During tumor progression, malignant cells must repeatedly survive microenvironmental stress. Hypoxia-inducible factor-1 (HIF-1) signaling has emerged as one major pathway allowing cellular adaptation to stress. Recent findings led to the hypothesis that HIF-1␣ may enhance the metastatic potential of tumor cells by a survival-independent mechanism. So far it has not been shown that HIF-1␣ also directly regulates invasive processes during metastasis in addition to conferring a survival advantage to metastasizing tumor cells. In a hypoxia-tolerant tumor cell line (L-CI.5s), which did not rely on HIF-1 signaling for viability in vitro and in vivo, knockdown of Hif-1␣ reduced invasiveness of the tumor cells in vitro as well as extravasation and secondary infiltration in vivo. Liver metastases associated induction of proinvasive receptor tyrosine kinase Met phosphorylation as well as gelatinolytic activity were Hif-1␣-dependent. Indeed, promoter activity of the matrix metalloproteinase-9 (mmp-9) was shown to be Hif-1␣-dependent. This study uncovers a new survival-independent biological function of HIF-1␣ contributing to the efficacy of metastases formation.During the metastatic cascade, tumor cells encounter several kinds of microenvironmental stress, namely lack of oxygen and nutrients (1). Hypoxia-inducible factor-1 (HIF-1) 2 is a transcription factor known to mediate the adaptation to microenvironmental stress in general (2) as well as during tumor progression in particular (1). HIF-1 consists of a constitutively expressed ␤-subunit and a highly regulated ␣-subunit that is, under physiological conditions, degraded by the proteasome (3, 4). During stress situations such as hypoxia, HIF-1␣ is stabilized and translocates to the nucleus where it forms together with HIF-1␤ the heterodimeric transcription factor HIF-1 (5). Via its interaction with hypoxia-responsive elements, HIF-1 regulates the expression of molecules such as the major pH-regulating enzyme carbonic anhydrase IX (CAIX) (6), which allows the metabolic adaptation on the cellular level (7,8). Furthermore, HIF-1 signaling is also a major determinant of adaptation on the tissue level by induction of the "angiogenic switch," thereby overcoming the limited supply of oxygen and nutrients in expanding neoplasias (1).Recent findings led us to the hypothesis that HIF-1␣ not only impacts on metastasis formation by securing survival but also directly impacts on metastasis in a survival-independent manner. This hypothesis was based on the following findings. Reduced levels of HIF-1␣ correlate with decreased invasive potential of tumor cells in vitro (9 -11). HIF-1␣ knock-out reduced primary tumor onset and growth in a transgenic model of cancer initiation (12). This correlated with a later onset of pulmonary metastasis (12). Furthermore, constitutive expression of HIF-1␣ has been shown to enhance bone metastasis in a breast cancer model (13). Although these data have suggested that HIF-1␣ increases the metastatic potential of tumor cells, recent reviews by Bertout et ...

The "protease web", representing the network of proteases, their inhibitors, and effector molecules, arises as a pivotal determinant of tissue homeostasis. Imbalances of this network, for instance caused by elevated host levels of tissue inhibitor of metalloproteinases-1 (TIMP-1), have been shown to increase the susceptibility of target organs to scattered metastasis by inducing the hepatocyte growth factor (HGF) pathway. Increased expression of the hypoxia-inducible factor-1α-subunit (HIF-1α) is also associated with tumour progression and is also known to induce HGF-signaling via up-regulation of the HGF-receptor Met, namely under canonical stress conditions like lack of oxygen. Here, we aimed to identify a possible metastasis-promoting connection between TIMP-1, HIF-1α, and HGF-signaling. We found that HIF-1α and HIF-1-signaling were increased during liver metastasis of L-CI.5s T-lymphoma cells in TIMP-1 overexpressing syngeneic DBA/2 mice. In vitro, exposure of L-CI.5s cells to recombinant TIMP-1 revealed that TIMP-1 itself was able to induce HIF-1α and HIF-1-signaling. Knock-down of HIF-1α identified tumour cell-derived HIF-1α as mediator of this TIMP-1-induced invasiveness in vitro. In vivo, HIF-1α knock-down significantly impaired Met expression as well as Met phosphorylation and inhibited scattered liver metastasis. Furthermore, HGF-dependent TIMP-1-promoted Met phosphorylation and HGF-dependent TIMP-1-induced invasiveness in vitro was mediated by HIF-1α. We conclude that elevated levels of TIMP-1 in the microenvironment of tumour cells can promote metastasis by inducing HIF-1α-dependent HGF-signaling. This connection between a protease inhibitor (TIMP-1) and a classically stress-related factor (HIF-1α) is a so far undiscovered impact of the "protease web" on tissue homeostasis with important implications for metastasis.

Paradoxically, not only proteinases but also their inhibitors can correlate with bad prognosis of cancer patients, underlining the evolving concept of the protease web as the complex interplay between proteinases, their inhibitors and effector molecules. Elevated levels of tissue inhibitor of metalloproteinases-1 (TIMP-1) render the liver more susceptible to metastasis by triggering urokinase plasminogen activator (uPA) expression as well as hepatocyte growth factor (HGF) signalling, thereby leading to the fatal scattered infiltration of metastasizing tumour cells throughout the parenchyma of the target organ. Here, we investigated whether host uPA is a crucial protagonist for the TIMP-1-induced modulation of a pro-metastatic microenvironment in the liver. Indeed, in livers of uPA-ablated mice elevated TIMP-1 levels did not trigger HGF signalling and did not promote metastasis of a murine T-lymphoma cell line. In contrast, lack of tumour cell-derived uPA induced by gene silencing did not interfere with this pro-metastatic pathway. Furthermore, host uPA was necessary for the recruitment of neutrophilic granulocytes and the associated increase of HGF in livers with elevated TIMP-1 levels. This newly identified co-operation between TIMP-1 and host uPA suggests that therapies, simultaneously interfering with pro- and anti-proteolytic pathways may be beneficial for patients with metastatic disease.