Increased Arginase1 expression in tumor microenvironment promotes mammary carcinogenesis via multiple mechanisms.

Arginine metabolism plays a significant role in regulating cell function, affecting tumor growth and metastatization. To study the effect of the arginine-catabolizing enzyme Arginase1 (ARG1) on tumor microenvironment, we generated a mouse model of mammary carcinogenesis by crossbreeding a transgenic mouse line overexpressing ARG1 in macrophages (FVBArg+/+) with the MMTV-Neu mouse line (FVBNeu+/+). This double transgenic line (FVBArg+/-;Neu+/+) showed a significant shortening in mammary tumor latency, and an increase in the number of mammary nodules. Transfer of tumor cells from FVBNeu+/+ into either FVB wild type or FVBArg+/+ mice resulted in increase regulatory T cells in the tumor infiltrate, suggestive of an impaired antitumor immune response. However, we also found increased frequency of tumor stem cells in tumors from FVBArg+/-;Neu+/+ transgenic compared with FVBNeu+/+ mice, suggesting that increased arginine metabolism in mammary tumor microenvironment may supports the cancer stem cells niche. We provide in vivo evidence of a novel, yet unexploited, mechanism through which ARG1 may contribute to tumor development.

Multifocal Signal Modulation Therapy by Celecoxib: A Strategy for Managing Castration-Resistant Prostate Cancer.

BACKGROUND: Prostate cancer (PCa) is a significant health concern throughout the world. Standard therapy for advanced disease consists of anti-androgens, however, almost all prostate tumors become castration resistant (CRPC). Progression from androgen-sensitive PCa to CRPC is promoted by inflammatory signaling through cyclooxygenase-2 (COX-2) expression and ErbB family receptors/AKT activation, compensating androgen receptor inactivity. METHODS: Making use of CRPC cell lines, we investigated the effects of the anti-inflammatory drug celecoxib. Biochemical data obtained using immunoblotting, enzyme-linked immunosorbent assay (ELISA), invasion, and xenografts were further integrated by bioinformatic analyses. RESULTS: Celecoxib reduced cell growth and induced apoptosis through AKT blockade, cleavage of poly (ADP-ribose) polymerase-1 (PARP-1), and proteasomal degradation of the anti-apoptotic protein Mcl-1. Epidermal growth factor receptor (EGFR), ErbB2, and ErbB3 degradation, and heterogeneous nuclear ribonucleoprotein K (hnRNP K) downregulation, further amplified the inhibition of androgen signaling. Celecoxib reduced the invasive phenotype of CRPC cells by modulating NF-κB activity and reduced tumor growth in mice xenografts when administered in association with the anti-EGFR receptor antibody cetuximab. Bioinformatic analyses on human prostate cancer datasets support the relevance of these pathways in PCa progression. CONCLUSIONS: Signaling nodes at the intersection of pathways implicated in PCa progression are simultaneously modulated by celecoxib treatment. In combination therapies with cetuximab, celecoxib could represent a novel therapeutic strategy to curb signal transduction during CRPC progression.

Dlx5 and Dlx6 control uterine adenogenesis during post-natal maturation: possible consequences for endometriosis.

Dlx5 and Dlx6 are two closely associated homeobox genes which code for transcription factors involved in the control of steroidogenesis and reproduction. Inactivation of Dlx5/6 in the mouse results in a Leydig cell defect in the male and in ovarian insufficiency in the female. DLX5/6 are also strongly expressed by the human endometrium but their function in the uterus is unknown. The involvement of DLX5/6 in human uterine pathology is suggested by their strong downregulation in endometriotic lesions and upregulation in endometrioïd adenocarcinomas. We first show that Dlx5/6 expression begins in Müllerian ducts epithelia and persists then in the uterine luminal and glandular epithelia throughout post-natal maturation and in the adult. We then use a new mouse model in which Dlx5 and Dlx6 can be simultaneously inactivated in the endometrium using a Pgr(cre/+) allele. Post-natal inactivation of Dlx5/6 in the uterus results in sterility without any obvious ovarian involvement. The uteri of Pgr(cre/+); Dlx5/6(flox/flox) mice present very few uterine glands and numerous abnormally large and branched invaginations of the uterine lumen. In Dlx5/6 mutant uteri, the expression of genes involved in gland formation (Foxa2) and in epithelial remodelling during implantation (Msx1) is significantly reduced. Furthermore, we show that DLX5 is highly expressed in human endometrial glandular epithelium and that its expression is affected in endometriosis. We conclude that Dlx5 and Dlx6 expression determines uterine architecture and adenogenesis and is needed for implantation. Given their importance for female reproduction, DLX5 and DLX6 must be regarded as interesting targets for future clinical research.

Systemic alkalinisation delays prostate cancer cell progression in TRAMP mice.

The microenvironment of solid tumours is extremely acidic and this condition arises since the precancerous stage. This acidic milieu could therefore provide a useful target for both prophylactic and therapeutic approaches. In TRAMP transgenic mice, an in vivo model of prostate adenocarcinoma (AC), oral administration of alkaline water was devoid of unwanted side effects, and when started from an early age was as effective as NaHCO3 in significantly delaying tumour progression, while when started when prostate tumours were already present, a nonstatistically significant trend in the same direction was detected. These findings indicate that the use of alkalinizing drugs should be considered for chemoprevention and, in association with standard chemotherapy, for treatment of human prostate AC.

Transgenic mice overexpressing arginase 1 in monocytic cell lineage are affected by lympho-myeloproliferative disorders and disseminated intravascular coagulation.

Arginase (ARG) is a metabolic enzyme present in two isoforms that hydrolyze l-arginine to urea and ornithine. In humans, ARG isoform 1 is also expressed in cells of the myeloid lineage. ARG activity promotes tumour growth and inhibits T lymphocyte activation. However, the two ARG transgenic mouse lines produced so far failed to show such effects. We have generated, in two different genetic backgrounds, transgenic mice constitutively expressing ARG1 under the control of the CD68 promoter in macrophages and monocytes. Both heterozygous and homozygous transgenic mice showed a relevant increase in mortality at early age, compared with wild-type siblings (67/267 and 48/181 versus 8/149, respectively, both P < 0.005). This increase was due to high incidence of haematologic malignancies, in particular myeloid leukaemia, myeloid dysplasia, lymphomas and disseminated intravascular coagulation (DIC), diseases that were absent in wild-type mice. Atrophy of lymphoid organs due to reduction in T-cell compartment was also detected. Our results indicate that ARG activity may participate in the pathogenesis of lymphoproliferative and myeloproliferative disorders, suggest the involvement of alterations of L-arginine metabolism in the onset of DIC and confirm a role for the enzyme in regulating T-cell homeostasis.

In vivo generation of decidual natural killer cells from resident hematopoietic progenitors.

Decidual natural killer cells accumulate at the fetal-maternal interface and play a key role in a successful pregnancy. However, their origin is still unknown. Do they derive from peripheral natural killer cells recruited in decidua or do they represent a distinct population that originates in situ? Here, we identified natural killer precursors in decidua and uterus of pregnant mice. These precursors underwent rapid in situ differentiation and large proportions of proliferating immature natural killer cells were present in decidua and uterus as early as gestation day 4.5. Here, we investigated the origin of decidua- and uterus-natural killer cells by performing transfer experiments of peripheral mature natural killer cells or precursors from EGFP(+) mice. Results showed that mature natural killer cells did not migrate into decidua and uterus, while precursors were recruited in these organs and differentiated towards natural killer cells. Moreover, decidua- and uterus-natural killer cells displayed unique phenotypic and functional features. They expressed high levels of the activating Ly49D receptor in spite of their immature phenotype. In addition, decidua- and uterus-natural killer cells were poorly cytolytic and produced low amounts of IFN-γ, while they released factors (GM-CSF, VEGF, IP-10) involved in neo-angiogenesis and tissue remodeling. Our data reveal in situ generation of decidual natural killer cells and provide an important correlation between mouse and human decidual natural killer cells, allowing further studies to be carried out on their role in pregnancy-related diseases.

Allelic reduction of Dlx5 and Dlx6 results in early follicular depletion: a new mouse model of primary ovarian insufficiency.

Primary ovarian insufficiency (POI) is characterized by the loss of ovarian function before the age of 40 in humans. Although most cases of POI are idiopathic, many are familial, underlying a genetic origin of the disease. Mutations in genes involved in the control of steroidogenesis, such as NR5A1 (SF-1, Steroidogenic Factor 1), CYP17, CYP19A1 (aromatase), StAR (Steroidogenic Acute Regulatory), and the forkhead transcription factor FOXL2 have been associated with different forms of POI. In males, the homeobox transcription factors Dlx5 and Dlx6 are involved in the control of steroidogenesis through the activation of GATA4-induced-StAR transcription. Here, we analyze the potential involvement of Dlx5 and Dlx6 in female reproduction. To this end, we make use of an existing mouse model in which Dlx5 and Dlx6 are simultaneously disrupted. We show that: (i) allelic reduction of Dlx5 and Dlx6 in the mouse results in a POI-like phenotype, characterized by reduced fertility and early follicular exhaustion; (ii) in granulosa cell lines, a reciprocal regulation exists between Dlx5 and Foxl2; (iii) in the mouse ovary, allelic reduction of Dlx5/6 results in the upregulation of Foxl2. We propose that the mutual regulation between Dlx5/6 and Foxl2 and their opposite effects on StAR expression might contribute to determine the homeostatic control of steroidogenesis within the ovary. Dysregulation of this homeostatic control would result in abnormal follicular maturation and reduced fertility. Our results bring new elements to our conceptual model of follicle maturation and maintenance and provide new potential genetic targets for cases of familial POI.

Curcumin inhibits prostate cancer metastasis in vivo by targeting the inflammatory cytokines CXCL1 and -2.

In America and Western Europe, prostate cancer is the second leading cause of death in men. Emerging evidence suggests that chronic inflammation is a major risk factor for the development and metastatic progression of prostate cancer. We previously reported that the chemopreventive polyphenol curcumin inhibits the expression of the proinflammatory cytokines CXCL1 and -2 leading to diminished formation of breast cancer metastases. In this study, we analyze the effects of curcumin on prostate carcinoma growth, apoptosis and metastasis. We show that curcumin inhibits translocation of NFκB to the nucleus through the inhibition of the IκB-kinase (IKKß, leading to stabilization of the inhibitor of NFκB, IκBα, in PC-3 prostate carcinoma cells. Inhibition of NFκB activity reduces expression of CXCL1 and -2 and abolishes the autocrine/paracrine loop that links the two chemokines to NFκB. The combination of curcumin with the synthetic IKKß inhibitor, SC-541, shows no additive or synergistic effects indicating that the two compounds share the target. Treatment of the cells with curcumin and siRNA-based knockdown of CXCL1 and -2 induce apoptosis, inhibit proliferation and downregulate several important metastasis-promoting factors like COX2, SPARC and EFEMP. In an orthotopic mouse model of hematogenous metastasis, treatment with curcumin inhibits statistically significantly formation of lung metastases. In conclusion, chronic inflammation can induce a metastasis prone phenotype in prostate cancer cells by maintaining a positive proinflammatory and prometastatic feedback loop between NFκB and CXCL1/-2. Curcumin disrupts this feedback loop by the inhibition of NFκB signaling leading to reduced metastasis formation in vivo.

Regulation of neuroendocrine differentiation by AKT/hnRNPK/AR/ß-catenin signaling in prostate cancer cells.

Current diagnostic tools cannot predict clinical failure and androgen-independent disease progression for patients with prostate cancer (PC). The survival signaling pathways of prostate cells play a central role in the progression of tumors to a neuroendocrine (NE) phenotype. NE cells demonstrate attributes that suggest that they are an integral part of the signaling cascade leading to castration-resistant PC. In this study, making use of in vitro neuroendocrine differentiation (NED) of human LNCaP and mouse TRAMP-C2 cells after androgen withdrawal, and of the transgenic adenocarcinoma of mouse prostate (TRAMP) model, we characterized a sequence of molecular events leading to NED and identified a number of markers that could be detectable by routine analyses not only in castration resistant PC but also in hormone naïve PC at the time of initial diagnosis. We found that NED associates with AKT activation that in turn regulates heterogeneous nuclear ribonucleoprotein K (hnRNP K), androgen receptor (AR) and ß-catenin levels. Addition of molecules targeting membrane-bound receptors and protein kinases blocks NE differentiation in LNCaP and TRAMP-C2 cells. The extent of AKT phosphorylation and hnRNP K, AR and ß-catenin levels may have a potential value as prognostic indicators discriminating between androgen-responsive and unresponsive cells and could be used as molecular targets to monitor the anti-tumor action of new therapeutic protocols based on antireceptor agents and/or neuroendocrine hormone antagonists.

Effect of Eribulin on Angiogenesis and the Expression of Endothelial Adhesion Molecules.

BACKGROUND/AIM: Tumor vasculature is an important component of the tumor microenvironment and deeply affects anticancer immune response. Eribulin is a non-taxane inhibitor of the mitotic spindle. However, off-target effects interfering with the tumor vasculature have been reported. The mechanisms responsible of this effect are still unclear. MATERIALS AND METHODS: We designed an in vitro study to investigate the effect of eribulin, with or without TGF-ß, on neo-angiogenesis, and on the expression of the adhesion molecules ICAM-1 and VCAM-1. We also investigated the effects of paclitaxel and vinorelbine under the same experimental conditions. RESULTS: Eribulin up-regulated the epithelial markers VE-cadherin and CD-31 in HUVEC and inhibited tube formation in HUVEC cells cultured in Matrigel. The drug effectively arrested tube formation even in the presence of TGF-ß and counteracted the TGF-ß-induced change in cell shape from the endothelial cobblestone-like morphology to an elongated spindle-shaped morphology. We also observed that eribulin was able to upregulate ICAM-1 and to counteract its down-regulation induced by TGF-ß. CONCLUSION: Eribulin exerts different off-label effects: increases vascular remodeling, counteracts the endothelial-to-mesenchymal transition (EndMT) mediated by TGF-ß and promotes tumor infiltration by immune cells via increasing the expression of ICAM-1 and transcription of CD31 and VE-cadherin. Moreover, eribulin was able to inhibit vasculature remodeling and the induction of EndMT mediated by TGF-ß better than vinorelbine and paclitaxel. The effects observed in this study might have important therapeutic consequence if the drug is combined with immunotherapy.

Treatment of newborn G6pc(-/-) mice with bone marrow-derived myelomonocytes induces liver repair.

BACKGROUND & AIMS: Several studies have shown that bone marrow-derived committed myelomonocytic cells can repopulate diseased livers by fusing with host hepatocytes and can restore normal liver function. These data suggest that myelomonocyte transplantation could be a promising approach for targeted and well-tolerated cell therapy aimed at liver regeneration. We sought to determine whether bone marrow-derived myelomonocytic cells could be effective for liver reconstitution in newborn mice knock-out for glucose-6-phosphatase-α. METHODS: Bone marrow-derived myelomonocytic cells obtained from adult wild type mice were transplanted in newborn knock-out mice. Tissues of control and treated mice were frozen for histochemical analysis, or paraffin-embedded and stained with hematoxylin and eosin for histological examination or analyzed by immunohistochemistry or fluorescent in situ hybridization. RESULTS: Histological sections of livers of treated knock-out mice revealed areas of regenerating tissue consisting of hepatocytes of normal appearance and partial recovery of normal architecture as early as 1 week after myelomonocytic cells transplant. FISH analysis with X and Y chromosome paints indicated fusion between infused cells and host hepatocytes. Glucose-6-phosphatase activity was detected in treated mice with improved profiles of liver functional parameters. CONCLUSIONS: Our data indicate that bone marrow-derived myelomonocytic cell transplant may represent an effective way to achieve liver reconstitution of highly degenerated livers in newborn animals.

Spatio-temporal dynamics of gene expression of the Edn1-Dlx5/6 pathway during development of the lower jaw.

The morphogenesis of the vertebrate skull results from highly dynamic integrated processes involving the exchange of signals between the ectoderm, the endoderm, and cephalic neural crest cells (CNCCs). Before migration CNCCs are not committed to form any specific skull element, molecular signals exchanged in restricted regions of tissue interaction are crucial in providing positional identity to the CNCCs mesenchyme and activate the specific morphogenetic process of different skeletal components of the head. In particular, the endothelin-1 (Edn1)-dependent activation of Dlx5 and Dlx6 in CNCCs that colonize the first pharyngeal arch (PA1) is necessary and sufficient to specify maxillo-mandibular identity. Here, to better analyze the spatio-temporal dynamics of this process, we associate quantitative gene expression analysis with detailed examination of skeletal phenotypes resulting from combined allelic reduction of Edn1, Dlx5, and Dlx6. We show that Edn1-dependent and -independent regulatory pathways act at different developmental times in distinct regions of PA1. The Edn1-->Dlx5/6-->Hand2 pathway is already active at E9.5 during early stages of CNCCs colonization. At later stages (E10.5) the scenario is more complex: we propose a model in which PA1 is subdivided into four adjacent territories in which distinct regulations are taking place. This new developmental model may provide a conceptual framework to interpret the craniofacial malformations present in several mouse mutants and in human first arch syndromes. More in general, our findings emphasize the importance of quantitative gene expression in the fine control of morphogenetic events.

Mutually exclusive expression of DLX2 and DLX5/6 is associated with the metastatic potential of the human breast cancer cell line MDA-MB-231.

BACKGROUND: The DLX gene family encodes for homeobox transcription factors involved in the control of morphogenesis and tissue homeostasis. Their expression can be regulated by Endothelin1 (ET1), a peptide associated with breast cancer invasive phenotype. Deregulation of DLX gene expression was found in human solid tumors and hematologic malignancies. In particular, DLX4 overexpression represents a possible prognostic marker in ovarian cancer. We have investigated the role of DLX genes in human breast cancer progression. METHODS: MDA-MB-231 human breast carcinoma cells were grown in vitro or injected in nude mice, either subcutaneously, to mimic primary tumor growth, or intravenously, to mimic metastatic spreading. Expression of DLX2, DLX5 and DLX6 was assessed in cultured cells, either treated or not with ET1, tumors and metastases by RT-PCR. In situ hybridization was used to confirm DLX gene expression in primary tumors and in lung and bone metastases. The expression of DLX2 and DLX5 was evaluated in 408 primary human breast cancers examining the GSE1456 and GSE3494 microarray datasets. Kaplan-Meier estimates for disease-free survival were calculated for the patients grouped on the basis of DLX2/DLX5 expression. RESULTS: Before injection, or after subcutaneous growth, MDA-MB-231 cells expressed DLX2 but neither DLX5 nor DLX6. Instead, in bone and lung metastases resulting from intravenous injection we detected expression of DLX5/6 but not of DLX2, suggesting that DLX5/6 are activated during metastasis formation, and that their expression is alternative to that of DLX2. The in vitro treatment of MDA-MB-231 cells with ET1, resulted in switch from DLX2 to DLX5 expression. By data mining in microarray datasets we found that expression of DLX2 occurred in 21.6% of patients, and was significantly correlated with prolonged disease-free survival and reduced incidence of relapse. Instead, DLX5 was expressed in a small subset of cases, 2.2% of total, displaying reduced disease-free survival and high incidence of relapse which was, however, non-significantly different from the other groups due to the small size of the DLX+ cohort. In all cases, we found mutually exclusive expression of DLX2 and DLX5. CONCLUSIONS: Our studies indicate that DLX genes are involved in human breast cancer progression, and that DLX2 and DLX5 genes might serve as prognostic markers.

Insulin-independent stimulation of skeletal muscle glucose uptake by low-dose abscisic acid via AMPK activation.

Abscisic acid (ABA) is a plant hormone active also in mammals where it regulates, at nanomolar concentrations, blood glucose homeostasis. Here we investigated the mechanism through which low-dose ABA controls glycemia and glucose fate. ABA stimulated uptake of the fluorescent glucose analog 2-NBDG by L6, and of [18F]-deoxy-glucose (FDG) by mouse skeletal muscle, in the absence of insulin, and both effects were abrogated by the specific AMPK inhibitor dorsomorphin. In L6, incubation with ABA increased phosphorylation of AMPK and upregulated PGC-1α expression. LANCL2 silencing reduced all these ABA-induced effects. In vivo, low-dose oral ABA stimulated glucose uptake and storage in the skeletal muscle of rats undergoing an oral glucose load, as detected by micro-PET. Chronic treatment with ABA significantly improved the AUC of glycemia and muscle glycogen content in CD1 mice exposed to a high-glucose diet. Finally, both acute and chronic ABA treatment of hypoinsulinemic TRPM2-/- mice ameliorated the glycemia profile and increased muscle glycogen storage. Altogether, these results suggest that low-dose oral ABA might be beneficial for pre-diabetic and diabetic subjects by increasing insulin-independent skeletal muscle glucose disposal through an AMPK-mediated mechanism.

IL-8 induces exocytosis of arginase 1 by neutrophil polymorphonuclears in nonsmall cell lung cancer.

Arginase 1 (ARG1) inhibits T-cell proliferation by degrading extracellular arginine, which results in decreased responsiveness of T cells to CD3/TCR stimulation. In humans, ARG1 is stored in inactive form within granules of polymorphonuclear neutrophils (PMNs) and gets activated on release. We studied the role of PMNs-related ARG1 activity in nonsmall cell lung cancer (NSLC), in which tumor-infiltrating lymphocytes showed reduced proliferation in response to CD3/TCR triggering. Patients with NSCLC had increased ARG1 plasma levels as compared to healthy controls. Furthermore, immunohistochemistry showed that tumor-infiltrating PMNs display reduced intracellular ARG1, in comparison to intravascular or peritumoral PMNs, suggesting a role of tumor microenvironment in ARG1 release. Indeed, supernatants of NSCLC cell lines induced exocytosis of ARG1 from PMNs. All (4/4) NSCLC cell lines and all (7/7) CD14- cell samples from NSCLC expressed interleukin (IL)-8 mRNA, whereas TNFalpha mRNA was expressed by 1 cell line and by 2 tumor specimens. Furthermore, all NSCLC cell lines secreted immunoreactive IL-8, albeit at different levels. IL-8 was as effective as TNFalpha in triggering ARG1 release and the 2 cytokines acted synergistically. Secreted ARG1 was biologically active and catabolized extracellular arginine. The supernatant of IL-8 gene-silenced NSCLC cells did not mediate ARG1 release by PMNs. Altogether these findings demonstrate a role of IL-8 in ARG1 exocytosis by PMNs and indicate that, due at least in part to IL-8 secreted by NSCLC cells, PMNs infiltrating NSCLC release ARG1. This phenomenon could contribute to local immune suppression.

The carboxyl terminal trimer of procollagen I induces pro-metastatic changes and vascularization in breast cancer cells xenografts.

BACKGROUND: The COOH terminal peptide of Pro-collagen type I (PICP, also called C3) is chemotactic for endothelial melanoma and breast cancer cells. PICP induces the expression of Metalloproteinases-2 and -9, of Vascular endothelial growth factor and of the chemokine CXCL-12 receptor CXCR4 in MDA MB231 breast carcinoma cells in vitro. METHODS: We used a model of xenografts in BalbC/nude mice obtaining tumors by implanting in contro-lateral subcutaneous positions MDA MB231 cells added or not with purified PICP and studied the earlier phases of tumor development, up to 48 days from implant, by histology, immunostain and in situ hybridization. RESULTS: Addition of PICP promotes rapid vascularization of the tumors while does not affect mitotic and apoptotic indexes and overall tumor growth. PICP-treated, relative to control tumors, show up-modulation of Vascular endothelial factor, Metalloproteinase-9 and CXCR4, all tumor prognostic genes; they also show down-modulation of the endogenous Metalloproteinase inhibitor, reversion-inducing-cysteine-rich protein with kazal motifs, and a different pattern of modulation of Tissue Inhibitor of Metalloproteinase-2. These changes occur in absence of detectable expression of CXCL-12, up to 38 days, in control and treated tumors. CONCLUSION: PICP has an early promoting effect in the acquisition by the tumors of prometastatic phenotype. PICP may be play a relevant role in the productive interactions between stroma and tumor cells by predisposing the tumor cells to respond to the proliferation stimuli ensuing the activation of signaling by engagement of CXCR4 by cytokines and by fostering their extravasion, due to the induction of increased vascular development.

Overexpression of the cohesin-core subunit SMC1A contributes to colorectal cancer development.

BACKGROUND: Cancer cells are characterized by chromosomal instability (CIN) and it is thought that errors in pathways involved in faithful chromosome segregation play a pivotal role in the genesis of CIN. Cohesin forms a large protein ring that binds DNA strands by encircling them. In addition to this central role in chromosome segregation, cohesin is also needed for DNA repair, gene transcription regulation and chromatin architecture. Though mutations in both cohesin and cohesin-regulator genes have been identified in many human cancers, the contribution of cohesin to cancer development is still under debate. METHODS: Normal mucosa, early adenoma, and carcinoma samples deriving from 16 subjects affected by colorectal cancer (CRC) were analyzed by OncoScan for scoring both chromosome gains and losses (CNVs) and loss of heterozygosity (LOH). Then the expression of SMC1A was analyzed by immunochemistry in 66 subjects affected by CRC. The effects of SMC1A overexpression and mutated SMC1A were analyzed in vivo using immunocompromised mouse models. Finally, we measured global gene expression profiles in induced-tumors by RNA-seq. RESULTS: Here we showed that SMC1A cohesin core gene was present as extra-copies, mutated, and overexpressed in human colorectal carcinomas. We then demonstrated that cohesin overexpression led to the development of aggressive cancers in immunocompromised mice through gene expression dysregulation. CONCLUSION: Collectively, these results support a role of defective cohesin in the development of human colorectal cancer.

Abnormal urethra formation in mouse models of split-hand/split-foot malformation type 1 and type 4.

Urogenital birth defects are one of the common phenotypes observed in hereditary human disorders. In particular, limb malformations are often associated with urogenital developmental abnormalities, as the case for Hand-foot-genital syndrome displaying similar hypoplasia/agenesis of limbs and external genitalia. Split-hand/split-foot malformation (SHFM) is a syndromic limb disorder affecting the central rays of the autopod with median clefts of the hands and feet, missing central fingers and often fusion of the remaining ones. SHFM type 1 (SHFM1) is linked to genomic deletions or rearrangements, which includes the distal-less-related homeogenes DLX5 and DLX6 as well as DSS1. SHFM type 4 (SHFM4) is associated with mutations in p63, which encodes a p53-related transcription factor. To understand that SHFM is associated with urogenital birth defects, we performed gene expression analysis and gene knockout mouse model analyses. We show here that Dlx5, Dlx6, p63 and Bmp7, one of the p63 downstream candidate genes, are all expressed in the developing urethral plate (UP) and that targeted inactivation of these genes in the mouse results in UP defects leading to abnormal urethra formation. These results suggested that different set of transcription factors and growth factor genes play similar developmental functions during embryonic urethra formation. Human SHFM syndromes display multiple phenotypes with variations in addition to split hand foot limb phenotype. These results suggest that different genes associated with human SHFM could also be involved in the aetiogenesis of hypospadias pointing toward a common molecular origin of these congenital malformations.

Msx1 and Dlx5 act independently in development of craniofacial skeleton, but converge on the regulation of Bmp signaling in palate formation.

Msx and Dlx homeoproteins control the morphogenesis and organization of craniofacial skeletal structures, specifically those derived from the pharyngeal arches. In vitro Msx and Dlx proteins have opposing transcriptional properties and form heterodimeric complexes via their homeodomain with reciprocal functional repression. In this report we examine the skeletal phenotype of Msx1; Dlx5 double knock-out (DKO) mice in relationship with their expression territories during craniofacial development. Co-expression of Dlx5 and Msx1 is only observed in embryonic tissues in which these genes have independent functions, and thus direct protein interactions are unlikely to control morphogenesis of the cranium. The DKO craniofacial phenotypes indicate a complex interplay between these genes, acting independently (mandible and middle ear), synergistically (deposition of bone tissue) or converging on the same morphogenetic process (palate growth and closure). In the latter case, the absence of Dlx5 rescues in part the Msx1-dependent defects in palate growth and elevation. At the basis of this effect, our data implicate the Bmp (Bmp7, Bmp4)/Bmp antagonist (Follistatin) signal: in the Dlx5(-/-) palate changes in the expression level of Bmp7 and Follistatin counteract the reduced Bmp4 expression. These results highlight the importance of precise spatial and temporal regulation of the Bmp/Bmp antagonist system during palate closure.

Inhibition of BUB1 results in genomic instability and anchorage-independent growth of normal human fibroblasts.

The relative contribution of aneuploidy and gene mutations to human tumorigenesis is not yet known. Studies in mice have demonstrated that even single point mutations in oncogenes and tumor suppressor genes can dramatically increase tumor frequency. However, models to evaluate the definitive role of aneuploidy and genomic instability are not yet available. Human fibroblast cells have long been used as a tool for investigating proliferation, senescence, immortalization, and tumorigenesis, all processes that are strongly interrelated. We have now used antisense and ribozyme-mediated temporary inhibition of BUB1 to study the consequences of mitotic checkpoint failure on the development of aneuploidy. The analysis of cell colonies selected by soft agar growth showed evidence of chromosome instability and delayed senescence, without being tumorigenic in nude mice. Our data suggest that chromosomal instability and aneuploidy are early changes that precede tumorigenicity in the multistep process leading to neoplastic transformation.