Youjun Li

Onzin, a c-Myc-repressed target, promotes survival and transformation by modulating the Akt-Mdm2-p53 pathway.

The c-Myc oncoprotein is a general transcription factor whose target genes dictate the c-Myc phenotype. One such target of c-Myc, ‘onzin’, is normally expressed at high levels in myeloid cells and is dramatically downregulated in response to c-Myc overexpression. We show here that short hairpin interfering RNA-mediated knockdown of endogenous onzin results in a reduced growth rate and a proapoptotic phenotype. In contrast, onzin overexpression in fibroblasts is associated with an increased growth rate, resistance to apoptotic stimuli, loss of the G2/M checkpoint, and tumorigenic conversion. Onzin-overexpressing cells fail to induce p53 in response to apoptotic stimuli and contain higher levels of the active, phosphorylated forms of Akt1 and, more strikingly, of Mdm2. Using yeast two-hybrid and coimmunoprecipitation assays, we show that onzin directly interacts with both proteins. Green fluorescent protein tagging also confirms directly that Akt1 and Mdm2 colocalize with onzin, although the precise subcellular distribution of each protein is dependent on its relative abundance. Collectively, our results identify onzin as a novel regulator of several p53-dependent aspects of the c-Myc phenotype via its dramatic effect on Mdm2. This is reminiscent of the c-Myc → p19ARF--∣ Mdm2 pathway and might function as a complementary arm to ensure the proper cellular response to oncogenic and/or apoptotic stimuli.

The ever expanding role for c-myc in promoting genomic instability

Genomic instability (GI) is a hallmark of many cancers. GI is believed toconfer upon impending neoplastic cells the ability to accumulate all of therequisite mutations for transformation within the relatively short time-frame of anorganism’s lifespan. Recently described properties of the c-Myc oncoproteinshow that, in addition to its directly transforming role, it can mediate GI via theinduction of reactive oxygen species and by promoting whole chromosomeinstability leading to tetraploidy and aneuploidy. Mediators of both propertieshave been identified and have begun to provide a framework within which tounderstand not only how c-Myc alters the genome but how it might alsocooperate with its more traditional transforming activities. These genome-alteringproperties of c-Myc suggest that they provide the protein with the ability to confera “mutator phenotype” to cells in which its expression is de-regulated.

The Negative c-Myc Target Onzin Affects Proliferation and Apoptosis via Its Obligate Interaction with Phospholipid Scramblase I

ABSTRACT Onzin, the product of a negatively c-Myc-regulated target gene, is highly expressed in myeloid cells. As a result of its interaction with and activation of Akt1 and Mdm2, onzin down-regulates p53. The apoptotic sensitivity of several cell lines is thus directly related to onzin levels. We have conducted a search for additional onzin-interacting proteins and identified phospholipid scramblase 1 (PLSCR1), an endofacial membrane protein, which is proposed to mediate the bidirectional movement of plasma membrane phospholipids during proliferation and apoptosis. PLSCR1 interacts with the same cysteine-rich domain of onzin as do Akt1 and Mdm2, whereas the onzin-interacting domain of PLSCR1 centers around, but does not require, a previously identified palmitoylation signal. Depletion of endogenous PLSCR1 in myeloid cells leads to a phenotype that mimics that of onzin overexpression, providing evidence that PLSCR1 is a physiologic regulator of onzin. In contrast, PLSCR1 overexpression in fibroblasts, which normally do not express onzin, affects neither growth nor apoptosis unless onzin is coexpressed, in which case PLSCR1 completely abrogates onzins positive effects on proliferation and survival. These findings demonstrate a functional interdependence between onzin and PLSCR1. They further suggest a contiguous link between the earliest events mediated by c-Myc and the latest ones, which culminate at the cell surface and lead to phospholipid reshuffling and cell death.

c-Myc-mediated genomic instability proceeds via a megakaryocytic endomitosis pathway involving Gp1bα

Genomic instability (GI) is essential for the initiation and evolution of many cancers and often precedes frank neoplastic conversion. Although GI can occur at several levels, the most conspicuous examples involve gains or losses of entire chromosomes (aneuploidy), the antecedent of which may be whole genome duplication (tetraploidy). Through largely undefined mechanisms, the c-Myc oncoprotein and its downstream target, MTMC1, promote tetraploidy and other forms of GI. In myeloid cells, c-Myc and MTMC1 also regulate a common, small subset of c-Myc target genes including GP1Bα, which encodes a subunit of the von Willebrands factor receptor complex that mediates platelet adhesion and aggregation. Gp1bα also participates in megakaryocyte endomitosis, a form of controlled and precise whole-genome amplification. In this article, we show that both c-Myc and MTMC1 strongly up-regulate Gp1bα concurrent with their promotion of tetraploidy. shRNA-mediated inhibition of Gp1bα prevents tetraploidy by both c-Myc and MTMC1, whereas Gp1bα overexpression alone is sufficient to induce tetraploidy in established and primary cells. Once acquired, tetraploidy persists in most cases examined. Our results indicate that chromosome-level GI, induced by c-Myc overexpression, proceeds by means of the sequential up-regulation of MTMC1 and Gp1bα and further suggest that the pathways leading to megakaryocytic endomitosis and c-Myc-induced tetraploidy are mechanistically linked by their reliance on Gp1bα.

Dual role for SUMO E2 conjugase Ubc9 in modulating the transforming and growth-promoting properties of the HMGA1b architectural transcription factor.

Members of the HMGA1 (high mobility group A1) family of architectural transcription factors, HMGA1a and HMGA1b, play important roles in many normal cellular processes and in tumorigenesis. We performed a yeast two-hybrid screen for HMGA1-interacting proteins and identified the SUMO E2 conjugase Ubc9 as one such partner. The Ubc9-interacting domain of HMGA1 is bipartite, consisting of a proline-rich region near the N terminus and an acidic domain at the extreme C terminus, whereas the HMGA1-interacting domain of Ubc9 comprises a single region previously shown to associate with and SUMOylate other transcription factors. Consistent with these findings, endogenous HMGA1 proteins and Ubc9 could be co-immunoprecipitated from several human cell lines. Studies with HMGA1b proteins containing mutations of either or both Ubc9-interacting domains and with Ubc9-depleted cell lines indicated that the proline-rich domain of HMGA1b positively influences transformation and growth, whereas the acidic domain negatively influences these properties. None of the changes in HMGA1 protein functions mediated by Ubc9 appears to require SUMOylation. These findings are consistent with the idea that Ubc9 can act as both a positive and negative regulator of proliferation and transformation via its non-SUMO-dependent interaction with HMGA1 proteins.

Transformation, genomic instability and senescence mediated by platelet/megakaryocyte glycoprotein Ibα

GpIbα, a subunit of the von Willebrand factor receptor, functions during blood clotting to promote platelet adhesion and activation. GpIbα is widely expressed, is positively regulated by c-Myc and is essential for the promotion of c-Myc-mediated chromosomal instability. We now show that GpIbα is also a classical oncoprotein in which its deregulated expression leads to transformation, reduced growth factor requirements, increased resistance to apoptosis, and, in primary cells, p53-dependent senescence. Finally, GpIbα also promotes double-stranded DNA breaks, and induces profound nuclear dysmorphology, indicating that, in addition to its direct transforming function, it displays genotoxicity at several distinct levels. These findings identify novel functions for GpIbα and pathways through which c-Myc mediates transformation and global genomic destabilization.

Widespread Genomic Instability Mediated by a Pathway Involving Glycoprotein Ibα and Aurora B Kinase

c-Myc (Myc) oncoprotein induction of genomic instability (GI) contributes to its initial transforming function and subsequent tumor cell evolution. We describe here a pathway by which Myc, via its target protein glycoprotein Ibα (GpIbα), mediates GI. Proteomic profiling revealed that the serine/threonine kinase Aurora B is down-regulated by GpIbα in p53-deficient primary human fibroblasts. The phenotypes of Aurora B deficiency are strikingly reminiscent of Myc or GpIbα overexpression and include double-stranded DNA breaks, altered nuclear size and morphology, chromatin bridges, cleavage furrow regression, and tetraploidy. During mitosis, GpIbα and Aurora B redistribute to the cleavage furrow along with other cleavage furrow proteins. GpIbα overexpression at levels comparable with those seen in some tumor cells causes the dispersal of these proteins but not Aurora B, resulting in furrow regression and cytokinesis failure. Aurora B normalization redirects the mislocalized furrow proteins to their proper location, corrects the cleavage furrow abnormalities, and restores genomic stability. Aurora B thus appears necessary for a previously unrecognized function in guiding and positioning a number of key proteins, including GpIbα to the cleavage furrow. These findings underscore the importance of maintaining a delicate balance among cleavage furrow-associated proteins during mitosis. Suppression of Aurora B via GpIbα provides a unifying and mechanistic explanation for several types of Myc-mediated GI.

microRNA-206 impairs c-Myc-driven cancer in a synthetic lethal manner by directly inhibiting MAP3K13

c-Myc (Myc) is one of the most frequently dysregulated oncogenic transcription factors in human cancer. By functionally screening a microRNA (miR) library, we identified miR-206 as being a synthetic lethal in Myc over-expressing human cancer cells. miR-206 inhibited MAP3K13, which resulted in Myc protein de-stabilization, and an inhibition of anchorage-independent growth and in vivo tumorigenesis by Myc over-expressing human cancer cells. Eliminating MAP3K13 by shRNA recapitulated the effects caused by miR-206, thus supporting the idea that miR-206s effect on Myc was mediated through MAP3K13. Conversely, enforced expression of MAP3K13 stabilized Myc by promoting its N-terminal phosphorylation and enhancing its transcriptional activity. Gene expression analyses of breast cancers expressing high levels of Myc indicated that low miR-206 expression and high MAP3K13 expression correlated with poor patient survival. The critical link between miR-206 and MAP3K13 in the development of Myc over-expressing human cancers suggests potential points of therapeutic intervention for this molecular sub-category.

Modularity of the Oncoprotein-like Properties of Platelet Glycoprotein Ibα

Glycoprotein Ib α (GpIbα), a trans-membrane glycoprotein, is expressed on the surface of megakaryocytes and platelets, where, in association with glycoprotein Ib β, glycoprotein V, and glycoprotein IX, it normally forms the von Willebrand factor receptor (vWFR). A fully functional vWFR is necessary for platelet attachment, aggregation, and activation and has also been shown to regulate megakaryocyte ploidy. We have recently shown that the gene encoding GpIbα is a transcriptional target for the c-Myc oncoprotein and is more widely expressed than previously thought, with particularly high levels occurring in transformed cells. Indeed, GpIbα can substitute for c-Myc in promoting growth, transformation, and genomic instability. In the current work, we have demonstrated that, despite the promiscuous expression of GpIbα, other vWFR subunits remain largely restricted to megakaryocytes. We have characterized a panel of GpIbα mutants and shown that some regions of the protein essential for vWFR activity are not necessary for c-Myc-like functions. Specifically, the six C-terminal amino acids of the cytoplasmic domain, which mediate vWFR signaling, are entirely dispensible for the c-Myc-like functions of GpIbα. Instead, these require a more membrane-proximal filamin-binding domain. Also important is the GpIbα signal peptide, which, in the absence of other vWFR subunits, directs GpIbα to the endoplasmic reticulum rather than the membrane. Together, these results provide strong evidence that the domains of GpIbα mediating c-Myc-like functions are modular, genetically distinct, and independent of those involved in vWFR signaling.

Ribosomopathy-like properties of murine and human cancers

Ribosomopathies comprise a heterogeneous group of hematologic and developmental disorders, often characterized by bone marrow failure, skeletal and other developmental abnormalities and cancer predisposition. They are associated with mutations and/or haplo-insufficiencies of ribosomal proteins (RPs) and inefficient ribosomal RNA (rRNA) processing. The resulting ribosomal stress induces the canonical p19ARF/Mdm2/p53 tumor suppressor pathway leading to proliferative arrest and/or apoptosis. It has been proposed that this pathway is then inactivated during subsequent neoplastic evolution. We show here that two murine models of hepatoblastoma (HB) and hepatocellular carcinoma (HCC) unexpectedly possess features that mimic the ribosomopathies. These include loss of the normal stoichiometry of RP transcripts and proteins and the accumulation of unprocessed rRNA precursors. Silencing of p19ARF, cytoplasmic sequestration of p53, binding to and inactivation of Mdm2 by free RPs, and up-regulation of the pro-survival protein Bcl-2 may further cooperate to drive tumor growth and survival. Consistent with this notion, re-instatement of constitutive p19ARF expression in the HB model completely suppressed tumorigenesis. In >2000 cases of human HCC, colorectal, breast, and prostate cancer, RP transcript deregulation was a frequent finding. In HCC and breast cancer, the severity of this dysregulation was associated with inferior survival. In HCC, the presence of RP gene mutations, some of which were identical to those previously reported in ribosomopathies, were similarly negatively correlated with long-term survival. Taken together, our results indicate that many if not all cancers possess ribosomopathy-like features that may affect their biological behaviors.