Oral Squamous Cell Carcinoma (OSCC) is currently the 6th most common type of cancer worldwide. It is generally aggressive and closely associated with recurrence and poor survival. Surgical resection in combination with chemotherapy and radiotherapy are the primary approaches taken to treat OSCC [1]. Unfortunately, there has been little improvement in survival trends in recent years indicating that new treatment protocols are urgently required.

Cisplatin is the most common platinum-based chemotherapeutic drug used clinically in the treatment of OSCC [2]. Cisplatin induces cytotoxicity through the formation of DNA adducts after its spontaneous activation by a series of intracellular aquation reactions. This results in DNA damage and activation of a range of signalling pathways including ATM (ataxia telangiectasia mutated) and ATR (ATM and Rad3-related), MAPK (mitogen-activated protein kinase) and p53, which in turn lead to apoptotic cell death [3]. Resistance to chemotherapeutic agents, such as cisplatin, is a major problem associated with OSCC leading to therapeutic failure and tumour progression and more study is required to understanding the mechanisms underlying drug resistance. Cisplatin efficacy is also reduced due to dose-limiting side effects including neurotoxicity and nephrotoxicity. One strategy to overcome these problems and to improve response to cisplatin is to use drug combinations. For example, cisplatin in combination with 5-fluouracil, is the current standard of care for treatment of unresectable locally advanced oesophageal cancer [4].

Evasion of apoptosis is a key hallmark of cancer and one important mechanism of suppression of apoptosis and resistance to anticancer chemo/radiotherapy has been attributed to the inhibitor of apoptosis protein (IAP) family [5,6]. Several members of the IAP family including X-linked inhibitor of apoptosis (XIAP), cellular inhibitor of apoptosis proteins 1 and 2 (cIAP1/cIAP2), and livin have been implicated in cancer progression and have been shown to be overexpressed in various malignancies including oesophageal, breast, lung and colorectal with expression levels correlated with disease severity [[7], [8], [9], [10]]. All members of the IAP family are characterised by at least one baculovirus-IAP-repeat (BIR) domain, an approximately 70-residue-large region which plays a crucial role in the activity of these proteins (Birnbaum, Clem and Miller, 1994). These BIR domains mediate protein-protein interactions and have been implicated in the binding to and inhibition of pro-apoptotic caspases. XIAP along with cIAPs 1/2 are the most widely studied and best characterised IAPs. XIAP is the only IAP family member that directly binds to and inhibits initiator caspase 9 and the effector caspases 3 and 7 through its BIR2 and BIR3 domains. In addition to the BIR domains, XIAP along with cIAP1/2 proteins contain a C-terminal RING domain which confers E3 ubiquitin ligase activity on these proteins. This RING domain induces the degradation of the endogenous IAP antagonist Smac which is released from the mitochondria during apoptosis, and of XIAP and cIAPs themselves thus further inhibiting apoptosis [11].

There is accumulating evidence for the involvement of IAPs in mediating chemotherapy resistance including resistance to cisplatin. Pre-clinical and patient studies have demonstrated a correlation between acquired cisplatin resistance and IAP overexpression [[12], [13], [14]]. Various strategies for targeting and inhibiting IAPs have been designed and tested in recent years including antisense oligonucleotides, silencing RNA and the use of IAP inhibitors. Embelin, is a naturally occurring molecule found in traditional Japanese herbal remedies. It binds to XIAP with high affinity and promotes apoptosis by relieving caspase 9 inhibition and inhibiting pro-survival NFkB signalling [15]. Embelin has also been shown to be effective at inducing apoptosis in pancreatic cancer, nasopharyngeal carcinoma, and breast cancer cells [16]. Small-molecule Smac mimetics are IAP inhibitors that mimic the N-terminal region of Smac which is necessary to bind to the BIR domains of IAPs [17]. This region comprises four conserved hydrophobic amino acid residues: Ala-Val-Pro-Ile (AVPI) and is essential for binding to the hydrophobic environment of the BIR2 and BIR3 domains of IAP proteins. Monovalent (e.g. Xevinapant and LCL161) as well as bivalent Smac mimetics have been developed (e.g. BV6 and birinapant). In pre-clinical studies, BV6 has been demonstrated to dose-dependently promote apoptosis by inducing the degradation of XIAP, cIAP1 and cIAP2 in several malignant cell lines, including multiple myeloma, colorectal adenocarcinoma, fibrosarcoma, cervical carcinoma and T cell lymphoma (El-Mesery, Shaker and Elgaml, 2016). Furthermore, a wide range of research has demonstrated the synergistic effect between Smac mimetics and other therapies for cancer. For example, the Smac mimetic LBW242 promoted the efficacy of cytarabine and doxorubicin in AML [18]. More recently, both Embelin and BV6 were shown to enhance both TRAIL and cisplatin mediated apoptosis in malignant rhabdoid tumours (MRT) ([19]; Coyle, O'Sullivan and Zisterer, 2022). Xevinapant has shown promising results in sensitising chemoresistant ovarian cancer cells to carboplatin [20] and in sensitising NSCLC cells to radiotherapy [21].

Overexpression of IAP family members such as XIAP and cIAP1/2 has been correlated with OSCC occurrence and lymph node metastasis [22,23], indicating that these proteins play an important role in OSCC development and are linked with poor prognosis. Further research to assess the role of the IAP family in mediating chemoresistance in OSCC is warranted and further evaluation of the use of IAP antagonists to combat resistance is required. This pre-clinical study assessed and compared the potential of various IAP inhibitors both as stand-alone treatments and as cisplatin sensitising agents in OSCC. Sensitivity of paired cisplatin-sensitive tongue carcinoma SCC4 and -resistant SCC4CisR cells to IAP inhibition via the Smac mimetics BV6 and Xevinapant along with XIAP inhibitor Embelin was examined. Both BV6 and Embelin synergistically enhanced cisplatin-mediated apoptotic cell death in resistant SCC4CisR cells. This enhanced apoptosis was mediated in part by XIAP inhibition. Furthermore, siRNA knockdown of XIAP enhanced cisplatin-mediated cell death, demonstrating the importance of targeting XIAP in this sensitisation. The current findings thus suggest potential novel therapeutic options for OSCC.