Pro-apoptotic Function Of Par-4 Research Articles

Abstract 1042: Activation of AKT negates the pro-apoptotic function of Par-4 in castration resistant prostate cancer cells

Abstract Activation of AKT signaling pathway is a common aberrations in advanced castration resistant prostate cancer (CRPC). PTEN, a tumor suppressor is known to negatively regulate AKT function, however, in most cancer deletion or mutation of PTEN facilitates AKT mediated survival signaling in most cancer types. In this study, we have determined the clinical and pathological significance of AKT signaling in CaP tissues and CRPC cell lines. AKT and pAKT (ser473) were measured in prostate tumor tissues (Normal = 23, Grade I=13, Grade II-24 and Grade III=26) by immunohistochemistry analysis. Phosphorylated AKT is significantly higher in tumors (Grades-I, II and III) as compared to the normal/hyperplastic tissues; however, there no significant differences were seen in AKT expression between normal and tumor tissues. In CRPC cell lines, stable expression of AKT increases proliferation, epithelial mesenchymal transition and angiogenesis in cancer cells. Injection of over expressed AKT, CRPC cells resulted in tumor growth in athymic mice as compared to vector cells. We observed AKT tumors grow rapidly, are larger in size and showed 100% tumorigenecity as compared to the control tumors (40%), which had slow growth, as well as small tumors. Interestingly, subcutaneous injection of AKT/CRPC cells in athymic mice showed hyperplasia in anterior and a ventral lateral lobe of the prostate, which was absent in control tumors. We also found that AKT tumors are highly vascularized than the control tumors; however no micrometastasis were observed in our H &E analysis. Our in vitro studies have revealed that treatment of Withaferin-A, an herbal compound inhibits AKT activation and up regulates Par-4 mediated apoptosis by activating caspase cascades in CRPC cells. In addition, WA overcomes AKT induced resistance in CRPC cells and restores Par-4 function that resulted in growth inhibition in AKT over expressed CRPC (DU-145 and C4-2B) cells. Similarly, oral administration of WA (4mg/kg) significantly inhibited the growth of both control and AKT over expressed tumors (P< 0.001). Currently, we are observing tumor regrowth after stopping the WA treatment and immunohistochemistry analysis to confirm AKT mediated pro-survival signaling and EMT markers in the tumor tissues. These finding suggest WA could be a potent agent which overrides AKT mediated pro-survival and EMT signaling in CRPC cells, hence it can be used as a chemopreventive and/or chemotherapeutic agent for highly aggressive and metastatic prostate cancer. Note: This abstract was not presented at the meeting. Citation Format: Trinath P. Das, Suman Suman, Chendil Damodaran. Activation of AKT negates the pro-apoptotic function of Par-4 in castration resistant prostate cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1042. doi:10.1158/1538-7445.AM2014-1042

Abstract 5457: FOXO3a transcriptionally regulates pro-apoptotic function of Par-4 and induces apoptosis in prostate cancer cells.

Abstract Deletion and mutation of tumor suppressor genes (PTEN and p53) may play an important role in the carcinogenesis of prostate cancer (CaP). Constitutive activation of Akt promotes cell proliferation and survival by regulating a number of transcription factors/genes in many cancer cells including CaP. Activation of AKT negatively regulates the pro-apoptotic functions of Fork head transcription factor (FOXO3a) and prostate apoptosis response-4 (Par-4) in CaP cells. In our studies, we found a molecular interplay between FOXO3a and Par-4 in CaP cells and more precisely, how, FOXO3a regulates Par-4 function in CaP cells. Knockin and knockout assays in CaP cells suggest that FOXO3a is upstream of Par-4 and FOXO3a may responsible for the transactivation of Par-4 and its pro-apoptotic function in CaP cells. Further, Immunofluorescence assays suggest during apoptotic stimuli, Par-4 and FOXO3A colocalization in nucleus, which in turn initiates pro-apoptotic signaling and induction of apoptosis. These results were further confirmed by pull down assays, which supports the physical interaction of these two proteins. These findings encouraged us to explore the FOXO3a binding sites in the promoter region of Par-4. We identified four FOXO3a binding sites at -2075 to -2069, -1810 to -1804, -1561 to -1555, and -1340 to -1334 in the promoter region of Par-4 gene. Luciferase reporter constructs of Par-4 promoter (2.1 kb) spanning the FOXO3a binding sites and sequentially deleted constructs were utilized to identify the crucial FOXO3a binding site for the regulation of Par-4. Sequential deletion of FOXO3a binding sites in Par-4 promoter resulted in inhibition of Par-4 function and induction of apoptosis in CaP cells. Further, confirm the role of FOXO3a, we specifically activated Par-4 function by small molecules and simultaneously co-transfect with dominant-negative FOXO3a constructs. As expected dominant negative FOXO3a abrogates Par-4 function in CaP cells, confirming FOXO3a is an upstream of Par-4 signaling and it is required for Par-4 induced apoptosis in CaP cells. We believe, these results decipher the mechanisms associated with FOXO3a induced Par-4 gene activation and apoptosis in CaP cells and activating of FOXO3a may result Par-4 mediated apoptosis in CaP cells. Citation Format: Trinath P. Das, Chendil Damodaran. FOXO3a transcriptionally regulates pro-apoptotic function of Par-4 and induces apoptosis in prostate cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5457. doi:10.1158/1538-7445.AM2013-5457 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Targeted Ablation of Par-4 Reveals a Cell Type–Specific Susceptibility to Apoptosis-Inducing Agents

The prostate apoptosis response-4 (Par-4) protein has been shown to function as an effector of cell death in response to various apoptotic stimuli, and down-regulation of this protein has been suggested to be a key event during tumorigenesis. Several studies suggest an essential function for the COOH-terminal leucine repeats/death domain of Par-4 in mediating apoptosis. We investigated the biological role of this domain in vivo by generating knock-out mice expressing a Par-4 mutant protein lacking the COOH terminus domain. We found that the Par-4 mutant mice are viable and fertile with no overt phenotype, thus excluding an essential role for the COOH terminus domain of Par-4 in embryogenesis and developmental apoptosis. To determine the requirement of Par-4 for apoptosis, we treated primary fibroblasts with various stimuli that trigger mitochondria and membrane receptor cell death pathways. Fibroblasts isolated from Par-4 mutant mice are as sensitive as the wild-type cells to these apoptosis-inducing agents. Similar effects were observed following RNA interference (RNAi)-mediated knockdown of Par-4 in these cells. In contrast, RNAi-mediated depletion of Par-4 in HeLa cells resulted in a significant inhibition of apoptosis induced by various proapoptotic agents. Taken together, our findings provide strong genetic evidence that the proapoptotic function of Par-4 is dependent on the cellular context and raise the possibility that alterations of Par-4 function may occur during carcinogenesis.

Ectopic expression of Par-4 leads to induction of apoptosis in CNS tumor cell lines

Prostate apoptosis response-4 (Par-4) is a pro-apoptotic protein originally identified as a gene product upregulated in prostate tumor cells undergoing apoptosis. Down-regulation of Par-4 has been linked to several cancers. Since Par-4 also plays a crucial role in neuronal apoptosis, we investigated the expression of Par-4 in tumor cell lines derived from representative tumor types of the CNS, including primitive neuroectodermal tumor (PNET), medulloblastoma, neuroblastoma and glioma of human, rat and murine origin. We show that Par-4 is frequently down-regulated, either transcriptionally or post-transcriptionally in the CNS tumor cell lines. Moreover, we demonstrate that ectopic expression of Par-4 is sufficient to directly induce apoptosis in these CNS tumor cells, in contrast to other cancer cells where replenishment of Par-4 levels only sensitizes the cells to apoptotic stimuli. Induction of apoptosis by Par-4 in the neural tumor cell lines is independent of endogenous Bcl-2 levels and PKCzeta activity, although it has been proposed that Par-4 can exert its pro-apoptotic function by down-modulation of Bcl2 expression and inhibition of PKCzeta. Co-expression of Par-4 and a dominant-negative mutant of FADD resulted in a slight reduction of apoptosis in some tumor cell lines, indicating that Par-4 may partially induce apoptosis via the Fas death pathway. Furthermore, these data suggested that the pro-apoptotic function of Par-4 involves (an)other yet unidentified apoptotic pathway(s) in the CNS tumor cell lines. Since Par-4 by itself is not sufficient to induce apoptosis in non-tumor cells, reintroduction of Par-4 into primary CNS tumors or reactivation of the pathways of Par-4-mediated apoptosis represent promising targets in anti-tumor therapy.

Pro-apoptotic action of PAR-4 involves inhibition of NF-kappaB activity and suppression of BCL-2 expression.

Par-4(1) (prostate apoptosis response 4) is known to function at an early stage in apoptosis in several different cell types, including neurons. On the other hand, activation of the transcription factor NF-kappaB can prevent apoptosis in various cancer cells and neurons. We now report that overexpression of full-length Par-4 in cultured PC12 cells results in a suppression of basal NF-kappaB DNA-binding activity and NF-kappaB activation following trophic factor withdrawal (TFW). The decreased NF-kappaB activity is correlated with enhanced apoptosis. Conversely, NF-kappaB activity is increased and vulnerability to apoptosis reduced in cells overexpressing a dominant-negative form of Par-4. Par-4 overexpression or functional blockade had no effect on AP-1 DNA-binding activity. Expression of the antiapoptotic protein Bcl-2 was dramatically reduced in PC12 cells overexpressing Par-4. Our data suggest that suppression of NF-kappaB activation plays a major role in the proapoptotic function of Par-4.

Apoptosis mediated by a novel leucine zipper protein Par-4.

The prostate apoptosis response-4 (par-4) gene was isolated in a differential screen for immediate-early genes that are up-regulated during apoptosis of prostate cancer cells. Unlike most other immediate-early genes, par-4 is exclusively induced during apoptosis. The expression or induction of par-4 is not restricted to prostatic cells. The par-4 gene is widely expressed in diverse normal tissues and cell types and conserved during evolution. Par-4 protein contains a leucine zipper domain that is essential for sensitization of cells to apoptosis. Functional studies indicate that par-4 expression is necessary to induce apoptosis. Par-4 protein may induce apoptosis by a p53-independent pathway that involves cytoplasmic inactivation of atypical protein kinase C isoforms resulting in down-regulation of MAP kinase activity and an up-regulation of p38 kinase activity. However, Par-4 is detected in the cytoplasm and in the nucleus, suggesting both cytoplasmic and nuclear roles for the pro-apoptotic protein. Interestingly, Par-4 is predicted to contain a death domain homologous to that of Fas or TRADD, and may therefore trigger a death cascade analogous to that of the death domain proteins. Par-4-dependent apoptosis is abrogated by Bcl-2 and by caspase inhibitors. Identification of the components of the p53-independent apoptosis pathway induced by Par-4 may help to further elucidate the mechanism of Par-4 action. Moreover, in view of the pro-apoptotic function of Par-4, its role in diseases, such as cancer and neurogenerative disorders, whose pathophysiology involves apoptotic cell death needs further investigation.