BackgroundThe Hedgehog (Hh) signaling pathway is vital to animal development as it mediates the differentiation of multiple cell types during embryogenesis. In adults, Hh signaling can be activated to facilitate tissue maintenance and repair. Moreover, stimulation of the Hh pathway has shown therapeutic efficacy in models of neuropathy. The underlying mechanisms of Hh signal transduction remain obscure, however: little is known about the communication between the pathway suppressor Patched (Ptc), a multipass transmembrane protein that directly binds Hh, and the pathway activator Smoothened (Smo), a protein that is related to G-protein-coupled receptors and is capable of constitutive activation in the absence of Ptc.ResultsWe have identified and characterized a synthetic non-peptidyl small molecule, Hh-Ag, that acts as an agonist of the Hh pathway. This Hh agonist promotes cell-type-specific proliferation and concentration-dependent differentiation in vitro, while in utero it rescues aspects of the Hh-signaling defect in Sonic hedgehog-null, but not Smo-null, mouse embryos. Biochemical studies with Hh-Ag, the Hh-signaling antagonist cyclopamine, and a novel Hh-signaling inhibitor Cur61414, reveal that the action of all these compounds is independent of Hh-protein ligand and of the Hh receptor Ptc, as each binds directly to Smo.ConclusionsSmo can have its activity modulated directly by synthetic small molecules. These studies raise the possibility that Hh signaling may be regulated by endogenous small molecules in vivo and provide potent compounds with which to test the therapeutic value of activating the Hh-signaling pathway in the treatment of traumatic and chronic degenerative conditions.

We report the identification and expression pattern of a full-length human cDNA and a partial mouse cDNA encoding cyclin B3. Cyclin B3 (CCNB3) is conserved from Caenorhabditis elegans to Homo sapiens and has an undefined meiotic function in female, but not male Drosophila melanogaster. We show that H. sapiens cyclin B3 interacts with cdk2, is localized to the nucleus, and is degraded during anaphase entry after the degradation of cyclin B1. Degradation is dependent on sequences conserved in a destruction box motif. Overexpression of nondegradable cyclin B3 blocks the mitotic cell cycle in late anaphase, and at higher doses it can interfere with progression through G 1 and entry into S phase. H. sapiens cyclin B3 mRNA and protein are detected readily in developing germ cells in the human testis and not in any other tissue. The mouse cDNA has allowed us to further localize cyclin B3 mRNA to leptotene and zygotene spermatocytes. The expression pattern of mammalian cyclin B3 suggests that it may be important for events occurring in early meiotic prophase I.Cyclins are positive regulatory subunits of the cyclin-dependent kinases (cdks).1 Cyclins increase kinase activity by inducing structural changes in the cdk (1). Cyclins also provide substrate binding motifs (2, 3), signals that localize cdks (4, 5), and motifs that allow the incorporation of the cdk into higher order molecular complexes (6, 7). Although first identified as regulators of mitotic cell cycle transitions, cyclin-cdk complexes have other roles: cyclin C-cdk8 and cyclin H-cdk7 regulate transcription as part of the polymerase II holoenzyme (8 -10), pho80-pho85 regulates the localization of the pho4 transcription factor (11), p35-cdk5 regulates neuronal function (12, 13), and cyclin A1, with an unidentified cdk, regulates the pachytene to diplotene transition during male gametogenesis (14).Originally cloned from a chicken cDNA library, cyclin B3 is expressed at very low levels in virally transformed hematopoietic cell lines and early embryonic stages of chicken. When Gallus gallus cyclin B3 is expressed in HeLa cells, it is nuclear, interacts with cdk2 and cdc2, and is associated with a modest histone H1 kinase activity (15). In Drosophila, cyclin B3 is expressed in both mitotic and meiotic cells. In mitotic cells, it is only required when either cyclin A or cyclin B1 is absent. Its abundance is down-regulated during the metaphase to anaphase transition, following degradation of cyclin B1 (16). Overexpression of a nondegradable cyclin B3 is associated with chromatin decondensation defects at the end of mitosis (17). Cyclin B3 is essential for fertility; but unlike cyclin B1 deficiency, infertility of cyclin B3-deficient flies is restricted to the female germ line and does not appear to involve defects in ovary structure or in meiosis I entry (16).We report the cloning of a full-length human cyclin B3 cDNA and a partial mouse cyclin B3 cDNA. Like the previously described chicken and Drosophila homologs, when mammalian cyclin B3 expression is enforced in m...

Breast cancer development is a complex pathobiological process involving sequential genetic alterations in normal epithelial cells that results in uncontrolled growth in a permissive microenvironment. Accordingly, physiologically relevant models of human breast cancer that recapitulate these events are needed to study cancer biology and evaluate therapeutic agents. Here, we report the generation and utilization of the human breast cancer in mouse (HIM) model, which is composed of genetically engineered primary human breast epithelial organoids and activated human breast stromal cells. By using this approach, we have defined key genetic events required to drive the development of human preneoplastic lesions as well as invasive adenocarcinomas that are histologically similar to those in patients. Tumor development in the HIM model proceeds through defined histological stages of hyperplasia, DCIS to invasive carcinoma. Moreover, HIM tumors display characteristic responses to targeted therapies, such as HER2 inhibitors, further validating the utility of these models in preclinical compound testing. The HIM model is an experimentally tractable human in vivo system that holds great potential for advancing our basic understanding of cancer biology and for the discovery and testing of targeted therapies.cancer model ͉ human in mouse ͉ tissue reconstitution

Purpose: ERBB3 is overexpressed in a broad spectrum of human cancers, and its aberrant activation is associated with tumor pathogenesis and therapeutic resistance to various anticancer agents. Neuregulin 1 (NRG1) is the predominant ligand for ERBB3 and can promote the heterodimerization of ERBB3 with other ERBB family members, resulting in activation of multiple intracellular signaling pathways. AV-203 is a humanized IgG1/k ERBB3 inhibitory antibody that completed a first-in-human phase I clinical trial in patients with advanced solid tumors. The purpose of this preclinical study was to identify potential biomarker(s) that may predict response to AV-203 treatment in the clinic.Experimental Design: We conducted in vivo efficacy studies using a broad panel of xenograft models representing a wide variety of human cancers. To identify biomarkers that can predict response to AV-203, the relationship between tumor growth inhibition (TGI) by AV-203 and the expression levels of ERBB3 and NRG1 were evaluated in these tumor models.Results: A significant correlation was observed between the levels of NRG1 expression and TGI by AV-203. In contrast, TGI was not correlated with ERBB3 expression. The correlation between the levels of NRG1 expression in tumors and their response to ERBB3 inhibition by AV-203 was further validated using patient-derived tumor explant models.Conclusions: NRG1 is a promising biomarker that can predict response to ERBB3 inhibition by AV-203 in preclinical human cancer models. NRG1 warrants further clinical evaluation and validation as a potential predictive biomarker of response to AV-203.

Colon cancer is the second most common cause of cancer mortality in the Western world with metastasis commonly present at the time of diagnosis. Screening for propagation and metastatic behavior in a novel chimeric-mouse colon cancer model, driven by mutant p53 and β-Catenin, led to the identification of a unique, invasive adenocarcinoma. Comparison of the genome of this tumor, CB42, with genomes from non-propagating tumors by array CGH and sequencing revealed an amplicon on chromosome five containing CDK6 and CDK14, and a KRAS mutation, respectively. Single agent small molecule inhibition of either CDK6 or MEK, a kinase downstream of KRAS, led to tumor growth inhibition in vivo whereas combination therapy not only led to regression of the subcutaneous tumors, but also near complete inhibition of lung metastasis; thus, genomic analysis of this tumor led to effective, individualized treatment.