Par-4 Induced Apoptosis Ashley Feng and Lilly Holman

Par-4 Overexpression of the prostate apoptosis response gene-4 (Par-4) causes apoptosis in hormone-independent cancer cells, but does not affect hormone-dependent cancer cells, or immortalized and primary normal cells (6). Dr. Vivek Rangnekar and other researchers at the University of Kentucky identified Par-4 in 1993 and have found that mice that overexpress the Par-4 gene are resistant to cancer (2). Par-4 could potentially be used to treat cancer, since it kills only cancer cells and has no known side effects (3).

Par-4 Injection Par-4 being injected into a fertilized mouse egg cell (courtesy Mike Green, Markey Cancer Center, University of Kentucky)

Par-4 Structure and Location The Par-4 gene maps to human chromosome 12q21 (5). This region is often destroyed in pancreatic and gastric cancer cells (5). Par-4 is induced by apoptic signals which are not necrotic, and do not affect growth (6). The protein expressed by this gene is proapoptic and has a leucine zipper domain at its carboxy terminus (5).

Mechanism The ability of Par-4 to cause apoptosis depends upon overexpression (6), phosphorylation of T155 on Par-4 by Protein Kinase A (PKA) (5), the leucine zipper domain on Par-4, the presence of thapsigargin (TG) (6,9), and nuclear localization signal domains (5,6). When any of these factors was mutated or otherwise nullified, Par-4 could not trigger apoptosis (5,6).

Overexpression Normal levels of Par-4 do not cause apoptosis; this is easily proven because basal levels of Par-4 in animals with prostate cancer do not cause apoptosis (6). Ectopic overexpression of Par-4 facilitates TG-inducible apoptosis in prostate cancer and melanoma cells (6).

TG-Induced Apoptosis Overexpression of Par-4 does not cause apoptosis on its own. Par-4 only sensitizes cancer cells to thapsigargin (6). Apoptosis can be induced in androgen-dependent cells by upregulation of intracellular calcium with calcium ionophores or with thapsigargin (TG), which activates the capacitative calcium channel (6,7). After a hormone-dependent cancer cell has been sensitized to TG by par-4, exposure to TG triggers apoptosis (6). Thapsigargin

TG-Induced Apoptosis (cont.) Apoptosis involves chromatin condensation, cell membrane blebbing, DNA fragmentation, and collapse of cellular structure (6). Apoptosis

Protein Kinase A (PKA) Protein Kinase A (PKA) phosphorylates and activates the selective for apoptosis induction in cancer cells (SAC) domain (5). The SAC domain is called that because it does not trigger apoptosis in normal or immortal cells (1). Par-4 does not affect normal cells because it must be phosphorylated by PKA in order to trigger apoptosis (5). PKA 1 is overexpressed by primary tumors and cancer cells (5).

Protein Kinase A (PKA) Production of PKA 1 is related to transformation by growth factors in cancer cells, and high levels of PKA activate Par-4, which leads to high levels of apoptosis only in cancer cells (5).

Leucine Zipper Domain The leucine zipper domain must be able to modulate the cellular functions of other proteins (6). The leucine zipper protein is the part of Par-4 that the protein uses to bond to the zinc finger domain of Wilms’ tumor protein WT1 (8), and it is essential for Par-4 to induce apoptosis (6). Leucine zipper domain

Nuclear Localization Nuclear localization signal (NLS) domains are also necessary for Par-4 to function (5). In experiments, Par-4 without a NSL domain could not induce apoptosis (5). NLS domains allow Par-4 to enter the nucleus of cancer cells, but not normal or immortalized cells (5). This is another reason why Par-4 does not affect normal cells. The SAC domain mutant of Par-4 can cause apoptosis in an even wider range of cancer cells, but still does not affect normal or immortalized cells (5).

Par-4 as Cancer Treatment Researchers believe Par-4 would be an ideal cancer treatment because it causes apoptosis in only cancer cells (3). Par-4 has no harmful effects on normal cells, and no known side effects, unlike current treatments such as chemotherapy (1). Mice injected with the Par-4 gene experienced significant shrinking of tumors and even had a longer lifespan than normal mice with prostate cancer (4). Par-4 treatment has not been tested on humans, but researchers believe Par-4 could be administered to human patients through bone marrow transplants (3).

Cost of Treatment The potential cost of this treatment is yet unknown, since there is a significant amount of research to be done until Par-4 treatment can become available to cancer patients.

Image References Apoptosis: Technische Universiteit Eindhoven http://www.bmt.tue.nl/research/unit%20descriptions/apoptosis.GIF Leucine Zipper Domain: Wikipedia http://upload.wikimedia.org/wikipedia/commons/thumb/e/e8/Leucine_zipper.png/180px-Leucine_zipper.png Par-4: ScienCentral News http://www.sciencentral.com/articles/view.php3?article_id=218393034 Thapsigargin: http://upload.wikimedia.org/wikipedia/en/thumb/9/94/Thapsigargin.png/200px-Thapsigargin.png

References 1. Cancer Resistance in Transgenic Mice Expressing the SAC Module of Par-4 http://cancerres.aacrjournals.org/cgi/content/abstract/67/19/9276 2. Dr. Rangnekar http://www.mc.uky.edu/microbiology/rangnekar-pubs.asp http://www.mc.uky.edu/microbiology/rangnekar.asp 3. Future Drugs- Expert Review of Anticancer Therapy http://www.future-drugs.com/doi/abs/10.1586/14737140.8.1.5 4. Anti-Cancer Mouse: Science Videos http://www.sciencentral.com/articles/view.php3?article_id=218393034 5. Phosphorylation of Par-4 by Protein Kinase A Is Critical for Apoptosis http://mcb.asm.org/cgi/content/full/25/3/1146?view=long&pmid=15657440

References (cont.) 6. Expression and function of the leucine zipper protein Par-4 in apoptosis http://mcb.asm.org/cgi/content/abstract/17/7/3823?maxtoshow=&HITS=10&hits=10&RESULTFORMAT=&searchid=1&FIRSTINDEX=0&minscore=4000&resourcetype=HWCIT 7. Thapsigargin, a Tumor Promoter, Discharges Intracellular Ca2+ Stores by Specific Inhibition of the Endoplasmic Reticulum Ca2+-ATPase http://www.pnas.org/cgi/content/abstract/87/7/2466 8. Par-4 transcriptionally regulates Bcl-2 through a WT1-binding site on the bcl-2 promoter http://www.ncbi.nlm.nih.gov/pubmed/12644474 9. Role of EGR-1 in Thapsigargin-Inducible Apoptosis in the Melanoma Cell Line A375-C6 http://mcb.asm.org/cgi/content/abstract/15/11/6262