Wnt-5a has been shown to influence the metastatic behavior of human breast cancer cells, and the loss of Wnt-5a expression is associated with metastatic disease. We show here that NFAT1, a transcription factor connected with breast cancer metastasis, is activated by Wnt-5a through a Ca 2؉ signaling pathway in human breast epithelial cells. This activation was simultaneously counteracted by a Wnt-5a-induced Yes/Cdc42 signaling pathway. The observation that inhibition of the Wnt-5a/Yes/Cdc42 signal prolonged the duration of ionomycin-induced NFAT1 activation revealed the general importance of this pathway. The Wnt-5a-induced inhibition of NFAT1 did not require glycogen synthase kinase 3␤, JNK, or Pak1 activity or modulation of the cytoskeleton. Instead, we observed that Wnt-5a induced a complex formation of NFAT1/casein kinase 1␣, even upon treatment with ionomycin, which was blocked upon inhibition of the Wnt-5a/Yes/Cdc42 signaling pathway. Our results explain why Wnt-5a/Ca 2؉ -induced NFAT activity is hard to detect and suggest a novel mechanism by which Wnt-5a can suppress tumor-specific, agonist-induced NFAT activity and thus the metastatic behavior of breast cancer cells.

Loss of Wnt-5a protein expression is associated with shorter recurrence-free survival in breast carcinoma patients and increased motility in mammary cell lines. Based on sequence analysis of Wnt5a, we identified 14 peptide fragments and investigated their ability to mimic the effects of Wnt-5a on mammary cell adhesion and migration. Two of these peptides significantly increased adhesion and impaired migration in the non-tumorigenic HB2 breast epithelial cell line and in the MDA-MB-468 breast cancer cell line, both of which show little endogenous expression of the Wnt-5a protein. We removed two amino acids at a time from the N terminus of the shorter of these two peptides to identify the shortest peptide that still inhibited migration. The influence on tumor cell adhesion was gradually lost and was no longer detectable when only six amino acids remained. However, formylation of the N-terminal methionine of this hexapeptide restored its effect on adhesion and reduced tumor cell motility via a Frizzled-5 receptor-dependent mechanism, even at a low pH such as encountered in breast tumor tissue. This formylated hexapeptide ligand induced a rapid cytosolic calcium signal, whereas it did not affect the cellular levels of unphosphorylated ␤-catenin or active JNK. The novel formyl-Met-AspGly-Cys-Glu-Leu peptide ligand is not only a valuable experimental tool but has also a potential role in antimetastatic treatment of the 50% of human breast cancer patients that have reduced endogenous Wnt-5a protein expression.The Wnts are a family of secreted glycoproteins that have molecular masses of 39 -46 kDa and participate in development and tumorigenesis via autocrine or paracrine routes (for reviews see Refs. 1-3). Secreted Wnt proteins bind to and activate G-protein-coupled receptors of the Frizzled (Frz) 2 family (4, 5), and it is presumed that the low density lipoprotein (LDL) receptor-related proteins LRP5 and LRP6 act as coreceptors in that context (6, 7). Based on differences in the ability to transform mouse mammary epithelial cells (8), the Wnt proteins can in the present context be divided into the following three distinct classes: Wnt-1, Wnt-3a, and Wnt-7a have the greatest transforming capacity; Wnt-2, Wnt-5b, and Wnt-7b have an intermediate transforming capacity; and Wnt-4, Wnt-5a, and Wnt-6 are non-transforming.We have previously reported that low level expression of Wnt-5a protein in primary invasive breast carcinomas is associated with higher histological grade (poor differentiation) and shortened recurrence-free survival because of more rapid development of distant metastases (9). This association cannot be caused by an effect of the protein on proliferation, because no correlation has been found between loss of Wnt-5a protein expression and presence of the proliferative marker Ki67 (10). Currently, the only available explanation for the ability of Wnt-5a to reduce the metastatic capacity of invasive breast cancer is that this protein enhances adhesion and thus reduces the migration of these tumor cells. However...

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The purinergic P2X7 receptor is implicated in both neuropathic and inflammatory pain, and has been suggested as a possible target in pain treatment. However, the specific role of the P2X7 receptor in bone cancer pain is unknown. We demonstrated that BALB/cJ P2X7 receptor knockout (P2X7R KO) mice were susceptible to bone cancer pain and moreover had an earlier onset of pain-related behaviours compared with cancer-bearing, wild-type mice. Furthermore, acute treatment with the selective P2X7 receptor antagonist, A-438079, failed to alleviate pain-related behaviours in models of bone cancer pain with and without astrocyte activation (BALB/cJ or C3H mice inoculated with 4T1 mammary cancer cells or NCTC 2472 osteosarcoma cells, respectively), suggesting that astrocytic P2X7 receptors play a negligible role in bone cancer pain. The results support the hypothesis that bone cancer pain is a separate pain state compared with those of neuropathic and inflammatory pain. However, the recent discovery of a P2X7 receptor splice variant expressed in the knockout mice used for this study complicates the interpretation of the results. The P2X7 splice variant receptor was detected in the spinal cord but not in osteoclasts of the P2X7R KO mouse. Further experiments are needed to elucidate the exact role of the P2X7 receptors in bone cancer pain.

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