Clustering of αvβ3 integrin after interaction with the RGD-like integrin-binding sequence present in neuronal Thy-1 triggers formation of focal adhesions and stress fibers in astrocytes via RhoA activation. A putative heparin-binding domain is present in Thy-1, raising the possibility that this membrane protein stimulates astrocyte adhesion via engagement of an integrin and the proteoglycan syndecan-4. Indeed, heparin, heparitinase treatment and mutation of the Thy-1 heparin-binding site each inhibited Thy-1-induced RhoA activation, as well as formation of focal adhesions and stress fibers in DI TNC1 astrocytes. These responses required both syndecan-4 binding and signaling, as evidenced by silencing syndecan-4 expression and by overexpressing a syndecan-4 mutant lacking the intracellular domain, respectively. Furthermore, lack of RhoA activation and astrocyte responses in the presence of a PKC inhibitor or a dominant-negative form of PKCα implicated PKCα and RhoA activation in these events. Therefore, combined interaction of the astrocyte αvβ3-integrin–syndecan-4 receptor pair with Thy-1, promotes adhesion to the underlying matrix via PKCα- and RhoA-dependent pathways. Importantly, signaling events triggered by such receptor cooperation are shown here to be the consequence of cell-cell rather than cell-matrix interactions. These observations are likely to be of widespread biological relevance because Thy-1–integrin binding is reportedly relevant to melanoma invasion, monocyte transmigration through endothelial cells and host defense mechanisms.

Caveolin-1 reportedly acts as a tumor suppressor and promotes events associated with tumor progression, including metastasis. The molecular mechanisms underlying such radical differences in function are not understood. Recently, we showed that caveolin-1 inhibits expression of the inhibitor of apoptosis protein survivin via a transcriptional mechanism involving the ␤-catenin-Tcf/Lef pathway. Surprisingly, while caveolin-1 expression decreased survivin mRNA and protein levels in HT29 (

Caveolin-1 is suggested to act as a tumor suppressor. We tested the hypothesis that caveolin-1 does so by repression of survivin, an Inhibitor of apoptosis protein that regulates cell-cycle progression as well as apoptosis and is commonly overexpressed in human cancers. Ectopic expression of caveolin-1 in HEK293T and ZR75 cells or siRNA-mediated silencing of caveolin-1 in NIH3T3 cells caused downregulation or upregulation of survivin mRNA and protein, respectively. Survivin downregulation in HEK293T cells was paralleled by reduced cell proliferation, increases in G0-G1 and decreases in G2-M phase of the cell cycle. In addition, apoptosis was evident, as judged by several criteria. Importantly, expression of green fluorescent protein-survivin in caveolin-1-transfected HEK293T cells restored cell proliferation and viability. In addition, expression of caveolin-1 inhibited transcriptional activity of a survivin promoter construct in a β-catenin-Tcf/Lef-dependent manner. Furthermore, in HEK293T cells caveolin-1 associated with β-catenin and inhibited Tcf/Lef-dependent transcription. Similar results were obtained upon caveolin-1 expression in DLD1 cells, where APC mutation leads to constitutive activation of β-catenin-Tcf/Lef-mediated transcription of survivin. Taken together, these results suggest that anti-proliferative and pro-apoptotic properties of caveolin-1 may be attributed to reduced survivin expression via a mechanism involving diminished β-catenin-Tcf/Lef-dependent transcription.

Early in cancer development, tumour cells express vascular endothelial growth factor (VEGF), a secreted molecule that is important in all stages of angiogenesis, an essential process that provides nutrients and oxygen to the nascent tumor and thereby enhances tumor-cell survival and facilitates growth. Survivin, another protein involved in angiogenesis, is strongly expressed in most human cancers, where it promotes tumor survival by reducing apoptosis as well as favoring endothelial cell proliferation and migration. The mechanisms by which cancer cells induce VEGF expression and angiogenesis upon survivin up-regulation remain to be fully established. Since the PI3K/Akt signalling and β-catenin-Tcf/Lef dependent transcription have been implicated in the expression of many cancer-related genes, including survivin and VEGF, we evaluated whether survivin may favor VEGF expression, release from tumor cells and induction of angiogenesis in a PI3K/Akt-β-catenin-Tcf/Lef-dependent manner. Here, we provide evidence linking survivin expression in tumor cells to increased β-catenin protein levels, β-catenin-Tcf/Lef transcriptional activity and expression of several target genes of this pathway, including survivin and VEGF, which accumulates in the culture medium. Alternatively, survivin downregulation reduced β-catenin protein levels and β-catenin-Tcf/Lef transcriptional activity. Also, using inhibitors of PI3K and the expression of dominant negative Akt, we show that survivin acts upstream in an amplification loop to promote VEGF expression. Moreover, survivin knock-down in B16F10 murine melanoma cells diminished the number of blood vessels and reduced VEGF expression in tumors formed in C57BL/6 mice. Finally, in the chick chorioallantoid membrane assay, survivin expression in tumor cells enhanced VEGF liberation and blood vessel formation. Importantly, the presence of neutralizing anti-VEGF antibodies precluded survivin-enhanced angiogenesis in this assay. These findings provide evidence for the existance of a posititve feedback loop connecting survivin expression in tumor cells to PI3K/Akt enhanced β-catenin-Tcf/Lef-dependent transcription followed by secretion of VEGF and angiogenesis.Electronic supplementary materialThe online version of this article (doi:10.1186/1476-4598-13-209) contains supplementary material, which is available to authorized users.

Increased expression of casein kinase 2 (CK2) is associated with hyperproliferation and suppression of apoptosis in cancer. Mutations in the tumor suppressor APC (adenomatous polyposis coli) are frequent in colon cancer and often augment ␤-catenin-T cell factor (Tcf)͞lymphoid enhancer binding factor (Lef)-dependent transcription of genes such as c-myc and cyclin-D1. CK2 has also been implicated recently in the regulation of ␤-catenin stability. To identify mechanisms by which CK2 promotes survival, effects of the specific CK2 inhibitors 4,5,6,7-tetrabromobenzotriazole (TBB) and 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole were assessed. TBB and 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole significantly decreased proliferation and increased apoptosis of HT29(US) colon cancer cells. RT-PCR and immunoblot analysis revealed that both inhibitors decreased survivin mRNA and protein levels in HT29(US) cells. Similar effects were observed with TBB in human DLD-1 and SW-480 colorectal cells as well as ZR-75 breast cancer cells and HEK-293T embryonic kidney cells. Expression of GFP-CK2␣ in HEK-293T cells resulted in ␤-catenin-Tcf͞Lef-dependent up-regulation of survivin and increased resistance to anticancer drugs. Augmented ␤-catenin-Tcf͞Lef-dependent transcription and resistance to apoptosis observed upon GFP-CK2␣ expression were abolished by TBB. Alternatively, HEK-293T cells expressing GFP-survivin were resistant to TBB-induced apoptosis. Finally, siRNA-mediated down-regulation of CK2␣ in HEK-293T cells coincided with reduced ␤-catenin and survivin levels. Taken together, these results suggest that CK2 kinase activity promotes survival by increasing survivin expression via ␤-catenin-Tcf͞Lef-mediated transcription. Hence, selective CK2 inhibition or down-regulation in tumors may provide an attractive opportunity for the development of novel cancer therapies.apoptosis ͉ cancer ͉ cell cycle