Supplementary Material and Methods

Supplementary Figures S1 - S9 Table S1 and S2

The emerging cytokine tissue inhibitor of metalloproteinases-1 (TIMP-1) correlates with the progression of inflammatory diseases, including cancer. However, the effects of TIMP-1 on immune cell activation and underlying molecular mechanisms are largely unknown. Unbiased ligand-receptor-capture-screening revealed TIMP-1-interaction with Amyloid Precursor Protein (APP) family members, namely APP and Amyloid Precursor-like Protein-2 (APLP2), which was confirmed by pull-down assays and confocal microscopy. We found that TIMP-1 triggered glucose uptake and proinflammatory cytokine expression in human monocytes. In cancer patients, TIMP-1 expression positively correlated with proinflammatory cytokine expression and processes associated with monocyte activation. In pancreatic cancer, TIMP-1 plasma levels correlated with the monocyte activation marker sCD163, and the combined use of both clinically accessible plasma proteins served as a powerful prognostic indicator. Mechanistically, TIMP-1 triggered monocyte activation by its C-terminal domain and via APP as demonstrated by in vitro interference, in silico docking, and the employment of recombinant TIMP-1 variants. Identification of TIMP-1 as a trigger of monocyte activation opens new therapeutic perspectives for inflammatory diseases.

Multifunctionality of tissue inhibitor of metalloproteinases-1 (TIMP-1) comprising antiproteolytic as well as cytokinic activity has been attributed to its N-terminal and C-terminal domains, respectively. The molecular basis of the emerging proinflammatory cytokinic activity of TIMP-1 is still not completely understood. The cytokine receptor invariant chain (CD74) is involved in many inflammation-associated diseases and is highly expressed by immune cells. CD74 triggers zeta chain-associated protein kinase-70 (ZAP-70) signaling-associated activation upon interaction with its only known ligand, the macrophage migration inhibitory factor. Here, we demonstrate TIMP-1-CD74 interaction by coimmunoprecipitation and confocal microscopy in cells engineered to overexpress CD74. In silico docking in HADDOCK predicted regions of the N-terminal domain of TIMP-1 (N-TIMP-1) to interact with CD74. This was experimentally confirmed by confocal microscopy demonstrating that recombinant N-TIMP-1 lacking the entire C-terminal domain was sufficient to bind CD74. Interaction of TIMP-1 with endogenously expressed CD74 was demonstrated in the Namalwa B lymphoma cell line by dot blot binding assays as well as confocal microscopy. Functionally, we demonstrated that TIMP-1-CD74 interaction triggered intracellular ZAP-70 activation. N-TIMP-1 was sufficient to induce ZAP-70 activation and interference with the cytokine-binding site of CD74 using a synthetic peptide-abrogated TIMP-1-mediated ZAP-70 activation. Altogether, we here identified CD74 as a receptor and mediator of cytokinic TIMP-1 activity and revealed TIMP-1 as moonlighting protein harboring both cytokinic and antiproteolytic activity within its N-terminal domain. Recognition of this functional TIMP-1-CD74 interaction may shed new light on clinical attempts to therapeutically target ligand-induced CD74 activity in cancer and other inflammatory diseases.

AbstractTumor-derived protein tissue inhibitor of metalloproteinases-1 (TIMP1) correlates with poor prognosis in many cancers, including highly lethal pancreatic ductal adenocarcinoma (PDAC). The noncanonical signaling activity of TIMP1 is emerging as one basis for its contribution to cancer progression. However, TIMP1–triggered progression-related biological processes are largely unknown. Formation of neutrophil extracellular traps (NET) in the tumor microenvironment is known to drive progression of PDAC, but factors or molecular mechanisms initiating NET formation in PDAC remain elusive. In this study, gene-set enrichment analysis of a human PDAC proteome dataset revealed that TIMP1 protein expression most prominently correlates with neutrophil activation in patient-derived tumor tissues. TIMP1 directly triggered formation of NETs in primary human neutrophils, which was dependent on the interaction of TIMP1 with its receptor CD63 and subsequent ERK signaling. In genetically engineered PDAC-bearing mice, TIMP1 significantly contributed to NET formation in tumors, and abrogation of TIMP1 or NETs prolonged survival. In patient-derived PDAC tumors, NETs predominantly colocalized with areas of elevated TIMP1 expression. Furthermore, TIMP1 plasma levels correlated with DNA-bound myeloperoxidase, a NET marker, in the blood of patients with PDAC. A combination of plasma levels of TIMP1 and NETs with the clinically established marker CA19–9 allowed improved identification of prognostically distinct PDAC patient subgroups. These observations may have a broader impact, because elevated systemic levels of TIMP1 are associated with the progression of a wide range of neutrophil-involved inflammatory diseases.Significance:These findings highlight the prognostic relevance of TIMP1 and neutrophil extracellular traps in highly lethal pancreatic cancer, where a noncanonical TIMP1/CD63/ERK signaling axis induces NET formation.

Sex disparity in cancer is so far inadequately considered, and components of its basis are rather unknown. We reveal that male versus female pancreatic cancer (PC) patients and mice show shortened survival, more frequent liver metastasis, and elevated hepatic metastasis-promoting gene expression. Tissue inhibitor of metalloproteinases 1 (TIMP1) was the secreted factor with the strongest male-biased expression in patient-derived pancreatic tumors. Male-specific up-regulation of systemic TIMP1 was demonstrated in PC mouse models and patients. Using TIMP1-competent and TIMP1-deficient PC mouse models, we established a causal role of TIMP1 in determining shortened survival and increased liver metastasis in males. Observing TIMP1 expression as a risk parameter in males led to identification of a subpopulation exhibiting increased TIMP1 levels (T1HI males) in both primary tumors and blood. T1HI males showed increased risk for liver metastasis development not only in PC but also in colorectal cancer and melanoma. This study reveals a lifestyle-independent sex disparity in liver metastasis and may open new avenues toward precision medicine.

The members of the tissue inhibitor of metalloproteinase (TIMP) family (TIMP-1, 2, 3, 4) are prominently appreciated as natural inhibitors of cancer-promoting metalloproteinases. However, clinical and recent functional studies indicate that some of them correlate with bad prognosis and contribute to the progression of cancer and metastasis, pointing towards mechanisms beyond inhibition of cancer-promoting proteases. Indeed, it is increasingly recognized that TIMPs are multi-functional proteins mediating a variety of cellular effects including direct cell signaling. Our aim was to provide comprehensive information towards a better appreciation and understanding of the biological heterogeneity and complexity of the TIMPs in cancer. Comparison of all four members revealed distinct cancer-associated expression patterns and distinct prognostic impact including a clear correlation of TIMP-1 with bad prognosis for almost all cancer types. For the first time, we present the interactomes of all TIMPs regarding overlapping and non-overlapping interaction partners. Interestingly, the overlap was maximal for metalloproteinases (e.g., matrix metalloproteinase 1, 2, 3, 9) and decreased for non-protease molecules, especially cell surface receptors (e.g., CD63, overlapping only for TIMP-1 and 4; IGF-1R unique for TIMP-2; VEGFR2 unique for TIMP-3). Finally, we attempted to identify and summarize experimental evidence for common and unique structural traits of the four TIMPs on the basis of amino acid sequence and protein folding, which account for functional disparities. Altogether, the four TIMPs have to be appreciated as molecules with commonalities, but, more importantly, functional disparities, which need to be investigated further in the future, since those determine their distinct roles in cancer and metastasis.