Inhibition of c-MET increases the antitumour activity of PARP inhibitors in gastric cancer models

Abstract

Gastric cancer is the fifth most common malignancy and the third leading cause of cancer-related death worldwide. Activation of c-MET increases tumour cell survival through the initiation of the DNA damage repair pathway. PARP is an essential key in the DNA damage repair pathway. The primary role of PARP is to detect and initiate an immediate cellular response to single-strand DNA breaks. Tumours suppressor genes such as BRCA1/2 are closely associated with the DNA repair pathway. In BRCA1/2 mutations or deficiency status, cells are more likely to develop additional genetic alterations and chromosomal instability and can lead to cancer. In this study, we investigate the role of c-MET and PARP inhibition in a gastric cancer model. We exploited functional in vitro and in vivo experiments to assess the antitumour potential of co-inhibition of c-MET (SU11274) and PARP (NU1025). This leads to a reduction of gastric cancer cells viability, especially after knockdown of BRCA1/2 through apoptosis and induction of γ-Η2ΑΧ. Moreover, in AGS xenograft models, the combinatorial treatment of NU1025 plus SU11274 reduced tumour growth and triggers apoptosis. Collectively, our data may represent a new therapeutic approach for GC thought co-inhibition of c-MET and PARP, especially for patients with BRCA1/2 deficiency tumours.

Keywords: BRCA1; BRCA2; PARP inhibitor; c-Met inhibitor; gastric cancer.

FIGURE 1

Steady‐state levels of gastric cancer cell lines. Using Western blot assay, steady protein levels of BRCA1, BRCA2 and c‐MET are analysed in primary gastric cancer cell lines HS746T and AGS. Protein levels were normalized against actin

FIGURE 2

Low levels of c‐MET partially sensitize GC cell lines in PARP inhibition. A, HS746T/AGS cells, control‐siRNA‐Hs746T/AGS cells and si‐c‐MET Hs746T/AGS cells were exposed to increasing doses (0‐40 µmol/L) of NU1025 for 48 h for determination of cell viability (MTT metabolic activity assay). The protein levels of c‐MET expression (by Western blot analysis) revealed down‐regulation of the c‐MET receptor in both cell lines (HS746T and AGS); (B) HS746T cells, control‐siRNA‐Hs746T cells and si‐BRCA1/2 Hs746T cells were exposed to increasing doses (0‐40 µmol/L) of NU1025 for 48 h for determination of cell viability (MTT metabolic activity assay). The protein levels of BRCA1 and BRCA2 expression (by Western blot analysis) revealed down‐regulation of the BRCA1/2 in HS746T cell line; (C) HS746T cells, control‐siRNA‐Hs746T, siBRCA1/2‐Hs746T and siMET/BRCA1/2‐Hs746T cells were cultured with the indicated concentrations of NU1025 (5, 10 and 20 μmol/L) for 48 h for determination of cell viability (MTT metabolic activity assay). Error bars represent SD

FIGURE 3

Co‐inhibition of c‐MET (SU11274) and PARP (NU1025) sensitizes GC cells after knockdown BRCA1/2. Knocking down BRCA1 or BRCA2 sensitizes cells to PARP and c‐MET inhibition in HS746T cells expressing low levels of c‐MET (AGS cells, c‐MET knockdown Hs746T cells) to PARP inhibition. A, HS746T cells, control‐siRNA‐Hs746T cells and siBRCA1/2‐Hs746T (upper panel) and AGS (lower panel) cells were exposed to 5 µmol/L of NU1025 and/or 5 µmol/L of SU11274 for 48 h for determination of cell viability (MTT metabolic activity assay). Results are expressed as percentages. Average values of three experiments ± SD are shown; (B) Western blot analysis of PARP and cl.caspase‐3 in Hs746T‐control‐siRNA, siBRCA1/2‐Hs746T (upper panel) and AGS (lower panel) cell lines. Cells were cultured with the indicated drugs (5 μmol/L NU1025, 5 μmol/L SU11274 alone or in combination for 24 h of treatment). Protein levels were normalized against actin

FIGURE 4

NU1025 plus SU11274 increases DNA damage in GC cell line. A, Western blot analysis of γ‐H2AX in Hs746T‐control‐siRNA, siBRCA1/2‐Hs746T and AGS cell lines. Cells were cultured with the indicated drugs (5 μmol/L NU1025, 5 μmol/L SU11274 alone or in combination after 24 h of treatment). Protein levels were normalized against actin; (B) confocal microscope images of two‐dimensional culture of HS746T (left panel) and AGS (right panel) cell lines. Hs746T‐control‐siRNA, siBRCA1/2‐Hs746T and AGS cells were cultured with the indicated drugs and concentrations for 24 h. Representative images of Hs746T and AGS nuclei (DAPI‐blue staining) and of γ‐H2AX (green) are shown in the figure

FIGURE 5

Effect of combinatorial treatment of c‐MET and PARP inhibition in tumour xenograft (AGS) model. AGS cells were inoculated into SCID mice (5 mice per group) on day 0. Mice were inoculated subcutaneously in the right flank with 0.1 mL PBS containing 3 × 106 AGS human gastric cancer cells. When the tumour volume reached ~ 100 mm3, mice were administered with NU1025 (1 mg/mouse) or SU11274 (1 mg/kg), alone or in combination every 4 d for 20 d. Tumour growth was calculated at the indicated time points. Tumour volume was measured 5 d using a calliper and calculated as (width) 2 × length/2. Photographs of excised AGS tumours after receiving different treatments (control, NU1025, SU11274 and NU1025 + SU11274) were captured at the end of 20 d of therapy (A‐left panel) and AGS tumour size progression as a function of time after administration (A‐right panel). Western blot showing the levels of PARP and cleaved caspase‐3 in the subcutaneous tumour tissues isolated from the mice after 20 d of therapy (B)