The goal of this project is to target a tumor suppressor (p53) to 2 different cellular compartments as new dual gene therapy approach for breast and other types of cancers. In normal cells, p53 protein is located mostly in the nucleus of the cell where it can act as a tumor suppressor and cause apoptosis. Under certain conditions it is also apoptotically active when directed to the mitochondria. In many types of cancers, p53 is mislocalized to the cytoplasm or inactivated. Indeed, p53 has emerged as a master switch for cancer prevention and is actively pursued as the ultimate cancer therapeutic target. To target p53 to the nucleus, we will use our emerging protein switch technology to capture mislocalized p53 in the cell cytoplasm, which can be dragged to the nucleus with addition of an external drug. Once in the nucleus, p53 will cause death of the cancer cell. To target p53 to the mitochondria, an improved mitochondrially directed version of p53 will be created. This 2- gene therapy approach is likely to increase the potency of p53 cell-killing ability. The initial target to demonstrate this technology is breast cancer, with future use in inflammatory breast carcinoma (IBC), a very aggressive and deadly form of breast cancer. IBC has mislocalized or mutated p53 and therefore should readily respond to this type of therapy. This approach is applicable to all types of cancers involving p53 mislocalization, nuclear exclusion, mutation, or inactivation.
The aims of this project are as follows: 1) Demonstrate that protein switch versions of p53 with nuclear localization signal (NLS) will translocate from the cytoplasm to the nucleus upon ligand addition and intrinsically cause apoptosis, or initiate enhanced apoptosis when binding endogenous mislocalized p53;2) Prove that a nuclear-localization deficient version of p53 engineered with an improved mitochondrial targeting signal will trigger apoptosis via the intrinsic apoptotic pathway;3) Assess the ability of the nuclear targeted protein switch-p53 from Aim 1, and the mitochondrially optimized p53 from Aim 2 in combination therapies to induce apoptosis in breast cancer cells, resulting in increased potency compared to the action of either construct alone. A cancer-specific promoter and delivery in cells using an adenoviral vector will also be tested;4) Validate that the combination therapy from Aim 3 delivered via adenovirus vector will eradicate or reduce breast cancer in a human xenograft solid tumor murine model in vivo. Finally, the approach described here is more advantageous that current strategies (including administering wt p53 or small molecule inhibitors that can restore tumor suppressor function in some cases) because targeting of p53 directly to active compartments of the cell will allow triggering of both intrinsic and extrinsic pathways of apoptosis in a specific and simultaneous or synergistic manner. This dual gene therapy is also expected to be beneficial for other types of aggressive cancers that currently have no effective therapies.

Public Health Relevance

The tumor suppressor p53 is inactive or malfunctioning in most types of cancer, and making restoration of p53 a prime candidate for cancer therapy. Our goal is to add p53 back into cancer cells, and simultaneously target p53 to its most active cellular compartments, the nucleus and mitochondria. Our long term goal is to use targeted delivery of p53 as a potent cancer therapeutic.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA151847-03
Application #
8274895
Study Section
Gene and Drug Delivery Systems Study Section (GDD)
Program Officer
Fu, Yali
Project Start
2010-07-01
Project End
2014-05-31
Budget Start
2012-06-01
Budget End
2013-05-31
Support Year
3
Fiscal Year
2012
Total Cost
$300,906
Indirect Cost
$99,631
Name
University of Utah
Department
Pharmacology
Type
Schools of Pharmacy
DUNS #
009095365
City
Salt Lake City
State
UT
Country
United States
Zip Code
84112
Okal, A; Matissek, K J; Matissek, S J et al. (2014) Re-engineered p53 activates apoptosis in vivo and causes primary tumor regression in a dominant negative breast cancer xenograft model. Gene Ther 21:903-12
Matissek, Karina J; Okal, Abood; Mossalam, Mohanad et al. (2014) Delivery of a monomeric p53 subdomain with mitochondrial targeting signals from pro-apoptotic Bak or Bax. Pharm Res 31:2503-15
Okal, Abood; Cornillie, Sean; Matissek, Stephan J et al. (2014) Re-engineered p53 chimera with enhanced homo-oligomerization that maintains tumor suppressor activity. Mol Pharm 11:2442-52
Mossalam, Mohanad; Soto, Jamie; Lim, Carol S et al. (2013) Solid phase synthesis of mitochondrial triphenylphosphonium-vitamin E metabolite using a lysine linker for reversal of oxidative stress. PLoS One 8:e53272
Davis, James R; Mossalam, Mohanad; Lim, Carol S (2013) Controlled access of p53 to the nucleus regulates its proteasomal degradation by MDM2. Mol Pharm 10:1340-9
Matissek, Karina J; Mossalam, Mohanad; Okal, Abood et al. (2013) The DNA binding domain of p53 is sufficient to trigger a potent apoptotic response at the mitochondria. Mol Pharm 10:3592-602
Reaz, Shams; Mossalam, Mohanad; Okal, Abood et al. (2013) A single mutant, A276S of p53, turns the switch to apoptosis. Mol Pharm 10:1350-9
Okal, Abood; Mossalam, Mohanad; Matissek, Karina J et al. (2013) A chimeric p53 evades mutant p53 transdominant inhibition in cancer cells. Mol Pharm 10:3922-33