Regulation of PD-L1 expression by matrix stiffness in lung cancer cells

Expression of programmed death-ligand 1 (PD-L1) in tumor cells such as lung cancer cells plays an important role in mechanisms underlying evasion of an immune check point system. Lung cancer tissue with increased deposition of extracellular matrix is much stiffer than normal lung tissue. There is emerging evidence that the matrix stiffness of cancer tissue affects the phenotypes and properties of cancer cells. Nevertheless, the effects of substrate rigidity on expression of PD-L1 in lung cancer cells remain elusive. We evaluated the effects of substrate stiffness on PD-L1 expression in HCC827 lung adenocarcinoma cells by using polyacrylamide hydrogels with stiffnesses of 2 and 25?kPa. Expression of PD-L1 protein was higher on the stiffer substrates (25?kPa gel and plastic dish) than on the soft 2?kPa gel. PD-L1 expression was reduced by detachment of cells adhering to the substrate. Interferon-γ enhanced expression of PD-L1 protein cultured on stiff (25?kPa gel and plastic dishes) and soft (2?kPa gel) substrates and in the cell adhesion-free condition. As the stiffness of substrates increased, formation of actin stress fiber and cell growth were enhanced. Transfection of the cells with short interfering RNA for PD-L1 inhibited cell growth without affecting stress fiber formation. Treatment of the cells with cytochalasin D, an inhibitor of actin polymerization, significantly reduced PD-L1 protein levels. Taken together, a stiff substrate enhanced PD-L1 expression via actin-dependent mechanisms in lung cancer cells. It is suggested that stiffness as a tumor environment regulates PD-L1 expression, which leads to evasion of the immune system and tumor growth.     

Endothelial actin and cell stiffness is modulated by substrate stiffness in 2D and 3D

There is a growing appreciation of the profound effects that passive mechanical properties, especially the stiffness of the local environment, can have on cellular functions. Many experiments are conducted in a 2D geometry (i.e., cells grown on top of substrates of varying stiffness), which is a simplification of the 3D environment often experienced by cells in vivo. To determine how matrix dimensionality might modulate the effect of matrix stiffness on actin and cell stiffness, endothelial cells were cultured on top of and within substrates of various stiffnesses. Endothelial cells were cultured within compliant (1.0–1.5 mg/ml, 124±8 to 202±27 Pa) and stiff (3.0 mg/ml, 502±48 Pa) type-I collagen gels. Cells elongated and formed microvascular-like networks in both sets of gels as seen in previous studies. Cells in stiffer gels exhibited more pronounced stress fibers and ～1.5-fold greater staining for actin. As actin is a major determinant of a cell's mechanical properties, we hypothesized that cells in stiff gels will themselves be stiffer. To test this hypothesis, cells were isolated from the gels and their stiffness was assessed using micropipette aspiration. Cells isolated from relatively compliant gels were 1.9-fold more compliant than cells isolated from relatively stiff gels (p<0.05). Similarly, cells cultured on top of 1700 Pa polyacrylamide gels were 2.0-fold more compliant that those cultured on 9000 Pa (p<0.05). These data demonstrate that extracellular substrate stiffness regulates endothelial stiffness in both three- and two-dimensional environments, though the range of stiffnesses that cells respond to vary significantly in different environments.     

Gomisin A ameliorates metastatic melanoma by inhibiting AMPK and ERK/JNK-mediated cell survival and metastatic phenotypes

BackgroundGomisin A (G.A), a lignan compound extracted from the fruits of Schisandra chinensis, is known to exert anti-tumor effects on hepatocarcinoma and colorectal cancer cells. Suppression of proliferation and metastatic abilities of cancer cells are some effective cancer treatment methods.PurposeThe objective of this study is to investigate the effects of G.A on metastatic melanoma, and the mechanism by which it affects metastatic melanoma.Study designThe anti-proliferative and anti-metastatic effects of G.A were observed in in vitro and in vivo.MethodsWST assay and flow cytometry were conducted to investigate the effect of G.A on proliferation, cell cycle arrest, and apoptosis in metastatic melanoma cell lines. Migration and invasion abilities of G.A-treated melanoma cells were observed by wound healing and invasion assays.ResultsG.A (25–100 μM) decreased the viability of melanoma cells by inducing cell cycle arrest and apoptosis. These anti-proliferative effects of G.A were found to be mediated by AMPK, ERK, and JNK activation. G.A (5–20 μM) decreased the migration and invasion of melanoma cells by suppressing epithelial-mesenchymal transition (EMT). Consequently, G.A (2–50 mg/kg) inhibited lung metastasis by suppressing EMT and inducing cell cycle arrest and apoptosis in melanoma cells.ConclusionThese results conclude that G.A has the potential to reduce metastatic melanoma through its anti-proliferative and anti-metastatic effects.     

Thrombospondin-1 overexpression stimulates loss of Smad4 and accelerates malignant behavior via TGF-β signal activation in pancreatic ductal adenocarcinoma

IntroductionPancreatic ductal adenocarcinoma (PDAC) is characterized by abundant stroma and cancer-associated fibroblasts (CAFs) provide a favorable tumor microenvironment. Smad4 is known as tumor suppressor in several types of cancers including PDAC, and loss of Smad4 triggers accelerated cell invasiveness and metastatic potential. The thrombospondin-1 (TSP-1) can act as a major activator of latent transforming growth factor-β (TGF-β) in vivo. However, the roles of TSP-1 and the mediator of Smad4 loss and TGF-β signal activation during PDAC progression have not yet been addressed. The aim is to elucidate the biological role of TSP-1 in PDAC progression.Methods and resultsHigh substrate stiffness stimulated TSP-1 expression in CAFs, and TSP-1 knockdown inhibited cell proliferation with suppressed profibrogenic and activated stroma-related gene expressions in CAFs. Paracrine TSP-1 treatment for PDAC cells promoted cell proliferation and epithelial mesenchymal transition (EMT) with activated TGF-β signals such as phosphorylated Akt and Smad2/3 expressions. Surprisingly, knockdown of DPC4 (Smad4 gene) induced TSP-1 overexpression with TGF-β signal activation in PDAC cells. Interestingly, TSP-1 overexpression also induced downregulation of Smad4 expression and enhanced cell proliferation in vitro and in vivo. Treatment with LSKL peptide, which antagonizes TSP-1-mediated latent TGF-β activation, attenuated cell proliferation, migration and chemoresistance with enhanced apoptosis in PDAC cells.ConclusionsTSP-1 derived from CAFs stimulates loss of Smad4 expression in cancer cells and accelerates malignant behavior by TGF-β signal activation in PDAC. TSP-1 could be a novel therapeutic target, not only for CAFs in stiff stroma, but also for cancer cells in the PDAC microenvironment.     

A Novel 2.5D Culture Platform to Investigate the Role of Stiffness Gradients on Adhesion-Independent Cell Migration

Current studies investigating the role of biophysical cues on cell migration focus on the use of culture platforms with static material parameters. However, migrating cells in vivo often encounter spatial variations in extracellular matrix stiffness. To better understand the effects of stiffness gradients on cell migration, we developed a 2.5D cell culture platform where cells are sandwiched between stiff tissue culture plastic and soft alginate hydrogel. Under these conditions, we observed migration of cells from the underlying stiff substrate into the alginate matrix. Observation of migration into alginate in the presence of integrin inhibition as well as qualitative microscopic analyses suggested an adhesion-independent cell migration mode. Observed migration was dependent on alginate matrix stiffness and the RhoA-ROCK-myosin-II pathway; inhibitors specifically targeting ROCK and myosin-II arrested cell migration. Collectively, these results demonstrate the utility of the 2.5D culture platform to advance our understanding of the effects of stiffness gradients and mechanotransductive signaling on adhesion-independent cell migration.     

Integrin-β4 regulates the dynamic changes of phenotypic characteristics in association with epithelial-mesenchymal transition (EMT) and RhoA activity in airway epithelial cells during injury and repair

Background: In airway disease such as asthma a hyperactive cellular event of epithelial-mesenchymal transition (EMT) is considered as the mechanism of pathological airway tissue remodeling after injury to the airway epithelium. And the initiation of EMT in the airways depends on the epithelial disruption involving dissolution and/or destabilization of the adhesive structures between the cells and ECM. Previously, we have shown that integrin-β4, an epithelial adhesion molecule in bronchial epithelium is an important regulator of cell proliferation and wound repair in human airway epithelial cells. Therefore, in this study we aimed to investigate whether integrin-β4 also regulates EMT phenotypes during injury and repair in airway epithelial cells of both wild type/integrin-β4^-/- mice in vivo and cultured cells treated with integrin-β4/nonsense siRNA in vitro.Methods: We induced injury to the airway epithelial cells by either repeated exposure to ozone and mechanical scratch wound, and subsequently examined the EMT-related phenotypic features in the airway epithelial cells including biomarkers expression, adhesion and cytoskeleton reorganization and cell stiffness.Results: The results show that in response to injury (ozone exposure/scratch wound) and subsequent spontaneous repair (ozone withdrawal/wound healing) both in vivo and in vitro, the airway epithelial cells underwent dynamic changes in the epithelial and mesenchymal biomarkers expression, adhesion and cytoskeleton structures as well as cell stiffness, all together exhibiting enhanced EMT phenotypic features after injury and reversal of the injury-induced effects during repair. Importantly, these injury/repair-associated EMT phenotypic changes in airway epithelial cells appeared to be dependent on integrin-β4 expression. More specifically, when integrin-β4 was deficient in mice (integrin-β4^-/-) the repair of ozone-injured airway epithelium was impaired and the recovery of ozone-enhanced EMT biomarkers expression in the airway epithelium was delayed. Similarly, in the scratch wounded airway epithelial cells with integrin-β4 knockdown, the cells were impaired in all aspects related to EMT during wound and repair including cell proliferation, wound closure rate, adhesion and cytoskeleton protein expression (vinculin and vimentin), mesenchymal-like F-actin reorganization, cell stiffness and RhoA activation.Conclusion: Taken together, these results suggested that integrin-β4 may be essential in regulating the effects of injury and repair on EMT in airway epithelial cells via influencing both the cell adhesion to ECM and cells'' physical phenotypes through RhoA signaling pathway.     

B7H1 Expression and Epithelial-To-Mesenchymal Transition Phenotypes on Colorectal Cancer Stem-Like Cells

Cancer stem cells (CSCs) can invade and metastasize by epithelial-to-mesenchymal transition (EMT). However, how they escape immune surveillance is unclear. B7H1 is crucial negative co-stimulatory molecule but little information about whether it works in CSCs. Therefore, we determined the expression of B7H1 and EMT-associated markers in colorectal cancer stem-like cells to investigate a possible immunoevasion way of CSCs. We enriched CD133⁺ colorectal cancer cells which manifested the CSCs-like properties such as higher levels of other stem cell markers Oct-4 and Sox-2, tumor sphere forming ability and more tumorigenic in NOD/SCID mice. These CD133⁺ cells possess EMT gene expression profile including higher level of Snail, Twist, vimentin, fibronectin and lower level of E-cadherin. Moreover, CD133⁺ cells in both cell line and colorectal cancer tissues expressed high level of negative co-stimulate molecule B7H1. Furthermore, some B7H1⁺ cancer cells also showed the characteristic of EMT, indicating EMT cells could escape immune attack during metastasis. B7H1 expression and EMT phenotypes on CSCs indicates a possible immunoevasion way.     

Integrin-linked kinase tunes cell–cell and cell-matrix adhesions to regulate the switch between apoptosis and EMT downstream of TGFβ1

Epithelial-mesenchymal transition (EMT) is a morphogenetic process that endows epithelial cells with migratory and invasive potential. Mechanical and chemical signals from the tumor microenvironment can activate the EMT program, thereby permitting cancer cells to invade the surrounding stroma and disseminate to distant organs. Transforming growth factor β1 (TGFβ1) is a potent inducer of EMT that can also induce apoptosis depending on the microenvironmental context. In particular, stiff microenvironments promote EMT while softer ones promote apoptosis. Here, we investigated the molecular signaling downstream of matrix stiffness that regulates the phenotypic switch in response to TGFβ1 and uncovered a critical role for integrin-linked kinase (ILK). Specifically, depleting ILK from mammary epithelial cells precludes their ability to sense the stiffness of their microenvironment. In response to treatment with TGFβ1, ILK-depleted cells undergo apoptosis on both soft and stiff substrata. We found that knockdown of ILK decreases focal adhesions and increases cell–cell adhesions, thus shifting the balance from cell–matrix to cell–cell adhesion. High cell–matrix adhesion promotes EMT whereas high cell–cell adhesion promotes apoptosis downstream of TGFβ1. These results highlight an important role for ILK in controlling cell phenotype by regulating adhesive connections to the local microenvironment.     

SWI/SNF Chromatin-Remodeling Factor Smarcd3/Baf60c Controls Epithelial-Mesenchymal Transition by Inducing Wnt5a Signaling

We previously identified a gene signature predicted to regulate the epithelial-mesenchymal transition (EMT) in both epithelial tissue stem cells and breast cancer cells. A phenotypic RNA interference (RNAi) screen identified the genes within this 140-gene signature that promoted the conversion of mesenchymal epithelial cell adhesion molecule-negative (EpCAM⁻) breast cancer cells to an epithelial EpCAM^+/high phenotype. The screen identified 10 of the 140 genes whose individual knockdown was sufficient to promote EpCAM and E-cadherin expression. Among these 10 genes, RNAi silencing of the SWI/SNF chromatin-remodeling factor Smarcd3/Baf60c in EpCAM⁻ breast cancer cells gave the most robust transition from the mesenchymal to epithelial phenotype. Conversely, expression of Smarcd3/Baf60c in immortalized human mammary epithelial cells induced an EMT. The mesenchymal-like phenotype promoted by Smarcd3/Baf60c expression resulted in gene expression changes in human mammary epithelial cells similar to that of claudin-low triple-negative breast cancer cells. These mammary epithelial cells expressing Smarcd3/Baf60c had upregulated Wnt5a expression. Inhibition of Wnt5a by either RNAi knockdown or blocking antibody reversed Smarcd3/Baf60c-induced EMT. Thus, Smarcd3/Baf60c epigenetically regulates EMT by activating WNT signaling pathways.     

Early-Stage Induction of SWI/SNF Mutations during Esophageal Squamous Cell Carcinogenesis

The SWI/SNF chromatin remodeling complex is frequently inactivated by somatic mutations of its various components in various types of cancers, and also by aberrant DNA methylation. However, its somatic mutations and aberrant methylation in esophageal squamous cell carcinomas (ESCCs) have not been fully analyzed. In this study, we aimed to clarify in ESCC, what components of the SWI/SNF complex have somatic mutations and aberrant methylation, and when somatic mutations of the SWI/SNF complex occur. Deep sequencing of components of the SWI/SNF complex using a bench-top next generation sequencer revealed that eight of 92 ESCCs (8.7%) had 11 somatic mutations of 7 genes, ARID1A, ARID2, ATRX, PBRM1, SMARCA4, SMARCAL1, and SMARCC1. The SMARCA4 mutations were located in the Forkhead (85Ser>Leu) and SNF2 family N-terminal (882Glu>Lys) domains. The PBRM1 mutations were located in a bromodomain (80Asn>Ser) and an HMG-box domain (1,377Glu>Lys). For most mutations, their mutant allele frequency was 31–77% (mean 61%) of the fraction of cancer cells in the same samples, indicating that most of the cancer cells in individual ESCC samples had the SWI/SNF mutations on one allele, when present. In addition, a BeadChip array analysis revealed that a component of the SWI/SNF complex, ACTL6B, had aberrant methylation at its promoter CpG island in 18 of 52 ESCCs (34.6%). These results showed that genetic and epigenetic alterations of the SWI/SNF complex are present in ESCCs, and suggested that genetic alterations are induced at an early stage of esophageal squamous cell carcinogenesis.     

Migration in Confined 3D Environments Is Determined by a Combination of Adhesiveness,Nuclear Volume,Contractility, and Cell Stiffness

In cancer metastasis and other physiological processes, cells migrate through the three-dimensional (3D) extracellular matrix of connective tissue and must overcome the steric hindrance posed by pores that are smaller than the cells. It is currently assumed that low cell stiffness promotes cell migration through confined spaces, but other factors such as adhesion and traction forces may be equally important. To study 3D migration under confinement in a stiff (1.77 MPa) environment, we use soft lithography to fabricate polydimethylsiloxane (PDMS) devices consisting of linear channel segments with 20 μm length, 3.7 μm height, and a decreasing width from 11.2 to 1.7 μm. To study 3D migration in a soft (550 Pa) environment, we use self-assembled collagen networks with an average pore size of 3 μm. We then measure the ability of four different cancer cell lines to migrate through these 3D matrices, and correlate the results with cell physical properties including contractility, adhesiveness, cell stiffness, and nuclear volume. Furthermore, we alter cell adhesion by coating the channel walls with different amounts of adhesion proteins, and we increase cell stiffness by overexpression of the nuclear envelope protein lamin A. Although all cell lines are able to migrate through the smallest 1.7 μm channels, we find significant differences in the migration velocity. Cell migration is impeded in cell lines with larger nuclei, lower adhesiveness, and to a lesser degree also in cells with lower contractility and higher stiffness. Our data show that the ability to overcome the steric hindrance of the matrix cannot be attributed to a single cell property but instead arises from a combination of adhesiveness, nuclear volume, contractility, and cell stiffness.