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BioAssay: AID 651824

Characterization of Small Molecules that Restore E-cadherin Expression

Background: Small molecules have been discovered that promote transcription of CDH1, the gene that encodes E-cadherin, a protein involved in normal epithelial cell-cell adhesion (Stoops, et al. 2011). When epithelial cells become transformed and progress to an invasive and aggressive phenotype, they undergo what is generally referred to an Epithelial to Mesenchymal Transition (EMT) (Savagner, et more ..
AID: 651824
Data Source: Vanderbilt Specialized Chemistry Center (E_Cadherin_Inhibitor_Summary)
BioAssay Type: Summary, Candidate Probes/Leads with Supporting Evidence
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2012-11-29
Modify Date: 2013-02-14
Sequence: E-cadherin [Homo sapiens]
Description ..   
Protein Family: Cadherin cytoplasmic region

Gene:CDH1     Related Protein 3D Structures     More BioActivity Data..
Related Experiments
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651826ML327 Competition in Radioligand Binding assays (Ricerca)Other1 depositor-specified cross reference: Ricerca ML327 Competition in Radioligand Binding assays
651862Small Molecules that Restore E-cadherin ExpressionConfirmatory5 depositor-specified cross reference: Quantitative Western Blot for E-cadherin expression (Odyssey Western)
651906ML327 E-Cadherin HDAC Spectrum Assay_HDAC6Confirmatory depositor-specified cross reference: HDAC6 Profiling Assay
651907ML327 E-Cadherin HDAC Spectrum Assay_HDAC7Confirmatory depositor-specified cross reference: HDAC7 Profiling Assay
651908ML327 E-Cadherin HDAC Spectrum Assay_HDAC8Confirmatory depositor-specified cross reference: HDAC8 Profiling Assay
651909ML327 E-Cadherin HDAC Spectrum Assay_HDAC9Confirmatory depositor-specified cross reference: HDAC9 Profiling Assay
651910ML327 E-Cadherin HDAC Spectrum Assay_HDAC10Confirmatory depositor-specified cross reference: HDAC10 Profiling Assay
651911ML327 E-Cadherin HDAC Spectrum Assay_HDAC11Confirmatory depositor-specified cross reference: HDAC11 Profiling Assay
651915ML327 E-Cadherin HDAC Spectrum Assay_HDAC5Confirmatory depositor-specified cross reference: HDAC5 Profiling Assay
651916ML327 E-Cadherin HDAC Spectrum Assay_HDAC3Confirmatory depositor-specified cross reference: HDAC3 Profiling Assay
651918ML327 E-Cadherin HDAC Spectrum Assay_HDAC1Confirmatory depositor-specified cross reference: HDAC1 Profiling Assay
651926ML327 E-Cadherin HDAC Spectrum Assay_HDAC2Confirmatory depositor-specified cross reference: HDAC2 Profiling Assay
651928ML327 E-Cadherin HDAC Spectrum Assay_HDAC4Confirmatory depositor-specified cross reference: HDAC4 Profiling Assay
Background: Small molecules have been discovered that promote transcription of CDH1, the gene that encodes E-cadherin, a protein involved in normal epithelial cell-cell adhesion (Stoops, et al. 2011). When epithelial cells become transformed and progress to an invasive and aggressive phenotype, they undergo what is generally referred to an Epithelial to Mesenchymal Transition (EMT) (Savagner, et al. 2001; Thiery, et al. 2009). One of the characteristics of EMT is loss of E-cadherin expression (Christiphori, et al. 1999, Brabletz, et al. 2005). The reversal of EMT, along with the restoration of E-cadherin expression, can revert cancer cells to a more epithelial and less aggressive phenotype (Yoo, et al., 2006).

Purpose: The primary goal is to identify the molecular target and to elucidate the mechanism of action of compounds that restore E-cadherin expression. The secondary goal is to further optimize the activity of these compounds in cancer cells.

Methods: The primary screen for compound activity is an in-cell Western (ICW) blot assay that measures changes in the expression of E-Cadherin. The screen is performed in colon (SW620) or non-small cell lung (H520) cancer cell lines. Active compounds from the primary screen are further tested for their ability to induce E-cadherin mRNA expression and transcription from a minimal promoter element. Histone modification assays are used to screen for compound activity affecting chromatin structure.

Results: Treatment of cancer cells with an active compound induces an 8-fold increase in E-cadherin mRNA levels within 3 hours and up to a 50-fold increase 24 hours after treatment. Additionally, treatment of the same cells with compound activates expression of a transfected E-cadherin promoter-luciferase reporter vector up to 20-fold. Using the luciferase-based E-cadherin reporter assay, we have been able to narrow the site of action to a 200 bp fragment of the proximal E-cadherin promoter, which consists of a limited number of known transcription factor binding sites. This region contains two E-box consensus binding sites for the Snail transcriptional repressor complex. The assembly and function of this transcriptional repressor complex involves changes in histone acetylation and methylation states. Based on direct analysis of the probe and related molecules, we have determined that active compounds do not directly inhibit HDAC 1-11 activity or SIRT activity. However, the compound does increase transcriptionally relevant histone acetylation in intact cells, suggesting an indirect effect on HDAC activity, or possibly an effect on histone acetylase activity. Thus, we are using biological techniques to narrow the search for the molecular target while simultaneously uncovering the mechanism of action.

Conclusions and Directions: The robustness of the E-cadherin expression response to compound will greatly facilitate further SAR studies to optimize probe potency and efficacy. Further, narrowing the active region of the E-cadherin promoter that is responsive to compound will help to identify its molecular target. With knowledge of the molecular target, we will be able to develop a biochemical assay to test the activity of newly synthesized analogs directly at the site of action. With a target-specific biochemical assay in hand, we will be better able to aggressively pursue additional SAR studies to drive our best analogs to nanomolar (nM) potency, while retaining selectivity over other biological targets. Selected analogs will also be subjected to drug metabolism and pharmacokinetic (DMPK) profiling to find a candidate compound for in vivo testing in cancer mouse models. Additionally, we will use these compounds as probes to address the role of the molecular target during tumor progression and (EMT). These probes may provide additional insights into the role of EMT during tumor progression and the mechanisms by which cancer cells revert between epithelial and mesenchymal states. The probes will also allow us to investigate the suitability of the molecular target as a potential cancer therapeutic target by itself or in combination with modulators of other known therapeutic targets.

1. Stoops, S.L., et al., Identification and optimization of small molecules that restore E-cadherin expression and reduce invasion in colorectal carcinoma cells. ACS Chem Biol. 2011. 6 (5): p. 452-65.
2. Christofori, G. and H. Semb, The role of the cell-adhesion molecule E-cadherin as a tumour-suppressor gene. Trends Biochem Sci, 1999. 24(2): p. 73-6.
3. Brabletz, T., et al., Opinion: migrating cancer stem cells - an integrated concept of malignant tumour progression. Nat Rev Cancer, 2005. 5(9): p. 744-9.
4. Savagner, P., Leaving the neighborhood: molecular mechanisms involved during epithelial-mesenchymal transition. Bioessays, 2001. 23(10): p. 912-23.
5. Thiery, J.P., et al., Epithelial-mesenchymal transitions in development and disease. Cell, 2009. 139: p. 871-890.
6. Yoo, C.B. and P.A. Jones, Epigenetic therapy of cancer: past, present and future. Nat Rev Drug Discov, 2006. 5(1): p. 37-50.
Categorized Comment - additional comments and annotations
From PubChem:
Assay Cell Type: H520
From MLP Probe Report:
Probe count: 1
MLP Probe ML# for probe 1: ML327
PubChem Substance ID (SID) for probe 1: 144116736
PubChem Compound ID (CID) for probe 1: 60167648
Probe type for probe 1: Agonist
IC50/EC50 (nM) for probe 1: 1000
Target for probe 1: E-cadherin (gi: 31073)
Disease relevance for probe 1: Cancer
Anti-target for probe 1: HDACs 1-11
Fold selectivity for probe 1: >30
Grant number for probe 1: CA95103-09
Additional Information
Grant Number: 5P50 CA95103-09