VEGF-mediated angiogenesis links EMT-induced cancer stemness to tumor initiation.

An epithelial-mesenchymal transition (EMT) underlies malignant tumor progression and metastatic spread by enabling cancer cells to depart from the primary tumor, invade surrounding tissue, and disseminate to distant organs. EMT also enriches for cancer stem cells (CSC) and increases the capacity of cancer cells to initiate and propagate tumors upon transplantation into immune-deficient mice, a major hallmark of CSCs. However, the molecular mechanisms promoting the tumorigenicity of cancer cells undergoing an EMT and of CSCs have remained widely elusive. We here report that EMT confers efficient tumorigenicity to murine breast cancer cells by the upregulated expression of the proangiogenic factor VEGF-A and by increased tumor angiogenesis. On the basis of these data, we propose a novel interpretation of the features of CSCs with EMT-induced, VEGF-A-mediated angiogenesis as the connecting mechanism between cancer cell stemness and tumor initiation.

First application of a microsphere-based immunoassay to the detection of genetically modified organisms (GMOs): quantification of Cry1Ab protein in genetically modified maize.

An innovative covalent microsphere immunoassay, based on the usage of fluorescent beads coupled to a specific antibody, was developed for the quantification of the endotoxin Cry1Ab present in MON810 and Bt11 genetically modified (GM) maize lines. In particular, a specific protocol was developed to assess the presence of Cry1Ab in a very broad range of GM maize concentrations, from 0.1 to 100% [weight of genetically modified organism (GMO)/weight]. Test linearity was achieved in the range of values from 0.1 to 3%, whereas fluorescence signal increased following a nonlinear model, reaching a plateau at 25%. The limits of detection and quantification were equal to 0.018 and 0.054%, respectively. The present study describes the first application of quantitative high-throughput immunoassays in GMO analysis.

Mouse models of breast cancer metastasis.

Metastatic spread of cancer cells is the main cause of death of breast cancer patients, and elucidation of the molecular mechanisms underlying this process is a major focus in cancer research. The identification of appropriate therapeutic targets and proof-of-concept experimentation involves an increasing number of experimental mouse models, including spontaneous and chemically induced carcinogenesis, tumor transplantation, and transgenic and/or knockout mice. Here we give a progress report on how mouse models have contributed to our understanding of the molecular processes underlying breast cancer metastasis and on how such experimentation can open new avenues to the development of innovative cancer therapy.

The coiled-coil domain is the structural determinant for mammalian homologues of Drosophila Sina-mediated degradation of promyelocytic leukemia protein and other tripartite motif proteins by the proteasome.

Mammalian homologues of Drosophila Seven in Absentia (SIAHs) target for proteasome-mediated degradation several factors involved in cell growth and tumorigenesis. Here we show that SIAH-1/2 binds and targets for proteasome-mediated degradation the putative tumor suppressor and tripartite motif (TRIM) family member PML, leading to the loss of its transcriptional co-activating properties and a reduction in the number of endogenous PML nuclear bodies. Association with PML requires the substrate-binding domain (SBD) of SIAH-1/2 through an interacting surface apparently distinct from those predicted by the structural studies, or shown experimentally to mediate binding to SIAH-associated factors. Within PML, the coiled-coil domain is required for Siah- and proteasome-mediated degradation, and deletions of regions critical for the integrity of this region impair the ability of Siah to trigger PML-RAR degradation. Fusion of the coiled-coil domain to heterologous proteins resulted in the capacity of mSiah-2 to target their degradation. All of the TRIM proteins tested were degraded upon mSiah-2 overexpression. Finally, we show that the fusion protein PML-RAR (that retains the coiled-coil domain), which causes acute promyelocytic leukemias, is also a potential substrate of mSiah-2. As a result of mSiah-2 overexpression and subsequent degradation of the fusion protein, the arrest in hematopoietic differentiation because of expression of PML-RAR is partially rescued. These results identify PML and other TRIMs as new factors post-translationally regulated by SIAH and involve the coiled-coil region of PML and of other SIAH substrates as a novel structural determinant for targeted degradation.

Acute myeloid leukemia fusion proteins deregulate genes involved in stem cell maintenance and DNA repair.

Acute myelogenous leukemias (AMLs) are genetically heterogeneous and characterized by chromosomal rearrangements that produce fusion proteins with aberrant transcriptional regulatory activities. Expression of AML fusion proteins in transgenic mice increases the risk of myeloid leukemias, suggesting that they induce a preleukemic state. The underlying molecular and biological mechanisms are, however, unknown. To address this issue, we performed a systematic analysis of fusion protein transcriptional targets. We expressed AML1/ETO, PML/RAR, and PLZF/RAR in U937 hemopoietic precursor cells and measured global gene expression using oligonucleotide chips. We identified 1,555 genes regulated concordantly by at least two fusion proteins that were further validated in patient samples and finally classified according to available functional information. Strikingly, we found that AML fusion proteins induce genes involved in the maintenance of the stem cell phenotype and repress DNA repair genes, mainly of the base excision repair pathway. Functional studies confirmed that ectopic expression of fusion proteins constitutively activates pathways leading to increased stem cell renewal (e.g., the Jagged1/Notch pathway) and provokes accumulation of DNA damage. We propose that expansion of the stem cell compartment and induction of a mutator phenotype are relevant features underlying the leukemic potential of AML-associated fusion proteins.

Reduced hepatic tumor incidence in cyclin G1-deficient mice.

Cyclin G1 is a transcriptional target of the tumor suppressor p53, and its expression is increased after DNA damage. Recent data show that cyclin G1 can regulate the levels of p53 by a mechanism that involves dephosphorylation of Mdm2 by protein phosphatase 2A. To understand the biologic role of cyclin G1, we have generated cyclin G1-deficient mice. In agreement with previous results, we showed that these mice develop normally, and that proliferation and induction of cellular senescence in cyclin G1-deficient mouse embryo fibroblasts are indistinguishable from wild-type fibroblasts. However, we found that the p53 levels in the cyclin G1-deficient mice are 2-fold higher that in wild-type mice. Moreover, we showed that treatment of mice with the alkylating agent 1,4-bis[N,N'-di(ethylene)-phosphamide]piperazine (Dipin), followed by partial hepatectomy, decreased G1-S transition in cyclin G1-null hepatocytes as compared with wild type. Finally, we found a significant decrease in tumor incidence, mass, and malignancy in both male and female cyclin G1-null mice after treatment with the potent hepatocarcinogen N-diethylnitrosamine. Taken with recent published data, our results suggest that cyclin G1, together with Mdm2, constitute a part of a negative feedback system that attenuates the activity of p53. In conclusion, our data suggest that the decreased tumor susceptibility after loss of cyclin G1 function is caused by the increased tumor suppressor action of p53.