Table 1.
Subtypes and mutation status of colorectal cancer cell lines.
Fig 1.
CRC subtype response to MEK162 and neratinib.
(A) and (B): NCI-H747, SW837, SW1116, SW1463, NCI-H508, SNU-C1, SW480, SW620, C2BBE1, Hs675.T, and HCT116 cells were treated with (A) a constant dose of neratinib (0.5 μM) in combination with different doses of MEK162, and (B) a constant dose of MEK162 (0.5 μM) in combination with different doses of neratinib for 72 hours. Dimethyl sulfoxide (DMSO) (0.01%) was used as the control treatment. Each data point is the mean of five replicates ± SD.
Fig 2.
MEK162 plus neratinib inhibits pERK in sensitive (inflammatory) cell lines.
Protein profiling of inflammatory cell lines: (A) NCI-H747 and (B) SW-837, in the absence and presence of MEK162 plus neratinib for 48 hours; protein lysates were prepared and analyzed with the PathScan RTK Signaling Antibody Array Kit. The chemiluminescent film image (upper panel) and the quantification of that image (lower panel) are shown. ERK = extracellular signal-regulated kinase.
Fig 3.
pERK in resistant (stem-like) cell lines after treatment with MEK162 plus neratinib.
Protein profiling of stem-like cells lines (A) SW 480 and (B) C2BBE1 were performed as described in Fig 2.
Fig 4.
Consistent activation of pERK in stem-like subtype responsible for resistance to the MEK162 plus neratinib combination.
(A) Effects of MEK162 alone, neratinib alone, and the combination of MEK162 plus neratinib were assessed in sensitive, inflammatory subtype cell lines (NCI-H747, SW-837), (B) resistant, stem-like subtype cell lines (SW480, SW620), and (C) resistant stem-like subtype cell lines (C2BBE1, Hs.675.T, and HCT-116). Cell lines were cultured with the indicated dosage of MEK162 or neratinib alone, or in combination for 48 hours. Protein expressions were evaluated by western blot analysis with the indicated antibodies. ß-actin was used as a loading control. (D) NCI-H747 and SW837 (inflammatory cell lines) and SW-480, C2BBE1, HCT-116, and HS.675.T (stem-like cell lines) were cultured with normal medium as control or medium containing combination of constant dose of 0.5 μM MEK162 with neratinib doses ranging from 0.062 μM to 1 μM. Apoptotic cells were determined by evaluating caspase 3/7 activity using Promega Apo-ONETM Homogeneous Caspase-3/7 assay. Each data point represents the mean of five replicates; error bars for standard deviation are shown.
Fig 5.
In-vivo tumor growth inhibited by combination in inflammatory subtype but not in stem-like.
(A) (B): NCI-H747 and (C) (D): SW480 cells, respectively, were implanted into athymic nude mice. Once xenografts reached ~100 mm3, mice were grouped into 4 different cohorts (7 mice/cohort) (control [untreated], neratinib-only (10mg/kg), MEK162-only (3 mg/kg), or MEK162 plus neratinib groups). MEK162 and neratinib were suspended in 0.5% carboxymethyl cellulose and orally administered q.d. for 28 days, respectively. Tumor volumes were measured three times weekly. Volumes represent the mean ± SD of tumor volumes from 7 mice/group. Respective body weights were measured once weekly.
Table 2.
Combination of MEK162 plus neratinib inhibits tumor growth in KRAS-mutant inflammatory cell line xenograft.
Fig 6.
Western blot analysis of pERK, total ERK, and ß-actin in tumor xenografts of the (A) immunoblot image, and (B) representative digitized values for pERK/total ERK normalized to β-actin from inflammatory subtype cell line NCI-H747, and (C) immunoblot image and (D) representative digitized values for pERK/total ERK normalized to β-actin from stem-like cell line SW480. Each lane contains a lysate from a single mouse.
Fig 7.
ERK inhibitor SCH772984 inhibits the cell viability of stem-like subtype cell lines.
(A) (B) NCI-H747, SW837, SW480, and SW620 cells were treated with the ERK inhibitor, (A) SCH772984, alone or in combination with (B) neratinib at indicated concentrations for 72 hours. Dimethyl sulfoxide (DMSO) (0.01%) was used as the control treatment. Each data point represents the mean of five replicates; error bars indicate one SD. (C) Western blot analysis of pERK and total ERK in inflammatory and stem-like cell lines after 48 hours of treatment with SCH772984.