Integrin-YAP/TEAD Feedback Loop Promotes Tumor Invasion by Enhanced Cell Adhesion and Cell Proliferation in Hepatocellular Carcinoma

doi:10.21203/rs.3.rs-3587839/v1

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Integrin-YAP/TEAD Feedback Loop Promotes Tumor Invasion by Enhanced Cell Adhesion and Cell Proliferation in Hepatocellular Carcinoma

https://doi.org/10.21203/rs.3.rs-3587839/v1

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Hepatocellular carcinoma (HCC) is the most common form of liver cancer with a high mortality rate. Hepatocellular carcinoma cells are known to invade surrounding tissues and organs which is a complicated process involving a number of unknown variables, such as many signaling pathways (e.g., integrins and IL-6/STAT3) or mechanical factors (e.g., stiffness). Finding successful therapies would benefit from knowing the specifics of the molecular mechanisms behind cellular activities (cell adhesion, migration, and proliferation). In this paper, we found that ITGB1, YAP1, TEAD1/3 expressions were all upregulated in tumor tissue, and ITGB1 had a significant positive correlation with YAP1 and TEAD1/3. We demonstrated through experiments and mathematical modeling that YAP/TEAD could regulate the expression of ITGB1, thus forming an obvious ITGB1-YAP/TEAD positive feedback loop. The experimental results also showed that PFL significantly enhanced the cell proliferation and migration ability. Finally, we used the cellular Potts model (CPM) to demonstrate that tumor cells with ITGB1-YAP/TEAD positive feedback loop can significantly have a stronger invasive capacity.

Integrin

Hepatocellular Carcinoma

Tumor invasion

YAP

TEAD

Hepatocellular carcinoma (HCC) is the most common form of liver cancer, accounting for about 90% of cases¹. Currently, the mortality rate of HCC remains high, especially in cases diagnosed at an advanced stage². According to the American Cancer Society, the 5-year survival rate for all stages of HCC combined is only about 20%³. HCC metastasis occur when liver cancer cells migration from the liver tissue to other parts of the body (such as bone or breast tissue). The process of metastasis is complex and involves many factors, including the ability of cancer cells to invade surrounding tissue (i.e., invasion) through cell-matrix interactions (such as cell adhesion) and to survive in a new environment. In addition, certain genetic mutations can promote metastasis by allowing cancer cells to grow and divide uncontrollably (such as increased cell proliferation). Overall, liver cancer metastasis in HCC is a complex process involving many factors that are not fully understood. Understanding the details of the molecular mechanism of cellular behaviors (cell adhesion, migration and proliferation) would help to find effective treatments.

From perspective of biomechanics and mechanobiology, HCC is a typical cancer because it occurs in an environment that is already very stiff. For instance, eighty to ninety percent of HCC cases occur in stiff, cirrhotic livers, which are substantial risk factors for the development of the disease⁴. This suggests that mechanical changes in the liver tissue may be a source of abnormal state of cancer cells leading to cancer development and metastasis. During the development of HCC, mechanical forces are transmitted from the extracellular matrix (ECM) via cell adhesion (such as integrins) of hepatocytes and hepatic stellate cells (HSCs) to the nucleus, ultimately influencing cell behaviour⁵. In liver sinusoidal endothelial cells (LSECs), VE-cadherin and PECAM-1 transmit shear stress between cells, leading to the activation of integrins⁵. In addition, cation channels are activated by mechanical stretch on LSECs⁶. In addition, increased substrate stiffness was found to promote proliferation and chemotherapy resistance through β1-integrin, FAK and YAP in hepatocellular carcinoma (HCC) cells^5,7. Thus, a mechanosensitive signaling network (such as integrin-FAK-YAP axis) is established and plays key roles in HCC. Therefore, it is now crucial to identify the relationship between mechanosensitive signaling pathways and HCC progression.

Studies have shown that the typical mechanosensitive pathway, integrin-YAP axis, plays a key role in tumor progression by promoting tumor cell survival, proliferation, migration, invasion and drug resistance^8,9. For example, α2β1 integrin activation through cellular adhesion affects the Hippo pathway in solid tumors and modulates the MST1-YAP signaling cascade¹⁰. Besides, integrin-αV (ITGAV) is a novel TAZ-specific target gene whose overexpression in human HCC patients correlates with poor clinical outcome, TAZ expression in HCCs, and abundance of YAP/TAZ target genes¹¹. In addition, ITGAV contributes to actin stress fiber assembly, tumor cell migration and invasion. Thus, targeting integrin holds promise for the treatment of HCC. However, whether liver cancer cell metastasis is related to integrin-mediated cell adhesion and enhanced cell proliferation is still unclear.

In this paper, we firstly found that ITGB1, YAP1, TEAD1/3 expressions were all upregulated in HCC in the database. Furthermore, ITGB1 had a significant positive correlation with YAP1 and TEAD1/3, and the correlation was further enhanced with tumor progression. Subsequently, we demonstrated through experiments and mathematical modeling that YAP/TEAD could regulate the expression of ITGB1, thus forming an obvious ITGB1-YAP/TEAD positive feedback loop (PFL). The experimental results also showed that PFL significantly enhanced the proliferation and migration ability of liver cancer cells (SNU-739 and LM3 cell lines). Finally, we used the cellular Potts model (CPM) to demonstrate that tumor cells with ITGB1-YAP/TEAD positive feedback loop can significantly have a stronger invasive capacity.

Cell culture

The HCC cell lines SNU-739 and LM3 were cultured in DMEM supplemented with 10% fetal bovine serum (Invitrogen, CA, USA) and 1% penicillin-streptomycin in an incubator at 37°C with 5% CO2.

Western blotting analysis

The harvested cells were lysed with RIPA on ice for 30 min. Then, the proteins were harvested by low-temperature high-speed centrifugation for 10 min. The primary antibodies used in our study include Integrin Beta 1 polyclonal antibody (1:1000; Proteintech, 12594-1-AP, China), β-Actin monoclonal antibody (1:2000; Sigma-Aldrich, A5316, USA). Goat anti-rabbit HRP-conjugated antibody (1:5000; Beyotime, A0181, China) and goat anti-mouse HRP-conjugated antibody (1:5000; Beyotime, A0216, China) were used as secondary antibodies. Then, the ultra-high sensitivity ECL kit (Mishushengwu, MI00607A, China) was used for chromogenic development for 1 to 30 seconds. The bands were detected using a chemiluminescence system Tanon 5500 (Tanon Science & Technology, China). The ImageJ program was used to quantify the detected protein bands.

Wound healing assay

Cells were seeded into 6-well plates. When cells reached confluence, a wound was made with a 100-µL sterile pipette tip and photographed (0 h). The rate of wound closure was measured at different time points. Each experiment was performed in triplicate.

Plate colony formation assay

The trypsinized cells were suspended and seeded into 6-well plates peer well. two weeks later, the cells were washed with PBS and fixed with 4% paraformaldehyde for 15 minutes and stained with 0.5% (w/v) crystal violet (Sigma-Aldrich) for 15 minutes. Colons in the plate were scanned using Odyssey Scanner (LI-COR, Lincoln, NE, USA) and the number of colons was quantified by Image J software.

The soft agar colony formation assay

cells were resuspended in PRMI 1640 containing 10% fetal bovine serum along with 0.3% low-melting agarose and plated onto a layer of 0.5% agrarose-containing medium in 6-wells plate (5 000 to 10 000 cells per well). Colonies were counted after 2–3 weeks, a phase-contrast microscopic pictures were taken for each sample using a digital camera coupled to a microscope.

TIMER (Tumor Immune Estimation Resource)

TIMER (https://cistrome.shinyapps.io/timer/) is a web server that provides immune infiltrates’ abundances estimated by multiple immune deconvolution methods, and allows users to generate high-quality figures dynamically to explore tumor immunological, clinical and genomic features. In this study, we analyzed the correlation of ITGB1-YAP1, ITGB1-TEAD1 and ITGB1-TEAD3 in HCC by gene modules. The correlation analysis is the Spearman’s correlation, statistical significance with p < 0.05 was indicated with ∗; p < 0.01 was indicated with ∗∗; p < 0.001 were indicated with ∗∗∗; and p < 0.0001 were indicated with ∗∗∗∗.

GEPIA (Gene Expression Profiling Analysis) 2 Database

GEPIA2 (http://gepia2.cancer-pku.cn/#index) is an enhanced web server for large-scale expression profiling and interactive analysis. It has extended gene expression quantification from the gene level to the transcript level, and supports analysis of a specific cancer subtype, and comparison between subtypes. It features 198 619 isoforms and 84 cancer subtypes. In this paper, for the expression of ITGB1/YAP1/TEAD1/3 in figure, the threshold of |log2FC| was 1, p-value cutoff was 0.05. Statistical significance with p < 0.05 was indicated with ∗, p < 0.01 was indicated with ∗∗, p < 0.001 were indicated with ∗∗∗, and p < 0.0001 were indicated with ∗∗∗∗. Besides, in this study, we also generated the survival map of ITGB1/YAP1/TEAD1/3 in the “survival analysis” module, and the significance level was 0.05.

cBioPortal

The cBio Cancer Genomics Portal (www.cbioportal.org/) is an open-access platform for interactive study of multidimensional cancer genomics data sets, with data from over 5,000 tumor samples from 20 cancer studies now available. This study looked at copy number variation (CNV) of ITGB1 and YAP1 in HCC.

ODE (Ordinary differential equation) modeling

In this work, we have created a mathematical model of a positive feedback loop between integrin and YAP (or TEAD1/3). In particular, we have built a rudimentary dynamical model consisting of two molecules: YAP/TEAD (for the ability of cell proliferation) and ITGB1 (for the ability of cell adhesion). According to our experimental results, there are two main regulatory pathways: (1) YAP1 expression is enhanced by integrin, and (2) integrin expression is enhanced by YAP/TEAD. Michaelis-Menten kinetics can be used to characterize the dynamics of various chemical processes. To further mimic the dynamics of positive feedback loops, we used a coupled ordinary differential equation. The mathematical equations are as follows:

$$\frac{d\left[{C}_{ITGB1}\right]}{dt}=\left[{k}_{1f}+\delta \left(t\right)\frac{{{C}_{YAP-TEAD}}^{n}}{1+{{C}_{YAP-TEAD}}^{n}}\right]\left(1-\left[{C}_{ITGB1}\right]\right)-{k}_{1r}\left[{C}_{ITGB1}\right]$$

$$\frac{d\left[{C}_{YAP-TEAD}\right]}{dt}={k}_{2f}\left(1-\left[{C}_{YAP-TEAD}\right]\right)-{k}_{2r}\left[{C}_{YAP-TEAD}\right]$$

where ${k}_{1f}$ (1 s^− 1) and ${k}_{2f}$ (1 s^− 1) represent the basal values of protein production; and ${k}_{1r}$ (0.1 s^− 1) and ${k}_{2r}$ (0.1 s^− 1). Here, we assumed that $\delta \left(t\right)$ is the positive feedback strength (from 0 and finally to 20); n (5) is Hill coefficient which represents the activation threshold. $\left[{C}_{ITGB1}\right]$ and $\left[{C}_{YAP-TEAD}\right]$ represent the concentrations of integrin, and YAP/TEAD molecules, respectively. All these concentrations are normalized to 1. Above all equations are solved numerically by Matlab.

Cellular Potts Model

The Cellular Potts Model (CPM) is a computational model of cells and tissues used to simulate individual and collective cell behavior, tissue morphogenesis and cancer development1. It is a spatial lattice-based formalism for the study of spatiotemporal behavior of biological cell populations. The details of intercellular interaction are essentially determined by the shape and the size of the individual cells as well as the length of the contact area between neighboring cells. Here is a general outline of how to build a Cellular Potts Model (CPM) for tumor cells entering the matrix microchannel from the left to the right: (1) Define the geometry of the microchannel and the initial position of the tumor cells. (2) Define the parameters of the CPM such as cell-cell adhesion energy, cell-substrate adhesion energy, and cell-cell surface tension. (3) Initialize the CPM lattice with cells and their properties. (4) Simulate the movement of cells using Monte Carlo methods. (5) Implement boundary conditions that reflect the geometry of the microchannel. (6) Analyze simulation results.

For each configuration of cells, the CPM utilizes a function known as the Hamiltonian H to characterize the energy (favorable behaviors). By selecting a random lattice site x, a section of a cell-cell interface, or a cell-media interface, cell motility evolved by trying to duplicate that site to a random nearby lattice site x'. The Hamiltonian was described as the product of four constraints, each of which stands for one of the four physical characteristics of simulated stem cells: 1) conservation of cell area, 2) locally polarized cell migration, 3) cell-cell adhesion, 4) and cell membrane length, which is frequently used to represent cortical tension. In the CPM, the objective was to reduce the Hamiltonian or the number of times that the ideal cellular behaviors were violated.

The free energy for a configuration of cells was defined as the sum of four constraints: local cell-cell/cell-ECM adhesion and cell area conservation:

$$H={H}_{adhesion}+{H}_{area}$$

For a configuration of cells, the free energy due to cell adhesion was

$${H}_{adhesion}=J(1-\delta ({\sigma }_{x},{\sigma }_{{x}^{{\prime }}}\left)\right)$$

where J represented the cell adhesion strength between lattice sites ${\sigma }_{x}$ and ${\sigma }_{{x}^{{\prime }}}$ that was defined by their cell type. The energy due to cells resisting changes from theirresting area was defined as,

$${H}_{area}=\sum \lambda {(a-A)}^{2}$$

where A represented the target area of a cell, a represented the current area of a cell, and $\lambda$ was the relative strength of area conservation term.

Statistical analysis

GraphPad Prism 8 (GraphPad Software, San Diego, CA, USA) was used for statistical analysis. Data were presented as mean ± standard deviation (SD). Two-tailed Student's t-test was used to compare the difference between two samples. One-way analysis of variance (ANOVA) with Tukey post hoc test and Kruskal-Wallis test was performed for comparisons between three or four samples (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001).

1. Positive correlation between ITGB1 and YAP/TEAD

Current studies have shown that liver tissue is accompanied by elevated stiffness during the development of HCC, and therefore, cellular mechanosensing behaviour plays an important role in this. Therefore, to comprehend the function of integrin-YAP mechanosensitive signalling pathway in HCC, we first compared transcriptional expression of ITGB1 gene between autopsy normal ovarian sample from GTEx (Genotype Tissue Expression) and HCC samples from TCGA (The Cancer Genome Atlas) by GEPIA2. We found that ITGB1, YAP1, TEAD1/3 were all significantly higher in HCC tumor tissue than in normal liver tissue (Fig. 1A). Then, we evaluated the relationship between expressions of ITGB1, YAP1, TEAD1 and HCC cancer patient outcomes using the GEPIA2 database. As shown in Fig. 1B, we could found that only ITGB1 was significantly associated with poor prognosis in HCC. However, interestingly, by prognostic analysis YAP1 and TEAD1 was not associated with patient prognosis (Fig. 1. C-D).

To further explore the mechanism of differential expression of ITGB1 in HCC and normal liver tissue, we analysis the correlations between these mechanosensitive molecules from TIMER and cBioPortal. Subsequently, we can see that ITGB1-YAP1 all showed significant positive correlations in HCC samples from TCGA (Fig. 1E) and normal ovarian sample from GTEx (Fig. 1F). Besides, ITGB1-CTGF, ITGB1-CYR61, ITGB1-TEAD1, ITGB1-TEAD3 all showed significant positive correlations from TIMER (Fig. 1G-H). Finally, to investigate the roles of such positive correlations in the development of HCC, we calculates the values of positive correlations in different stages of HHC. Interestingly, we found that the positive correlation of ITGB1-YAP1 gradually increased with the development of HCC (from 0.56 in Grade 1 to 0.88 in Grade 4) (Fig. 1I). Since matrix stiffness also gradually increased with the progression of HCC, we tentatively suggest that ITGB1-YAP mechanosensing signals play an important role in the development of HCC.

2. YAP/TEAD regulate ITGB1 expression in liver cancer cells

Next, we further explore how and why ITGB1 and YAP/TEAD can form a positive correlation during development in HCC. We developed an ordinary differential equation (ODE) model to describe the mutual regulation of the two genes (Fig. 2A). In this mathematical model, the ITGB1 and YAP/TEAD proteins are produced by their genes with rates λ₁ and λ₂ and degraded with rates α₁ and α₂, respectively. Here, we used the parameter of δ to represent the strength of YAP/TEAD protein activating the ITGB1 gene (Fig. 2B). In our simulation, for the case δ = 0 which means that YAP/TEAD does not regulate ITGB1, the results showed that there is no significant correlation between ITGB1 and YAP/TEAD (Fig. 2C). However, when the values of δ gradually increases (i.e., the activating strength of ITGB1 gene by YAP/TEAD protein increasing), the correlation coefficient of ITGB1 and YAP/TEAD gradually increases (Fig. 2D). These simulation results suggest that ITGB1 and YAP/TEAD show a significant positive correlation when there is a significant regulatory effect of YAP/TEAD protein on ITGB1 gene expression.

Subsequently, we will further validate results of mathematical model through experiments. Modeling results led us to hypothesize that YAP/TEAD may regulate ITGB1 at the transcriptional level in liver cancer cells. We then analyzed the public-opened ChIP-seq database and found the obvious binding peaks of TEAD1/3 on the proximal region of ITGB1 locus in hepatocellular carcinoma cell lines (Fig. 2E). For further verification, we subsequently knocked down YAP1 and TEAD1/3 separately through Western blot (WB) experiments, and the cell line validation results showed that we successfully knocked them down in LM3 cell lines (Fig. 2F). Importantly, we found that ITGB1 was significantly down-regulated in the knockout cells (Fig. 2F).

Subsequently, we have labelled the nucleus, cytoskeleton and integrin β1 by immunofluorescence assay and the results are consistent with WB assay (Fig. 2G). Knockdown of YAP/TEAD significantly prevented the formation of cell adhesion. For now, there is a large body of literature showing that integrin significantly activates the expression of YAP protein. Overall, therefore, by combining the experiments on YAP regulation of ITGB1 expression in this paper, we demonstrate that there is a significant positive feedback loop (PFL) in liver cancer cells. This ITGB1-YAP/TEAD positive feedback loop predicts that cells may be in two states, the normal state (low force-sensitive signalling activity) and the abnormal state (high force-sensitive activity state).

3. ITGB1-YAP/TEAD PFL promotes the cell migration and proliferation

Next, we explore the role of ITGB1-YAP/TEAD PFL in HCC invasion. First, we used two independent shRNAs to stably knock down YAP and test the migration and proliferation in SNU-739 and LM3 cells respectively. Subsequently, we performed Transwell analysis to observe cell migration behaviour at time (0 h) and (24 h), respectively (Fig. 3A-B). The results showed that YAP knockdown effectively inhibited the migration of SNU-739 and LM3 cells. And the further clonogenic formation assay showed that in both 2D and 3D cell culture system, the number and the size of cell clones obviously decrease in YAP-knockdown cells when compared with that in control group (Fig. 3C-F). In summary, we can find that the positive feedback loop formed by ITGB1-YAP can effectively enhance the cell proliferation and migration ability of hepatocellular carcinoma cells. Specially, cancer cells with ITGB1-YAP/TEAD PFL have strong proliferation and migration capabilities, while conversely, they do not.

4. Integrin-mediated cell-ECM interaction promotes HCC invasion

Next, we further explore the role of integrins in tumor invasion. Cellular Potts model (CPM) are often used to describe processes such as multicellular tissue development and cancer migration with some advantage¹². We thus developed a CPM to describe the invasive process of tumor cells (Fig. 4A). In the simulated region of CPM, a population of tumor cells is set up on the left, with the extracellular matrix microchannels in somewhat green, through which the tumor cells can metastasize into the blood vessels. Integrins can form significant cell adhesions with the extracellular matrix and here we hypothesis that expression level of integrins (ITGB1) is positively correlated with strength of cell-ECM interactions (J = 1, 5, 10). Also, the rise in J suggests a gradual development of positive feedback loop in cells.

Later, we performed simulations using our CPM. The simulation results showed that enhanced cell-ECM interaction (integrin mediated PFL) significantly enhanced the invasive capacity of the tumor cells (Fig. 4A). When J = 10, tumor cells did not enter the microchannel significantly with increasing time. And when J decreased (i.e., cell-ECM interaction increased), the invasion length of tumor cells in the microchannels gradually increased (Fig. 4B). This result suggests that as HCC gradually develops, ITGB1-YAP/TEAD PFL can maintain cells in a state of higher integrin concentration (more cell adhesion and greater cell-ECM interaction); and the higher integrin concentration can produce stronger cell adhesion to the extracellular matrix, which in turn produces stronger invasive ability.

5. Increased cell proliferation promotes HCC invasion

Our above experiments revealed that ITGB1-YAP/TEAD PFL (increased YAP) significantly enhanced the cell proliferation of tumor cells. And this may be caused by high expression of YAP¹³. Therefore, next, in the CPM, we mirrored the cell proliferation process of the cells by introducing the cell mitosis module with a cell division rate 0.1 and 0.5. We can find that the simulation results show that when the proliferation capacity of tumor cells is enhanced, the tumor cells are able to access more extracellular matrix microchannels, and in turn are able to enter more extracellular matrix and cause more cell invasion (Fig. 5A-B). The length of tumor invasion increased significantly with time; the average length of tumor invasion in microchannels increased significantly with increased cell-ECM adhesion (Fig. 5C). Finally, we can see that in the presence of ITGB1-YAP/TEAD PFL, cancer cells exhibit a state of high adhesion-high proliferation capacity, and such a state is conducive to the invasion and metastasis of liver cancer cells, leading an invasion of HCC.

This study firstly found that typical mechanosensing pathway (that is integrin-YAP axis)

demonstrates that YAP/TEAD can directly promote the expression of integrin β1. Besides, other studies have shown that integrin also can induce the increase of expression of YAP activation. Thus, a significant positive feedback loop between integrin and YAP/TEAD can form in liver cancer cells here. Later, we further experimentally found that such PFL can also promote the cell migration and proliferation in LM3 and SUN cell lines. Finally, we developed a CPM to investigate the role of integrin mediated cell adhesion and enhanced cell proliferation in HCC invasion.

ITGAV was identified as a TAZ specific target gene, and TAZ-induced ITGAV expression contributes to a positive feedback loop regulating YAP/TAZ activation by the modulation of actin stress fiber assembly. In other words, TAZ regulates ITGAV expression, which in turn affects the assembly of actin stress fibers and activates YAP/TAZ signaling. This mechanism is involved in promoting cancer cell migration and invasion. However, the relationship between YAP/TEAD and other integrin isoform is still unclear.

Prior exposure to stimuli can hasten or improve reactions to future stimuli in many biological systems. PFL, which are known to provide "bistability" and so permit long-term or permanent recall of previous environmental circumstances, frequently act as a mediating factor in this process. PFL can play distinctly different functions in many regulatory systems, including eukaryotic cell cycle transitions and the development of vertebrate oocytes. In the context of metabolic changes or animal growth, several studies have demonstrated that genetically identical cells can respond differently to the same environmental signals. For instance, factors that cause cell differentiation may also cause other cells to proliferate.

The metabolic or developmental status of the cell, which is dependent on the environmental history of the cell, often determines the result of stimulation. This kind of history-dependent behavior, referred to as "hysteresis," often results from bistability, which is mediated by a PFL. In this situation, the cell can be irreversibly switched to an induced state by the prior environmental circumstances, which results in an irreversible cellular phenotype. Therefore, more research on PFL's function in liver cells is required.

The differentiation and activation of fibroblasts and myofibroblasts, which in turn promotes matrix synthesis and stiffening, are important factors in the development of fibrosis. A progressive feedforward loop, or PFL, is produced by these compositional and mechanical modifications. Through mechanotransduction pathways, which enable tissue-resident cells to monitor changes in mechanical cues and convert mechanical signals to downstream biochemical signals, biomechanical features of tissues are recognized and reacted to. Recent research has made it possible for us to start elucidating the precise processes that sustain the feedforward loops connected to cancer and fibrosis, even if our knowledge of the mechanotransduction pathways involved in the development of cancer and fibrosis is still limited.

The growth of cancer or fibrosis can currently be slowed down, but neither scarring nor inducing total death of cancer cells is now possible. therapy approaches targeting cellular mechanosensing may be essential targets for integration with current therapy regimens given the importance of mechanical microenvironment driven PFL in the start and progression of cancer and fibrosis. As a result, this study explores the effects of PFL on biological behavior at various scales as well as potential therapeutic insights based on mechanistically mediated PFL.

In summary, our results highlight the roles of ITGB1-YAP/TEAD positive feedback loop in liver cancer cell adhesion and proliferation, leading to an enhanced HCC invasion. Inhibiting integrin and YAP/TEAD simultaneously may disrupt the formation of such PFL, thereby inhibiting HCC invasion. Our results thus provide a theoretical basis for the development of potential clinical drugs.

Acknowledgements

This work was financially supported by National Natural Science Foundation of China (12302411), the Natural Science Foundation of Shaanxi Province (2023-JC-QN-0224, 2023-JC-QN-0851), the Province Key R&D Program of Shaanxi Province (2021ZDLSF01-07), Young Elite Scientists Sponsorship Program by Shaanxi Association for Science and Technology (SWYY202221), the Science and Technology Incubation Fund and Talent Program Projects of Shaanxi Provincial People’s Hospital (2022JY-44, 2022YJY-30, 2021LJ-02).

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No competing interests reported.

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Integrin-YAP/TEAD Feedback Loop Promotes Tumor Invasion by Enhanced Cell Adhesion and Cell Proliferation in Hepatocellular Carcinoma

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Abstract

Figures

Introduction

Methods and materials

Cell culture

Western blotting analysis

Wound healing assay

Plate colony formation assay

The soft agar colony formation assay

TIMER (Tumor Immune Estimation Resource)

GEPIA (Gene Expression Profiling Analysis) 2 Database

cBioPortal

ODE (Ordinary differential equation) modeling

Cellular Potts Model

Statistical analysis

Results

1. Positive correlation between ITGB1 and YAP/TEAD

2. YAP/TEAD regulate ITGB1 expression in liver cancer cells

3. ITGB1-YAP/TEAD PFL promotes the cell migration and proliferation

4. Integrin-mediated cell-ECM interaction promotes HCC invasion

5. Increased cell proliferation promotes HCC invasion

Discussion

Declarations

Acknowledgements

References

Additional Declarations

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