Abstract 1252: The mechanism of the drug, SHetA2, in cervical cancer cells involves growth inhibition, mitochondria damage and release of AIF to cause caspase-independent cell death
Authors: Rai Rajani , Chandra Vishal , Benbrook Doris M. 
Published: July 2021

Introduction SHetA2 is an orally-bioavailable, novel small-molecule drug that is entering first in human trials for gynecologic cancers. Upon binding to three related heat shock protein A chaperones (HSPA 5, 8 and 9 genes encoding BiP, hsc70 and mortalin proteins, respectively), SHetA2 causes release of their client proteins and inhibition of mortalin import into mitochondria. The purpose of this study was to evaluate the potential of SHetA2 for cervical cancer therapy and to explore its mechanism of action.

Methods: The cytotoxic effect of SHetA2 on cervical cancer cell lines, C33a, C-4 II, CaSki, ME-180 and SiHa was assessed using a MTT metabolic viability assay. The effects of SHetA2 on cell cycle arrest were evaluated by flow cytometric analysis of PI staining and western blots. SHetA2 effects on anchorage-independent growth and cell migration were analyzed using soft-agar colony formation and wound healing scratch assays, respectively. The effect of SHetA2 on mitochondria was assessed using the JC-1 dye, measurement of ATP, electron microscopy, confocal microscopy and Seahorse XF Cell Mito Stress.

Result: SHetA2 significantly reduced proliferation of all cervical cell lines in time- and dose- dependent manners. This drug also inhibited anchorage-independent growth, cell migration and cell cycle progression through G1-phase cell-cycle arrest in C-33 A and CaSki, and G2- arrest in SiHa, in association with relevant alterations of cell-cycle regulatory proteins. SHetA2 significantly decreased mitochondrial membrane potential and ATP production indicating mitochondrial toxicity. Electron microscopy demonstrated mitochondrial swelling and increased autophagic vesicles in SHetA2-treated SiHa cells. Confocal microscopy using mitochondria-specific dye demonstrated loss of mitochondrial networking and structural damage. Consistent with this, our western blot results demonstrated significant downregulation in the expression of proteins involved in mitochondrial fusion indicating that SHetA2 causes inhibition of mitochondrial fusion. Metabolic studies demonstrated that SHetA2 caused attenuation of efficient oxidative phosphorylation, ATP production, and elevation of basal glycolysis. Eventual SHetA2-induced cell death was not affected by a caspase inhibitor. Further mechanistic study demonstrated translocation of apoptosis inducing factor (AIF) from the mitochondria to the nucleus, along with nuclear DNA damage.

Conclusions: SHetA2 inhibits growth, migration and invasion of cervical cancer cell lines in association with cell cycle arrest and mitochondrial toxicity. SHetA2-induced mitochondrial damage in cervical cancer cell lines likely mediates release of AIF to cause cell death in a caspase-independent manner.

Citation Format: Rajani Rai, Vishal Chandra, Doris M. Benbrook. The mechanism of the drug, SHetA2, in cervical cancer cells involves growth inhibition, mitochondria damage and release of AIF to cause caspase-independent cell death [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1252.