Small-molecule modulators of Hedgehog signaling: identification and characterization of Smoothened agonists and antagonists

BACKGROUND: The 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. RESULTS: We 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. CONCLUSIONS: Smo 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

Small-molecule modulators of Hedgehog signaling: identification and characterization of Smoothened agonists and antagonists

<p>Abstract</p> <p>Background</p> <p>The 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.</p> <p>Results</p> <p>We 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 <it>in vitro,</it> while <it>in utero</it> it rescues aspects of the Hh-signaling defect in <it>Sonic hedgehog</it>-null, but not <it>Smo</it>-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.</p> <p>Conclusions</p> <p>Smo 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 <it>in vivo</it> 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.</p

Dissecting genetic requirements of human breast tumorigenesis in a tissue transgenic model of human breast cancer in mice

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

Spontaneous Genomic Alterations in a Chimeric Model of Colorectal Cancer Enable Metastasis and Guide Effective Combinatorial Therapy

<div><p>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.</p></div

Characterization of the β-Catenin-driven chimeric colon model.

<p>A). Bioluminescent imaging of three chimeric mice after three months of doxycycline induction. Luminescent signals were detected in the lower abdominal region in two mice. B). Kaplan-Meyer curve showing the latency of colon tumor development in chimeric mice. C). Histological comparison of normal gastrointestinal tract, benign polyps, and adenocarcinoma from chimeric mice. D). Chemical colorimetric staining for Mucin and immunohistochemical staining for β-Catenin, Ki-67, and Cyclin D1 in normal GI epithelium, polyps and adenocarcinoma.</p

Treatment of CB42 subcutaneous allografts (n = 10 per treatment arm) and subsequent metastasis to the lung in nude mice with targeted therapy.

<p>A). MET inhibitor crizotinib did not inhibit CB42 tumor growth. B). CDK4/6 inhibitor PD0332991 significantly inhibited CB42 tumor growth (***P<0.001). C). MEK1/2 inhibitor PD0325901 completely blocked CB42 tumor growth (***P<0.001). D). Combination of CDK4/6 and MEK1/2 inhibitor induced CB 42 tumor regression (****P<0.0001). E). Subcutaneous tumor volumes of the three treatment arms at the end of study. The combination is significantly more potent than CDK4/6 inhibitor alone (****P<0.0001) or the MEK inhibitor alone (***P<0.001). F). Gross examination of lung lobes for metastatic nodules revealed that both CDK4/6 inhibitor and MEK inhibitor significantly inhibited CB42 lung metastasis (*P<0.05), but the combination was significantly more effective than either agent alone (****P<0.0001) and almost completely eliminated lung metastasis.</p

CBP1, which lack Cdk6 amplification and KRAS mutation, did not respond to either the CDK4/6 inhibitor or the MEK inhibitor.

<p>The mild tumor growth inhibition is not statistically significant from the vehicle for all three treatment arms (P>0.05).</p

Metastasis of CB42 from subcutaneous space to lung and lymph nodes.

<p>Lung metastasis H&E at 5X (A) and 20X (C) objective magnification; lymph node metastasis H&E at 5X (B) and 20X (D) objective magnification; pan-cytokeratin immunohistochemistry of lung (E) and lymph node (F) metastases.</p

List of mutations identified in CB42P3 by next gen sequencing.

<p>List of mutations identified in CB42P3 by next gen sequencing.</p

Schematic representation of genetic engineering of the β-Catenin-driven chimeric colon model.

<p>A). In ES cells: 3 transgenic cassettes were inserted: Villin-Cre, GAPDH-lox-stop-lox-rtTA-IRES-Luciferase, and TetO- β-CateninΔN131; all three remain silent in ES cells. In addition, one p53 allele was floxed and expresses wild type p53 in ES cells and the other allele was targeted with lox-stop-lox and R172H point mutation, which is functionally null in ES cells. B). In the gastro-intestinal epithelium, expression of Cre leads to deletion of the two lox-stop-lox cassettes as well as the floxed p53 allele. p53 expression will switch from wild type to the mutant form, and expression of rtTA and Luciferase will switch on. C). in the presence of doxycycline, rtTA will bind to the TetO promoter and activate transcription of β-CateninΔN131.</p