Par-4 Research Articles

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting (LIMACS): a Novel Method to Analyze Protein-lipid Interaction

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification procedures. As an alternative, lipids can be coated on flat surfaces as used for lipid ELISA and Plasmon resonance spectroscopy. However, surface coating lipids do not form microdomain structures, which may be important for the lipid binding properties. Further, these methods do not allow for the purification of larger amounts of proteins binding to their target lipids. To overcome these limitations of testing lipid protein interaction and to purify lipid binding proteins we developed a novel method termed lipid vesicle-mediated affinity chromatography using magnetic-activated cell sorting (LIMACS). In this method, lipid vesicles are prepared with the target lipid and phosphatidylserine as the anchor lipid for Annexin V MACS. Phosphatidylserine is a ubiquitous cell membrane phospholipid that shows high affinity to the protein Annexin V. Using magnetic beads conjugated to Annexin V the phosphatidylserine-containing lipid vesicles will bind to the magnetic beads. When the lipid vesicles are incubated with a cell lysate the protein binding to the target lipid will also be bound to the beads and can be co-purified using MACS. This method can also be used to test if recombinant proteins reconstitute a protein complex binding to the target lipid. We have used this method to show the interaction of atypical PKC (aPKC) with the sphingolipid ceramide and to co-purify prostate apoptosis response 4 (PAR-4), a protein binding to ceramide-associated aPKC. We have also used this method for the reconstitution of a ceramide-associated complex of recombinant aPKC with the cell polarity-related proteins Par6 and Cdc42. Since lipid vesicles can be prepared with a variety of sphingo- or phospholipids, LIMACS offers a versatile test for lipid-protein interaction in a lipid environment that resembles closely that of the cell membrane. Additional lipid protein complexes can be identified using proteomics analysis of lipid binding protein co-purified with the lipid vesicles.

Assessment of the pathway of apoptosis involving PAR-4, DAXX and ZIPK proteins in CLL patients and its relationship with the principal prognostic factors

Par-4 (prostate apoptosis response-4) protein was originally found upregulated in prostate tumor cells undergoing apoptosis. Then it was further identified as a proapoptotic protein upregulated both in normal and leukemic lymphocytes. The aim of our study was to assess PAR-4 protein expression in the B cells of CLL patients and to examine its relationship with the expression of other proteins involved in the apoptosis process, such as DAXX, ZIPK and BCL-2. We found a positive relationship between PAR-4 and BCL-2 protein expression. Additionally, there was a positive correlation between PAR-4 and both DAXX and ZIPK protein expression. The results of our research were also analyzed in association with the principal CLL prognostic factors. There was a positive correlation between the expression of PAR-4 protein and the lactate dehydrogenase (LDH) serum concentration (p < 0.005). The expression of PAR-4 protein in B cells correlated positively with the percentage of CD38(+) cells (p < 0.05), as well as with CD38(+)/ZAP-70(+) cells (p < 0.05). Moreover, we found a close relationship between LPL protein expression or LPL/ADAM29 MFI ratio and PAR-4 protein expression. Our results confirm the significance of apoptosis deregulation in CLL, and suggest a possible relationship between PAR-4 expression and the clinical course of the disease. This however requires further investigation.

Abstract 2891: Gastrin signaling and Par-4 in pancreatic cancer

Abstract Background: The gastrointestinal peptide, gastrin, has been identified as a growth factor in pancreatic cancer. Gastrin stimulates growth of pancreatic cancer through the cholecystokinin (CCK)-2 receptor. Prostate apoptosis response-4 (Par-4) is a tumor-suppressor protein that induces apoptosis in cancer cells but not in normal cells. Par-4 is down-regulated in a variety of cancers, including pancreatic where it is correlated with lower rate of survival. The anti-apoptotic activity of Par-4 is reduced when active Akt1 binds and phosphorylates it. In colon cancer cells, Par-4 also binds to NFκB and repressed NFκB gene transcription. Gastrin stimulation mediated through the CCK-2R regulates NFκB and Akt activity. Hypothesis: Stimulation of human pancreatic cancer cells with gastrin affects Par-4 activity which may be mediated through Akt. Methods: PANC-1 cells were serum starved for 16-hours, pretreated with a CCK-2R specific inhibitor (YM022) or vehicle for 1hr, and then stimulated with 10-8 M gastrin-17 for 20min. Cells were immediately lysed and the whole cell lysate was subjected to immunoprecipitation with magnetic Dynabeads bound to antibodies specific for Par-4 or NFκB(p65). Immunoprecipitation samples were probed for Par-4, and NFκB(p65), and cell lysates were probed for Par-4, NFκB(p65), Akt, phospho-p65 (Ser536), and phospho-Akt (Ser473). PANC-1 cells were transfected with siRNA specific to the CCK-2R or appropriate scrambled control delivered by Lipofectamine 2000. Protein was isolated 72 hours after transfection. Results: Pretreatment of PANC-1 cells with YM022 before gastrin stimulation blocked phosphorylation of Akt while total levels of Akt remained unchanged. siRNA knock-down of CCK-2R also resulted in a decrease of Akt activation. Phosphorylation of NFκB(p65) was increased in cells pretreated with YM022 compared to cells treated with gastrin alone; total levels of NFκB(p65) remained unchanged. Binding of Par-4 to NFκB(p65) was enhanced in cells treated with both gastrin and YM022, compared to cells treated with gastrin alone. Total levels of Par-4 remained unchanged. Conclusion: For the first time we provide evidence supporting that gastrin stimulation of pancreatic cancer cells alters activity of Par-4. Gastrin stimulation decreases binding of Par-4 to NFκB(p65) in PANC-1 cells while pretreatment with YM022 blocks this decrease in binding. The change in Par-4 activity may be mediated through Akt because YM022 and gastrin stimulation alters their activity and they are known binding partners of Par-4. Since Par-4 sensitizes cells to apoptosis induction as exemplified by sensitization of colon cancer cells to the chemotherapeutic agent, 5-fluorouracil, when Par-4 is delivered in vivo, understanding of the mechanism by which Par-4 is down-regulated is important in developing therapies to take advantage of Par-4 induced sensitization to apoptosis. Supported by: R01 CA117926 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2891. doi:10.1158/1538-7445.AM2011-2891

Abstract 4093: Prostate apoptosis response-4 (Par-4): A novel substrate of caspase-3 during apoptosis

Abstract Prostate apoptosis response-4 (Par-4) is a ubiquitously expressed pro-apoptotic tumor suppressor protein. Par-4 is known to selectively induce apoptosis in cancer cells either by activating apoptosis or by inhibiting cell survival. Here, we show, for the first time, that Par-4 is a novel substrate of caspase-3 during apoptosis. We found that Par-4 is cleaved during cisplatin-induced apoptosis in human normal and cancer cell lines. Caspase-3-deficient MCF-7 cells did not show Par-4 cleavage in response to cisplatin treatment. This Par-4 cleavage was concomitant with Parp cleavage and generated a C-terminal fragment of approx. 25kDa. The cleavage of Par-4 was completely inhibited by Z-VAD-fmk (an inhibitor of caspase like protease) suggesting that caspase-3 like activity is involved in the cleavage of Par-4. Consistently, recombinant caspase-3 efficiently cleaved Par-4 in vitro for all cell lines tested, suggesting the involvement of this protease in Par-4 processing in vivo. Overexpression of caspase-3 in PC-3 cells increased caspase-3 activity, which further resulted in the cleavage of Par-4 in these cells. However, overexpression of a catalytic mutant caspase-3 (C163A) failed to induce Par-4 cleavage. Further, restoration of caspase-3 in MCF-7 cells showed a decrease in Par-4 levels with the appearance of the cleaved fragment. The cleavage of Par-4 in caspase-3 expressing MCF-7 cells was efficiently blocked by a caspase-3 inhibitor. These results establish for the first time that Par-4 is a novel substrate of caspase-3 both in vitro and in vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4093. doi:10.1158/1538-7445.AM2011-4093

Par-4 gene silence inhibited hydrogen peroxide-induced apoptosis in alveolar epithelial cells

Objective To investigate the effects of Par-4 gene silence on hydrogen peroxide-induced apoptosis in alveolar epithelial cells. Method The alveolar epithelial cells A549 were cultured and exposed to hydrogen peroxide. The siRNA sequences targeted Par-4 gene was chemically synthesized and transfected to A549 cells with or without the exposure of hydrogen peroxide. The cells were divided into normal control groups, hydrogen peroxide-treated group(The cells were treated with 0. 1 mmol/L hydrogen peroxide), hydrogen peroxide and Par-4-siRNA-treated group(The cells were treated with 0. 1 mmol/L hydrogen peroxide after transfection of Par-4-siRNA), Non-specific DNA sequence transfection control group. The apoptosis of A549 cells was quantified by flow cytometry. The expression of Smac protein was detected by Western blot.Electrophoretic mobility shift assay was applied for evaluating the change of E2F1 DNA binding activity. Relative activity of Caspase-3 was detected by clolorimetric assay. Results The percent of apoptotic cells in hydrogen peroxide and Par-4-siRNA-treated group was (29.7 ± 2.3) %, which was significantly lower than that of hydrogen peroxide-treated group [(54.2 ± 4.1)%, q= 8.91, P ＜ 0.01)]. Par-4 siRNA could significantly suppress the increase of Smac protein, E2F1 DNA binding activity and caspase-3 activity induced by hydrogen peroxide in A549 cells. Conclusions Par-4 gene silence induced by siRNA might inhibit the apoptosis of alveolar epithelial cells, which might be resulted from suppression of the up-regulation of Smac gene expression, E2F1 DNA binding activity and caspase-3 activity. Key words: Par-4; Small RNA interference; Hydrogen peroxide; Alveolar epithelial cells; Apoptosis

Expression Pattern of the Pro-apoptotic Gene PAR-4 During the Morphogenesis of MCF-10A Human Mammary Epithelial Cells

The histological organization of the mammary gland involves a spatial interaction of epithelial and myoepithelial cells with the specialized basement membrane (BM), composed of extra-cellular matrix (ECM) proteins, which is disrupted during the tumorigenic process. The interactions between mammary epithelial cells and ECM components play a major role in mammary gland branching morphogenesis. Critical signals for mammary epithelial cell proliferation, differentiation, and survival are provided by the ECM proteins. Three-dimensional (3D) cell culture was developed to establish a system that simulates several features of the breast epithelium in vivo; 3D cell culture of the spontaneously immortalized cell line, MCF10A, is a well-established model system to study breast epithelial cell biology and morphogenesis. Mammary epithelial cells grown in 3D form spheroids, acquire apicobasal polarization, and form lumens that resemble acini structures, processes that involve cell death. Using this system, we evaluated the expression of the pro-apoptotic gene PAWR (PKC apoptosis WT1 regulator; also named PAR-4, prostate apoptosis response-4) by immunofluorescence and quantitative real time PCR (qPCR). A time-dependent increase in PAR-4 mRNA expression was found during the process of MCF10A acinar morphogenesis. Confocal microscopy analysis also showed that PAR-4 protein was highly expressed in the MCF10A cells inside the acini structure. During the morphogenesis of MCF10A cells in 3D cell culture, the cells within the lumen showed caspase-3 activation, indicating apoptotic activity. PAR-4 was only partially co-expressed with activated caspase-3 on these cells. Our results provide evidence, for the first time, that PAR-4 is differentially expressed during the process of MCF10A acinar morphogenesis.

PAR-4 and hTERT expression are negatively correlated after RNA interference targeting hTERT in laryngocarcinoma cells

We investigated the relationship between telomerase activity and the expression of human telomerase reverse transcriptase (hTERT) and prostate apoptosis response 4 (Par-4) proteins in laryngocarcinoma cells. Following treatment with short hairpin RNA (shRNA) against hTERT, hTERT protein expression was inhibited, while Par-4 expression was increased. These changes were statistically significant ( p < 0.05), with Par-4 and hTERT expression being negatively correlated ( p < 0.05, r = −0.908). However, we found no correlation between telomerase activity and hTERT expression. Our findings suggest that hTERT exerts its anti-apoptotic effects through a telomerase-independent pathway, which is likely to intersect with the Par-4 apoptosis pathway.

Prostate Apoptosis Response-4 Is Expressed in Normal Cholangiocytes, Is Down-Regulated in Human Cholangiocarcinoma, and Promotes Apoptosis of Neoplastic Cholangiocytes When Induced Pharmacologically

Prostate apoptosis response-4 (Par-4) is a tumor suppressor protein that sensitizes cells to apoptosis; therefore, Par-4 modulation has therapeutic potential. No data currently exist on Par-4 expression in cholangiocarcinoma (CCA). We evaluated the expression of Par-4 in normal and neoplastic cholangiocytes and the effects of its pharmacological or genetic modulation. The study was performed in human and rat liver, CCA patient biopsies, and two CCA cell lines. PAR-4 was expressed in normal rat and human cholangiocytes, but its expression levels decreased in both human CCA and CCA cell lines. In both intrahepatic and extrahepatic CCA, Par-4 expression (as shown by immunohistochemistry) was inversely correlated with markers of proliferation (eg, proliferating cellular nuclear antigen) and directly correlated with apoptotic markers (eg, Bax and Bax/BCL2 ratio). Par-4 expression was decreased during CCA cell proliferation but was enhanced after apoptosis induction. Pharmacological induction of Par-4 expression in CCA cell lines by diindolymethane or withaferin A promoted activation of apoptosis and inhibition of proliferation. In contrast, specific Par-4 silencing by small-interfering RNA determined activation of CCA cell line proliferation. Par-4 is expressed in rat and human cholangiocytes and is down-regulated in both human CCA and CCA cell lines. Par-4 protein levels decrease during cell proliferation but increase during apoptosis. Pharmacological or genetic induction of Par-4 determines apoptosis of CCA cells, suggesting Par-4 targeting as a CCA treatment strategy.

Secretory expression of Par-4 SAC-HA2TAT following adeno-associated virus-mediated gene transfer induces apoptosis in HepG2 cells

Prostate apoptosis response-4 (Par-4) is a tumor-suppressor protein that induces apoptosis in cancer cells, but not in normal cells. The cancer-specific pro-apoptotic action of Par-4 is encoded in its centrally located SAC domain. In this study, to further enhance the anti-cancer effect of Par-4 in order to overcome the limitations of peptide therapy, a recombinant adeno-associated virus was constructed using the following strategies: the secretory expression of therapeutic peptide, a HA2TAT-mediated cytosolic delivery technique, and an adeno-associated virus gene transfer system. To test the hypothesis that Par-4 has an additive bystander effect as an anti-cancer therapy, we designed a secretory protein by adding a secretory signal peptide NT4(Si) to the Par-4 SAC-HA2TAT peptide gene sequence [NT4(Si)-Par-4 SAC-HA2TAT]. The results indicated that, compared to the normal NIH3T3 cell line, AAV-NT4(Si)-Par-4 SAC-HA2TAT significantly suppressed cell growth and induced rapid cell death in HepG2 cells in a time-dependent manner through successful gene transfer and secretory expression of therapeutic peptide at 48 h post-transfection. In addition, the secretory properties of Par-4 may greatly increase its effectiveness in cancer therapy when delivered in vivo.

Two‐state conformational equilibrium in the Par‐4 leucine zipper domain

Prostate apoptosis response factor-4 (Par-4) is a pro-apoptotic and tumor-suppressive protein. A highly conserved heptad repeat sequence at the Par-4 C-terminus suggests the presence of a leucine zipper (LZ). This C-terminal region is essential for Par-4 self-association and interaction with various effector proteins. We have used nuclear magnetic resonance (NMR) spectroscopy to fully assign the chemical shift resonances of a peptide comprising the LZ domain of Par-4 at neutral pH. Further, we have investigated the properties of the Par-4 LZ domain and two point mutants under a variety of conditions using NMR, circular dichroism (CD), light scattering, and bioinformatics. Results indicate an environment-dependent conformational equilibrium between a partially ordered monomer (POM) and a predominantly coiled coil dimer (CCD). The combination of techniques used allows the time scales of the equilibrium to be probed and also helps to identify features of the amino acid sequence that may influence the equilibrium.

Widespread expression of prostate apoptosis response‐4 in nasopharyngeal carcinoma

Prostate apoptosis response-4 (Par-4) augments apoptosis in various tumors, either during apoptotic insult or by ectopic overexpression. However, investigation of Par-4 expression in nasopharyngeal carcinoma (NPC) is lacking. Specimens from patients with NPC, hypopharyngeal carcinoma (HPC), or oral cavity cancer were examined for Par-4 expression using immunohistochemistry. NPC cell proliferation and apoptosis were analyzed using immunohistochemical staining for Ki67, B-cell lymphoma 2 (Bcl-2), and in situ terminal deoxyribonucleotidyl transferase (TdT)-mediated biotin-16-dUTP nick end-labeling (TUNEL) assay, respectively. Par-4 was ubiquitously expressed in NPC biopsies (96.2%, 25/26) and was significantly higher than in HPC (47.6%, 50/105, p < .0001) and oral cavity cancers (38.7%, 12/31, p < .0001). Remarkably, apoptosis of NPC cells was absent and Par-4 expression was associated with obvious expression of Bcl-2 and Ki67 in all patients tested with NPC. Immunohistochemistry results showed widespread expression of Par-4 in NPC and revealed sustainable proliferation of NPC cells regardless of Par-4 expression.

231 Application of non-steroidal anti-inflammatory drugs to enhance 5-fluorouracil efficacy on experimental systems

Background & Aims: Many reports indicate that nonsteroidal anti-inflammatory drugs (NSAIDs) have antineoplastic effects, but the precise molecular mechanism(s) responsible are unclear. We evaluated the effect of cyclooxygenase (COX) inhibitors (NSAIDs) on human colon carcinoma cells (HCA-7) and identified several genes that are regulated after treatment with NS-398, a selective COX-2 inhibitor. Methods: Differential display polymerase chain reaction cloning techniques were used to identify genes regulated by treatment with NSAIDs and selective COX-2 inhibitors. Results: A prostate apoptosis response 4 (Par-4) gene was up-regulated after NSAID treatment. Par-4 was first isolated from prostate carcinoma cells undergoing apoptosis, and expression of Par-4 sensitized cancer cells to apoptotic stimuli. Par-4 levels were increased in cells treated with COX inhibitors such as NS-398, nimesulide, SC-58125, and sulindac sulfide. Treatment of HCA-7 cells with these agents also induced apoptotic cell death. Conclusions: The results suggest that regulation of Par-4 contributes to the proapoptotic effects of high-dose COX inhibitors (NSAIDs) by serving as a downstream mediator leading to initiation of programmed cell death.GASTROENTEROLOGY 2000;118:1012-1017

The Ras Effector RASSF2 Controls the PAR-4 Tumor Suppressor

RASSF2 is a novel proapoptotic effector of K-Ras. Inhibition of RASSF2 expression enhances the transforming effects of K-Ras, and epigenetic inactivation of RASSF2 is frequently detected in mutant Ras-containing primary tumors. Thus, RASSF2 is implicated as a tumor suppressor whose inactivation facilitates transformation by disconnecting apoptotic responses from Ras. The mechanism of action of RASSF2 is not known. Here we show that RASSF2 forms a direct and endogenous complex with the prostate apoptosis response protein 4 (PAR-4) tumor suppressor. This interaction is regulated by K-Ras and is essential for the full apoptotic effects of PAR-4. RASSF2 is primarily a nuclear protein, and shuttling of PAR-4 from the cytoplasm to the nucleus is essential for its function. We show that RASSF2 modulates the nuclear translocation of PAR-4 in prostate tumor cells, providing a mechanism for its biological effects. Thus, we identify the first tumor suppressor signaling pathway emanating from RASSF2, we identify a novel mode of action of a RASSF protein, and we provide an explanation for the extraordinarily high frequency of RASSF2 inactivation we have observed in primary prostate tumors.

Down-regulation of the candidate tumor suppressor gene PAR-4 is associated with poor prognosis in breast cancer

Substantial experimental evidence indicates that PAWR gene (PKC apoptosis WT1 regulator; also named PAR-4, prostate apoptosis response-4) is a central player in cancer cell survival and a potential target for cancer-selective targeted therapeutics. However, little is known about the role of PAR-4 in breast cancer. We investigated the possible role of PAR-4 expression in breast cancer. IHC results on tissue microarrays containing 1,161 primary breast tumor samples showed that 57% (571/995) of analyzable cases were negative for PAR-4 nuclear staining. Down-regulation of nuclear PAR-4 protein expression predicted a poor prognosis for breast cancer patients (OS; P=0.041, log-rank test). PAR-4 down-regulation also correlates with poor survival in the group of patients with luminal A subtype breast cancer (P=0.028). Additionally, in this large series of breast cancer patients, we show that ERBB2/HER2, EGFR and pAKT protein expression are significantly associated with shorter disease-free survival and overall survival, but the prognosis was even worse for HER2-positive, EGFR-positive or pAKT-positive breast cancer patients with tumors negative for nuclear PAR-4 expression. Furthermore, using three-dimensional (3D) cell culture we provide preliminary results showing that PAR-4 is highly expressed in the MCF10A cells inside the acini structure, suggesting that PAR-4 might have a role in the lumen acini formation. Taken together, our results provide, for the first time, evidence that PAR-4 may have a role in the process of the mammary gland morphogenesis and its functional inactivation is associated with tumor aggressive phenotype and might represent an additional prognostic and predictive marker for breast cancer.

Prenatal alcohol exposure triggers ceramide-induced apoptosis in neural crest-derived tissues concurrent with defective cranial development

Fetal alcohol syndrome (FAS) is caused by maternal alcohol consumption during pregnancy. The reason why specific embryonic tissues are sensitive toward ethanol is not understood. We found that in neural crest-derived cell (NCC) cultures from the first branchial arch of E10 mouse embryos, incubation with ethanol increases the number of apoptotic cells by fivefold. Apoptotic cells stain intensely for ceramide, suggesting that ceramide-induced apoptosis mediates ethanol damage to NCCs. Apoptosis is reduced by incubation with CDP-choline (citicoline), a precursor for the conversion of ceramide to sphingomyelin. Consistent with NCC cultures, ethanol intubation of pregnant mice results in ceramide elevation and increased apoptosis of NCCs in vivo. Ethanol also increases the protein level of prostate apoptosis response 4 (PAR-4), a sensitizer to ceramide-induced apoptosis. Prenatal ethanol exposure is concurrent with malformation of parietal bones in 20% of embryos at day E18. Meninges, a tissue complex derived from NCCs, is disrupted and generates reduced levels of TGF-β1, a growth factor critical for bone and brain development. Ethanol-induced apoptosis of NCCs leading to defects in the meninges may explain the simultaneous presence of cranial bone malformation and cognitive retardation in FAS. In addition, our data suggest that treatment with CDP-choline may alleviate the tissue damage caused by alcohol.

Immunohistochemical Analysis of Prostate Apoptosis Response-4 (Par-4) in Mexican Women with Breast Cancer: A Preliminary Study

We undertook this study to compare the expression level of prostate apoptosis response-4 (Par-4) among patient outcome in two groups of women with breast cancer (short and long survival) and two groups without breast cancer (benign lesion and control). We included breast specimens with nonhistological abnormalities (eight samples) as a control group. Semiquantitative and quantitative analysis of immunohistochemical staining by image analysis software were used to study the intensity of Par-4 expression. Both methods produced similar results (p>0.05). No significant expression of Par-4 was observed in normal breast tissue. Benign lesions and breast cancer tissue showed strong nuclear expression of Par-4, predominantly on epithelial cells and specifically in ductal cells. Par-4 expression was lower in myoepithelial cells and there was no appreciable stromal staining. Significantly less Par-4 reactivity was detected in tissue from patients with a short survival compared with patients with benign lesions and those with a long survival. Our findings suggest that a lower expression level of Par-4 is related to an unfavorable prognosis. A larger prospective study of samples of all patient groups with a longer follow-up is needed to validate this finding.

Prostate apoptosis response protein 4 sensitizes human colon cancer cells to chemotherapeutic 5-FU through mediation of an NFκB and microRNA network

BackgroundDiminished expression or activity of prostate apoptosis response protein 4 (Par-4) has been demonstrated in a number of cancers, although reports on Par-4 expression during colon cancer progression are lacking. An understanding of the molecular events in conjunction with the genetic networks affected by Par-4 is warranted.ResultsColon cancer specimens derived from patients have significantly diminished expression of Par-4 mRNA relative to paired normal colon. Hence, the functional consequences of reintroducing Par-4 into HT29 colon cancer cells were assessed. Overexpression augmented the interaction of Par-4 with NFκB in the cytosol but not nucleus, and facilitated apoptosis in the presence of 5-fluorouracil (5-FU). Analogous findings were obtained when AKT1 pro-survival signaling was inhibited. Transcriptome profiling identified ~700 genes differentially regulated by Par-4 overexpression in HT29 cells. Nearly all Par-4-regulated genes were shown by promoter analysis to contain cis-binding sequences for NFκB, and meta-analysis of patient expression data revealed that one-third of these genes exist as a recurrent co-regulated network in colon cancer specimens. Sets of genes involved in programmed cell death, cell cycle regulation and interestingly the microRNA pathway were found overrepresented in the network. Noteworthy, Par-4 overexpression decreased NFκB occupancy at the promoter of one particular network gene DROSHA, encoding a microRNA processing enzyme. The resulting down-regulation of DROSHA was associated with expression changes in a cohort of microRNAs. Many of these microRNAs are predicted to target mRNAs encoding proteins with apoptosis-related functions. Western and functional analyses were employed to validate several predictions. For instance, miR-34a up-regulation corresponded with a down-regulation of BCL2 protein. Treating Par-4-overexpressing HT29 cells with a miR-34a antagomir functionally reversed both BCL2 down-regulation and apoptosis by 5-FU. Conversely, bypassing Par-4 overexpression by direct knockdown of DROSHA expression in native HT29 cells increased miR-34a expression and 5-FU sensitivity.ConclusionOur findings suggest that the initiation of apoptotic sensitivity in colon cancer cells can be mediated by Par-4 binding to NFκB in the cytoplasm with consequential changes in the expression of microRNA pathway components.

PAR-4 as a possible new target for pancreatic cancer therapy

Importance of the field: Pancreatic cancer (PC) is a deadly disease that is intractable to currently available treatment regimens. Although well described in different tumors types, the importance of apoptosis inducer prostate apoptosis response-4 (Par-4) in PC has not been appreciated. PC is an oncogenic kras driven disease, which is known to downregulate Par-4. Therefore, this review highlights its significance and builds a strong case supporting the role of Par-4 as a possible therapeutic target in PC.Areas covered in this review: Literature-based evidence spanning the last 15 years on Par-4 and its significance in PC.What the reader will gain: This review provides comprehensive knowledge of the significance of Par-4 and its association with kras status in PC, along with the crosstalk with crucial resistance and survival molecules NF-κB and Bcl-2 that ultimately are responsible for the overall poor outcome of different therapeutic approaches in this disease.Take home message: Par-4 holds promise as a potential therapeutic target that can be induced by chemopreventive agents and small-molecule inhibitors either alone or in combination with standard chemotherapeutics leading to selective apoptosis in PC cells. It also acts as a chemosensitizer and therefore warrants further clinical investigations in this disease.

Abstract 1209: Par-4 as therapy for colon cancer

Abstract Par-4 (Prostate apoptosis response-4) is a tumor suppressor that induces apoptosis in tumor cells in response to apoptotic stimuli. It has been reported to be reduced in a number of human cancers. We examined the expression levels of Par-4 in human colon cancers and explored the possibility of using Par-4 as a treatment for colon cancer. We found that expression of Par-4 is reduced in human colon cancers 2.65-fold compared to paired normal colon. To assess the role of Par-4 in colon cancer growth, the human colon cancer cell line, HT29, with low native Par-4 expression, was transfected to overexpress Par-4. It has been shown that apoptosis was higher in the transfected cells than in the mock transfected cells. It was also shown that tumors growing in nude mice were smaller when the cells were transfected to overexpress Par-4. Importantly for therapeutic considerations, it has been reported that Par-4 expressing cells can have a bystander effect on tumor cells that are not transfected, also inducing apoptosis in the untransfected cells. We hypothesized that HT29 colon cancer cells producing Par-4 in vivo would secrete Par-4 into the animal system and result in lower tumor volume of tumors created with wild type cells growing distally. Methods: To show proof of concept, nude mice were injected subcutaneously with wild type HT29 cells. Half of the mice were also injected with HT29 cells overexpressing Par-4 in the opposite flank. Mice were treated with 5-fluorouracil to test the sensitivity of tumors to the chemotherapy. Results: At the end of the first week, the wild type tumors in the mice with Par-4 tumors also were 1.8-fold smaller (p=0) than those tumors in mice who received only wild type tumor cell injections. Over the course of three weeks, the wild type tumors growing in mice without Par-4 tumors increased in volume 12-fold more than those wild type tumors growing in mice with Par-4 tumors. In addition, in the event that Par-4 tumors resolved completely (5% of the cases) the wild type tumors in those mice showed increased growth rates. This data suggests that overexpression of Par-4 in tumor cells in vivo not only sensitized those cells to chemotherapy, but had a bystander effect on tumor cells growing distally to the Par-4 overexpressing cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1209.

Par-4: A New Activator of Myosin Phosphatase

Myosin phosphatase (MP) is a key regulator of myosin light chain (LC20) phosphorylation, a process essential for motility, apoptosis, and smooth muscle contractility. Although MP inhibition is well studied, little is known about MP activation. We have recently demonstrated that prostate apoptosis response (Par)-4 modulates vascular smooth muscle contractility. Here, we test the hypothesis that Par-4 regulates MP activity directly. We show, by proximity ligation assays, surface plasmon resonance and coimmunoprecipitation, that Par-4 interacts with the targeting subunit of MP, MYPT1. Binding is mediated by the leucine zippers of MYPT1 and Par-4 and reduced by Par-4 phosphorylation. Overexpression of Par-4 leads to increased phosphatase activity of immunoprecipitated MP, whereas small interfering RNA knockdown of endogenous Par-4 significantly decreases MP activity and increases MYPT1 phosphorylation. LC20 phosphorylation assays demonstrate that overexpression of Par-4 reduces LC20 phosphorylation. In contrast, a phosphorylation site mutant, but not wild-type Par-4, interferes with zipper-interacting protein kinase (ZIPK)-mediated MP inhibition. We conclude from our results Par-4 operates through a "padlock" model in which binding of Par-4 to MYPT1 activates MP by blocking access to the inhibitory phosphorylation sites, and inhibitory phosphorylation of MYPT1 by ZIPK requires "unlocking" of Par-4 by phosphorylation and displacement of Par-4 from the MP complex.