Amplification of the MYCN gene leads to its overexpression at both the mRNA and protein levels. Overexpression of MYCN mRNA may also have an important role in promoting neuroblastoma (NB) beyond the translation of MYCN protein. In the present study, we report a small molecule compound (MX25-1) that was able to bind to the 3’UTR of MYCN mRNA and induce MYCN mRNA degradation; this resulted in potent cell-growth inhibition and cell death specifically in MYCN-amplified or MYCN 3’UTR overexpressing NB cells. To evaluate the role of MYCN 3’UTR-mediated signals in contributing to the anticancer activity of MX25-1, we examined the status and activation of the tumor suppressor microRNA (miRNA) let-7, which is a target of MYCN 3’UTR in MYCN-amplified NB. We first observed that overexpression of MYCN mRNA was associated with high-level expression of the let-7 oncogenic targets DICER1, ARID3B and HMGA2. Following MYCN mRNA degradation, the expression of DICER1, ARID3B and HMGA2 was downregulated in MX25-1-treated cells. Inhibition of let-7 reversed the downregulation of these oncogenic mRNAs and significantly increased resistance of NB cells to MX25-1. Our results from this study supported the notion that overexpression of MYCN mRNA due to gene amplification has an independent function in NB cell growth and disease progression and suggest that targeting MYCN mRNA may represent an attractive strategy for therapy of MYCN amplified NB, both by inhibiting MYCN’s cell-survival effects and activating the tumor-suppressor effect of let-7.
Background: It is known that the MDM2 protein is stabilized when it forms a heterodimer with its partner MDM4, but MDM2 protein stability in its homodimer form is not known. The MDM2 protein contains a C-terminal RING domain that not only functions as an E3 ligase to regulate ubiquitination of p53 and MDM2 itself, but also is characterized to be able to bind several specific cellular mRNAs to regulate gene expression. In this study, we evaluate whether the MDM2 protein stability is regulated by the binding of a specific small RNA (XIAP IRES mRNA).
Methods: We performed chemical cross-linking and bimolecular fluorescence complementation (BiFC) assay to measure the human MDM2 protein stability in its homodimer form and the effect of XIAP IRES on MDM2 homodimerization and protein stabilization. Ubiquitination and pulse-chase assays were used to detect MDM2 self-ubiquitination and protein turn-over. Fluorescent titration and ITC were used to examine the binding between MDM2 RING protein and XIAP IRES. Western blot assay was used for determining protein expression. Clonogenic assay, WST and flow cytometry were used to test the effects of XIAP IRES, siXIAP and IR on cancer cell growth and apoptosis.
Results: We found that self-association (homodimerization) of MDM2 occurs through the C-terminal RING domain of MDM2 and that the MDM2 protein becomes unstable when it is homodimerized. MDM2 homodimerization resulted in an increased function of the RING domain for MDM2 self-ubiquitination. Binding of XIAP IRES to the RING domain inhibited MDM2 homodimerization and self-ubiquitination, which resulted in stabilization of MDM2, as well as increased XIAP expression. Upregulation of XIAP and MDM2 that led to inhibition of p53 by the XIAP IRES resulted in cell growth and survival in both p53-normal and -deficient cancer cells.
Conclusions: Our study identified a new IRES RNA that interacts with MDM2 protein and regulates its stabilization, which suggested that targeting of MDM2 through disruption of MDM2 protein-RNA interaction might be a useful strategy for developing novel anti-cancer therapeutics.
Inactivation of the tumor suppressor Ras-association domain family 1 isoform A (RASSF1A) due to epigenetic silencing occurs in a variety of human cancers, and still largely unknown are the regulators and mechanisms underlying RASSF1A gene promoter methylation. Herein, we report that this methylation is regulated by p53 and death-associated protein 6 (DAXX) in acute lymphoblastic leukemia (ALL). We found that p53 bound to the RASSF1A promoter, recruiting DAXX as well as DNA methyltransferase 1 (DNMT1) for DNA methylation, which subsequently resulted in inactivation of RASSF1A in wild-type p53 ALL cells. Although the presence of p53 was required for the recruitment of DAXX and DNMT1 to the RASSF1A promoter, fluctuation in p53 protein levels did not affect the rates of RASSF1A methylation. Conversely, methylation of RASSF1A promoter was critically controlled by DAXX, as the enforced overexpression of DAXX led to enhanced RASSF1A promoter methylation, whereas inhibition of DAXX reduced RASSF1A methylation. Interestingly, we found that the p53/DAXX-mediated RASSF1A methylation regulated murine double minute 2 (MDM2) protein stability in ALL. Our results reveal a novel function for p53 in the methylation of RASSF1A promoter by its interaction with DAXX. Discovery of this mechanism provides new insight into the interactions among the tumor-related factors p53, RASSF1A, DAXX, and MDM2 in cancer pathogenesis.—Zhang, H., He, J., Li, J., Tian, D., Gu, L., Zhou, M. Methylation of RASSF1A gene promoter is regulated by p53 and DAXX.
Previous studies show that the MYCN and MDM2-p53 signal pathways are mutually regulated: MYCN stimulates MDM2 and p53 transcription, whereas MDM2 stabilizes MYCN mRNA and induces its translation. Herein, we report that the interaction between MDM2 and MYCN plays a critical role in MYCN-amplified neuroblastoma tumor cell growth and survival. Distinct from the known role that MDM2 has in regulating tumor promotion in non-MYCN-amplified neuroblastoma, in which MDM2 inhibits p53, we found that MDM2 stimulated tumor growth in MYCN-amplified neuroblastoma in a p53-independent manner. In MYCN-amplified neuroblastoma cells, enforced expression of MDM2 further enhanced MYCN expression, yet no p53 inhibition was observed by MDM2 due to upregulation of MYCN that stimulated p53 transcription. Similarly, p53 expression remained unchanged in MDM2-silenced MYCN-amplified neuroblastoma cells because MDM2 inhibition resulted in a downregulation of MYCN that decreased p53 transcription, although the MDM2-mediated degradation of p53 was reduced. Also, we found that the enforced overexpression of MDM2, or conversely, the inhibition of overexpressed endogenous MDM2, led to either a remarkable increase or decrease in tumor growth, respectively, in MYCN-amplified neuroblastoma (even though no p53 function was involved). These results suggest that p53 that is reciprocally regulated by MDM2 and MYCN is dispensable for suppression of MYCN-amplified neuroblastoma, and that the direct interaction between MDM2 and MYCN may contribute significantly to MYCN-amplified neuroblastoma growth and disease progression.
Nilotinib is a selective BCR-ABL tyrosine kinase inhibitor related to imatinib that is more potent than imatinib. Nilotinib is widely used to treat chronic myelogenous leukemia (CML) and Philadelphia-positive (Ph+) acute lymphoblastic leukemia (ALL). The present study identifies Mouse double minute 2 homolog (MDM2) as a target of nilotinib. In studying ALL cell lines, we found that the expression of MDM2 in both Philadelphia positive (Ph+) and Philadelphia negative (Ph-) ALL cells was remarkably inhibited by nilotinib, in a dose- and time-dependent manner. Further studies demonstrated that nilotinib inhibited MDM2 at the post-translational level by inducing MDM2 self-ubiquitination and degradation. Nilotinib-mediated MDM2 downregulation did not result in accumulation and activation of p53. Inhibition of MDM2 in nilotinib-treated ALL cells led to downregulation of the anti-apoptotic protein X-linked inhibitor of apoptosis protein (XIAP), a translational target of MDM2, resulting in activation of caspases. Inhibition of XIAP following nilotinib-mediated downregulation of MDM2 resulted in apoptosis of MDM2-expressing ALL; however, similar nilotinib treatment induced stronger apoptosis in Ph+/MDM2+ ALL than in Ph-/MDM2+ or Ph+/MDM2- ALL. The ALL cells that were Ph-/MDM2- were totally resistant to nilotinib. These results suggested that nilotinib can inhibit MDM2 and induce a p53-independent apoptosis pathway by downregulating XIAP; thus, nilotinib can treat not only Ph+, but also Ph- ALL patients whose cancer cells overexpress MDM2.
Background
Tissue factor (TF) is a transmembrane protein that acts as a receptor for activated coagulation factor VII (FVIIa), initiating the coagulation cascade. Recent studies demonstrate that expression of tumor-derived TF also mediates intracellular signaling relevant to tumor growth and apoptosis. Our present study investigates the possible mechanism by which the interaction between TF and FVIIa regulates chemotherapy resistance in neuroblastoma cell lines.
Methods
Gene and siRNA transfection was used to enforce TF expression in a TF-negative neuroblastoma cell line and to silence endogenous TF expression in a TF-overexpressing neuroblastoma line, respectively. The expression of TF, Bcl-2, STAT5, and Akt as well as the phosphorylation of STAT5 and Akt in gene transfected cells or cells treated with JAK inhibitor and LY294002 were determined by Western blot assay. Tumor cell growth was determined by a clonogenic assay. Cytotoxic and apoptotic effect of doxorubicin on neuroblastoma cell lines was analyzed by WST assay and annexin-V staining (by flow cytometry) respectively.
Results
Enforced expression of TF in a TF-negative neuroblastoma cell line in the presence of FVIIa induced upregulation of Bcl-2, leading to resistance to doxorubicin. Conversely, inhibition of endogenous TF expression in a TF-overexpressing neuroblastoma cell line using siRNA resulted in down-regulation of Bcl-2 and sensitization to doxorubicin-induced apoptosis. Additionally, neuroblastoma cells expressing high levels of either endogenous or transfected TF treated with FVIIa readily phosphorylated STAT5 and Akt. Using selective pharmacologic inhibitors, we demonstrated that JAK inhibitor I, but not the PI3K inhibitor LY294002, blocked the TF/FVIIa-induced upregulation of Bcl-2.
Conclusion
This study shows that in neuroblastoma cell lines overexpressed TF ligated with FVIIa produced upregulation of Bcl-2 expression through the JAK/STAT5 signaling pathway, resulting in resistance to apoptosis. We surmise that this TF-FVIIa pathway may contribute, at least in part, to chemotherapy resistance in neuroblastoma.
MDM4 and topoisomerase IIα (TOP2A) are overexpressed in various human cancers. MDM4 acts as an oncoprotein which promotes cancer progression by inhibiting tumor suppressor p53. As a DNA replication- and cell division-regulating enzyme, TOP2A is the main target of many anticancer therapy regimens; however, the exact role of TOP2A in cancer remains elusive. Herein, we report that MDM4 and TOP2A bind to each other and are mutually upregulated at the post-translational level, leading to TOP2A protein stabilization, inhibition of p53, and increased tumor-cell proliferation. We demonstrate that the C-terminal region (CTR) of TOP2A binds to a unique sequence (residues: 188–238) of MDM4, which contains an auto-inhibitory segment regulating the MDM4-p53 interaction. TOP2A binding in turn activates MDM4 for p53 binding, resulting in enhanced inhibition of p53 and cancer cell proliferation. Conversely, binding of the MDM4 sequence to the CTR of TOP2A stabilizes TOP2A protein, leading to increased TOP2A protein expression. These results reveal novel functions of MDM4 and TOP2A as well as their interactions in oncogenesis, suggesting that inhibition of the MDM4-TOP2A interaction may represent a novel strategy in specifically and simultaneously targeting TOP2A and MDM4 for cancer treatment.
TRAF1 is a member of the TRAF family, which plays important roles in signal transduction that mediate cell life and death in the immune response, inflammatory and malignant diseases. It is known that TRAF1 transcription is inducible by various cytokines, but little is known about the regulation of its mRNA translation. In the present study, we demonstrated that the human TRAF1 mRNA has an unusually long 5′-UTR that contains internal ribosome entry segment (IRES) regulating its translation. By performing gene transfection and reporter assays, we revealed that this IRES sequence is located within the 572 nt upstream from the AUG start codon. An element between nt −392 and −322 was essential for the IRES activity. Interestingly, we found that the TRAF1 expression is induced in cancer cells by chemotherapeutic drug vincristine that regulates cytoplasmic localization of polypyrimidine tract binding protein, which may contribute to the IRES-dependent translation of TRAF1 during vincristine treatment. These results indicate that TRAF1 translation is initiated via the IRES and regulated by vincristine, and suggest that regulation of the IRES-dependent translation of TRAF1 may be involved in effecting the cancer cell response to vincristine treatment.
The FK506-binding protein 12 (FKBP12) is a cytoplasmic protein and has been reported to possess multiple functions in signaling transduction based on its interaction with different cellular targets. Here, we report that FKBP12 interacts with oncoprotein MDM2 and induces MDM2 degradation. We demonstrate that FKBP12 degrades MDM2 through binding to MDM2 protein, disrupting MDM2/MDM4 interaction and inducing MDM2 self-ubiquitination. The FKBP12-mediated MDM2 degradation was significantly enhanced when the transfected MDM2 was localized in the cytoplasm. The endogenous MDM2, when it was induced by p53 subjecting to DNA-damaging stimuli such as treatment with doxorubicin, was also significantly inhibited by FKBP12. This is due to translocation of p53-induced MDM2 from the nucleus to the cytoplasm, which facilitates interaction with cytoplasmic FKBP12. Furthermore, the enhanced level of MDM2 following p53 activation in nutlin-3 treated cells was also inhibited by FKBP12. The FKBP12-mediated downregulation of MDM2 in response to doxorubicin or nutlin-3 results in continuing and constitutive activation of p53, inhibition of XIAP and sensitization of cancer cells to apoptosis. These results identify a novel function for FKBP12 in downregulating MDM2, which directly enhances sensitivity of cancer cells to chemotherapy and nutlin-3 treatment.
A novel small-molecule anthraquinone (AQ) analogue, AQ-101, which was synthesized through chemical modification of the core structures of rhein, exhibited potent anticancer activity. In the present study, we evaluated the cancer-inhibiting mechanism of AQ-101 and tested the therapeutic potential of this compound for treating cancer in mice. We found that AQ-101 was able to induce MDM2 protein degradation through a selfubiquitination and proteasome-mediated mechanism. This AQ-101-induced MDM2 downregulation led to activation of p53, which contributed to apoptosis of acute lymphoblastic leukemia (ALL), especially those with a wild-type p53 phenotype andMDM2 expression in vitro and in vivo. When given for a period of 2 weeks (20 mg/kg/day, 3/week), AQ-101 inhibited development of ALL in nude or SCID mice with a human ALL xenograft and achieved cure by the end of the 5-month experiment. Importantly, AQ-101 showed minimal or no inhibitory effect on normal human hematopoiesis in vitro and was well tolerated in vivo in animal models. Given that MDM2-overexpressing cancers are commonly refractory to current treatment options, our study results suggest that further development of AQ-101 is warranted, as it represents a potentially new, safe anticancer drug with a novel strategy for targeting MDM2.