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Angiomodulin (AGM/IGFBP-rP1), a glycoprotein of about 30 kDa, is overexpressed in tumor vasculature as well as some human cancer cell lines, but it has been suggested to be a tumor suppressor. To elucidate roles of angiomodulin (AGM) in tumor progression, we here examined distribution of AGM in three types of human cancer tissues by immunohistochemistry. The results showed that AGM was overexpressed in the stroma as well as the vasculature surrounding tumor cells in the human cancer tissues. AGM and α-smooth muscle actin (α-SMA) as an activated fibroblast marker were often colocalized in cancer-associated fibroblasts (CAFs). In vitro analysis indicated that transforming growth factor (TGF)-β1 might be an important inducer of AGM in normal human fibroblasts. AGM strongly stimulated the expression of fibronectin and weakly that of α-SMA in normal fibroblasts. AGM significantly stimulated the proliferation and migration of fibroblasts. The AGM-induced expression of fibronectin and α-SMA was blocked by a TGF-β signal inhibitor but neither the stimulation of cell growth nor migration. These results imply that AGM activates normal fibroblasts by TGF-β-dependent and independent mechanisms. These findings also suggest that AGM and TGF-β1 cooperatively or complementarily contribute to the stromal activation and connective tissue formation in human cancer tissues, contributing to tumor progression. (Cancer Sci 2012; 103: 691–699)
It has recently been accepted that tumor microenvironment plays critical roles in tumor progression.[1-3] Fibroblasts, vascular endothelial cells, inflammatory cells and their secreted protein products including extracellular matrix proteins are major components present in the tumor microenvironment. The behavior of tumor cells is regulated by complex interaction between the tumor cells and surrounding stromal cells and secreted factors.[2, 3]
Angiomodulin (AGM), a secretory glycoprotein of about 30 kDa, is a member of the insulin-like growth factor binding protein (IGFBP) superfamily. However, the amino acid identity of AGM to IGFBPs is as low as about 20%, and its affinity for IGFs is far lower than that of IGFBPs.[4-6] Therefore, the names “IGFBP-related protein-1 (IGFBP-rP1)” and “IGFBP-7” are often used for this protein. AGM was originally identified as tumor-derived cell adhesion factor (TAF) secreted by human bladder carcinoma cells and as prostacyclin-stimulating factor (PSF) from human fibroblasts. The cDNA of AGM was cloned as mac25, which was expressed in normal human leptomeningeal cells but scarcely in meningiomas. Because AGM is highly expressed in tumor blood vessels, the name “angiomodulin (AGM)” was proposed. A recent study has shown that AGM is mainly expressed in developmental and adult vascular systems and plays a synergistic role with VEGF in angiogenesis. The AGM message or protein is detected in a wide range of normal tissues such as the heart, spleen, ovary, small intestine and colon and cells such as vascular endothelial cells, high endothelial cells, smooth muscle cells, fibroblasts and cancer cells.[4, 7-10]
There are many contradictory reports on the pathological roles of AGM in cancer. Although our earlier studies showed that AGM is highly expressed in some cancer cell lines, invading tumor cells in colon cancer and tumor vasculature, other studies showed that AGM may be a tumor-suppressing protein. Expression of AGM is associated with cell senescence in normal human mammary epithelial cells.[15, 16] In breast, prostate and lung cancers, reduced expression of AGM is correlated with worse prognosis of patients compared to higher expression. Forced expression of exogenous AGM in breast, lung and prostate[20, 21] cancer cells inhibited the cell growth by inducing senescence or apoptosis in culture. Moreover, forced expression of exogenous AGM in colon carcinoma, lung carcinoma and melanoma cell lines suppressed the tumor growth in xenograft models. In the case of melanoma cells, the administration of recombinant AGM protein suppresses tumor cell growth by inducing apoptosis both in vivo and in vitro.[23, 24] Thus, AGM might play positive and negative roles in tumor progression depending on some unknown conditions.
As past studies have not clearly shown the distribution of AGM in human cancer tissues, here we again analyzed expression and distribution of AGM in human cancer tissues of the lung, colon and uterus by immunohistochemistry. We found that AGM was overexpressed in not only the vasculature but also stromal fibroblasts of the cancers. The biological activity of AGM towards human fibroblasts was also investigated in vitro.
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In the present study, we found that AGM is overexpressed in the stroma as well as the vasculature surrounding tumor cells in human cancer tissues. In vitro analysis indicated that TGF-β1 might be an important inducer of AGM in human fibroblasts. AGM significantly stimulated the growth of human normal fibroblasts and their fibronectin production in vitro. In addition, AGM weakly stimulated the expression of α-SMA, a representative marker of myofibroblasts, and promoted the migration of fibroblasts. It is well known that TGF-β1 plays a critical role in the activation of fibroblasts in the tumor stroma. TGF-β1 strongly stimulates fibroblasts to express α-SMA and ECM proteins such as fibronectin and type I collagen.[27-29] Indeed, in this study the activities to induce α-SMA and fibronectin were obviously much higher with TGF-β1 than AGM. However, their growth activities towards fibroblasts were contrasted: TGF-β1 significantly suppressed the cell growth, whereas AGM stimulated it. Therefore, it is likely that TGF-β1 plays a major role in the activation and ECM production of fibroblasts, while AGM plays a specific role in the fibroblast proliferation. Thus, it is expected that AGM and TGF-β1 cooperatively or complementarily contribute to the stromal activation and connective tissue formation in cancer tissues. It should also be noted that the effective concentration of AGM was about 1000-times higher than that of TGF-β1. Our sandwich ELISA analysis has shown that the AGM concentration is approximately 30 ng/mL in normal human serum and exceeded 200 ng/mL in the culture medium of a cancer cell line highly expressing AGM (Kayano Moriyama and Kaoru Miyazaki, unpublished data, 2005). AGM is an extracellular matrix protein rather than a cytokine. Just like fibronectin and laminins, AGM exerted its biological activities at concentrations ranging 1–10 μg/mL. As shown in this study, it is highly deposited on vascular basement membrane and stromal tissues near cancer cells. Therefore, the apparently high concentrations of AGM seem to be pathologically relevant in some tumor microenvironments.
The activity of TGF-β1 is mainly mediated by the Smad signaling. Unexpectedly, the Smad signal inhibitor SB431542 inhibited the α-SMA and fibronectin expression induced not only by TGF-β1 but also by AGM. On the other hand, this inhibitor significantly enhanced the growth-simulating activity of AGM on fibroblasts. Thus, AGM activities are separated into the TGF-β-independent and TGF-β-like activities. The latter activity may be mediated by TGF-β1 or related factors. We could not detect TGF-β1 in the AGM preparation as analyzed by immunoblotting (data not shown). It is possible that AGM enhances the activity of the endogenous TGF-β1 produced by fibroblasts, leading to the elevated expression of α-SMA and fibronectin. The growth-stimulatory effect of the Smad inhibitor suggests that AGM-induced endogenous TGF-β1 has growth-inhibitory activity on fibroblasts. There are many possible mechanisms for the effect of AGM on TGF-β action, for example, stimulation of TGF-β transcription, activation of the latent TGF-β protein, promotion of the TGF-β binding to the receptor, and co-stimulation of TGF-β signaling. Moreover, we cannot yet exclude the possibility that our AGM preparation contained a trace amount of TGF-β bound to AGM. Further studies are required for clarifying these possibilities. We have reported that AGM interacts with syndecan-1 on cell surface. In this study, we were unable to identify the receptor and cell signaling that are involved in the growth-stimulatory activity of AGM.
Our early study on AGM showed that it stimulates the growth of mouse fibroblasts. Recent studies by other groups suggested that AGM/IGFBP-rP1 might be involved in liver fibrosis.[30, 31] These studies also show that AGM plays some roles in connective tissue formation. Similarly, there are reports showing that some other IGFBP super families such as IGFBP-3, IGFBP-5 and CTGF (IGFBP-rP2) are expressed in and contribute to pathological fibrosis.[30, 32-34] In addition, CTGF has been shown to promote transdifferentiation of mesenchymal stem cells to fibroblasts. Therefore, the connective tissue formation may be a common function of IGFBP-related proteins. However, no previous studies have reported overexpression of AGM in CAFs. The enhanced proliferation of fibroblasts and accumulation of ECM in tumor stroma, i.e. desmoplasia, is known to be a typical feature of solid tumors. The reactive stroma is also a key feature in some pathological conditions such as fibrosis, inflammation and wound healing. In such reactive stroma, activated fibroblasts, i.e. myofibroblasts, secrete various cytokines and acquire the capabilities of migration, proliferation and contraction.[3, 36] The mutual interaction between cancer cells and myofibroblasts through cell–cell interaction and secreted proteins is essential for cancer invasion and causes a poor clinical outcome.[37, 38] For example, tumor-derived fibroblasts stimulate tumor cell growth in coculture experiments. When colon cancer cells are cocultured with TGF-β-treated fibroblasts, the cancer cells acquire invasive potential within collagen gel. Similarly, animal experiments demonstrated that CAFs or other types of fibroblast enhance the efficiency of tumor growth when co-injected with tumor cells.[39, 41, 42] However, it is unclear in these studies what factors in the activated fibroblasts are responsible for the enhanced invasive growth of tumor cells. Because the number of cancer specimens analyzed in this study was very low, the relationship between the AGM expression in CAFs and its clinical output in patients is unknown. Based on the facts found in many past studies, it is supposed that AGM expressed in CAFs activates the fibroblasts by an autocrine mechanism and contribute to tumor progression.
The present study also showed the elevated expression of AGM in vasculature in all cases and in tumor cells of some cancer tissues. Past studies have suggested that AGM in blood vessels may be related to elevated vascular permeability[10, 43] and angiogenesis. On the other hand, a considerable number of studies have suggested the tumor-suppressive activity of AGM.[15-23] In this regard, it is noted that in the present histochemical analysis, AGM expression was often found in invading carcinoma cells but not normal epithelial cells. It is unknown whether AGM expressed in invading tumor cells has positive or negative activity for tumor growth. AGM is known to have post-transcriptional modifications such as proteolytic cleavage and glycosylation. Such modifications, as well as differences in AGM-expressing cells, may explain the two opposite effects of AGM on tumor cells in future studies.