Dual role of ER stress in response to metabolic co-targeting and radiosensitivity in head and neck cancer cells

Abstract

Arginine deprivation therapy (ADT) is a new metabolic targeting approach with high therapeutic potential for various solid cancers. Combination of ADT with low doses of the natural arginine analog canavanine effectively sensitizes malignant cells to irradiation. However, the molecular mechanisms determining the sensitivity of intrinsically non-auxotrophic cancers to arginine deficiency are still poorly understood. We here show for the first time that arginine deficiency is accompanied by global metabolic changes and protein/membrane breakdown, and results in the induction of specific, more or less pronounced (severe vs. mild) ER stress responses in head and neck squamous cell carcinoma (HNSCC) cells that differ in their intrinsic ADT sensitivity. Combination of ADT with canavanine triggered catastrophic ER stress via the eIF2α-ATF4(GADD34)-CHOP pathway, thereby inducing apoptosis; the same signaling arm was irrelevant in ADT-related radiosensitization. The particular strong supra-additive effect of ADT, canavanine and irradiation in both intrinsically more and less sensitive cancer cells supports the rational of ER stress pathways as novel target for improving multi-modal metabolic anti-cancer therapy.

Efficient Heat Shock Response Affects Hyperthermia-Induced Radiosensitization in a Tumor Spheroid Control Probability Assay

“…Activation of the IRE1α-XBP1 pathway usually constitutes a pro-survival response during UPR [52]. Recently published data from our group demonstrated that SAS cells are in general more sensitive to the XBP1 splicing reaction than FaDu cells, implying that SAS cells require a lower stress threshold [38]. This also holds true for the reaction to HT, i.e., XBP1-splicing was more pronounced at lower thermal doses in the SAS than the FaDu spheroid model.…”

“…This also holds true for the reaction to HT, i.e., XBP1-splicing was more pronounced at lower thermal doses in the SAS than the FaDu spheroid model. In the previous study, we found that via knockdown of the ER stress sensor IRE1, the IRE1α-XBP1 pathway does not play a role in metabolic stress-induced radiosensitization in SAS cells [38]. We, therefore, hypothesize that the upregulation of XPB1 splicing upon HT/HT + RT, further enhanced by 4-PBA, also just reflects the cells' attempt for survival but supposedly fails.…”

“…Two human HNSCC cell lines were used in this study: SAS cells obtained from the HSRRB/JCRB (Osaka, Japan) and FaDu cells, a subline of the FaDu-ATCC HTB-43 [37]. Both cell lines were routinely tested free of mycoplasms using a PCR Mycoplasma Kit (AppliChem, Darmstadt, Germany), and the genetic profile was verified before use via microsatellite analyses using multiplex PCR kits as detailed previously [38]. Cultures were routinely grown from validated frozen stocks for ≥2 to ≤20 passages (<120 cumulative population doublings).…”

“…All spheroids were imaged individually directly before treatment and every 48-72 h thereafter until they reached~900 µm in diameter. Spheroid sizes and subsequent volumes were determined from the images as described [29,38]. Relative growth delay was calculated as a time interval of each spheroid in a treatment arm to reach 5-times its initial volume before the onset of treatment (5 × V 0 ) divided by the average time required by an untreated spheroid control population.…”

“…xenograft experiments [11,12,30,38]. Figure 2a illustrates the increasing loss in growth recovery in an SAS spheroid population when RT is applied after 30 or 60 min of HT at 42.5 • C. Figure 2b depicts the HT-dependent re-growth capacity of FaDu and SAS spheroids as a function of time post-treatment for one irradiation dose of interest.…”