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Timp3 is commonly silenced in breast cancer, but mechanistic studies have identified both tumor promotion and suppression effects of this gene. We have taken a genetic approach to determine the impact of Timp3 loss on two mouse models of breast cancer. Interestingly, MMTV-PyMT Timp3-⁄- mice have delayed tumor onset and 36% of MMTV-Neu Timp3-⁄- mice remain tumor free. TIMP3 is a regulator of TNF signaling and similar to Timp3, Tnf or Tnfr1 loss delays early tumorigenesis. The tumor suppression in Timp3 null mice requires Tnfr1, but does not result in alterations in the local immune compartment. In the mammary gland, Timps are highly expressed in the stroma and through the transplantation of tumor cells we observe that Timp3 deficiency in the host is sufficient to delay the growth of early, but not advanced tumor cells. Together our data is the first to identify a tumor promoting role of endogenous Timp3 in vivo, the spatial and temporal windows of this effect, and its dependence on Tnfr1.

Identifying versatile inhibitors of metastasis that operate in multiple sites against distinct cancer cell types is important for designing novel therapeutics for metastasis. We show that multiple tissues of timp-3-/- mice are more susceptible to metastatic colonization. Overall, a 5-14-fold increase in liver and kidney colonization occurred by EL-4 lymphoma cells, and a twofold increase upon targeting B16F10 melanoma cells to the bone or lung of timp-3-/- mice. There was a general lack of macrophage or neutrophil localization to metastases in the liver, kidney and lung, and of osteoclasts to bone in both genotypes. Analysis of lung showed that proliferation or angiogenesis were unaltered within the metastatic colonies. Lung-trap assays revealed that initial tumor cell trapping was similar in the lung vasculature of timp-3-/- and wild-type mice. However, more tumor cells were found in timp-3-/- lungs at 48 and 96 h after tumor cell injection indicating more efficient extravasation and initial proliferation. Activation of pro-MMP-2 was greater in timp-3-/- lungs at these time points. These data demonstrate TIMP-3 functions to inhibit metastatic dissemination of diverse cancer cells to multiple organs. TIMP-3 regulates MMP-2 activation to limit tumor cell extravasation and subsequent colonization of the lung, without augmenting inflammatory cell response.

Bone metastases are a frequent complication of many cancers that result in severe disease burden and pain. Since the late nineteenth century, it has been thought that the microenvironment of the local host tissue actively participates in the propensity of certain cancers to metastasize to specific organs, and that bone provides an especially fertile 'soil'. In the case of breast cancers, the local chemokine milieu is now emerging as an explanation for why these tumours preferentially metastasize to certain organs. However, as the inhibition of chemokine receptors in vivo only partially blocks metastatic behaviour, other factors must exist that regulate the preferential metastasis of breast cancer cells. Here we show that the cytokine RANKL (receptor activator of NF-kappaB ligand) triggers migration of human epithelial cancer cells and melanoma cells that express the receptor RANK. RANK is expressed on cancer cell lines and breast cancer cells in patients. In a mouse model of melanoma metastasis, in vivo neutralization of RANKL by osteoprotegerin results in complete protection from paralysis and a marked reduction in tumour burden in bones but not in other organs. Our data show that local differentiation factors such as RANKL have an important role in cell migration and the tissue-specific metastatic behaviour of cancer cells.

Tumor-necrosis factor (TNF), a pleiotropic cytokine, triggers physiological and pathological responses in several organs. Here we show that deletion of the mouse gene Timp3 resulted in an increase in TNF-alpha converting enzyme activity, constitutive release of TNF and activation of TNF signaling in the liver. The increase in TNF in Timp3(-/-) mice culminated in hepatic lymphocyte infiltration and necrosis, features that are also seen in chronic active hepatitis in humans. This pathology was prevented when deletion of Timp3 was combined with Tnfrsf1a deficiency. In a liver regeneration model that requires TNF signaling, Timp3(-/-) mice succumbed to liver failure. Hepatocytes from Timp3(-/-) mice completed the cell cycle but then underwent cell death owing to sustained activation of TNF. This hepatocyte cell death was completely rescued by a neutralizing antibody to TNF. Dysregulation of TNF occurred specifically in Timp3(-/-), and not Timp1(-/-) mice. These data indicate that TIMP3 is a crucial innate negative regulator of TNF in both tissue homeostasis and tissue response to injury.

A myriad of novel mediators in neoplastic development and progression are currently being explored. Of significance are those that directly explain clinical manifestations of cancer, because understanding these may lead to new diagnostic, preventive, and therapeutic strategies. This review focuses on novel mediators that address how cancer, before it is treated, can induce cachexia, pain, hematological, and immune alterations. It highlights two concepts: first, that a synergy between tumor and stromal cells may be partly responsible for these manifestations, and second, that soluble factors, and in particular cytokines are being identified as major players in tumor-induced local and systemic effects.

Elevated levels of insulin-like growth factor (IGF)-II are associated with a poor prognosis in human pulmonary adenocarcinoma; however, a causal role for IGF-II in pulmonary adenocarcinoma has not been demonstrated. Here, we show that transgenic overexpression of IGF-II in lung epithelium induces lung tumors in 69% of mice older than 18 months of age. These tumors displayed morphological characteristics of human pulmonary adenocarcinoma such as their epithelial origin, tubulo-acinar architecture and expression of TTF-1, SP-B and proSP-C. Examination of signaling molecules downstream of the IGF-IR showed the activation of either the Erk1/Erk2 or p38 MAPK pathways, but not both, within the lung tumors. Notably, all lung tumors contained high levels of phosphorylated CREB, suggesting that both the Erk1/Erk2 and p38 MAPK pathways converged on this transcription factor. Moreover, IGF-II induced proliferation and CREB phosphorylation in human lung cancer cell lines, suggesting that IGF-II and CREB also contribute to the growth of human lung tumors. Thus, IGF-II is an important genetic factor in the development of lung tumorigenesis, in which activation of CREB is a ubiquitous event. The MMTV-IGF-II transgenic mice provide a critical model for elucidating the role of IGF-II in this fatal human disease.

Tissue inhibitors of metalloproteinases regulate ECM degradation by matrix metalloproteinases (MMPs). We have developed a mouse line deficient for tissue inhibitor of metalloproteinases-3 (TIMP-3), the only TIMP known to reside within the ECM. Homozygous Timp-3-null animals develop spontaneous air space enlargement in the lung that is evident at 2 weeks after birth and progresses with age of the animal. As early as 13 months of age animals become moribund. Lung function, measured by carbon monoxide uptake, is impaired in aged null animals. Lungs from aged null animals have reduced abundance of collagen, enhanced degradation of collagen in the peribronchiolar space, and disorganization of collagen fibrils in the alveolar interstitium, but no increase in inflammatory cell infiltration or evidence of fibrosis in comparison with controls. Using in situ zymography, we show that lungs from aged null animals have heightened MMP activity over wild-type and heterozygotic animals. Finally, TIMP-3-null fibroblast cultures demonstrate enhanced destruction of ECM molecules in vitro. We propose that the deletion of TIMP-3 results in a shift of the TIMP/MMP balance in the lung to favor ECM degradation, culminating in incapacitating illness and a shorter life span.