cell-ECM Adhesion Research Articles

766 Keratinocyte differentiation is coupled to mechanical cues through the LINC complex

Skin tissue is resilient to mechanical forces, but also can respond to prolonged mechanical forces. For example, tissue expanders are used to generate more skin for breast reconstruction surgery. At the cellular level, multiple mechanical forces are exerted on epidermal keratinocytes. Progenitor keratinocytes, unlike differentiated keratinocytes, have both cell-cell adhesions and cell-extracellular matrix (ECM) adhesions that keep progenitor keratinocytes attached to the basement membrane. During keratinocyte differentiation, progenitor keratinocytes lose cell-ECM adhesions and detach from the basement membrane, but the exact mechanism of how this occurs is not fully understood. External forces must be communicated from the plasma membrane at the outside of the cell to the nucleus, and the Linker of Nucleoskeletal and Cytoskeletal (LINC) complex is poised on the nuclear membrane to sense these mechanical forces. We were interested in determining if there is tension on the LINC complex in keratinocytes and if this tension influences the function of epidermal keratinocytes. Tension on the LINC complex was measured using a FRET-based Nesprin tension sensor, which showed that keratinocytes undergoing differentiation have less tension on the LINC complex compared to progenitor keratinocytes. To investigate the functional consequences of tension on the LINC complex, mice lacking LINC complex components SUN1 and SUN2 proteins were generated. Multiple lines of evidence indicate that Sun1/2 null keratinocytes undergo precocious differentiation both in culture and in vivo. In conclusion, this work demonstrates that mechanical tension on the LINC complex maintains keratinocytes in a progenitor state and identifies tension on the LINC complex as a new regulator of keratinocyte differentiation.

4078 Identification of distinct fibroblast populations with unique roles in pancreatic cancer progression and tumor immunity

OBJECTIVES/GOALS: The desmoplastic reaction in PDAC involves a significant accumulation of immune cells and fibroblasts.The functional diversity of carcinoma associated fibroblasts (CAFs) remains largely unknown, and identification of immune regulating subsets would have a substantial impact in augmentation of immunotherapy efficacy. METHODS/STUDY POPULATION: Employing histology, FACs, multiplex immunohistochemistry, single cell RNA sequencing (sc-RNA-seq) and genetically engineered mouse models, we demonstrate that aSMA+ cells are a dominant CAF population in PDAC with tumor restraining properties (TS-CAFs), as opposed those of the FAP+ CAFs, which demonstrate tumor promoting activity (TP-CAFs). RESULTS/ANTICIPATED RESULTS: Analysis of bulk tumor depleted of either TS-CAFs or TP-CAFs showed that TS-CAFs predominantly modulate extracellular matrix (ECM) production, facilitate cell-ECM adhesion and regulate adaptive immunity, while TP-CAFs exhibit a lineage that is skewed towards a pro-inflammatory, chemokine secreting phenotype. Further, scRNA-Seq analyses demonstrate that CAFs share distinct gene expression profiles characteristic of lymphocytic and myeloid lineages. Together our data distinguish two populations of CAFs, one which is tumor suppressing with roles in ECM remodeling and another which is tumor promoting with roles in cytokine production, both with immune modulating capabilities. DISCUSSION/SIGNIFICANCE OF IMPACT: Our study identifies a complex network of functionally heterogeneous fibroblasts during PDAC progression with significant immunotherapeutic implication. The identification of distinct fibroblast subsets will allow us to discriminately target fibroblast populations to augment immunotherapy efficacy in pancreatic cancer.

Global phosphotyrosinylated protein profile of cell-matrix adhesion complexes of trabecular meshwork cells.

Dysregulation of the mechanical properties and cell adhesive interactions of trabecular meshwork (TM) are known to impair aqueous humor drainage and elevate intraocular pressure in glaucoma patients. The identity of regulatory mechanisms underlying TM mechanotransduction, however, remains elusive. Here we analyzed the phosphotyrosine proteome of human TM cell-extracellular matrix (ECM) adhesion complexes, which play a key role in sensing and transducing extracellular chemical and mechanical cues into intracellular activities, using a two-level affinity pull-down (phosphotyrosine antibody and titanium dioxide beads) method and mass spectrometry. This analysis identified ~1,000 tyrosine-phosphorylated proteins of TM cell-ECM adhesion complexes. Many consensus adhesome proteins were found to be tyrosine phosphorylated. Interestingly, several of the phosphotyrosinylated proteins found in TM cell-ECM adhesion complexes are known to be required for podocyte glomerular filtration, indicating the existence of molecular parallels that are likely relevant to the shared fluid barrier and filtration functions of the two mechanosensitive cell types.

114 Galectin-1 Is Overexpressed in Post-Burn Hypertrophic Scar

Abstract Introduction Upon healing, burn wounds often leave hypertrophic scars (HTS), a significant morbidity for patients that can have lasting psychosocial impacts. HTS is marked by excess collagen deposition, dermal and epidermal thickening, and an increased concentration of fibroblasts. The Galectins, a family of lectins with a conserved carbohydrate-recognition-domain, function intracellularly and extracellularly as mediators of a multitude of biological processes including inflammatory responses, cell migration and differentiation, and cell-ECM adhesion. Galectin-1 (Gal-1) has been associated with several fibrotic diseases and can induce keratinocyte and fibroblast proliferation and migration as well as differentiation into fibroproliferative myofibroblasts. Gal-1 transcription is driven by the PI3K/AKT pathway, and AKT activation is increased in HTS compared to normal skin (NS). In this study, Gal-1 expression was assessed in human and porcine HTS and NS tissues. Methods mRNA was isolated from biopsies of human or porcine HTS and NS tissue. Total transcriptome microarray analysis was performed in porcine HTS vs. NS. Transcription of the Galectin-1 gene (LGALS1) was assessed by qRT-PCR using primers specific to human or porcine LGALS1 and GAPDH genes. A Gal-1 antibody was used for immunofluorescent (IF) staining to corroborate gene expression data. Results LGALS1, 4, 8, and 12 were differentially regulated genes in pig HTS vs. NS by microarray analysis (fold change= 3.58, 6.11, 1.89, and 3.03, p< 0.01). Confirmatory qRT-PCR results showed significant upregulation of LGALS1 transcription in HTS compared to NS of both human and porcine tissues (Fold change= 6.11 and 7.70, n=15 and n=3 scars respectively, p< .001). In pig HTS, this upregulation was maintained throughout scar development and remodeling (days 49, 56, 77, 99, and 112 post-wounding). IF Gal-1 in human HTS showed significantly increased fluorescence (503±58 vs 346±23, p< .001, n=5 scars) compared to NS, and Gal-1 was visualized in both intracellular and extracellular areas of the dermis. Conclusions Galectin-1 is overexpressed in HTS at the mRNA and protein levels and may have a role in the development of scar phenotypes due to fibroblast over-proliferation, collagen secretion, and dermal thickening. The role of galectins shows promise for future study and may lead to development of new pharmacotherapies. Applicability of Research to Practice A better understanding of galectin signaling and its effects on the ECM and cell growth may lead to new treatments aimed at reducing scar formation. Changes in galectin expression could be induced as possible downstream effects of anti-scarring gene therapies aimed at AKT-inactivation.

Abstract A108: Identification of distinct fibroblast populations with unique roles in PDAC progression and tumor immunity

Abstract The desmoplastic reaction in pancreatic ductal adenocarcinomas (PDAC) involves a significant accumulation of immune cells and fibroblasts. Although carcinoma-associated fibroblasts (CAFs) have been associated with immune suppression and immunotherapy resistance in PDAC mouse models, some studies have shown immune-promoting and tumor-suppressing roles of CAFs. The functional diversity of carcinoma-associated fibroblasts (CAFs) remains largely unknown, and identification of immune-regulating subsets would have a substantial impact in augmentation of immunotherapy efficacy. Employing histology, FACs, multiplex immunohistochemistry, single-cell RNA sequencing (sc-RNA-seq), and genetically engineered mouse models, we demonstrate that SMA+ cells are a dominant CAF population in PDAC with tumor-restraining properties (TS-CAFs), as opposed those of the FAP+ CAFs, which demonstrate tumor-promoting activity (TP-CAFs). Analysis of bulk tumor depleted of either TS-CAFs or TP-CAFs showed that TS-CAFs predominantly modulate extracellular matrix (ECM) production, facilitate cell-ECM adhesion, and regulate adaptive immunity, while TP-CAFs exhibit a lineage that is skewed towards a proinflammatory, chemokine-secreting phenotype. Further, scRNA-Seq analyses demonstrate that CAFs share distinct gene expression profiles characteristic of lymphocytic and myeloid lineages. Together our data distinguish two populations of CAFs, one that is tumor suppressing with roles in ECM remodeling and another that is tumor promoting with roles in cytokine production, both with immune-modulating capabilities. Collectively, our study identifies a complex network of functionally heterogeneous fibroblasts during PDAC progression with significant immunotherapeutic implication. Citation Format: J. Kebbeh Darpolor, Xiaofeng Zheng, Sujuan Yang, Kathleen M. McAndrews, Julienne L. Carstens, Patricia E. Phillips, Hikaru Sugimoto, Pedro Correa De Sampaio, Chia-Chin Wu, Valerie S. LeBleu, Raghu Kalluri. Identification of distinct fibroblast populations with unique roles in PDAC progression and tumor immunity [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A108.

Tissue Regeneration from Mechanical Stretching of Cell-Cell Adhesion.

Cell-cell adhesion complexes are macromolecular adhesive organelles that integrate cells into tissues. This mechanochemical coupling in cell-cell adhesion is required for a large number of cell behaviors, and perturbations of the cell-cell adhesion structure or related mechanotransduction pathways can lead to critical pathological conditions such as skin and heart diseases, arthritis, and cancer. Mechanical stretching has been a widely used method to stimulate the mechanotransduction process originating from the cell-cell adhesion and cell-extracellular matrix (ECM) complexes. These studies aimed to reveal the biophysical processes governing cell proliferation, wound healing, gene expression regulation, and cell differentiation in various tissues, including cardiac, muscle, vascular, and bone. This review explores techniques in mechanical stretching in two-dimensional settings with different stretching regimens on different cell types. The mechanotransduction responses from these different cell types will be discussed with an emphasis on their biophysical transformations during mechanical stretching and the cross talk between the cell-cell and cell-ECM adhesion complexes. Therapeutic aspects of mechanical stretching are reviewed considering these cellular responses after the application of mechanical forces, with a focus on wound healing and tissue regeneration. Impact Statement Mechanical stretching has been proposed as a therapeutic option for tissue regeneration and wound healing. It has been accepted that mechanotransduction processes elicited by mechanical stretching govern cellular response and behavior, and these studies have predominantly focused on the cell-extracellular matrix (ECM) sites. This review serves the mechanobiology community by shifting the focus of mechanical stretching effects from cell-ECM adhesions to the less examined cell-cell adhesions, which we believe play an equally important role in orchestrating the response pathways.

523 MITF-mediated extracellular matrix changes control intratumour heterogeneity in melanoma

Differential tumour cell behaviour caused by environmental conditions, termed dynamic heterogeneity, is a prime cause of drug resistance and understanding its underlying mechanisms is crucial to design effective therapies. We have demonstrated cell-cycling heterogeneity in both melanoma xenograft tumours and melanoma spheroids. This was characterized by the presence within the same tumour/spheroid of clusters of proliferating cells and clusters of G1-arrested cells. The location of the quiescent zones supported oxygen/nutrient deprivation as the cause of cell cycle arrest and the G1-arrested cells reversed to cycling when cultured under normoxia in 2D cultures. Here we demonstrate that this heterogeneity is consistently decreased in vivo and in vitro by high expression of MITF, a transcription factor strongly associated with melanoma development, progression and therapy response. This phenomenon is not associated with reduced hypoxia, indicating that high MITF expression allows a portion of hypoxic cells to maintain cell cycling despite oxygen deprivation. Conversely, modulation of MITF expression lead to changes of spheroid architecture and in extracellular matrix (ECM) and cell-ECM adhesion and crosstalk proteins. Furthermore, ECM swapping and inhibition of the Rho/ROCK signalling pathway, respectively, rescues and mimics the morphology and cell cycle effects of high MITF expression. These findings support a novel role of MITF in controlling intratumour melanoma heterogeneity through changes in cell-ECM crosstalk.

Key roles of Rho GTPases, YAP, and Mutant P53 in anti‐neoplastic effects of statins

Emerging epidemiological and preclinical studies have focused on statins and mevalonate pathway to identify potential therapeutic target and clarify the underlying mechanism of the anti-neoplastic effects. Reductions of mevalonate or isoprenoids, caused by statins, would further decrease the isoprenylation of Rho GTPases which is the crucial step for Rho GTPases to anchor on inner cellular membrane. Following anchoring, activated Rho GTPases can mediate a series of cellular activities such as cytoskeleton reprogramming, front-rear polarity, and cell-ECM adhesion. These changes not only facilitate tumor cell detachment and migration but also bring great mechanical changes to directly activate YAP, the major nuclear mechanotransducer, to translocate into nucleus. Recently, statins have been identified as potent inhibitors of YAP. Once entering nucleus, YAP would combine TEADs to promote the transcription of about 100 genes, which are involved in cell proliferation, cell cycle regulation, stemness, invasion, and metastasis. Besides, statins are able to promote the degradation of misfolded mutant p53 (mutp53), which is an oncogene in a variety of human malignancies. Reduction in mevalonate-5-phosphate (MVP), also induced by statins, would impair the stability of DNAJA1-mutp53 complex; then, elevated C terminus of Hsc70-interacting protein (CHIP) mediates the nuclear export and degradation of misfolded mutp53 through ubiquitin-proteasome pathway. It is worth noted that YAP, mutp53, and mevalonate pathway form two positive feedback loops. It is reasonable to believe that Rho GTPases, YAP, and mutp53 are determinants for statins as anti-cancer agents: tumor cells harboring mutp53 and nuclear-located YAP would be more sensitive to statins.

Depletion of Ras Suppressor-1 (RSU-1) promotes cell invasion of breast cancer cells through a compensatory upregulation of a truncated isoform

Extracellular matrix (ECM)-adhesion proteins and actin cytoskeleton are pivotal in cancer cell invasion. Ras Suppressor-1 (RSU-1), a cell-ECM adhesion protein that interacts with PINCH-1, thus being connected to Integrin Linked Kinase (ILK), alpha-parvin (PARVA), and actin cytoskeleton, is up-regulated in metastatic breast cancer (BC) samples. Apart from the originally-identified gene (RSU-1L), an alternatively-spliced isoform (RSU-1-X1) has been reported. We used non-invasive MCF-7 cells, expressing only RSU-1L, and highly invasive MDA-MB-231-LM2 expressing both isoforms and generated stable shRNA-transduced cells lacking RSU-1L, while the truncated RSU-1-X1 isoform was depleted by siRNA-mediated silencing. RSU-1L depletion in MCF-7 cells resulted in complete abrogation of tumor spheroid invasion in three-dimensional collagen gels, whereas it promoted MDA-MB-231-LM2 invasion, through a compensatory upregulation of RSU-1-X1. When RSU-1-X1 was also eliminated, RSU-1L-depletion-induced migration and invasion were drastically reduced being accompanied by reduced urokinase plasminogen activator expression. Protein expression analysis in 23 human BC samples corroborated our findings showing RSU-1L to be upregulated and RSU-1-X1 downregulated in metastatic samples. We demonstrate for the first time, that both RSU-1 isoforms promote invasion in vitro while RSU-1L elimination induces RSU-1-X1 upregulation to compensate for the loss. Hence, we propose that both isoforms should be blocked to effectively eliminate metastasis.

Abstract 1299: Potential antitumor effects of a Golgi disrupting agent, M-COPA, via targeting cell-extracellular matrix interaction under the spheroid culture conditions

Abstract The Golgi apparatus plays an essential role in the transport, processing, and sorting of cell surface proteins. We previously demonstrated that a Golgi disrupting agent, 2-methylcoprophilinamide (M-COPA), exhibited antitumor effect in human cancer cells in vitro and in vivo; however, in vivo antitumor efficacy of M-COPA did not simply reflect its in vitro efficacy, probably due to the lack of microenvironmental features of tumor tissues in the monolayer culture conditions. To bridge the gap between in vitro and in vivo efficacies, we exploited 3-dimensional (3D) spheroid culture models, which can recapitulate physiological characteristics of tumor tissues; i.e., nutrient/oxygen gradient, cell-cell interaction and cell-extracellular matrix (ECM) interaction, if cancer cells synthesize their own ECM molecules. Interestingly, human gastric cancer MKN1 cells displayed hypersensitivity to M-COPA under the 3D culture conditions compared to those in the monolayer culture conditions. Especially, we found that M-COPA caused loss of spheroid integrity. We previously demonstrated that M-COPA exerted antitumor effects via inhibiting intracellular transport of receptor tyrosine kinases onto the cell surface. Therefore, we examined the involvement of cell adhesion molecules in the antitumor effect of M-COPA. As a result, we found that specific integrins, which serve as cell surface receptors responsible for cell-ECM adhesion, were overexpressed and their downstream signaling molecules such as Src, Akt and MEK/ERK were highly phosphorylated in the cells under the 3D culture conditions compared to those under the monolayer culture conditions. Upon exposure to M-COPA, cell surface expression of these integrins was efficiently decreased and the downstream signaling molecules were dephosphorylated along with loss of spheroid integrity. Moreover, expression knockdown or neutralizing antibody of the integrins clearly suppressed sphere formation of MKN1 cells. These results suggest that M-COPA exerted in vivo antitumor effects via targeting specific integrins that mediate cell-matrix adhesion and its downstream intracellular signaling pathway observed in 3D spheroid culture models. Citation Format: Yoshimi Ohashi, Kazuma Takeuchi, Mutsumi Okamura, Akinobu Akatsuka, Isamu Shiina, Kentaro Yoshimatsu, Shingo Dan. Potential antitumor effects of a Golgi disrupting agent, M-COPA, via targeting cell-extracellular matrix interaction under the spheroid culture conditions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1299.

Abstract 5184: N-CoR2 epigenetically regulates adhesion-dependent branching morphogenesis and its loss correlates with malignant progression of the mammary gland

Abstract Branching morphogenesis depends upon extracellular matrix remodeling and dynamic cell-cell and cell-extracellular matrix interactions, although how this developmental program is coordinated remains unclear. Expression profiling of mammary tissue at different stages of branching morphogenesis combined with histological and functional studies identified the histone deacetylase (HDAC) regulator, nuclear receptor co-repressor 2 (N-CoR2), as a key orchestrator of adhesion-dependent branching morphogenesis in the mammary gland. Molecular studies revealed that N-CoR2 actively represses fibronectin (FN) and thrombospondin-1 (THBS-1) promoter accessibility in an HDAC-dependent manner. Consistently, histological analysis revealed that N-CoR2 is abundantly expressed in the luminal epithelial cells localized along the outer rim of invading terminal end buds (TEB) and that its expression is lowest at sites of TEB branching bifurcation. In addition, reducing N-CoR2 expression drove the scattering of human mammary acini in vitro and promoted promiscuous branching of humanized mouse mammary tissue in vivo, suggesting that downregulation of N-CoR2 is required for branching morphogenesis in mammary gland. Furthermore, downregulation of N-CoR2 in breast cancer cells increased the expression of both FN and THBS-1 and reduced the expression of E-cadherin and enhanced the migratory capability of breast cancer cells. Intriguingly, immunohistochemical and clinical correlative analysis indicated that loss of N-CoR2 correlates with perturbed cell-cell and enhanced cell-ECM adhesion and acquisition of an invasive phenotype and local lymph node metastasis in a subset of breast carcinomas. These findings suggest N-CoR2 may epigenetically regulate adhesion-dependent branching morphogenesis in the mammary gland and that its loss could promote malignant progression of some breast cancers. Citation Format: Shenq-Shyang Huang, Valerie M. Weaver, Kelvin K.C. Tsai. N-CoR2 epigenetically regulates adhesion-dependent branching morphogenesis and its loss correlates with malignant progression of the mammary gland [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5184.

Using a continuum model to decipher the mechanics of embryonic tissue spreading from time-lapse image sequences: An approximate Bayesian computation approach.

Advanced imaging techniques generate large datasets capable of describing the structure and kinematics of tissue spreading in embryonic development, wound healing, and the progression of many diseases. These datasets can be integrated with mathematical models to infer biomechanical properties of the system, typically identifying an optimal set of parameters for an individual experiment. However, these methods offer little information on the robustness of the fit and are generally ill-suited for statistical tests of multiple experiments. To overcome this limitation and enable efficient use of large datasets in a rigorous experimental design, we use the approximate Bayesian computation rejection algorithm to construct probability density distributions that estimate model parameters for a defined theoretical model and set of experimental data. Here, we demonstrate this method with a 2D Eulerian continuum mechanical model of spreading embryonic tissue. The model is tightly integrated with quantitative image analysis of different sized embryonic tissue explants spreading on extracellular matrix (ECM) and is regulated by a small set of parameters including forces on the free edge, tissue stiffness, strength of cell-ECM adhesions, and active cell shape changes. We find statistically significant trends in key parameters that vary with initial size of the explant, e.g., for larger explants cell-ECM adhesion forces are weaker and free edge forces are stronger. Furthermore, we demonstrate that estimated parameters for one explant can be used to predict the behavior of other similarly sized explants. These predictive methods can be used to guide further experiments to better understand how collective cell migration is regulated during development.

Determination of Fibroblast Polarization under the Combination of Physical, Molecular, and Genetic Cues

Cell polarization plays a crucial role in dynamic cellular events such as cell proliferation, differentiation, and directional migration in response to diverse extracellular and intracellular cues. While cell polarization involves highly orchestrated intracellular molecular reorganization, however, its underlying mechanism is largely missing in presence of multiple stimuli. Here, we show that the front-rear cell polarization, systematically assessed by relative position of nucleus and microtubule organizing center (MTOC), is precisely controlled by intracellular mechanical interactions encompassing cell-ECM adhesion and nucleus-cytoskeletal connection. By modulating the size and distribution of fibronectin-coated adhesion spots located in the polarized cell shape mimicking micro-contact printed teardrop patterns, we could monitor the alteration of cell polarization in a single cell level. We note that the localization of adhesion spots is more dominant than cell shape itself to induce intracellular polarization. Moreover, degree of cell polarization is significantly diminished by disrupting nuclear lamin A/C that mediates nucleus-cytoskeletal connection. We further tested with human dermal fibroblasts (HDFs) derived from variously aged donors that represent intrinsic decline of lamin A/C expression. As expected, cell polarization is proportionally decreased as biological aging progresses. These results further demonstrate that nuclear lamin A/C-mediated nucleus-cytoskeletal interplay is essential in single cell polarization. Our study demonstrates that aging-related LMNA deficiency leads to mis-positioning of intracellular organelles including nucleus, MTOC, and cytoskeletal organization. Our results provide a novel insight into understanding of single cell polarization under the combined physical, molecular and genetic cues.

Identification of molecular genetic contributants to canine cutaneous mast cell tumour metastasis by global gene expression analysis.

Cutaneous mast cell tumours are one of the most common canine cancers. Approximately 25% of the tumours metastasise. Activating c-kit mutations are present in about 20% of tumours, but metastases occur in the absence of mutations. Tumour metastasis is associated with significantly diminished survival in spite of adjuvant chemotherapy. Available prognostic tests do not reliably predict whether a tumour will metastasise. In this study we compared the global expression profiles of 20 primary cutaneous mast cell tumours that metastasised with those of 20 primary tumours that did not metastasise. The objective was to identify genes associated with mast cell tumour metastatic progression that may represent targets for therapeutic intervention and biomarkers for prediction of tumour metastasis. Canine Gene 1.1 ST Arrays were employed for genome-wide expression analysis of formalin-fixed, paraffin-embedded biopsies of mast cell tumours borne by dogs that either died due to confirmed mast cell tumour metastasis, or were still alive more than 1000 days post-surgery. Decreased gene expression in the metastasising tumours appears to be associated with a loss of cell polarity, reduced cell-cell and cell-ECM adhesion, and increased cell deformability and motility. Dysregulated gene expression may also promote extracellular matrix and base membrane degradation, suppression of cell cycle arrest and apoptosis, and angiogenesis. Down-regulation of gene expression in the metastasising tumours may be achieved at least in part by small nucleolar RNA-derived RNA and microRNA-effected gene silencing. Employing cross-validation, a linear discriminant analysis-based classifier featuring 19 genes that displayed two-fold differences in expression between metastasising and non-metastasising tumours was estimated to classify metastasising and non-metastasising tumours with accuracies of 90–100% and 70–100%, respectively. The differential expression of 9 of the discriminator genes was confirmed by quantitative reverse transcription-PCR.

Direct binding of Talin to Rap1 is required for cell-ECM adhesion in Drosophila.

Attachment of cells to the extracellular matrix (ECM) via integrins is essential for animal development and tissue maintenance. The cytoplasmic protein Talin (encoded by rhea in flies) is necessary for linking integrins to the cytoskeleton, and its recruitment is a key step in the assembly of the adhesion complex. However, the mechanisms that regulate Talin recruitment to sites of adhesion in vivo are still not well understood. Here, we show that Talin recruitment to, and maintenance at, sites of integrin-mediated adhesion requires a direct interaction between Talin and the GTPase Rap1. A mutation that blocks the direct binding of Talin to Rap1 abolished Talin recruitment to sites of adhesion and the resulting phenotype phenocopies that seen with null alleles of Talin. Moreover, we show that Rap1 activity modulates Talin recruitment to sites of adhesion via its direct binding to Talin. These results identify the direct Talin-Rap1 interaction as a key in vivo mechanism for controlling integrin-mediated cell-ECM adhesion.

Talin Autoinhibition Regulates Cell-ECM Adhesion Dynamics and Wound Healing In Vivo

Cells in multicellular organisms are arranged in complex three-dimensional patterns. This requires both transient and stable adhesions with the extracellular matrix (ECM). Integrin adhesion receptors bind ECM ligands outside the cell and then, by binding the protein talin inside the cell, assemble an adhesion complex connecting to the cytoskeleton. The activity of talin is controlled by several mechanisms, but these have not been well studied invivo. By generating mice containing the activating point mutation E1770A in talin (Tln1), which disrupts autoinhibition, we show that talin autoinhibition controls cell-ECM adhesion, cell migration, and wound healing invivo. In particular, blocking autoinhibition gives rise to more mature, stable focal adhesions that exhibit increased integrin activation. Mutant cells also show stronger attachment to ECM and decreased traction force. Overall, these results demonstrate that modulating talin function via autoinhibition isan important mechanism for regulating multiple aspects of integrin-mediated cell-ECM adhesion invivo.

Abstract A037: C1orf116, a gene of unknown function, promotes tight junctions and epithelial cell phenotype in prostate cancer

Abstract While advances in the treatment of localized prostate cancer (PCa) have improved five-year survival to near 100%, metastatic disease remains incurable. Lethal metastases are the end-result of a cancer cell that escapes the primary tumor, travels through the vasculature, and eventually invades and colonizes a secondary site. Thus, in order for a cancer cell to metastasize from the primary tumor, it must move. In a critical early event of metastasis, a rare population of cells in the primary tumor undergoes a phenotype switch from a proliferating epithelial cell to gain mesenchymal cell characteristics, including high migratory capacity, during the process of epithelial-to-mesenchymal transition (EMT). Using a multistudy gene expression discovery analysis, we previously identified C1orf116 as a novel candidate driver of EMT (doi: 10.1186/s12885-017-3413-3). C1orf116 is an unnamed gene of unknown function that remains largely uncharacterized in any disease state, including cancer or EMT. To substantiate its potential role in EMT, we queried the Cancer Cell Line Encyclopedia dataset (GSE36133) and found that C1orf116 expression is associated with classical epithelial markers and inversely associated with mesenchymal markers. To specifically interrogate the role of C1orf116 in EMT, we overexpressed C1orf116 in otherwise C1orf116low PC3 cells (PC3-oe). PC3-oe cells demonstrated distinctive cobblestone epithelial cellular morphology. Correspondingly, PC3-oe cultures showed increased epithelial marker expression (including OVOL1, CDH1, ESRP), indicating that C1orf116 expression was sufficient to induce an epithelial transition. To determine whether C1orf116 influenced migration, we performed a scratch-closure assay. PC3-oe cultures showed reduced motility compared to control, suggesting that C1orf116 restricts motility. Cellular migration is influenced by many factors, including cell-ECM adhesion, “go vs. grow” switching, and cell-cell adhesion. We observed that PC-oe established cell-cell contacts more rapidly than cells in the control cultures (time-lapse photography immediately following culture seeding) and hypothesized that C1orf116 promoted cell-cell adhesion. We found that PC3-oe had increased expression of tight junction complexes (including ZO1, ZO2, ZO3). Likewise, siRNA-mediated knockdown of C1orf116 expression in C42b (C1orf116high) cells resulted in decreased expression of tight junction proteins. These findings demonstrate that C1orf116 is a novel driver of the epithelial phenotype. In particular, C1orf116 expression promotes cell-cell tight junctions, thereby restricting the active emigrant phenotype of potentially lethal metastatic cells. Citation Format: Sarah R. Amend, James Hernandez, Princy Parsana, Kenneth J. Pienta. C1orf116, a gene of unknown function, promotes tight junctions and epithelial cell phenotype in prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr A037.

Erybraedin A is a potential Src inhibitor that blocks the adhesion and viability of non-small-cell lung cancer cells

The adhesion of cancer cells to the extracellular matrix (ECM) is crucial for cell proliferation, survival, and metastasis. Thus, it is necessary to inhibit cell-ECM adhesion by blocking the activation of the associated signaling to control cancer. Here, we identify erybraedin A (EBA) as a potential Src inhibitor that blocks cell adhesion and viability in non-small-cell lung cancer (NSCLC). EBA significantly inhibited the adhesion of NSCLC cells to fibronectin. EBA also markedly inhibited the activation of Src and its downstream targets, including FAK and Akt. The interaction between integrin β1 or integrin β3 and Src was inhibited by EBA treatment. A docking study revealed the bindings of EBA to the ATP-binding pocket and the allosteric regulatory site of the Src kinase. Additionally, EBA markedly inhibited the viability and the colony formation of NSCLC cells and induced apoptotic cell death. These results describe novel biological properties of EBA, which can block the Src-mediated adhesion and survival of NSCLC cells, suggesting the potential of EBA as an anticancer Src inhibitor that warrants further development in advanced preclinical and clinical settings.

Cell Adhesion and Matrix Stiffness: Coordinating Cancer Cell Invasion and Metastasis.

Metastasis is a multistep process in which tumor extracellular matrix (ECM) and cancer cell cytoskeleton interactions are pivotal. ECM is connected, through integrins, to the cell’s adhesome at cell–ECM adhesion sites and through them to the actin cytoskeleton and various downstream signaling pathways that enable the cell to respond to external stimuli in a coordinated manner. Cues from cell-adhesion proteins are fundamental for defining the invasive potential of cancer cells, and many of these proteins have been proposed as potent targets for inhibiting cancer cell invasion and thus, metastasis. In addition, ECM accumulation is quite frequent within the tumor microenvironment leading in many cases to an intense fibrotic response, known as desmoplasia, and tumor stiffening. Stiffening is not only required for the tumor to be able to displace the host tissue and grow in size but also contributes to cell–ECM interactions and can promote cancer cell invasion to surrounding tissues. Here, we review the role of cell adhesion and matrix stiffness in cancer cell invasion and metastasis.

Matching material and cellular timescales maximizes cell spreading on viscoelastic substrates

Recent evidence has shown that, in addition to rigidity, the viscous response of the extracellular matrix (ECM) significantly affects the behavior and function of cells. However, the mechanism behind such mechanosensitivity toward viscoelasticity remains unclear. In this study, we systematically examined the dynamics of motor clutches (i.e., focal adhesions) formed between the cell and a viscoelastic substrate using analytical methods and direct Monte Carlo simulation. Interestingly, we observe that, for low ECM rigidity, maximum cell spreading is achieved at an optimal level of viscosity in which the substrate relaxation time falls between the timescale for clutch binding and its characteristic binding lifetime. That is, viscosity serves to stiffen soft substrates on a timescale faster than the clutch off-rate, which enhances cell-ECM adhesion and cell spreading. On the other hand, for substrates that are stiff, our model predicts that viscosity will not influence cell spreading, since the bound clutches are saturated by the elevated stiffness. The model was tested and validated using experimental measurements on three different material systems and explained the different observed effects of viscosity on each substrate. By capturing the mechanism by which substrate viscoelasticity affects cell spreading across a wide range of material parameters, our analytical model provides a useful tool for designing biomaterials that optimize cellular adhesion and mechanosensing.