Mikael S. Lindström

An ARF-independent c-Myc-activated tumor suppression pathway mediated by ribosomal protein-Mdm2 interaction

In vitro studies have shown that inhibition of ribosomal biogenesis can activate p53 through ribosomal protein (RP)-mediated suppression of Mdm2 E3 ligase activity. To study the physiological significance of the RP-Mdm2 interaction, we generated mice carrying a cancer-associated cysteine-to-phenylalanine substitution in the zinc finger of Mdm2 that disrupted its binding to RPL5 and RPL11. Mice harboring this mutation, retain normal p53 response to DNA damage, but lack of p53 response to perturbations in ribosome biogenesis. Loss of RP-Mdm2 interaction significantly accelerates Eμ-Myc-induced lymphomagenesis. Furthermore, ribosomal perturbation-induced p53 response does not require tumor suppressor p19ARF. Collectively, our findings establish RP-Mdm2 interaction as a genuine p53 stress-signaling pathway activated by aberrant ribosome biogenesis and essential for safeguarding against oncogenic c-MYC-induced tumorigenesis.

NPM1/B23: A Multifunctional Chaperone in Ribosome Biogenesis and Chromatin Remodeling

At a first glance, ribosome biogenesis and chromatin remodeling are quite different processes, but they share a common problem involving interactions between charged nucleic acids and small basic proteins that may result in unwanted intracellular aggregations. The multifunctional nuclear acidic chaperone NPM1 (B23/nucleophosmin) is active in several stages of ribosome biogenesis, chromatin remodeling, and mitosis as well as in DNA repair, replication and transcription. In addition, NPM1 plays an important role in the Myc-ARF-p53 pathway as well as in SUMO regulation. However, the relative importance of NPM1 in these processes remains unclear. Provided herein is an update on the expanding list of the diverse activities and interacting partners of NPM1. Mechanisms of NPM1 nuclear export functions of NPM1 in the nucleolus and at the mitotic spindle are discussed in relation to tumor development. It is argued that the suggested function of NPM1 as a histone chaperone could explain several, but not all, of the effects observed in cells following changes in NPM1 expression. A future challenge is to understand how NPM1 is activated, recruited, and controlled to carry out its functions.

Emerging functions of ribosomal proteins in gene-specific transcription and translation.

Ribosomal proteins have remained highly conserved during evolution presumably reflecting often critical functions in ribosome biogenesis or mature ribosome function. In addition, several ribosomal proteins possess distinct extra-ribosomal functions in apoptosis, DNA repair and transcription. An increasing number of ribosomal proteins have been shown to modulate the trans-activation function of important regulatory proteins such as NF-kappaB, p53, c-Myc and nuclear receptors. Furthermore, a subset of ribosomal proteins can bind directly to untranslated regions of mRNA resulting in transcript-specific translational control outside of the ribosome itself. Collectively, these findings suggest that ribosomal proteins may have a wider functional repertoire within the cell than previously thought. The future challenge is to identify and validate these novel functions in the background of an often essential primary function in ribosome biogenesis and cell growth.

PDGF and PDGF receptors in glioma

Abstract The family of platelet-derived growth factors (PDGFs) plays a number of critical roles in normal embryonic development, cellular differentiation, and response to tissue damage. Not surprisingly, as it is a multi-faceted regulatory system, numerous pathological conditions are associated with aberrant activity of the PDGFs and their receptors. As we and others have shown, human gliomas, especially glioblastoma, express all PDGF ligands and both the two cell surface receptors, PDGFR-α and -β. The cellular distribution of these proteins in tumors indicates that glial tumor cells are stimulated via PDGF/PDGFR-α autocrine and paracrine loops, while tumor vessels are stimulated via the PDGFR-β. Here we summarize the initial discoveries on the role of PDGF and PDGF receptors in gliomas and provide a brief overview of what is known in this field.

Cancer-associated mutations in the MDM2 zinc finger domain disrupt ribosomal protein interaction and attenuate MDM2-induced p53 degradation.

ABSTRACT The p53-inhibitory function of the oncoprotein MDM2 is regulated by a number of MDM2-binding proteins, including ARF and ribosomal proteins L5, L11, and L23, which bind the central acidic domain of MDM2 and inhibit its E3 ubiquitin ligase activity. Various human cancer-associated MDM2 alterations targeting the central acidic domain have been reported, yet the functional significance of these mutations in tumor development has remained unclear. Here, we show that cancer-associated missense mutations targeting MDM2s central zinc finger disrupt the interaction of MDM2 with L5 and L11. We found that the zinc finger mutant MDM2 is impaired in undergoing nuclear export and proteasomal degradation as well as in promoting p53 degradation, yet retains the function of suppressing p53 transcriptional activity. Unlike the wild-type MDM2, whose p53-suppressive activity can be inhibited by L11, the MDM2 zinc finger mutant escapes L11 inhibition. Hence, the MDM2 central zinc finger plays a critical role in mediating MDM2s interaction with ribosomal proteins and its ability to degrade p53, and these roles are disrupted by human cancer-associated MDM2 mutations.

Role of genetic and epigenetic changes in Burkitt lymphoma

All Burkitt lymphomas (BLs) carry reciprocal chromosomal translocations that activate the c-myc oncogene through juxtaposition to one of the immunoglobulin (Ig) loci. Many BL carry point mutation in the p53 tumor suppressor gene or other defects in the p14ARF-MDM2-p53 pathway, and inactivation of the p16INK4a gene by promoter methylation or homozygous deletion. This indicates that disruption of both the pRb and p53 tumor suppressor pathways is critical for BL development. Alterations of other genes, including Bax, p73, and BCL-6, may provide further growth stimulation and apoptosis protection. Thus, BL development involves multiple genetic and epigenetic changes that drive cell cycle progression and avert cell death by apoptosis.

p14ARF homozygous deletion or MDM2 overexpression in Burkitt lymphoma lines carrying wild type p53

The hallmark of Burkitt lymphoma (BL) is a constitutively activated c-myc gene that drives tumor cell growth. A majority of BL-derived cell lines also carry mutant p53. In addition, the p16INK4a promoter is hypermethylated in most BL biopsies and BL cell lines, leading to silencing of this gene. Activation of c-myc and/or cell cycle dysregulation can induce ARF expression and p53-dependent apoptosis. We therefore investigated the p14ARF-MDM2-p53 pathway in BL cell lines. p14ARF was expressed and localized to nucleoli in all BL carrying mutant p53. Three out of seven BL carrying wt p53 had a homozygous deletion of the CDKN2A locus that encodes both p14ARF and p16INK4a. Three BL carrying wild type p53 retained the CDKN2A locus and overexpressed MDM2. DNA sequencing revealed a point mutation in CDKN2A exon 2 in one of these BL, Seraphine. However, this point mutation did not affect p14ARFs nucleolar localization or ability to induce p53. The Bmi-1 protein that negatively regulates the p14ARF promoter and co-operates with c-myc in tumorigenesis was expressed at low to moderate levels in all BL analysed. Our results indicate that inactivation of the ARF-MDM2-p53 pathway is an essential step during the development of Burkitt lymphoma, presumably as a mechanism to escape c-myc induced apoptosis.

Predictive Significance of the Alterations of p16INK4A, p14ARF, p53, and Proliferating Cell Nuclear Antigen Expression in the Progression of Cervical Cancer

Purpose: The purpose of this research was to evaluate the clinical significance of p16INK4A, p14ARF, p53, and proliferating cell nuclear antigen (PCNA) expression in tumor progression of cervical cancer. Design: Seventeen patients (40 samples) with consecutive cervical lesions from normal squamous epithelium, inflammation of the cervix to cervical intraepithelial neoplasm (CIN) and invasive cervical squamous cell cancer (SCC), or from CIN to SCC were collected for this study. Expression of p16INK4A, p14ARF, p53, and PCNA were detected by immunohistochemistry on paraffin-embedded sections. Human papillomavirus DNA was detected simultaneously with PCR and typed according to its DNA sequence. Results: p16INK4A overexpression was significantly higher in CIN (75%) and in SCC (75%) than in normal or inflammation of the cervix (12.5%; P < 0.01, P < 0.05, respectively). The positive rate of p14ARF expression was higher in SCC (83%) than in normal/inflammation of the cervix (25%; P < 0.05). PCNA expression was negative in normal or inflammation of the cervix, but an increased in expression was seen in 63.2% in CIN and 100% in SCC (P < 0.01, P < 0.05). When the time interval for disease progression from initial biopsy to CIN 3 or invasive cancer was compared with states of p16INK4A expression, cases stained positive for p16INK4A progressed within 64.2 months as compared with 122.3 months among those stained negatively (P < 0.01). Cases with increased p14ARF expression also had a short time interval for disease progression of 78.8 months as compared with 108.3 months in cases that were p14ARF negative. Cases with stable or decreased p53 expression had the shortest time interval for progression of 32.3 months in contrast to cases with no p53 expression (113.9 months). However, cases with increasing p53 expression progressed within 60.8 months. Conclusions: Our results suggested that altered states of p16INK4A, p14ARF, p53, and PCNA may be valuable markers to predict the progression of cervical neoplasia.

Ribosomal Protein S9 Is a Novel B23/NPM-binding Protein Required for Normal Cell Proliferation

B23 (NPM/nucleophosmin) is a multifunctional nucleolar protein and a member of the nucleoplasmin superfamily of acidic histone chaperones. B23 is essential for normal embryonic development and plays an important role in genomic stability, ribosome biogenesis, and anti-apoptotic signaling. Altered protein expression or genomic mutation of B23 is encountered in many different forms of cancer. Although described as multifunctional, a genuine molecular function of B23 is not fully understood. Here we show that B23 is associated with a protein complex consisting of ribosomal proteins and ribosome-associated RNA helicases. A novel, RNA-independent interaction between ribosomal protein S9 (RPS9) and B23 was further investigated. We found that S9 binding requires an intact B23 oligomerization domain. Depletion of S9 by small interfering RNA resulted in decreased protein synthesis and G1 cell cycle arrest, in association with induction of p53 target genes. We determined that S9 is a short-lived protein in the absence of ribosome biogenesis, and proteasomal inhibition significantly increased S9 protein level. Overexpression of B23 facilitated nucleolar storage of S9, whereas knockdown of B23 led to diminished levels of nucleolar S9. Our results suggest that B23 selectively stores, and protects ribosomal protein S9 in nucleoli and therefore could facilitate ribosome biogenesis.

Targeting of MCL-1 kills MYC-driven mouse and human lymphomas even when they bear mutations in p53

The transcriptional regulator c-MYC is abnormally overexpressed in many human cancers. Evasion from apoptosis is critical for cancer development, particularly c-MYC-driven cancers. We explored which anti-apoptotic BCL-2 family member (expressed under endogenous regulation) is essential to sustain c-MYC-driven lymphoma growth to reveal which should be targeted for cancer therapy. Remarkably, inducible Cre-mediated deletion of even a single Mcl-1 allele substantially impaired the growth of c-MYC-driven mouse lymphomas. Mutations in p53 could diminish but not obviate the dependency of c-MYC-driven mouse lymphomas on MCL-1. Importantly, targeting of MCL-1 killed c-MYC-driven human Burkitt lymphoma cells, even those bearing mutations in p53. Given that loss of one allele of Mcl-1 is well tolerated in healthy tissues, our results suggest that therapeutic targeting of MCL-1 would be an attractive therapeutic strategy for MYC-driven cancers.