Stefano Confalonieri

Biological and Molecular Heterogeneity of Breast Cancers Correlates with Their Cancer Stem Cell Content

Pathways that govern stem cell (SC) function are often subverted in cancer. Here, we report the isolation to near purity of human normal mammary SCs (hNMSCs), from cultured mammospheres, on the basis of their ability to retain the lipophilic dye PKH26 as a consequence of their quiescent nature. PKH26-positive cells possess all the characteristics of hNMSCs. The transcriptional profile of PKH26-positive cells (hNMSC signature) was able to predict biological and molecular features of breast cancers. By using markers of the hNMSC signature, we prospectively isolated SCs from the normal gland and from breast tumors. Poorly differentiated (G3) cancers displayed higher content of prospectively isolated cancer SCs (CSCs) than did well-differentiated (G1) cancers. By comparing G3 and G1 tumors in xenotransplantation experiments, we directly demonstrated that G3s are enriched in CSCs. Our data support the notion that the heterogeneous phenotypical and molecular traits of human breast cancers are a function of their CSC content.

Tip60 is a haplo-insufficient tumour suppressor required for an oncogene-induced DNA damage response

The acetyl-transferase Tip60 might influence tumorigenesis in multiple ways. First, Tip60 is a co-regulator of transcription factors that either promote or suppress tumorigenesis, such as Myc and p53. Second, Tip60 modulates DNA-damage response (DDR) signalling, and a DDR triggered by oncogenes can counteract tumour progression. Using Eμ–myc transgenic mice that are heterozygous for a Tip60 gene (Htatip) knockout allele (hereafter denoted as Tip60+/– mice), we show that Tip60 counteracts Myc-induced lymphomagenesis in a haplo-insufficient manner and in a time window that is restricted to a pre- or early-tumoral stage. Tip60 heterozygosity severely impaired the Myc-induced DDR but caused no general DDR defect in B cells. Myc- and p53-dependent transcription were not affected, and neither were Myc-induced proliferation, activation of the ARF–p53 tumour suppressor pathway or the resulting apoptotic response. We found that the human TIP60 gene (HTATIP) is a frequent target for mono-allelic loss in human lymphomas and head-and-neck and mammary carcinomas, with concomitant reduction in mRNA levels. Immunohistochemical analysis also demonstrated loss of nuclear TIP60 staining in mammary carcinomas. These events correlated with disease grade and frequently concurred with mutation of p53. Thus, in both mouse and human, Tip60 has a haplo-insufficient tumour suppressor activity that is independent from—but not contradictory with—its role within the ARF–p53 pathway. We suggest that this is because critical levels of Tip60 are required for mounting an oncogene-induced DDR in incipient tumour cells, the failure of which might synergize with p53 mutation towards tumour progression.

Evolution of Shc functions from nematode to human

The Shc protein family is characterized by the (CH2)-PTB-CH1-SH2 modularity. Its complexity increased during evolution from one locus in Drosophila (dShc), to at least three loci in mammals (shc, rai and sli). The three mammalian loci encode, because of alternative initiation codon usage and splicing pattern, at least six Shc-like proteins. Genetic and biological evidence indicates that the mammalian Shc isoforms regulate functions as diverse as growth (p52/p46Shc), apoptosis (p66Shc) and life-span (p66Shc). Available structure-function data and analysis of sequence similarities of Shc-like genes and proteins suggest complex diversification of Shc functions during evolution. Notably, Ras activation, the best-characterized Shc activity, appears to be a recent evolutionary acquisition.

IRSp53: crossing the road of membrane and actin dynamics in the formation of membrane protrusions

A tight spatiotemporal coordination of the machineries controlling membrane bending and trafficking, and actin dynamics is crucial for the generation of cellular protrusions. Proteins that are simultaneously capable of regulating actin dynamics and sensing or inducing membrane curvature are predicted to have a prominent role. A prototypical example of this type of proteins is the insulin receptor tyrosine kinase substrate of 53kDa, the founding member of a recently discovered family of proteins, including missing-in-metastasis and ABBA (actin-bundling protein with BAIAP2 homology). Structural, biochemical and cell biological experiments support the unique role of this family as transducers of signalling, linking the protruding membrane to the underlying actin cytoskeleton.

Molecular mechanisms of coupled monoubiquitination

Many proteins contain ubiquitin-binding domains or motifs (UBDs), such as the UIM (ubiquitin-interacting motif) and are referred to as ubiquitin receptors. Ubiquitin receptors themselves are frequently monoubiquitinated by a process that requires the presence of a UBD and is referred to as coupled monoubiquitination. Using a UIM-containing protein, eps15, as a model, we show here that coupled monoubiquitination strictly depends on the ability of the UIM to bind to monoubiquitin (mUb). We found that the underlying molecular mechanism is based on interaction between the UIM and a ubiquitin ligase (E3), which has itself been modified by ubiquitination. Furthermore, we demonstrate that the in vivo ubiquitination of members of the Nedd4 family of E3 ligases correlates with their ability to monoubiquitinate eps15. Thus, our results clarify the mechanism of coupled monoubiquitination and identify the ubiquitination of E3 ligases as a critical determinant in this process.

Eps8 controls actin-based motility by capping the barbed ends of actin filaments

Actin filament barbed-end capping proteins are essential for cell motility, as they regulate the growth of actin filaments to generate propulsive force. One family of capping proteins, whose prototype is gelsolin, shares modular architecture, mechanism of action, and regulation through signalling-dependent mechanisms, such as Ca2+ or phosphatidylinositol-4,5-phosphate binding. Here we show that proteins of another family, the Eps8 family, also show barbed-end capping activity, which resides in their conserved carboxy-terminal effector domain. The isolated effector domain of Eps8 caps barbed ends with an affinity in the nanomolar range. Conversely, full-length Eps8 is auto-inhibited in vitro, and interaction with the Abi1 protein relieves this inhibition. In vivo, Eps8 is recruited to actin dynamic sites, and its removal impairs actin-based propulsion. Eps8-family proteins do not show any similarity to gelsolin-like proteins. Thus, our results identify a new family of actin cappers, and unveil novel modalities of regulation of capping through protein–protein interactions. One established function of the Eps8–Abi1 complex is to participate in the activation of the small GTPase Rac, suggesting a multifaceted role for this complex in actin dynamics, possibly through the participation in alternative larger complexes.

Determinants of conformational dimerization of Mad2 and its inhibition by p31comet

The spindle assembly checkpoint (SAC) monitors chromosome attachment to spindle microtubules. SAC proteins operate at kinetochores, scaffolds mediating chromosome‐microtubule attachment. The ubiquitous SAC constituents Mad1 and Mad2 are recruited to kinetochores in prometaphase. Mad2 sequesters Cdc20 to prevent its ability to mediate anaphase onset. Its function is counteracted by p31comet (formerly CMT2). Upon binding Cdc20, Mad2 changes its conformation from O‐Mad2 (Open) to C‐Mad2 (Closed). A Mad1‐bound C‐Mad2 template, to which O‐Mad2 binds prior to being converted into Cdc20‐bound C‐Mad2, assists this process. A molecular understanding of this prion‐like property of Mad2 is missing. We characterized the molecular determinants of the O‐Mad2:C‐Mad2 conformational dimer and derived a rationalization of the binding interface in terms of symmetric and asymmetric components. Mutation of individual interface residues abrogates the SAC in Saccharomyces cerevisiae. NMR chemical shift perturbations indicate that O‐Mad2 undergoes a major conformational rearrangement upon binding C‐Mad2, suggesting that dimerization facilitates the structural conversion of O‐Mad2 required to bind Cdc20. We also show that the negative effects of p31comet on the SAC are based on its competition with O‐Mad2 for C‐Mad2 binding.

Recognition specificity of individual EH domains of mammals and yeast

The Eps homology (EH) domain is a recently described protein binding module that is found, in multiple or single copies, in several proteins in species as diverse as human and yeast. In this work, we have investigated the molecular details of recognition specificity mediated by this domain family by characterizing the peptide‐binding preference of 11 different EH domains from mammal and yeast proteins. Ten of the eleven EH domains could bind at least some peptides containing an Asn‐Pro‐Phe (NPF) motif. By contrast, the first EH domain of End3p preferentially binds peptides containing an His‐Thr/Ser‐Phe (HT/SF) motif. Domains that have a low affinity for the majority of NPF peptides reveal some affinity for a third class of peptides that contains two consecutive amino acids with aromatic side chains (FW or WW). This is the case for the third EH domain of Eps15 and for the two N‐terminal domains of YBL47c. The consensus sequences derived from the peptides selected from phage‐displayed peptide libraries allows for grouping of EH domains into families that are characterized by different NPF‐context preference. Finally, comparison of the primary sequence of EH domains with similar or divergent specificity identifies a residue at position +3 following a conserved tryptophan, whose chemical characteristics modulate binding preference.

EH and UIM: Endocytosis and More

Exogenously and endogenously originated signals are propagated within the cell by functional and physical networks of proteins, leading to numerous biological outcomes. Many protein-protein interactions take place between binding domains and short peptide motifs. Frequently, these interactions are inducible by upstream signaling events, in which case one of the two binding surfaces may be created by a posttranslational modification. Here, we discuss two protein networks. One, the EH-network, is based on the Eps15 homology (EH) domain, which binds to peptides containing the sequence Asp-Pro-Phe (NPF). The other, which we define as the monoubiquitin (mUb) network, relies on monoubiquitination, which is emerging as an important posttranslational modification that regulates protein function. Both networks were initially implicated in the control of plasma membrane receptor endocytosis and in the regulation of intracellular trafficking routes. The ramifications of these two networks, however, appear to extend into many other aspects of cell physiology as well, such as transcriptional regulation, actin cytoskeleton remodeling, and DNA repair. The focus of this review is to integrate available knowledge of the EH- and mUb networks with predictions of genetic and physical interactions stemming from functional genomics approaches. This STKE Review, with 4 figures, 7 tables, and 181 references, concerns protein interaction networks built through two classes of protein-protein interactions, those involving the Eps15 homology (EH) domain and those that depend on monoubiquitination, a posttranslational modification that creates the monoubiquitin (mUb)-network. Functional and physical protein networks linked by protein interaction domains--regions with conserved structure and amino acid sequence--can be created through signaling events and serve functions critical to intracellular signaling pathways. The protein networks involving EH domains and ubiquitin (Ub)-interacting motifs (UIMs) were initially implicated in the regulation of receptor endocytosis and in the trafficking of intracellular vesicles. More recent data, however, indicate that both networks serve other important functions as well. This review integrates available knowledge on the EH- and mUb networks with predictions based on functional genomics.

Frequent Alterations in the Expression of Serine/Threonine Kinases in Human Cancers

Protein kinases constitute a large family of regulatory enzymes involved in the homeostasis of virtually every cellular process. Subversion of protein kinases has been frequently implicated in malignant transformation. Within the family, serine/threonine kinases (STK) have received comparatively lesser attention, vis-a-vis tyrosine kinases, in terms of their involvement in human cancers. Here, we report a large-scale screening of 125 STK, selected to represent all major subgroups within the subfamily, on nine different types of tumors ( approximately 200 patients), by using in situ hybridization on tissue microarrays. Twenty-one STK displayed altered levels of transcripts in tumors, frequently with a clear tumor type-specific dimension. We identified three patterns of alterations in tumors: (a) overexpression in the absence of expression in the normal tissues (10 kinases), (b) overexpression in the presence of expression by normal tissues (8 kinases), and (c) underexpression (3 kinases). Selected members of the three classes were subjected to in-depth analysis on larger case collections and showed significant correlations between their altered expression and biological and/or clinical variables. Our findings suggest that alteration in the expression of STK is a relatively frequent occurrence in human tumors. Among the overexpressed kinases, 10 were undetectable in normal controls and are therefore ideal candidates for further validation as potential targets of molecular cancer therapy.