Aggressive cancer cells often upregulate GRP78, a major endoplasmic reticulum (ER) chaperone and key
regulator of the unfolded protein response (UPR) to augment protein folding capacity and maintain ER
homeostasis, thereby promoting survival and acquiring therapeutic resistance. A high-throughput drug screen
revealed that a class of compounds referred to as cardiac glycosides (CGs) such as digoxin (DIG), in current
use to treat heart failure, as novel inhibitors of GRP78 stress induction in a wide range of human cancer. The
discovery that CGs can suppress stress induction of GRP78, which is pivotal for cancer survival, invasion and
oncogenic signaling, opens up a new mechanism for the antineoplastic action of the CGs. Among the CGs,
oleandrin (OLN) with 100-fold higher affinity for the a3 versus the a1 subunit of Na-K ATPase and high tolerability,
is most potent compared to DIG and other CGs. Here we propose to test the efficacy of OLN to suppress GRP78
and cell viability, taking advantage of unique sets of preclinical patient-derived breast and colon cancer models
that closely recapitulate the in vivo milieu developed by our collaborative team. We hypothesize that OLN, acting
through the a3 subunit of the Na-K ATPase, acutely suppresses stress induction of both intracellular and the cell
surface form of GRP78 through blocking its acute translation following ER stress. OLN may also preferentially
suppress translation of other proteins critical for survival under stress. As a consequence, ER homeostasis is
perturbed and ER-stress mediated apoptosis is triggered. These contribute majorly to the antineoplastic
mechanism of OLN. In this proposal, we will: 1) establish the efficacy of OLN and for comparison, DIG, in
suppressing GRP78 expression, cell viability, stemness and invasion in patient-derived breast circulating tumor
cells both in vitro and in vivo; 2) establish the efficacy of OLN and DIG in lowering GRP78 expression and viability
of patient-derived colorectal cancer cells and enhancing standard therapy in organoids. Promising treatment
schedules will be validated in PDX models in vivo. The GRP78 expression levels, the status of the a3 subunit of
Na-K ATPase and the OLN/DIG concentration in the plasma and tumor in the model systems will be monitored;
and 3) examine the mechanisms whereby OLN and DIG suppress stress induction of GRP78 and its impact on
the UPR and apoptosis as well as the importance of GRP78 in the anti-cancer effect of OLN/CG. In summary,
our studies will provide critical preclinical evaluation of the efficacy of OLN/DIG in cancer leveraging novel
patient-derived breast and colon cancer model systems, and a high content imaging platform that mimics the
hypoxic tumor microenvironment for monitoring the response. Our studies will also explore the utility of GRP78
and a3 subunit of Na-K ATPase as biomarkers for stratifying patients for OLN/DIG therapy and drug response,
and provide proof-of-concept for further development of CG agents in combination or targeted therapy settings
in suppressing GRP78, impacting the UPR and apoptosis. Our results will have far-reaching impact as they are
also applicable to other solid and blood cancers dependent on GRP78 for growth, invasion and resistance.