Comparison of Tepotinib, Paclitaxel, or Ramucirumab Efficacy According to the Copy Number or Phosphorylation Status of the MET Gene: Doublet Treatment versus Single Agent Treatment

Abstract

Although conventional combination chemotherapies for advanced gastric cancer (GC) increase survival, such therapies are associated with major adverse effects; more effective and less toxic treatments are required. Combinations of different anti-cancer drugs, for example, paclitaxel plus ramucirumab, have recently been used as second-line treatments for advanced GC. This study evaluated how copy number variations of the MET gene, MET mutations, and MET gene and protein expression levels in human GC cells modulate the susceptibility of such cells to single-agent (tepotinib, ramucirumab, or paclitaxel) and doublet (tepotinib-plus-paclitaxel or ramucirumab-plus-paclitaxel treatment regimens. Compared with ramucirumab-plus-paclitaxel, tepotinib-plus-paclitaxel better inhibited the growth of GC cells with MET exon 14 skipping mutations and those lacking MET amplification but containing phosphorylated MET; such inhibition was dose-dependent and associated with cell death. Tepotinib-plus-paclitaxel and ramucirumab-plus-paclitaxel similarly inhibited the growth of GC cells lacking MET amplification or MET phosphorylation, again in a dose-dependent manner, but without induction of cell death. However, tepotinib alone or tepotinib-plus-ramucirumab was more effective against c-MET-positive GC cells (>30 copy number variations) than was ramucirumab or paclitaxel alone or ramucirumab-plus-paclitaxel. These in vitro findings suggest that compared with ramucirumab-plus-paclitaxel, tepotinib-plus-paclitaxel better inhibits the growth of c-MET-positive GC cells, cells lacking MET amplification but containing phosphorylated MET, and cells containing MET mutations. Clinical studies are required to confirm the therapeutic effects of these regimens.

Keywords: copy number variation; paclitaxel; phosphorylated MET; ramucirumab; tepotinib.

Figure 1

Mutations in 286 genes in five GC cell lines. Mutations were quantitated via targeted next-generation sequencing. All GC-related mutations are shown. Figure 1 was created using Excel 2016 software.

Figure 2

MET gene expression levels and copy numbers, and the MET protein expression levels, in five GC cell lines. (a) MET mRNA expression levels were determined via quantitative RT-PCR. Quantities of MET and GAPDH mRNAs were determined in the 5 GC cells using all the tested methods of real-time PCR data analysis. The amount of MET mRNA in the 5 GC cells was then divided by the normalization factor (geometric mean of GAPDH amounts) of the sample. The values are arithmetical means ± S.E.M., n = 3. (b) MET copy numbers were assessed via targeted NGS. (c) The phospho-MET and total MET protein levels were determined via Western blotting.

Figure 3

The effects of ramucirumab, paclitaxel, and tepotinib alone on GC viability. Five GC cell lines were treated with various concentrations of ramucirumab, paclitaxel, or tepotinib for 48 h.

Figure 4

The effects of drug combinations on GC cell viability. GC cells were treated with various concentrations of paclitaxel with or without 10 nM ramucirumab or 10 nM tepotinib for 48 h. Tepo, tepotinib; Ramu, ramucirumab, PTX, paclitaxel.

Figure 5

The effects of drug combinations on apoptosis and necrosis in GC cell lines. Hs746T, SNU620, AGS, SNU638, and MKN45 cell lines were treated with 20 nM paclitaxel, 20 nM ramucirumab, or 10 nM tepotinib for 48 h. Con, control; Te, tepotinib; Ramu, ramucirumab.

Figure 6

Effects of drug combinations on GC cell migration. A wound-healing assay was used to assess the effects of drugs on the migration of Hs746T, MKN45, SNU638, and AGS cell lines treated with 20 nM paclitaxel, 20 nM ramucirumab, or 10 nM tepotinib for 2–6 days. Con, control.

Figure 7

The effects of drug combinations on the expression levels of carcinogenesis-related proteins in GC cells. Con, control; T, tepotinib; Ramu, ramucirumab, P, paclitaxel.