TLR4 Activates the β-catenin pathway to cause intestinal neoplasia

Colonic bacteria have been implicated in the development of colon cancer. We have previously demonstrated that toll-like receptor 4 (TLR4), the receptor for bacterial lipopolysaccharide (LPS), is over-expressed in humans with colitis-associated cancer. Genetic epidemiologic data support a role for TLR4 in sporadic colorectal cancer (CRC) as well, with over-expression favoring more aggressive disease. The goal of our study was to determine whether TLR4 played a role as a tumor promoter in sporadic colon cancer. Using immunofluorescence directed to TLR4, we found that a third of sporadic human colorectal cancers over-express this marker. To mechanistically investigate this observation, we used a mouse model that over-expresses TLR4 in the intestinal epithelium (villin-TLR4 mice). We found that these transgenic mice had increased epithelial proliferation as measured by BrdU labeling, longer colonic crypts and an expansion of Lgr5+ crypt cells at baseline. In addition, villin-TLR4 mice developed spontaneous duodenal dysplasia with age, a feature that is not seen in any wild-type (WT) mice. To model human sporadic CRC, we administered the genotoxic agent azoxymethane (AOM) to villin-TLR4 and WT mice. We found that villin-TLR4 mice showed an increased number of colonic tumors compared to WT mice as well as increased β-catenin activation in non-dysplastic areas. Biochemical studies in colonic epithelial cell lines revealed that TLR4 activates β-catenin in a PI3K-dependent manner, increasing phosphorylation of β-catenin, a phenomenon associated with activation of the canonical Wnt pathway. Our results suggest that TLR4 can trigger a neoplastic program through activation of the Wnt/β-catenin pathway. Our studies highlight a previously unexplored link between innate immune signaling and activation of oncogenic pathways, which may be targeted to prevent or treat CRC

Repurposing the FDA-Approved Pinworm Drug Pyrvinium as a Novel Chemotherapeutic Agent for Intestinal Polyposis

<div><p>Mutations in the WNT-pathway regulator ADENOMATOUS POLYPOSIS COLI (APC) promote aberrant activation of the WNT pathway that is responsible for APC-associated diseases such as Familial Adenomatous Polyposis (FAP) and 85% of spontaneous colorectal cancers (CRC). FAP is characterized by multiple intestinal adenomas, which inexorably result in CRC. Surprisingly, given their common occurrence, there are few effective chemotherapeutic drugs for FAP. Here we show that the FDA-approved, anti-helminthic drug Pyrvinium attenuates the growth of WNT-dependent CRC cells and does so via activation of CK1α. Furthermore, we show that Pyrvinium can function as an <i>in vivo</i> inhibitor of WNT-signaling and polyposis in a mouse model of FAP: <i>APC^min</i> mice. Oral administration of Pyrvinium, a CK1α agonist, attenuated the levels of WNT-driven biomarkers and inhibited adenoma formation in <i>APC^min</i> mice. Considering its well-documented safe use for treating enterobiasis in humans, our findings suggest that Pyrvinium could be repurposed for the clinical treatment of APC-associated polyposes.</p></div

Pyrvinium reduces CRC cell viability.

<p><b>A</b>. Structure of Pyrvinium. <b>B</b>. MTT assay of various CRC cells treated with increasing amounts of Pyrvinium or vehicle control (<b>V</b>). Treated plates were incubated with 10% MTT, lysed and MTT reduction assessed. <b>C</b>. Similar assay as B was performed using WT MEF cells. <b>D</b>. Clonogenic assay of Pyrvinium treated CRC cell lines. The CRC cell lines SW480, SW620, HT29 and HCT116 were treated with increasing concentrations of Pyrvinium or vehicle control for 2 weeks. Colonies were fixed with gluteraldehyde and stained with crystal violet. Mean number of colonies was quantitated relative to vehicle controls. Error bars indicate ± S.E.M of three experiments.</p

Pyrvinium inhibits WNT signaling in CRC cells.

<p><b>A</b>. Pyrvinium inhibits WNT reporter gene activity in CRC cells. Cells were treated with Pyrvinium at the indicated concentrations and relative WNT reporter luciferase activity determined. Immunoblots show that Pyrvinium decreases PYGOPUS2 levels in both SW480 and HCT116 WTKO cells in a dose-dependent manner. Error bars indicate ± S.E.M of three experiments. <b>B</b>. Expression of WNT regulated genes <i>AXIN2</i>, <i>DKK1</i>, <i>CCND1</i> and <i>LGR5</i> in CRC cell lines treated with 100 nM Pyrvinium or vehicle control was determined using real-time RT-PCR. Error bars indicate ± S.E.M of three experiments. *p<0.05. <b>C</b>. A nuclear or cytosolic fraction was extracted from vehicle or 100 nM Pyrvinium treated SW480 and HT29 cells and CTNNB1 protein levels were determined by immunoblot. <b>D</b>. Immunoblot of the WNT associated biomarkers CTNNB1 and CCND1 in CRC cell lines treated with Pyrvinium or vehicle control. Cells were treated, lysed in protein sample buffer and immunoblotted with the cognate antibodies for CTNNB1, CCND1 and GAPDH control.</p

Pyrvinium inhibits WNT signaling in a CK1α dependent manner.

<p><b>A</b>. The expression of the WNT target gene: <i>AXIN2</i> was determined from NIH 3T3 cells expressing control or <i>CK1α</i>-specific shRNAs. Cells were treated with control or WNT3a conditioned media with or without 100 nM Pyrvinium. <i>AXIN2</i> expression was quantified using real-time RT-PCR. Error bars indicate ± S.E.M of three experiments. *p<0.05. <b>B</b>. Pyrvinium suppressed WNT signaling biomarkers in a CK1α dependent manner. Upper left, <i>CK1α</i> expression was assessed by real-time RT-PCR in control shRNA or <i>CK1α</i> shRNA expressing HCT116 cells. The three other panels show control shRNA or <i>CK1α</i> shRNA infected HCT116 cells treated with or without 100 nM Pyrvinium for 24 hours and <i>AXIN2, DKK1 and LGR5</i> expression. Error bars indicate ± S.E.M of three experiments. *p<0.05. <b>C</b>. Overexpression of CK1α inhibits WNT reporter activity and decreases steady state levels of PYGOPUS2 in CRC cells. SW480 and HCT116 WTKO cells harboring the TOPflash reporter were transfected with a control plasmid or one expressing <i>HA-CK1α</i>. Immunoblots show decreased PYGOPUS2 levels for SW480 and HCT116 WTKO cells. Error bars indicate ± S.E.M of three experiments. *p<0.05. <b>D</b>. HCT116 stably expressing the indicated shRNA were treated with or without 100 nM Pyrvinium followed by determination of their colony forming capacity. Error bars indicate ± S.E.M of three experiments. *p<0.05.</p

Pyrvinium treated CRC cell lines: summary.

<p>Pyrvinium treated CRC cell lines: summary.</p

Pyrvinium reduces adenoma formation <i>in vivo</i>.

<p><b>A</b>. Mean number of intestinal adenomas in Pyrvinium (25 mg/kg) or vehicle treated mice. Mice were treated via oral gavage once every 48 h for 10 weeks. Intestines were removed from euthanized mice, fixed in phosphate buffered formalin and then adenomatous polyps were quantitated. Error bars represent S.E.M, n = 12. *p<0.05. <b>B</b>. Expression levels of the WNT target genes <i>AXIN2</i> and <i>LGR5</i> in the intestinal epithelium of <i>APC^min</i> mice chronically treated with vehicle control or 25 mg/Kg Pyrvinium. Total RNA from intestinal tissue sections of euthanized mice was converted to cDNA and subjected to real-time RT-PCR analysis. <b>C</b>. Mice treated with Pyrvinium for 10 weeks via oral gavage showed no significant weight loss in response to Pyrvinium treatment. <b>D</b>. Upper panels, TUNEL assays of intestinal sections from chronic Pyrvinium or vehicle treated <i>APC^min</i> mice. Intestinal tissue sections were isolated from euthanized mice embedded in paraffin and subjected to an in situ TUNEL assay using the DeadEnd colorimetric TUNEL assay (N: nucleus). Lower panels, Immunohistochemistry showing CTNNB1 expression and localization in intestinal sections from Pyrvinium and vehicle treated mice. Mice were treated for 7 days. Intestinal tissues isolated from euthanized mice were fixed using buffered formalin, paraffin embedded and subjected to immunohistochemistry using an antibody for CTNNB1 (N: nucleus). Scale = 50 µm.</p

Inhibition of WNT signaling by Pyrvinium is independent of UPR or AKT inhibition.

<p><b>A</b>. HCT116 cells under normal culture conditions were treated with vehicle control and either 100 nM Pyrvinium, 1 µg/mL Tunicamycin or a combination of 100 nM Pyrvinium and 1 µg/mL Tunicamycin for 24 h. cDNA from these cells was assessed for <i>XBP-1</i> mRNA splicing using <i>XBP-1</i> specific probes. XBP-1 full-length transcript produces a 380 bp PCR product, while the XBP-1S shortened splice variant produces a 350 bp PCR product. <b>B</b>. Pyrvinium inhibits WNT signaling at concentrations that does not block AKT activation. HEK 293 cells stably expressing a WNT responsive reporter gene were stimulated with or without WNT3a and the indicated concentrations of Pyrvinium or the pan-PI3K inhibitor BKM120. Error bars indicate ± S.E.M of four experiments. *p<0.05. As a control, we show that while BKM120 is capable of attenuating AKT activation using a phospho-specfic antibody to AKT [phospho-AKT (T308)], Pyrvinium does not inhibit AKT activation at concentrations sufficient to inhibit WNT signaling (Upper panels). Lysates from HEK 293 cells treated with indicated drugs were immunoblotted for phospho-AKT (T308) or total AKT, as indicated. <b>C</b>. SW480 cells were treated with 100 nM Pyrvinium for the indicated time points and S45 phosphorylated CTNNB1 levels, a known CK1α substrate, were detected by immunoblot.</p