Yiwei Zhang

A small molecule Inauhzin inhibits SIRT1 activity and suppresses tumour growth through activation of p53

Although ∼50% of all types of human cancers harbour wild‐type TP53, this p53 tumour suppressor is often deactivated through a concerted action by its abnormally elevated suppressors, MDM2, MDMX or SIRT1. Here, we report a novel small molecule Inauhzin (INZ) that effectively reactivates p53 by inhibiting SIRT1 activity, promotes p53‐dependent apoptosis of human cancer cells without causing apparently genotoxic stress. Moreover, INZ stabilizes p53 by increasing p53 acetylation and preventing MDM2‐mediated ubiquitylation of p53 in cells, though not directly in vitro. Remarkably, INZ inhibits cell proliferation, induces senescence and tumour‐specific apoptosis, and represses the growth of xenograft tumours derived from p53‐harbouring H460 and HCT116 cells without causing apparent toxicity to normal tissues and the tumour‐bearing SCID mice. Hence, our study unearths INZ as a novel anti‐cancer therapeutic candidate that inhibits SIRT1 activity and activates p53.

Inauhzin and Nutlin3 synergistically activate p53 and suppress tumor growth

Several proteins have been suggested in promoting tumor formation in numerous human tissues by inactivating the tumor suppressor p53. This has generated interest in the development of small molecules to block these inhibitors of p53 and to regain p53 activity. Recently, we identified a small molecule, Inauhzin, which can inhibit SIRT1 activity and activate p53. SIRT1 is a deacetylase that deacetylates p53 and facilitates Mdm2 mediated p53 destabilization. In this study, we tested if combining Inauhzin with Nutlin-3, an inhibitor of MDM2-p53 binding, might synergistically activate p53 to suppress tumor growth. Indeed, at lower doses, combination of Inauhzin and Nutlin-3 exhibited a synergistic effect on inhibiting cell growth and promoting apoptosis in human colon and lung cancer cell lines in a p53-dependent fashion. Minimal effects were observed with treatment of either compound alone. Using a xenograft tumor model, we also showed a synergistic effect with both compounds. Thus, to fully regain p53 activity, targeting its multiple inhibitory proteins might be a better approach. Our study provides evidence supporting this concept for achieving better therapeutic efficacy in tumors that possess wild type p53.

Nerve growth factor receptor negates the tumor suppressor p53 as a feedback regulator

Cancer develops and progresses often by inactivating p53. Here, we unveil nerve growth factor receptor (NGFR, p75NTR or CD271) as a novel p53 inactivator. p53 activates NGFR transcription, whereas NGFR inactivates p53 by promoting its MDM2-mediated ubiquitin-dependent proteolysis and by directly binding to its central DNA binding domain and preventing its DNA-binding activity. Inversely, NGFR ablation activates p53, consequently inducing apoptosis, attenuating survival, and reducing clonogenic capability of cancer cells, as well as sensitizing human cancer cells to chemotherapeutic agents that induce p53 and suppressing mouse xenograft tumor growth. NGFR is highly expressed in human glioblastomas, and its gene is often amplified in breast cancers with wild type p53. Altogether, our results demonstrate that cancers hijack NGFR as an oncogenic inhibitor of p53. DOI: http://dx.doi.org/10.7554/eLife.15099.001

Nucleostemin stabilizes ARF by inhibiting the ubiquitin ligase ULF

Upregulated expression of nucleolar GTPase nucleostemin (NS) has been associated with increased cellular proliferation potential and tumor malignancy during cancer development. Recent reports attribute the growth regulatory effects of NS protein to its role in facilitating ribosome production. However, the oncogenic potential of NS remains unclear, as imbalanced levels of NS have been reported to exert growth inhibitory effect by modulating p53 tumor-suppressor activity. It also remains in questions if aberrant NS levels might have a p53-independent role in regulation of cell proliferation and growth. In this study, we performed affinity purification and mass spectrometry analysis to explore protein–protein interactions influencing NS growth regulatory properties independently of p53 tumor suppressor. We identified the alternative reading frame (ARF) protein as a key protein associating with NS and further verified the interaction through in vitro and in vivo assays. We demonstrated that NS is able to regulate cell cycle progression by regulating the stability of the ARF tumor suppressor. Furthermore, overexpression of NS suppressed ARF polyubiquitination by its E3 ligase Ubiquitin Ligase for ARF and elongated its half-life, whereas knockdown of NS led to the decrease of ARF levels. Also, we found that NS can enhance NPM stabilization of ARF. Thus, we propose that in the absence of p53, ARF can be stabilized by NS and nucleophosmin to serve as an alternative tumor-suppressor surveillance, preventing potential cellular transformation resulting from the growth-inducing effects of NS overexpression.

Aberrant activation of p53 due to loss of MDM2 or MDMX causes early lens dysmorphogenesis

Although forming a heterodimer or heterooligomer is essential for MDM2 and MDMX to fully control p53 during early embryogenesis, deletion of either MDM2 or MDMX in specific tissues using the loxp-Cre system reveals phenotypic diversity during organ morphogenesis, which can be completely rescued by loss of p53, suggesting the spatiotemporal independence and specificity of the regulation of p53 by MDM2 and MDMX. In this study, we investigated the role of the MDM2-MDMX-p53 pathway in the developing lens that is a relatively independent region integrating cell proliferation, differentiation and apoptosis. Using the mice expressing Cre recombinase specifically in the lens epithelial cells (LECs) beginning at E9.5, we demonstrated that deletion of either MDM2 or MDMX induces apoptosis of LEC and reduces cell proliferation, resulting in lens developmental defect that finally progresses into aphakia. Specifically, the lens defect caused by MDM2 deletion was evident at E10, occurring earlier than that caused by MDMX deletion. These lens defects were completely rescued by loss of two alleles of p53, but not one allele of p53. These results demonstrate that both MDM2 and MDMX are required for monitoring p53 activity during lens development, and they may function independently or synergistically to control p53 and maintain normal lens morphogenesis.

Ccdc3: A new P63 target involved in regulation of liver lipid metabolism

TAp63, a member of the p53 family, has been shown to regulate energy metabolism. Here, we report coiled coil domain-containing 3 (CCDC3) as a new TAp63 target. TAp63, but not ΔNp63, p53 or p73, upregulates CCDC3 expression by directly binding to its enhancer region. The CCDC3 expression is markedly reduced in TAp63-null mouse embryonic fibroblasts and brown adipose tissues and by tumor necrosis factor alpha that reduces p63 transcriptional activity, but induced by metformin, an anti-diabetic drug that activates p63. Also, the expression of CCDC3 is positively correlated with TAp63 levels, but conversely with ΔNp63 levels, during adipocyte differentiation. Interestingly, CCDC3, as a secreted protein, targets liver cancer cells and increases long chain polyunsaturated fatty acids, but decreases ceramide in the cells. CCDC3 alleviates glucose intolerance, insulin resistance and steatosis formation in transgenic CCDC3 mice on high-fat diet (HFD) by reducing the expression of hepatic PPARγ and its target gene CIDEA as well as other genes involved in de novo lipogenesis. Similar results are reproduced by hepatic expression of ectopic CCDC3 in mice on HFD. Altogether, these results demonstrate that CCDC3 modulates liver lipid metabolism by inhibiting liver de novo lipogenesis as a downstream player of the p63 network.

Monitoring p53 by MDM2 and MDMX is required for endocrine pancreas development and function in a spatio-temporal manner

Although p53 is not essential for normal embryonic development, it plays a pivotal role in many biological and pathological processes, including cell fate determination-dependent and independent events and diseases. The expression and activity of p53 largely depend on its two biological inhibitors, MDM2 and MDMX, which have been shown to form a complex in order to tightly control p53 to an undetectable level during early stages of embryonic development. However, more delicate studies using conditional gene-modification mouse models show that MDM2 and MDMX may function separately or synergistically on p53 regulation during later stages of embryonic development and adulthood in a cell and tissue-specific manner. Here, we report the role of the MDM2/MDMX-p53 pathway in pancreatic islet morphogenesis and functional maintenance, using mouse lines with specific deletion of MDM2 or MDMX in pancreatic endocrine progenitor cells. Interestingly, deletion of MDM2 results in defects of embryonic endocrine pancreas development, followed by neonatal hyperglycemia and lethality, by inducing pancreatic progenitor cell apoptosis and inhibiting cell proliferation. However, unlike MDM2-knockout animals, mice lacking MDMX in endocrine progenitor cells develop normally. But, surprisingly, the survival rate of adult MDMX-knockout mice drastically declines compared to control mice, as blockage of neonatal development of endocrine pancreas by inhibition of cell proliferation and subsequent islet dysfunction and hyperglycemia eventually lead to type 1 diabetes-like disease with advanced diabetic nephropathy. As expected, both MDM2 and MDMX deletion-caused pancreatic defects are completely rescued by loss of p53, verifying the crucial role of the MDM2 and/or MDMX in regulating p53 in a spatio-temporal manner during the development, functional maintenance, and related disease progress of endocrine pancreas. Also, our study suggests a possible mouse model of advanced diabetic nephropathy, which is complementary to other established diabetic models and perhaps useful for the development of anti-diabetes therapies.

Abstract 891: Dual defects of MDM2/MDMX-p53 pathways cause global metabolic disruption and enhance tumorigenesis

Although recent discoveries have linked the tumor suppressing functions of p53 to its regulation on cellular metabolism, the exact signaling pathways underlying how p53 is activated in response to various metabolism stresses, and how p53 coordinates the systemic metabolism homeostasis and further blocks tumorigenesis remain largely unclear. Previous studies have shown that the mice containing a single knock-in at cysteine residue 305 of MDM2 (MDM2C305F) is defective in ribosomal stress (RS) induced p53 activation and display severe nutrient shortage-induced hepatosteatosis. And very recently we found that in response to metabolic stress (MeS), AMPK directly phosphorylates serine 342 of MDMX, leading to p53 activation, which is defective in MDMX3SA mice harboring a mutant MDMX with S341A, S367A and S402A mutations. In the current study, we want to determine if the dual defects in the MDM2/MDMX-p53 pathways might thoroughly impair p539s ability to regulate metabolic homeostasis and thus favor metabolism-related tumorigenesis, by generating a double knock-in (DKI) MDMX3SA/MDM2C305F mouse line. Our results indicate that DKI mice on normal chow diet exhibit mild, but significant, metabolism abnormalities, including increased body weight, higher fasting-glucose level and impaired glucose tolerance, which is exacerbated by high-fat-diet feeding. Consistently, MEFs isolated from DKI mice possess higher capacity of differentiation into adipocytes, and less beige fat is induced in the subcutaneous white adipose tissue of DKI mice after chronic cold exposure. Furthermore, we observe that the survival rate of DKI mice is significantly lower compared to either control or single knock-in groups, which may be due to the higher morbidity of cancers in DKI mice, including hepatoma, lymphoma and lung carcinoma. Because of these observations, we further specifically investigate if dual defects of these p53 activation pathways will accelerate hepatocellular carcinogenesis or not. We indeed find that treatment of carcinogen DEN causes more and bigger tumor nodules in the liver of DKI mice. These results suggest that both RS-MDM2-p53 and MeS-MDMX-p53 pathways play important roles in regulation of global metabolism, and dual defects of these pathways benefit metabolism-related tumor formation and growth. Further study is required to establish the direct effect of the p53 pathway defects-mediated metabolic disruption on tumorigenesis, and determine the underlying mechanisms. Citation Format: Hua Lu, Yi-wei zhang, Peng Liao, Wenjuan Liao, Jiaxiang Chen, Shelya X. Zeng. Dual defects of MDM2/MDMX-p53 pathways cause global metabolic disruption and enhance tumorigenesis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 891.

Abstract 4474: Dual targeting of the stress-p53 pathway as a potential anti-cancer therapy

The “ribosomal stress (RS)-p53 pathway” is triggered by any stressor or genetic alteration that disrupts ribosomal biogenesis, and mediated by several ribosomal proteins (RPs), such as RPL11 and RPL5, which inhibit MDM2 and activate p53. IMPDH2 is a rate-limiting enzyme in de novo guanine nucleotide biosynthesis and crucial for maintaining cellular guanine deoxy- and ribonucleotide pools needed for DNA and RNA synthesis. It is highly expressed in many malignancies. We previously showed that inhibition of IMPDH2 leads to p53 activation by causing RS. Surprisingly, our current study reveals that Inauzhin (INZ), a novel non-genotoxic p53 activator by inhibiting SIRT1, can also inhibit cellular IMPDH2 activity, and reduce the levels of cellular GTP and GTP-binding nucleostemin that is essential for rRNA processing. Consequently, INZ induces RS and the RPL11/RPL5-MDM2 interaction, activating p53. These results support the new notion that INZ suppresses cancer cell growth by dually targeting SIRT1 and IMPDH2. Note: This abstract was not presented at the meeting. Citation Format: Hua Lu, Qi Zhang, Shelya X. Zeng, Rui-Zhi Wu, Yiwei Zhang, Daniel Nguyen, Xiang Zhou, Jun-ming Liao, Bo Cao. Dual targeting of the stress-p53 pathway as a potential anti-cancer therapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4474. doi:10.1158/1538-7445.AM2015-4474