Investigation into the Molecular Mechanisms Underlying Cisplatin Resistance in Oral Squamous Cell Carcinoma

Abstract

Oral Squamous Cell Carcinoma (OSCC) is the 6th most common cancer worldwide. It is generally aggressive and closely associated with chemoresistance and poor survival. Cisplatin serves as one of the mainstay drugs in the treatment of advanced OSCC. It induces apoptotic cell death in tumour cells by directly binding to nuclear and mitochondrial DNA and by promoting the generation of cellular reactive oxygen species (ROS). Although a significant anti-cancer response is observed initially in response to cisplatin treatment, patients often relapse due to the acquirement of chemoresistance. This study sought to examine some of the molecular mechanisms underlying chemoresistance to cisplatin and to establish novel therapeutic strategies to sensitise OSCC cells to cisplatin. More specifically, this study aimed to characterise differences in the Nrf-2/HO-1 antioxidant axis, anti-apoptotic Bcl-2 protein and inhibitor of apoptosis protein (IAP) expression in an isogenic cell culture model of acquired cisplatin resistance in OSCC in order to identify novel targets and treatment strategies. Given the link between cisplatin and mitochondrial function, seahorse bioenergetic analyses were first conducted to determine whether acquired cisplatin resistance was impacting mitochondrial function. No differences in basal oxygen consumption rate (OCR), ATP-linked OCR and extracellular acidification rate (ECAR), an index of glycolysis, were observed between the paired cisplatin-sensitive tongue squamous carcinoma SCC4 and cisplatin-resistant SCC4cisR cell lines, suggesting that the development of resistance to cisplatin was not associated with alterations in oxidative metabolism and mitochondrial function. Alterations in the balance between oxidative stress mediated by ROS and the antioxidant system, mediated by the transcription factor nuclear erythroid-related factor-2 (Nrf-2) and its downstream enzyme heme oxygenase-1 (HO-1), has been reported to be involved in the mechanism of chemoresistance in a number of tumour types. The role of Nrf-2/HO-1 axis in mediating chemoresistance of OSCC was next evaluated. Basal expression levels of Nrf-2 and HO-1 were observed to be significantly upregulated in SCC4cisR cells when compared to parental cells. However, the inhibition of Nrf-2 and HO-1, mediated by the small molecule Nrf-2 inhibitor ML385, failed to sensitise the SCC4cisR cells to cisplatin, even though ML385 treatment successfully promoted cellular ROS, suggesting that the balance between ROS and Nrf-2/HO-1 does not play a significant role in mediating cisplatin resistance in these OSCC cells. Dysregulation of apoptosis is a hallmark of cancer and deregulated expression of members of the Bcl-2 family of proteins is a key mechanism leading to apoptosis evasion and drug resistance. This study next characterised the expression levels of a series of pro-and anti-apoptotic members of the Bcl-2 family in the paired OSCC cell lines. The value of the BH3-mimetics, venetoclax and obatoclax, as sensitisers for cisplatin treatment in OSCC was also evaluated. Obatoclax was shown to synergistically enhance cisplatin-induced apoptosis, and this enhancement was associated with a marked degradation in pro-survival Mcl-1 and upregulation in conformationally active form of pro-apoptotic Bak. This study demonstrates that targeted therapy with BH-3 mimetics, such as obatoclax, may represent a novel strategy for OSCC therapy. Overexpression of IAP family members such as XIAP and cIAP1/2 has been previously correlated with OSCC occurrence and lymph node metastasis indicating that these proteins play an important role in OSCC development and are linked with poor prognosis. This study finally assessed and compared the potential of various IAP inhibitors both as stand-alone treatments and as cisplatin sensitising agents in OSCC. Sensitivity of SCC4 and 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 cells which was mediated in part by depletion of XIAP. In addition, knockdown of XIAP using siRNA enhanced cisplatin-mediated cell death, demonstrating the importance of targeting XIAP in this sensitisation. In conclusion, this study has provided novel insights into the molecular mechanisms underlying cisplatin resistance in OSCC and proposes a number of novel therapeutic options for this disease.