Xiongbin Lu

p53 mutant mice that display early ageing-associated phenotypes.

The p53 tumour suppressor is activated by numerous stressors to induce apoptosis, cell cycle arrest, or senescence. To study the biological effects of altered p53 function, we generated mice with a deletion mutation in the first six exons of the p53 gene that express a truncated RNA capable of encoding a carboxy-terminal p53 fragment. This mutation confers phenotypes consistent with activated p53 rather than inactivated p53. Mutant (p53+/m) mice exhibit enhanced resistance to spontaneous tumours compared with wild-type (p53+/+) littermates. As p53+/m mice age, they display an early onset of phenotypes associated with ageing. These include reduced longevity, osteoporosis, generalized organ atrophy and a diminished stress tolerance. A second line of transgenic mice containing a temperature-sensitive mutant allele of p53 also exhibits early ageing phenotypes. These data suggest that p53 has a role in regulating organismal ageing.

The ATM Kinase Induces MicroRNA Biogenesis in the DNA Damage Response

The DNA damage response involves a complex network of processes that detect and repair DNA damage. Here we show that miRNA biogenesis is globally induced upon DNA damage in an ATM-dependent manner. About one-fourth of miRNAs are significantly upregulated after DNA damage, while loss of ATM abolishes their induction. KH-type splicing regulatory protein (KSRP) is a key player that translates DNA damage signaling to miRNA biogenesis. The ATM kinase directly binds to and phosphorylates KSRP, leading to enhanced interaction between KSRP and pri-miRNAs and increased KSRP activity in miRNA processing. Mutations of the ATM phosphorylation sites of KSRP impaired its activity in regulating miRNAs. These findings reveal a mechanism by which DNA damage signaling is linked to miRNA biogenesis.

The type 2C phosphatase Wip1: An oncogenic regulator of tumor suppressor and DNA damage response pathways

The Wild-type p53-induced phosphatase 1, Wip1 (or PPM1D), is unusual in that it is a serine/threonine phosphatase with oncogenic activity. A member of the type 2C phosphatases (PP2Cδ), Wip1 has been shown to be amplified and overexpressed in multiple human cancer types, including breast and ovarian carcinomas. In rodent primary fibroblast transformation assays, Wip1 cooperates with known oncogenes to induce transformed foci. The recent identification of target proteins that are dephosphorylated by Wip1 has provided mechanistic insights into its oncogenic functions. Wip1 acts as a homeostatic regulator of the DNA damage response by dephosphorylating proteins that are substrates of both ATM and ATR, important DNA damage sensor kinases. Wip1 also suppresses the activity of multiple tumor suppressors, including p53, ATM, p16INK4a and ARF. We present evidence that the suppression of p53, p38 MAP kinase, and ATM/ATR signaling pathways by Wip1 are important components of its oncogenicity when it is amplified and overexpressed in human cancers.

Oncogenic Wip1 Phosphatase Is Inhibited by miR-16 in the DNA Damage Signaling Pathway

Wild-type p53-induced phosphatase 1 (Wip1) was identified as an oncogene amplified and overexpressed in several human cancers. Recent evidence suggested that Wip1 is a critical inhibitor in the ATM/ATR-p53 DNA damage signaling pathway. Wip1 dephosphorylates several key DNA damage-responsive proteins and reverses DNA damage-induced cell cycle checkpoints. Previous reports showed that Wip1 was transcriptionally induced by p53 at the early stage of the DNA damage response. To investigate the temporal and functional regulation of Wip1, we identified a microRNA, miR-16, that specifically targets the mRNA of Wip1 and thus negatively regulates the expression level of Wip1. miR-16 itself is induced immediately after DNA damage. Therefore, the increase in Wip1 protein level is significantly postponed compared with that of its mRNA level, preventing a premature inactivation of ATM/ATR signaling and allowing a functional completion of the early DNA damage response. To better understand miR-16 biological functions in the context of cancer cells, we examined its expression in mammary tumor stem cells and found it to be markedly downregulated in mammary tumor stem cells. Overexpression of miR-16 or inhibition of Wip1 suppresses the self-renewal and growth of mouse mammary tumor stem cells and sensitizes MCF-7 human breast cancer cells to the chemotherapeutic drug doxorubicin. Together, our results suggest an important role of miR-16 in the regulation of Wip1 phosphatase in the DNA damage response and mammary tumorigenesis.

Synthesis and characterization of thermally responsive pluronic F127-chitosan nanocapsules for controlled release and intracellular delivery of small molecules

In this study, we synthesized empty core-shell structured nanocapsules of Pluronic F127 and chitosan and characterized the thermal responsiveness of the nanocapsules in size and wall-permeability. Moreover, we determined the feasibility of using the nanocapsules to encapsulate small molecules for temperature-controlled release and intracellular delivery. The nanocapsules are ∼37 nm at 37 °C and expand to ∼240 nm when cooled to 4 °C in aqueous solutions, exhibiting >200 times change in volume. Moreover, the permeability of the nanocapsule wall is high at 4 °C (when the nanocapsules are swollen), allowing free diffusion of small molecules (ethidium bromide, MW = 394.3 Da) across the wall, while at 37 °C (when the nanocapsules are swollen), the wall-permeability is so low that the small molecules can be effectively withheld in the nanocapsule for hours. As a result of their thermal responsiveness in size and wall-permeability, the nanocapsules are capable of encapsulating the small molecules for temperature-controlled release and intracellular delivery into the cytosol of both cancerous (MCF-7) and noncancerous (C3H10T1/2) mammalian cells. The cancerous cells were found to take up the nanocapsules much faster than the noncancerous cells during 45 min incubation at 37 °C. Moreover, toxicity of the nanocapsules as a delivery vehicle was found to be negligible. The Pluronic F127-chitosan nanocapsules should be very useful for encapsulating small therapeutic agents to treat diseases particularly when it is combined with cryotherapy where the process of cooling and heating between 37 °C and hypothermic temperatures is naturally done.

Medulloblastomas overexpress the p53-inactivating oncogene WIP1/PPM1D

Medulloblastoma is the most common malignant brain tumor of childhood. Despite numerous advances, clinical challenges range from recurrent and progressive disease to long-term toxicities in survivors. The lack of more effective, less toxic therapies results from our limited understanding of medulloblastoma growth. Although TP53 is the most commonly altered gene in cancers, it is rarely mutated in medulloblastoma. Accumulating evidence, however, indicates that TP53 pathways are disrupted in medulloblastoma. Wild-typep53-induced phosphatase 1 (WIP1 or PPM1D) encodes a negative regulator of p53. WIP1 amplification (17q22-q23) and its overexpression have been reported in diverse cancer types. We examined primary medulloblastoma specimens and cell lines, and detected WIP1 copy gain and amplification prevalent among but not exclusively in the tumors with 17q gain and isochromosome 17q (i17q), which are among the most common cytogenetic lesions in medulloblastoma. WIP1 RNA levels were significantly higher in the tumors with 17q gain or i17q. Immunoblots confirmed significant WIP1 protein in primary tumors, generally higher in those with 17q gain or i17q. Under basal growth conditions and in response to the chemotherapeutic agent, etoposide, WIP1 antagonized p53-mediated apoptosis in medulloblastoma cell lines. These results indicate that medulloblastoma express significant levels of WIP1 that modulate genotoxic responsiveness by negatively regulating p53.

Long non-coding RNA ANRIL (CDKN2B-AS) is induced by the ATM-E2F1 signaling pathway

The maintenance of genome integrity is essential for the proper function and survival of all organisms. Human cells have evolved prompt and efficient DNA damage response to eliminate the detrimental effects of DNA lesions. The DNA damage response involves a complex network of processes that detect and repair DNA damage, in which long non-coding RNAs (lncRNAs), a new class of regulatory RNAs, may play important roles. Recent studies have identified a large number of lncRNAs in mammalian transcriptomes. However, little is known about the regulation and function of lncRNAs in the DNA damage response. In the present study, we demonstrate that one specific lncRNA, ANRIL, is transcriptionally up-regulated by the transcription factor E2F1 in an ATM-dependent manner following DNA damage, and elevated levels of ANRIL suppress the expression of INK4a, INK4b and ARF at the late-stage of DNA damage response, allowing the cell to return to normal at the completion of the DNA repair.

miRNA response to DNA damage.

Faithful transmission of genetic material in eukaryotic cells requires not only accurate DNA replication and chromosome distribution but also the ability to sense and repair spontaneous and induced DNA damage. To maintain genomic integrity, cells undergo a DNA damage response using a complex network of signaling pathways composed of coordinate sensors, transducers and effectors in cell cycle arrest, apoptosis and DNA repair. Emerging evidence has suggested that miRNAs play a crucial role in regulation of DNA damage response. In this review, we discuss the recent findings on how miRNAs interact with the canonical DNA damage response and how miRNA expression is regulated after DNA damage.

Chitosan-Decorated Doxorubicin-Encapsulated Nanoparticle Targets and Eliminates Tumor Reinitiating Cancer Stem-like Cells.

Tumor reinitiating cancer stem-like cells are responsible for cancer recurrence associated with conventional chemotherapy. We developed a doxorubicin-encapsulated polymeric nanoparticle surface-decorated with chitosan that can specifically target the CD44 receptors of these cells. This nanoparticle system was engineered to release the doxorubicin in acidic environments, which occurs when the nanoparticles are localized in the acidic tumor microenvironment and when they are internalized and localized in the cellular endosomes/lysosomes. This nanoparticle design strategy increases the cytotoxicity of the doxorubicin by six times in comparison to the use of free doxorubicin for eliminating CD44(+) cancer stem-like cells residing in 3D mammary tumor spheroids (i.e., mammospheres). We further show these nanoparticles reduced the size of tumors in an orthotopic xenograft tumor model with no evident systemic toxicity. The development of nanoparticle system to target cancer stem-like cells with low systemic toxicity provides a new treatment arsenal for improving the survival of cancer patients.

USP4 inhibits p53 through deubiquitinating and stabilizing ARF-BP1.

Tumour suppressor p53 levels in the cell are tightly regulated by controlled degradation through ubiquitin ligases including Mdm2, COP1, Pirh2, and ARF‐BP1. The ubiquitination process is reversible via deubiquitinating enzymes, such as ubiquitin‐specific peptidases (USPs). In this study, we identified ubiquitin‐specific peptidase 4 (USP4) as an important regulator of p53. USP4 interacts directly with and deubiquitinates ARF‐BP1, leading to the stabilization of ARF‐BP1 and subsequent reduction of p53 levels. Usp4 knockout mice are viable and developmentally normal, but showed enhanced apoptosis in thymus and spleen in response to ionizing radiation. Compared with wild‐type mouse embryonic fibroblasts (MEFs), Usp4−/− MEFs exhibited retarded growth, premature cellular senescence, resistance to oncogenic transformation, and hyperactive DNA damage checkpoints, consistent with upregulated levels and activity of p53 in the absence of USP4. Finally, we showed that USP4 is overexpressed in several types of human cancer, suggesting that USP4 is a potential oncogene.