Signaling In Colon Cancer Research Articles

Wnt5A Activates the Calpain-Mediated Cleavage of Filamin A

We have previously shown that Wnt5A and ROR2, an orphan tyrosine kinase receptor, interact to mediate melanoma cell motility. In other cell types, this can occur through the interaction of ROR2 with the cytoskeletal protein filamin A. Here, we found that filamin A protein levels correlated with Wnt5A levels in melanoma cells. Small interfering RNA (siRNA) knockdown of WNT5A decreased filamin A expression. Knockdown of filamin A also corresponded to a decrease in melanoma cell motility. In metastatic cells, filamin A expression was predominant in the cytoplasm, which western analysis indicated was due to the cleavage of filamin A in these cells. Treatment of nonmetastatic melanoma cells with recombinant Wnt5A increased filamin A cleavage, and this could be prevented by the knockdown of ROR2 expression. Further, BAPTA-AM chelation of intracellular calcium also inhibited filamin A cleavage, leading to the hypothesis that Wnt5A/ROR2 signaling could cleave filamin A through activation of calcium-activated proteases, such as calpains. Indeed, WNT5A knockdown decreased calpain 1 expression, and by inhibiting calpain 1 either pharmacologically or using siRNA, it decreased cell motility. Our results indicate that Wnt5A activates calpain-1, leading to the cleavage of filamin A, which results in a remodeling of the cytoskeleton and an increase in melanoma cell motility.

Abstract B84: NADPH oxidase 1 (NOX1) and related NOX isoforms as key mediators of NFκB signaling in colon cancer

Abstract B84 NADPH oxidase/dual-oxidase (Nox/Duox) family members have been implicated in nuclear factor kappa-B (NFκB)-mediated inflammation and inflammation-associated pathologies. In rats given 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) alternating with a high-fat diet, Nox1, Nox4 and Duo2, but not Nox2, were over-expressed in colon tumors at 1 year, as shown by quantitative RT-PCR and immunoblotting. Over-expression of Nox/duox isoforms coincided with increased nuclear NFκB protein, and elevated levels of downstream targets IL-1β, IL6, TNF-α and TNFR1. Over-expression of Nox1 and activation of NFκB also occurred at early stages of colon carcinogenesis, prior to tumor development. Nox/Duox isoforms were over-expressed in primary human colon cancers and in several human colorectal cancer cell lines. In HT29 cells, silencing of Nox1 inhibited nuclear translocation and DNA binding activity of NFκB, attenuated downstream targets IL1β, c-myc, and cyclin D1, and blocked lipopolysaccharide-induced phosphorylation of IκB kinaseα/β, resulting in G1 arrest. We conclude that the Nox1-NFκB pathway is important in colon tumorigenesis, and that Nox1 may provide a target for novel therapeutic strategies against colon cancer development. Citation Information: Cancer Prev Res 2008;1(7 Suppl):B84.

Inhibitory Gli3 Activity Negatively Regulates Wnt/β-Catenin Signaling

The Hedgehog (Hh) and Wingless (Wnt) families of secreted signaling molecules have key roles in embryonic development and adult tissue homeostasis [1-3]. In the developing neural tube, Wnt and Shh, emanating from dorsal and ventral regions, respectively, have been proposed to govern the proliferation and survival of neural progenitors [4-10]. Surprisingly, Shh is required for the growth and survival of cells in both ventral and dorsal neural tube [11]. Here we demonstrate that inhibition of Shh signaling causes a reduction in Wnt-mediated transcriptional activation. This reduction requires Gli3. Assays in embryos and cell lines indicate that repressor forms of the Hh-regulated transcription factor, Gli3 (Gli3R), which are generated in the absence of Hh signaling, inhibit canonical Wnt signaling. Gli3R acts by antagonizing active forms of the Wnt transcriptional effector, beta-catenin. Consistent with this, Gli3R appears to physically interact with the carboxy-terminal domain of beta-catenin, a region that includes the transactivation domain. These data offer an explanation for the proliferative defects in Shh null embryos and suggest a novel mechanism for crosstalk between the Hh and Wnt pathways.

The Metastasis-Associated Gene S100A4 Is a Novel Target of β-catenin/T-cell Factor Signaling in Colon Cancer

Activation of the Wnt/beta-catenin pathway is frequently observed in colorectal cancers. Our aim was to elucidate the impact of gain-of-function beta-catenin on the metastasis-associated gene S100A4 in human colon cancer cell lines and tumors. We analyzed cell lines heterozygous for gain-of-function and wild-type beta-catenin, and variants homozygous for gain- or loss-of-function mutation in beta-catenin, for S100A4 expression, cell motility, and in vivo metastasis. beta-catenin-mediated S100A4 promoter activation was tested by reporter assays. For human colon carcinomas, S100A4 expression, beta-catenin genotype, and metachronous metastasis were correlated. We identified S100A4 as the most regulated gene by gain-of-function beta-catenin using a 10K microarray. Cell lines with gain-of-function beta-catenin expressed up to 60-fold elevated S100A4 levels, displayed strongly increased migration and invasion in vitro, and induced metastasis in mice. S100A4 small interfering RNA, beta-catenin small interfering RNA, or dominant negative T-cell factor (TCF) knocked down S100A4 and blocked biological effects. S100A4 complementary DNA transfection increased migration and invasion. We identified a TCF binding site within the S100A4 promoter and demonstrated the direct binding of heterodimeric beta-catenin/TCF complexes. Reporter assays confirmed the beta-catenin-induced S100A4 promoter activity. Furthermore, S100A4 mRNA expression was increased in primary colon cancers, which later developed distant metastases, compared to non-metastasizing tumors. Colon tumors heterozygous for gain-of-function beta-catenin showed concomitant nuclear beta-catenin localization, high S100A4 expression, and metastases. S100A4 is a direct beta-catenin/TCF target, induces migration and invasion in vitro and metastasis in vivo, and has value for prognosis of metastasis formation in colon cancer patients.

β-Catenin Interacts With the FUS Proto-oncogene Product and Regulates Pre-mRNA Splicing

beta-Catenin is a downstream effector of the Wnt signaling pathway and is believed to exert its oncogenic function by activating T-cell factor (TCF)/lymphoid enhancer factor (LEF) family transcriptional factors. However, it is still uncertain whether the diverse effects of beta-catenin are caused solely by aberrant gene transactivation. In this study, we used a proteomics approach to obtain further insight into the functional properties of nuclear beta-catenin. The protein assembly of a native beta-catenin-containing complex in nuclear extracts from a colorectal cancer cell line, DLD-1, was identified using immunoprecipitation and mass spectrometry. beta-Catenin physically interacted with fusion (FUS)/translocated in liposarcoma (TLS) and various RNA-binding proteins. The expression of FUS/TLS was closely associated with the accumulation of beta-catenin and with the undifferentiated status of intestinal epithelial cells. The transient transfection of FUS suppressed beta-catenin-evoked gene transactivation of TCF/LEF, and beta-catenin transfection affected the splicing pattern of the E1A minigene and induced a novel splicing variant of estrogen receptor (ER)-beta exerting a dominant-negative activity. Human cancer expresses a large variety of alternatively spliced messenger RNA (mRNA), but the precise molecular mechanisms responsible for cancer-related alternative splicing are largely unknown. In this study, we demonstrated the interaction of beta-catenin with FUS/TLS and other RNA-binding proteins involved in the regulation of pre-mRNA splicing. Certain mRNA splicing abbreviations seen in human cancers may be induced by the activation of the Wnt signaling pathway.

Use of a promoterless Renilla luciferase vector as an internal control plasmid for transient co-transfection assays of Ras-mediated transcription activation.

Use of a promoterless Renilla luciferase vector as an internal control plasmid for transient co-transfection assays of Ras-mediated transcription activation.

Activation of β-Catenin-Tcf Signaling in Colon Cancer by Mutations in β-Catenin or APC

Inactivation of the adenomatous polyposis coli (APC) tumor suppressor gene initiates colorectal neoplasia. One of the biochemical activities associated with the APC protein is down-regulation of transcriptional activation mediated by beta-catenin and T cell transcription factor 4 (Tcf-4). The protein products of mutant APC genes present in colorectal tumors were found to be defective in this activity. Furthermore, colorectal tumors with intact APC genes were found to contain activating mutations of beta-catenin that altered functionally significant phosphorylation sites. These results indicate that regulation of beta-catenin is critical to APC's tumor suppressive effect and that this regulation can be circumvented by mutations in either APC or beta-catenin.