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 ..
Table of Contents
Target
| Sequence: E-cadherin [Homo sapiens] Description ..   Protein Family: Cadherin cytoplasmic region Gene:CDH1 Related Protein 3D Structures |
Depositor Specified Assays
Description:
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.
REFERERENCES
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.
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.
REFERERENCES
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.
Additional Information
Grant Number: 5P50 CA95103-09
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