Matrix viscoelasticity promotes liver cancer progression in the pre-cirrhotic liver.

Type 2 diabetes mellitus is a major risk factor for hepatocellular carcinoma (HCC). Changes in extracellular matrix (ECM) mechanics contribute to cancer development1,2, and increased stiffness is known to promote HCC progression in cirrhotic conditions3,4. Type 2 diabetes mellitus is characterized by an accumulation of advanced glycation end-products (AGEs) in the ECM; however, how this affects HCC in non-cirrhotic conditions is unclear. Here we find that, in patients and animal models, AGEs promote changes in collagen architecture and enhance ECM viscoelasticity, with greater viscous dissipation and faster stress relaxation, but not changes in stiffness. High AGEs and viscoelasticity combined with oncogenic ß-catenin signalling promote HCC induction, whereas inhibiting AGE production, reconstituting the AGE clearance receptor AGER1 or breaking AGE-mediated collagen cross-links reduces viscoelasticity and HCC growth. Matrix analysis and computational modelling demonstrate that lower interconnectivity of AGE-bundled collagen matrix, marked by shorter fibre length and greater heterogeneity, enhances viscoelasticity. Mechanistically, animal studies and 3D cell cultures show that enhanced viscoelasticity promotes HCC cell proliferation and invasion through an integrin-ß1-tensin-1-YAP mechanotransductive pathway. These results reveal that AGE-mediated structural changes enhance ECM viscoelasticity, and that viscoelasticity can promote cancer progression in vivo, independent of stiffness.

Phase transition of tensin-1 during the focal adhesion disassembly and cell division.

Biomolecular condensates are nonmembranous structures that are mainly formed through liquid-liquid phase separation. Tensins are focal adhesion (FA) proteins linking the actin cytoskeleton to integrin receptors. Here, we report that GFP-tagged tensin-1 (TNS1) proteins phase-separate to form biomolecular condensates in cells. Live-cell imaging showed that new TNS1 condensates are budding from the disassembling ends of FAs, and the presence of these condensates is cell cycle dependent. TNS1 condensates dissolve immediately prior to mitosis and rapidly reappear while postmitotic daughter cells establish new FAs. TNS1 condensates contain selected FA proteins and signaling molecules such as pT308Akt but not pS473Akt, suggesting previously unknown roles of TNS1 condensates in disassembling FAs, as the storage of core FA components and the signaling intermediates.

Tensins - emerging insights into their domain functions, biological roles and disease relevance.

Tensins are a family of focal adhesion proteins consisting of four members in mammals (TNS1, TNS2, TNS3 and TNS4). Their multiple domains and activities contribute to the molecular linkage between the extracellular matrix and cytoskeletal networks, as well as mediating signal transduction pathways, leading to a variety of physiological processes, including cell proliferation, attachment, migration and mechanical sensing in a cell. Tensins are required for maintaining normal tissue structures and functions, especially in the kidney and heart, as well as in muscle regeneration, in animals. This Review discusses our current understanding of the domain functions and biological roles of tensins in cells and mice, as well as highlighting their relevance to human diseases.

Force-induced recruitment of cten along keratin network in epithelial cells.

The cytoskeleton provides structural integrity to cells and serves as a key component in mechanotransduction. Tensins are thought to provide a force-bearing linkage between integrins and the actin cytoskeleton; yet, direct evidence of tensin's role in mechanotransduction is lacking. We here report that local force application to epithelial cells using a micrometer-sized needle leads to rapid accumulation of cten (tensin 4), but not tensin 1, along a fibrous intracellular network. Surprisingly, cten-positive fibers are not actin fibers; instead, these fibers are keratin intermediate filaments. The dissociation of cten from tension-free keratin fibers depends on the duration of cell stretch, demonstrating that the external force favors maturation of cten-keratin network interactions over time and that keratin fibers retain remarkable structural memory of a cell's force-bearing state. These results establish the keratin network as an integral part of force-sensing elements recruiting distinct proteins like cten and suggest the existence of a mechanotransduction pathway via keratin network.

Tensin regulates pharyngeal pumping in Caenorhabditis elegans.

Tensin is a focal adhesion molecule that is known to regulate cell adhesion, migration, and proliferation. Although there are four tensin homologs (TNS1, TNS2, TNS3, and CTEN/TNS4) in mammals, only one tensin gene is found in Caenorhabditis elegans. Sequence analysis suggests that Caenorhabditis elegans tensin is slightly closer aligned with human TNS1 than with other human tensins. To establish the role of TNS1 in Caenorhabditis elegans, we have generated TNS1 knockout (KO) worms by CRISPR-Cas9 and homologous recombination directed repair approaches. Lack of TNS1 does not appear to affect the development or gross morphology of the worms. Nonetheless, defecation cycles are significantly longer in TNS1 KO worms. In addition, their pharyngeal pumping rate is markedly faster, which is likely due to a shorter pump duration in the KO worms. These findings indicate that TNS1 is not required for the development and survival of Caenorhabditis elegans but point to a critical role in modulating defecation and pharyngeal pumping rates.

Tensin1 positively regulates RhoA activity through its interaction with DLC1.

DLC1 is a RhoGAP-containing tumor suppressor and many of DLC1's functions are absolutely dependent on its RhoGAP activity. Through its RhoGAP domain, DLC1 inhibits the activity of RhoA GTPase, which regulates actin cytoskeleton networks and dis/assembly of focal adhesions. Tensin1 (TNS1) is a focal adhesion molecule that links the actin cytoskeleton to integrins and forms signaling complexes through its multiple binding domains. Here, we report that TNS1 enhances RhoA activity in a DLC1-dependent manner. This is accomplished by binding to DLC1 through TNS1's C2, SH2, and PTB domains. Point mutations at these three sites disrupt TNS1's interaction with DLC1 as well as its effect on RhoA activity. The biological relevance of this TNS1-DLC1-RhoA signaling axis is investigated in TNS1 knockout (KO) cells and mice. Endothelial cells isolated from TNS1 KO mice or those silenced with TNS1 siRNA show significant reduction in proliferation, migration, and tube formation activities. Concomitantly, the RhoA activity is down-regulated in TNS1 KO cells and this reduction is restored by further silencing of DLC1. Furthermore, the angiogenic process is compromised in TNS1 KO mice. These studies demonstrate that TNS1 binds to DLC1 and fine-tunes its RhoGAP activity toward RhoA and that the TNS1-DLC1-RhoA signaling axis is critical in regulating cellular functions that lead to angiogenesis.

An integrin binding-defective mutant of insulin-like growth factor-1 (R36E/R37E IGF1) acts as a dominant-negative antagonist of the IGF1 receptor (IGF1R) and suppresses tumorigenesis but still binds to IGF1R.

Insulin-like growth factor-1 (IGF1) is a major therapeutic target for cancer. We recently reported that IGF1 directly binds to integrins (αvß3 and α6ß4) and induces ternary complex formation (integrin-IGF1-IGF1 receptor (IGF1R)) and that the integrin binding-defective mutant of IGF1 (R36E/R37E) is defective in signaling and ternary complex formation. These findings predict that R36E/R37E competes with WT IGF1 for binding to IGF1R and inhibits IGF signaling. Here, we described that excess R36E/R37E suppressed cell viability increased by WT IGF1 in vitro in non-transformed cells. We studied the effect of R36E/R37E on viability and tumorigenesis in cancer cell lines. We did not detect an effect of WT IGF1 or R36E/R37E in cancer cells under anchorage-dependent conditions. However, under anchorage-independent conditions, WT IGF1 enhanced cell viability and induced signals, whereas R36E/R37E did not. Notably, excess R36E/R37E suppressed cell viability and signaling induced by WT IGF1 under anchorage-independent conditions. Using cancer cells stably expressing WT IGF1 or R36E/R37E, we determined that R36E/R37E suppressed tumorigenesis in vivo, whereas WT IGF1 markedly enhanced it. R36E/R37E suppressed the binding of WT IGF1 to the cell surface and the subsequent ternary complex formation induced by WT IGF1. R36E/R37E suppressed activation of IGF1R by insulin. WT IGF1, but not R36E/R37E, induced ternary complex formation with the IGF1R/insulin receptor hybrid. These findings suggest that 1) IGF1 induces signals under anchorage-independent conditions and that 2) R36E/R37E acts as a dominant-negative inhibitor of IGF1R (IGF1 decoy). Our results are consistent with a model in which ternary complex formation is critical for IGF signaling.

Up-regulated cten by FGF2 contributes to FGF2-mediated cell migration.

Cten is a focal adhesion molecule that is expressed at very low levels in most normal tissues. Nonetheless, its expression has been found to increase dramatically in many types of cancer including colorectal, breast, gastric, and pancreatic cancer, suggesting that cten may play a critical role during tumorigenesis. To study the mechanisms that induce cten expression and the function of up-regulated cten, we examined the effects of several cancer-associated growth factors and cytokines on cten expression. We found that EGF, FGF2, NGF, PDGF, TGF-ß, IGF-1, IL-6, and IL-13 were able to induce cten expression in a dose- and time-dependent manner. The Mek-Erk and PI3K-Akt pathways were two main signaling cascades responsible for cten up-regulation, whereas the Jak-Stat pathway could contribute to the increase in some conditions. Since many of these factors are known to promote cell migration, we hypothesized that up-regulated cten might contribute to this process. This hypothesis was investigated in FGF2-mediated cell migration. Silencing of cten not only reduced regular cell motility but also FGF2-mediated cell migration. Overexpression of cten promoted cell migration and FGF2 treatment failed to further enhance cell migration. Our findings that (1) cten is a common downstream molecule of these cancer-associated growth factors and cytokines; and that (2) up-regulated cten modulates cell migration induced by FGF2 and likely other growth factors as well, strongly suggest that cten could be a potential downstream therapeutic target for treating cancers associated with aberrant signaling of these growth factors and cytokines.

A reciprocal tensin-3-cten switch mediates EGF-driven mammary cell migration.

Cell migration driven by the epidermal growth factor receptor (EGFR) propels morphogenesis and involves reorganization of the actin cytoskeleton. Although de novo transcription precedes migration, transcript identity remains largely unknown. Through their actin-binding domains, tensins link the cytoskeleton to integrin-based adhesion sites. Here we report that EGF downregulates tensin-3 expression, and concomitantly upregulates cten, a tensin family member that lacks the actin-binding domain. Knockdown of cten or tensin-3, respectively, impairs or enhances mammary cell migration. Furthermore, cten displaces tensin-3 from the cytoplasmic tail of integrin beta1, thereby instigating actin fibre disassembly. In invasive breast cancer, cten expression correlates not only with high EGFR and HER2, but also with metastasis to lymph nodes. Moreover, treatment of inflammatory breast cancer patients with an EGFR/HER2 dual-specificity kinase inhibitor significantly downregulated cten expression. In conclusion, a transcriptional tensin-3-cten switch may contribute to the metastasis of mammary cancer.

Tensins in Kidney Function and Diseases.

Tensins are focal adhesion proteins that regulate various biological processes, such as mechanical sensing, cell adhesion, migration, invasion, and proliferation, through their multiple binding activities that transduce critical signals across the plasma membrane. When these molecular interactions and/or mediated signaling are disrupted, cellular activities and tissue functions are compromised, leading to disease development. Here, we focus on the significance of the tensin family in renal function and diseases. The expression pattern of each tensin in the kidney, their roles in chronic kidney diseases, renal cell carcinoma, and their potentials as prognostic markers and/or therapeutic targets are discussed in this review.

Nanostructures of designed geometry and functionality enable regulation of cellular signaling processes.

Extracellular matrices (ECM) triggered cellular signaling processes often begin with the clustering of the cellular receptors such as integrin and FcεRI. The sizes of these initial protein complexes or clusters are tens to 100 nm in dimension; therefore, engineered nanostructures could provide effective mimics of ECM for investigation and control of the initial and downstream specific signaling processes. This current topic discusses recent advances in nanotechnology in the context of design and production of matching chemical functionality and geometry for control of specific cellular signaling processes. Two investigations are reported to demonstrate this concept: (a) how the presentation of antigen at the nanometer scale would influence the aggregation of FcεRI, which would impact the formation of activation complexes, leading to the rearrangement of actin in cytoskeleton and degranulation or activation of mast cells; (b) how the engineered nanostructure could guide the initial integrin clustering, which would impact the formation of focal adhesion and downstream cell signaling cascades, leading to polarization, migration, and morphological changes. Complementary to engineered ECMs using synthetic ligands or peptides, or topographic control at the micrometer scale, nanostructures of designed geometry and chemical functionality provide new and effective biochemical cues for regulation of cellular signaling processes and downstream behaviors.

The phosphotyrosine-independent interaction of DLC-1 and the SH2 domain of cten regulates focal adhesion localization and growth suppression activity of DLC-1.

The tensin family member cten (C-terminal tensin like) is an Src homology 2 (SH2) and phosphotyrosine binding domain-containing focal adhesion molecule that may function as a tumor suppressor. However, the mechanism has not been well established. We report that cten binds to another tumor suppressor, deleted in liver cancer 1 (DLC-1), and the SH2 domain of cten is responsible for the interaction. Unexpectedly, the interaction between DLC-1 and the cten SH2 domain is independent of tyrosine phosphorylation of DLC-1. By site-directed mutagenesis, we have identified several amino acid residues on cten and DLC-1 that are essential for this interaction. Mutations on DLC-1 perturb the interaction with cten and disrupt the focal adhesion localization of DLC-1. Furthermore, these DLC-1 mutants have lost their tumor suppression activities. When these DLC-1 mutants were fused to a focal adhesion targeting sequence, their tumor suppression activities were significantly restored. These results provide a novel mechanism whereby the SH2 domain of cten-mediated focal adhesion localization of DLC-1 plays an essential role in its tumor suppression activity.

Endothelial DLC1 is dispensable for liver and kidney function in mice.

DLC1 is a focal adhesion molecule that regulates cell polarity, proliferation, migration, and survival. DLC1 functions as a tumor suppressor and its expression is often down-regulated in various malignant neoplasms of epithelial origin. Recent studies have suggested that lack of DLC1 in endothelial cells may contribute to the development of angiosarcoma, and that DLC1 mutations have been identified in patients with nephrotic syndrome, a disease mainly due to leaky glomerular filtration barriers. To demonstrate whether lack of endothelial DLC1 induces angiosarcoma and/or damages glomerular capillaries leading to nephrotic syndrome, we have extended our analyses on endothelial cell-specific DLC1 knockout mice with focuses on their liver and kidney function. Mice were monitored up to 24 months of age. However, no histological or clinical difference was found between DLC1 knockout and wild type mice, indicating that lack of endothelial DLC1 alone does not compromise kidney and liver function in mice.

Transient regulation of focal adhesion via Tensin3 is required for nascent oligodendrocyte differentiation.

The differentiation of oligodendroglia from oligodendrocyte precursor cells (OPCs) to complex and extensive myelinating oligodendrocytes (OLs) is a multistep process that involves large-scale morphological changes with significant strain on the cytoskeleton. While key chromatin and transcriptional regulators of differentiation have been identified, their target genes responsible for the morphological changes occurring during OL myelination are still largely unknown. Here, we show that the regulator of focal adhesion, Tensin3 (Tns3), is a direct target gene of Olig2, Chd7, and Chd8, transcriptional regulators of OL differentiation. Tns3 is transiently upregulated and localized to cell processes of immature OLs, together with integrin-ß1, a key mediator of survival at this transient stage. Constitutive <i>Tns3</i> loss of function leads to reduced viability in mouse and humans, with surviving knockout mice still expressing Tns3 in oligodendroglia. Acute deletion of <i>Tns3</i> in vivo, either in postnatal neural stem cells (NSCs) or in OPCs, leads to a twofold reduction in OL numbers. We find that the transient upregulation of Tns3 is required to protect differentiating OPCs and immature OLs from cell death by preventing the upregulation of p53, a key regulator of apoptosis. Altogether, our findings reveal a specific time window during which transcriptional upregulation of Tns3 in immature OLs is required for OL differentiation likely by mediating integrin-ß1 survival signaling to the actin cytoskeleton as OL undergo the large morphological changes required for their terminal differentiation.

Direct Observations of Silver Nanowire-Induced Frustrated Phagocytosis among NR8383 Lung Alveolar Macrophages.

The interaction of long nanowires and living cells is directly related to nanowires' nanotoxicity and health impacts. Interactions of silver nanowires (AgNWs) and macrophage cell lines (NR8383) were investigated using laser scanning confocal microscopy and single cell compression (SCC). With high-resolution imaging and mechanics measurement of individual cells, AgNW-induced frustrated phagocytosis was clearly captured in conjunction with structural and property changes of cells. While frustrated phagocytosis is known for long microwires and long carbon nanotubes, this work reports first direct observations of frustrated phagocytosis of AgNWs among living cells in situ. In the case of partial penetration of AgNWs into NR8383 cells, confocal imaging revealed actin participation at the entry sites, whose behavior differs from microwire-induced frustrated phagocytosis. The impacts of frustrated phagocytosis on the cellular membrane and cytoskeleton were also quantified by measuring the mechanical properties using SCC. Taken collectively, this study reveals the structural and property characteristics of nanowire-induced frustrated phagocytosis, which deepens our understanding of nanowire-cell interactions and nanocytotoxicity.

Conditional loss of PTEN leads to skeletal abnormalities and lipoma formation.

To understand the role of tumor suppressor PTEN in cartilage development, we have generated chondrocyte specific PTEN deletion mice using Col2a1Cre and PTEN(loxp/loxp) mice. PTEN mutant mice are viable and fertile, nonetheless, develop kyphosis over time. Histological analyses show mutant vertebrae and intervertebral discs are larger and therefore the spines are longer than in control mice. In addition, the growth plates are thicker, invading trabecular bone areas are deeper, and marrow adipocyte populations are higher in PTEN mutant mice. Furthermore, the growth plates, not normally fused in mouse long bones, are fused in PTEN mutants. Intriguingly, PTEN mice develop lipomas and show abnormal accumulation of fat tissues along spines. Cell tracking assays have confirmed that lipomas and a portion of fat tissues were derived from Col2a1Cre PTEN(loxp/loxp) cells. Further analyses have suggested that the phenotypes of PTEN mutant likely attribute to PTEN's negatively regulating role in PI3K/Akt pathway.

Cell tracing dyes significantly change single cell mechanics.

Cell tracing dyes are very frequently utilized in cellular biology research because they provide highly sensitive fluorescent tags that do not compromise cellular functions such as growth and proliferation. In many investigations concerning cellular adhesion and mechanics, fluorescent dyes have been employed with the assumption of little impact on the results. Using the single cell compression technique developed by our team, the single cell mechanics of MDA-MB-468 and MLC-SV40 cells were investigated as a function of dye uptake. Cell tracing dyes increase living cell stiffness 3-6 times and cell-to-probe adhesion up to 7 times. These results suggest a more significant effect than toxins, such as thrombin. A simple analytical model was derived to enable the extraction of the Young's moduli of the cell membrane and cytoskeleton from the force-deformation profiles measured for individual cells. The increase in Young's modulus of the membrane is 3-7 times, which is more significant than that of the cytoskeleton (1.1-3.4 times). We propose that changes in cell mechanics upon the addition of fluorescent tracing dye are primarily due to the incorporation of amphiphilic dye molecules into the cellular plasma membrane, which increases the lateral interaction among phospholipid chains and thus enhances their rigidity and adhesion.

Identification of subcellular targeting sequences of Cten reveals its role in cell proliferation.

Spatial and temporal subcellular localization plays critical roles in regulating protein function. Cten (C-terminal tensin like) is a member of the tensin family. Cten recruits signaling molecules, such as DLC1, to focal adhesions, modulates homeostasis of receptor tyrosine kinases, including EGFR and c-Met, and promotes cell migration. These functions are likely controlled by Cten localization at focal adhesions and/or in the cytoplasm. In addition, Cten has been detected in the nucleus by which mechanism is unknown. To this end, we have examined the distribution of Cten in various cell lines, determined primary sequence requirements for its nuclear and focal adhesion localizations, and analyzed potential roles of nuclear Cten. Our results show that a proportion of Cten translocates to nuclei in cancer cell lines and that nuclear exporting of Cten is a CRM1-dependent process. A nuclear localization sequence and a nuclear export sequence are identified within Cten. In addition, like other tensins, Cten contains two independent focal adhesion binding sites. Although further expression of recombinant Cten showed no effect on cancer cell proliferation, silencing of Cten significantly reduced cell growth. Furthermore, expression of Cten mutants either with defective nuclear export sequence or tagged with SV40 nuclear localization sequence promoted cell growth. These results suggest that nuclear Cten contributes to cancer cell proliferation. Our findings identify a molecular mechanism for regulating Cten protein trafficking in mammalian cells and provide new insights into the dynamics of focal adhesion complexes in health and disease.

Hyperactivity of Mek in TNS1 knockouts leads to potential treatments for cystic kidney diseases.

Cystic kidney disease is the progressive development of multiple fluid-filled cysts that may severely compromise kidney functions and lead to renal failure. TNS1 (tensin-1) knockout mice develop cystic kidneys and die from renal failure. Here, we have established TNS1-knockout MDCK cells and applied 3D culture system to investigate the mechanism leading to cyst formation. Unlike wild-type MDCK cells, which form cysts with a single lumen, TNS1-knockout cysts contain multiple lumens and upregulated Mek/Erk activities. The multiple lumen phenotype and Mek/Erk hyperactivities are rescued by re-expression of wild-type TNS1 but not the TNS1 mutant lacking a fragment essential for its cell-cell junction localization. Furthermore, Mek inhibitor treatments restore the multiple lumens back to single lumen cysts. Mek/Erk hyperactivities are also detected in TNS1-knockout mouse kidneys. Treatment with the Mek inhibitor trametinib significantly reduces the levels of interstitial infiltrates, fibrosis and dilated tubules in TNS1-knockout kidneys. These studies establish a critical role of subcellular localization of TNS1 in suppressing Mek/Erk signaling and maintaining lumenogenesis, and provide potential therapeutic strategies by targeting the Mek/Erk pathway for cystic kidney diseases.