Tumor Associated Macrophages Protect Colon Cancer Cells from TRAIL-Induced Apoptosis through IL-1β- Dependent Stabilization of Snail in Tumor Cells

We recently reported that colon tumor cells stimulate macrophages to release IL-1beta, which in turn inactivates GSK3beta and enhances Wnt signaling in colon cancer cells, generating a self-amplifying loop that promotes the growth of tumor cells.Here we describe that macrophages protect HCT116 and Hke-3 colon cancer cells from TRAIL-induced apoptosis. Inactivation of IL-1beta by neutralizing IL-1beta antibody, or silencing of IL-1beta in macrophages inhibited their ability to counter TRAIL-induced apoptosis. Accordingly, IL-1beta was sufficient to inhibit TRAIL-induced apoptosis. TRAIL-induced collapse of the mitochondrial membrane potential (Delta psi) and activation of caspases were prevented by macrophages or by recombinant IL-1beta. Pharmacological inhibition of IL-1beta release from macrophages by vitamin D(3), a potent chemopreventive agent for colorectal cancer, restored the ability of TRAIL to induce apoptosis of tumor cells cultured with macrophages. Macrophages and IL-1beta failed to inhibit TRAIL-induced apoptosis in HCT116 cells expressing dnIkappaB, dnAKT or dnTCF4, confirming that they oppose TRAIL-induced cell death through induction of Wnt signaling in tumor cells. We showed that macrophages and IL-1beta stabilized Snail in tumor cells in an NF-kappaB/Wnt dependent manner and that Snail deficient tumor cells were not protected from TRAIL-induced apoptosis by macrophages or by IL-1beta, demonstrating a crucial role of Snail in the resistance of tumor cells to TRAIL.We have identified a positive feedback loop between tumor cells and macrophages that propagates the growth and promotes the survival of colon cancer cells: tumor cells stimulate macrophages to secrete IL-1beta, which in turn, promotes Wnt signaling and stabilizes Snail in tumor cells, conferring resistance to TRAIL. Vitamin D(3) halts this amplifying loop by interfering with the release of IL-1beta from macrophages. Accordingly, vitamin D(3) sensitizes tumor cells to TRAIL-induced apoptosis, suggesting that the therapeutic efficacy of TRAIL could be augmented by this readily available chemopreventive agent

Activating mutations in β-catenin in colon cancer cells alter their interaction with macrophages; the role of snail.

Tumor cells become addicted to both activated oncogenes and to proliferative and pro-survival signals provided by the abnormal tumor microenvironment. Although numerous soluble factors have been identified that shape the crosstalk between tumor cells and stroma, it has not been established how oncogenic mutations in the tumor cells alter their interaction with normal cells in the tumor microenvironment.We showed that the isogenic HCT116 and Hke-3 cells, which differ only by the presence of the mutant kRas allele, both stimulate macrophages to produce IL1β. In turn, macrophages enhanced Wnt signaling, proliferation and survival in both HCT116 and Hke-3 cells, demonstrating that signaling by oncogenic kRas in tumor cells does not impact their interaction with macrophages. HCT116 cells are heterozygous for β-catenin (HCT116(WT/MT)), harboring one wild type (WT) and one mutant (MT) allele, but isogenic lines that carry only the WT (HCT116(WT)) or MT β-catenin allele (HCT116(MT)) have been generated. We showed that macrophages promoted Wnt signaling in cells that carry the MT β-catenin allele, but not in HCT116(WT) cells. Consistent with this observation, macrophages and IL1β failed to stabilize Snail in HCT116(WT) cells, and to protect these cells from TRAIL-induced apoptosis. Finally, we demonstrated that HCT116 cells expressing dominant negative TCF4 (dnTCF4) or HCT116 cells with silenced Snail failed to stimulate IL1β production in macrophages, demonstrating that tumor cells activate macrophages via a Wnt-dependent factor.Our data demonstrate that oncogenic β-catenin mutations in tumor cells, and subsequent activation of Wnt signaling, not only trigger cell-intrinsic alterations, but also have a significant impact on the crosstalk of tumor cells with the tumor associated macrophages

GM-CSF RESCUES TF-1 CELLS FROM GROWTH FACTOR WITHDRAWAL-INDUCED, BUT NOT DIFFERENTIATION-INDUCED APOPTOSIS: THE ROLE OF BCL-2 AND MCL-1

Cytokines such as granulocyte–macrophage colony-stimulating factor (GM-CSF) and IL-3 promote the survival and stimulate the proliferation of haematopoietic cells. Using the GM-CSF-dependent TF-1 myeloid leukaemia cell line, the authors show that the endogenous levels of BCL-2 and MCL-1 are downregulated upon GM-CSF withdrawal, whereas the levels of BCL-xLand Bax are unchanged. Re-exposure of growth factor deprived cells to GM-CSF resulted in an early and transient increase in MCL-1 expression, and prolonged induction of BCL-2, which prevented apoptosis. In contrast, the expression of BCL-2 and MCL-1 were not modulated during TPA-induced differentiation of TF-1 cells, which was followed by apoptosis despite the presence of GM-CSF. TF-1 cells overexpressing BCL-2 or MCL-1 underwent delayed apoptosis upon growth factor withdrawal, but displayed no impaired apoptosis in response to TPA. Erythropoietin (Epo) induced the expression of BCL-2 and MCL-1 protein in TF-1 cells, however it did not support their long term proliferation, further demonstrating that upregulation of these anti-apoptotic genes is insufficient for the long term proliferation of TF-1 cells

A proposed model for the interaction of tumor cells with macrophages: Colon cancer cells produce a soluble factor, a product of a Wnt regulated gene, which through binding to an unknown receptor stimulates macrophages to release IL1β.

<p>IL1β in turn binds IL1R on tumor cells and stimulates Wnt signaling. We showed that macrophage-derived factor stabilize Snail in tumor cells, which further promotes Wnt signaling and is required for the ability of tumor cells to activate macrophages to produce IL1β.</p

Sodium Salicylate Activates Caspases and Induces Apoptosis of Myeloid Leukemia Cell Lines

Abstract Nonsteroidal antiinflammatory agents (NSAIA) have been shown to exert potent chemopreventive activity against colon, lung, and breast cancers. In this study, we show that at pharmacological concentrations (1 to 3 mmol/L) sodium salicylate (Na-Sal) can potently induce programmed cell death in several human myeloid leukemia cell lines, including TF-1, U937, CMK-1, HL-60, and Mo7e. TF-1 cells undergo rapid apoptosis on treatment with Na-Sal, as indicated by increased annexin V binding capacity, cpp-32 (caspase-3) activation, and cleavage of poly (ADP-ribose) polymerase (PARP) and gelsolin. In addition, the expression of MCL-1, an antiapoptotic member of the BCL-2 family, is downregulated during Na-Sal–induced cell death, whereas the expression of BCL-2, BAX, and BCL-XL is unchanged. Z-VAD, a potent caspase inhibitor, prevents the cleavage of PARP and gelsolin and rescues cells from Na-Sal–induced apoptosis. In addition, we show that Na-Sal accelerates growth factor withdrawal-induced apoptosis and synergizes with daunorubicin to induce apoptosis in TF-1 cells. Thus, our data provide a potential mechanism for the chemopreventive activity of NSAIA and suggest that salicylates may have therapeutic potential for the treatment of human leukemia

ORIGINAL PAPER The NF-κB/AKT-dependent Induction of Wnt Signaling in Colon Cancer Cells by Macrophages and IL-1β

Abstract Progression of colon cancer from microadenoma to macroscopic tumors is coupled to augmentation of canonical Wnt signaling. We recently reported that tumor associated macrophages, through interleukin 1β (IL-1β) dependent phosphorylation of GSK3β, promote Wnt signaling in colon cancer cells, demonstrating that proinflammatory cytokines can enhance TCF4/β-catenin transcriptional activity in tumor cells. Here we investigated the pathway whereby IL-1β inactivates GSK3β and promotes Wnt signaling in colon cancer cells. We showed that normal human monocytes, THP1 macrophages and IL-1 failed to induce Wnt signaling in tumor cells expressing dominant negative IκB (dnIκB), demonstrating that macrophages and IL-1 activate Wnt signaling in a NF-κB-dependent manner. NF-κB activity was required for macrophages and IL-1 to activate PDK1 and AKT in tumor cells and thereby inhibit GSK3β activity. Consistently, dominant negative AKT (dnAKT), or pharmacological inhibition of AKT in tumor cells, prevented macrophage/IL-1 mediated phosphorylation of GSK3β, activation of Wnt signaling, and induction of c-jun and c-myc, confirming that macrophages and IL-1 promote Wnt signaling in an AKT dependent manner. Finally, we showed IL-1 and macrophages failed to promote growth of colon cancer cells with impaired NFκB or AKT signaling, confirming the requirement for NFκB and AKT activation for the protumorigenic activity of tumor associated macrophages. Thus, we showed that IL-1 and tumor associated macrophages activate NF-κBdependent PDK1/AKT signaling in tumor cells, and thereb