Stiffer Matrix Accelerates Migration of Hepatocellular Carcinoma Cells through Enhanced Aerobic Glycolysis Via the MAPK-YAP Signaling

Abstract

Increased extracellular matrix (ECM) stiffness and metabolic reprogramming of cancer cells are two fundamental mediators of tumor progression, including hepatocellular carcinoma (HCC). Yet, the correlation between ECM stiffness and excessive aerobic glycolysis in promoting the development of HCC remains unknown. Here, we demonstrated that stiffer ECM promotes HCC cell migration depending on their accelerated aerobic glycolysis. Our results also indicated that stiffer ECM-induced YAP activation plays a major role in promoting aerobic glycolysis of HCC cells. Moreover, we showed that JNK and p38 MAPK signaling are critical for mediating YAP activation in HCC cells. Together, our findings established that the MAPK-YAP signaling cascade that act as a mechanotransduction pathway is essential for promoting HCC cell aerobic glycolysis and migration in response to ECM stiffness.

Keywords: ECM stiffness; aerobic glycolysis; hepatocellular carcinoma; mechanotransduction; migration.

Figure 1

Migration of HCC cells is regulated by ECM stiffness. (a,b) Transwell assay analysis (a) and wound scratch assay analysis (b) of HepG2 and MHCC97L cells migration after cultured on different stiffness of hydrogel for 48 h; quantitative analysis is shown below. (Scale bar: 100 µm; n = 3, * p < 0.05, ** p < 0.01).

Figure 2

Aerobic glycolysis of HCC cells is regulated by ECM stiffness. (a,b) Measurement of glucose consumption (a) and lactate production (b) of HepG2 and MHCC97L cells cultured on different stiffness of hydrogel for 48 h. (c) qRT-PCR analysis of indicated genes mRNA levels in HCC cells. (d) Western blot analysis of indicated protein levels in HCC cells. (n = 3, * p < 0.05, ** p < 0.01).

Figure 3

Aerobic glycolysis is responsible for stiffer ECM-mediated migration. (a) Western blot analysis showed the protein expression of HKII in HepG2 and MHCC97L after knockdown of HKII (n = 3, ** p < 0.01 versus control-siRNA group). (b) Transwell analysis of HKII-knockdown HepG2 and MHCC97L cells migration (Scale bar: 100 µm; n = 3, ** p < 0.01). (c) Wound scratch assay analysis of HepG2 and MHCC97L cells after treatment with 2-DG (20 mM) (Scale bar: 100 µm; n = 3).

Figure 3

Aerobic glycolysis is responsible for stiffer ECM-mediated migration. (a) Western blot analysis showed the protein expression of HKII in HepG2 and MHCC97L after knockdown of HKII (n = 3, ** p < 0.01 versus control-siRNA group). (b) Transwell analysis of HKII-knockdown HepG2 and MHCC97L cells migration (Scale bar: 100 µm; n = 3, ** p < 0.01). (c) Wound scratch assay analysis of HepG2 and MHCC97L cells after treatment with 2-DG (20 mM) (Scale bar: 100 µm; n = 3).

Figure 4

YAP activity is regulated by ECM stiffness. (a) Western blot analysis of total YAP and p-YAP protein levels in HepG2 and MHCC97L cells cultured on different stiffness of hydrogel for 48 h (n = 3, * p < 0.05, ** p < 0.01). (b) qRT-PCR analysis of indicated genes mRNA levels in HepG2 and MHCC97L cells (n = 3, * p < 0.05, ** p < 0.01). (c) Confocal immunofluorescence images of YAP in HepG2 and MHCC97L cells cultured on different stiffness of hydrogel for 48 h (Scale bar: 25 µm).

Figure 4

YAP activity is regulated by ECM stiffness. (a) Western blot analysis of total YAP and p-YAP protein levels in HepG2 and MHCC97L cells cultured on different stiffness of hydrogel for 48 h (n = 3, * p < 0.05, ** p < 0.01). (b) qRT-PCR analysis of indicated genes mRNA levels in HepG2 and MHCC97L cells (n = 3, * p < 0.05, ** p < 0.01). (c) Confocal immunofluorescence images of YAP in HepG2 and MHCC97L cells cultured on different stiffness of hydrogel for 48 h (Scale bar: 25 µm).

Figure 5

YAP is responsible for ECM stiffness-mediated aerobic glycolysis. (a) Western blot analysis of YAP expression in HepG2 and MHCC97L cells after YAP knockdown (n = 3, ** p < 0.01 versus control-siRNA group). (b) qRT-PCR analysis of indicated genes mRNA levels in YAP-knockdown HepG2 and MHCC97L cells. (c) Western blot analysis of indicated proteins in YAP-knockdown HepG2 and MHCC97L cells. (d,e) Measurement of glucose consumption (d) and lactate production (e) in HepG2 and MHCC97L cells after YAP knockdown (n = 3, * p < 0.05, ** p < 0.01).

Figure 5

YAP is responsible for ECM stiffness-mediated aerobic glycolysis. (a) Western blot analysis of YAP expression in HepG2 and MHCC97L cells after YAP knockdown (n = 3, ** p < 0.01 versus control-siRNA group). (b) qRT-PCR analysis of indicated genes mRNA levels in YAP-knockdown HepG2 and MHCC97L cells. (c) Western blot analysis of indicated proteins in YAP-knockdown HepG2 and MHCC97L cells. (d,e) Measurement of glucose consumption (d) and lactate production (e) in HepG2 and MHCC97L cells after YAP knockdown (n = 3, * p < 0.05, ** p < 0.01).

Figure 6

JNK and p38 MAPK signaling regulate stiffer ECM-induced YAP activation. (a) Western blot analysis of the indicated proteins in HepG2 and MHCC97L cells cultured on different stiffness of hydrogels for 48 h. (b,c,d) Western blot analysis of the indicated proteins in HepG2 and MHCC97L cells treated with U0126 (10 μM), SP600125 (20 μM), SP600125 (10 μM) or vehicle. (e) Confocal immunofluorescence images of YAP in HepG2 and MHCC97L cells after treated with U0126, SP600125, SP600125 or vehicle (Scale bar: 25 µm). (f) qRT-PCR analysis of indicated genes mRNA levels in HepG2 and MHCC97L cells after treated with U0126, SP600125, SP600125 or vehicle (n = 3, * p < 0.05, ** p < 0.01). (g) Transwell assay analysis of HepG2 and MHCC97L cells migration after indicated treatment (n = 3, * p < 0.05, ** p < 0.01).

Figure 6

JNK and p38 MAPK signaling regulate stiffer ECM-induced YAP activation. (a) Western blot analysis of the indicated proteins in HepG2 and MHCC97L cells cultured on different stiffness of hydrogels for 48 h. (b,c,d) Western blot analysis of the indicated proteins in HepG2 and MHCC97L cells treated with U0126 (10 μM), SP600125 (20 μM), SP600125 (10 μM) or vehicle. (e) Confocal immunofluorescence images of YAP in HepG2 and MHCC97L cells after treated with U0126, SP600125, SP600125 or vehicle (Scale bar: 25 µm). (f) qRT-PCR analysis of indicated genes mRNA levels in HepG2 and MHCC97L cells after treated with U0126, SP600125, SP600125 or vehicle (n = 3, * p < 0.05, ** p < 0.01). (g) Transwell assay analysis of HepG2 and MHCC97L cells migration after indicated treatment (n = 3, * p < 0.05, ** p < 0.01).

Figure 7

Schematic model for stiffer ECM-induced MAPK and YAP activation for metabolic reprogramming and migration of HCC cells. Stiffer ECM stimulates a signaling cascade through activation of MAPK and YAP to transduce biomechanical force. Stiffer ECM activates JNK and p38 and promotes their phosphorylation. The activated JNK and p38 dephosphorylate YAP and promote its localization in the nucleus. Then, activated YAP increases the expression of glycolysis-related genes, which in turn promotes cell glycolysis and migration.