Unravelling the distinct biological functions and potential therapeutic applications of TIMP2 in cancer.

Tissue inhibitors of metalloproteinases (TIMPs) are a conserved family of proteins that were originally identified as endogenous inhibitors of matrixin and adamalysin endopeptidase activity. The matrixins and adamalysins are the major mediators of extracellular matrix (ECM) turnover, thus making TIMPs important regulators of ECM structure and composition. Despite their high sequence identity and relative redundancy in inhibitory profiles, each TIMP possesses unique biological characteristics that are independent of their regulation of metalloproteinase activity. As our understanding of TIMP biology has evolved, distinct roles have been assigned to individual TIMPs in cancer progression. In this respect, data regarding TIMP2's role in cancer have borne conflicting reports of both tumor suppressor and, to a lesser extent, tumor promoter functions. TIMP2 is the most abundant TIMP family member, prevalent in normal and diseased mammalian tissues as a constitutively expressed protein. Despite its apparent stable expression, recent work highlights how TIMP2 is a cell stress-induced gene product and that its biological activity can be dictated by extracellular posttranslational modifications. Hence an understanding of TIMP2 molecular targets, and how its biological functions evolve in the progressing tumor microenvironment may reveal new therapeutic opportunities. In this review, we discuss the continually evolving functions of TIMP proteins, future perspectives in TIMP research, and the therapeutic utility of this family, with a particular focus on TIMP2.

Extracellular Proximity Labeling Reveals an Expanded Interactome for the Matrisome Protein TIMP2.

Classical methods of investigating protein-protein interactions (PPIs) are generally performed in non-living systems, yet in recent years new technologies utilizing proximity labeling (PL) have given researchers the tools to explore proximal PPIs in living systems. PL has distinct advantages over traditional protein interactome studies, such as the ability to identify weak and transient interactions in vitro and in vivo. Most PL studies are performed on targets within the cell or on the cell membrane. We have adapted the original PL method to investigate PPIs within the extracellular compartment, using both BioID2 and TurboID, that we term extracellular PL (ePL). To demonstrate the utility of this modified technique, we investigate the interactome of the widely expressed matrisome protein tissue inhibitor of metalloproteinases 2 (TIMP2). Tissue inhibitors of metalloproteinases (TIMPs) are a family of multi-functional proteins that were initially defined by their ability to inhibit the enzymatic activity of metalloproteinases (MPs), the major mediators of extracellular matrix (ECM) breakdown and turnover. TIMP2 exhibits a broad expression profile and is often abundant in both normal and diseased tissues. Understanding the functional transformation of matrisome regulators, like TIMP2, during the evolution of tissue microenvironments associated with disease progression is essential for the development of ECM-targeted therapeutics. Using carboxyl- and amino-terminal fusion proteins of TIMP2 with BioID2 and TurboID, we describe the TIMP2 proximal interactome. We also illustrate how the TIMP2 interactome changes in the presence of different stimuli, in different cell types, in unique culture conditions (2D vs 3D), and with different reaction kinetics (BioID2 vs. TurboID); demonstrating the power of this technique versus classical PPI methods. We propose that the screening of matrisome targets in disease models using ePL will reveal new therapeutic targets for further comprehensive studies.

Whole organism profiling of the Timp gene family.

Tissue inhibitor of metalloproteinases (TIMPs/Timps) are an endogenous family of widely expressed matrisome-associated proteins that were initially identified as inhibitors of matrix metalloproteinase activity (Metzincin family proteases). Consequently, TIMPs are often considered simply as protease inhibitors by many investigators. However, an evolving list of new metalloproteinase-independent functions for TIMP family members suggests that this concept is outdated. These novel TIMP functions include direct agonism/antagonism of multiple transmembrane receptors, as well as functional interactions with matrisome targets. While the family was fully identified over two decades ago, there has yet to be an in-depth study describing the expression of TIMPs in normal tissues of adult mammals. An understanding of the tissues and cell-types that express TIMPs 1 through 4, in both normal and disease states are important to contextualize the growing functional capabilities of TIMP proteins, which are often dismissed as non-canonical. Using publicly available single cell RNA sequencing data from the Tabula Muris Consortium, we analyzed approximately 100,000 murine cells across eighteen tissues from non-diseased organs, representing seventy-three annotated cell types, to define the diversity in Timp gene expression across healthy tissues. We describe the unique expression profiles across tissues and organ-specific cell types that all four Timp genes display. Within annotated cell-types, we identify clear and discrete cluster-specific patterns of Timp expression, particularly in cells of stromal and endothelial origins. RNA in-situ hybridization across four organs expands on the scRNA sequencing analysis, revealing novel compartments associated with individual Timp expression. These analyses emphasize a need for specific studies investigating the functional significance of Timp expression in the identified tissues and cell sub-types. This understanding of the tissues, specific cell types and microenvironment conditions in which Timp genes are expressed adds important physiological context to the growing array of novel functions for TIMP proteins.

Tissue inhibitors of metalloproteinases are proteolytic targets of matrix metalloproteinase 9.

Extracellular proteolysis and turnover are core processes of tissue homeostasis. The predominant matrix-degrading enzymes are members of the Matrix Metalloproteinase (MMP) family. MMPs extensively degrade core matrix components in addition to processing a range of other factors in the extracellular, plasma membrane, and intracellular compartments. The proteolytic activity of MMPs is modulated by the Tissue Inhibitors of Metalloproteinases (TIMPs), a family of four multi-functional matrisome proteins with extensively characterized MMP inhibitory functions. Thus, a well-regulated balance between MMP activity and TIMP levels has been described as critical for healthy tissue homeostasis, and this balance can be chronically disturbed in pathological processes. The relationship between MMPs and TIMPs is complex and lacks the constraints of a typical enzyme-inhibitor relationship due to secondary interactions between various MMPs (specifically gelatinases) and TIMP family members. We illustrate a new complexity in this system by describing how MMP9 can cleave members of the TIMP family when in molar excess. Proteolytic processing of TIMPs can generate functionally altered peptides with potentially novel attributes. We demonstrate here that all TIMPs are cleaved at their C-terminal tails by a molar excess of MMP9. This processing removes the N-glycosylation site for TIMP3 and prevents the TIMP2 interaction with latent proMMP2, a prerequisite for cell surface MMP14-mediated activation of proMMP2. TIMP2/4 are further cleaved producing ∼14 kDa N-terminal proteins linked to a smaller C-terminal domain through residual disulfide bridges. These cleaved TIMP2/4 complexes show perturbed MMP inhibitory activity, illustrating that MMP9 may bear a particularly prominent influence upon the TIMP:MMP balance in tissues.

Timp2 loss-of-function mutation and TIMP2 treatment in murine model of NSCLC: modulation of immunosuppression and oncogenic signaling.

Mounting evidence suggests that the tissue inhibitor of metalloproteinases-2 (TIMP2) can reduce tumor burden and metastasis. However, the demonstration of such anti-tumor activity and associated mechanisms using in vivo tumor models is lacking. The effects of a Timp2 functional mutation and administration of recombinant TIMP2 were examined in both orthotopic and heterotopic murine models of lung cancer using C57Bl/6 syngeneic Lewis Lung 2-luciferase 2 cells (LL2-luc2) cells. Mice harboring a functional mutation of TIMP2 (mT2) display markedly increased primary lung tumor growth, increased mortality, enriched vasculature, and enhanced infiltration of pro-tumorigenic, immunosuppressive myeloid cells. Treatment with recombinant TIMP2 reduced primary tumor growth in both mutant and wild-type (wt) mice. Comparison of transcriptional profiles of lung tissues from tumor-free, wt versus mT2 mice reveals only minor changes. However, lung tumor-bearing mice of both genotypes demonstrate significant genotype-dependent changes in gene expression following treatment with TIMP. In tumor-bearing wt mice, TIMP2 treatment reduced the expression of upstream oncogenic mediators, whereas treatment of mT2 mice resulted in an immunomodulatory phenotype. A heterotopic subcutaneous model generating metastatic pulmonary tumors demonstrated that daily administration of recombinant TIMP2 significantly downregulates the expression of heat shock proteins, suggesting a reduction of cell-stress responses. In summary, we describe how TIMP2 exerts novel, anti-tumor effects in a murine model of lung cancer and that rTIMP2 treatment supports a normalizing effect on the tumor microenvironment. Our findings show that TIMP2 treatment demonstrates significant potential as an adjuvant in the treatment of NSCLC.