Differential response of head and neck cancer cell lines to TRAIL or Smac mimetics is associated with the cellular levels and activity of caspase-8 and caspase-10.

BACKGROUND: Current treatment strategies for head and neck cancer are associated with significant morbidity and up to 50% of patients relapse, highlighting the need for more specific and effective therapeutics. Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) and Smac mimetics (SMs) are promising anticancer agents, but their effect on head and neck squamous cell carcinoma (HNSCC) remains unknown. METHODS: We examined the response of a panel of nine HNSCC cell lines to TRAIL and SMs and investigated the mechanism of cell type-specific response by functional analysis. RESULTS: Head and neck cancer cell lines revealed a converse response pattern with three cell lines being highly sensitive to Smac-164 (SM) but resistant to TRAIL, whereas the other six were sensitive to TRAIL but resistant to SM. Distinct protein expression and activation patterns were found to be associated with susceptibility of HNSCC cell lines to TRAIL and SM. Tumour necrosis factor-related apoptosis-inducing ligand sensitivity was associated with high caspase-8 and Bid protein levels, and TRAIL-sensitive cell lines were killed via the type II extrinsic apoptotic pathway. Smac mimetic-sensitive cells expressed low levels of caspase-8 and Bid but had high TNF-α expression. Smac mimetic-induced cell death was associated with caspase-10 activation, suggesting that in the absence of caspase-8, caspase-10 mediates response to SM. Cotreatment with TNF-α sensitised the resistant cells to SM, demonstrating a decisive role for TNF-α-driven feedback loop in SM sensitivity. CONCLUSIONS: Tumour necrosis factor-related apoptosis-inducing ligand and SMs effectively kill HNSCC cell lines and therefore represent potential targeted therapeutics for head and neck cancer. Distinct molecular mechanisms determine the sensitivity to each agent, with levels of TNF-α, caspase-8, Bid and caspase-10 providing important predictive biomarkers of response to these agents.

Annexin A1 regulates EGFR activity and alters EGFR-containing tumour-derived exosomes in head and neck cancers.

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) is the 6th most common cancer with approximately half a million cases diagnosed each year worldwide. HNSCC has a poor survival rate which has not improved for over 30 years. The molecular pathogenesis of HNSCCs remains largely unresolved; there is high prevalence of p53 mutations and EGFR overexpression; however, the contribution of these molecular changes to disease development and/or progression remains unknown. We have recently identified microRNA miR-196a to be highly overexpressed in HNSCC with poor prognosis. Oncogenic miR-196a directly targets Annexin A1 (ANXA1). Although increased ANXA1 expression levels have been associated with breast cancer development, its role in HNSCC is debatable and its functional contribution to HNSCC development remains unclear. METHODS: ANXA1 mRNA and protein expression levels were determined by RNA Seq analysis and immunohistochemistry, respectively. Gain- and loss-of-function studies were performed to analyse the effects of ANXA1 modulation on cell proliferation, mechanism of activation of EGFR signalling as well as on exosome production and exosomal phospho-EGFR. RESULTS: ANXA1 was found to be downregulated in head and neck cancer tissues, both at mRNA and protein level. Its anti-proliferative effects were mediated through the intracellular form of the protein. Importantly, ANXA1 downregulation resulted in increased phosphorylation and activity of EGFR and its downstream PI3K-AKT signalling. Additionally, ANXA1 modulation affected exosome production and influenced the release of exosomal phospho-EGFR. CONCLUSIONS: ANXA1 acts as a tumour suppressor in HNSCC. It is involved in the regulation of EGFR activity and exosomal phospho-EGFR release and could be an important prognostic biomarker.

Bortezomib sensitises TRAIL-resistant HPV-positive head and neck cancer cells to TRAIL through a caspase-dependent, E6-independent mechanism.

Human papillomavirus (HPV) is causative for a new and increasing form of head and neck squamous cell carcinomas (HNSCCs). Although localised HPV-positive cancers have a favourable response to radio-chemotherapy (RT/CT), the impact of HPV in advanced or metastatic HNSCC remains to be defined and targeted therapeutics need to be tested for cancers resistant to RT/CT. To this end, we investigated the sensitivity of HPV-positive and -negative HNSCC cell lines to TRAIL (tumour necrosis factor-related apoptosis-inducing ligand), which induces tumour cell-specific apoptosis in various cancer types. A clear correlation was observed between HPV positivity and resistance to TRAIL compared with HPV-negative head and neck cancer cell lines. All TRAIL-resistant HPV-positive cell lines tested were sensitised to TRAIL-induced cell death by treatment with bortezomib, a clinically approved proteasome inhibitor. Bortezomib-mediated sensitisation to TRAIL was associated with enhanced activation of caspase-8, -9 and -3, elevated membrane expression levels of TRAIL-R2, cytochrome c release and G2/M arrest. Knockdown of caspase-8 significantly blocked cell death induced by the combination therapy, whereas the BH3-only protein Bid was not required for induction of apoptosis. XIAP depletion increased the sensitivity of both HPV-positive and -negative cells to TRAIL alone or in combination with bortezomib. In contrast, restoration of p53 following E6 knockdown in HPV-positive cells had no effect on their sensitivity to either single or combination therapy, suggesting a p53-independent pathway for the observed response. In summary, bortezomib-mediated proteasome inhibition sensitises previously resistant HPV-positive HNSCC cells to TRAIL-induced cell death through a mechanism involving both the extrinsic and intrinsic pathways of apoptosis. The cooperative effect of these two targeted anticancer agents therefore represents a promising treatment strategy for RT/CT-resistant HPV-associated head and neck cancers.