Downregulation Of PUMA Research Articles

BH3-only proteins Puma and Beclin1 regulate autophagic death in neurons in response to Amyloid-\u03b2

Alzheimer’s disease (AD) is characterized by accumulation of senile amyloid-β (Aβ) plaques and hyperphosphorylated tau tangles causing progressive loss of synapse and neuronal death. Out of the various neuron death modalities, autophagy and apoptosis are reported to be the major death paradigms in AD. However, how these two processes lead to neuronal loss is still inconspicuous. Here we report that under Aβ toxicity, aberrant autophagy is induced with inefficient autophagic flux in neurons. Simultaneous activation of both autophagy and apoptosis are seen in primary cortical neurons as well as in transgenic mice brains. We found that induction of autophagy by rapamycin is detrimental for neurons; whereas downregulation of Beclin1, an important autophagy inducing protein, provides significant protection in Aβ treated neuronal cells by blocking cytochrome-c release from the mitochondria. We further report that downregulation of Puma, a BH3-only pro-apoptotic protein, inhibits the induction of aberrant autophagy and also ameliorates the autophagy flux under the influence of Aβ. Notably, stereotactic administration of shRNAs against Puma and Beclin1 in adult Aβ-infused rat brains inhibits both apoptotic and autophagic pathways. The regulation of both of the death processes is brought about by the direct interaction between Puma and Beclin1 upon Aβ treatment. We conclude that both Beclin1 and Puma play essential roles in the neuronal death caused by the induction of aberrant autophagy in AD and targeting their interaction could be vital to understand the crosstalk of autophagy and apoptosis as well as to develop a potential therapeutic strategy in AD.

Abstract A38: RNA-seq of diagnostic and relapse disseminated tumor cells (DTCs) in stage M neuroblastoma patients and first steps towards DTC-derived xenografts in zebrafish embryos

Abstract Neuroblastoma (NB) is the most common tumor diagnosed in the first year of life. More than 90% of stage M NB patients present with disseminated tumor cells (DTCs) in the bone marrow (BM). Although these cells represent a major obstacle to treatment, their global gene expression has, so far, been only poorly analyzed. GD2POS DTCs of stage M NB patients were separated from the remaining BM-derived nontumor mononuclear cells (MNCs) by applying magnetic associated cell sorting. mRNA of diagnostic DTCs (n=22) and MNCs (n=14), primary tumors (n=16), relapse DTCs (n=20) and MNCs (n=14) was isolated and used for RNA-seq (50bp, single-end) in order to characterize the biology of DTCs. Furthermore, four DTC-derived neuroblastoma cell lines were established and used to generate a robust patient-derived zebrafish xenograft model. Annexin V staining (apoptosis), EdU proliferation assays, and transwell migration assays of the four cell lines were performed in vitro at temperatures between 34°C and 37°C. 322 differently expressed genes (DEG) were identified comparing primary tumors and DTCs (q < 0.001, |log2FC|>2). Especially, genes encoded by mitochondrial DNA (mtDNA) were upregulated in DTCs. Furthermore, we found the gene expression signature of relapse DTCs largely resembling those of diagnostic DTCs, with only 113 DEG under relaxed cut-offs (q < 0.01, |log2FC|>0.5). Notably, relapse DTCs showed a positional enrichment of 31 downregulated genes on chromosome 19, including five tumor-suppressor genes: SIRT6, BBC3/PUMA, STK11, CADM4, and GLTSCR2. Genomic analysis of 34 DTC samples revealed clonal deletions affecting a portion of the 31 downregulated genes encoded by chromosome 19. Although these deletions occurred at a higher frequency in relapse patients (9 out of 17 samples) as compared to the diagnostic samples (4 out of 17 samples), the positional distribution of downregulated genes did not coincide with the deleted ones. While chromosomal deletions in relapse samples were mainly found on 19q (8 out of 20 samples), most of the downregulated genes were encoded by 19p, which rarely contained deletions (3 out of 20 samples). The downregulation of PUMA (p=0.002) and CADM4 (p=0.02) was associated with a worse event-free survival in stage M patients (survival analysis performed in R2 with the Oberthuer dataset). Furthermore, four DTC-derived cell lines were tested for suitability of injecting into zebrafish embryos and subsequent functional testing. In vitro analysis revealed that in two cell lines (STA-NB-8 and STA-NB-12) proliferation and apoptosis were not affected by lower temperatures (34°C), at which zebrafish embryos still can develop properly, while in the remaining two cell lines both were impaired. Fewer STA-NB-15 and STA-NB-19 cells were in the S phase at 34°C (18.6% of STA-NB-15 and 24.2% of STA-NB-19 cells) as compared to 37°C (37.8% of STA-NB-15 and 45.1% of STA-NB-19 cells). Additionally, more of the STA-NB-15 cells were undergoing apoptosis at 34°C (50.3% Annexin V positive cells) as compared to 37°C (33.9% Annexin V positive cells). This first RNA-seq analysis of neuroblastoma DTCs revealed their unique expression profile in contrast to tumors, but showed less pronounced differences between diagnostic and relapse DTCs. The latter preferentially affected the downregulation of genes encoded by chromosome 19, which might be associated with treatment failure and disease progression. The establishment of xenografts in zebrafish embryos with DTC-derived cells will enable in vivo functional studies and optimize the generation of patient-derived xenografts in zebrafish embryos, which will allow drug screening and functional studies for personalized treatment approaches. Citation Format: Fikret Rifatbegovic, Sabine Taschner-Mandl, Martin Distel, M. Reza Abbasi, Christian Frech, Andrea Ziegler, Inge M. Ambros, Peter F. Ambros. RNA-seq of diagnostic and relapse disseminated tumor cells (DTCs) in stage M neuroblastoma patients and first steps towards DTC-derived xenografts in zebrafish embryos [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr A38.

Hepatocyte growth factor renders BRAF mutant human melanoma cell lines resistant to PLX4032 by downregulating the pro-apoptotic BH3-only proteins PUMA and BIM.

A large proportion of melanomas harbour the activating BRAFV600E mutation that renders these cells dependent on MAPK signalling for their survival. Although the highly specific and clinically approved BRAFV600E kinase inhibitor, PLX4032, induces apoptosis of melanoma cells bearing this mutation, the underlying molecular mechanisms are not fully understood. Here, we reveal that PLX4032-induced apoptosis depends on the induction of the pro-apoptotic BH3-only protein PUMA with a minor contribution of its relative BIM. Apoptosis could be significantly augmented when PLX4032 was combined with an inhibitor of the pro-survival protein BCL-XL, whereas neutralization of the pro-survival family member BCL-2 caused no additional cell death. Although the initial response to PLX4032 in melanoma patients is very potent, resistance to the drug eventually develops and relapse occurs. Several factors can cause melanoma cells to develop resistance to PLX4032; one of them is the activation of the receptor tyrosine kinase cMET on melanoma cells by its ligand, hepatocyte growth factor (HGF), provided by the tumour microenvironment or the cancer cells themselves. We found that HGF mediates resistance of cMET-expressing BRAF mutant melanoma cells to PLX4032-induced apoptosis through downregulation of PUMA and BIM rather than by increasing the expression of pro-survival BCL-2-like proteins. These results suggest that resistance to PLX4032 may be overcome by specifically increasing the levels of PUMA and BIM in melanoma cells through alternative signalling cascades or by blocking pro-survival BCL-2 family members with suitable BH3 mimetic compounds.

Constitutive BAK activation as a determinant of drug sensitivity in malignant lymphohematopoietic cells.

Mitochondrial outer membrane permeabilization (MOMP), a key step in the intrinsic apoptotic pathway, is incompletely understood. Current models emphasize the role of BH3-only BCL2 family members in BAX and BAK activation. Here we demonstrate concentration-dependent BAK autoactivation under cell-free conditions and provide evidence that this autoactivation plays a key role in regulating the intrinsic apoptotic pathway in intact cells. In particular, we show that up to 80% of BAK (but not BAX) in lymphohematopoietic cell lines is oligomerized and bound to anti-apoptotic BCL2 family members in the absence of exogenous death stimuli. The extent of this constitutive BAK oligomerization is diminished by BAK knockdown and unaffected by BIM or PUMA down-regulation. Further analysis indicates that sensitivity of cells to BH3 mimetics reflects the identity of the anti-apoptotic proteins to which BAK is constitutively bound, with extensive BCLXL•BAK complexes predicting navitoclax sensitivity, and extensive MCL1•BAK complexes predicting A1210477 sensitivity. Moreover, high BAK expression correlates with sensitivity of clinical acute myelogenous leukemia to chemotherapy, whereas low BAK levels correlate with resistance and relapse. Collectively, these results inform current understanding of MOMP and provide new insight into the ability of BH3 mimetics to induce apoptosis without directly activating BAX or BAK.

Slug mediates nasopharyngeal carcinoma radioresistance via downregulation of PUMA in a p53-dependent and -independent manner.

Slug is involved in the radioresistance and chemoresistance of several types of cancers. In the present study, we first studied the effect of Slug on the radioresistance of nasopharyngeal carcinoma (NPC). We established radioresistant CNE-2 cells (CNE-2-RES) by exposing CNE-2 cells to gradually increasing doses of irradiation (IR). We used lentiviral infection technique to stably knock down Slug and then studied the effects in vitro and in vivo. Western blotting and RT-PCR were applied to detect the protein and mRNA expression in NPC cells or xenograft tumor tissues, respectively. Colony forming assay was applied to detect the cell survival after IR. As a result, CNE-2-RES cells were successfully established, CNE-2-RES cells showed relatively higher expression of Slug, higher expression of p53 and lower expression of PUMA. Following inhibition of Slug, the radiosensitivity of NPC was enhanced both in vitro and in vivo. Slug inversely regulated PUMA and p53 expression in both CNE-2 and CNE-2-RES cells. Animal experiments showed the same trend of protein expression as the in vitro results. In conclusion, our study demonstrated that Slug overexpression in CNE-2-RES cells may result in the radioresistance of cells. Slug mediates CNE-2 radioresistance via downregulation of PUMA in both a p53-dependent and p53-independent manner.

The Essential Role of p53-up-regulated Modulator of Apoptosis (Puma) and Its Regulation by FoxO3a Transcription Factor in β-Amyloid-induced Neuron Death

Neurodegeneration underlies the pathology of Alzheimer disease (AD). The molecules responsible for such neurodegeneration in AD brain are mostly unknown. Recent findings indicate that the BH3-only proteins of the Bcl-2 family play an essential role in various cell death paradigms, including neurodegeneration. Here we report that Puma (p53-up-regulated modulator of apoptosis), an important member of the BH3-only protein family, is up-regulated in neurons upon toxic β-amyloid 1-42 (Aβ(1-42)) exposure both in vitro and in vivo. Down-regulation of Puma by specific siRNA provides significant protection against neuron death induced by Aβ(1-42). We further demonstrate that the activation of p53 and inhibition of PI3K/Akt pathways induce Puma. The transcription factor FoxO3a, which is activated when PI3K/Akt signaling is inhibited, directly binds with the Puma gene and induces its expression upon exposure of neurons to oligomeric Aβ(1-42). Moreover, Puma cooperates with another BH3-only protein, Bim, which is already implicated in AD. Our results thus suggest that Puma is activated by both p53 and PI3K/Akt/FoxO3a pathways and cooperates with Bim to induce neuron death in response to Aβ(1-42).

Puma Deletion Delays Cardiac Dysfunction in Murine Heart Failure Models Through Attenuation of Apoptosis

Puma (p53-upregulated modulator of apoptosis) is a proapoptotic Bcl-2 family protein that serves as a general sensor in response to pathological apoptotic stimuli. In previous work, we demonstrated that puma ablation protects the heart from reperfusion injury in a Langendorff setting. Consistent with this, downregulation of Puma in isolated cardiac myocytes prevented apoptosis induced by different proapoptotic agents. Here, we extended our research to investigate the role of Puma, a downstream mediator of p53, in the development of heart failure using Puma(-/-) mice. Mice underwent transverse aortic constriction, and the characteristics of cardiac remodeling were analyzed by echocardiography, histology, and gene expression at multiple time points after surgery. Four weeks after the operation, puma deletion attenuated pressure overload-induced apoptosis and fibrosis; however, it did not affect hypertrophy and angiogenesis and maintained functional performance (fractional shortening, 39% versus 25.2% in Puma(-/-) versus WT mice, respectively). Even at 12 weeks after transverse aortic constriction, Puma(-/-) mice displayed only slightly reduced contractility. In addition, transverse aortic constriction induced puma expression in a partially p53-dependent manner. To corroborate these findings, we studied another heart failure model in which heart-specific mdm4 deletion leads to p53 activation and dilated cardiomyopathy. In these mice, Puma was upregulated and its deletion rescued the cardiomyopathy phenotype. Our data indicate that Puma might be a critical component of the apoptotic signaling pathways that contribute to ventricular remodeling and heart failure. Therefore, Puma inactivation may serve as a preferential target to prevent heart failure induced by cellular stress.

The Codon 47 Polymorphism in p53 Is Functionally Significant

In addition to a common polymorphism at codon 72, the p53 tumor suppressor gene also contains a rare single nucleotide polymorphism at amino acid 47. Wild type p53 encodes proline at this residue, but in <5% of African Americans, this amino acid is serine. Notably, phosphorylation of the adjacent serine 46 by the proline-directed kinase p38 MAPK is known to greatly enhance the ability of p53 to induce apoptosis. Here we showed that the serine 47 polymorphic variant, which replaces the proline residue necessary for recognition by proline-directed kinases, is a markedly poorer substrate for phosphorylation on serine 46 by p38 MAPK. Consistent with this finding, we showed that the serine 47 variant has up to 5-fold decreased ability to induce apoptosis compared with wild type p53. Mechanistically, we found that this variant has decreased ability to transactivate two p53 target genes, p53AIP1 and PUMA, but not other p53 response genes; this is the first time that phosphorylation of serine 46 has been implicated in transactivation of PUMA by p53. Down-regulation of PUMA in cells with wild type p53 using short interfering RNAs reduced apoptosis in these cells to a level comparable to that in cells containing the serine 47 variant. The combined data indicated that, like the codon 72 polymorphism, the codon 47 polymorphism of p53 is functionally significant and may play a role in cancer risk, progression, and the efficacy of therapy.