Apoptosis-stimulating Protein Of P53-2 Research Articles

Enhancing ASPP2 promotes acute liver injury via an inflammatory immunoregulatory mechanism.

Acute liver injury (ALI), which is a type of inflammation-mediated hepatocellular injury, is a clinical syndrome that results from hepatocellular apoptosis and hemorrhagic necrosis. Apoptosis stimulating protein of p53-2 (ASPP2) is a proapoptotic member of the p53 binding protein family. However, the role of ASPP2 in the pathogenesis of ALI and its regulatory mechanisms remain unclear. The expression of ASPP2 were compared between liver biopsies derived from patients with CHB, patients with ALI, and normal controls. Acute liver injury was modelled in mice by administration of D-GalN/LPS. Liver injury was demonstrated by serum transaminases and histological assessment of liver sections. ASPP2-knockdown mice (ASPP2+/-) were used to determine its role in acute liver injury. Mouse bone marrow macrophages (BMMs) were isolated from wildtype and ASPP2+/- mice and stimulated with LPS, and the supernatant was collected to incubate with the primary hepatocytes. Quantitative real-time PCR and western blot were used to analyze the expression level of target. The expression of ASPP2 was significantly upregulated in the liver tissue of ALI patients and acute liver injury mice. ASPP2+/- mice significantly relieved liver injury through reducing liver inflammation and decreasing hepatocyte apoptosis. Moreover, the conditioned medium (CM) of ASPP2+/- bone marrow-derived macrophages (BMMs) protected hepatocytes against apoptosis. Mechanistically, we revealed that ASPP2 deficiency in BMMs specifically upregulated IL-6 through autophagy activation, which decreased the level of TNF-α to reduce hepatocytes apoptosis. Furthermore, up-regulation of ASPP2 sensitizes hepatocytes to TNF-α-induced apoptosis. Our novel findings show the critical role of ASPP2 in inflammatory immunoregulatory mechanism of ALI and provide a rationale to target ASPP2 as a refined therapeutic strategy to ameliorate acute liver injury.

RDIVpSGP motif of ASPP2 binds to 14-3-3 and enhances ASPP2/k18/14-3-3 ternary complex formulation to promote BRAF/MEK/ERK signal inhibited cell proliferation in hepatocellular carcinoma.

The Apoptosis Stimulating Protein of p53 2 (ASPP2) is a heterozygous insufficient tumor suppressor; however, its molecular mechanism(s) in tumor suppression is not completely understood. ASPP2 plays an essential role in cell growth, as shown by liver hepatocellular carcinoma (LIHC) RNA-seq assay using the Cancer Genome Atlas (TCGA) and High-Throughput-PCR assay using ASPP2 knockdown cells. These observations were further confirmed by in vivo and in vitro experiments. Mechanistically, N-terminus ASPP2 interacted with Keratin 18 (k18) in vivo and in vitro. Interestingly, the RDIVpSGP motif of ASPP2 associates with 14-3-3 and promotes ASPP2/k18/14-3-3 ternary-complex formation which promotes MEK/ERK signal activation by impairing 14-3-3 and BRAF association. Additionally, ASPP2-rAd injection promotes paclitaxel-suppressed tumor growth by suppressing cell proliferation in the BALB/c nude mice model. ASPP2 and k18 were preferentially downregulated in Hepatocellular Carcinoma (HCC), which predicted poor prognosis in HCC patients. Overall, these findings suggested that ASPP2 promoted BRAF/MEK/ERK signal activation by promoting the formation of an ASPP2/k18/14-3-3 ternary complex via the RDIVpSGP motif at the N terminus. Moreover, this study provides novel insights into the molecular mechanism of tumor suppression in HCC patients.

ASPP2 k, a Dominant-Negative Splicing Variant of the Apoptosis-Stimulating Protein of p53-2 (ASPP2), Modulates Treatment Response Towards BCL-Signaling Inhibitors in Acute Myeloid Leukemia

In recent years, it has become increasingly apparent that BCL-2 inhibition in AML is a clinically highly effective approach - and the first BCL-2 inhibitor, venetoclax, has gained FDA and EMA approval. However, drug resistance frequently occurs and mechanisms or biological markers to predict response to BCL-2 inhibition are urgently needed. ASPP2 plays a central role to orchestrate induction of apoptosis via binding of p53 - but also via binding of antiapoptotic BCL-2, allowing release of proapoptotic proteins. We recently described an alternatively spliced oncogenic ASPP2 isoform (ASPP2k) (Schittenhelm et al., 2019), which is characterized by truncation of the C-terminus allocating the BCL2- as well as the p53 binding sites (in analogy to major TP53 mutations lacking the ASPP2 binding motifs). We therefore hypothesized that expression of ASPP2k attenuates efficacy of pro-apoptotic compounds:AML cell line models, including MOLM13/14 (FLT3 ITD+), OCI-AML3 (NPM1 A+) and HL60, as well as freshly isolated native leukemia blasts (n=40) are used in dose-dilution assays to assess for pro-apoptotic efficacy in annexin V-based assays. Bone marrow donors served as a control population. A lentiviral approach was used for isoform-specific ASPP2k-shRNA transduction. A HisMax vector was used to forcely express ASPP2k. Several compounds targeting BCL-2 signaling, which are under clinical investigation, were tested (BCL-2: venetoclax, BCL-2/Xl: AZD4320, MCL-1: AZD5991 and CDK9: AZD4573).To summarize, leukemia cells demonstrated variable and preferential sensitivity profiles towards the tested compounds: MOLM cells were sensitive towards all tested compounds. In contrast, OCI-AML3 proofed reduced sensitivity towards BCL-2/Xl inhibition - whereas MCL-1 and CDK9 inhibition (indirectly targeting MCL-1 signaling) showed IC50s in the nanomolar range (AZD5991: 307nM, AZD4573: 16nM). Furthermore, HL60 were relatively resistant towards both BCL-2/Xl and MCL-1 inhibition - however remained high sensitivity towards CDK9 inhibition (IC50 25nM). Priming with a hypomethylating agent (HMA, decitabine) resulted in additive (CI close to 1) to synergistic (CI 0.25 - 0.7) proapoptotic effects in isobologram analysis and led to a release of drug resistance in primary resistant cell lines.Exposure of native leukemia cells towards all inhibitors confirmed (individually differing) sensitivity in the nanomolar ranges - whereas bone marrow donor controls were relatively resistant towards the tested compounds, which argues for a therapeutic clinical window.In a last step, we addressed, whether ASPP2k functionally impedes the proapoptotic efficacy observed for the tested compounds. Indeed, we demonstrate that isoform-specific ASPP2k interference similarly results in a significant increase of pro-apoptotic capacities of all tested BCL-2, BCL-2/XL, MCL-1 and CDK9 inhibitors, (resp. attenuation thereof after forcely expressing the dominant-negative splicing variant).To summarize, we show that inhibition of BCL-2 signaling is a promising approach to target acute leukemia - as a monoagent as well as in combination with HMA: Further, we provide a path for exploration of ASPP2k as a predictive tool as well as a therapeutic sensitizer of pro-apoptotic drugs, which will be addressed in future studies. DisclosuresSchittenhelm: BMS: Other: advisory board; Astellas: Other: advisory board; Takeda: Other: advisory board; University of Tuebingen: Patents & Royalties: patent for ASPP2k. Kampa-Schittenhelm: University of Tuebingen: Patents & Royalties: patent related to ASPP2k.

ASPP2 inhibits hepatitis B virus replication by preventing nucleus translocation of HSF1 and attenuating the transactivation of ATG7.

Hepatitis B virus (HBV) is a kind of virus with the capability to induce autophagy, thereby facilitating its replication. Reducing hepatocyte autophagy is proved to be a useful way to inhibit HBV replication. Herein, we reported that p53‐binding protein 2 (apoptosis‐stimulating protein of p53‐2, ASPP2) could attenuate HBV‐induced hepatocyte autophagy in a p53‐independent manner. Mechanistically, overexpressed ASPP2 binds to HSF1 in cytoplasm of HBV‐infected cells, which prevents the translocation of HSF1 to nuclei, thereby inhibiting the transactivation of Atg7. By regulating the transcription of Atg7, ASPP2 reduces hepatocyte autophagy, thereby inhibiting HBV replication. Therefore, ASPP2 is a key regulator of cell autophagy, and overexpression of ASPP2 could be a novel method to inhibit HBV replication in hepatocytes.

CagA–ASPP2 complex mediates loss of cell polarity and favors H. pylori colonization of human gastric organoids

The main risk factor for stomach cancer, the third most common cause of cancer death worldwide, is infection with Helicobacter pylori bacterial strains that inject cytotoxin-associated gene A (CagA). As the first described bacterial oncoprotein, CagA causes gastric epithelial cell transformation by promoting an epithelial-to-mesenchymal transition (EMT)-like phenotype that disrupts junctions and enhances motility and invasiveness of the infected cells. However, the mechanism by which CagA disrupts gastric epithelial cell polarity to achieve its oncogenicity is not fully understood. Here we found that the apoptosis-stimulating protein of p53 2 (ASPP2), a host tumor suppressor and an important CagA target, contributes to the survival of cagA-positive H. pylori in the lumen of infected gastric organoids. Mechanistically, the CagA-ASPP2 interaction is a key event that promotes remodeling of the partitioning-defective (PAR) polarity complex and leads to loss of cell polarity of infected cells. Blockade of cagA-positive H. pylori ASPP2 signaling by inhibitors of the EGFR (epidermal growth factor receptor) signaling pathway-identified by a high-content imaging screen-or by a CagA-binding ASPP2 peptide, prevents the loss of cell polarity and decreases the survival of H. pylori in infected organoids. These findings suggest that maintaining the host cell-polarity barrier would reduce the detrimental consequences of infection by pathogenic bacteria, such as H. pylori, that exploit the epithelial mucosal surface to colonize the host environment.

HDAC1-induced epigenetic silencing of ASPP2 promotes cell motility, tumour growth and drug resistance in renal cell carcinoma

Renal cell carcinoma (RCC) is highly resistant to chemotherapies. The lack of efficacious treatment for metastatic RCC has led to a poor 5-year survival rate. Here, we found that Apoptosis-stimulating protein of p53-2(ASPP2) was frequently decreased in primary RCC tissues in comparison with non-tumoural kidney controls. Decreased ASPP2 was correlated with high grades and poor outcomes of RCC. Further studies revealed that ASPP2 downregulation promoted EMT and increased resistance to 5-Fluorouracil (5-FU)-induced apoptosis. To this end, the regulatory mechanisms of ASPP2 were further explored. Our data revealed that ASPP2 was inhibited by histone deacetylatlase 1 (HDAC1), which acted by preventing the binding between transcription factor (E2F1) and the ASPP2 promoter. Of particular importance, HDAC1 inhibitor vorinostat restored ASPP2 transcription and produced a synergistic effect with 5-FU in elevating ASPP2, promoting apoptosis and inhibiting EMT in both in vitro and in vivo RCC models. In summary, our data not only highlight an important role of ASPP2 in RCC progression and drug resistance, but also reveal new regulatory mechanisms of ASPP2, which provides important insights into novel treatment strategies by targeting ASPP2 dysregulation in RCC.

Expression and significance of ASPP2 in squamous carcinoma of esophagus

To study the significance of apoptosis stimulating protein of P53 2 (ASPP2) expression in esophageal squamous cell carcinoma (ESCC), immunohistochemistry S-P method was used to examine the expression of ASPP2 in 136 cases of ESCC, 35 cases of high grade intraepithelial neoplasia (HGIN), 29 cases of low grade intraepithelial neoplasia (LGIN) and 37 cases of normal esophageal epithelium (NEE). The associations of ASPP2 expression with clinicopathological data and overall survival (OS) were also analyzed. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to evaluate ASPP2 expression in a total of 20 matched human ESCC tumor tissues and normal adjacent tissues (NAT). In addition, EC109 cells were treated with cisplatin (CDDP) in vitro for 24 h (the intervention group) and the control group was set up at the same time. Western blot was used to examine the expression of ASPP2 protein between the two groups. The expression of ASPP2 decreased progressively from NEE to LGIN, to HGIN, and to ESCC, and it was related to TNM stage, histological differentiation and lymph node metastasis in ESCC (P < 0.05). ASPP2 was a protective factor of patients with ESCC (P = 0.008). The relative expression of ASPP2 mRNA was markedly downregulated in ESCC compared with the paired NAT (P < 0.01). Western blot results showed that cells in the intervention group could express ASPP2 while there was no expression of ASPP2 in the control group. Taken together, these results indicate that the abnormal expression of ASPP2 may play an important role for development and metastasis in ESCC.

ASPP2 Plays a Dual Role in gp120-Induced Autophagy and Apoptosis of Neuroblastoma Cells.

HIV invasion of the central nervous system (CNS) in the majority of patients infected with HIV-1, leads to dysfunction and injury within the CNS, showing a variety of neurological symptoms which was broadly termed HIV-associated neurocognitive disorder (HAND). But the molecular mechanisms are not completely understood. It has been suggested that apoptosis and autophagic dysfunction in neurons may play an important role in the development of HAND. Previous studies have indicated that p53 may be involved in the onset of neurological disorder in AIDS. Apoptosis-stimulating protein of p53-2 (ASPP2), a p53-binding protein with specific function of inducing p53, has been reported to modulate autophagy. In the present study, we observed that gp120 induces autophagy and apoptosis in SH-SY5Y neuroblastoma cells. Adenovirus-mediated overexpression of ASPP2 significantly inhibited autophagy and apoptosis induced by low dose of gp120 protein (50 ng/mL), but induced autophagy and apoptosis when treated by high dose of gp120 protein (200 ng/mL). Further, ASPP2 knockdown attenuated autophagy and apoptosis induced by gp120.Conclusion: ASPP2 had different effects on the autophagy and apoptosis of neurons induced by different concentration of gp120 protein. It may be a potential therapeutic agent for HAND through modulating autophagy and apoptosis in CNS.

Oncogenic Splicing of the Apoptosis-Stimulating Protein of p53-2 (ASPP2) Is an Initiating Step in Leukemogenesis Facilitating Acquisition of Structural Genomic Mutations

ASPP2 is an independent haploinsufficient tumor suppressor which initiates induction of apoptosis after cellular damage in a TP53-dependent manner. We recently reported the identification of two C-terminally truncated splice variants of ASPP2 with high frequency in acute leukemias. Tantalizingly, these variants lack the functionally relevant TP53 binding sites, suggesting a dominant-negative, oncogenic function. The most prevalent isoform lacking exon 17 (ASPP2k) is detected in up to 60% of acute leukemias. A cell sort for the CD34+ stem cell fraction confirmed ASPP2k-expression in the leukemia-initiating clone. Immunoblotting revealed translation of ASPP2k into a genuine protein isoform.Generation of isoform-specific antibodies allows to discriminate ASPP2k from the wildtype isoforms. Interestingly, ASPP2k is the sole isoform expressed in some native leukemia samples. Other samples reveal a heterozygous expression pattern suggesting a dominant-negative function of the C-terminally truncated ASPP2k isoform.Forced expression of ASPP2k using a HisMax plasmid vector in murine IL3-dependent pro-B Ba/F3 cells induced a more aggressive phenotype with mitotic failure and perturbed cellular proliferation compared to the empty vector controls. Of note, IL3 was successfully weaned in the ASPP2k cells - indicating that autoactivating genomic alterations must have occurred upholding cellular integrity and viability. Fractionated gamma irradiation accelerated this process. In contrast, the empty vector strains did not survive IL3 weaning. Chromosomal analyses of IL3-independent Ba/F3 ASPP2kcells revealed structural alterations including monosomies and additional marker chromosomes.Furthermore, we demonstrate that ASPP2k is stress-inducible. Changing culture conditions (e.g. temperature) of cell lines or ex vivo native AML samples resulted in a dramatic increase of the ASPP2k isoform. Consecutive treatment of cells with daunorubicin lead to relative resistance of pre-stressed cells compared to their counterparts cultured under standard conditions. Vice versa, specific knock-down of ASPP2k resulted in a significant increase in induction of apoptosis upon chemotherapy compared to empty vector controls.Our data demonstrate that ASPP2k plays a distinctive role as an anti-apoptotic regulator of the TP53 checkpoint rendering cells to a more aggressive phenotype as evidenced by proliferation and apoptosis rates - and facilitates acquisition of genomic mutations, a first initiating step in leukemogenesis. DisclosuresNo relevant conflicts of interest to declare.

Upregulation of MiR-205 under hypoxia promotes epithelial-mesenchymal transition by targeting ASPP2.

The epithelial–mesenchymal transition (EMT) is one of the crucial procedures for cancer invasion and distal metastasis. Despite undergoing intensive studies, the mechanisms underlying EMT remain to be completely elucidated. Here, we identified that apoptosis-stimulating protein of p53-2 (ASPP2) is a novel target of MiR-205 in various cancers. Interestingly, the binding site of MiR-205 at the 3′-untranslated region of ASPP2 was highly conserved among different species. An inverse correlation between MiR-205 and ASPP2 was further observed in vivo in cervical cancers, suggesting MiR-205 may be an important physiological inhibitor of ASPP2. Hypoxia is a hallmark of solid tumor microenvironment and one of such conditions to induce EMT. Notably, MiR-205 was remarkably induced by hypoxia in cervical and lung cancer cells. A marked suppression of ASPP2 was observed simultaneously. Further studies confirmed that hypoxia-induced ASPP2 suppression was mainly attributed to the elevated MiR-205. Interestingly, the alteration of MiR-205/ASPP2 under hypoxia was accompanied with the decreased epithelial marker E-cadherin and increased mesenchymal marker Vimentin, as well as a morphological transition from the typical cobblestone-like appearance to the mesenchymal-like structure. More importantly, MiR-205 mimics or ASPP2 silencing similarly promoted EMT process. By contrast, ASPP2 recovery or MiR-205 inhibitor reversed MiR-205-dependent EMT. Further studies demonstrated that the newly revealed MiR-205/ASPP2 axis promoted cell migration and also increased cell proliferation both in vivo and in vitro. These data together implicated a critical impact of MiR-205/ASPP2 on promoting EMT. MiR-205/ASPP2 may be potential diagnostic and therapeutic biomarkers in cervical and lung cancers.

ASPP2 deficiency causes features of 1q41q42 microdeletion syndrome.

Chromosomal abnormalities are implicated in a substantial number of human developmental syndromes, but for many such disorders little is known about the causative genes. The recently described 1q41q42 microdeletion syndrome is characterized by characteristic dysmorphic features, intellectual disability and brain morphological abnormalities, but the precise genetic basis for these abnormalities remains unknown. Here, our detailed analysis of the genetic abnormalities of 1q41q42 microdeletion cases identified TP53BP2, which encodes apoptosis-stimulating protein of p53 2 (ASPP2), as a candidate gene for brain abnormalities. Consistent with this, Trp53bp2-deficient mice show dilation of lateral ventricles resembling the phenotype of 1q41q42 microdeletion patients. Trp53bp2 deficiency causes 100% neonatal lethality in the C57BL/6 background associated with a high incidence of neural tube defects and a range of developmental abnormalities such as congenital heart defects, coloboma, microphthalmia, urogenital and craniofacial abnormalities. Interestingly, abnormalities show a high degree of overlap with 1q41q42 microdeletion-associated abnormalities. These findings identify TP53BP2 as a strong candidate causative gene for central nervous system (CNS) defects in 1q41q42 microdeletion syndrome, and open new avenues for investigation of the mechanisms underlying CNS abnormalities.

Attenuation of Apoptosis-Stimulating Protein of p53-2 (ASPP2) Promotes IL-3-Independent Growth and Enhances Cytogenetic Abnormalities after Gamma-Irradiation in Mouse Hematopoietic Cells - Mimicking Early Leukemogenesis of Therapy-Associated Human AML

Inactivation of tp53 by chromosome/gene loss or loss-of function mutation is frequently observed in therapy-associated acute myeloid leukemia. Tp53 mutations associate with complex and monosomal karyotypes and provide the strongest predictive risk factor in this subgroup. ASPP2 is an independent tumor suppressor. We have previously shown that ASPP2 expression is attenuated in acute leukemia via methylation by unknown mechanisms. We now provide evidence that attenuation of ASPP2 expression may act as an early leukemia-initiating event - facilitating acquisition of structural chromosomal aberrations and loss of its binding partner p53.We found low ASPP2 mRNA and protein expression levels in complex karyotype AML, arguing for a role of ASPP2 loss in leukemogenesis. To evaluate for malignant transformation we engineered IL3-dependant murine Ba/F3 cells and stably silenced ASPP2 expression using retroviral-induced shRNA, Interestingly, only ASPP2(ko) cell strains could be successfully weaned from IL3 dependence - suggesting that loss of ASPP2 expression promoted cellular transformation. In contrast, empty vector control cells did not survive the weaning process. As expected, cellular proliferation was enhanced in the ASPP2(ko) cell lines as measured by standard XTT and CSFE protocols. To study how loss of ASPP2 could promote transformation, we performed cytogenetic analysis of the chromosome set of parental (+IL3) vs. gamma-irradiated empty vector controls (+IL3) and ASPP2(ko) cell strains (-IL3) with and without irradiation (+/- stress enforcement using gamma irradiation, 2 x 5Gy). Gamma irradiation did not significantly alter cytogenic analysis between the empty vector controls compared to the parental cells. Tantalizingly however, we found a consistent chromosomal loss of chr8, der8 and der19 - and, most intriguingly, loss of chromosome 11 (which locates tp53 in mice), and a monosomal karyotype in the ASPP2-attenuated cells. In addition, several marker chromosomes (6-8 in ASPP2i vs. 0-2 in empty vector controls were identified. We are now in the process of verifying our findings in knockout native human bone marrow blasts.Conclusions: Our findings provide novel evidence that loss of ASPP2 contributes to malignant transformation by promoting genomic instability as measured by increased cytogenetic abnormalities after gamma-irradiation. These findings may have important relevance for the pathogenesis of therapy-associated AML - but also of hypodiploid acute lymphoblastic leukemia, which is as well associated with tp53 loss-of-function and predicted for a dismal outcome. Studies are ongoing to elucidate the mechanisms of how loss of ASPP2 expression promotes human AML, as well as restore ASPP2 function as a novel therapeutic pathway to exploit in treating AML. DisclosuresNo relevant conflicts of interest to declare.

ASPP2 Attenuation Is an Early Step in Leukemogenesis Facilitating Acquisition of Mutations

Inactivation of the p53 pathway is an universal event in human cancers. As p53 mutations are rare in de novo acute leukemias, the p53-pathway must be inactivated by other mechanisms instead. The Apoptosis stimulating Protein of p53-2 (ASPP2) is a haploinsufficient tumor suppressor, which we have shown to be attenuated in acute leukemia. We now provide evidence that ASPP2 regulates pathways involved in early hematopoiesis, and attenuated expression perturbs malignant transformation.Gene-chip microarrays were performed on ASPP2+/+ versus +/- mouse embryonic fibroblasts (MEFs) (-/- knockouts are not viable) and pathway analysis was performed using Affimetrix software. ASPP2 expression was silenced in IL3-dependent Ba/F3 pro-B cells and leukemia cell lines using standard siRNA protocols. Cellular proliferation was assessed using XTT-based assays. To evaluate for malignant transformation, long term cell cultures with stably retrovirally silenced Ba/F3 cells (+/- stress inforcement using gamma irradiation, 4x 5Gy) were employed. IL3 weaning was performed as an indicator of autoactivated cellular proliferation, and analysis of chromosomal defects or gene mutations were performed.Microarray mRNA analysis revealed that attenuated ASPP2 expression resulted in significant alterations of pathways involved in mediating cellular growth, proliferation and tissue differentiation. Subanalysis proofed involvement of genes playing a major role in embryogenesis, the development of the hematopoietic system and leukemogenesis (homeobox (HOX) family members, NOTCH, BCL, IRF7 and EGR1). Population doubling times were significantly increased in ASPP2+/- MEFs compared to the parental cells. Modeling ASPP2 attenuation via specific siRNA knockdown in leukemia cell lines (K562 and Kasumi1) resulted in significantly increased cellular proliferation. Similarly, ASPP2-interference in pro-B Ba/F3 cells lead to perturbed proliferation and an increase of polyploid cells with a dramatic change in morphology with megaloid cells, indicating mitotic failure. This effect was even more pronounced after stress induction with daunorubicin. Lentiviral shRNA-ASPP2 stable knock-out was performed in Ba/F3 cells to follow cells in long term cultures for malignant transformation. Again, we confirmed signs of mitotic failure as observed in the siRNA approach. Even more, gamma irradiated ASPP2-lacking strains were successfully IL3 weaned, which indicates autoactivated cellular proliferation. A screen for acquired oncogenic mutations was performed and analyses are ongoing.In summary, we provide evidence that ASPP2 has a pivotal role in early hematopoiesis, and loss of ASPP2 is a driver mechanism to fuel leukemogenesis. DisclosuresNo relevant conflicts of interest to declare.

CHOP mediates ASPP2-induced autophagic apoptosis in hepatoma cells by releasing Beclin-1 from Bcl-2 and inducing nuclear translocation of Bcl-2.

Apoptosis-stimulating protein of p53-2 (ASPP2) induces apoptosis by promoting the expression of pro-apoptotic genes via binding to p53 or p73; however, the exact mechanisms by which ASPP2 induces apoptotic death in hepatoma cells are still unclear. Here, we show that the transient overexpression of ASPP2 induces autophagic apoptosis in hepatoma cells by promoting p53- or p73-independent C/EBP homologous protein (CHOP) expression. CHOP expression decreases the expression of Bcl-2; this change releases Beclin-1 from cytoplasmic Bcl-2-Beclin-1 complexes and allows it to initiate autophagy. However, transient overexpression of Beclin-1 can induce autophagy but not apoptosis. Our results show that ASPP2 induces the expression of damage-regulated autophagy modulator (DRAM), another critical factor that cooperates with free Beclin-1 to induce autophagic apoptosis. The effect of CHOP on the translocation and sequestration of Bcl-2 in the nucleus, which requires the binding of Bcl-2 to ASPP2, is also critical for ASPP2-induced autophagic apoptosis. Although the role of nuclear ASPP2–Bcl-2 complexes is still unclear, our results suggest that nuclear ASPP2 can prevent the translocation of the remaining Bcl-2 to the cytoplasm by binding to Bcl-2 in a CHOP-dependent manner, and this effect also contributes to Beclin-1-initiated autophagy. Thus, CHOP is critical for mediating ASPP2-induced autophagic apoptosis by decreasing Bcl-2 expression and maintaining nuclear ASPP2–Bcl-2 complexes. Our results, which define a mechanism whereby ASPP2 overexpression induces autophagic apoptosis, open a new avenue for promoting autophagy in treatments to cure hepatocellular carcinoma.

Structure of the Helicobacter pylori CagA oncoprotein bound to the human tumor suppressor ASPP2

The Cytotoxin associated gene A (CagA) protein of Helicobacter pylori is associated with increased virulence and risk of cancer. Recent proteomic studies have demonstrated an association of CagA with the human tumor suppressor Apoptosis-stimulating Protein of p53-2 (ASPP2). We present here a genetic, biochemical, and structural analysis of CagA with ASPP2. Domain delineation of the 120-kDa CagA protein revealed a stable N-terminal subdomain that was used in a yeast two-hybrid screen that identified the proline-rich domain of ASPP2 as a host cellular target. Biochemical experiments confirm this interaction. The cocrystal structure to 2.0-Å resolution of this N-terminal subdomain of CagA with a 7-kDa proline-rich sequence of ASPP2 reveals that this domain of CagA forms a highly specialized three-helix bundle, with large insertions in the loops connecting the helices. These insertions come together to form a deep binding cleft for a highly conserved 20-aa peptide of ASPP2. ASPP2 forms an extended helix in this groove of CagA, burying more than 1,000 Å(2) of surface area. This interaction is disrupted in vitro and in vivo by structure-based, loss-of-contact point mutations of key residues in either CagA or ASPP2. Disruption of CagA and ASPP2 binding alters the function of ASPP2 and leads to the decreased survival of H. pylori-infected cells.

Aspp2 negatively regulates body growth but not developmental timing by modulating IRS signaling in zebrafish embryos

The growth and developmental rate of developing embryos and fetus are tightly controlled and coordinated to maintain proper body shape and size. The insulin receptor substrate (IRS) proteins, key intracellular transducers of insulin and insulin-like growth factor signaling, play essential roles in the regulation of growth and development. A short isoform of apoptosis-stimulating protein of p53 2 (ASPP2) was recently identified as a binding partner of IRS-1 and IRS-2 in mammalian cells in vitro. However, it is unclear whether ASPP2 plays any role in vertebrate embryonic growth and development. Here, we show that zebrafish Aspp2a and Aspp2b negatively regulate embryonic growth without affecting developmental rate. Human ASPP2 had similar effects on body growth in zebrafish embryos. Aspp2a and 2b inhibit Akt signaling. This inhibition was reversed by coinjection of myr-Akt1, a constitutively active form of Akt1. Zebrafish Aspp2a and Aspp2b physically bound with Irs-1, and the growth inhibitory effects of ASPP2/Aspp2 depend on the presence of their ankyrin repeats and SH3 domains. These findings uncover a novel role of Aspp2 in regulating vertebrate embryonic growth.

ASPP2 gene silence promotes hepatocellular carcinoma cell proliferation via regulating autophagy under starvation

Objective To study the effects of apoptosis-stimulating protein of p53-2(ASPP2) gene on hepatocellular carcinoma(HCC) cell proliferation and apoptosis under starvation and the related mechanism.MethodsLentivirus encoding shRNA against ASPP2 was constructed to knockdown ASPP2 expression in hepatoma cell HepG2.Cell proliferation and apoptosis were observed by transmission electron microscopy,MTS analysis,flow cytometry analysis and morphologic changes. The influence of silencing ASPP2 gene on the proliferation and apoptosis under amino acid-starvation and serum-deprivation culture was observed;autophagy inhibitor 3-methyladenine(3-MA) was added in the experiment so as to detect the involvement of autophagy in the changes induced by ASPP2 down-regulation. ResultsTransmission electron microscopy showed cytoplasmic accumulation of autophagosomes when ASPP2 was knocked down under amino acid-starvation and serum-deprivation(P0.05),with increased GFP-LC3 dots(P0.05).MTS analysis showed that silence of ASPP2 gene greatly enhanced the proliferation of HepG2 cells(P0.05),which could be inhibited by addition of 3-MA(P0.05).Microscope observation showed that silence of ASPP2 gene promoted the anti-apoptotic ability of HepG2 cells,which was reversed by treatment with 3-MA.Early sign of apoptosis was observed in shASPP2+3-MA group. Annexin Ⅴ-PI double staining showed that ASPP2 silence decreased the apoptotic rate of HepG2 cells from(38±5)% to(15±4)%(P0.05),and 3-MA treatment increased it to(36±3)%(P0.05).ConclusionDown-regulation of ASPP2 expression may facilitate the survival and proliferation of HCC cells through activating autophagy under starvation.

Attenuated Expression of Apoptosis Stimulating Protein of p53-2 (ASPP2) in Human Acute Leukemia Is Associated with Therapy Failure

Inactivation of the p53 pathway is a universal event in human cancers and promotes tumorigenesis and resistance to chemotherapy. Inactivating p53 mutations are uncommon in non-complex karyotype leukemias, thus the p53-pathway must be inactivated by other mechanisms. The Apoptosis Stimulating Protein of p53-2 (ASPP2) is a damage-inducible p53-binding protein that enhances apoptosis at least in part through a p53-mediated pathway. We have previously shown, that ASPP2 is an independent haploinsufficient tumor suppressor in vivo. Now, we reveal that ASPP2 expression is significantly attenuated in acute myeloid and lymphoid leukemia – especially in patients with an unfavorable prognostic risk profile and patients who fail induction chemotherapy. In line, knock down of ASPP2 in expressing leukemia cell lines and native leukemic blasts attenuates damage-induced apoptosis. Furthermore, cultured blasts derived from high-risk leukemias fail to induce ASPP2 expression upon anthracycline treatment. The mechanisms of ASPP2 dysregulation are unknown. We provide evidence that attenuation of ASPP2 is caused by hypermethylation of the promoter and 5′UTR regions in native leukemia blasts. Together, our results suggest that ASPP2 contributes to the biology of leukemia and expression should be further explored as a potential prognostic and/or predictive biomarker to monitor therapy responses in acute leukemia.

Downregulation of ASPP2 in choriocarcinoma contributes to increased migratory potential through Src signaling pathway activation

Gestational choriocarcinoma is a malignant tumor derived from placental trophoblast and the most aggressive member of gestational trophoblastic disease (GTD). Apoptosis-stimulating protein of p53-2 (ASPP2) is a member of ASPP family that transactivates p53 and thereby functions as a tumor suppressor. In this study, the expression profile of ASPP2 in choriocarcinoma was examined in comparison with normal placentas and hydatidiform moles, the latter being a type of GTD that carries malignant potential. Downregulation of ASPP2 messenger RNA and protein was demonstrated in choriocarcinoma by quantitative PCR and immunohistochemistry. ASPP2-transfected choriocarcinoma cells (JEG-3 and JAR) showed an increase in apoptosis and a decrease in cell migration as detected by TdT-mediated dUTP nick end labeling and wound healing assays, respectively, illustrating the complex action of ASPP2 on cell functions other than programmed cell death. Activated Src is known to be important in tumor progression. Transfection of ASPP2 but not ASPP1, another tumor-suppressive ASPP, was found to be related to subsequent decreased Src-pY416 phosphorylation, suggesting an inactivating effect of ASPP2 on Src. Moreover, this ASPP2-mediated inactivation of Src could be abolished by RNA interference with C-terminal Src kinase (Csk), a kinase that can inhibit Src activation. Our findings suggested that the ability of ASPP2 to attenuate Src activation was specific to ASPP2 in a Csk-dependent manner. Taken together, we demonstrated a loss of tumor-suppressive ASPP2 in choriocarcinoma with effects on cell migration and apoptosis. We also unveiled a possible mechanistic link between ASPP2 and Csk/Src signaling pathway, implicating the multiple cellular functions of ASPP2.

Abstract LB-49: Identification of C-terminal truncated splice variants of the apoptosis-stimulating protein of p53-2 (ASPP2) in acute leukemia.

Abstract ASPP2 is an independent haploinsufficient tumor suppressor (Kampa et al., PNAS2009) and studies in tumors including breast cancer and aggressive lymphoma have shown that attenuation/loss of ASPP2 is a common occurrence. In acute leukemia, we have evidence that attenuation of ASPP2 significantly associates with higher-risk disease and poor clinical outcome (Schittenhelm et al.,ASH2012). In an attempt to screen for ASPP2 gene/transcript aberrations we identified two novel splicing variants of ASPP2 in acute myeloid and lymphoid leukemia blasts from 80 consented patients: The more prevalent isoform lacking exon 17, named ASPP2κ, was detected in &amp;gt;30% of patients. ASPP2μ, lacking ex16/17, was identified in one patient. A quantitative (q)PCR assay on genomic DNA to compare exon 15 versus exon 17 expression levels did not reveal any significant differences arguing for a splicing event rather than mutation. Isolating CD34(+) leukemia blasts using cell sorting technology demonstrated ASPP2κ-expression in leukemia stem/progenitor cells. Lack of exon 16 (resp. exon 16/17) results in a reading frame-shift with a premature translation stop, omitting most of the C-terminus - which harbors the p53-binding sites. We generated isoform-specific antibodies directed against the hypothetical fusion site. Sequence-specificity was confirmed by BlastN search. Co-immunoprecipitation using antibodies targeting the ASPP2 N-terminal and probing for ASPP2κ-specific antibodies revealed genuine translation into two protein isoforms (ASPP2κ-1/2, corresponding to ASPP2-1/2) with the predicted sizes of 97 and 111 kDa. Western immunoblotting and intracellular semi-quantitative immunophenotyping of freshly isolated native leukemia as well as bone marrow donor samples demonstrated ASPP2κ-1/2 to be specifically expressed in leukemia. Additionally we developed a highly isoform—specific qRT-PCR assay running on a light-cycler platform: A restriction motif-specific ASPP2 exon 17 digest was performed to deplete “wildtype” ASPP2 and ASPP2κ-specific primer were generated. Although ASPP2κ amplicons were detectable in physiologic cells, high expression levels were exclusively found in leukemic blasts. Tantalizingly ASPP2κ as well as ASPP2μ dramatically decreased in patients achieving complete remission (CR) - whereas patients failing remission retained high transcript levels. Preliminary data suggest, that transfection of ASPP2κ in murine pro-B Ba/F3 cells renders cells to more aggressive biology with mitotic failure and perturbed cellular proliferation. Systematic analysis of other tumor entities and functional analysis including other models are ongoing. In summary, we identified two novel ASPP2 splice variants lacking the p53-binding site - which are predominantly detected in acute leukemia and may link to early tumorigenesis. Citation Format: Kerstin M. Kampa-Schittenhelm, Charles D. Lopez, Barbara Illing, Michael Walter, Marcus M. Schittenhelm. Identification of C-terminal truncated splice variants of the apoptosis-stimulating protein of p53-2 (ASPP2) in acute leukemia. [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 LB-49. doi:10.1158/1538-7445.AM2013-LB-49