Matrix stiffness-mediated effects on stemness characteristics occurring in HCC cells.
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ABSTRACT:
SUBMITTER: You Y
PROVIDER: S-EPMC5078009 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Our previous study has validated that higher matrix stiffness obviously improves vascular endothelial growth factor (VEGF) expression in HCC cells, highlighting a linkage between matrix stiffness and HCC angiogenesis. However, the effects of matrix stiffness on vascular endothelial cells in HCC and its underlying mechanism remain largely uncharacterized. Here we further analyzed the expression of vascular endothelial growth factor receptor 2 (VEGFR2) in human umbilical vein endothelial cells (HUVECs) grown on different stiffness substrates and explored its regulatory mechanism for better understanding matrix stiffness-regulated angiogenesis in HCC. Our results revealed that increased matrix stiffness significantly upregulated the expression of VEGFR2 in HUVECs, and the expression level of VEGFR2 was positively correlated with the expression levels of COL1 and lysyl oxidase in human HCC tissues and rat HCC tissue, moreover VEGFR2 and CD34 were co-localized at blood vessel of HCC tissues, indicating an obvious regulation role of matrix stiffness in VEGFR2 expression. Simultaneously, increased matrix stiffness also elevated the phosphorylation level of Akt and the expressions of integrin αV/β5 and nuclear Sp1 in HUVECs. Inhibition of integrin αVβ5 remarkably reversed the expression of VEGFR2 and phosphorylation level of Akt in HUVECs grown on higher stiffness substrate. Except that, PI3K inhibitor also suppressed the phosphorylation level of Akt and the expressions of VEGFR2 and nuclear Sp1 evidently. Taken together, higher matrix stiffness increased VEGFR2 expression in HUVECs, and integrin αVβ5/Akt/Sp1 pathway participated in stiffness-mediated effects on VEGFR2 upregulation. This study combining with our previous report discloses a new paradigm in which higher matrix stiffness as an initiator drives HCC angiogenesis via upregulating both VEGFR2 expression in vascular endothelial cells and VEGF expression in HCC cells.
| S-EPMC7471346 | biostudies-literature
Project description:BACKGROUND:Increased liver stiffness exerts a detrimental role in driving hepatocellular carcinoma (HCC) malignancy and progression, and indicates a high risk of unfavorable outcomes. However, it remains largely unknown how liver matrix stiffness as an independent cue triggers epithelial-mesenchymal transition (EMT) and facilitates HCC metastasis. METHODS:Buffalo rat HCC models with different liver stiffness backgrounds and an in vitro Col I-coated cell culture system with tunable stiffness were used in the study to explore the effects of matrix stiffness on EMT occurrence and its underlying molecular mechanism. Clinical significance of liver stiffness and key molecules required for stiffness-induced EMT were validated in HCC cohorts with different liver stiffness. RESULTS:HCC xenografts grown in higher stiffness liver exhibited worse malignant phenotypes and higher lung metastasis rate, suggesting that higher liver stiffness promotes HCC invasion and metastasis. Cell tests in vitro showed that higher matrix stiffness was able to strikingly strengthen malignant phenotypes and independently induce EMT occurrence in HCC cells, and three signaling pathways converging on Snail expression participated in stiffness-mediated effect on EMT including integrin-mediated S100A11 membrane translocation, eIF4E phosphorylation, and TGF ?1 autocrine. Additionally, the key molecules required for stiffness-induced EMT were highly expressed in tumor tissues of HCC patients with higher liver stiffness and correlated with poor tumor differentiation and higher recurrence. CONCLUSIONS:Higher matrix stiffness as an initiator triggers epithelial-mesenchymal transition (EMT) in HCC cells independently, and three signaling pathways converging on Snail expression contribute to this pathological process. This work highlights a significant role of biomechanical signal in triggering EMT and facilitating HCC invasion and metastasis.
| S-EPMC6839087 | biostudies-literature
Project description:BACKGROUND:Human mesenchymal stem cell (hMSC) differentiation into osteoblasts has important clinical significance in treating bone injury, and the stiffness of the extracellular matrix (ECM) has been shown to be an important regulatory factor for hMSC differentiation. The aim of this study was to further delineate how matrix stiffness affects intracellular signaling through integrin α5/β1, FAK, and Wnt signaling, subsequently regulating the osteogenic phenotype of hMSCs. METHODS:hMSCs were cultured on tunable polyacrylamide hydrogels coated with fibronectin with stiffness corresponding to a Young's modulus of 13-16 kPa and 62-68 kPa. After hMSCs were cultured on gels for 1 week, gene expression of alpha-1 type I collagen, BGLAP, and RUNX2 were evaluated by real-time PCR. After hMSCs were cultured on gels for 24 h, signaling molecules relating to integrin α5 (FAK, ERK, p-ERK, Akt, p-Akt, GSK-3β, p-GSK-3β, and β-catenin) were evaluated by western blot analysis. RESULTS:Osteogenic differentiation was increased on 62-68 kPa ECM, as evidenced by alpha-1 type I collagen, BGLAP, and RUNX2 gene expression, calcium deposition, and ALP staining. In the process of differentiation, gene and protein expression of integrin α5/β1 increased, together with protein expression of the downstream signaling molecules FAK, p-ERK, p-Akt, GSK-3β, p-GSK-3β, and β-catenin, indicating that these molecules can affect the osteogenic differentiation of hMSCs. An antibody blocking integrin α5 suppressed the stiffness-induced expression of all osteoblast markers examined. In particular, alpha-1 type I collagen, RUNX2, and BGLAP were significantly downregulated, indicating that integrin α5 regulates hMSC osteogenic differentiation. Downstream expression of FAK, ERK, p-ERK, and β-catenin protein was unchanged, whereas Akt, p-Akt, GSK-3β, and p-GSK-3β were upregulated. Moreover, expression of Akt and p-Akt was upregulated with anti-integrin α5 antibody, but no difference was observed for FAK, ERK, and p-ERK between the with or without anti-integrin α5 antibody groups. At the same time, expression of GSK-3β and p-GSK-3β was upregulated and β-catenin levels showed no difference between the groups with or without anti-integrin α5 antibody. Since Akt, p-Akt, GSK-3β, and p-GSK-3β displayed the same changes between the groups with or without anti-integrin α5 antibody, we then detected the links among them. Expression of p-Akt and p-GSK-3β was reduced effectively in the presence of the Akt inhibitor Triciribine. However, Akt, GSK-3β, and β-catenin were unchanged. These results suggested that expression of p-GSK-3β was regulated by p-Akt on 62-68 kPa ECM. CONCLUSIONS:Taken together, our results provide evidence that matrix stiffness (62-68 kPa) affects the osteogenic outcome of hMSCs through mechanotransduction events that are mediated by integrin α5.
| S-EPMC5831741 | biostudies-literature
Project description:Oncogenic transformation of mammary epithelial cells (MECs) is a critical step in epithelial-to-mesenchymal transition (EMT), but evidence also shows that MECs undergo EMT with increasing matrix stiffness; the interplay of genetic and environmental effects on EMT is not clear. To understand their combinatorial effects on EMT, premalignant MCF10A and isogenic Ras-transformed MCF10AT are cultured on polyacrylamide gels ranging from normal mammary stiffness, ≈150 Pa, to tumor stiffness, ≈5700 Pa. Though cells spread on stiff hydrogels independent of transformation, only 10AT cells exhibit heterogeneous spreading behavior on soft hydrogels. Within this mixed population, spread cells exhibit an elongated, mesenchymal-like morphology, disrupted localization of the basement membrane, and nuclear localization of the EMT transcription factor TWIST1. MCF10AT spreading is not driven by typical mechanosensitive pathways including YAP and TGF-β or by myosin contraction. Rather, ERK activation induces spreading of MCF10AT cells on soft hydrogels and requires dynamic microtubules. These findings indicate the importance of oncogenic signals, and their hierarchy with substrate mechanics, in regulating MEC EMT.
| S-EPMC7274860 | biostudies-literature
Project description:BackgroundMetformin, a traditional first-line anti-hyperglycemic agent for diabetes, recently exhibits better antitumor effect in hepatocellular carcinoma (HCC). However, its resistance and tolerance mechanism in HCC remains largely unknown. Here, we investigated whether increased matrix stiffness attenuated the intervention effects of metformin on HCC invasion and metastasis, and explored its underlying molecular mechanism.MethodsFN-coated gel substrates with 6, 10, and 16 kPa, which simulated the stiffness of normal, fibrotic, and cirrhotic liver tissues respectively, were established to evaluate matrix stiffness-mediated effects on HCC cells. Alterations in morphology, proliferation, motility, and invasive/metastatic-associated genes (PTEN, MMP2, MMP9) of HCC cells grown on different-stiffness substrates were comparatively analyzed before and after metformin intervention. Subsequently, the underlying molecular mechanism by which higher matrix stiffness attenuates antitumor effects of metformin in HCC was further elucidated.ResultsMetformin significantly inhibited proliferation, migration, and invasion of HCC cells. Compared with the controls on lower-stiffness substrate, HCC cells grown on higher-stiffness substrate exhibited an obvious resistance to intervention effects of metformin on proliferation, migration, invasion and metastasis. High stiffness stimulation significantly activated the miR-17-5p/PTEN/PI3K/Akt signaling pathway in HCC cells via integrin β1 and in turn resulted in MMP2 and MMP9 upregulation. Meanwhile, integrin β1 knockdown or PI3K inhibitor partially reversed the activation of the above signaling molecules. For HCC cells grown on the same-stiffness substrate, metformin remarkably upregulated PTEN expression and suppressed the activation of the PI3K/Akt/MMP pathway, but no effect on integrin β1 expression. Importantly, the increase in fold of PTEN expression and decrease in folds of Akt phosphorylation level and MMP2 and MMP9 expressions in the treated HCC cells with metformin on 16-kPa stiffness substrate were evidently weakened compared with those in the controls on the 6-kPa stiffness substrate.ConclusionsIncreased matrix stiffness significantly attenuates the inhibitory effect of metformin on HCC invasion and metastasis, and a common pathway of PTEN/PI3K/Akt/MMPs activated by mechanical stiffness signal and inactivated by metformin contributes to matrix stiffness-caused metformin resistance. To the best of our knowledge, this is the first report to clarify the mechanism of metformin intervention resistance from the perspective of tumor biophysical microenvironment.
| S-EPMC7466473 | biostudies-literature
Project description:The mechanical properties of the extracellular matrix have recently been shown to promote myofibroblast differentiation and lung fibrosis. Mechanisms by which matrix stiffness regulates myofibroblast differentiation are not fully understood. The goal of this study was to determine the intrinsic mechanisms of mechanotransduction in the regulation of matrix stiffness-induced myofibroblast differentiation. A well established polyacrylamide gel system with tunable substrate stiffness was used in this study. Megakaryoblastic leukemia factor-1 (MKL1) nuclear translocation was imaged by confocal immunofluorescent microscopy. The binding of MKL1 to the ?-smooth muscle actin (?-SMA) gene promoter was quantified by quantitative chromatin immunoprecipitation assay. Normal human lung fibroblasts responded to matrix stiffening with changes in actin dynamics that favor filamentous actin polymerization. Actin polymerization resulted in nuclear translocation of MKL1, a serum response factor coactivator that plays a central role in regulating the expression of fibrotic genes, including ?-SMA, a marker for myofibroblast differentiation. Mouse lung fibroblasts deficient in Mkl1 did not respond to matrix stiffening with increased ?-SMA expression, whereas ectopic expression of human MKL1 cDNA restored the ability of Mkl1 null lung fibroblasts to express ?-SMA. Furthermore, matrix stiffening promoted production and activation of the small GTPase RhoA, increased Rho kinase (ROCK) activity, and enhanced fibroblast contractility. Inhibition of RhoA/ROCK abrogated stiff matrix-induced actin cytoskeletal reorganization, MKL1 nuclear translocation, and myofibroblast differentiation. This study indicates that actin cytoskeletal remodeling-mediated activation of MKL1 transduces mechanical stimuli from the extracellular matrix to a fibrogenic program that promotes myofibroblast differentiation, suggesting an intrinsic mechanotransduction mechanism.
| S-EPMC3488695 | biostudies-literature
Project description:BMMSCs have drawn great interest in tissue engineering and regenerative medicine attributable to their multi-lineage differentiation capacity. Increasing evidence has shown that the mechanical stiffness of extracellular matrix is a critical determinant for stem cell behaviors. However, it remains unknown how matrix stiffness influences MSCs commitment with changes in cell morphology, adhesion, proliferation, self-renewal and differentiation. We employed fibronectin coated polyacrylamide hydrogels with variable stiffnesses ranging from 13 to 68 kPa to modulate the mechanical environment of BMMSCs and found that the morphology and adhesion of BMMSCs were highly dependent on mechanical stiffness. Cells became more spread and more adhesive on substrates of higher stiffness. Similarly, the proliferation of BMMSCs increased as stiffness increased. Sox2 expression was lower during 4h to 1 week on the 13-16 kPa and 62-68 kPa, in contrast, it was higher during 4h to 1 week on the 48-53 kPa. Oct4 expression on 13-16 kPa was higher than 48-53 kPa at 4h, and it has no significant differences at other time point among three different stiffness groups. On 62-68 kPa, BMMSCs were able to be induced toward osteogenic phenotype and generated a markedly high level of RUNX2, ALP, and Osteopontin. The cells exhibited a polygonal morphology and larger spreading area. These results suggest that matrix stiffness modulates commitment of BMMSCs. Our findings may eventually aid in the development of novel, effective biomaterials for the applications in tissue engineering.
| S-EPMC5820855 | biostudies-literature
Project description:Abnormal energy metabolism is one of the characteristics of tumours. In the last few years, more and more attention is being paid to the role and regulation of tumour aerobic glycolysis. Cancer cells display enhanced aerobic glycolysis, also known as the Warburg effect, whereby tumour cells absorb glucose to produce a large amount of lactic acid and energy under aerobic conditions to favour tumour proliferation and metastasis. In this study, we report that the haploinsufficient tumour suppressor ASPP2, can inhibit HCC growth and stemness characteristics by regulating the Warburg effect through the WNT/β-catenin pathway. we performed glucose uptake, lactate production, pyruvate production, ECAR and OCR assays to verify ASPP2 can inhibit glycolysis in HCC cells. The expression of ASPP2 and HK2 was significantly inversely correlated in 80 HCC tissues. Our study reveals downregulation of ASPP2 can promote the aerobic glycolysis metabolism pathway, increasing HCC proliferation, glycolysis metabolism, stemness and drug resistance. This ASPP2-induced inhibition of glycolysis metabolism depends on the WNT/β-catenin pathway. ASPP2-regulated Warburg effect is associated with tumour progression and provides prognostic value. and suggest that may be promising as a new therapeutic strategy in HCC.
| S-EPMC9983321 | biostudies-literature
Project description:Increased stromal stiffness is associated with hepatocellular carcinoma (HCC) development and progression. However, the molecular mechanism by which matrix stiffness stimuli modulate HCC progress is largely unknown. In this study, we explored whether matrix stiffness-mediated effects on osteopontin (OPN) expression occur in HCC cells. We used a previously reported in vitro culture system with tunable matrix stiffness and found that OPN expression was remarkably upregulated in HCC cells with increasing matrix stiffness. Furthermore, the phosphorylation level of GSK3? and the expression of nuclear ?-catenin were also elevated, indicating that GSK3?/?-catenin pathway might be involved in OPN regulation. Knock-down analysis of integrin ?1 showed that OPN expression and p-GSK3? level were downregulated in HCC cells grown on high stiffness substrate compared with controls. Simultaneously, inhibition of GSK-3? led to accumulation of ?-catenin in the cytoplasm and its enhanced nuclear translocation, further triggered the rescue of OPN expression, suggesting that the integrin ?1/GSK-3?/?-catenin pathway is specifically activated for matrix stiffness-mediated OPN upregulation in HCC cells. Tissue microarray analysis confirmed that OPN expression was positively correlated with the expression of LOX and COL1. Taken together, high matrix stiffness upregulated OPN expression in HCC cells via the integrin ?1/GSK-3?/?-catenin signaling pathway. It highlights a new insight into a pathway involving physical mechanical signal and biochemical signal molecules which contributes to OPN expression in HCC cells.
| S-EPMC4539226 | biostudies-literature
Project description:The clinical translation of mesenchymal stem cells (MSCs) is limited by population heterogeneity and inconsistent responses to engineered signals. Specifically, the extent in which MSCs respond to mechanical cues varies significantly across MSC lines. Although induced pluripotent stem cells (iPSCs) have recently emerged as a novel cell source for creating highly homogeneous MSC (iMSC) lines, cellular mechanosensing of iMSCs on engineered materials with defined mechanics is not well understood. Here, we tested the mechanosensing properties of three human iMSC lines derived from iPSCs generated using a fully automated platform. Stiffness-driven changes in morphology were comparable between MSCs and iMSCs cultured atop hydrogels of different stiffness. However, contrary to tissue derived MSCs, no significant changes in iMSC morphology were observed between iMSC lines atop different stiffness hydrogels, demonstrating a consistent response to mechanical signals. Further, stiffness-driven changes in mechanosensitive biomarkers were more pronounced in iMSCs than MSCs, which shows that iMSCs are more adaptive and responsive to mechanical cues than MSCs. This study reports that iMSCs are a promising stem cell source for basic and applied research due to their homogeneity and high sensitivity to engineered mechanical signals.
| S-EPMC9119934 | biostudies-literature