JNK Phosphorylation of p53 Results in a p53-p73 Complex to Induce Apoptosis

poster abstractTumor protein p53 is the most commonly mutated tumor suppressor in human cancers. The p53 family consists of three proteins p53, p63, and p73, which are all transcription factors. Mutant p53 functions as a dominant negative through the interaction with either p63 or p73, while wild-type p53 has yet to be shown heterodimerize with these family members. In order to therapeutically target many human cancers, it is critical to understand how p53 functions in cells to bring about apoptosis. In this study, we first verified mutant p53 complexes with p73 in U373 human glioblastoma astrocytoma cells. In addition, we found that mutant p53 was phosphorylated at threonine 81 (T81), a site proximal to the proline rich domain of p53, which is responsible for induction of apoptosis. This led us to examine wild type p53. We treated U87 glioblastoma cells with chemotherapeutics agents and found wild-type p53 to interact with p73. Since T81 was phosphorylated in the mutant, we examined wild type p53 and found that it was also modified. We explored the possibility that phosphorylation at T81 was central to the heterodimerization of p53 and p73. The JNK kinase has been reported to phosphorylate T81 of p53. We found that activation of JNK resulted in p53-p73 interaction, while blockade of JNK prevented binding of p53 with p73. The interaction of phosphorylated T81 of p53 and the resultant p53-p73 complex binds to the apoptotic promoters of puma and bax and induces gene expression. Thus, our study provides the first evidence that wild-type p53 forms a complex with p73 to induce apoptosis

Emerging Non-Canonical Functions and Regulation by p53: p53 and Stemness

Since its discovery nearly 40 years ago, p53 has ascended to the forefront of investigated genes and proteins across diverse research disciplines and is recognized most exclusively for its role in cancer as a tumor suppressor. Levine and Oren (2009) reviewed the evolution of p53 detailing the significant discoveries of each decade since its first report in 1979. In this review, we will highlight the emerging non-canonical functions and regulation of p53 in stem cells. We will focus on general themes shared among p53's functions in non-malignant stem cells and cancer stem-like cells (CSCs) and the influence of p53 on the microenvironment and CSC niche. We will also examine p53 gain of function (GOF) roles in stemness. Mutant p53 (mutp53) GOFs that lead to survival, drug resistance and colonization are reviewed in the context of the acquisition of advantageous transformation processes, such as differentiation and dedifferentiation, epithelial-to-mesenchymal transition (EMT) and stem cell senescence and quiescence. Finally, we will conclude with therapeutic strategies that restore wild-type p53 (wtp53) function in cancer and CSCs, including RING finger E3 ligases and CSC maintenance. The mechanisms by which wtp53 and mutp53 influence stemness in non-malignant stem cells and CSCs or tumor-initiating cells (TICs) are poorly understood thus far. Further elucidation of p53's effects on stemness could lead to novel therapeutic strategies in cancer research

ELEVATED LEVELS OF PLATELETS AND MDM2 EXPRESSION ARE CONTRIB-UTING FACTORS TO FACILITATING THE METASTASIS OF OSTEOSARCOMA

poster abstractOsteosarcoma (OS) is the most common form of primary bone cancer and the 6th leading cause of cancer in pediatric patients. A chart review of OS patients treated at this institution suggests that a high platelet count at di-agnosis is significantly (p=0.023) and inversely associated with the first year of survival. As the effects of platelet interaction with OS have been exten-sively researched and suggest that platelets may facilitate tumor metastasis, and the most important prognostic factor for OS patient survival is metasta-sis to the lungs, we hypothesized that platelets increase metastasis to the lungs and reduce survival. Therefore, we sought to determine whether in-creasing platelet numbers in a well characterized OS mouse model would de-crease survival and/or increase metastasis to the lungs. We found that thrombopoietin (TPO) treated mice, had increased platelet numbers, died earlier than placebo treated controls, and that lungs from TPO treated mice contained a small number of large tumor cells (most metastatic lesions were 2-4 cells), whereas lungs from placebo treated controls showed no signs of metastases. Next, an OS tissue microarray (TMA) was built from OS patients seen at our institution over the past 10 years. Mdm2, p53, TPO, and c-mpl expression were evaluated by immunohistochemical (IHC) staining followed by quantitation using the Aperio Imaging system and analysis software. C-mpl (TPO receptor) expression was higher in the metastatic than the primary tumors, suggesting that platelets may contribute to the metastasis of OS. Elevated levels of Mdm2 correlated with metastasis and lower levels of p53, as detected by IHC. In conclusion, both the mouse model and the human OS data were similar, suggesting that both platelets and Mdm2 promote metas-tases in OS

The fate of murine double minute X (MdmX) is dictated by distinct signaling pathways through murine double minute 2 (Mdm2)

Mouse double minute 2 (Mdm2) and MdmX dimerize in response to low levels of genotoxic stress to function in a ubiquitinating complex, which signals for destabilization of p53. Under growth conditions, Mdm2 functions as a neddylating ligase, but the importance and extent of MdmX involvement in this process are largely unknown. Here we show that when Mdm2 functions as a neddylating enzyme, MdmX is stabilized. Furthermore, we demonstrate that under growth conditions, MdmX enhances the neddylation activity of Mdm2 on p53 and is a substrate for neddylation itself. Importantly, MdmX knockdown in MCF-7 breast cancer cells resulted in diminished neddylated p53, suggesting that MdmX is important for Mdm2-mediated neddylation. Supporting this finding, the lack of MdmX in transient assays or in p53/MdmX-/- MEFs results in decreased or altered neddylation of p53 respectively; therefore, MdmX is a critical component of the Mdm2-mediated neddylating complex. c-Src is the upstream activator of this Mdm2-MdmX neddylating pathway and loss of Src signaling leads to the destabilization of MdmX that is dependent on the RING (Really Interesting New Gene) domain of MdmX. Treatment with a small molecule inhibitor of neddylation, MLN4924, results in the activation of Ataxia Telangiectasia Mutated (ATM). ATM phosphorylates Mdm2, converting Mdm2 to a ubiquitinating enzyme which leads to the destabilization of MdmX. These data show how distinct signaling pathways engage neddylating or ubiquitinating activities and impact the Mdm2-MdmX axis

Src phosphorylation converts Mdm2 from a ubiquitinating to a neddylating E3 ligase

Murine double minute-2 protein (Mdm2) is a multifaceted phosphorylated protein that plays a role in regulating numerous proteins including the tumor suppressor protein p53. Mdm2 binds to and is involved in conjugating either ubiquitin or Nedd8 (Neural precursor cell expressed, developmentally down-regulated 8) to p53. Although regulation of the E3 ubiquitin activity of Mdm2 has been investigated, regulation of the neddylating activity of Mdm2 remains to be defined. Here we show that activated c-Src kinase phosphorylates Y281 and Y302 of Mdm2, resulting in an increase in Mdm2 stability and its association with Ubc12, the E2 enzyme of the neddylating complex. Mdm2-dependent Nedd8 conjugation of p53 results in transcriptionally inactive p53, a process that is reversed with a small molecule inhibitor to either Src or Ubc12. Thus, our studies reveal how Mdm2 may neutralize and elevate p53 in actively proliferating cells and also provides a rationale for using therapies that target the Nedd8 pathway in wild-type p53 tumors

Mutant and wild-type p53 form complexes with p73 upon phosphorylation by the kinase JNK

The transcription factors p53 and p73 are critical to the induction of apoptotic cell death, particularly in response to cell stress that activates c-Jun N-terminal kinase (JNK). Mutations in the DNA-binding domain of p53, which are commonly seen in cancers, result in conformational changes that enable p53 to interact with and inhibit p73, thereby suppressing apoptosis. In contrast, wild-type p53 reportedly does not interact with p73. We found that JNK-mediated phosphorylation of Thr81 in the proline-rich domain (PRD) of p53 enabled wild-type p53, as well as mutant p53, to form a complex with p73. Structural algorithms predicted that phosphorylation of Thr81 exposes the DNA-binding domain in p53 to enable its binding to p73. The dimerization of wild-type p53 with p73 facilitated the expression of apoptotic target genes [such as those encoding p53–up-regulated modulator of apoptosis (PUMA) and Bcl-2-associated X protein (BAX)] and, subsequently, the induction of apoptosis in response to JNK activation by cell stress in various cells. Thus, JNK phosphorylation of mutant and wild-type p53 promotes the formation of a p53/p73 complex that determines cell fate: apoptosis in the context of wild-type p53 or cell survival in the context of the mutant. These findings refine our current understanding of both the mechanistic links between p53 and p73 and the functional role for Thr81 phosphorylation

Mdm2 mediated neddylation of pVHL blocks the induction of anti-angiogenic factors.

Mutations in the tumor suppressor TP53 are rare in renal cell carcinomas. p53 is a key factor for inducing anti-angiogenic genes and RCC are highly vascularized, which suggests that p53 is inactive in these tumors. One regulator of p53 is the Mdm2 oncogene, which is correlated with high-grade, metastatic tumors. However, the sole activity of Mdm2 is not just to regulate p53, but it can also function independent of p53 to regulate the early stages of metastasis. Here, we report that the oncoprotein Mdm2 can bind directly to the tumor suppressor VHL, and conjugate nedd8 to VHL within a region that is important for the p53-VHL interaction. Nedd8 conjugated VHL is unable to bind to p53 thereby preventing the induction of anti-angiogenic factors. These results highlight a previously unknown oncogenic function of Mdm2 during the progression of cancer to promote angiogenesis through the regulation of VHL. Thus, the Mdm2-VHL interaction represents a pathway that impacts tumor angiogenesis

Megakaryocytes Regulate Expression of Pyk2 Isoforms and Caspase-mediated Cleavage of Actin in Osteoblasts

The proliferation and differentiation of osteoblast (OB) precursors are essential for elaborating the bone-forming activity of mature OBs. However, the mechanisms regulating OB proliferation and function are largely unknown. We reported that OB proliferation is enhanced by megakaryocytes (MKs) via a process that is regulated in part by integrin signaling. The tyrosine kinase Pyk2 has been shown to regulate cell proliferation and survival in a variety of cells. Pyk2 is also activated by integrin signaling and regulates actin remodeling in bone-resorbing osteoclasts. In this study, we examined the role of Pyk2 and actin in the MK-mediated increase in OB proliferation. Calvarial OBs were cultured in the presence of MKs for various times, and Pyk2 signaling cascades in OBs were examined by Western blotting, subcellular fractionation, and microscopy. We found that MKs regulate the temporal expression of Pyk2 and its subcellular localization. We also found that MKs regulate the expression of two alternatively spliced isoforms of Pyk2 in OBs, which may regulate OB differentiation and proliferation. MKs also induced cytoskeletal reorganization in OBs, which was associated with the caspase-mediated cleavage of actin, an increase in focal adhesions, and the formation of apical membrane ruffles. Moreover, BrdU incorporation in MK-stimulated OBs was blocked by the actin-polymerizing agent, jasplakinolide. Collectively, our studies reveal that Pyk2 and actin play an important role in MK-regulated signaling cascades that control OB proliferation and may be important for therapeutic interventions aimed at increasing bone formation in metabolic diseases of the skeleton

Early-Stage Metastasis Requires Mdm2 and Not p53 Gain of Function

Metastasis of cancer cells to distant organ systems is a complex process that is initiated with the programming of cells in the primary tumor. The formation of distant metastatic foci is correlated with poor prognosis and limited effective treatment options. We and others have correlated Mouse double minute 2 (Mdm2) with metastasis; however, the mechanisms involved have not been elucidated. Here, it is reported that shRNA-mediated silencing of Mdm2 inhibits epithelial–mesenchymal transition (EMT) and cell migration. In vivo analysis demonstrates that silencing Mdm2 in both post-EMT and basal/triple-negative breast cancers resulted in decreased primary tumor vasculature, circulating tumor cells, and metastatic lung foci. Combined, these results demonstrate the importance of Mdm2 in orchestrating the initial stages of migration and metastasis