KRAS/BRAF mutation status and ERK1/2 activation as biomarkers for MEK1/2 inhibitor therapy in colorectal cancer

Abstract

Phase II clinical trials of mitogen-activated protein/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitors are ongoing and ERK1/2 activation is frequently used as a biomarker. In light of the mutational activation of BRAF and KRAS in colorectal cancer, inhibitors of the Raf-MEK-ERK mitogen-activated protein kinase are anticipated to be promising. Previous studies in pancreatic cancer have found little correlation between BRAF/KRAS mutation status and ERK1/2 activation, suggesting that identifying biomarkers of MEK inhibitor response may be more challenging than previously thought. The purpose of this study was to evaluate the effectiveness of MEK inhibitor therapy for colorectal cancer and BRAF/KRAS mutation status and ERK1/2 activation as biomarkers for MEK inhibitor therapy. First, we found that MEK inhibitor treatment impaired the anchorage-independent growth of nearly all KRAS/BRAF mutant, but not wild-type, colorectal cancer cells. There was a correlation between BRAF, but not KRAS, mutation status and ERK1/2 activation. Second, neither elevated ERK1/2 activation nor reduction of ERK1/2 activity correlated with MEK inhibition of anchorage-independent growth. Finally, we validated our cell line observations and found that ERK1/2 activation correlated with BRAF, but not KRAS, mutation status in 190 patient colorectal cancer tissues. Surprisingly, we also found that ERK activation was elevated in normal colonic epithelium, suggesting that normal cell toxicity may be a complication for colorectal cancer treatment. Our results suggest that although MEK inhibitors show promise in colorectal cancer, KRAS/BRAF mutation status, but not ERK activation as previously thought, may be useful biomarkers for MEK inhibitor sensitivity.

Figure 1

CRC cell lines exhibit differential sensitivity to ERK and anchorage-independent growth inhibition by MEK inhibitor treatment. A, Inhibition of anchorage-independent growth of CRC cell lines by treatment with the U0126 MEK1/2 inhibitor. The indicated cells were suspended in soft agar with vehicle (DMSO) or 30 µM U0126. The number of colonies of proliferating cells was quantitated after 30 days. B, Inhibition of ERK1/2 activation in CRC cell lines by U0126 MEK inhibitor treatment. Cells were treated for 24 h with vehicle (DMSO) or 30 µM U0126, then lysed and immunoblotted with anti-pERK1/2, anti-total-ERK1/2 or anti-vinculin sera. C, Inhibition of anchorage-independent growth of CRC cells by treatment with the CI-1040 MEK inhibitor. The indicated cells were suspended in soft agar with vehicle (DMSO) or 1 µM CI-1040. The number of colonies of proliferating cells was quantitated after 30 days. D, Inhibition of ERK activation in CRC cell lines by CI-1040 MEK inhibitor treatment. Cells were treated for 24 h with vehicle (DMSO) or 1 µM CI-1040, then lysed and immunoblotted with anti-pERK, anti-total-ERK or anti-vinculin sera. Analyses with total anti-vinculin were done to verify equivalent loading of cellular protein. Fold change in the relative intensity of pERK was calculated for MEK inhibitor treatment compared to vehicle control (+/−) using total ERK1/2 as the standard. Data shown are representative of at least three independent experiments. (WT, KRAS/BRAF wild-type; KRAS, KRAS mutation positive, BRAF, BRAF mutation positive).

Figure 1

CRC cell lines exhibit differential sensitivity to ERK and anchorage-independent growth inhibition by MEK inhibitor treatment. A, Inhibition of anchorage-independent growth of CRC cell lines by treatment with the U0126 MEK1/2 inhibitor. The indicated cells were suspended in soft agar with vehicle (DMSO) or 30 µM U0126. The number of colonies of proliferating cells was quantitated after 30 days. B, Inhibition of ERK1/2 activation in CRC cell lines by U0126 MEK inhibitor treatment. Cells were treated for 24 h with vehicle (DMSO) or 30 µM U0126, then lysed and immunoblotted with anti-pERK1/2, anti-total-ERK1/2 or anti-vinculin sera. C, Inhibition of anchorage-independent growth of CRC cells by treatment with the CI-1040 MEK inhibitor. The indicated cells were suspended in soft agar with vehicle (DMSO) or 1 µM CI-1040. The number of colonies of proliferating cells was quantitated after 30 days. D, Inhibition of ERK activation in CRC cell lines by CI-1040 MEK inhibitor treatment. Cells were treated for 24 h with vehicle (DMSO) or 1 µM CI-1040, then lysed and immunoblotted with anti-pERK, anti-total-ERK or anti-vinculin sera. Analyses with total anti-vinculin were done to verify equivalent loading of cellular protein. Fold change in the relative intensity of pERK was calculated for MEK inhibitor treatment compared to vehicle control (+/−) using total ERK1/2 as the standard. Data shown are representative of at least three independent experiments. (WT, KRAS/BRAF wild-type; KRAS, KRAS mutation positive, BRAF, BRAF mutation positive).

Figure 2

ERK activity does not appear to be associated with KRAS and BRAF mutation status. Western blot analyses with phospho-specific anti-ERK1 and ERK2 antibody were done to evaluate ERK1/2 phosphorylation and activation. Parallel blotting analyses with total anti-vinculin were done to verify equivalent loading of cellular protein. Data shown are representative of at least three independent experiments. (WT, KRAS/BRAF wild-type; KRAS, KRAS mutation positive, BRAF, BRAF mutation positive).

Figure 3

ERK activation in matched normal and tumors from colorectal cancer patients. A, ERK is activated in normal tissue. Total cell lysates of matched normal (N) and tumors (T), identified by AJCC stage, were immunoblotted with anti-pERK and anti-GAPDH sera. Fold change in the relative intensity of pERK was calculated for matched tumors compared to normals (T/N) using total ERK as the standard. B, ERK is activated in normal colonic epithelial cells. CRC tissues, identified by AJCC stage, were immunohistochemically stained with anti-pERK serum. Arrows indicate pERK staining in normal adjacent colonic epithelial cells. Arrowheads indicate pERK staining in tumor. Paraffin embedded SW48 cell lines with high levels of pERK are shown as a positive control.

Figure 4

ERK activation in colorectal cancer patient normal and tumor tissues. Tissue microarrays of 190 matched normal colon and tumors were prepared as described in Materials and Methods and immunohistochemically stained with anti-P-ERK serum. Mean scores were expressed as the product of intensity (I) and proportion (P) of positive epithelial staining. A, ERK activation in matched normal mucosa and colorectal tumors. B, ERK activation in patients with KRAS and BRAF mutations.