CDK12 promotes tumorigenesis but induces vulnerability to therapies inhibiting folate one-carbon metabolism in breast cancer.

Cyclin-dependent kinase 12 (CDK12) overexpression is implicated in breast cancer, but whether it has a primary or only a cooperative tumorigenic role is unclear. Here, we show that transgenic CDK12 overexpression in the mouse mammary gland per se is sufficient to drive the emergence of multiple and multifocal tumors, while, in cooperation with known oncogenes, it promotes earlier tumor onset and metastasis. Integrative transcriptomic, metabolomic and functional data reveal that hyperactivation of the serine-glycine-one-carbon network is a metabolic hallmark inherent to CDK12-induced tumorigenesis. Consistently, in retrospective patient cohort studies and in patient-derived xenografts, CDK12-overexpressing breast tumors show positive response to methotrexate-based chemotherapy targeting CDK12-induced metabolic alterations, while being intrinsically refractory to other types of chemotherapy. In a retrospective analysis of hormone receptor-negative and lymph node-positive breast cancer patients randomized in an adjuvant phase III trial to 1-year low-dose metronomic methotrexate-based chemotherapy or no maintenance chemotherapy, a high CDK12 status predicts a dramatic reduction in distant metastasis rate in the chemotherapy-treated vs. not-treated arm. Thus, by coupling tumor progression with metabolic reprogramming, CDK12 creates an actionable vulnerability for breast cancer therapy and might represent a suitable companion biomarker for targeted antimetabolite therapies in human breast cancers.

Endocytosis and mitogenic signaling.

Is there mitogenic signaling during endocytosis or is receptor internalization mainly an attenuator of signals? Recent data indicate that the answer appears to be yes to both questions. Signal transduction occurs physiologically from the cell surface and endocytosis downregulates signaling by removing receptors from the plasma membrane. In cancer, the involvement of endocytic/sorting proteins points to dysregulation of apparently unrelated pathways, which might account for an important causative role in neoplasia.

The Eps15 C. elegans homologue EHS-1 is implicated in synaptic vesicle recycling.

Eps15 represents the prototype of a family of evolutionarily conserved proteins that are characterized by the presence of the EH domain, a protein-protein interaction module, and that are involved in many aspects of intracellular vesicular sorting. Although biochemical and functional studies have implicated Eps15 in endocytosis, its function in the endocytic machinery remains unclear. Here we show that the Caenorhabditis elegans gene, zk1248.3 (ehs-1), is the orthologue of Eps15 in nematodes, and that its product, EHS-1, localizes to synaptic-rich regions. ehs-1-impaired worms showed temperature-dependent depletion of synaptic vesicles and uncoordinated movement. These phenotypes could be correlated with a presynaptic defect in neurotransmission. Impairment of EHS-1 function in dyn-1(ky51) worms, which express a mutant form of dynamin and display a temperature-sensitive locomotion defect, resulted in a worsening of the dyn-1 phenotype and uncoordination at the permissive temperature. Thus, ehs-1 and dyn-1 interact genetically. Moreover, mammalian Eps15 and dynamin protein were shown to interact in vivo. Taken together, our results indicate that EHS-1 acts in synaptic vesicle recycling and that its function might be linked to that of dynamin.

Understanding biological dynamics: following cells and molecules to track functions and mechanisms.

The dissection of the molecular circuitries at the base of cell life and the identification of their abnormal transformation during carcinogenesis rely on the characterization of biological phenotypes generated by targeted overexpression or deletion of gene products through genetic manipulation. Fluorescence microscopy provides a wide variety of tools to monitor cell life with minimal perturbations. The observation of living cells requires the selection of a correct balance between temporal, spatial and "statistical" resolution according to the process to be analyzed. In the following paper ad hoc developed optical tools for dynamical tracking from cellular to molecular resolution will be presented. Particular emphasis will be devoted to discuss how to exploit light-matter interaction to selectively target specific molecular species, understanding the relationships between their intracellular compartmentalization and function.

Deliberative ethics in a biomedical institution: an example of integration between science and ethics.

The deliberative ethics guidelines elaborated and implemented by members of the IFOM-IEO Campus (Firc Institute of Molecular Oncology (IFOM) and the European Institute of Oncology (IEO)). These should serve the dual purpose of establishing a minimal set of standard rules for bioethical debate and any ensuing decision-making process, especially for the perspective of providing real instruments to foster public engagement and public awareness on the ethical issues involved in biomedical research. It is shown that these guidelines instantiate the scheme of one of the correct ways of debating formalised by the western thought.

Tyrosine phosphorylation of Eps15 is required for ligand-regulated, but not constitutive, endocytosis.

Membrane receptors are internalized either constitutively or upon ligand engagement. Whereas there is evidence for differential regulation of the two processes, little is known about the molecular machinery involved. Previous studies have shown that an unidentified kinase substrate is required for endocytosis of the epidermal growth factor receptor (EGFR), the prototypical ligand-inducible receptor, but not of the transferrin receptor (TfR), the prototypical constitutively internalized receptor. Eps15, an endocytic protein that is tyrosine phosphorylated by EGFR, is a candidate for such a function. Here, we show that tyrosine phosphorylation of Eps15 is necessary for internalization of the EGFR, but not of the TfR. We mapped Tyr 850 as the major in vivo tyrosine phosphorylation site of Eps15. A phosphorylation-negative mutant of Eps15 acted as a dominant negative on the internalization of the EGFR, but not of the TfR. A phosphopeptide, corresponding to the phosphorylated sequence of Eps15, inhibited EGFR endocytosis, suggesting that phosphotyrosine in Eps15 serves as a docking site for a phosphotyrosine binding protein. Thus, tyrosine phosphorylation of Eps15 represents the first molecular determinant, other than those contained in the receptors themselves, which is involved in the differential regulation of constitutive vs. regulated endocytosis.

Epsin 1 undergoes nucleocytosolic shuttling and its eps15 interactor NH(2)-terminal homology (ENTH) domain, structurally similar to Armadillo and HEAT repeats, interacts with the transcription factor promyelocytic leukemia Zn(2)+ finger protein (PLZF).

Epsin (Eps15 interactor) is a cytosolic protein involved in clathrin-mediated endocytosis via its direct interactions with clathrin, the clathrin adaptor AP-2, and Eps15. The NH(2)-terminal portion of epsin contains a phylogenetically conserved module of unknown function, known as the ENTH domain (epsin NH(2)-terminal homology domain). We have now solved the crystal structure of rat epsin 1 ENTH domain to 1.8 A resolution. This domain is structurally similar to armadillo and Heat repeats of beta-catenin and karyopherin-beta, respectively. We have also identified and characterized the interaction of epsin 1, via the ENTH domain, with the transcription factor promyelocytic leukemia Zn(2)+ finger protein (PLZF). Leptomycin B, an antifungal antibiotic, which inhibits the Crm1- dependent nuclear export pathway, induces an accumulation of epsin 1 in the nucleus. These findings suggest that epsin 1 may function in a signaling pathway connecting the endocytic machinery to the regulation of nuclear function.

Numb is an endocytic protein.

Numb is a protein that in Drosophila determines cell fate as a result of its asymmetric partitioning at mitosis. The function of Numb has been linked to its ability to bind and to biologically antagonize Notch, a membrane receptor that also specifies cell fate. The biochemical mechanisms underlying the action of Numb, however, are still largely unknown. The wide pattern of expression of Numb suggests a general function in cellular homeostasis that could be additional to, or part of, its action in fate determination. Such a function could be endocytosis, as suggested by the interaction of Numb with Eps15, a component of the endocytic machinery. Here, we demonstrate that Numb is an endocytic protein. We found that Numb localizes to endocytic organelles and is cotrafficked with internalizing receptors. Moreover, it associates with the appendage domain of alpha adaptin, a subunit of AP2, a major component of clathrin-coated pits. Finally, fragments of Numb act as dominant negatives on both constitutive and ligand-regulated receptor-mediated internalization, suggesting a general role for Numb in the endocytic process.

The eps15 homology (EH) domain-based interaction between eps15 and hrb connects the molecular machinery of endocytosis to that of nucleocytosolic transport.

The Eps15 homology (EH) module is a protein-protein interaction domain that establishes a network of connections involved in various aspects of endocytosis and sorting. The finding that EH-containing proteins bind to Hrb (a cellular cofactor of the Rev protein) and to the related protein Hrbl raised the possibility that the EH network might also influence the so-called Rev export pathway, which mediates nucleocytoplasmic transfer of proteins and RNAs. In this study, we demonstrate that Eps15 and Eps15R, two EH-containing proteins, synergize with Hrb and Hrbl to enhance the function of Rev in the export pathway. In addition, the EH-mediated association between Eps15 and Hrb is required for the synergistic effect. The interaction between Eps15 and Hrb occurs in the cytoplasm, thus pointing to an unexpected site of action of Hrb, and to a possible role of the Eps15-Hrb complex in regulating the stability of Rev.

Nucleocytoplasmic shuttling of endocytic proteins.

Many cellular processes rely on the ordered assembly of macromolecular structures. Here, we uncover an unexpected link between two such processes, endocytosis and transcription. Many endocytic proteins, including eps15, epsin1, the clathrin assembly lymphoid myeloid leukemia (CALM), and alpha-adaptin, accumulate in the nucleus when nuclear export is inhibited. Endocytosis and nucleocytoplasmic shuttling of endocytic proteins are apparently independent processes, since inhibition of endocytosis did not appreciably alter nuclear translocation of endocytic proteins, and blockade of nuclear export did not change the initial rate of endocytosis. In the nucleus, eps15 and CALM acted as positive modulators of transcription in a GAL4-based transactivation assay, thus raising the intriguing possibility that some endocytic proteins play a direct or indirect role in transcriptional regulation.

An effector region in Eps8 is responsible for the activation of the Rac-specific GEF activity of Sos-1 and for the proper localization of the Rac-based actin-polymerizing machine.

Genetic and biochemical evidence demonstrated that Eps8 is involved in the routing of signals from Ras to Rac. This is achieved through the formation of a tricomplex consisting of Eps8-E3b1-Sos-1, which is endowed with Rac guanine nucleotide exchange activity. The catalytic subunit of this complex is represented by Sos-1, a bifunctional molecule capable of catalyzing guanine nucleotide exchange on Ras and Rac. The mechanism by which Sos-1 activity is specifically directed toward Rac remains to be established. Here, by performing a structure-function analysis we show that the Eps8 output function resides in an effector region located within its COOH terminus. This effector region, when separated from the holoprotein, activates Rac and acts as a potent inducer of actin polymerization. In addition, it binds to Sos-1 and is able to induce Rac-specific, Sos-1-dependent guanine nucleotide exchange activity. Finally, the Eps8 effector region mediates a direct interaction of Eps8 with F-actin, dictating Eps8 cellular localization. We propose a model whereby the engagement of Eps8 in a tricomplex with E3b1 and Sos-1 facilitates the interaction of Eps8 with Sos-1 and the consequent activation of an Sos-1 Rac-specific catalytic ability. In this complex, determinants of Eps8 are responsible for the proper localization of the Rac-activating machine to sites of actin remodeling.

erbB-2 is a potent oncogene when overexpressed in NIH/3T3 cells.

A wide variety of human tumors contain an amplified or overexpressed erbB-2 gene, which encodes a growth factor receptor-like protein. When erbB-2 complementary DNA was expressed in NIH/3T3 cells under the control of the SV40 promoter, the gene lacked transforming activity despite expression of detectable levels of the erbB-2 protein. A further five- to tenfold increase in its expression under influence of the long terminal repeat of Moloney murine leukemia virus was associated with activation of erbB-2 as a potent oncogene. The high levels of the erbB-2 product associated with malignant transformation of NIH/3T3 cells were observed in human mammary tumor cells that overexpressed this gene. These findings demonstrate a new mechanism for acquisition of oncogenic properties by genes encoding growth factor receptor-like proteins and provide a functional basis for the role of their overexpression in the development of human malignancies.

EGF receptor and erbB-2 tyrosine kinase domains confer cell specificity for mitogenic signaling.

The epidermal growth factor (EGF) receptor (EGFR) can efficiently couple with mitogenic signaling pathways when it is transfected into interleukin-3 (IL-3)-dependent 32D hematopoietic cells. When expression vectors for erbB-2, which is structurally related to EGFR, or its truncated counterpart, delta NerbB-2, were introduced into 32D cells, neither was capable of inducing proliferation. This was despite overexpression and constitutive tyrosine kinase activity of their products at levels associated with potent transformation of fibroblast target cells. Thus, EGFR and erbB-2 couple with distinct mitogenic signaling pathways. The region responsible for the specificity of intracellular signal transduction was localized to a 270-amino acid stretch encompassing their respective tyrosine kinase domains. Thus, tissue- or cell-specific regulation of growth factor receptor signaling can occur at a point after the initial interaction of growth factor with receptor. Such specificity in signal transduction may account for the selection of certain oncogenes in some malignancies.

Signal transduction through the EGF receptor transfected in IL-3-dependent hematopoietic cells.

An expression vector for the epidermal growth factor (EGF) receptor was introduced into the 32D myeloid cell line, which is devoid of EGF receptors and absolutely dependent on interleukin-3 (IL-3) for its proliferation and survival. Expression of the EGF receptor conferred the ability to utilize EGF for transduction of a mitogenic signal. When the transfected cells were propagated in EGF, they exhibited a more mature myeloid phenotype than was observed under conditions of IL-3-directed growth. Moreover, exposure to EGF led to a rapid stimulation of phosphoinositide metabolism, while IL-3 had no detectable effect on phosphoinositide turnover either in control or EGF receptor-transfected 32D cells. Although the transfected cells exhibited high levels of functional EGF receptors, they remained nontumorigenic. In contrast, transfection of v-erbB, an amino-terminal truncated form of the EGF receptor with constitutive tyrosine kinase activity, not only abrogated the IL-3 growth factor requirement of 32D cells, but caused them to become tumorigenic in nude mice. These results show that a naïve hematopoietic cell expresses all of the intracellular components of the EGF-signaling pathway necessary to evoke a mitogenic response and sustain continuous proliferation.

Comparison of biological properties and transforming potential of human PDGF-A and PDGF-B chains.

Human platelet-derived growth factor (PDGF) consists of two distinct but related polypeptide chains designated PDGF-A and PDGF-B. The gene encoding PDGF-B has given rise to the v-sis oncogene. In the present study the transforming activities of PDGF-A and PDGF-B genes are compared. The PDGF-A chain gene is markedly less efficient in inducing transformation than the PDGF-B gene under the influence of the same promoter. There are significant differences in the secretory and growth stimulating properties of the two chains. These properties appear to account for the much more potent transforming ability of the PDGF-B gene. These findings provide insights into biologic properties of a growth factor responsible for potent autocrine stimulation of abnormal cell proliferation.

Gene expression analysis of early and advanced gastric cancers.

Gastric carcinoma is one of the major causes of cancer mortality worldwide. Early detection results in excellent prognosis for patients with early cancer (EGC), whereas the prognosis of advanced cancer (AGC) patients remains poor. It is not clear whether EGC and AGC are molecularly distinct, and whether they represent progressive stages of the same tumor or different entities ab initio. Gene expression profiles of EGC and AGC were determined by Affymetrix technology and quantitative polymerase chain reaction. Representative regulated genes were further analysed by in situ hybridization (ISH) on tissue microarrays. Expression analysis allowed the identification of a signature that differentiates AGC from EGC. In addition, comparison with normal gastric mucosa indicated that the majority of alterations associated with EGC are retained in AGC, and that further expression changes mark the transition from EGC to AGC. Finally, ISH analysis showed that representative genes, differentially expressed in the invasive areas of EGC and AGC, are not differentially expressed in the non-invasive areas of the same tumors. Our data are more directly compatible with a progression model of gastric carcinogenesis, whereby EGC and AGC may represent different molecular stages of the same tumor. Finally, the identification of an AGC-specific signature might help devising novel therapeutic strategies for advanced gastric cancer.

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.

eps15, a novel tyrosine kinase substrate, exhibits transforming activity.

An expression cloning method which allows direct isolation of cDNAs encoding substrates for tyrosine kinases was applied to the study of the epidermal growth factor (EGF) receptor (EGFR) signaling pathway. A previously undescribed cDNA was isolated and designated eps15. The structural features of the predicted eps15 gene product allow its subdivision into three domains. Domain I contains signatures of a regulatory domain, including a candidate tyrosine phosphorylation site and EF-hand-type calcium-binding domains. Domain II presents the characteristic heptad repeats of coiled-coil rod-like proteins, and domain III displays a repeated aspartic acid-proline-phenylalanine motif similar to a consensus sequence of several methylases. Antibodies specific for the eps15 gene product recognize two proteins: a major species of 142 kDa and a minor component of 155 kDa, both of which are phosphorylated on tyrosine following EGFR activation by EGF in vivo. EGFR is also able to directly phosphorylate the eps15 product in vitro. In addition, phosphorylation of the eps15 gene product in vivo is relatively receptor specific, since the erbB-2 kinase phosphorylates it very inefficiently. Finally, overexpression of eps15 is sufficient to transform NIH 3T3 cells, thus suggesting that the eps15 gene product is involved in the regulation of mitogenic signals.

Efficient coupling with phosphatidylinositol 3-kinase, but not phospholipase C gamma or GTPase-activating protein, distinguishes ErbB-3 signaling from that of other ErbB/EGFR family members.

Recombinant expression of a chimeric EGFR/ErbB-3 receptor in NIH 3T3 fibroblasts allowed us to investigate cytoplasmic events associated with ErbB-3 signal transduction upon ligand activation. An EGFR/ErbB-3 chimera was expressed on the surface of NIH 3T3 transfectants as two classes of receptors possessing epidermal growth factor (EGF) binding affinities comparable to those of the wild-type EGF receptor (EGFR). EGF induced autophosphorylation in vivo of the chimeric receptor and DNA synthesis of EGFR/ErbB-3 transfectants with a dose response similar to that of EGFR transfectants. However, the ErbB-3 and EGFR cytoplasmic domains exhibited striking differences in their interactions with several known tyrosine kinase substrates. We demonstrated strong association of phosphatidylinositol 3-kinase activity with the chimeric receptor upon ligand activation comparable in efficiency with that of the platelet-derived growth factor receptor, while the EGFR exhibited a 10- to 20-fold-lower efficiency in phosphatidylinositol 3-kinase recruitment. By contrast, both phospholipase C gamma and GTPase-activating protein failed to associate with or be phosphorylated by the ErbB-3 cytoplasmic domain under conditions in which they coupled with the EGFR. In addition, though certain signal transmitters, including Shc and GRB2, were recruited by both kinases, EGFR and ErbB-3 elicited tyrosine phosphorylation of distinct sets of intracellular substrates. Thus, our findings show that ligand activation of the ErbB-3 kinase triggers a cytoplasmic signaling pathway that hitherto is unique within this receptor subfamily.