Triptolide in Combination with the Gli1 Inhibitor GANT61 Eradicates Anaplastic Thyroid Cancer by Suppressing Cancer Stem Cell Self-Renewal and Enhancing Apoptosis and Pyroptosis.

Anaplastic thyroid cancer (ATC) is a lethal malignancy characterized by therapy resistance and rapid recurrence, driven in part by a population of thyroid cancer stem cells (CSCs). While the Gli1 inhibitor GANT61 targets CSC self-renewal, its efficacy is limited by compensatory activation of the pro-survival TAK1-AMPK pathway. Our present study aims to determine whether triptolide (TPL), a natural compound that inhibits TAK1 and induces reactive oxygen species (ROS), can overcome this resistance and achieve a synergistic anticancer effect when use with GANT61. Here, we report that TPL blocked GANT61-induced TAK1-AMPK activation. TPL alone effectively inhibited the proliferation of two ATC cell lines (8505C and SW1736) and achieved a synergistic anti-proliferative effect when used in combination with GANT61. Consistently, TPL alone effectively suppressed SW1736 xenograft tumor growth but, when used in combination with GANT61, prevented tumor recurrence. TPL in combination with GANT61 synergistically inhibited thyroid CSC self-renewal and induced apoptosis and pyroptosis. Mechanistically, TPL blocked GANT61-mediated activation of the TAK1-AMPK axis and synergistically induced ROS generation, which potentiated pyroptotic activity of Gasdermin D and E (GSDMD/E). This was confirmed by the evidence that TAK1 and AMPK inhibition by gene knockout or their specific inhibitors enhanced apoptotic and pyroptotic activity of GANT61, whereas ROS scavenge blocked apoptosis and pyroptosis induced by TPL and/or GANT61. Taken together, our study provides mechanistic insights into how TPL overcomes a key resistance pathway in CSC target therapy and suggests that the TPL-GANT61 combination could be a promising strategy to eradicate ATC.