Tissue inhibitor of metalloproteinase 1 (TIMP-1) is a major player in preserving tissue integrity and has recently also emerged as a decisive factor in several human pathologies. This appreciation ...has prompted this review addressing the largely underestimated complexity of the functions executed by TIMP-1 and their mechanistic basis. In fact, the versatile impact of TIMP-1 on cellular functions stems from its two-domain structure harboring metalloproteinase-inhibitory and cytokine-like signaling activities. This feature leads to functional interactions with numerous and distinct enzymatic and cell-surface proteins that initiate an exceptionally broad range of downstream effects. We propose here that this multifunctionality and the remarkably large interactome explain the diverse biological consequences of TIMP-1 expression in health and disease.
TIMP-1 controls major aspects of cell biology, and its deregulation contributes to various diseases such as inflammation, fibrosis, and cancer. However, functional misconceptions still hamper our understanding of its precise molecular role in complex contexts.
TIMP-1 is a two-domain protein with multiple functions, and the list of its interaction partners has rapidly expanded in recent years. This review develops the first integrated view of multifunctionality as an important biological feature of TIMP-1.
We propose that TIMP-1 is a regulatory hub in an exceptionally large protein-interaction network, and highlight several highly context-specific regulatory mechanisms that can affect the molecular mode of action of TIMP-1.
Appreciation of the interrelated functions of TIMP-1 as a protease inhibitor and signaling molecule facilitates integration of previously contradictory findings. This may aid the development of precise TIMP-1-directed therapeutic strategies and serve as a role model for other multifunctional proteins and their impact on cell biology.
Background
Cachexia, a devastating syndrome in cancer patients, critically determines survival and life quality. It is characterized by impaired homeostasis of multiple organs including the liver, ...involves tissue wasting, and is conventionally diagnosed and classified by weight loss (WL). However, recent studies pointed at the problem that WL is not sufficient for precise classification of cancer patients according to disease severity (i.e. prognosis). Tissue inhibitor of metalloproteinases‐1 (TIMP‐1) is an easily accessible cachexia‐associated biomarker in the blood, known to alter liver homeostasis. Here, we investigated the value of combining blood levels of TIMP‐1 with parameters of liver functionality towards establishment of a cachexia‐associated clinical score, which predicts survival of cancer patients, reflects the clinical manifestation of cachexia, and is easily accessible in the clinic.
Methods
The TIMP‐1/liver cachexia (TLC) score, expressed as numerical value ranging from 0 to 1, was calculated by categorizing the blood levels of TIMP‐1 and parameters of liver functionality (C‐reactive protein, ferritin, gamma‐glutamyl transferase, albumin, and total protein) for each patient as below/above a certain risk threshold. The TLC score was tested in a cohort of colorectal cancer (CRC) patients (n = 82, 35.4% women, 64.6% men, median age: 70 years) and validated in a cohort of pancreatic cancer (PC) patients (n = 84, 54.8% women, 45.2% men, median age: 69 years).
Results
In CRC patients, the TLC score positively correlated with presence of cachexia‐related symptoms (WL, impaired liver function), predicted survival P < 0.001, hazard ratio (HR): 96.91 (9.85–953.90), and allowed classification of three prognostically distinct patient subpopulations low (LO)‐risk, intermediate (IM)‐risk, and high (HI)‐risk groups; LO vs. IM: P = 0.003, LO vs. HI: P < 0.001, IM vs. HI: P = 0.029. The prognostic power of the cachexia‐associated TLC score P < 0.001, HR: 7.37 (2.80–19.49) and its application to define risk groups (LO vs. IM: P = 0.032, LO vs. HI: P < 0.001, IM vs. HI: P = 0.014) was confirmed in a cohort of PC patients. The prognostic power of the TLC score was independent of presence of liver metastases in CRC or PC patients and was superior to clinically established staging classifications.
Conclusions
The TLC score, a result of straightforward determination of blood parameters, is an objective cachexia‐associated clinical tool for precise survival prediction of gastrointestinal cancer patients.
Appreciation of the entire biological impact of an individual protein can be hampered by its original naming based on one function only. Tissue inhibitor of metalloproteinases-1 (TIMP-1), mostly ...known for its eponymous function to inhibit metalloproteinases, exhibits only a fraction of its cellular effects via this feature. Recently, TIMP-1 emerged as a potent cytokine acting via various cell-surface receptors, explaining a so-far under-appreciated role of TIMP-1-mediated signaling on immune cells. This, at least partly, resolved why elevated blood levels of TIMP-1 correlate with progression of numerous inflammatory diseases. Here, we emphasize the necessity of unbiased name-independent recognition of structure-function relationships to properly appreciate the biological potential of TIMP-1 and other cytokines in complex physiological processes such as inflammation.
The functional diversity of living cells is significantly determined by protein multifunctionality. This multifunctionality is based mostly on differential interaction of a given protein with different molecules, such as proteins or DNA, triggering very different downstream processes.Original naming of proteins is based mostly on one function only, and can obscure the appreciation of protein multifunctionality in the scientific community, for which tissue inhibitor of metalloproteinases-1 (TIMP-1) is a perfect example.From clinical evidence, TIMP-1 emerges as an important regulator of inflammatory processes.TIMP-1 is detected, at elevated levels, in the blood of patients with an exceptionally wide range of inflammation-associated diseases.Another interesting link of TIMP-1 with the immune system recently became evident when invariant chain (CD74) was discovered as a new signaling-inducing receptor of TIMP-1.
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.
Abstract
Tumor-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.
Tumor cells exhibiting the Warburg effect rely on aerobic glycolysis for ATP production and have a notable addiction to anaplerotic use of glutamine for macromolecular synthesis. This strategy ...maximizes cellular biosynthetic potential while avoiding excessive depletion of NAD
and provides an attractive anabolic environment for viral infection. Here, we evaluate infection of highly permissive and poorly permissive cancer cells with wild-type adenoviruses and the oncolytic chimeric adenovirus enadenotucirev (EnAd). All adenoviruses caused an increase in glucose and glutamine uptake along with increased lactic acid secretion. Counterintuitively, restricting glycolysis using 2-deoxyglucose or by limiting glucose supply strongly improved virus activity in both cell types. Antagonism of glycolysis also boosted EnAd replication and transgene expression within human tumor biopsies and in xenografted tumors
. In contrast, the virus life cycle was critically dependent on exogenous glutamine. Virus activity in glutamine-free cells was rescued with exogenous membrane-permeable α-ketoglutarate, but not pyruvate or oxaloacetate, suggesting an important role for reductive carboxylation in glutamine usage, perhaps for production of biosynthetic intermediates. This overlap between the metabolic phenotypes of adenovirus infection and transformed tumor cells may provide insight into how oncolytic adenoviruses exploit metabolic transformation to augment their selectivity for cancer cells. SIGNIFICANCE: This study describes changes in glucose and glutamine metabolism induced by oncolytic and wild-type adenoviruses in cancer cells, which will be important to consider in the preclinical evaluation of oncolytic viruses.
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.
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 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.
Cells in pancreatic ductal adenocarcinoma (PDAC) undergo autophagy, but its effects vary with tumor stage and genetic factors. We investigated the consequences of varying levels of the autophagy ...related 5 (Atg5) protein on pancreatic tumor formation and progression.
We generated mice that express oncogenic Kras in primary pancreatic cancer cells and have homozygous disruption of Atg5 (A5;Kras) or heterozygous disruption of Atg5 (A5+/–;Kras), and compared them with mice with only oncogenic Kras (controls). Pancreata were analyzed by histology and immunohistochemistry. Primary tumor cells were isolated and used to perform transcriptome, metabolome, intracellular calcium, extracellular cathepsin activity, and cell migration and invasion analyses. The cells were injected into wild-type littermates, and orthotopic tumor growth and metastasis were monitored. Atg5 was knocked down in pancreatic cancer cell lines using small hairpin RNAs; cell migration and invasion were measured, and cells were injected into wild-type littermates. PDAC samples were obtained from independent cohorts of patients and protein levels were measured on immunoblot and immunohistochemistry; we tested the correlation of protein levels with metastasis and patient survival times.
A5+/–;Kras mice, with reduced Atg5 levels, developed more tumors and metastases, than control mice, whereas A5;Kras mice did not develop any tumors. Cultured A5+/–;Kras primary tumor cells were resistant to induction and inhibition of autophagy, had altered mitochondrial morphology, compromised mitochondrial function, changes in intracellular Ca2+ oscillations, and increased activity of extracellular cathepsin L and D. The tumors that formed in A5+/–;Kras mice contained greater numbers of type 2 macrophages than control mice, and primary A5+/–;Kras tumor cells had up-regulated expression of cytokines that regulate macrophage chemoattraction and differentiation into M2 macrophage. Knockdown of Atg5 in pancreatic cancer cell lines increased their migratory and invasive capabilities, and formation of metastases following injection into mice. In human PDAC samples, lower levels of ATG5 associated with tumor metastasis and shorter survival time.
In mice that express oncogenic Kras in pancreatic cells, heterozygous disruption of Atg5 and reduced protein levels promotes tumor development, whereas homozygous disruption of Atg5 blocks tumorigenesis. Therapeutic strategies to alter autophagy in PDAC should consider the effects of ATG5 levels to avoid the expansion of resistant and highly aggressive cells.
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