pp32 (ANP32A) expression inhibits pancreatic cancer cell growth and induces gemcitabine resistance by disrupting HuR binding to mRNAs

The expression of protein phosphatase 32 (PP32, ANP32A) is low in poorly differentiated pancreatic cancers and is linked to the levels of HuR (ELAV1), a predictive marker for gemcitabine response. In pancreatic cancer cells, exogenous overexpression of pp32 inhibited cell growth, supporting its long-recognized role as a tumor suppressor in pancreatic cancer. In chemotherapeutic sensitivity screening assays, cells overexpressing pp32 were selectively resistant to the nucleoside analogs gemcitabine and cytarabine (ARA-C), but were sensitized to 5-fluorouracil; conversely, silencing pp32 in pancreatic cancer cells enhanced gemcitabine sensitivity. The cytoplasmic levels of pp32 increased after cancer cells are treated with certain stressors, including gemcitabine. pp32 overexpression reduced the association of HuR with the mRNA encoding the gemcitabine-metabolizing enzyme deoxycytidine kinase (dCK), causing a significant reduction in dCK protein levels. Similarly, ectopic pp32 expression caused a reduction in HuR binding of mRNAs encoding tumor-promoting proteins (e.g., VEGF and HuR), while silencing pp32 dramatically enhanced the binding of these mRNA targets. Low pp32 nuclear expression correlated with high-grade tumors and the presence of lymph node metastasis, as compared to patients' tumors with high nuclear pp32 expression. Although pp32 expression levels did not enhance the predictive power of cytoplasmic HuR status, nuclear pp32 levels and cytoplasmic HuR levels associated significantly in patient samples. Thus, we provide novel evidence that the tumor suppressor function of pp32 can be attributed to its ability to disrupt HuR binding to target mRNAs encoding key proteins for cancer cell survival and drug efficacy.     

pp32/PHAPI determines the apoptosis response of non-small-cell lung cancer

During malignant transformation, cancer cells have to evade cell-intrinsic tumor suppressor mechanisms including apoptosis, thus acquiring a phenotype that is relatively resistant to clinically applied anticancer therapies. Molecular characterization of apoptotic signal transduction defects may help to identify prognostic markers and to develop novel therapeutic strategies. To this end we have undertaken functional analyses of drug-induced apoptosis in human non-small cell-lung cancer (NSCLC) cells. We found that primary drug resistance correlated with defects in apoptosome-dependent caspase activation in vitro. While cytochrome c-induced apoptosome formation was maintained, the subsequent activation of caspase-9 and -3 was abolished in resistant NSCLC. The addition of recombinant pp32/putative human HLA class II-associated protein (pp32/PHAPI), described as a putative tumor suppressor in prostate cancer, successfully restored defective cytochrome c-induced caspase activation in vitro. Conditional expression of pp32/PHAPI sensitized NSCLC cells to apoptosis in vitro and in a murine tumor model in vivo. Immunohistochemical analyses of tumor samples from NSCLC patients revealed that the expression of pp32/PHAPI correlated with an improved outcome following chemotherapy. These results identify pp32/PHAPI as regulator of the apoptosis response of cancer cells in vitro and in vivo, and as a predictor of survival following chemotherapy for advanced NSCLC.     

pp32 overexpression induces nuclear pleomorphism in rat prostatic carcinoma cells

Abstract. Nuclear pleomorphism is an important diagnostic factor in tumour pathology. Traditionally, nuclear pleomorphism is evaluated qualitatively or semiquantitatively, often as a component of tumour grade; the molecular basis of nuclear pleomorphism, however, remains unclear. In this study, we investigated the quantitative effects on nuclear morphology of overexpressing pp32, a recently described nuclear phosphoprotein highly expressed in self-renewing and neoplastic cell populations. Assessment of Feulgen-stained transfected and control lines of AT3.1, a rat prostatic carcinoma cell line, using a computerized Cellular Image Analysis System (BD CAS-200) showed that stable overexpression of human pp32 in AT3.1 cells is accompanied by marked increases in the coefficient of variation of nuclear shape, nuclear size and chromatin textures but not in DNA content. In contrast, stable transfection with control vector, with ras , or with bcl-2 failed to affect nuclear morphology. Cell cycle analysis further showed that pp32-related increases in variation of nuclear structure manifested principally in G_1. These studies suggest that pp32 plays a role either directly or indirectly in the control of nuclear shape of G₁ cells.     

Identification of sequences required for inhibition of oncogene-mediated transformation by pp32.

Oncogenic potential in prostate cancer is modulated in part by alternative use of genes of the pp32 family. This family includes the tumor suppressor pp32, expressed in normal tissue, and the pro-oncogenic genes pp32r1 and pp32r2 that are found principally in neoplastic cells. At the protein level, pp32, pp32r1, and pp32r2 are approximately 90% identical, yet they subsume opposite functions. In this study, we identify the region of pp32 associated with the ability to inhibit oncogene-mediated transformation in a rat embryo fibroblast system, an in vitro correlate of tumor-suppressive activity. Deletion and truncation analysis define a region spanning pp32 amino acids 150-174 as absolutely required for inhibition of transformed foci elicited by RAS and MYC. Comparison of pp32 with the pp32r1 sequence by moving averages of sequence identity reveals divergence over this region; pp32r2 also differs in this region through truncation after pp32 amino acid 131. The deletion experiments and the experiments of nature therefore converge to demonstrate that tumor-suppressive functions of pp32 reside in amino acids 150-174. Identification of this minimal tumor-suppressive region should help elaborate the pathways and mechanisms through which pp32 family members exert their functions.     

Persistent Inhibition of ABL Tyrosine Kinase Causes Enhanced Apoptotic Response to TRAIL and Disrupts the Pro-Apoptotic Effect of Chloroquine

TNF-Related Apoptosis Inducing Ligand (TRAIL) binds to and activates death receptors to stimulate caspase-8 and apoptosis with higher efficiency in cancer than normal cells but the development of apoptosis resistance has limited its clinical efficacy. We found that stable, but not transient knockdown of the ABL tyrosine kinase enhanced the apoptotic response to TRAIL. Re-expression of Abl, but not its nuclear import- or kinase-defective mutant, in the ABL-knockdown cells re-established apoptosis suppression. TRAIL is known to stimulate caspase-8 ubiquitination (Ub-C8), which can facilitate caspase-8 activation or degradation by the lysosomes. In the ABL-knockdown cells, we found a higher basal level of Ub-C8 that was not further increased by lysosomal inhibition. Re-expression of Abl in the ABL-knockdown cells reduced the basal Ub-C8, correlating with apoptosis suppression. We found that lysosomal inhibition by chloroquine (CQ) could also enhance TRAIL-induced apoptosis. However, this pro-apoptotic effect of CQ was lost in the ABL-knockdown cells but restored by Abl re-expression. Interestingly, kinase inhibition at the time of TRAIL stimulation was not sufficient to enhance apoptosis. Instead, persistent treatment for several days with imatinib, an ABL kinase inhibitor, was required to cause the enhanced and the CQ-insensitive apoptotic response to TRAIL. Together, these results show that persistent loss of nuclear ABL tyrosine kinase function can sensitize cells to TRAIL and suggest that long-term exposure to the FDA-approved ABL kinase inhibitors may potentiate apoptotic response to TRAIL-based cancer therapy.     

Intracellular localization and activity state of tissue transglutaminase differentially impacts cell death

Tissue transglutaminase (tTG) is a unique member of the transglutaminase family as it is both a transamidating enzyme and a GTPase. In the cell tTG is mostly cytosolic, however it is also found in the nucleus and associated with the plasma membrane. tTG can be proapoptotic, however anti-apoptotic activities of the enzyme have also been reported. To determine how the intracellular localization and transamidating activity of tTG modulates its effects on apoptosis, HEK293 cells were transiently transfected with tTG or [C277S]tTG (which lacks transamidating activity) constructs that were targeted to different intracellular compartments. Apoptosis was induced by thapsigargin treatment, which results in increased intracellular calcium concentrations. Cytosolic tTG was pro-apoptotic, while nuclear localization of [C277S]tTG attenuated apoptosis. Membrane-targeted tTG had neither pro- nor anti-apoptotic functions. This finding indicates for the first time that intracellular localization is an important determinant of the effect of tTG on apoptosis. Previous studies have suggested that tTG may modulate retinoblastoma (Rb) protein, an important suppressor of apoptosis. tTG interacted with Rb and after induction of apoptosis, the interaction of nuclear-targeted [C277S]tTG with Rb was increased significantly concomitant with an attenuation of apoptosis. In contrast, the interaction of nuclear-targeted tTG with Rb was significantly decreased and apoptosis was not attenuated. These data suggest that tTG protects cells against apoptosis in response to stimuli that do not result in increased transamidating activity by translocating to the nucleus, and that complexing with Rb may be an important aspect of the protective effects of tTG.     

p32, a novel binding partner of Mcl-1, positively regulates mitochondrial Ca uptake and apoptosis

Mcl-1 is a major anti-apoptotic Bcl-2 family protein. It is well known that Mcl-1 can interact with certain pro-apoptotic Bcl-2 family proteins in normal cells to neutralize their pro-apoptotic functions, thus prevent apoptosis. In addition, it was recently found that Mcl-1 can also inhibit mitochondrial calcium uptake. The detailed mechanism, however, is still not clear. Based on Yeast Two-Hybrid screening and co-immunoprecipitation, we identified a mitochondrial protein p32 (C1qbp) as a novel binding partner of Mcl-1. We found that p32 had a number of interesting properties: (1) p32 can positively regulate UV-induced apoptosis in HeLa cells. (2) Over-expressing p32 could significantly promote mitochondrial calcium uptake, while silencing p32 by siRNA suppressed it. (3) In p32 knockdown cells, Ruthenium Red treatment (an inhibitor of mitochondrial calcium uniporter) showed no further suppressive effect on mitochondrial calcium uptake. In addition, in Ruthenium Red treated cells, Mcl-1 also failed to suppress mitochondrial calcium uptake. Taken together, our findings suggest that p32 is part of the putative mitochondrial uniporter that facilitates mitochondrial calcium uptake. By binding to p32, Mcl-1 can interfere with the uniporter function, thus inhibit the mitochondrial Ca²⁺ uploading. This may provide a novel mechanism to explain the anti-apoptotic function of Mcl-1.     

Cytochrome c activation of CPP32-like proteolysis plays a critical role in a Xenopus cell-free apoptosis system.

In a cell-free system based on Xenopus egg extracts, Bcl-2 blocks apoptotic activity by preventing cytochrome c release from mitochondria. We now describe in detail the crucial role of cytochrome c in this system. The mitochondrial fraction, when incubated with cytosol, releases cytochrome c. Cytochrome c in turn induces the activation of protease(s) resembling caspase-3 (CPP32), leading to downstream apoptotic events, including the cleavage of fodrin and lamin B1. CPP32-like protease activity plays an essential role in this system, as the caspase inhibitor, Ac-DEVD-CHO, strongly inhibited fodrin and lamin B1 cleavage, as well as nuclear morphology changes. Cytochrome c preparations from various vertebrate species, but not from Saccharomyces cerevisiae, were able to initiate all signs of apoptosis. Cytochrome c by itself was unable to process the precursor form of CPP32; the presence of cytosol was required. The electron transport activity of cytochrome c is not required for its pro-apoptotic function, as Cu- and Zn-substituted cytochrome c had strong pro-apoptotic activity, despite being redox-inactive. However, certain structural features of the molecule were required for this activity. Thus, in the Xenopus cell-free system, cytosol-dependent mitochondrial release of cytochrome c induces apoptosis by activating CPP32-like caspases, via unknown cytosolic factors.     

Apoptosis and restriction of G(1)/S cell cycle by fenretinide in Burkitt's lymphoma mutu I cell line accessed with bcl-6 down-regulation

Fenretinide (4-HPR) is a synthetic retinoid with cancer chemopreventative potential and clinically manageable side effects, compared to the prototype retinoid, all-trans retinoic acid (RA). 4-HPR has been shown to modulate cell proliferation and induce apoptosis in a variety of human tumor cell types, but its effects on B-cell non-Hodgkin's lymphomas (NHL-B) have not been explored. Treatment of Burkitt's lymphoma Mutu I cells with 3 microM 4-HPR is accompanied by growth arrest, induction of apoptosis, and restricted progression of the cell cycle at the G(1)/S checkpoint. We also observed that 4-HPR elicited a reduced expression of bcl-6 in these cells, which supports the proposed role of bcl-6 as an anti-apoptotic gene. While 4-HPR treatment had no effect on total Rb gene expression, it significantly reduced the state of hyperphosphorylation of Rb, resulting in the predominant existence of Rb in the underphosphorylated state.     

The retinoblastoma protein (Rb) as an anti-apoptotic factor: expression of Rb is required for the anti-apoptotic function of BAG-1 protein in colorectal tumour cells

Although the retinoblastoma-susceptibility gene RB1 is inactivated in a wide range of human tumours, in colorectal cancer, the retinoblastoma protein (Rb) function is often preserved and the RB locus even amplified. Importantly, we have previously shown that Rb interacts with the anti-apoptotic Bcl-2 associated athanogene 1 (BAG-1) protein, which is highly expressed in colorectal carcinogenesis. Here we show for the first time that Rb expression is critical for BAG-1 anti-apoptotic activity in colorectal tumour cells. We demonstrate that Rb expression not only increases the nuclear localisation of the anti-apoptotic BAG-1 protein, but that expression of Rb is required for inhibition of apoptosis by BAG-1 both in a γ-irradiated Saos-2 osteosarcoma cell line and colorectal adenoma and carcinoma cell lines. Further, consistent with the fact that nuclear BAG-1 has previously been shown to promote cell survival through increasing nuclear factor (NF)-κB activity, we demonstrate that the ability of BAG-1 to promote NF-κB activity is significantly inhibited by repression of Rb expression. Taken together, data presented suggest a novel function for Rb, promoting cell survival through regulating the function of BAG-1. As BAG-1 is highly expressed in the majority of colorectal tumours, targeting the Rb–BAG-1 complex to promote apoptosis has exciting potential for future therapeutic development.     

Modulation of oncogenic potential by alternative gene use in human prostate cancer

Only a small percentage of primary prostate cancers have genetic changes. In contrast, nearly 90% of clinically significant human prostate cancers seems to express high levels of the nuclear phosphoprotein pp32 by in situ hybridization. Because pp32 inhibits oncogene-mediated transformation, we investigated its paradoxical expression in cancer by comparing the sequence and function of pp32 species from paired benign prostate tissue and adjacent prostatic carcinoma from three patients. Here we demonstrate that pp32 is expressed in benign prostatic tissue, but pp32r1 and pp32r2, closely-related genes located on different chromosomes, are expressed in prostate cancer. Although pp32 is a tumor suppressor, pp32r1 and pp32r2 are tumorigenic. Alternative use of the pp32, pp32r1 and pp32r2 genes may modulate the oncogenic potential of human prostate cancer.     

Adenovirus E4orf6 targets pp32/LANP to control the fate of ARE-containing mRNAs by perturbing the CRM1-dependent mechanism

E4orf6 plays an important role in the transportation of cellular and viral mRNAs and is known as an oncogene product of adenovirus. Here, we show that E4orf6 interacts with pp32/leucine-rich acidic nuclear protein (LANP). E4orf6 exports pp32/LANP from the nucleus to the cytoplasm with its binding partner, HuR, which binds to an AU-rich element (ARE) present within many protooncogene and cytokine mRNAs. We found that ARE-mRNAs, such as c-fos, c-myc, and cyclooxygenase-2, were also exported to and stabilized in the cytoplasm of E4orf6-expressing cells. The oncodomain of E4orf6 was necessary for both binding to pp32/LANP and effect for ARE-mRNA. C-fos mRNA was exported together with E4orf6, E1B-55kD, pp32/LANP, and HuR proteins. Moreover, inhibition of the CRM1-dependent export pathway failed to block the export of ARE-mRNAs mediated by E4orf6. Thus, E4orf6 interacts with pp32/LANP to modulate the fate of ARE-mRNAs by altering the CRM1-dependent export pathway.     

Resveratrol causes COX-2- and p53-dependent apoptosis in head and neck squamous cell cancer cells

Cyclooxygenase-2 (COX-2) content is increased in many types of tumor cells. We have investigated the mechanism by which resveratrol, a stilbene that is pro-apoptotic in many tumor cell lines, causes apoptosis in human head and neck squamous cell carcinoma UMSCC-22B cells by a mechanism involving cellular COX-2. UMSCC-22B cells treated with resveratrol for 24 h, with or without selected inhibitors, were examined: (1) for the presence of nuclear activated ERK1/2, p53 and COX-2, (2) for evidence of apoptosis, and (3) by chromatin immunoprecipitation to demonstrate p53 binding to the p21 promoter. Stilbene-induced apoptosis was concentration-dependent, and associated with ERK1/2 activation, serine-15 p53 phosphorylation and nuclear accumulation of these proteins. These effects were blocked by inhibition of either ERK1/2 or p53 activation. Resveratrol also caused p53 binding to the p21 promoter and increased abundance of COX-2 protein in UMSCC-22B cell nuclei. Resveratrol-induced nuclear COX-2 accumulation was dependent upon ERK1/2 activation, but not p53 activation. Activation of p53 and p53-dependent apoptosis were blocked by the COX-2 inhibitor, NS398, and by transfection of cells with COX-2-siRNA. In UMSCC-22B cells, resveratrol-induced apoptosis and induction of nuclear COX-2 accumulation share dependence on the ERK1/2 signal transduction pathway. Resveratrol-inducible nuclear accumulation of COX-2 is essential for p53 activation and p53-dependent apoptosis in these cancer cells.     

Review of <Emphasis Type="Italic">Molecular Biology of Human Cancers</Emphasis> (<Emphasis Type="Italic">An Advanced Student’s Text</Emphasis>, by Wolfgang Schulz (Department of Urology and Center for Biological and Medical Research,Heinrich Heine University,Düsseldorf) ISBN 1-4020-3185-8

Postmenopausal women with estrogen receptor positive (ER⁺) breast cancer frequently respond paradoxically to estrogen administration with tumor regression. Using both LTED and E8CASS cells derived from MCF-7 breast cancer cells by long-term estrogen-deprivation, we previously reported that 17 -estradiol (estradiol) is a powerful, pro-apoptotic hormone which kills the cancer cells through activation of the Fas/FasL death receptor pathway. We postulated that the mitochondrial interactive protein Bcl-2 might play a role in the regulation of estradiol-induced apoptosis in both LTED and E8CASS cells. In this study, we assessed estradiol effects on cell growth, proliferation and apoptosis. Additionally we investigated the effect of estradiol on caspase activation, NF-KB and Bcl-2 expression. The functional role of Bcl-2 in estradiol-induced apoptosis was further studied by knockdown or decrease of Bcl-2 with siRNA. Our results show that estradiol significantly inhibited cell growth primarily through a pro-apoptotic action involving caspase-7 and 9 activations (p < 0.01). Basal Bcl-2 and NF-KB levels were greatly elevated and estradiol decreased NF-KB, but not Bcl-2 expression. Knockdown of Bcl-2 expression with siRNA decreased the levels of this protein by 9 fold (p < 0.01). This reduction markedly sensitized both LTED and E8CASS cells to the pro-apoptotic action of estradiol, leading to a synergistic induction of apoptosis and a concomitant reduction in cell number (p < 0.01). Therefore, down-regulation of Bcl-2 synergistically enhanced estradiol-induced apoptosis in ER⁺ postmenopausal breast cancer cells.     

Avian Retrovirus pp32 DNA-Binding Protein I. Recognition of Specific Sequences on Retrovirus DNA Terminal Repeats

The avian retrovirus pp32 protein possesses a DNA-nicking activity which prefers supercoiled DNA as substrate. We have investigated the binding of pp32 to avian retrovirus long terminal repeat (LTR) DNA present in both supercoiled and linear forms. The cloned viral DNA was derived from unintegrated Schmidt-Ruppin A (SRA) DNA. A subclone of the viral DNA in pBR322 (termed pPvuII-DG) contains some src sequences, tandem copies of LTR sequences, and partial gag sequences in the order src-U(3) U(5):U(3) U(5)-gag. Binding of pp32 to supercoiled pPvuII-DG DNA followed by digestion of this complex with a multicut restriction enzyme (28 fragments total) permitted pp32 to preferentially retain on nitrocellulose filters two viral DNA fragments containing only LTR DNA sequences. In addition, pp32 also preferentially retained four plasmid DNA fragments containing either potential promoters or Tn3 "left-end" inverted repeat sequences. Mapping of the pp32 binding sites on viral LTR DNA was accomplished by using the DNase I footprinting technique. The pp32 protein, but not the avian retrovirus alphabeta DNA polymerase, is able to form a unique protein-DNA complex with selected regions of either SRA or Prague A LTR DNAs. Partial DNase I digestion of a 275-base pair SRA DNA fragment complexed with pp32 gives upon electrophoresis in denaturing gels a unique ladder pattern, with regions of diminished DNase I susceptibility from 6 to 10 nucleotides in length, in comparison with control digests in the absence of protein. The binding of pp32 to this fragment also yields enhanced DNase I-susceptible sites that are spaced between the areas protected from DNase I digestion. The protected region of this unique complex was a stretch of 170 +/- 10 nucleotides that encompasses the presumed viral promoter site in U(3), which is adjacent to the src region, extends through U(5), and proceeds past the joint into U(3) for about 34 base pairs. No specific protection or DNase I enhancement by pp32 was observed in experiments with a 435-base pair SRA DNA fragment derived from a part of U(3) and the adjacent src region or a 55-base pair DNA fragment derived from another part of U(3). The DNA sequence of Prague A DNA at the fused LTRs differs from that of SRA DNA. The alteration in the sequence at the juncture of the LTRs prevented pp32 from forming a stable complex in this region of the LTR. Our results are relevant to two aspects of the interaction between pp32 and LTR DNA. First, the pp32 protein in the presence of selected viral DNA restriction fragments possibly forms a higher order oligomer analogous to Escherichia coli DNA gyrase-DNA complexes or eucaryotic nucleosome structures. Second, the specificity of the binding suggests a role for pp32 and the protected DNA sequences in the retrovirus life cycle. The preferred sequences to which pp32 binds include two adjacent 15-base pair inverted terminal repeats at the joint between U(5) and U(3) in SRA DNA. This region is involved in circularization of linear DNA and is perhaps the site that directs integration into cellular DNA.