In the present study, we investigated the contribution of ASM to the suppression of tumor growth in liver metastasis of colon cancer cells. Our results indicated that liver ASM inhibits tumor growth through S1P formation and subsequent macrophage accumulation and TIMP1 production from hMFs. These results suggest novel therapeutic possibilities for treating metastatic liver tumors of colon cancer.
ASM in cancer cells is involved in cell death and plays an important role in the host response to a variety of anticancer treatments, including those for colon cancer (1
). In the present study, metastatic tumors of colon cancer cells stimulated ASM expression and ceramide in liver cells. Moreover, host ASM deficiency increased, while overexpression of ASM in the liver decreased, the growth of liver tumors after inoculation with colon cancer cells, which suggests that ASM in host cells contributes to antitumor defense. Among the roles that ASM plays in liver cells, ASM in hepatocytes stimulates glucose uptake, resulting in improvement of glucose tolerance in mice through S1P production (27
). ASM has pleiotropic signaling functions related to posttranslational processing of ASM, and secretory and lysosomal ASM (35
) may have distinct functions. Although it is unclear which type of ASM was involved in S1P production in our study, hepatocytes represented one source of S1P, as the S1P elevation by AdASM was inhibited in both SphK1-deficient livers and SphK1-deficient primary cultured rat hepatocytes (data not shown). Previous studies have shown that the generated S1P can be secreted and act as a ligand for S1P receptors (S1PRs; refs. 27
), while the S1P/S1PR1 axis controls the trafficking and migration of immune cells, including macrophages (16
). In accordance with these prior reports, S1P increased the migration of peritoneal macrophages in vitro, with less and more macrophage accumulation, respectively, in the tumors of Asm–/–
and AdASM-infected mice compared with controls. These findings indicate that ASM in hepatocytes leads to macrophage accumulation in tumors via S1P production and that this process exerts an antitumor effect, in contrast to macrophage depletion. Although it has been reported that TAMs, which resemble regulatory M2 macrophages (13
), function as promoters of tumor progression (7
), the macrophages in the tumors inoculated with SL4 colon cancer cells in this study were M1 dominant. It has been proposed that a gradual shift of TAM polarization from the M1 to the M2 subtype occurs during different stages of tumor progression, due to dynamic changes in the tumor microenvironment (38
), and is paralleled by the gradual inhibition of NF-κB (39
). In this study, we observed immunofluorescent staining of NF-κB p65 in the nuclei of F4/80+
cells in the tumors (data not shown), which suggests that NF-κB is activated in these macrophages. This finding may indicate that in metastatic liver tumors characterized by rapid growth, such as by increased numbers of SL4 cells (40
), the accumulated TAMs may retain an M1 phenotype that displays a cytotoxic proinflammatory phenotype. Thus, the number of macrophages present in the tumor is inversely correlated with tumor growth.
Myofibroblasts represent another important component of the tumor stroma; these cells are driven by migration and proliferation-stimulating factors released from cancer cells. Whereas some studies have reported a supportive role for myofibroblasts in cancer cell development (41
) and a direct correlation between their presence and poor prognosis (42
), others have reported a protective role of myofibroblasts against cancer cells (43
), with p53 in stromal fibroblasts demonstrated to inhibit tumor growth and malignant progression (45
). In the current study using SL4 cell inoculation, hMF accumulation was found to be dependent on macrophage accumulation and inversely correlated with tumor growth. Specifically, hMF accumulation was increased only around the invasive margins of the tumors, and no hMF infiltration or collagen deposition was observed in the central part of tumors (data not shown). These findings indicate that the supportive effects of hMFs in tumor growth may be limited. However, the observation of enhanced tumor growth in Timp1–/–
mice indicates that TIMP1 produced from hMFs, stimulated by S1P, might inhibit tumor growth through inhibition of ECM degradation. Although previous studies reported a protumor role for TIMP1, TIMP1 staining was observed only around the tumor invasive margin in the current study, which indicates that the supportive effects of TIMP1 in tumor growth may also be limited.
Whereas ceramide acts as a signaling molecule in the induction of cell responses, such as apoptosis and growth arrest (3
), S1P acts as a potent mitogen for a variety of cell types (48
), with SphK1/S1P contributing to colon carcinogenesis (49
). Ceramide levels in colon carcinoma tissue are significantly lower than those in normal tissue (50
), reflecting the fact that the dynamic balance between intracellular levels of ceramide and S1P (the “ceramide/S1P rheostat”) determines cell growth and cell death in colon cancer. In the present study, adenoviral overexpression of SphK1 in the liver inhibited tumor growth when colon cancer cells were inoculated after infection. In contrast to AdASM, AdSphK did not exert an inhibitory effect when administered after cell inoculation, which suggests that its inhibitory effect on existing tumors is less than that of AdASM. The observation that AdSphK increased the proliferation of cultured SL4 cells suggests that it decreased the ceramide/S1P ratio in the cancer cells, which may promote their proliferation. In contrast, AdASM might increase the ceramide/S1P ratio, which may inhibit SL4 cell proliferation. We therefore conclude that AdASM, but not AdSphK, may be a useful candidate for gene therapy against metastatic liver tumors of colon cancer.
mice, B16C2M melanoma cells formed small metastatic lesions in the liver (4 of 9, 44.4%) whereas none of the Asm+/+
mice developed metastases (0 of 11, 0%; Supplemental Figure 7), which suggests that the antitumor effect of ASM is not specific for colon cancer cells. However, use of the Asm
knockout may have secondary effects due to the severe lipid storage defect in these mice, particularly in macrophages. Asm–/–
mice are known to have severe immune defects (51
) and display severe neurocutaneous disease by 8 weeks of age. Thus, Asm–/–
mice may have various abnormalities in the liver as a normal pathology. In our study, the livers of Asm–/–
mice without injection of SL4 cells did not show the nodular appearance. In addition, liver weight of Asm–/–
mice without injection of SL4 cells was comparable to that of Asm+/+
mice. Thus, it seems unlikely that the changes are due to normal Asm–/–
mouse pathology. In addition, besides ASM, ceramide synthesized by the de novo pathway can be involved in TIMP1 regulation. These data do not provide direct evidence that S1P production in hepatocytes recruits macrophages, and experiments using mice with conditional knockout of SphK are also required to confirm the involvement of SphK and S1P in macrophage recruitment and TIMP1 regulation. Moreover, the mechanism by which tumor cells stimulate ASM expression in hepatocytes remains unclear, and there is no direct evidence of a positive correlation between ASM activity in hepatocytes and TIMP1 expression in hMFs; further studies are needed to resolve these uncertainties. In conclusion, we found here that ASM in hepatocytes inhibited tumor growth via S1P formation and subsequent cytotoxic macrophage accumulation. This S1P formation in turn stimulated TIMP1 production by hMFs, leading to tumor suppression. Thus, targeting ASM may represent a new therapeutic strategy for treating liver metastasis of colon cancer.