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ABSTRACT
The irreversible ERBB1/2/4 inhibitor neratinib has been shown to rapidly down-regulate the expression of ERBB1/2/4 as well as the levels of c-MET, PDGFRα and mutant RAS proteins via autophagic degradation. Neratinib interacted in an additive to synergistic fashion with the approved PARP1 inhibitor niraparib to kill ovarian cancer cells. Neratinib and niraparib caused the ATM-dependent activation of AMPK which in turn was required to cause mTOR inactivation, ULK-1 activation and ATG13 phosphorylation. The drug combination initially increased autophagosome levels followed later by autolysosome levels. Preventing autophagosome formation by expressing activated mTOR or knocking down of Beclin1, or knock down of the autolysosome protein cathepsin B, reduced drug combination lethality. The drug combination caused an endoplasmic reticulum stress response as judged by enhanced eIF2α phosphorylation that was responsible for reducing MCL-1 and BCL-XL levels and increasing ATG5 and Beclin1 expression. Knock down of BIM, but not of BAX or BAK, reduced cell killing. Expression of activated MEK1 prevented the drug combination increasing BIM expression and reduced cell killing. Downstream of the mitochondrion, drug lethality was partially reduced by knock down of AIF, but expression of dominant negative caspase 9 was not protective. Our data demonstrate that neratinib and niraparib interact to kill ovarian cancer cells through convergent DNA damage and endoplasmic reticulum stress signaling. Cell killing required the induction of autophagy and was cathepsin B and AIF -dependent, and effector caspase independent.
Abbreviations
| ca | = | constitutively active |
| CMV | = | empty vector plasmid or virus |
| dn | = | dominant negative |
| ER | = | endoplasmic reticulum |
| ERK | = | extracellular regulated kinase |
| HDAC | = | histone deacetylase. |
| mTOR | = | mammalian target of rapamycin |
| MAPK | = | mitogen activated protein kinase |
| PI3K | = | phosphatidyl inositol 3 kinase |
| PTEN | = | phosphatase and tensin homologue on chromosome ten |
| ROS | = | reactive oxygen species |
| si | = | small interfering |
| SCR | = | scrambled |
| VEH | = | vehicle |
Disclosure of potential conflicts of interest
No potential conflicts of interest were disclosed.
Acknowledgments
Support for the present study was funded from philanthropic funding from Massey Cancer Center, the Universal Inc. Chair in Signal Transduction Research and PHS R01-CA192613. Thanks to Dr. H.F. Young and the Betts family fund for support in the purchase of the Hermes Wiscan instrument. The authors have no conflicts of interest to report.