CD147-targeted siRNA in A375 malignant melanoma cells induces the phosphorylation of EGFR and downregulates cdc25C and MEK phosphorylation

Abstract

The Raf-MEK-ERK signaling pathway is important during oncogenesis. An activating mutation of BRAF constitutively activates the Raf-MEK-ERK signaling cascade, and has been identified in ~70% of malignant melanomas (MMs). Cluster of differentiation 147 (CD147)/basigin is an integral plasma membrane protein belonging to the immunoglobulin superfamily. The protein is highly expressed on MM cells, and promotes cellular proliferation and tumor growth. The present study investigated the correlation between CD147 expression and Raf-MEK-ERK signaling in MM using the A375 human MM cell line, which harbors the activating mutation of BRAF. The phosphorylation of epidermal growth factor receptor (EGFR) was upregulated, and mitogen-activated protein kinase kinase (MEK) and cell division cycle 25C phosphorylation was downregulated by CD147 silencing in the A375 cells. Cell growth was inhibited by the EGFR inhibitor erlotinib and by CD147 silencing, and additive growth inhibition was observed when these techniques were combined. The results of the present study indicate that the combination of EGFR and CD147 inhibition may be useful in BRAF-mutated MM.

Keywords: BRAF; CD147; EGFR; erlotinib; malignant melanoma.

Figure 1.

(A) shRNA-mediated silencing of CD147 expression in A375 human malignant melanoma cells. CD147 expression was lower in the A375 cells transfected with CD147 shRNA (shCD147) compared with the control (shCtrl). The CD147 shRNA was delivered using a lentivirus and the control was empty lentiviral vector. (B) GAPDH was used as a loading control. (C) Quantification of CD147 expression levels in shCD147 and shCtrl cells. *P< 0.05 vs. control. CD147, cluster of differentiation 147; sh, short hairpin.

Figure 2.

EGFR phosphorylation was induced in A375 human malignant melanoma cells by exogenous EGF (100 ng/ml) in a time-dependent manner. (A) Western blot analysis revealed that EGFR phosphorylation was upregulated by shRNA knockdown of CD147 (shCD147) compared with the control (shCtrl; empty lentiviral vector). The highest level of EGFR phosphorylation was recorded at 10 min. GAPDH was used as a loading control. (B) Quantification of EGFR phosphorylation levels in shCtrl and shCD147 cells. *P<0.05 vs. control. EGFR, epidermal growth factor; pEGFR, phosphorylated EGF receptor; CD147, cluster of differentiation 147; sh, short hairpin.

Figure 3.

(A) EGFR phosphorylation was induced in A375 human malignant melanoma cells by exogenous EGF (100 ng/ml) at different time-points. Western blot analysis demonstrates that knockdown of CD147 using lentivirus-delivered shRNA (shCD147) inhibits the phosphorylation levels of (B) MEK and (C) cdc25C, compared with the control (shCtrl; empty lentiviral vector). GAPDH was used as an internal housekeeping control. *P<0.05 vs. control. EGF; epidermal growth factor receptor; pMEK, phosphorylated mitogen-activated protein kinase kinase; CD147, cluster of differentiation 147; sh, short hairpin.

Figure 4.

Additive growth inhibition of A375 human malignant melanoma cells by CD147 knockdown (shCD147), treatment with erlotinib (an EGFR inhibitor), or the two techniques in combination. Inhibition of growth was measured using the MTT assay, and the absorbances at 570 nm and 630 nm were recorded for test and reference wavelengths, respectively. Growth inhibition was significantly lower in shCD147 cells than control (shCtrl) cells. Erlotinib significantly inhibited the growth of shCtrl cells. Growth inhibition by erlotinib was significantly higher in shCD147 cells than shCtrl cells. *P<0.01 and **P<0.05 vs. shCtrl; ^†P<0.01 vs. shCtrl plus erlotinib. MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; EGFR, epidermal growth factor receptor; CD147, cluster of differentiation 147; sh, short hairpin.