The Jackson Laboratory, Sacramento, CA
Appointment period: 1/06/2015 to 1/05/2017
Mechanisms of Hexamethylene Amiloride Induced Depletion of Breast Cancer Cells
Accumulating evidence suggests a subpopulation of stem-like, undifferentiated cancer cells potentiate tumor initiation, growth, therapeutic resistance and disease recurrence. As such, directed targeting of cancer stem cells provides a rational approach to overcoming tumor recurrence and therapeutic resistance to improve patient outcomes. Our preliminary data suggests derivatives of the potassium sparing diuretic, amiloride, as strong candidates for the depletion of therapy resistant breast cancer cells. In particular, amiloride analogs stimulated a nonapoptotic form of cell death resembling programmed necrosis (type III programmed cell death (PCD) with potent activity against docetaxel resistant breast cancer cells but little activity against non-transformed breast cells. Intriguingly, amiloride induced cytotoxicity appears to involve the nuclear translocation of apoptosis initiating factor (AIF) independent of the previously ascribed mediators of type III PCD. These data suggest that certain amiloride derivatives may induce a novel form of AIF dependent-type III PCD specifically in cancer cells.
We propose a comprehensive set of experiments to elucidate the mechanism by which amiloride induces type III PCD in breast cancer cells. Our studies suggest that a key amiloride derivative induced cytotoxicity dependent on elevated calcium (Ca2+) and reactive oxygen species (ROS). While the mechanisms driving elevated Ca2+ and ROS are currently unknown, our studies and the work of others suggests that amiloride facilitates a Ca2+ and ROS type III PCD dependent upon direct interactions with ER- and mitochondrial associated proteins. We propose to (a) delineate the ER- and mitochondria associated affects of the amiloride analog, (b) evaluate potential interactions of the amiloride derivative with ER and/or mitochondrial associated proteins and (c) evaluate which of these ER and mitochondrial dependent effects contribute to cell death. These data will be used to compliment an ongoing study on the affect of amiloride derivatives on cancer stem cells in vitro and in vivo and inform our overarching goal of validating type III PCD as a therapeutic strategy for chemotherapy resistant cancer to improve patient outcomes.