Basement collagens in their native molecular structure form a scaffold for cell homeostasis. In this study, we have demonstrated that tumstatin, a BM Col IVα3 chain-derived NC1 domain, can be detected in the circulation of normal mice and functions as an endogenous regulator of pathologic angiogenesis and that the activity of tumstatin is dependent on αVβ3 integrin and the ECM-degrading enzyme, MMP-9.
The Col IVα3 chain is localized to many VBM and is most abundant in the GBM of the kidney and the BM of the testis (Frojdman et al., 1998
; Hudson et al., 1993
; Kahsai et al., 1997
). It is likely that physiological turnover of GBM and other BM mediated by ECM-degrading MMPs contributes to the circulating physiological levels of tumstatin. Col IVα3 chain-deficient mice do not have circulating tumstatin, and syngeneic LLC tumors larger than 500 mm3
on these mice grew at more than twice the rate of tumors on wild-type mice. This difference in growth could be restored to wild-type levels when the null mice were provided with physiological concentrations of tumstatin in recombinant form. These results suggest that the acceleration of tumor growth in the null mice is not due to the absence of α3 (IV) chain, but is likely due the lack of the tumstatin domain. Our study distinguishes the physiological role of tumstatin from its potential pharmacological applications. Providing tumstatin (300 ng/day, i.v.) to the null mice slowed the tumor growth to the wild-type level, but did not cause regression or inhibition of tumor growth. The pharmacological doses needed to achieve inhibition of LLC tumor growth are much higher, in the range of 5–15 μg/day, i.v. (data not shown). Further studies are required to understand what mechanisms drive the switch between the physiological and pharmacological functions of tumstatin.
An interesting insight into the physiological role of tumstatin in the suppression of tumstatin can be made from the observation that, until the tumors reach 500 mm3
, the difference in tumor growth between the wild-type mice and Col IVα3/tumstatin- deficient mice is insignificant. Our results suggest that lack of significant vascular expression of β3 integrin until the tumors reach 500 mm3
is a likely explanation for the insignificant difference in the growth of tumor in the absence of tumstatin before tumors reach 500 mm3
. Alternatively, it is possible that during the early growth phase of the tumors, absence of tumstatin can be compensated by other inhibitors of angiogenesis, such as thrombospondin-1, angiostatin, and endostatin (Hawighorst et al., 2001
; O’Reilly et al., 1994
; Rodriguez-Manzaneque et al., 2001
; Volpert et al., 2002
). But once the tumor reaches a critical size and β3 integrin expression increases significantly, the entire antiangiogenic arsenal including tumstatin is required for the control of tumor growth.
We previously implicated αVβ3 integrin as a putative receptor for tumstatin and the binding between them (Maeshima et al., 2000a
) is independent of RGD sequence, vitronectin, and fibronectin binding. In the present study, we used genetic experiments with β3-deficient mice to demonstrate that αVβ3 integrin mediates tumstatin action. Tumors also grow faster in β3-deficient mice than in wild-type mice (Reynolds et al., 2002
). Collectively, these observations strongly suggest a role for αVβ3 integrin as a negative regulator of angiogenesis (Hynes, 2002a
; Hynes et al., 2002b
; Kalluri, 2002
; Maeshima et al., 2001a
Our experiments suggest that tumor angiogenesis, but not angiogenesis associated with the repair of skin wound and regeneration of liver, is associated with robust expression of β3 integrins, including αVβ3 integrin. This is likely the explanation for the lack of difference on the skin wound repair and liver regeneration in the Col IVα3/tumstatin-deficient mice and also in pharmacological studies with recombinant human tumstatin (data not shown). Both Col IVα3/tumstatin-deficient mice and β3 integrin-deficient mice on a C57BL/6 background do not exhibit developmental angiogenesis defects (Andrews et al., 2002
; Cosgrove et al., 1996
; Hodivala-Dilke et al., 1999
), suggesting that β3 integrin and tumstatin may not be essential for the regulation of developmental angiogenesis.
MMP-9 has been implicated in the degradation of type IV collagen present in the BM (Egeblad and Werb, 2002
; McCawley and Matrisian, 2001
), and fragments of Col IV and laminin have been detected in the serum of normal individuals and cancer patients (Gabrielli et al., 1988
; Yudoh et al., 1994
). Tumstatin is a proteolytic product that has to be liberated from the parent Col IVα3 chain. MMP-9 was the most effective enzyme in the generation of tumstatin domain from BM and type IV collagen preparations from the human kidney, testis, and placenta and amnion. We demonstrated that mice deficient in MMP-9 have lower circulating levels of tumstatin, and that this results in increased tumor growth. The accelerated tumor growth can be restored to wild-type levels by supplementing the null mice with the missing physiological concentration of tumstatin. Moreover, Col IV is elevated in the tumor tissue and also in the GBM of MMP-9-deficient mice (data not shown). Such increased accumulation is likely due to reduced Col IV turnover in the mice. These results provide further evidence for the endogenous tumor-suppressive action of tumstatin.
However, MMP-9 has also been implicated as a positive regulator of the angiogenic switch, which leads to early stage tumor differences resulting in lower tumor size and volume (Bergers et al., 2000
). But, in the same mice at a later stage, the tumor size and volume catch up with the wild-type levels, suggesting an acceleration of tumor growth in the absence of MMP-9, as observed in the present study (personal communication, G. Bergers and D. Hanahan). In this regard, Pozzi et al. recently demonstrated that pharmacological inhibition of MMP-9 in mice results in acceleration of tumor growth, further supporting our observation (Pozzi et al., 2000
). Thus, MMP-9 has opposing actions on neoplasia at different stages of tumor progression. While it may mediate the angiogenic switch leading to the initial burst of tumor growth, it also restrains tumor development by generating endogenous inhibitors of angiogenesis such as tumstatin. After the angiogenic switch has been achieved by MMP-9, the degradation products resulting from digestion of BM by the increased amount of MMP-9 negatively influence the growth of tumors. Therefore, the two opposite properties of MMP-9 are integral to the overall progression of tumor growth. In this regard, recent clinical trial failures with MMP inhibitors could be explained by our data, although early intervention of tumor growth by MMP inhibitors is still potentially feasible (Bramhall et al., 2002
What roles do the other physiological angiogenesis inhibitors play? Interestingly, endostatin (type XVIII collagen)-deficient mice have altered retinal vascular development, but do not exhibit accelerated tumor growth (Fukai et al., 2002
). Recent experiments have shown that thrombospondin-1-deficient mice also exhibit increased tumor growth compared to the wild-type mice, as shown here for Col IVα3/tumstatin-deficient mice (Lawler et al., 2001
; Rodriguez-Manzaneque et al., 2001
). Whether the antiangiogenic function of endostatin is compensated by Col XV or other molecules in the type XVIII collagen-deficient mice remains to be determined (Eklund et al., 2001
; Marneros and Olsen, 2001
In conclusion, this study identifies tumstatin as a physiologically functional protein domain that circulates in the blood. Tumstatin is not a gene product by itself, but is produced when MMP-9 cleaves it from Col IV in BM. The physiological level of tumstatin is dependent on MMP-9, and in its absence, the physiological concentration in the blood declines, potentially facilitating pathological angiogenesis and increased growth of tumors. The tumor suppressor activity of tumstatin seen at supraphysiological concentrations is associated with inhibition of protein synthesis, specifically mediated by mTOR, in the endothelial cells (Maeshima et al., 2002
). Based on these results, we propose that endogenous inhibitors like tumstatin function as tumor suppressors, constituting an additional line of defense against tumor growth in the body, similar to tumor suppressor genes such as p53.