1. Factors secreted by macrophages induce canonical Wnt signaling in colon tumor cells and promote their proliferation
We developed an in vitro
model to evaluate crosstalk between macrophages and colon cancer cells. Experiments were performed using HCT116 and Hke-3 cells, isogenic colon cancer cell lines that differ only by the presence of the mutant kRas allele (Shirasawa et al., 1993
), and THP1 cells which display several characteristics of tumor associated macrophages (TAMs), such as defective activation of NF-κB, lack of nitric oxide production in response to LPS/IFNγ (not shown) and high constitutive STAT1 signaling (see below).
Both colon cell lines secrete factors that induce differentiation of THP1 cells into cells that exhibit characteristics of mature macrophages. Tumor cell - derived factors inhibited growth of THP1 macrophages (Supplemental Fig. 1 A, B, C, D) and induced morphological changes comparable to those induced by TPA, a known inducer of differentiation of the THP1 monocytic line into macrophages (Tsuchiya et al., 1982
) (Supplemental Fig. 1E). THP1 cells grown in the presence of conditioned medium from tumor cells, like TPA- treated cells, displayed enlarged cytoplasm and reorganized actin cytoskeleton, and became irregular in shape, flat and adherent (Supplemental Fig. 1E). Tumor derived factors also induced similar changes in primary human monocytes, the precursors of tumor associated macrophages (not shown), consistent with the concept that infiltration of monocytes into the tumor microenvironment is followed by their differentiation into mature macrophages (Allavena et al., 2008
To identify pathways altered in tumor cells exposed to macrophages, we transfected HCT116 cells with luciferase (LUC) – labeled reporter vectors that measure the activity of major signaling pathways known to be perturbed in colon cancer. We compared the activity of NF-κB, Notch, AP1, Wnt and STAT1 in HCT116 cells that were either maintained as monocultures, or were co-cultured with THP1 cells. At conditions tested, the presence of THP1 macrophages did not modulate the activity of the Notch, AP1 or STAT1 signaling, marginally affected NFκB activity (Supplemental Fig. 2), and significantly increased canonical Wnt signaling, measured by activity of the TOP-FLASH reporter. The THP1 macrophages increased Wnt signaling by 40% in HCT116 cells and by 39% in Hke-3 cells (). The activity of TOP-FOP, a reporter gene with a mutated TCF4 binding site, was not affected by factors secreted by the macrophages (not shown). Peripheral blood monocytes, precursors of the tumor associated macrophages, also increased Wnt signaling in both HCT116 and Hke-3 cells ().
Macrophages activate Wnt signaling in colon cancer cells and promote their clonogenic growth
Consistent with increased TCF4 transcriptional activity, the levels of both unphosphorylated (active) β–catenin and total β–catenin, and the expression of c-myc and cyclin D1, two key target genes of Wnt signaling, were elevated in tumor cells that were cultured in the presence of macrophages ().
To determine whether macrophages alter the clonogenic potential of tumor cells, HCT116 and HKe-3 cells were plated at a low density alone or together with an increasing number of macrophages. We showed that the presence of THP1 macrophages () or primary peripheral blood monocytes (, lower panel) significantly increased the clonogenic growth of colon cancer cells.
To determine whether direct contact between macrophages and tumor cells is required for enhanced proliferation of tumor cells, we co-cultured HCT116 and THP1 cells in transwells that allowed the exchange of soluble factors, but were impermeable for cells themselves (0.4 μM pore membrane, schematically shown in Supplemental Fig. 3A). THP1 macrophages enhanced proliferation of both HCT116 and Hke-3 cells and increased the fraction of tumor cells in the S phase of the cell cycle (not shown). Consistently, both HCT116 and Hke-3 cells co-cultured with THP1 cells displayed increased expression of cyclin D1, cyclin E and cyclin A (Supplemental Fig. 3B).
Together, these data demonstrated that macrophage-derived factors increased Wnt signaling and enhanced clonogenic potential and proliferation of colonic epithelial cells and showed that the presence of activated Ras in the epithelial cells does not affect the interaction between tumor cells and macrophages.
2. IL-1β is sufficient to activate Wnt signaling and is required for increased Wnt signaling in tumor cells
Because the crosstalk between the tumor cells and macrophages was mediated by soluble factors, we compared the levels of chemokines and cytokines in conditioned medium collected from monocultures and cocultures of macrophages with HCT116 cells. Using a cytokine antibody array that assessed 36 soluble mediators (R&D Systems) we showed that the most abundant chemokine secreted by THP1 macrophages was CCL5 (Rantes), while HCT116 and Hke-3 cells expressed detectable levels of MIF and PAI-1 (). Interaction of THP1 macrophages with HCT116 cells provoked a marked elevation in the expression of several chemokines including CCL5, CXCL1, CCL3, IL-8, and IL-1β, a major proinflammatory cytokine (). IL-1β was also induced by co-culturing the primary peripheral blood monocytes, the precursors of tumor associated macrophages, with tumor cells ().
IL-1β is sufficient and required to activate Wnt signaling
Which of these soluble factors mediated increased Wnt signaling in the tumor cells? We tested the activity of Rantes, IL-1β, and TNF, a macrophage-derived cytokine recently shown to activate Wnt signaling in gastric cancer (Oguma et al., 2008
). However, unlike in gastric cells, IL-1β was a more potent inducer of TCF4/β-catenin transcriptional activity than TNF, and CCL5, a predominant cytokine secreted by THP1 cells, did not modulate Wnt signaling in HCT116 cells (not shown). IL-1β induced TCF4/β-catenin dependent reporter gene activity in a concentration dependent manner and did not affect the activity of the TOP-FOP reporter which harbors a mutant TCF4 binding site (). Consistent with increased Wnt signaling, IL-1β increased the levels of active β–catenin in HCT116 cells (, the inset). Treatment of HCT116 cells with IL-1β increased phosphorylation of GSK3β (), which is known to inhibit its activity (Cross et al., 1995
). Concomitant with inactivation of GSK3β and resulting stabilization of β-catenin, the levels of c-myc were induced by IL-1β ().
The ability of IL-1β to activate Wnt signaling was inhibited by neutralizing IL-1β antibody, confirming efficacy and specificity of the antibody (). Neutralizing IL-1β also reversed the ability of THP1 macrophages to induce Wnt signaling in HCT116 cells, demonstrating that IL-1β is required for macrophage-mediated increased Wnt signaling in the tumor cells. The neutralizing antibody against IL-1β did not affect basal Wnt signaling in HCT116 cells ()
The ability of THP1 macrophages to increase the clonogenic growth of tumor cells was inhibited by neutralization of IL-1β (), demonstrating that macrophages increase the growth of tumor cells mostly through IL-1β. Consistently, IL-1β was sufficient to increase the clonogenic growth of HCT116 cells ().
To confirm that macrophages are the source of IL-1β in cocultures, the IL-1β gene was silenced in either the tumor cells or the THP1 macrophages. Silencing of IL-1β in macrophages, but not in HCT116 cells completely inhibited the ability of macrophages to induce Wnt signaling () and to induce the clonogenic growth of tumor cells (). Thus, inhibition of the IL-1β release from macrophages attenuates the protumorigenic activity of macrophages.
3. 1,25(OH)2D3 inhibits the release of IL-1β and interrupts the crosstalk between macrophages and tumor cells
Next we examined whether vitamin D3
, a potent chemopreventive agent for colorectal cancer, can modulate the cross talk between macrophages and tumor cells. Consistent with published results (Kumagai et al., 2003
), vitamin D3
did not inhibit the growth of HCT116 cells (Supplemental Fig. 4A). Although oncogenic kRas alters the responsiveness of cells to a variety of chemopreventive and chemotherapeutic agents (Klampfer et al., 2003
; Klampfer et al., 2004
; Klampfer et al., 2007
; Klampfer et al., 2005
), the isogenic Hke-3 cells, which lack the mutant kras allele, also displayed resistance to 1,25(OH)2
(Supplemental Fig. 4A). In contrast, proliferation of THP1 macrophages was markedly inhibited by 1,25(OH)2
, demonstrated by the MTT assay and BrdU incorporation (Supplemental Fig. 4B).
Because vitamin D3 has anti-inflammatory properties, we tested whether 1,25(OH)2D3 modulates the release of IL-1β from macrophages. Co-culturing of HCT116 cells and THP1 macrophages induced significant secretion of IL-1β and 1,25(OH)2D3 inhibited the release of IL-1β from macrophages induced by tumor cells, and also in response to LPS stimulation (Supplemental Fig. 4C). Surprisingly, the release of IL-8 induced by the interaction of tumor cells with macrophages was enhanced by 1,25(OH)2D3(Supplemental Fig. 4C). This suggested that 1,25(OH)2D3 may regulate the cross-talk between macrophages and tumor cells.
To test whether 1,25(OH)2D3 alters the ability of THP1 cells to activate Wnt signaling in tumor cells, HCT116 cells transfected with the TOP-FLASH reporter gene were cultured alone or together with THP1 cells, and were treated with vitamin D3. 1,25(OH)2D3 did not affect endogenous Wnt signaling in HCT116 cells, but completely prevented the ability of THP1 macrophages to enhance Wnt signaling in HCT116 cells () and inhibited the increased levels of active β–catenin and total β–catenin in HCT116 cells cultured in the presence of THP1 macrophages (). Coculture of tumor cells with THP1 macrophages, like treatment with IL-1β, resulted in phosphorylation of GSK3β and treatment with 1,25(OH)2D3 prevented THP-1 mediated phosphorylation of GSK3β and induction of c-myc (, right panel). THP-1 macrophages transfected with IL-1β specific siRNA failed to inactivate GSK3β and to induce c-myc (, right panel), confirming the crucial role of IL-1β in the crosstalk between tumor cells and macrophages. 1,25(OH)2D3 did not inhibit butyrate-driven Wnt signaling in HCT116 cells (Supplemental Fig. 5), demonstrating that it does not act as a general inhibitor of Wnt signaling.
Vitamin D3 inhibits macrophage-mediated increase in Wnt signaling
The ability of vitamin D3 to inhibit macrophage-mediated Wnt signaling in tumor cells was rescued by exogenous IL-1β (), suggesting that 1,25(OH)2D3 acts predominantly by inhibiting the release of IL-1β from THP1 macrophages induced by tumor cells. Indeed, 1,25(OH)2D3 did not interfere with the ability of IL-1β to increase Wnt signaling in tumor cells (, Supplemental Fig. 6).
Consistent with the ability to abrogate Wnt signaling, 1,25(OH)2D3 blocked the ability of THP1 macrophages to promote the clonogenic growth of HCT116 cells (). Exogenous IL-1β partially restored the ability of vitamin D3 treated THP1 macrophages to promote clonogenic growth of tumor cells (). Vitamin D3 did not affect the ability of IL-1β or TNF to promote the clonogenic growth of HCT116 cells (, Supplemental Fig. 6), confirming that 1,25(OH)2D3 does not inhibit signaling by IL-1β. These data therefore demonstrate that 1,25(OH)2D3 interrupts the crosstalk between tumor cells and the tumor microenvironment through its ability to inhibit the release of IL-1β from macrophages.
To confirm that 1,25(OH)2D3 regulates the interactions between tumor cells and macrophages, we showed that untreated THP1 cells, but not 1,25(OH)2D3 treated THP1 macrophages, promoted proliferation of HCT116 cells (Supplementary Fig. 7A). Consistently, the increased expression of cyclin D1, cyclin A and cyclin E in HCT116 cultured with macrophages, returned to basal level upon treatment of cultures with 1,25(OH)2D3 (Supplementary Fig. 7B).
4. 1,25(OH)2D3 inhibits THP1 driven Wnt signaling and THP1-driven proliferation of tumor cells in a VDR dependent manner
To examine whether vitamin D3 exerts its biological activity in a VDR dependent manner, we silenced VDR expression in THP1 macrophages (inset, ). VDR deficient macrophages retained the ability to increase Wnt signaling upon 1,25(OH)2D3treatment, in contrast to nontransfected THP1 cells or THP1 cells transfected with nontargeting siRNA (NSP) (). Accordingly, 1,25(OH)2D3 failed to inhibit macrophage mediated increased expression of c-myc and cyclin D1 in tumor cells upon silencing of VDR in THP1 cells (). Finally, we showed that VDR deficient THP1 macrophages failed to respond to vitamin D3 and continued to secrete factors that promote proliferation of HCT116 cells upon treatment with vitamin D3 (). 1,25(OH)2D3 also failed to inhibit the growth promoting activity of VDR deficient THP1 macrophages in co-culture experiments (). These data established that the ability of vitamin D3 to regulate the cross-talk between THP1 macrophages and colon cancer cells requires the expression of VDR on the THP1 cells, confirming that macrophages, and not epithelial cells, were targeted by 1,25(OH)2D3. Consistent with this notion, silencing of VDR expression in HCT116 cells did not impact the interplay between epithelial cells and macrophages, or influence the ability of 1,25(OH)2D3 to interfere with this crosstalk (data not shown).
Inhibition of Wnt signaling by vitamin D3 requires VDR expression on macrophages
5. p-STAT1 mediates the ability of THP1 macrophages to induce Wnt signaling and to promote proliferation of colon cancer cells
Tumor associated macrophages often express constitutively activated STAT1 (Biswas et al., 2006
; Kusmartsev and Gabrilovich, 2005
), as we demonstrated for the THP1 cells () and tumor cells have been recently shown to induce STAT1 phoshorylation on Y701 in primary macrophages (Hagemann et al., 2008
). Since the expression of IL-1β is regulated by STAT1 (Lee et al., 2006
; Tsukada et al., 1996
), we investigated whether STAT1 was required for the release of IL-1β from macrophages. HCT116 and HKe-3 cells were co-cultured with THP1 cells transfected with nonspecific (NSP) or STAT1 specific siRNA ( inset). As shown in , STAT1 deficient THP1 cells failed to release IL-1β in response to tumor cells or in response to stimulation with LPS. In contrast, induction of IL-8 in response to LPS was STAT1 independent ().
Secretion of IL-1β, but not IL-8, from THP1 macrophages, requires STAT1
Consistently, STAT1 deficient THP1 macrophages failed to promote proliferation of the tumor cells (), did not activate Wnt signaling in tumor cells (, right panel) and failed to stabilize β–catenin and to increase the expression of c-myc and cyclin D1 in tumor cells ().
Activated STAT1 is required for pro-tumorigenic activity of THP1 macrophages
To examine whether 1,25(OH)2D3 regulates STAT1 in macrophages, THP1 cells were treated with vitamin D3 and tested for the expression of both p-STAT1 and total STAT1. As shown in , 1,25(OH)2D3 inhibited the expression of constitutively active STAT1 (pSTAT1Y701), without a significant and reproducible effect on the expression of total STAT1. Consistently, while most of the STAT1 in untreated THP1 cells appeared to be nuclear, treatment of cells with vitamin D3 resulted in translocation of STAT1 from the nucleus to the cytoplasm ().
Therefore, our data established that tumor associated macrophages secrete IL-1β in a pSTAT1 dependent manner, activate Wnt signaling and thereby promote proliferation of the tumor cells. 1,25(OH)2D3, through its ability to inhibit pSTAT1 in macrophages and thus IL-1β production, inhibits their capability to induce Wnt signaling and to promote proliferation of tumor cells.