Matrix stiffness modulates hepatic stellate cell activation into tumor-promoting myofibroblasts via E2F3-dependent signaling and regulates malignant progression

Abstract

The hepatic stellate cells (HSCs) activation by myofibroblastic differentiation is critical for liver fibrosis. Crosstalk between stromal cells and tumor cells in the microenvironment alters the properties and facilitates the growth and metastasis of tumor cells. How mechanical stimuli originally stiffness of extracellular matrix (ECM) contribute to tumor development remains poorly understood. Here, we demonstrated that stiffness contributes to mechanosignal transduction in HSCs, which promotes hepatocellular carcinoma (HCC) cells growth and metastasis through secretion of FGF2. On stiffness matrix, HSCs activation was confirmed by immunofluorescence (IF) and Western blot (WB) for α-smooth muscle actin (SMA). Increasing matrix stiffness promoted HSCs activation by CD36-AKT-E2F3 mechanosignaling through shRNA-mediated E2F3 knockdown, AKT inhibitors, and CD36 shRNA. Moreover, ChIP-qPCR. Confirmed that E2F3 combined the promoter of FGF2, and stiffness promoted FGF2 expression. On a stiff matrix, HCC cells cultured with conditioned media (CM) from HSCs increased HCC cells growth and metastasis by binding FGFR1 to activate PI3K/AKT and MEK/ERK signaling pathways. Moreover, conditional E2F3 knockout mice were subjected to CCl4 treatment to assess the role of E2F3 in HSC activation. Additionally, the DEN-induced HCC model was also used to evaluate the role of E2F3 in liver fibrosis and HCC growth. In conclusion, we demonstrated that stiffness-induced HSC activation by E2F3 dependent. Stiffness activated CD36-AKT-E2F3 signaling and targeted FGF2 transcription, subsequently, activated HCC growth and metastasis by FGFR1-mediated PI3K/AKT and MEK/ERK signaling.

Fig. 1. Substrate stiffness promotes E2F3 expression and MF activation of HSCs.

A HSCs were plated on a 1 or 32 kPa hydrogel and subjected to phase-contrast microscopy. B IF for α-SMA detected HSC activation-induced by increasing stiffness. C HSCs were plated on a 1 or 32 kPa hydrogel and subjected to WB with quantitative data shown. 32 kPa stiffness promoted HSC activation and E2F3 protein level. *P < 0.05 by t-test, n = 3 repeats. D IF assay was used to analyze stiffness-induced E2F3 nuclear expression. Cell nuclei were counterstained with DAPI. E LX2 cells plated on polyacrylamide hydrogels were collected for WB. F IF revealed nuclear expression of E2F3 in LX2 cells on 32 kPa. Cell nuclei were counterstained with DAPI.

Fig. 2. Stiffness induces HSC activation by an E2F3-dependent mechanism.

A Primary human HSCs on 1 or 32 kPa were transduced with lentiviruses encoding NT shRNA or E2F3 shRNA. Stiffness-mediated HSC activation was abrogated by E2F3 knockdown. *P < 0.05 by ANOVA, n = 3. B Primary HSCs seeded on a 1 or 32 kPa gel were transduced with NT shRNA or E2F3 shRNA for IF. Stiffness-mediated upregulation of α-SMA and E2F3 was abrogated by E2F3 gene deletion. Cell nuclei were counterstained by DAPI. C WB revealed that stiffness-mediated HSC activation was abrogated by E2F3 gene deletion. *P < 0.05 by ANOVA, n = 3 repeats.

Fig. 3. Stiffness promoted E2F3 expression is mediated by CD36.

A Quantitative real-time PCR after reverse transcription revealed that stiffness significantly increased CD36 mRNA level (P > 0.05 by t-test, n = 6). B Primary human HSCs and LX2 cells on hydrogels were collected for WB. CD36 protein was increased by stiffness. *P < 0.05 by ANOVA, n = 3 for WB. C Primary human HSCs or LX2 cells on 1 or 32 kPa were treated with vehicle or CD36 inhibitor SSO. Stiffness-mediated E2F3 upregulation and HSC activation was abrogated by SSO. *P < 0.05 by ANOVA, n = 3 for WB. D Primary human HSCs or LX2 transduced with retroviruses encoding LacZ (Retro-vector) or CD36 (Retro-CD36) were seeded on 1 or 32 kPa for WB. CD36 led to HSC activation, E2F3 upregulation in HSCs on 1 kPa. *P < 0.05 by ANOVA, n = 3 repeats.

Fig. 4. Stiffness promoted E2F3 expression by activating a CD36-AKT pathway.

A WB revealed that stiffness-induced AKT phosphorylation at S473. B AKT inhibitor MK2206 reduced p-AKT(S473), total E2F3, and HSC activation markers, α-SMA, CTGF level of HSCs. Data represent multiple repeats with similar results. C IF showed that MK2206 suppressed stiffness-mediated E2F3 overexpression and activation of HSCs. D CD36 inhibitor SSO reduced p-AKT, and E2F3 levels of HSCs.

Fig. 5. Stiffness-E2F3 axis promotes transcription of tumor-promoting factors FGF2 of HSCs.

A Stiffness increased FGF2 mRNA as revealed by qPCR after reverse transcription. *P < 0.05 by t-test, n = 6. B Stiffness increased FGF2 protein through HSC E2F3. *P < 0.05 by t-test, n = 3. C Conditioned medium (CM) of HSCs on different substrates were collected for ELISA to quantitate HSC-released FGF2. Stiffness promoted HSCs to release FGF2 through HSC E2F3. *P < 0.05 by ANOVA, n = 6. D E2F3/DNA complexes were pulled down by control IgG or anti-E2F3 for ChIP-qPCR and qPCR was performed with primers for FGF2 promoter. The stiffness increased E2F3 on the FGF2 promoter. *P < 0.05 by ANOVA, n = 3. E Luciferase promoter assays in HEK-293A cells of the FGF2 promoter reveal that the addition of an activating E2F3 induces an obvious increase of FGF2 promoter activity. F SSO or MK2206 reduced E2F3 on FGF2 promoter of HSCs. *P < 0.05 by ANOVA, n = 3.

Fig. 6. Stiffness potentiates tumor-promoting effects of HSCs through HSC E2F3.

A HSC conditioned media (CMs) were used as stimulants for Hep3B proliferation assays. HSC 32 kPa CM promoted Hep3B proliferation as compared to 1 kPa CM. *p < 0.05 by ANOVA, n = 6. B HSC CMs were used as attractants for Hep3B cells in the Transwell assay. CM of HSCs on 32 kPa promoted Hep3B migration as compared with. that of HSCs on 1 kPa. *p < 0.05 by ANOVA, n = 3. C Recombinant FGF2 dose-dependently promoted Hep3B proliferation. *p < 0.05 by ANOVA, n = 4. D CM of HSCs on 32 kPa promoted Hep3B proliferation and this effect of 32 kPa CM was dose-dependently reduced by a neutralizing anti-FGF2 antibody. *p < 0.05 by ANOVA, n = 3. E The effects of HSC CM on Hep3B proliferation were abrogated by E2F3 knockdown in HSCs. *p < 0.05 by ANOVA, n = 5. F CMs were used as attractants in the Transwell assay. CM of control HSCs on 32 kPa promoted Hep3B cell migration as compared to that of control HSCs on 1 kPa, and this effect of 32 kPa CM was abrogated by E2F3 knockdown in HSCs. *p < 0.05 by ANOVA, n = 6. G Serum-starved Hep3B pretreated with a CM at 37 °C for 2 h were resuspended in the CM and Hep3B/CM were then co-injected into SCID mice subcutaneously. Tumor nodules were isolated and quantitated on day 10. *P < 0.05 by ANOVA, n = 5 per group. H Tumor lysates were subjected to WB for α-SMA and Collagen I. *P < 0.05 by ANOVA, n = 3.

Fig. 7. E2F3 is overexpressed in the HSCs/MF murine and patient’s HCC.

IHC of α-SMA, collagen, and E2F3 showed strong staining in the activated-HSC/MF surrounding Hep3B/CM (A) and patients’ (B) tumors. C Atomic force microscopy (AFM) was used to measure Young’s modulus of liver cryosections. The stroma of HCC is significantly stiffer than that of normal liver. D Representative images of mouse livers from the indicated genotype induced by DEN. E WB revealed that CD36 and p-Akt levels of E2F3 F/F Cre HCC were reduced as compared to those of E2F3 + / + Cre HCC.

Fig. 8. CCl4-induced HSC activation and liver fibrosis are impaired in E2F3 F/F cre mice as compared to E2F3 + / + cre mice.

A Livers of olive oil-treated mice or CCl4-treated mice were collected for WB. CCl4 treatment upregulated α-SMA and E2F3 in murine liver. Densitometry is shown on the bottom. *p < 0.05 by t-test. B E2F3 F/F cre mice and E2F3 + / + cre mice were treated with CCl4 for 6 weeks and their livers were. collected for IF for HSC activation markers and collagen I. Quantitative IF data are shown on the right. E2F3 F/F cre livers showed reduced α-SMA and collagen I level, as compared to E2F3 + / + cre livers after CCl4 treatment. *p < 0.05 by t-test. C CCl4-treated livers were subjected to WB and densitometry data were shown on the bottom. CCl4-treated E2F3 F/F cre livers contained reduced α-SMA and collagen I level as compared to E2F3 + / + cre livers. *p < 0.05 by t-test. D, E Sirius red staining and Trichrome staining of liver sections are shown. Collagen deposition was reduced in CCl4-treated E2F3 F/F cre livers as compared to E2F3 + / + cre livers. *p < 0.05 by t-test.