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.

Circulating microRNAs: next-generation biomarkers for early lung cancer detection.

Early diagnosis of lung cancer by low-dose computed tomography is an effective strategy to reduce cancer mortality in high-risk individuals. However, recruitment of at-risk individuals with asymptomatic lung cancer still remains challenging. We developed a minimal invasive serum test, based on the detection of circulating microRNAs, which can identify at-risk individuals with asymptomatic early stage non-small cell lung carcinomas with 80% accuracy.

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.

Prep1 (pKnox1)-deficiency leads to spontaneous tumor development in mice and accelerates EmuMyc lymphomagenesis: a tumor suppressor role for Prep1.

The Prep1 homeodomain transcription factor is essential for embryonic development. 25% of hypomorphic Prep1(i/i) embryos, expressing the gene at 2% of the normal levels, survive pregnancy and live a normal-length life. Later in life, however, these mice develop spontaneous pre-tumoral lesions or solid tumors (lymphomas and carcinomas). In addition, transplantation of E14.5 fetal liver (FL) Prep1(i/i) cells into lethally irradiated mice induces lymphomas. In agreement with the above data, haploinsufficiency of a different Prep1-deficient (null) allele accelerates EmuMyc lymphoma growth. Therefore Prep1 has a tumor suppressor function in mice. Immunohistochemistry on tissue micrroarrays (TMA) generated from three distinct human cohorts comprising a total of some 1000 human tumors revealed that 70% of the tumors express no or extremely low levels of Prep1, unlike normal tissues. Our data in mice are thus potentially relevant to human cancer.

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.

Alterations of ubiquitin ligases in human cancer and their association with the natural history of the tumor.

Protein ubiquitination is critical for many cellular processes, through its ability to regulate protein degradation and various signaling mechanisms. In the ubiquitin (Ub) system, substrate specificity is achieved through the E3 family of Ub ligases. Because alterations of the ubiquitination machinery have been reported in human cancers, the selective interference with Ub ligases might represent a powerful therapeutic tool. Here, we report the first wide survey of misregulation of Ub ligases in cancer. We analysed 82 Ub ligases in nine types of cancer by in situ hybridization on tissue microarrays. We found 27 instances in which an Ub ligase was altered in a given type of tumor, when compared with normal tissues: 21 cases of overexpression and 6 cases of underexpression. We further analysed selected Ub ligases in large cohorts of breast and non-small-cell lung carcinomas. In five, of six, of these extended analyses (HUWE1, CCNB1IP1, SIAH1 and SIAH2 in breast cancer and CCNB1IP1 in lung cancer), we found that the levels of Ub ligases correlated significantly with relevant prognostic factors, and with clinical outcome. Our findings show that the alteration of Ub ligases is a frequent event in cancer and identify candidate targets for molecular therapies.

Breast cancer metastases are molecularly distinct from their primary tumors.

Metastases have been widely thought to arise from rare, selected, mutation-bearing cells in the primary tumor. Recently, however, it has been proposed that breast tumors are imprinted ab initio with metastatic ability. Thus, there is a debate over whether 'phenotypic' disease progression is really associated with 'molecular' progression. We profiled 26 matched primary breast tumors and lymph node metastases and identified 270 probesets that could discriminate between the two categories. We then used an independent cohort of breast tumors (81 samples) and unmatched distant metastases (32 samples) to validate and refine this list down to a 126-probeset list. A representative subset of these genes was subjected to analysis by in situ hybridization, on a third independent cohort (57 primary breast tumors and matched lymph node metastases). This not only confirmed the expression profile data, but also allowed us to establish the cellular origin of the signals. One-third of the analysed representative genes (4 of 11) were expressed by the epithelial component. The four epithelial genes alone were able to discriminate primary breast tumors from their metastases. Finally, engineered alterations in the expression of two of the epithelial genes (SERPINB5 and LTF) modified cell motility in vitro, in accordance with a possible causal role in metastasis. Our results show that breast cancer metastases are molecularly distinct from their primary tumors.

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.

Prognostic implications of NUMB immunoreactivity in salivary gland carcinomas.

The gene numb encodes for a protein (Numb) involved in cell fate decisions in Drosophila, with proposed endocytic and developmental functions in mammalians. The distribution pattern of Numb in human tissues however, has not been fully characterized. We set out to explore the immunohistochemical expression of Numb in normal and neoplastic (28 adenoid cystic and 34 mucoepidermoid carcinomas) salivary glands, and correlated the results with the clinico-pathologic features of the neoplasms. Intense Numb immunoreactivity was detected in normal ductal cells and in a subset of acinar cells. In salivary carcinomas, we detected diffuse and intense Numb immunostaining in 5 adenoid cystic and 8 mucoepidermoid carcinomas. By contrast, the majority of adenoid cystic and mucoepidermoid cancers showed only moderate (14 and 5 cases) or focal staining (9 and 21 cases), respectively. The corresponding expression of Numb mRNA was documented in normal parotid gland and adenoid cystic carcinoma. Numb immunoreactivity was inversely correlated with the histological grade and Ki-67 immunoreactivity of both adenoid cystic and mucoepidermoid carcinomas. In addition, while tumor grade, stage, Ki-67 and Numb immunoreactivity were associated with disease-free survival in univariate analysis, only Numb and Ki-67 immunoreactivities retained independent prognostic significance in multivariate analysis. These data suggest that loss of Numb is implicated in aberrant differentiation programs of salivary gland carcinomas and may serve as a prognostic indicator in patients treated for these neoplasms.

A JC virus-induced signal is required for infection of glial cells by a clathrin- and eps15-dependent pathway.

Infectious entry of JC virus (JCV) into human glial cells occurs by receptor-mediated clathrin-dependent endocytosis. In this report we demonstrate that the tyrosine kinase inhibitor genistein blocks virus entry and inhibits infection. Transient expression of dominant-negative eps15 mutants, including a phosphorylation-defective mutant, inhibited both virus entry and infection. We also show that the JCV-induced signal activates the mitogen-activated protein kinases ERK1 and ERK2. These data demonstrate that JC virus binding to human glial cells induces an intracellular signal that is critical for entry and infection by a ligand-inducible clathrin-dependent mechanism.

Signaling through monoubiquitination.

Ubiquitination is a post-translational modification in which a small conserved peptide, ubiquitin, is appended to target proteins in the cell, through a series of complex enzymatic reactions. Recently, a particular form of ubiquitination, monoubiquitination, has emerged as a nonproteolytic reversible modification that controls protein function. In this review, we highlight recent findings on monoubiquitination as a signaling-induced modification, controlled, among others, by pathways originating from active receptor tyrosine kinases. Furthermore, we review the major cellular processes controlled by ubiquitin modification, including membrane trafficking, histone function, transcription regulation, DNA repair, and DNA replication.

A repertoire library that allows the selection of synthetic SH2s with altered binding specificities.

Tyrosine phosphorylation is one of the major mechanisms involved in the intracellular propagation of external signals. Strategies aimed at interfering with this process might allow the control of several cellular phenotypes. SH2 domains mediate protein-protein interactions by recognizing phosphotyrosine (pY) residues in the context of specific phosphopeptides. We created an SH2-scaffolded repertoire library by randomly mutagenizing five critical amino acid positions in the specificity-determining region of the PLCgamma C-terminal SH2 domain. Synthetic SH2 domains were selected from the library using biotinylated phosphopeptides derived from a natural PLCgamma-SH2 ligand as well as unrelated SH2 ligands. The isolated SH2s displayed high binding affinity constants for the selecting peptides and were capable of interacting with the corresponding proteins.

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.

The Eps8 protein coordinates EGF receptor signalling through Rac and trafficking through Rab5.

How epidermal growth factor receptor (EGFR) signalling is linked to EGFR trafficking is largely unknown. Signalling and trafficking involve small GTPases of the Rho and Rab families, respectively. But it remains unknown whether the signalling relying on these two classes of GTPases is integrated, and, if it is, what molecular machinery is involved. Here we report that the protein Eps8 connects these signalling pathways. Eps8 is a substrate of the EGFR, which is held in a complex with Sos1 by the adaptor protein E3bl (ref. 2), thereby mediating activation of Rac. Through its src homology-3 domain, Eps8 interacts with RN-tre. We show that RN-tre is a Rab5 GTPase-activating protein, whose activity is regulated by the EGFR. By entering in a complex with Eps8, RN-tre acts on Rab5 and inhibits internalization of the EGFR. Furthermore, RN-tre diverts Eps8 from its Rac-activating function, resulting in the attenuation of Rac signalling. Thus, depending on its state of association with E3b1 or RN-tre, Eps8 participates in both EGFR signalling through Rac, and trafficking through Rab5.

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.

Epidermal growth factor pathway substrate 15, Eps15.

Eps15 was originally identified as a substrate for the kinase activity of the epidermal growth factor receptor (EGFR). Eps15 has a tripartite structure comprising a NH2-terminal portion, which contains three EH domains, a central putative coiled-coil region, and a COOH-terminal domain containing multiple copies of the amino acid triplet Aspartate-Proline-Phenylalanine. A pool of Eps15 is localized at clathrin coated pits where it interacts with the clathrin assembly complex AP-2 and a novel AP-2 binding protein, Epsin. Perturbation of Eps15 and Epsin function inhibits receptor-mediated endocytosis of EGF and transferrin, demonstrating that both proteins are components of the endocytic machinery. Since the family of EH-containing proteins is implicated in various aspects of intracellular sorting, biomolecular strategies aimed at interfering with these processes can now be envisioned. These strategies have potentially far reaching implications extending to the control of cell proliferation. In this regard, it is of note that Eps15 has the potential of transforming NIH-3T3 cells and that the eps15 gene is rearranged with the HRX/ALL/MLL gene in acute myelogeneous leukemias, thus implicating this protein in the subversion of cell proliferation in neoplasia.

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.