Erik Meulmeester

The dynamic roles of TGF‐β in cancer

The transforming growth factor‐β (TGF‐β) signalling pathway plays a critical and dual role in the progression of human cancer. During the early phase of tumour progression, TGF‐β acts as a tumour suppressor, exemplified by deletions or mutations in the core components of the TGF‐β signalling pathway. On the contrary, TGF‐β also promotes processes that support tumour progression such as tumour cell invasion, dissemination, and immune evasion. Consequently, the functional outcome of the TGF‐β response is strongly context‐dependent including cell, tissue, and cancer type. In this review, we describe the molecular signalling pathways employed by TGF‐β in cancer and how these, when perturbed, may lead to the development of cancer. Concomitantly with our increased appreciation of the molecular mechanisms that govern TGF‐β signalling, the potential to therapeutically target specific oncogenic sub‐arms of the TGF‐β pathway increases. Indeed, clinical trials with systemic TGF‐β signalling inhibitors for treatment of cancer patients have been initiated. However, considering the important role of TGF‐β in cardiovascular and many other tissues, careful screening of patients is warranted to minimize unwanted on‐target side effects. Copyright

Amplification of Mdmx (or Mdm4) directly contributes to tumor formation by inhibiting p53 tumor suppressor activity

ABSTRACT Human tumors are believed to harbor a disabled p53 tumor suppressor pathway, either through direct mutation of the p53 gene or through aberrant expression of proteins acting in the p53 pathway, such as p14ARF or Mdm2. A role for Mdmx (or Mdm4) as a key negative regulator of p53 function in vivo has been established. However, a direct contribution of Mdmx to tumor formation remains to be demonstrated. Here we show that retrovirus-mediated Mdmx overexpression allows primary mouse embryonic fibroblast immortalization and leads to neoplastic transformation in combination with HRasV12. Furthermore, the human Mdmx ortholog, Hdmx, was found to be overexpressed in a significant percentage of various human tumors and amplified in 5% of primary breast tumors, all of which retained wild-type p53. Hdmx was also amplified and highly expressed in MCF-7, a breast cancer cell line harboring wild-type p53, and interfering RNA-mediated reduction of Hdmx markedly inhibited the growth potential of these cells in a p53-dependent manner. Together, these results make Hdmx a new putative drug target for cancer therapy.

ATM-Mediated Phosphorylations Inhibit Mdmx/Mdm2 Stabilization by HAUSP in Favor of p53 Activation

The p53 tumor suppressor protein has a major role in protecting genome integrity. Under normal circumstances Mdmx and Mdm2 control the activity of p53. Both proteins inhibit the transcriptional regulation by p53, while Mdm2 also functions as an E3 ubiquitin ligase to target both p53 and Mdmx for proteasomal degradation. HAUSP counteracts the destabilizing effect of Mdm2 by direct deubiquitination of p53. Subsequently, HAUSP was shown to deubiquitinate Mdm2 and Mdmx, thereby stabilizing these proteins. The ATM protein kinase is a key regulator of the p53 pathway in response to double strand breaks (DSBs) in the DNA. ATM fine-tunes p53s response to DNA damage by directly phosphorylating it, by regulating additional post-translational modifications of this protein, and by affecting two p53 regulators: Mdm2 and Mdmx. ATM directly and indirectly induces Mdm2 and Mdmx phosphorylation, resulting in decreased activity and stability of these proteins. We recently provided a mechanism for the reduced stability of Mdm2 and Mdmx by showing that ATM-dependent phosphorylation lowers their affinity for the deubiquitinating enzyme HAUSP. Altogether, the emerging picture portrays an elaborate, but fine-tuned, ATM-mediated control of p53 activation and stabilization following DNA damage. Further insight into the mechanism by which ATM switches the interactions between HAUSP, Mdmx, Mdm2 and p53, to favor p53 activation may offer new tools for therapeutic intervention in the p53 pathway for cancer treatment.

Sumoylation inhibits α-synuclein aggregation and toxicity

Sumoylation of α-synuclein decreases its rate of aggregation and its deleterious effects in vitro and in vivo.

Hdmx Protein Stability Is Regulated by the Ubiquitin Ligase Activity of Mdm2

The stability of the p53 tumor suppressor protein is critically regulated by the Hdm2 and Hdmx proteins. Hdm2 protein levels are auto-regulated by the self-ubiquitination activity of Hdm2 and on the transcriptional level by p53-activated transcription of the hdm2 gene. Little is known about the regulation of Hdmx expression levels, apart from the observation that the Mdmx protein can be cleaved by caspase-3 in a p53-inducible manner. In the functional analysis of two mutant Hdmx proteins, products of two alternatively spliced mRNAs, it was found that Hdmx proteins are targets for ubiquitination by Mdm2. The stability of the Hdmx protein is partly dependent on the presence of its internal acidic domain. Mdm2 appears only to require an intact RING domain to be able to ubiquitinate Hdmx and target it for proteasomal degradation. These findings highlight the intricate functional relationships between p53, Mdm2, and Hdmx.

Critical Role for a Central Part of Mdm2 in the Ubiquitylation of p53

ABSTRACT The stability of the p53 protein is regulated by Mdm2. By acting as an E3 ubiquitin ligase, Mdm2 directs the ubiquitylation of p53 and its subsequent degradation by the 26S proteasome. In contrast, the Mdmx protein, although structurally similar to Mdm2, cannot ubiquitylate or degrade p53 in vivo. To ascertain which domains determine this functional difference between Mdm2 and Mdmx and consequently are essential for p53 ubiquitylation and degradation, we generated Mdm2-Mdmx chimeric constructs. Here we show that, in addition to a fully functional Mdm2 RING finger, an internal domain of Mdm2 (residues 202 to 302) is essential for p53 ubiquitylation. Strikingly, the function of this domain can be fulfilled in trans, indicating that the RING domain and this internal region perform distinct activities in the ubiquitylation of p53.

Differential Roles of ATM- and Chk2-Mediated Phosphorylations of Hdmx in Response to DNA Damage

ABSTRACT The p53 tumor suppressor plays a major role in maintaining genomic stability. Its activation and stabilization in response to double strand breaks (DSBs) in DNA are regulated primarily by the ATM protein kinase. ATM mediates several posttranslational modifications on p53 itself, as well as phosphorylation of p53s essential inhibitors, Hdm2 and Hdmx. Recently we showed that ATM- and Hdm2-dependent ubiquitination and subsequent degradation of Hdmx following DSB induction are mediated by phosphorylation of Hdmx on S403, S367, and S342, with S403 being targeted directly by ATM. Here we show that S367 phosphorylation is mediated by the Chk2 protein kinase, a downstream kinase of ATM. This phosphorylation, which is important for subsequent Hdmx ubiquitination and degradation, creates a binding site for 14-3-3 proteins which controls nuclear accumulation of Hdmx following DSBs. Phosphorylation of S342 also contributed to optimal 14-3-3 interaction and nuclear accumulation of Hdmx, but phosphorylation of S403 did not. Our data indicate that binding of a 14-3-3 dimer and subsequent nuclear accumulation are essential steps toward degradation of p53s inhibitor, Hdmx, in response to DNA damage. These results demonstrate a sophisticated control by ATM of a target protein, Hdmx, which itself is one of several ATM targets in the ATM-p53 axis of the DNA damage response.

“ChopNSpice,” a Mass Spectrometric Approach That Allows Identification of Endogenous Small Ubiquitin-like Modifier-conjugated Peptides

Conjugation of small ubiquitin-like modifier (SUMO) to substrates is involved in a large number of cellular processes. Typically, SUMO is conjugated to lysine residues within a SUMO consensus site; however, an increasing number of proteins are sumoylated on non-consensus sites. To appreciate the functional consequences of sumoylation, the identification of SUMO attachment sites is of critical importance. Discovery of SUMO acceptor sites is usually performed by a laborious mutagenesis approach or using MS. In MS, identification of SUMO acceptor sites in higher eukaryotes is hampered by the large tryptic fragments of SUMO1 and SUMO2/3. MS search engines in combination with known databases lack the possibility to search MSMS spectra for larger modifications, such as sumoylation. Therefore, we developed a simple and straightforward database search tool (“ChopNSpice”) that successfully allows identification of SUMO acceptor sites from proteins sumoylated in vivo and in vitro. By applying this approach we identified SUMO acceptor sites in, among others, endogenous SUMO1, SUMO2, RanBP2, and Ubc9.

Identification of endogenous SUMO1 accepter sites by mass spectrometry.

Posttranslational modification (PTM) by the covalent conjugation of small ubiquitin-like modifier (SUMO) plays an important role in many biological processes, such as cell cycle progression, transcriptional regulation, subcellular transport, and other processes. An in-depth understanding of the function of SUMOylation requires the discovery of SUMO accepter sites. However, identification of endogenous SUMO-conjugated sites in higher eukaryotes by MS-based proteomic strategies is hampered by the low abundance of SUMO conjugates, the large tryptic fragments of SUMO1 or SUMO2/3 and the inability to match MS/MS spectra by protein database search engine. In this chapter, we describe a powerful method to overcome at least some of these challenges. To identify SUMO acceptor sites in endogenous SUMO1 conjugated protein, the SUMO1 conjugates are purified by immunoprecipitation with anti-SUMO1 antibodies followed by SDS-PAGE separation and in-gel tryptic digestion. The resulting peptides are either performed using standard data dependent acquisition (DDA) for protein identification or high mass DDA to enhance the sensitivity of detection on the LTQ-Orbitrap mass spectrometer. Finally, a Web-based database tool, ChopNSpice, coupled with a protein database search engine is introduced to ease the identification of SUMO1 attachment sites. Although this method was initially used to identify SUMO1 accepter sites, it can be readily adapted to study SUMO2/3 conjugates or even other Ubiquitin-like modifiers.

Integration of transcriptional signals at the tumor cell invasive front.

Comment on: Fuxe J et al. Cell Cycle 2010; 9:2362-74.