F-box protein FBXO31 directs degradation of MDM2 to facilitate p53-mediated growth arrest following genotoxic stress

The tumor suppressor p53 plays a critical role in maintaining genomic stability. In response to genotoxic stress, p53 levels increase and induce cell-cycle arrest, senescence, or apoptosis, thereby preventing replication of damaged DNA. In unstressed cells, p53 is maintained at a low level. The major negative regulator of p53 is MDM2, an E3 ubiquitin ligase that directly interacts with p53 and promotes its polyubiquitination, leading to the subsequent destruction of p53 by the 26S proteasome. Following DNA damage, MDM2 is degraded rapidly, resulting in increased p53 stability. Because of the important role of MDM2 in modulating p53 function, it is critical to understand how MDM2 levels are regulated. Here we show that the F-box protein FBXO31, a candidate tumor suppressor encoded in 16q24.3 for which there is loss of heterozygosity in various solid tumors, is responsible for promoting MDM2 degradation. Following genotoxic stress, FBXO31 is phosphorylated by the DNA damage serine/threonine kinase ATM, resulting in increased levels of FBXO31. FBXO31 then interacts with and directs the degradation of MDM2, which is dependent on phosphorylation of MDM2 by ATM. FBXO31-mediated loss of MDM2 leads to elevated levels of p53, resulting in growth arrest. In cells depleted of FBXO31, MDM2 is not degraded and p53 levels do not increase following genotoxic stress. Thus, FBXO31 is essential for the classic robust increase in p53 levels following DNA damage

Oncogenic RAS directs silencing of tumor suppressor genes through ordered recruitment of transcriptional repressors

We previously identified 28 cofactors through which a RAS oncoprotein directs transcriptional silencing of Fas and other tumor suppressor genes (TSGs). Here we performed RNAi-based epistasis experiments and found that RAS-directed silencing occurs through a highly ordered pathway that is initiated by binding of ZFP354B, a sequence-specific DNA-binding protein, and culminates in recruitment of the DNA methyltransferase DNMT1. RNAi and pharmacological inhibition experiments reveal that silencing requires continuous function of RAS and its cofactors and can be rapidly reversed, which may have therapeutic implications for reactivation of silenced TSGs in RAS-positive cancers

F-box protein FBXO16 functions as a tumor suppressor by attenuating nuclear beta-catenin function

Aberrant activation of beta-catenin has been implicated in a variety of human diseases, including cancer. In spite of significant progress, the regulation of active Wnt/beta-catenin-signaling pathways is still poorly understood. In this study, we show that F-box protein 16 (FBXO16) is a putative tumor suppressor. It is a component of the SCF (SKP1-Cullin1-F-box protein) complex, which targets the nuclear beta-catenin protein to facilitate proteasomal degradation through the 26S proteasome. FBXO16 interacts physically with the C-terminal domain of beta-catenin and promotes its lysine 48-linked polyubiquitination. In addition, it inhibits epithelial-to-mesenchymal transition (EMT) by attenuating the level of beta-catenin. Therefore, depletion of FBXO16 leads to increased levels of beta-catenin, which then promotes cell invasion, tumor growth, and EMT of cancer cells. Furthermore, FBXO16 and beta-catenin share an inverse correlation of cellular expression in clinical breast cancer patient samples. In summary, we propose that FBXO16 functions as a putative tumor suppressor by forming an SCF(FBXO16) complex that targets nuclear beta-catenin in a unique manner for ubiquitination and subsequent proteasomal degradation to prevent malignancy. This work suggests a novel therapeutic strategy against human cancers related to aberrant beta-catenin activation

Tumor suppressor SMAR1 represses IκBα expression and inhibits p65 transactivation through matrix attachment regions

Aberrant NF-κB activity promotes tumorigenesis. However, NF-κB also inhibits tumor growth where tumor suppressor pathways remain unaltered. Thus, its role in tumorigenesis depends upon the function of other cellular factors. Tumor suppressor SMAR1 down-modulated in high grade breast cancers is regulated by p53 and is reported to interact and stabilize p53. Because both SMAR1 and NF-κB are involved in tumorigenesis, we investigated the effect of SMAR1 upon NF-κB activity. We show that SMAR1 induction by doxorubicin or overexpression produces functional NF-κB complexes that are competent for binding to NF-κB consensus sequence. However, SMAR1 induced p65-p50 complex is phosphorylation- and transactivation-deficient. Induction of functional NF-κB complexes stems from down-regulation of IκBα transcription through direct binding of SMAR1 to the matrix attachment region site present in IκBα promoter and recruitment of corepressor complex. Real time PCR array for NF-κB target genes revealed that SMAR1 down-regulates a subset of NF-κB target genes that are involved in tumorigenesis. We also show that SMAR1 inhibits tumor necrosis factor α-induced induction of NF-κB suggesting that activation of NF-κB by SMAR1 is independent and different from classical pathway. Thus, for the first time we report that a tumor suppressor protein SMAR1 can modulate NF-κB transactivation and inhibit tumorigenesis by regulating NF-κB target genes

Gene regulation by SMAR1: role in cellular homeostasis and cancer

Changes in the composition of nuclear matrix associated proteins contribute to alterations in nuclear structure, one of the major phenotypes of malignant cancer cells. The malignancy-induced changes in this structure lead to alterations in chromatin folding, the fidelity of genome replication and gene expression programs. The nuclear matrix forms a scaffold upon which the chromatin is organized into periodic loop domains called matrix attachment regions (MAR) by binding to various MAR binding proteins (MARBPs). Aberrant expression of MARBPs modulates the chromatin organization and disrupt transcriptional network that leads to oncogenesis. Dysregulation of nuclear matrix associated MARBPs has been reported in different types of cancers. Some of these proteins have tumor specific expression and are therefore considered as promising diagnostic or prognostic markers in few cancers. SMAR1 (scaffold/matrix attachment region binding protein 1), is one such nuclear matrix associated protein whose expression is drastically reduced in higher grades of breast cancer. SMAR1 gene is located on human chromosome 16q24.3 locus, the loss of heterozygosity (LOH) of which has been reported in several types of cancers. This review elaborates on the multiple roles of nuclear matrix associated protein SMAR1 in regulating various cellular target genes involved in cell growth, apoptosis and tumorigenesis

Coordinated regulation of p53 apoptotic targets BAX and PUMA by SMAR1 through an identical MAR element

How tumour suppressor p53 bifurcates cell cycle arrest and apoptosis and executes these distinct pathways is not clearly understood. We show that BAX and PUMA promoters harbour an identical MAR element and are transcriptional targets of SMAR1. On mild DNA damage, SMAR1 selectively represses BAX and PUMA through binding to the MAR independently of inducing p53 deacetylation through HDAC1. This generates an anti-apoptotic response leading to cell cycle arrest. Importantly, knockdown of SMAR1 induces apoptosis, which is abrogated in the absence of p53. Conversely, apoptotic DNA damage results in increased size and number of promyelocytic leukaemia (PML) nuclear bodies with consequent sequestration of SMAR1. This facilitates p53 acetylation and restricts SMAR1 binding to BAX and PUMA MAR leading to apoptosis. Thus, our study establishes MAR as a damage responsive cis element and SMAR1-PML crosstalk as a switch that modulates the decision between cell cycle arrest and apoptosis in response to DNA damage

Comparative Evaluation of Löwenstein-Jensen Proportion Method, BacT/ALERT 3D System, and Enzymatic Pyrazinamidase Assay for Pyrazinamide Susceptibility Testing of Mycobacterium tuberculosis

Pyrazinamide (PZA) is an important first-line antituberculosis drug because of its sterilizing activity against semidormant tubercle bacilli. In spite of its very high in vivo activity, its in vitro activity is not apparent unless an acidic environment is available, which makes PZA susceptibility testing difficult by conventional methods. The present study was, therefore, planned to assess the performance of the colorimetric BacT/ALERT 3D system and compare the results with those from conventional tests, i.e., the Löwenstein-Jensen (LJ) proportion method (pH 4.85) and Wayne's pyrazinamidase (PZase) assay, using 107 clinical isolates. The concordance among all of these tests was 89.71% after the first round of testing and reached 92.52% after resolution of the discordant results by retesting. Prolonged incubation of the PZase tube for up to 10 days was found to increase the specificity of the PZase test. The concordances between LJ proportion and BacT/ALERT 3D, LJ proportion and the PZase assay, and BacT/ALERT 3D and the PZase assay were found to be 99.06%, 93.46%, and 92.52%, respectively. Using the LJ results as the gold standard, the sensitivities of BacT/ALERT 3D and the PZase assay were 100 and 82.85%, respectively, while the specificity was 98.61% for both of the tests. The difference between the sensitivities of BacT/ALERT 3D and the PZase assay was significant (P = 0.025). The mean turnaround times for the detection of resistant and susceptible results by BacT/ALERT 3D were 8.04 and 11.32 days, respectively. While the major limitations associated with the PZase assay and the LJ proportion method are lower sensitivity in previously treated patients and a longer time requirement, respectively, the BacT/ALERT 3D system was found to be rapid, highly sensitive, and specific