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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Oncogene. Author manuscript; available in PMC 2011 January 1.
Published in final edited form as:
PMCID: PMC2900490
NIHMSID: NIHMS212522

Cancer-Selective Apoptotic Effects of Extracellular and Intracellular Par-4

Abstract

Selectivity toward cancer cells is the most desirable element in cancer therapeutics. Par-4 is a cancer cell-selective pro-apoptotic protein that functions intracellularly in the cytoplasmic and nuclear compartments, as a tumor suppressor. Moreover, recent findings indicate that the Par-4 protein is secreted by cells, and extracellular Par-4 induces cancer cell-specific apoptosis by interaction with the cell-surface receptor GRP78. This review describes the mechanisms underlying the apoptotic effects of both extracellular and intracellular Par-4 acting via its effector domain SAC.

Keywords: Par-4, cell surface GRP78, apoptosis

Introduction

Cancer ranks as the second leading cause of death in males and females of all age groups in the United States, and currently, 1 out of 4 deaths in the U.S. is due to cancer (Jemal et al., 2008). The severe adverse effects and morbidity due to many therapeutic strategies currently in use are associated with their non cancer cell-specific mode of action. The general effort at present, therefore, is directed toward identifying mechanism-based molecular targets in carcinogenic pathways for therapy. To this end, recent developments in the study of the cancer cell-selective pro-apoptotic protein Par-4 present an attractive therapeutic potential. The purpose of this review is to examine the apoptotic actions of intracellular Par-4 in the light of the new findings related to Par-4 secretion, and its implications and possibilities within cancer therapeutics.

Identification of Par-4

Prostate tumors consist of a heterogeneous mixture of both androgen-dependent and –independent cells (Isaacs and Coffey, 1981). Androgen-dependent prostate tumor cells are susceptible to androgen ablation therapy, whereupon these cells, in the absence of the hormone, undergo apoptosis in response to elevated levels of intracellular calcium ([Ca2+]i), thus resulting in involution of the tissue and regression of the tumor. This process is calcium-dependent and accordingly, the androgen-independent cells, which fail to exhibit raised [Ca2+]i in response to androgen withdrawal, are resistant to apoptosis despite possessing fully functional apoptotic machinery. The recalcitrant fraction of androgen-independent cells is often responsible for the relapse of a more aggressive tumor. However, apoptosis can be induced in these androgen-independent cells with forced elevation of [Ca2+]i (Martikainen et al., 1991).

The prostate apoptosis response-4 (par-4) gene was first identified by the differential hybridization technique as an immediate early apoptotic gene upregulated in response to elevated intracellular Ca2+ concentration ([Ca2+]i) in the androgen-independent rat prostate cancer cells AT-3 treated with ionomycin. Par-4 expression was also observed in apoptotic androgen-dependent rat ventral prostate following androgen ablation induced by castration. Moreover, pre-treatment with a calcium channel blocker such as nifedipine could effectively inhibit the upregulation of the par-4 gene, indicating the event is [Ca2+] dependent. Furthermore, par-4 was not upregulated in liver, kidneys or other androgen receptor containing organs that do not undergo apoptosis upon castration. These observations, combined with the fact that par-4 gene upregulation occurs only downstream to apoptotic signaling, and not in other processes such as growth stimulation, necrosis, oxidative stress or growth arrest recognized par-4 as an apoptosis-specific gene (Sells et al., 1994).

Human Par-4, which was found to share significant sequence similarity with its rat counterpart, was subsequently identified in yeast-two hybrid studies as a partner of the atypical Protein Kinase C (aPKC) (Diaz-Meco et al., 1996) and tumor suppressor Wilm’s tumor-1 (WT1) (Johnstone et al., 1996). Ubiquitous Par-4 mRNA and protein expression has also been observed in nearly all tissues of mice, horses, pigs, and cows. Par-4 is also known to be expressed in other vertebrates such as fish, birds, and Xenopus (El-Guendy and Rangnekar, 2003).

Structure

The human par-4 gene has been mapped to the minus strand of chromosome 12q21 (Johnstone et al., 1998). The gene comprises seven exons and six introns, and a total of 99.06 kb of DNA (Zhao and Rangnekar, 2008). The product of the par-4 gene is an approximately 40 kDa protein consisting of 342 amino acids in humans; 332 amino acids in rats; and 333 amino acids in mice (El-Guendy and Rangnekar, 2003). Par-4 is known to be evolutionarily conserved among all vertebrates (Boghaert et al., 1997). Rat and mouse Par-4 proteins exhibit a 93% amino acid homology, and rat and human Par-4 proteins share about 75% identical and 84% functionally similar amino acids. Moreover, the protein domains that are presumably of high functional importance are 100% conserved across these three species (El-Guendy and Rangnekar, 2003).

Some of the conserved functional domains of Par-4 include: a) two putative nuclear localization sequences (NLS), designated NLS1 (amino acid residues 20–25) and NLS2 (a bipartite sequence comprising residues 137–153) in the N-terminal region, b) a leucine-zipper domain spanning amino acids 290–332 in the C-terminal region, and c) a nuclear export sequence (NES) in the C-terminus. The protein also possesses several consensus sites for phosphorylation by kinases such as protein kinases A and C (PKA and PKC, respectively). These domains appear to be important in the regulation, localization, dimerization, and post-translational modification of the Par-4 protein (El-Guendy and Rangnekar, 2003).

Localization

Studies with a wide array of cell lines transiently transfected with GFP-Par-4 suggest a strong correlation between intracellular localization of Par-4 and its apoptotic function. In most cancer cells, ectopic Par-4 readily translocates to the nucleus to induce apoptosis. In contrast, in normal cells, ectopic Par-4 is predominantly localized to the cytoplasm and does not induce apoptosis unless a second apoptotic insult occurs. Similarly, Par-4 does not induce apoptosis in the hormone-dependent prostate and breast cancer cell lines, LNCaP and MCF-7 respectively, in which its localization is primarily cytoplasmic. However, Par-4 can translocate to the nucleus to directly induce apoptosis in hormone-independent derivatives of these cell lines, namely, MCF-7 cells stably expressing Ras (MCF-7 Ras) or LNCaP cells stably expressing IL-6 (LNCaP-IL6). These studies also provided the evidence that NLS2, but not NLS1, is essential for nuclear localization, inhibition of NF-κB activity and induction of apoptosis by Par-4 (El-Guendy et al., 2003). Moreover, a recently published study provides evidence that intracellular Par-4 is spontaneously secreted by mammalian cells, and therefore is also present in the extracellular compartment, e.g. in cell culture conditioned medium or circulating in the serum (Burikhanov et al., 2009).

Par-4 is a tumor suppressor

From the functional viewpoint, tumor suppressors are responsible for preserving the genomic integrity (i.e. the ‘caretaker’); and for regulating cell cycle, cell proliferation, differentiation and apoptosis (i.e. the ‘gatekeeper’). For practical purposes, identification of a tumor suppressor gene should satisfy the following parameters: a) a ‘loss-of-function’ mutation in the gene should result in a cancer phenotype, and b) inactivation of the gene in vivo should enhance tumor initiation, growth or progression (Paige, 2003).

Consistent with these criteria, Par-4 is down-regulated in many cancers, such as renal cell carcinoma (Cook et al., 1999), neuroblastoma (Kogel et al., 2001), endometrial cancer (Moreno-Bueno et al., 2007), and breast cancer (Zapata-Benavides et al., 2009). In about 32% of endometrial carcinomas, the Par-4 gene was observed to be silenced by promoter hypermethylation. A single base mutation in exon 3 of the Par-4 gene, converting an arginine residue (CGA) to a stop codon (TGA) has also been reported in endometrial cancer (Moreno-Bueno et al., 2007).

The role of the ras oncogene in human cancers has been well established, and mutational activation of the ras gene is frequently encountered in most cancers (Bos, 1989). K-Ras mutations are the most frequent mutations in pancreatic cancer (Almoguera et al., 1988), and Par-4 is significantly downregulated in pancreatic cancers harboring K-Ras point mutations (Ahmed et al., 2008). In addition, higher levels of Par-4 were correlated with better prognosis in terms of survival (Ahmed et al., 2008). Oncogenic Ras has been shown to downregulate Par-4 in a variety of cells via the MEK-ERK pathway, and this is considered as an important step towards Ras-induced transformation. Restoration of Par-4 levels either by MEK inhibition or by stable expression of ectopic Par-4 abrogates cellular transformation. This tumor-suppressor action of Par-4 appears to be distinct from its apoptotic function (Barradas et al., 1999; Pruitt et al., 2005; Qiu et al., 1999). Furthermore, in Ras-transformed epithelial cells, long-term reduction in Par-4 expression is achieved by MEK-dependent hypermethylation of the Par-4 promoter (Pruitt et al., 2005). If Par-4 is reinstated in these cells by transient transfection, it will selectively kill only those cells that express oncogenic Ras; this action of Par-4 is achieved by inhibition of NF-κB-mediated transcriptional activation (Nalca et al., 1999). The tumor-suppressor function of Par-4 has also been demonstrated in hematopoietic stem cells where it antagonizes the activation of oncogenic Ras, and disrupts BCR-Abl signaling to produce anti-transformation outcomes (Kukoc-Zivojnov et al., 2004).

Pancreatic and gastric cancers frequently show a deletion or instability in chromosome 12q21, the region where Par-4 is located (Kimura et al., 1998; Schneider et al., 2003). The aberrant expression of this region may also contribute to Wilm’s tumorigenesis (Johnstone et al., 1998). In the case of prostate cancer, Par-4 is not down-regulated, silenced or mutated, but inactivated due to phosphorylation by Akt1, which prevents nuclear translocation of Par-4, thereby retaining it in the cytoplasm and rendering it incapable of causing apoptosis (Goswami et al., 2005).

Animal studies have demonstrated that Par-4 knockout mice are prone to spontaneous development of tumors in various tissues, e.g. lungs, liver, urinary bladder and endometrium, and also exhibit prostatic intraepithelial neoplasia (PIN). Par-4 knockout mice are also more susceptible to chemical- or hormone-induced lesions. The endometrium and the prostate gland appear to be relatively sensitive to Par-4 loss, suggesting a role for Par-4 in the development of hormone-dependent tissues. Par-4 knockout mice also have a significantly shorter life span compared to wild-type animals, due to death by spontaneous tumors (Garcia-Cao et al., 2005).

Apoptosis by Par-4

The pro-apoptotic action of Par-4 is indicated by its increased expression in actively apoptosing cells, e.g. the granulosa cells of atretic ovarian follicles; interdigitating web cells of the mouse embryo; involuting tadpole tail; and dying neurons during neuronal development (El-Guendy and Rangnekar, 2003). The fact that the prostatic ductal cells do not normally express Par-4, but do so when forced to undergo apoptosis following testosterone ablation, further underscores the pro-apoptotic function of Par-4 (Boghaert et al., 1997).

Studies conducted in cell culture models show that over-expression of Par-4 is sufficient to directly induce apoptosis in many cancer cell types. The ability of Par-4 to directly cause apoptosis is associated with its nuclear translocation. Moreover, the apoptotic action of Par-4 can overcome cell protective mechanisms, such as the presence of Bcl-xL, Bcl-2, or absence of wild-type p53 or PTEN function. Interestingly, Par-4 is incapable of directly inducing apoptosis in normal or immortalized cells, and in hormone-responsive cancer cells. However, such Par-4-resistant cells are sensitized by Par-4 over-expression to a wide array of apoptotic signal(s), such as increased intracellular Ca2+, growth factor withdrawal, TNF-α, or UV, X-ray and gamma radiation (Chakraborty et al., 2001; El-Guendy et al., 2003; Nalca et al., 1999). The “apoptosis-sensitizing” function of Par-4 in some of the cancer cells is attributed to its accumulation in the cytoplasm and inability to translocate into the nucleus, due to phosphorylation by Akt1 which renders Par-4 subject to sequestration in the cytoplasm by complexing it with chaperone proteins such as 14-3-3 (Goswami et al., 2005); however, treatment with the other apoptotic signals translocates Par-4 into the nucleus to produce apoptosis. The mechanism underlying Par-4 retention in the cytoplasm in normal/immortalized cells remains unidentified. Thus, the difference between “apoptosis-inducing” and “apoptosis-sensitizing’ function of Par-4 is associated with its ability or inability, respectively, to translocate to the nucleus in the absence of another apoptotic stimulus.

The apoptotic effect of Par-4 is evident in vivo as well. A single injection of adenoviral construct of Par-4 into solid tumors produced in mice by implanting PC-3 cells resulted in a drastic reduction in tumor volume within 3 weeks. This outcome was largely due to an increase in apoptosis caused by Par-4 (Chakraborty et al., 2001). In tumors generated by xenotransplanted A375-C6 melanoma cells in SCID mice, over-expression of Par-4 by transfection correlated with decreased tumor development, and an increase in apoptosis (Lucas et al., 2001).

Mechanism of apoptosis

The apoptotic effect of Par-4 involves either an activation of the cellular apoptotic machinery or inhibition of the cellular pro-survival mechanisms. Par-4 induces apoptosis in hormone-independent cancer cells by enabling the translocation of Fas and Fas ligand (Fas/FasL) to the plasma membrane, which recruits the adapter protein Fas-dependent death domain (FADD); induces the formation of the death-inducing signaling complex (DISC); and thereby initiates the caspase cascade. Therefore, in these cancer cells, over-expression of Par-4 is a sufficient signal for cell death (Chakraborty et al., 2001). In parallel, Par-4 translocates to the nucleus and inhibits NF-κB-mediated cell survival mechanisms; this constitutes one of the essential mechanisms of Par-4-induced apoptosis (Figure 1) (Nalca et al., 1999). NF-κB regulates a number of pro-survival genes, including but not limited to, cell-protective genes such as those of the Bcl-2 family, e.g. Bcl-xL, Al/Bfl1; and anti-apoptotic genes such as X-linked inhibitor of apoptosis (XIAP) that can protect the cell from TNF-induced apoptosis (Barkett and Gilmore, 1999). In other studies, Par-4 has been shown to function in the cytoplasm, wherein it represses NF-κB-dependent gene transcription by inhibiting the TNFα-induced nuclear translocation of the p65 (Rel A) subunit by blocking the atypical protein kinase C (aPKC) (Figure 1), or IκB kinase (IKKβ)-mediated phosphorylation of the NF-κB inhibitory protein IκB (Diaz-Meco et al., 1999).

Figure 1
Key intracellular and extracellular components of Par-4-mediated apoptosis

Phosphorylation of the threonine 155 (T155) residue of Par-4 by Protein Kinase A (PKA), as well as its nuclear translocation, is essential for the apoptotic action of Par-4. PKA-mediated phosphorylation of Par-4 at T155 is required both for trafficking of Fas/FasL to the membrane, and for NF-κB inhibition. The precise role of T155 phosphorylation in nuclear translocation of Par-4 is not yet resolved, and it is possible that this phosphorylation event prevents intra-molecular folding of the leucine zipper domain that may otherwise mask the NLS2 domain, and thereby promotes nuclear entry. However, PKA phosphorylation is necessary but not sufficient for nuclear entry of Par-4, implying multiple factors may regulate Par-4 translocation to the nucleus. Elevated PKA activity in diverse cancer cells, relative to normal and immortalized cells, is one of the underlying mechanisms for cancer cell-specific apoptosis by Par-4 (Gurumurthy et al., 2005).

Identification of a domain selective for apoptosis in cancer cells

Analysis of several mutants resulting from serial deletion of the full-length Par-4 protein from both the N- and C-termini led to the identification of a unique core domain (spanning amino acids 137–195) which, when over-expressed, induces apoptosis specifically in cancer cells, and therefore is called the ‘selective for apoptosis of cancer cells’ (SAC) domain. This segment contains the NLS2 domain, which facilitates its nuclear translocation, and the T155 PKA phosphorylation site, which is responsible for its activation. Interestingly, the SAC domain localizes to the nucleus in normal/immortalized and cancer cell types, and is capable of inducing apoptosis not only in Par-4-sensitive cancer cells, but also in those cancer cells that show primarily cytoplasmic localization of full-length Par-4 and are resistant to Par-4-inducible apoptosis. However, in normal and immortalized cells, the SAC domain fails to be adequately phosphorylated at the T155 residue, because of low level PKA activity in these cells, and hence fails to causes apoptosis (El-Guendy et al., 2003). Consistent with the cancer-selective apoptotic action of the SAC domain of Par-4, green fluorescent protein (GFP)-tagged SAC transgenic mice show remarkable resistance toward formation of spontaneous tumors, as well as toward oncogene-induced tumor growth (Zhao et al., 2007).

Par-4 is secreted and shows cancer cell-specific apoptosis

While most of the studies on Par-4 focused on the apoptotic effect mediated by intracellular Par-4, an entirely new dimension to the Par-4 paradigm was added by our recent findings on its secretion by mammalian cells in general, and the apoptosis caused by this secreted Par-4 via the cell-surface protein GRP78. Endogenous Par-4, as well as the ectopically introduced GFP-Par-4 and GFP-SAC proteins, were observed to be spontaneously secreted by a broad range of normal or immortalized cells, as well as by cancer cells. In addition, endogenous Par-4 secretion could also be induced by exogenous stimuli capable of causing stress in the endoplasmic reticulum (ER) (Burikhanov et al., 2009). The findings also indicate that secretion occurs by a mechanism that is independent of the apoptotic action of Par-4 or the SAC domain. The conditioned medium (CM) obtained from Par-4-GFP and SAC-GFP transfectants induced apoptosis in hormone-independent prostate cancer PC-3 cells, but not in non-transformed BPH-1 cells, demonstrating cancer cell-specific apoptosis (Burikhanov et al., 2009).

Following up on the observations of secreted Par-4 on cancer-cell specific apoptosis in vitro, Burikhanov et al. (2009) demonstrated that Par-4 is also secreted in vivo and can be detected in the serum. Aliquots of serum samples obtained from Par-4 transgenic mice were found to contain detectable amounts of Par-4, as judged by Western blot analysis. In addition, Par-4 activity in serum was able to induce apoptosis specifically in cancer cells. In view of these findings, both systemic (extracellular) and intracellular Par-4 may contribute to tumor-resistance in SAC- and Par-4-transgenic mice (Zhao et al., 2007).

Par-4 secretion occurs via the classical pathway and is associated with ER stress

Immunocytochemical studies showing Par-4 localization in the ER and plasma membrane, and a substantial inhibition of secretion observed with Brefeldin A (BFA) pre-treatment of cells show that secretion of Par-4 takes place via the conventional ER-Golgi secretory pathway (Burikhanov et al., 2009). BFA blocks protein trafficking from the ER to cis-Golgi cisternae, resulting in the accumulation of normally secreted proteins within the ER (Fujiwara et al., 1988).

Par-4 secretion seems to be strongly influenced by agents that cause ER stress. Calcium depletion in the ER lumen, inhibition of asparagine (N)-linked glycosylation, reduction of disulfide bonds, etc., are some of the ways that disrupt ER homeostasis, resulting in an increase in unfolded proteins and the induction of an unfolded protein response (UPR) launched by the ER. Under moderate conditions of stress, the UPR causes a reduction in general protein synthesis, while selectively upregulating protein-folding enzymes and ER chaperone proteins like the glucose-regulated proteins (GRPs), including GRP78 (Kaufman, 1999). GRP78, also known as BiP (immunoglobulin heavy-chain binding protein), is a member of the HSP70 protein family, and is perceived as the “master regulator” of ER function. Intracellularly, GRP78 serves as an ER chaperone protein, facilitating protein folding and as an ER stress signaling regulator (Lee, 2007).

In response to severe ER stress, the UPR causes a strong induction of CHOP/GADD153 (CCAAT/enhancer-binding protein homologous protein/Growth Arrest and DNA Damage inducible), a transcription factor that negatively regulates cell growth and may promote apoptosis (Kaufman, 1999). GRP78 and CHOP/GADD153, therefore, serve as molecular indicators of ER stress. Tunicamycin (TU), an inhibitor of N-linked glycosylation (Zong et al., 2003), and thapsigargin (TG), blocker of the sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA) (Inesi et al., 1998), act as potent inducers of ER stress. Treatment of cells with ER stress inducers TU and TG for short time periods caused enhanced secretion of Par-4 and up-regulation of GRP78 and CHOP/GADD153 (Burikhanov et al., 2009). In order to characterize the functions and mechanisms of apoptosis induced by secreted Par-4 and SAC, Burikhanov et al. sought to identify binding partners for these secreted proteins. In so doing, the authors uncovered an integral role of the ER chaperone protein GRP78 in extracellular Par-4 and SAC-mediated apoptosis.

Cell-surface GRP78 serves as a receptor, and is essential for apoptosis by extracellular Par-4 and SAC

GST-pull down assays and subsequent mass spectrometry of PC-3 whole cell extracts led to the identification of GRP78 as a binding partner of Par-4 and SAC. Co-immunoprecipitation and Western blotting data further confirmed that the SAC protein as well as the endogenous Par-4 bind to the GRP78 protein. Presence of neutralizing antibody against the N-terminus of GRP78, knock-down of GRP78 expression with RNA-interference (RNAi), or neutralization of Par-4 in the CM with recombinant GRP78 inhibited apoptosis by extracellular Par-4 (Burikhanov et al., 2009). These findings suggested that cell-surface GRP78 serves as a receptor for secreted Par-4 acting via the SAC domain, and its interaction with the N-terminus of GRP78 is a prerequisite to apoptotic signaling initiation.

Immunocytochemical studies show that TRAIL treatment causes a marked increase in membrane expression of GRP78 and in its colocalization with Par-4 in PC-3 cells, but elicits no such change in BPH cells. In contrast, Par-4 and GRP78 colocalize in the ER in vehicle-treated PC-3 and BPH cells. These observations suggest that ER stress results in the translocation of GRP78-Par-4 complex from the ER to the plasma membrane in cancer cells, but not in normal or immortalized cells (Burikhanov et al., 2009).

intracellular Par-4 is essential for apoptosis by secreted Par-4

Both GRP78 and intracellular Par-4 seem to function in concert to initiate the apoptotic machinery triggered by extracellular Par-4 (Burikhanov et al., 2009). Knock-down of endogenous Par-4 by RNA-interference (RNAi) results in resistance to recombinant Par-4- or SAC-induced apoptosis. Par-4 knock-down also results in a reduction in ER stress-related translocation of GRP78 to the cell surface, implying that endogenous Par-4 is required for trafficking the GRP78 protein to the cell membrane. Further, sensitivity to recombinant Par-4 was restored when these Par-4 knockdown cells were transfected with membrane-directed full length GRP78 (mGRP78), whereas transfection with a membrane-directed N-terminal mutant (mΔN-GRP78, from which 66 amino acids at the N-terminus have been deleted) did not undergo recombinant Par-4-induced apoptosis. In fact, transfection with full-length mGRP78 also rendered otherwise resistant immortalized BPH cells sensitive to recombinant Par-4-induced apoptosis (Burikhanov et al., 2009). Thus, apoptosis by extracellular Par-4 is dependent on both intracellular Par-4 and membrane-localized GRP78.

Mechanism of extracellular Par-4 induced apoptosis

Interaction of extracellular Par-4 with cell-surface GRP78 induces apoptosis in a FADD-dependent manner (Figure 1). Co-localization of caspase-8 with GRP78 at the cell membrane, and detection of activated caspase-8 and caspase-3 upon treatment with recombinant Par-4 suggests that the adapter protein FADD is then able to recruit caspase-8 at the cell membrane, which initiates the apoptotic machinery by activating downstream caspase-3. As mentioned above, ER stress is observed to play a role in translocation of GRP78 to the cell membrane, as well as in extracellular Par-4- or TRAIL-mediated apoptosis. PKR-like ER Kinase (PERK), which is an ER-stress activated protein known to promote FADD- and caspase-8-dependent apoptosis (Park et al., 2008), seems to be the molecular link between ER stress and extracellular Par-4-induced apoptosis. Burikhanov et al. (2009) noted that phospho-PERK levels are significantly increased in cells treated with recombinant Par-4 or TRAIL, and knock-down of PERK expression (by RNAi) leads to inhibition of caspase-8-dependent apoptosis, suggesting that the apoptotic process is regulated by PERK. The novel findings that mammalian cells show spontaneous, as well as ER-stress-induced secretion of the pro-apoptotic protein Par-4, and that secreted Par-4 exerts apoptotic action selectively on cancer cells, without harming the normal cells, hold immense promise for the future. Further studies aimed at understanding the molecular players and regulators in the extrinsic apoptotic pathway evoked by secreted Par-4 may help define the potential for targeting Par-4 secretion in therapeutic application.

Role of GRP78

Given the involvement of GRP78 in extracellular Par-4-induced apoptosis, its role in cellular homeostasis appears extremely intriguing. Intracellularly, the functions of GRP78 promote cell growth, survival, and are anti-apoptotic in nature. Consistent with this observation, GRP78 is over-expressed in multiple cancers, and elevated levels are associated with tumor severity and chemoresistance (Lee, 2007). In addition, GRP78 expression has also been detected on the surface of cancer cells (Schwarze and Rangnekar, 2010). On the cell surface, GRP78 acts as a receptor for α2-macroglobulin (Misra et al., 2005) and for the extracellular signaling protein Cripto (Kelber et al., 2009), and generates pro-survival and pro-oncogenic signals. On the contrary, GRP78 also shows pro-apoptotic functions as a receptor for the angiogenesis inhibitor Kringle 5 (Davidson et al., 2005), and, as discussed here, for Par-4 (Burikhanov et al., 2009). Thus, GRP78 appears to play a central role in determining the cellular outcomes in response to binding by diverse ligands.

Future directions

This discovery of secreted Par-4 adds a new dimension to its apoptotic function and its role in cancer. Both Par-4 secretion and membrane translocation of the normally ER-resident protein GRP78 are remarkably induced by ER stress. Cancer cells often exhibit higher ER stress due to elevated glucose metabolism and exposure to tumor hypoxia (Lee, 2007). Thus, in cancer cells in particular, ER stress-induced upregulation in membrane-bound GRP78 expression and Par-4 secretion, and their interaction at the cell-surface feed forward to produce more ER stress, leading to increased trafficking and elevated expression of GRP78 on the cell membrane in an intracellular Par-4-dependent manner. These findings emphasize the concept of a strong ER stress loop involving GRP78 and both extracellular and intracellular Par-4. The biochemical details of the interaction between GRP78 and Par-4, along with their specific roles in this feed-forward loop remain to be investigated. Future studies will resolve whether Par-4 membrane translocation and secretion occurs by a common pathway, or whether the pathways diverge and Par-4 is routed to its different destinations through separate mechanisms. Since Par-4 lacks a typical signal peptide, the molecular basis for its transport into the ER and its secretion process remains unclear. Therefore, the mechanism of Par-4 secretion and characterization of the secretion pathway warrants further investigation.

A deeper understanding of the dynamics of Par-4 and GRP78 interaction as it relates to apoptosis shows considerable promise in terms of revealing new strategies for cancer therapeutics. Moreover, GRP78 figures as a major therapeutic target for drugs that can activate it in the same way as Par-4. Similarly, identifying key regulators in Par-4 secretion and/or extracellular Par-4 mediated apoptotic pathways can reveal new therapeutic targets.

Nuclear localization of intracellular Par-4 is essential for apoptosis to occur. In a recent report (Shareef et al., 2007), TRAIL has been shown to cause the nuclear translocation of Par-4 in H460 cells, and this mechanism is purportedly responsible for ionizing radiation-induced bystander effect triggered in response to high-dose X-rays. Interestingly, Par-4 is reported to interact with DAP-like kinase (Dlk), and subsequently recruit Dlk to the cytoplasmic actin filaments; this relocation of the Par-4/Dlk complex to actin is considered to be essential for Dlk-mediated apoptosis (Boosen et al., 2009; Page et al., 1999). It is possible that the putative NES present at the C-terminus of Par-4 plays a role in relocating the Dlk/Par-4 complex to the cytoplasm. These observations suggest a role for the NLS and NES domains of Par-4 in this cytoplasm-nucleus-cytoplasm translocation loop. It is unclear whether this intracellular loop is linked to the ER loop activated by extracellular Par-4. Elucidation of other biochemical and molecular players in these two loops constitutes an important area of investigation.

Considering the cancer cell-specific apoptotic nature and tumor suppressive function of Par-4 in the Par-4-transgenic mice, it is possible that there is a correlation between secreted Par-4 and cancer risk. In general, serum Par-4 levels may be diminished in cancer compared to normal conditions. What causes this reduction in the secretion of Par-4 in cancer? Is there a particular ‘source organ’ responsible for secreting Par-4 into the serum? Could Par-4 secretion be regulated by cross-talk among multiple organs, or by other molecules in circulation? Is down-regulation of Par-4 secretion specific to certain types of cancer, or is it common to all tumors? These represent some of the intriguing challenges that need to be addressed in future investigations regarding the role of extracellular Par-4 in normal and cancerous states. Such studies may offer new insights into systemic Par-4 and its association with predisposition to cancer.

Since Par-4 secretion is related to ER stress and occurs via the classical secretory pathway, it is important to identify drugs that can modulate Par-4 secretion. Compounds with satisfactory outcomes can then be used to enhance Par-4 secretion as a cancer-preventive strategy. Contrary to most therapeutic agents currently in clinical use, the cancer cell-specific induction of apoptosis by Par-4 and lack of adverse effects in normal cells, make Par-4 an attractive molecule for cancer therapeutics.

Acknowledgments

This study was supported by NIH/NCI grants CA60872, CA105453, and CA84511 (to VMR).

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