Endoplasmic reticulum stress and unfolded protein response in oral squamous cell carcinoma

Abstract

Oral squamous cell carcinoma (OSCC) is the most frequent form of cancer of the head and neck region, with high rates of morbidity and mortality. It significantly impacts the individual patients affected, as well as society at large, at multiple levels.

In squamous epithelial malignancies, the malignant keratinocytes and surrounding stromal cells form a tumour microenvironment (TME), which is constantly subjected to the effects of intrinsic and extrinsic stressful stimuli, including endoplasmic reticulum (ER) stress. ER stress leads to the activation of the unfolded protein response (UPR). This is an evolutionarily conserved set of mechanisms designed to ameliorate the ER homeostatic imbalance or to induce cellular termination through apoptosis when ER stress cannot be mitigated. The UPR is extensively intertwined with other vital processes, including those that influence development and progression of cancer. However, at this stage, the understanding of the role of ER stress-induced UPR in the pathogenesis and progression of OSCC is still in its infancy, requiring further elucidation. In this series of investigations, it was hypothesised that: a) ER stress would differentially affect the regulation of fundamental cellular processes, i.e. the maintenance of OSCC cell viability and apoptosis and b) UPR genes and proteins would be differentially regulated, expressed and activated under the effect of ER stress in OSCC, compared to normal and dysplastic counterparts.

In order to test these hypotheses the effects of ER stress on OSCC in relation to: a) cell viability, b) direct and indirect measures of apoptosis, c) the differential regulation of 84 key UPR and ER stress-associated genes, d) the differential expression of UPR and ER stress-associated counterpart proteins and e) the resultant transcriptional activation were assessed in an established in vitro model using the potent inducer of ER stress, tunicamycin. Specifically seven extensively substantiated cell lines derived from normal, dysplastic and malignant oral keratinocytes were used. They were subjected to tunicamycin-induced ER stress of varying intensity and chronicity. The effects on the cell viability were determined by examining the metabolic activity of cells using a resazurin/resorufin-based cell viability assay and apoptotic responses were directly (TUNEL assay for the quantification of DNA fragmentation) and indirectly (Caspase-3/7 activity) assessed. A comprehensive set of 84 UPR-related genes were studied using a highly sensitive quantitative real-time reverse transcriptase polymerase chain reaction (qRT2-PCR) and the expression of significantly regulated protein counterparts were profiled by means of enzyme-linked immunosorbent assays (ELISA) and also by examining the DNA binding activity using a transcription factor assay (TFA).

In the cell viability experiments it was demonstrated that OSCC cells maintained cell viability in the presence of ER stress at a significantly greater level, compared to normal oral keratinocytes. Furthermore, caspase-3/7 activity and DNA fragmentation, hallmarks of cell death, were suppressed in OSCC. It was also discovered, for the first time, that UPR-induced apoptosis-related factors, most notably DDIT3, were significantly up-regulated in OSCC. Also, the master regulator of lipid metabolism, SREBP1, and CREB3L3, an ER-resident transcription factor closely related to ATF6, which plays an important role in linking ER stress with immune-inflammatory responses, were significantly up-regulated in OSCC. Both SREBP1 and CREB3L3 influence metabolic reprogramming processes, one of key hallmarks of cancer. Subsequent protein studies further substantiated the involvement of SREBP1 through the demonstration of a significantly higher level of SREBP1 activation in OSCC under ER stress. This is the first time that DDIT3, SREBP1 and CREB3L3 have been shown to be implicated in the pathogenesis of OSCC.

For the first time, this study has highlighted the importance of, and the influence of ER stress and UPR, on the pathogenesis and pathobiology of OSCC, especially centred around factors that influence apoptosis, TME and lipid metabolism. The identified factors should be further studied and validated ex vivo and, eventually, in vivo, in view of their potential diagnostic and prognostic role in improving the diagnosis, treatment and management of oral cancer.