| Description | Human Par-4/PAWR is a about 40 kDa protein containing 340 amino acids. Rat Par-4 has 332 amino acids whereas mouse Par-4 has 333 amino acids. Par-4/PAWR has two putative nuclear localization sequences in the N-terminal region and a leucine zipper domain and a nuclear export sequence in the C-terminal portion. There is a SAC domain (147-206 amino acids), selective for apoptosis induction in cancer cells. SAC domain is the effecter domain of Par-4/PAWR. These domains are 100% conserved in human, rat and mouse homologs. |
| Expression | Par-4/PAWR is ubiquitously expressed in normal mammalian tissues. However, Par-4/PAWR is diminished in a majority (>75% specimens) of renal cell carcinoma specimens. Par-4/PAWR expression is also decreased in endometrial tumors, lung cancer, neuroblastoma and in cells of patients with acute lymphatic leukemia and chronic lymphocytic leukemia. Par-4 is down-regulated in breast cancer, nasopharyngeal tumors and glioblastoma, and this is associated with therapy resistance and reduced patient survival. |
| Localisation | Immonocytochemical analysis indicates that Par-4/PAWR is predominantly localized in cytoplasm in normal cells and is strongly localized in cytoplasm and nucleus in most cancer cell lines. However, Western blot analysis indicates that Par-4/PAWR is also in the nuclear fraction of normal cells implying it is masked in the nucleus. Par-4 is spontaneously secreted by normal and cancer cells, and secreted Par-4 induces cancer-cell specific apoptosis through cell surface receptor GRP78, expressed at the surface of only cancer cells, not normal cells. |
| Function | Par-4/PAWR, a pro-apoptotic protein, was first identified in prostate cancer cells that were induced to undergo apoptosis. Par-4 knockout mice spontaneously develop tumors of the liver, lung, and endometrium; prostatic intraepithelial neoplasia, and an increased frequency of estrogen-inducible tumors in the endometrium and BBN-inducible tumors in the bladder. Endogenous Par-4/PAWR expressed in normal and cancer cells does not, by itself, causes apoptosis, yet is essential for apoptosis via diverse cell death pathways. Par-4/PAWR sensitizes cells to apoptosis by wide variety of pro-apoptotic stimuli, such as growth factor withdrawal, agents that elevate intracellular Ca2+, TNF, TRAIL, UV, X-ray and gamma irradiation, or IFN-gamma. Ectopic Par-4/PAWR over-expression is by itself sufficient to induce apoptosis in most cancer cells, but not in normal or immortalized cells. The cancer selective pro-apoptotic function of Par-4/PAWR is localized in its central core SAC (Selective for Apoptosis-induction in Cancer cells) domain (amino acids 147-206 in human Par-4/PAWR; or 137-195 in rat Par-4) which is 100% conserved in human, mouse and rat. Apoptosis by ectopic Par-4/PAWR requires Par-4/PAWR nuclear translocation and involves both activation of the Fas death receptor signaling pathway and NF-kappaB inhibition. Par-4/PAWR also inhibits the prosurvival protein Bcl-2 and down regulates ERK-2 expression. Neither p53 nor PTEN are directly required for apoptosis by Par-4/PAWR or the SAC domain. Par-4/PAWR has been shown to be i nvolved in suppression of transformation by down-regulation of Ras. Overexpression of Par-4/PAWR results in apoptosis of cells expressing oncogenic Ras.
Several partner proteins of Par-4/PAWR have been identified and partner interaction requires an intact Par-4/PAWR leucine zipper domain. Par-4/PAWR associates with aPKC resulting in inhibition of NF-kappaB activity, interaction with WT1 results in transcriptional repression of Bcl-2, whereas binding to and phosphorylation by Akt1 results in Par-4/PAWR cytoplasm retention by 14-3-3, thus isolating Par-4/PAWR from its nuclear targets. Par-4/PAWR also binds to DLK/ZIP kinase (ZIPK) and induces DAAX/ZIPK-mediated apoptosis. In addition, THAP1 (a novel nuclear pro-apoptotic factor) interacts with Par-4/PAWR and potentiates both serum withdrawal and TNF-induced apoptosis in endothelial cells.
Par-4/PAWR is also involved in sensitization of neurons to apoptosis. Endogenous Par-4/PAWR is reported to be up-regulated in different neurodegenerative diseases including Alzheimer's, Huntington's and Parkinson's diseases and amyotrophic lateral sclerosis.
Post-translational modifications: The apoptosis of Par-4/PAWR requires phosphorylation of the threonine residue (T155 in rat Par-4/PAWR) in the SAC domain by PKA, which is elevated in cancer cells. Amino acid S249 in rat Par-4/PAWR is phosphorylated by AKT for Par-4/PAWR cytoplasm retention and inactivation.
Par-4 is secreted via classical pathway and is associated with ER-stress
Most of the studies until now have been focused on the intracellular role of Par-4 but Burikhanov et al. (2009) provided a new direction to the field with their discovery that Par-4 is secreted by normal or immortalized cells, as well as cancer cells and that secreted Par-4 shows cancer cell-specific apoptosis through cell surface HSPA5 (GRP78), which is an endoplasmic reticulum (ER)-resident protein expressed at the cell surface selectively in cancer cells. Par-4 secretion is observed in vivo and plasma or serum samples obtained from Par-4 transgenic mice contain detectable amounts of Par-4 that are adequate to induce apoptosis ex vivo specifically in cancer cells. Cells treated with Brefeldin A (BFA, an antiviral antibiotic, which blocks protein trafficking from the ER to cis-Golgi cisternae), decreased secretion of Par-4, suggesting that the secretion of Par-4 occurs through a BFA-sensitive classical pathway involving the ER-Golgi network. Also, Par- 4 secretion is increased upon treatment of cells with ER stress inducers tunicamycin (an inhibitor of N-linked glycosylation) and thapsigargin (an inhibitor of the sarcoplasmic/Endoplasmic Reticulum Ca+2 ATPase), which also caused upregulation of GRP78.
Par-4 is involved in the translocation of GRP78 from ER to the cell surface
The correlation between intracellular Par-4, GRP78 and extracellular Par-4 in activating apoptosis has been further confirmed through RNAi studies. Knocking down Par-4 resulted in decreased translocation of GRP78 to the cell surface, indicating that endogenous Par-4 is required for cell surface expression of GRP78. This finding was further supported by Cohen et al. who confirmed the role of endogenous Par-4 in binding and translocation of GRP78 in trophoblastic cells. For the first time, this study showed the presence of Par-4 in both villous and extra villous cytotrophoblastic cells (CTB) and expression of Par-4 correlated with expression of membrane GRP78. Also, Par-4 overexpression led to an increase in cell surface expression of GRP78, whereas reduced Par-4 expression resulted in reduced cell surface localization of GRP78. The observations corroborate the role of Par-4 in translocation of GRP78 from ER to the cell surface. Decreased membrane GRP78 expression has been observed in preeclamptic (PE) cytotrophoblastic cells (CTB). This could be due to an altered expression of GRP78 transporting proteins. Interestingly, Par-4 protein expression was found to be reduced in PE CTB. This finding will help in better understanding of Par-4 expression, localization and posttranslational modifications in CTB to prevent PE.
Par-4/SAC-activity is transferable by bone marrow transplantation
To analyze the functional efficacy of SAC domain, transgenic mice that ubiquitously express the SAC domain were generated. To confirm the functional activity of this secreted Par-4/SAC in transgenic mice, bone marrow of SAC-GFP-transgenic mice, littermate control mice or GFP-transgenic control mice was transferred to irradiated littermate control mice. Serum samples obtained from mice that received bone marrow cells from SAC-GFP-transgenic mice, but not from littermate control or GFP-transgenic mice, induced apoptosis of aggressive Lewis Lung Carcinoma (LLC1) cells in culture. This finding implies that Par-4/SAC is secreted and that bone marrow transplantation is effective in transferring cells capable of secreting transgenic SAC-GFP activity into recipient mice.
Inhibition of lung metastasis in mice by recombinant Par-4/SAC
To further confirm that Par-4/SAC activity in serum of transgenic mice had the potential to inhibit metastasis, recombinant TRX-Par-4 or TRX-SAC protein, which induced apoptosis of cancer cells but not non-transformed cells in culture, was used. TRX-Par-4 or TRX-SAC but not TRX control protein, induced apoptosis in LLC1 and PC-3 cell cultures but not in non-transformed BPH1 cell culture. Also, recombinant Par-4 or SAC protein when introduced intravenously in immunocompetent C57BL/6 mice significantly inhibited lung metastasis of LLC1 cells. These findings confirmed that Par-4/SAC is secreted by normal cells and is systemically active for an extended period of time in suppressing tumor growth and metastasis in mice. A majority of the tumor suppressor proteins have exclusive intracellular mode of action, this induction of both extracellular and intracellular apoptotic pathways by Par-4/SAC is unique and can be harnessed for cancer-selective therapeutics.
Empowering Normal Cells for Paracrine Inhibition of Cancer
I- P53 induces Par-4 secretion by downregulating UACA
TP53 also known as the "guardian of the genome" acts as a tumor suppressor by intracellular activation of growth arrest and apoptotic cell death pathways. Owing to its prominent role as a tumor suppressor and involvement in apoptotic pathways, p53 is mutated in over 50% of cancers. Mutant forms of p53 may render cancer cells resistant to both chemotherapy and radiation therapy. Burikhanov et al. (2014) have shown that activation of p53 function in normal cells causes paracrine growth inhibitory effects in cancer cells by systemically inducing secretion of Par-4. Interestingly, secreted Par-4 mediates a paracrine growth-inhibitory effect by inducing apoptosis of p53-deficient cancer cells. In a search for the direct target of p53 for inducing Par-4 secretion, UACA, which is a binding partner of Par-4, was identified. Previous studies have shown that UACA binds to Par-4 and prevents its secretion. UACA was lower in wild type p53 cells and mouse tissues relative to p53 null cells and mouse tissues, and UACA levels correlated inversely with the levels of Par-4 secreted in the CM. However, knocking down UACA in p53 null MEFs did not increase the secretion of Par-4 indicating that intracellular p53 function is necessary to regulate the secretion of Par-4.
II- Activating p53 induces systemic expression of Par-4 in mice
To investigate the role of p53 in Par-4 secretion in vivo, p53 wild-type mice, p53 null mice, or Par-4 null mice were treated with Nutlin-3a, an activator of p53, and with PS-1145, an inhibitor of NF-kB. Combined treatment of Nutlin-3a and PS-1145 caused a 5-fold increase in serum levels of Par-4 protein in p53 wild-type mice compared to basal levels in serum, whereas it failed to elevate systemic levels of Par-4 in p53 null mice, suggesting that p53 function was essential for upregulation of Par-4 secretion. Interestingly, the serum from Nutlin-3a plus PS-1145-treated p53 wild-type mice, but not p53 null mice or Par-4 null mice, induced ex vivo apoptosis of cancer cell cultures but not normal cell cultures. These findings suggest that p53 activation in normal mice induces adequate levels of systemic Par-4 protein that is functionally effective in inducing apoptosis of cancer cells.
Par-4 secretagogues
As normal cells express wild type p53 function, recent studies have been directed toward utilizing the potential of normal immune cells or non-immune cells to control the growth of tumors. Studies by Burikhanov et al. (2014) suggest a novel cross-talk between p53 and Par-4 where p53 regulates the secretion of Par-4 via classical pathway from normal cells and secreted Par-4 mediates the paracrine apoptotic effects of p53 in a cancer-cell specific manner. Since the levels of Par-4 secreted by normal cells are not adequate to cause massive apoptosis in cancer cell cultures, new compounds and FDA-approved drugs are being actively discovered as Par-4 secretagogues. Burikhanov et al. (2014) have identified a novel secretagogue, Arylquin-1, a 3-arylquinoline, which shows a dose dependent secretion of Par-4 from both normal fibroblasts and epithelial cells. This study identified VIM (vimentin) as a novel binding partner of Par-4 and showed that Arylquin-1 binds to vimentin to release vimentin-bound Par-4 for secretion. Interestingly, Arylquin-1 itself at low nanomolar concentrations (100-500 nM) did not kill normal cells and most cancer cells but it caused robust secretion of Par-4 from normal cells to induce apoptosis of cancer cells only when they were co-cultured.
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| Homology | The Par-4/PAWR gene has been identified in various organisms, including mammals (Pan Troglodytes), rodents (mouse, rat), chicken (Gallus gallus), fish(Zebrafish and Pufferfish) and tadpole. The nuclear localization, leucine zipper, nuclear export and SAC domain sequences are highly conserved. |
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