Role of integrin β4 expression in glioblastoma invasion and proliferation

Abstract

Background: Glioblastoma (GBM) is the most malignant form of glioma and characterized by aggressive local invasiveness, making complete surgical resection of the cancer nearly impossible. To identify mechanisms of GBM invasion, we isolated highly invasive cells from the U87MG cell line via adhesion to a laminin-2 substrate and comparatively analyzed characteristics of U87MG and U373MG cells. Furthermore, we focused and analyzed the function, clinicopathologic features and prognosis of integrin β4 expression in GBM. Methods: We selected the first 10% of invading cells (U87-Inv) from the U87MG GBM cell line using laminin-2-coated Transwell filters. To characterize the highly invasive cells, we performed a wound-healing assay and proliferation assay. Also, we evaluated the expression of invasion-related factors, including gelatin zymography for matrix metalloproteinase-2 (MMP-2), immunofluorescence for fascin and actin, real-time quantitative polymerase chain reaction (RT-qPCR) for integrin subunits and western blot for fascin, Akt, and Erk. To focus and evaluate the function of integrin β4, we used the method of pRK5 β4 plasmid DNA transfection for U87MG and knockout with integrin β4 shRNA for U373MG. Moreover, integrin β4 expression was determined using immunohistochemistry (IHC) in a retrospective cohort of 89 consecutive patients with GBM. Cytoplasmic staining of moderate to strong intensity in neoplastic cells was considered positive staining. In addition, epidermal growth factor receptor (EGFR), p53, and isocitrate dehydrogenase 1 (IDH-1) expressions and Ki-67 were investigated by IHC. Results: The migration rate of U87-Inv cells increased approximately 20% compared with that of the relatively less invasive cells (U87-Non). U87-Inv cells demonstrated faster wound healing, but lower proliferative activity. U87-Inv cells also showed extensive lamellipodia with the expression of fascin and actin, an increase in the activity of MMP-2, but a decrease in the expression of Erk. The expression of integrins α1 and α7 in U87-Inv cells increased approximately 1.5-fold, whereas that of integrins α6 and β4 was reduced by approximately 0.4- and 0.6-fold, respectively. Interestingly, U373MG cells showed slower migration rate and more increased proliferation rate than that of the U87MG cells. Moreover, U373MG showed markedly increased integrin β4 expression compared with that of U87MG cells. The pRK5 β4 transfected U87MG cells showed slower migration rate and more increased proliferation rate than that of the control U87MG cells. The integrin β4 knockout U373MG cells showed faster migration rate and more decreased proliferation rate than that of the control U373MG cells. Our findings suggest that expression of integrin β4 is positively correlated with proliferation and negatively with invasiveness in GBM cell line. In GBM patients, positive staining for integrin β4 was observed in 33 (37.1%). Integrin β4 expression was significantly correlated with Ki-67 expression and negatively with tumor multiplicity and relapse. Therefore, integrin β4 expression seems to play an important role in proliferation and non-invasiveness in GBM patients. However, Kaplan–Meier analysis indicated that integrin β4 expression was not correlated with overall survival in GBM patients. Conclusion: Using a laminin-2 substrate, we successfully isolated a subpopulation of highly invasive GBM cells. The highly invasive GBM cells likely showed a decrease in proliferation because of Erk inactivation via integrins α6 and β4. Moreover, we focused on evaluation of role of integrin β4 in both GBM cell line and tissue. Notably, the clinicopathological feature and prognosis were investigated in integrin β4 expressed GBM patients. In conclusion, our findings suggest that expression of integrin β4 is negatively associated with invasiveness and positively with proliferation in GBM.