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Small molecule-mediated disruption of Wnt-dependent signaling in tissue regeneration and cancer.

Chen B, Dodge ME, Tang W, Lu J, Ma Z, Fan CW, Wei S, Hao W, Kilgore J, Williams NS, Roth MG, Amatruda JF, Chen C, Lum L - Nat. Chem. Biol. (2009)

Bottom Line: With these small molecules, we establish a chemical genetic approach for studying Wnt pathway responses and stem cell function in adult tissue.We achieve transient, reversible suppression of Wnt/beta-catenin pathway response in vivo, and we establish a mechanism-based approach to target cancerous cell growth.The signal transduction mechanisms shown here to be chemically tractable additionally contribute to Wnt-independent signal transduction pathways and thus could be broadly exploited for chemical genetics and therapeutic goals.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75390, USA.

ABSTRACT
The pervasive influence of secreted Wnt signaling proteins in tissue homeostasis and tumorigenesis has galvanized efforts to identify small molecules that target Wnt-mediated cellular responses. By screening a diverse synthetic chemical library, we have discovered two new classes of small molecules that disrupt Wnt pathway responses; whereas one class inhibits the activity of Porcupine, a membrane-bound acyltransferase that is essential to the production of Wnt proteins, the other abrogates destruction of Axin proteins, which are suppressors of Wnt/beta-catenin pathway activity. With these small molecules, we establish a chemical genetic approach for studying Wnt pathway responses and stem cell function in adult tissue. We achieve transient, reversible suppression of Wnt/beta-catenin pathway response in vivo, and we establish a mechanism-based approach to target cancerous cell growth. The signal transduction mechanisms shown here to be chemically tractable additionally contribute to Wnt-independent signal transduction pathways and thus could be broadly exploited for chemical genetics and therapeutic goals.

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Chemical inhibition of cancerous Wnt/β-catenin pathway activity(a) IWR-1 blocks β-catenin accumulation induced by loss of APC tumor suppressor. (b) IWRs block aberrant Wnt/βbcatenin pathway activity in the colorectal cancer (CRC) cells. Constitutive Wnt/β-catenin pathway activity in DLD-1 cells consequential to APC loss-of-function, is abrogated by IWR compounds as measured using the STF reporter. (c) β-catenin-dependent growth of several cancer cell lines. Cells from lung, colon, and prostate cancers transiently transfected with a β-catenin siRNA pool were seeded at clonal density and cell viability measured using Cell-Titer Glo assay 10 days later. (d) Growth-inhibitory effects of IWR compounds on cancerous cells. The same assay in (c) was performed except cells were treated with IWR-3 for 6 days. (e) Overexpression of β-catenin can rescue the growth-inhibitory effects of an IWR compound in DLD-1 cells as indicated by levels of Renilla luciferase (RL) activity in cells transfected with or without a β-catenin expression construct and the RL reporter DNA. (f) H460 cells lack aberrant Wnt/β-catenin pathway activity as measured by STF reporter assay. (g) IWR-3 induces Axin1 protein stabilization in H460 and DLD-1 cells. (h) IWR-1 stabilization of Axin2 results in decreased transcription of Axin2, a Wnt/β-catenin target gene as measured using RT-PCR. (i) The predicted utility of IWP and IWR compounds for inhibiting Wnt ligand-dependent and -independent pathway responses. For (b-f), data represent mean values ± s.d.
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Figure 7: Chemical inhibition of cancerous Wnt/β-catenin pathway activity(a) IWR-1 blocks β-catenin accumulation induced by loss of APC tumor suppressor. (b) IWRs block aberrant Wnt/βbcatenin pathway activity in the colorectal cancer (CRC) cells. Constitutive Wnt/β-catenin pathway activity in DLD-1 cells consequential to APC loss-of-function, is abrogated by IWR compounds as measured using the STF reporter. (c) β-catenin-dependent growth of several cancer cell lines. Cells from lung, colon, and prostate cancers transiently transfected with a β-catenin siRNA pool were seeded at clonal density and cell viability measured using Cell-Titer Glo assay 10 days later. (d) Growth-inhibitory effects of IWR compounds on cancerous cells. The same assay in (c) was performed except cells were treated with IWR-3 for 6 days. (e) Overexpression of β-catenin can rescue the growth-inhibitory effects of an IWR compound in DLD-1 cells as indicated by levels of Renilla luciferase (RL) activity in cells transfected with or without a β-catenin expression construct and the RL reporter DNA. (f) H460 cells lack aberrant Wnt/β-catenin pathway activity as measured by STF reporter assay. (g) IWR-3 induces Axin1 protein stabilization in H460 and DLD-1 cells. (h) IWR-1 stabilization of Axin2 results in decreased transcription of Axin2, a Wnt/β-catenin target gene as measured using RT-PCR. (i) The predicted utility of IWP and IWR compounds for inhibiting Wnt ligand-dependent and -independent pathway responses. For (b-f), data represent mean values ± s.d.

Mentions: Based on our biochemical evidence, IWR compounds likely inhibit Wnt-induced accumulation of β-catenin by targeting a pathway component that functions downstream of Lrp and Dvl proteins (see Fig. 2). The β-catenin destruction complex, which consists of Apc, Axin, Ck1, and Gsk3β, promotes proteasome-mediated proteolysis of phosphorylated β-catenin18. We examined the biochemical effects of IWR compounds on components of this destruction complex in the DLD-1 colorectal cancer (CRC) cell line. Interestingly, we observed a potent IWR-dependent induction of Axin2 protein with little change in levels of Apc or Gsk3β (Fig. 4a). Despite this increase in Axin protein levels, we did not observe a concomitant decrease in β-catenin levels as would be expected based on our results in L-cells (see Fig. 2). As the majority of β-catenin protein in colonic epithelial cells is sequestered in complexes with the cell-cell adhesion molecule E-cadherin19, we examined the pool of “free” β-catenin that is available for Wnt-mediated response. Indeed, levels of β-catenin not bound to E-cadherin are decreased in DLD-1 cells after addition of IWR-1 (Fig. 4b). The IWR-induced increase in Axin protein levels was accompanied by elevated levels of β-catenin phosphorylation, a prerequisite for proteasome-mediated destruction of β-catenin (Fig. 4c). Thus, IWR compounds promote β-catenin destruction likely by promoting stability of Axin-scaffolded destruction complexes. Consistent with this model, the IWR compounds did not induce de novo synthesis of Axin2 (Supp. Fig. 8a; see also Fig. 7h), a transcriptional target of the Wnt/β-catenin pathway20,21, inhibit the proteasome (Supp. Fig. 8b), alter the affinity of Axin2 for β-catenin or its ability to interact other pathway components (Supp. Fig. 8c-e), or disrupt subcellular localization of Axin (Supp. Fig. 8f).


Small molecule-mediated disruption of Wnt-dependent signaling in tissue regeneration and cancer.

Chen B, Dodge ME, Tang W, Lu J, Ma Z, Fan CW, Wei S, Hao W, Kilgore J, Williams NS, Roth MG, Amatruda JF, Chen C, Lum L - Nat. Chem. Biol. (2009)

Chemical inhibition of cancerous Wnt/β-catenin pathway activity(a) IWR-1 blocks β-catenin accumulation induced by loss of APC tumor suppressor. (b) IWRs block aberrant Wnt/βbcatenin pathway activity in the colorectal cancer (CRC) cells. Constitutive Wnt/β-catenin pathway activity in DLD-1 cells consequential to APC loss-of-function, is abrogated by IWR compounds as measured using the STF reporter. (c) β-catenin-dependent growth of several cancer cell lines. Cells from lung, colon, and prostate cancers transiently transfected with a β-catenin siRNA pool were seeded at clonal density and cell viability measured using Cell-Titer Glo assay 10 days later. (d) Growth-inhibitory effects of IWR compounds on cancerous cells. The same assay in (c) was performed except cells were treated with IWR-3 for 6 days. (e) Overexpression of β-catenin can rescue the growth-inhibitory effects of an IWR compound in DLD-1 cells as indicated by levels of Renilla luciferase (RL) activity in cells transfected with or without a β-catenin expression construct and the RL reporter DNA. (f) H460 cells lack aberrant Wnt/β-catenin pathway activity as measured by STF reporter assay. (g) IWR-3 induces Axin1 protein stabilization in H460 and DLD-1 cells. (h) IWR-1 stabilization of Axin2 results in decreased transcription of Axin2, a Wnt/β-catenin target gene as measured using RT-PCR. (i) The predicted utility of IWP and IWR compounds for inhibiting Wnt ligand-dependent and -independent pathway responses. For (b-f), data represent mean values ± s.d.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2628455&req=5

Figure 7: Chemical inhibition of cancerous Wnt/β-catenin pathway activity(a) IWR-1 blocks β-catenin accumulation induced by loss of APC tumor suppressor. (b) IWRs block aberrant Wnt/βbcatenin pathway activity in the colorectal cancer (CRC) cells. Constitutive Wnt/β-catenin pathway activity in DLD-1 cells consequential to APC loss-of-function, is abrogated by IWR compounds as measured using the STF reporter. (c) β-catenin-dependent growth of several cancer cell lines. Cells from lung, colon, and prostate cancers transiently transfected with a β-catenin siRNA pool were seeded at clonal density and cell viability measured using Cell-Titer Glo assay 10 days later. (d) Growth-inhibitory effects of IWR compounds on cancerous cells. The same assay in (c) was performed except cells were treated with IWR-3 for 6 days. (e) Overexpression of β-catenin can rescue the growth-inhibitory effects of an IWR compound in DLD-1 cells as indicated by levels of Renilla luciferase (RL) activity in cells transfected with or without a β-catenin expression construct and the RL reporter DNA. (f) H460 cells lack aberrant Wnt/β-catenin pathway activity as measured by STF reporter assay. (g) IWR-3 induces Axin1 protein stabilization in H460 and DLD-1 cells. (h) IWR-1 stabilization of Axin2 results in decreased transcription of Axin2, a Wnt/β-catenin target gene as measured using RT-PCR. (i) The predicted utility of IWP and IWR compounds for inhibiting Wnt ligand-dependent and -independent pathway responses. For (b-f), data represent mean values ± s.d.
Mentions: Based on our biochemical evidence, IWR compounds likely inhibit Wnt-induced accumulation of β-catenin by targeting a pathway component that functions downstream of Lrp and Dvl proteins (see Fig. 2). The β-catenin destruction complex, which consists of Apc, Axin, Ck1, and Gsk3β, promotes proteasome-mediated proteolysis of phosphorylated β-catenin18. We examined the biochemical effects of IWR compounds on components of this destruction complex in the DLD-1 colorectal cancer (CRC) cell line. Interestingly, we observed a potent IWR-dependent induction of Axin2 protein with little change in levels of Apc or Gsk3β (Fig. 4a). Despite this increase in Axin protein levels, we did not observe a concomitant decrease in β-catenin levels as would be expected based on our results in L-cells (see Fig. 2). As the majority of β-catenin protein in colonic epithelial cells is sequestered in complexes with the cell-cell adhesion molecule E-cadherin19, we examined the pool of “free” β-catenin that is available for Wnt-mediated response. Indeed, levels of β-catenin not bound to E-cadherin are decreased in DLD-1 cells after addition of IWR-1 (Fig. 4b). The IWR-induced increase in Axin protein levels was accompanied by elevated levels of β-catenin phosphorylation, a prerequisite for proteasome-mediated destruction of β-catenin (Fig. 4c). Thus, IWR compounds promote β-catenin destruction likely by promoting stability of Axin-scaffolded destruction complexes. Consistent with this model, the IWR compounds did not induce de novo synthesis of Axin2 (Supp. Fig. 8a; see also Fig. 7h), a transcriptional target of the Wnt/β-catenin pathway20,21, inhibit the proteasome (Supp. Fig. 8b), alter the affinity of Axin2 for β-catenin or its ability to interact other pathway components (Supp. Fig. 8c-e), or disrupt subcellular localization of Axin (Supp. Fig. 8f).

Bottom Line: With these small molecules, we establish a chemical genetic approach for studying Wnt pathway responses and stem cell function in adult tissue.We achieve transient, reversible suppression of Wnt/beta-catenin pathway response in vivo, and we establish a mechanism-based approach to target cancerous cell growth.The signal transduction mechanisms shown here to be chemically tractable additionally contribute to Wnt-independent signal transduction pathways and thus could be broadly exploited for chemical genetics and therapeutic goals.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75390, USA.

ABSTRACT
The pervasive influence of secreted Wnt signaling proteins in tissue homeostasis and tumorigenesis has galvanized efforts to identify small molecules that target Wnt-mediated cellular responses. By screening a diverse synthetic chemical library, we have discovered two new classes of small molecules that disrupt Wnt pathway responses; whereas one class inhibits the activity of Porcupine, a membrane-bound acyltransferase that is essential to the production of Wnt proteins, the other abrogates destruction of Axin proteins, which are suppressors of Wnt/beta-catenin pathway activity. With these small molecules, we establish a chemical genetic approach for studying Wnt pathway responses and stem cell function in adult tissue. We achieve transient, reversible suppression of Wnt/beta-catenin pathway response in vivo, and we establish a mechanism-based approach to target cancerous cell growth. The signal transduction mechanisms shown here to be chemically tractable additionally contribute to Wnt-independent signal transduction pathways and thus could be broadly exploited for chemical genetics and therapeutic goals.

Show MeSH
Related in: MedlinePlus