OBJECTIVE In patients with glioblastoma, local invasion of tumor cells causes recurrence and shortens survival. The goal of this study was to determine whether protein disulfide isomerase (PDI) A6 regulates migration and invasion of glioblastoma cells and the associated factors. METHODS U87MG cells were treated with either PDIA6 or ADAM17 small interfering RNA (siRNA) fragments or with both types of siRNA fragments, and expression was confirmed by reverse transcription-polymerase chain reaction and Western blot. Migration and invasion were assessed using a wound-healing assay, a Matrigel assay, and an organotypic culture system. After the U87MG cells were treated with siRNAs and epidermal growth factor receptor (EGFR) inhibitors, the expression of matrix metalloproteinase-2 (MMP-2), membrane Type 1-matrix metalloproteinase (MT1-MMP), integrin, phosphorylated focal adhesion kinase (pFAK), and phosphorylated EGFR (pEGFR) was detected by Western blotting and zymography. RESULTS U87MG cell migration and invasion increased significantly after inhibition of PDIA6. The MMP-2 activation ratio and ADAM17 activity (as a sheddase of the proligand) increased, and expression of pEGFR, pFAK, integrin α5β3, and MT1-MMP was induced, compared with control levels. Furthermore, heparin-binding epidermal growth factor (EGFR signaling ligand) was highly expressed in PDIA6-knockdown cells. After siPDIA6-transfected U87MG cells were treated with EGFR signaling inhibitors, expression of pFAK, MMP-2, and MT1-MMP decreased and invasion decreased significantly. Simultaneous double-knockdown of PDIA6 and ADAM17 reduced pEGFR and pFAK expression, compared with control levels. CONCLUSIONS The authors propose that inhibiting PDIA6 could transduce EGFR signaling by activating and inducing ADAM17 during migration and invasion of U87MG glioblastoma cells. The results of this study suggest that PDIA6 is an important component of EGFR-mediated migration and invasion of U87MG cells. This is the first report of the effects of PDIA6 on migration and invasion in glioblastoma.
Keywords: A+P = knockdown of both ADAM17 and PDIA6; ADAM = a disintegrin and metalloprotease; ADAM17; DMEM = Dulbecco's modified Eagle medium; EGFR; EGFR = epidermal growth factor receptor; F-actin = filamentous actin; FAK = focal adhesion kinase; HB-EGF = heparin-binding epidermal growth factor; MMP-2; MT1-MMP; MT1-MMP = membrane Type 1–matrix metalloproteinase; NC = negative control; PBS = phosphate-buffered saline; PDI = protein disulfide isomerase; PDIA6; RT-PCR = reverse transcription–polymerase chain reaction; SDS-PAGE = sodium dodecyl sulfate polyacrylamide gel electrophoresis; U87MG; glioblastoma; oncology; pEGFR = phosphorylated EGFR; pFAK = phosphorylated FAK; siRNA = small interfering RNA.