In examining the potent novel high affinity PPAR3 agonist, RS5444, as a candidate cancer therapeutic, we discovered that RhoB mRNA and protein were rapidly upregulated by RS5444 in concert with observed antineoplastic effects. This prompted us to examine the novel hypothesis that downstream RhoB effects in part mediate the contributions of PPAR3 to cancer pathogenesis. RhoB is a small GTPase regulating actin organization and vesicle transport not mutated in cancer. RhoB, however, inhibits proliferation. We had previously showed RS5444 and paclitaxel can be combined synergistically in anaplastic thyroid cancer (ATC) models in vitro and in vivo and that the cyclin kinase inhibitor, p21WAF1/CIP1 (p21), is necessary for PPAR3- mediated growth inhibition and for the apoptotic synergy induced by the combination. We now report that RhoB is critical for RS5444/PPAR3-mediated p21 mRNA and protein induction. Silencing RhoB in our laboratory resulted in loss of growth inhibitory activity by RS5444, bolstering our hypothesis, and leading us to demonstrate for the first time that forced RhoB upregulation leads to cancer cell death in ATC. These data, coupled with preliminary data of RhoB down-regulation in our archival ATC tissues, led us to hypothesize that RhoB is important in the chemosensitivity of ATC and in ATC pathogenesis. Based upon these findings, a Phase 1/2 clinical trial combining RS5444 and paclitaxel is under way in ATC in efforts to improve the single agent effects of paclitaxel 3 in this nearly uniformly fatal cancer. Tumor biopsies obtained in conjunction with this trial will allow measurement of RhoB and p21 induction as early response correlates to patient outcome. RS5444 alone induces G0/G1 cell cycle arrest without apoptosis in ATC and forced expression of RhoB (Tet RhoB) proves sufficient for induction of p21, G2/M arrest and apoptosis. These observations coupled with another discovery that RhoB mislocalizes to the nucleus, colocalizing with p21 in a nuclear complex that includes protein kinase C-related kinase one (PRK1), a known RhoB interacting protein and serine/threonine kinase. This complex forms as a result of RS5444 as well as forced RhoB expression leading us to hypothesize that this novel nuclear complex mediates RhoB antitumor activity via direct interaction with cell cycle machinery complexes and that post-translational modifications of p21 direct its physical association with either G0/G1 or G2/M cell cycle machinery thereby specifying cell cycle arrest or apoptosis.
In Aim 1, we will elaborate the mechanism(s) by which RhoB mediates cell cycle arrest and apoptosis via this novel nuclear complex.
In Aim 2, the role of RhoB in apoptotic and antitumor synergy will be defined in animal models as well as using other newly discovered drugs upregulating RhoB that may be useful for patients with PPAR3- negative ATC.
In Aim 3, we will develop prognostic indicators for response to therapy. Our data indicate RhoB as a novel molecular switch dictating cell fate. Thusly, proposed studies have potential not only to improve understanding of RhoB signaling, but to ultimately lead to improved therapeutic approaches in ATC.

Public Health Relevance

RhoB, a small GTPase, has been discovered as a critical link in a novel antitumor signaling pathway. It is critical for combinatorial therapy antitumor synergy using a novel PPARgamma agonist combined with paclitaxel. We now propose to extend our studies to more fully elucidate the mechanisms underlying the effects of Rho B on cell proliferation and cell death, antitumor synergy in combination therapy, and examine molecular correlates for response to therapy in a Phase 1/2 ATC clinical trial.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA136665-03
Application #
8250472
Study Section
Basic Mechanisms of Cancer Therapeutics Study Section (BMCT)
Program Officer
Forry, Suzanne L
Project Start
2010-07-01
Project End
2015-04-30
Budget Start
2012-05-01
Budget End
2013-04-30
Support Year
3
Fiscal Year
2012
Total Cost
$312,406
Indirect Cost
$101,955
Name
Mayo Clinic Jacksonville
Department
Type
DUNS #
153223151
City
Jacksonville
State
FL
Country
United States
Zip Code
32224
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