Abstract 3308: Mitophagy induction and interference with cancer-specific apoptosis in SHetA2-treated cells
Authors: Chandra Vishal , Long Andrew , Masamha Chioniso Patience , Benbrook Doris Mangiaracina 
Published: July 2017

Background: SHetA2 is a small molecule flexible heteroarotinoid (Flex-Het) compound that exhibited in-vivo cancer therapeutic and chemoprevention activity, lack of mutagenicity or teratogenicity, and a pharmacologic profile suitable for an oral drug. First-in-human clinical trials of SHetA2 for ovarian cancer prevention and cervical cancer treatment are in development. Autophagy is induced in cancer cells by a wide variety of therapeutic drugs and commonly interferes with the drugs efficacies. Because SHetA2 binds to mortalin and induces mitochondrial damage and apoptosis in cancer, but not in healthy cells, we hypothesized that this drug induces mitochondria-selective autophagy (mitophagy) in cancer, but not healthy cells; and that the mitophagy interferes with SHetA2-induction of apoptosis.

Methods: Cultures of human ovarian and cervical cancer cell lines and human ovarian surface epithelium (HOSE) were treated with various concentrations and combinations of SHetA2, 3-methyladenine (3-MA), Beclin-1 siRNA, and caspase inhibitors. Autophagy was evaluated by electron microscopy (EM) and fluorescent imaging of LC3B processing in cells and by Western blot (WB) analysis of p62, Beclin-1 and LC3B processing. Mitophagy was measured by EM of cells and WB analysis of BNIP3 total levels and co-immunoprecipitation with LC3B in protein extracts. Apoptosis was measured by Annexin-V and propidium iodide staining of cells and by WB analysis of caspase 9 and 3 and PARP-1 cleavage.

Result: Induction of autophagy by SHetA2 treatment was demonstrated by the development of double-membraned autophagosomes and accumulation of the processed LC3B II in cancer, but not in HOSE cells. Mitophagy was demonstrated by the presence of mitochondria inside the autophagosomes and by reduction of total BNIP3 in association with increased BNIP3/LC3B complexes in SHetA2 treated cells. Autophagic flux was demonstrated by gradual reduction of p62. Conversion of autophagy to apoptosis was associated with reappearance of basal p62 levels and cleavage of Beclin-1, PARP-1, and caspases 9 and 3 about 24 hours after initiation of treatment. Interference of the autophagy with apoptosis was demonstrated by increased levels of apoptosis in cells co-treated with SHetA2 and autophagy inhibitors (3-MA or Beclin-1 siRNA) along with reduced apoptosis in cells co-treated with SHetA2 and an autophagy inducer (rapamycin). This was observed in cell lines harboring mutant or no p53, but not in an ovarian cancer cell line with wild type p53.

Conclusion: SHetA2-induction of autophagy may be initiated by mitochondrial damage and reduces, but does prevent, SHetA2-mediated apoptosis in ovarian and cervical cancer cell lines, but not in a cell line with wild type p53 or in HOSE cells. Combination of SHetA2 with an autophagy inhibitor may improve the therapeutic efficacy in clinical trials of patients harboring mutant or loss of p53 protein.

Funded by: RO1CA196200 and RO1CA200126

Citation Format: Vishal Chandra, Andrew Long, Chioniso Patience Masamha, Doris Mangiaracina Benbrook. Mitophagy induction and interference with cancer-specific apoptosis in SHetA2-treated cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3308. doi:10.1158/1538-7445.AM2017-3308