Combinations of Tyrosine Kinase Inhibitor and ERAD Inhibitor Promote Oxidative Stress–Induced Apoptosis through ATF4 and KLF9 in Medullary Thyroid Cancer

Abstract

Medullary thyroid carcinoma (MTC) originates from the C cells of the thyroid gland, which secrete calcitonin. Lymph node and distant metastases are frequently present at diagnosis. Activating mutations of RET, a driver oncogene in MTC that encodes a tyrosine kinase receptor, prevents apoptosis through inhibition of ATF4, a key transcriptional regulator of endoplasmic reticulum (ER) stress. We hypothesized that the combination of a tyrosine kinase inhibitor (TKI) and an ATF4 inducer promotes cell death by triggering catastrophic oxidative stress and apoptotic cell death. Here, we report that the ERassociated protein degradation (ERAD) inhibitor eeyarestatin sensitized MTC cells to the TKIs, sunitinib and vandetanib, thereby leading to synergistic upregulation of ATF4 expression, accumulation of reactive oxygen species, and subsequent cell death. Genome-wide analysis of ATF4 interaction sites by chromatin immunoprecipitation (ChIP) sequencing revealed that among ATF4 target genes was KLF9 (Kruppel-like factor 9), which induces MTC apoptosis. ChIP assays revealed that ATF4 occupancy at the KLF9 promoter was increased in MTC cells treated with eeyarestatin or vandetanib alone and was further enhanced in cells treated with both drugs, leading to increased KLF9 transcription. Depletion of ATF4 by shRNA led to downregulation of KLF9 expression and prevented oxidative stress-induced cell death. Furthermore, we identified ATF4 target genes (LZTFL1, MKNK2, and SIAH1 with known tumor suppressor function) that were synergistically upregulated with the combination of TKI and ERAD inhibitor. Implications: These findings reveal a combination therapy that induces reactive oxygen species-dependent catastrophic cell death through induction of ATF4 and KLF9 transcriptional activity.

Modulation of ERQC and ERAD: A Broad-Spectrum Spanner in the Works of Cancer Cells?

“…For these reasons, terminal ERAD components inhibitors have been proposed as targets to specifically impair the survival of cancer cells [22, 91]. Blocking ERAD can also trigger cellular apoptosis [92].…”

Dexamethasone induces aberrant macrophage immune function and apoptosis

“…Although various modes of action are still debated, it is broadly accepted that GCs mediate their anti-inflammatory and immunosuppressive effects by affecting macrophage viability and antitumor functionality (4). Several studies have demonstrated that tumor-derived KLF9 may act as a tumor suppressor by promoting apoptosis (8,19,20). Decreased KLF9 suppresses oxidative stress and apoptosis, subsequently promoting cancer progression (7).…”

Involvement of Metabolic Lipid Mediators in the Regulation of Apoptosis

“…Additionally, ATF4 increases the production of ROS, which can also be proapoptotic. The importance of ATF4 was demonstrated by blocking its transcription, which promoted apoptosis [43,44]. In contrast, translation of CHOP stimulates the transcription of protein phosphatase 1 regulatory subunit 15 (PPP1R15A/GADD34), TNF-related apoptosis-inducing ligand (TRAIL2), tribbles homolog 3 (TRB3), and, as a consequence, endoplasmic reticulum disulphide oxidase 1α (Ero1α).…”