RT-induced oncogenic CXCL1-Snail-signalling, thus further advancing its therapeutic potential with RT. British Journal of Cancer; https://doi.org/10.1038/s41416-023-02195-0 INTRODUCTION Although radical cystectomy (RC) remains the gold standard for patients with muscle-invasive bladder cancer (MIBC) [1], bladderpreserving trimodality therapy (TMT), which combines maximal transurethral resection of bladder tumours followed by concurrent chemotherapy and radiotherapy (RT), has been shown to be an effective alternative to patients who are either not suitable surgical candidates or prefer organ preservation [2, 3]. A recent systematic review found similar overall survival but inferior cancer-specific survival in TMT patients compared with RC patients [4], and the chance of undergoing salvage cystectomy occurred in 10.7% of patients receiving TMT [5]. Nevertheless, the recommended chemotherapy regimens, such as cisplatin with fluorouracil or paclitaxel, fluorouracil with mitomycin C, or cisplatin alone (NCCN, preferred/2A) [6], have many well-recognised toxicities. Therefore, there is an unmet need to develop a more effective strategy to enhance the efficacy of RT in BC without the induction of unwanted toxicities. We have previously reported that afatinib, an EGFR/HER-2 dual inhibitor, can radiosensitize BC cells by enhancing radiationinduced DNA damage [7]. Interestingly, histone acetylation has been regarded as a determinant of the radioresponse through mechanisms that regulate chromatin structure and gene expression [8]. By interfering with DNA damage signalling and repair pathways, inhibitors of histone deacetylases (HDAC) decrease the ability of tumour cells to repair radiation-induced DNA damage [9]. Several HDAC inhibitors (HDACi) have entered clinical trials for evaluation of efficacy and toxicity in combination with RT or chemoradiation [10, 11]. In BC, panobinostat, a hydroxamate pan-HDACi, has been demonstrated to have radiosensitizing activity through E3 ligase 1 Department of Oncology, National Taiwan University Hospital, Taipei, Taiwan. 2Graduate Institute of Oncology, National Taiwan University College of Medicine, Taipei, Taiwan. Department of Medical Research, National Taiwan University Hospital, Taipei, Taiwan. 4Graduate Institute of Medical Genomics and Proteomics, National Taiwan University College of Medicine, Taipei, Taiwan. 5Department of Pathology, National Taiwan University Hospital, Taipei, Taiwan. 6Institute of Statistical Science, Academia Sinica, Taipei, Taiwan. 7Department of Urology, National Taiwan University Hospital, Taipei, Taiwan. 8Department of Medical Oncology, National Taiwan University Cancer Centre, Taipei, Taiwan. 9YongLin Institute of Health, YongLin Scholar, National Taiwan University, Taipei, Taiwan. ✉email: 3 Received: 17 July 2022 Revised: 27 January 2023 Accepted: 31 January 2023 Y.-C. Tsai et al. 2 cellular inhibitor of apoptosis protein 2 (cIAP2)-mediated posttranscriptional downregulation of meiotic recombination 11 homologue (MRE11) [12, 13]. An in vivo study by Groselj et al. further demonstrated that panobinostat and RT inhibited bladder tumour growth in RT112 xenografts better than did RT alone without a significant increase in radiation toxicity in the normal tissue [14]. In addition, panobinostat showed clinical benefit in a phase I trial for advanced UC [15]. However, dose-limiting toxicities (DLTs) were reported in recurrent glioma patients who had received a high dose (30 mg) of panobinostat combined with stereotactic irradiation [16]. Therefore, the study of selective HDACis is encouraged, as specific HDACis may be more efficacious and have fewer systemic side effects [14, 17, 18]. HDAC6 is a structurally and functionally unique deacetylase that targets both histone and nonhistone substrates, such as heat shock protein (Hsp90), cortactin, peroxiredoxin, α-tubulin, and heat shock transcription factor-1 (HSF-1) [19, 20], resulting in diverse biological effects. HDAC6 has the ability to promote cell motility, migration and invasion [21, 22]. Selective inhibition of HDAC6 induces DNA damage, suppresses tumour proliferation and sensitises transformed cells to anti-cancer agents [23–25]. Moreover, HDAC6-specific inhibitors (HDAC6i) have entered clinical investigation as part of a combination regimen for anticancer activity [19]. Ricolinostat and citarinostat are selective HDAC6is that were found to enhance efficacy in relapsed multiple myeloma [26, 27]. Interestingly, an increasing number of studies, including preclinical models, have reported the immunoregulatory effect of HDAC6is on cancer suppression and prolonged survival with no significant toxicity [28, 29]. Nevertheless, there is limited investigation of the effects of HDAC6is on radioresponse, especially in treating BC. In this study, we examined the role of HDAC6 and HDAC6 inhibition on BC radiosensitivity. We further performed transcriptomic analysis to elucidate the target genes affected by HDAC6 knockdown in (...truncated)