Thyroid cancer (TC) is the most common endocrine malignancy, generally successfully treated with standard therapies. However, aggressive forms remain resistant to current treatments. The concept of non-oncogene addiction (NOA) implies that cancer cells depend on non-mutated genes that are essential for their uncontrolled growth; thus NOA genes may represent potential targets for anti-cancer treatment. We have previously identified COPZ1, the ζ1 component of the coatomer complex, as a NOA vulnerability in TC. COPZ1 is involved in intracellular trafficking, endosome maturation, lipid homeostasis and autophagy. Inhibition of COPZ1 induced endoplasmic reticulum (ER) stress, inflammatory responses and immunogenic cell death in TC cells, without affecting normal cells. The translational value of our results requires in vivo experiments in immunocompetent murine models. Preliminary to the generation of in vivo preclinical models of COPZ1 silencing, we have characterized the susceptibility to COPZ1 inhibition of four murine cell lines.

COPZ1 was silenced through a siRNA transfection in four murine TC cell lines: T3531L, 3610R, T4888M and 3868. Cell proliferation was evaluated by crystal violet assay. Protein expression was analyzed by Western Blot. Cytokines secretion was quantified in conditioned media by ELISA Array. Gene expression was analyzed by Real Time PCR (RT-PCR), using TaqMan probes. The levels of intracellular oxidative stress were measured using commercial kits (ROS-Glo™ H2O2 Assay and GSH/GSSG-Glo™ Assay).

After COPZ1 depletion, all four murine TC cell lines underwent a reduction in relative proliferation with a different extent for each one. T3531L and 3610R activated a transcriptional program associated with viral mimicry response and IFN pathway. Moreover, both cell lines released several cytokines associated with immune components recruitment. On the contrary, COPZ1 silencing didn’t cause a modulation of inflammatory-related genes expression in T4888M and 3868 cell lines, but ER stress was induced as documented by the up-regulation of Chop and BiP. Emerging evidence suggests that ER stress can be involved in other forms of cell death, such as ferroptosis, a type of regulated cell death driven by oxidative stress and the iron-dependent accumulation of lipid peroxides. Oxidative stress increased in T4888M and 3868 cell lines after COPZ1 silencing, as demonstrated by higher levels of ROS and by lower GSH/GSSG ratio. Additionally, ferroptosis markers such as HO-1 and TFRC were overexpressed in COPZ1-silenced cells. These results suggest that ferroptosis could represent a cell death mechanism activated by COPZ1 depletion.

The described results demonstrate that COPZ1 silencing reduces cell proliferation in murine TC cells and that different cell death mechanisms are activated. Further analysis is still needed to confirm the activation of ferroptosis.