Interactions between neoplastic and stromal cells contribute to tumor progression. Wnt genes, involved in cell migration and often deregulated in cancers, are attractive candidates to regulate these effects. We have recently shown that coculture of breast cancer cells with macrophages enhances invasiveness via matrix metalloproteases and TNF-␣. Here we demonstrate that coculture of MCF-7 cells and macrophages leads to up-regulation of Wnt 5a in the latter. This was accompanied by activation of AP-1͞c-Jun in MCF-7. Recombinant Wnt 5a mimicked the coculture effect. Wnt 5a was also detectable in tumor-associated macrophages in primary breast cancers. Experiments with agonists and antagonists of Wnt signaling revealed that a functional canonical pathway in the tumor cells was a necessary prerequisite; however, noncanonical signaling via Wnt 5a and the Jun-N-terminal kinase pathway was critical for invasiveness. It was also responsible for induction of matrix metalloprotease-7, known to release TNF-␣. All these effects could be antagonized by dickkopf-1. Our results indicate that Wnt 5a is essential for macrophage-induced invasiveness, because it regulates tumor cell migration as well as proteolytic activity of the macrophages. The function of Wnt 5a as either a suppressor or promoter of malignant progression seems to be modulated by intercellular interactions. Wnt 5a detection in tumor-associated macrophages in breast cancer biopsies supports the assumption that similar events play a role in vivo.tumor microenvironment ͉ TNF-␣ ͉ matrix metalloproteases

Increased nuclear accumulation of b-catenin, a mediator of canonical Wnt signaling, is found in numerous tumors and is frequently associated with tumor progression and metastasis. Inhibition of Wnt/b-catenin signaling therefore is an attractive strategy for anticancer drugs. In this study, we have identified a novel small molecule inhibitor of the b-catenin signaling pathway, JW55, that functions via inhibition of the PARP domain of tankyrase 1 and tankyrase 2 (TNKS1/2), regulators of the b-catenin destruction complex. Inhibition of TNKS1/2 poly(ADP-ribosyl)ation activity by JW55 led to stabilization of AXIN2, a member of the b-catenin destruction complex, followed by increased degradation of b-catenin. In a dose-dependent manner, JW55 inhibited canonical Wnt signaling in colon carcinoma cells that contained mutations in either the APC (adenomatous polyposis coli) locus or in an allele of b-catenin. In addition, JW55 reduced XWnt8-induced axis duplication in Xenopus embryos and tamoxifen-induced polyposis formation in conditional APC mutant mice. Together, our findings provide a novel chemotype for targeting canonical Wnt/b-catenin signaling through inhibiting the PARP domain of TNKS1/2. Cancer Res; 72(11); 2822-32. Ó2012 AACR.

The ␤-catenin proto-oncogene, the vertebrate homolog to armadillo, links cell adhesion and cell differentiation; it stabilizes cell-cell adhesion by anchoring cadherins via ␣-catenin to the cytosekeleton (28, 55) and transduces the Wnt/Wg signal to target genes by interacting with transcription factors of the LEF-TCF family (1, 24, 39). The evolutionarily highly conserved Wnt/Wg signaling cascade includes a membrane-integrated receptor of the frizzled (fz) family, which activates the phosphoprotein dishevelled (dsh), which leads to inhibition of glycogen synthase kinase 3␤. Because ␤-catenin is a substrate of this serine/threonine kinase, it remains hypophosphorylated upon Wnt/Wg signaling and accumulates in the cytoplasm. This promotes its binding to LEF-TCF transcription factors. The ␤-catenin-LEF-TCF heterodimer enters the nucleus and is able to activate or repress gene transcription (for detailed reviews, see references 4, 15, and 32). Important developmental target genes, such as siamois, twin, and nodal-related 3 in Xenopus as well as Ubx (Ultrabithorax) in Drosophila, were found to be controlled by direct binding of ␤-catenin-LEF-TCF to their promoters (3,34,36,46).Abnormally high concentrations of ␤-catenin have been reported in several tumor and carcinoma cell lines caused by mutations in the adenomatous polyposis coli gene or ␤-catenin gene. These mutations prevent the degradation of ␤-catenin (41), which then contributes to the formation of a constitutively active ␤-catenin-LEF-TCF transcription complex (25,27,31,38,40,48,54). Most recently, He et al. (18) have identified the proto-oncogene c-myc as a direct target gene of the ␤-catenin-Tcf-4 complex in a human colorectal cancer cell line. This links upregulation of ␤-catenin to loss of proliferation control in tumorigenesis.The different functions of ␤-catenin, strengthening of cadherin-mediated cell adhesion and regulation of target genes of the Wnt/Wg signaling pathway, can compete with each other. When cytosolic or nuclear ␤-catenin is tethered to the plasma membrane by cadherin overexpression in Xenopus embryos, severe developmental defects are observed due to inhibition of ␤-catenin's nuclear function (9,19). Using a quite similar approach, we have previously shown that ectopic expression of cadherins in Xenopus XTC fibroblasts shifted ␤-catenin to the plasma membrane and led to downregulation of fibronectin and ␣ 3 ␤ 1 integrin synthesis. Additionally, the transfectants altered their adhesive properties, losing their ability to adhere to substrate molecules (10). These findings give evidence for a cross talk between cell-cell and cell-substrate adhesion regulated by ␤-catenin. Other examples of mutual interference between the two adhesion systems have been reported for different cell types. When keratinocytes were treated with cadherin antibodies, expression of ␣ 6 ␤ 1 integrin persisted during terminal differentiation (22), while dominant-negative expression of E-cadherin resulted in disappearance of ␣ 2 ␤ 1 and ␣ 3 ␤ 1 integrins (58). Recent...

Spinal muscular atrophy (SMA) is a motoneuron disease caused by reduced levels of survival motoneuron (SMN) protein. Previous studies have assigned SMN to uridine-rich small nuclear ribonucleoprotein particle (U snRNP) assembly, splicing, transcription, and RNA localization. Here, we have used gene silencing to assess the effect of SMN protein deficiency on U snRNP metabolism in living cells and organisms. In HeLa cells, we show that reduction of SMN to levels found in SMA patients impairs U snRNP assembly. In line with this, induced silencing of SMN expression in Xenopus laevis or zebrafish arrested embryonic development. Under less severe knock-down conditions, zebrafish embryos proceeded through development yet exhibited dramatic SMA-like motor axon degeneration. The same was observed after silencing two other essential factors in the U snRNP assembly pathway, Gemin2 and pICln. Importantly, the injection of purified U snRNPs into either SMN-or Gemin2-deficient embryos of Xenopus and zebrafish prevented developmental arrest and motoneuron degeneration, respectively. These findings suggest that motoneuron degeneration in SMA patients is a direct consequence of impaired production of U snRNPs.[Keywords: Survival motor neurons (SMN); U snRNP assembly; motoneuron; spinal muscular atrophy; zebrafish] Supplemental material is available at http://www.genesdev.org.

Canonical Wnt signaling is deregulated in several types of human cancer where it plays a central role in tumor cell growth and progression. Here we report the identification of 2 new small molecules that specifically inhibit canonical Wnt pathway at the level of the destruction complex. Specificity was verified in various cellular reporter systems, a Xenopus double-axis formation assay and a gene expression profile analysis. In human colorectal cancer (CRC) cells, the new compounds JW67 and JW74 rapidly reduced active b-catenin with a subsequent downregulation of Wnt target genes, including AXIN2, SP5, and NKD1. Notably, AXIN2 protein levels were strongly increased after compound exposure. Long-term treatment with JW74 inhibited the growth of tumor cells in both a mouse xenograft model of CRC and in Apc Min mice (multiple intestinal neoplasia, Min). Our findings rationalize further preclinical and clinical evaluation of these new compounds as novel modalities for cancer treatment. Cancer Res; 71(1); 197-205. Ó2011 AACR.