A novel mechanism regulates the tumor cell rigidity and cancer-associated fibroblast infiltration through the collagen IV/integrin-initiated signaling pathway

Abstract

It has been recognized that stromal cells and tumor microenvironment play pivotal roles in tumor progression Collagen IV deposition in tumor microenvironment can influence cancer cells invasive capacity stromal cell behaviors and tissue tension homeostasis To gain a better understanding of the underlying molecular mechanism we knocked-down the type IV collagen alpha 1 gene (Col4A?) of cells by the lentiviral-mediated RNA interference strategy designated as shCol cells Although there was no obvious effect on cell growth in vitro silencing the Col4A? gene decreased the tumorigenicity of B16F10 in C57BL/6 mice which was accompanied by a reduction in the stromal infiltration of alpha-smooth muscle actin-positive (?-SMA+) fibroblasts Silencing Col4-A??gene or disrupting integrin engagement by blocking antibody reduced the expression of platelet-derived growth factor A (PDGF-A) a potent chemotactic factor for fibroblasts Furthermore ectopic expression of the autoclustering integrin mutant significantly stimulated PDGF-A expression in murine B16F10 human U118MG and Huh7 cells PDGF-A-specific sh-RNA and neutralizing anti-PDGF-A antibody effectively inhibited the transwell migration of fibroblasts Adding recombinant PDGF-A back to shCol cells-conditioned media restored the fibroblast-attraction ability supporting the notion that PDGF-A is a major chemotactic factor for fibroblasts in the current model The integrin-associated PDGF-A production correlated with the activation of Src and ERK High type IV collagen staining intensity colocalized with elevated PDGF-A expression was observed in tumor tissues obtained from hepatoma and glioma patients The integrin signal pathway was activated by collagen engagement through Src and ERK leading to enhanced PDGF-A production which serves as a key regulator of fibroblast recruitment Further to understand how type IV collagen affects mechanical rigidity and migration Although having similar growth rates shCol cells featured a flatter morphology compared to that of the corresponding controls Notably knocking-down the Col4A1gene endowed the cells with higher levels of elasticity and lower motility Exposure to blocking antibodies against human β1integrin ?2β1integrin or the pharmacological inhibition of Src and ERK activity by PP1 and U0126 respectively effectively reduced cell motility and raised cell stiffness Reduced Src and ERK activities in shCol cells indicate the involvement of a collagen IV/integrin signaling pathway The forced expression of β1 integrin significantly stimulated Src and ERK phosphorylation reduced cell stiffness and accelerated cell motility In an experimental metastasis assay using C57BL/6 mice B16F10 shCol cells formed significantly fewer and smaller lung nodules confirming the contribution of collagen to metastasis In summary the integrin signaling pathway activated in a tumor environment with collagen deposition appears to be responsible for cancer-associated fibroblast recruitment low cell elasticity and high metastatic ability

Date of Award	2015 Feb 10
Original language	English
Supervisor	Bei-Chang Yang (Supervisor)