KRAS or BRAF mutation status is a useful predictor of sensitivity to MEK inhibition in ovarian cancer

Abstract

This study examined the status of KRAS and BRAF mutations, in relation to extracellular signal-regulated protein kinase (ERK) activation in 58 ovarian carcinomas to clarify the clinicopathological and prognostic significance of KRAS/BRAF mutations. Somatic mutations of either KRAS or BRAF were identified in 12 (20.6%) out of 58 ovarian carcinomas. The frequency of KRAS/BRAF mutations in conventional serous high-grade carcinomas (4.0% : 1/25) was significantly lower than that in the other histological type (32.3% : 10/31). Phosphorylated ERK1/2 (p-ERK1/2) expression was identified in 18 (38.2%) out of 45 ovarian carcinomas. KRAS/BRAF mutation was significantly correlated with International Federation of Gynecology and Obstetrics (FIGO) stage I, II (P<0.001), and p-ERK1/2 (P<0.001). No significant correlations between KRAS/BRAF mutations or p-ERK1/2 expression and overall survival were found in patients with ovarian carcinoma treated with platinum and taxane chemotherapy (P=0.2460, P=0.9339, respectively). Next, to clarify the roles of ERK1/2 activation in ovarian cancers harbouring KRAS or BRAF mutations, we inactivated ERK1/2 in ovarian cancer cells using CI-1040. Cl-1040 is a compound that selectively inhibits MAP kinase kinase (MEK), an upstream regulator of ERK1/2, and thus prevents ERK1/2 activation. Profound growth inhibition and apoptosis were observed in CI-1040-treated cancer cells with mutations in either KRAS or BRAF in comparison with the ovarian cancer cells containing wild-type sequences. This was evident in both in vitro and in vivo studies. The findings in this study indicate that an activated ERK1/2 pathway is critical to tumour growth and survival of ovarian cancers with KRAS or BRAF mutations. Furthermore, they suggest that the CI-1040-induced phenotypes depend on the mutational status of KRAS and BRAF in ovarian cancers. Therefore, ovarian cancer patients with KRAS or BRAF mutations may benefit from CI-1040 treatment.

Figure 1

Chromatograms of KRAS and BRAF mutational status in three representative ovarian cancer cells. (A) Left panel (MDAH2774) showing a point mutation in the KRAS gene. (B) Right panel (ES2) showing a point mutation in the BRAF gene. Arrows represent spike which indicates mutation.

Figure 2

Immunohistochemical staining of phosphorylated extracellular-regulated kinase (p-ERK1/2). (A) Intense immunoreactivity is present in both the nucleus and the cytoplasm in this ovarian carcinoma. (B) A case with negative staining of phosphorylated ERK1/2 (p-ERK1/2).

Figure 3

Kaplan–Meier survival curve in 45 patients with ovarian carcinoma according to KRAS/BRAF mutation and phosphorylated ERK (p-ERK) expression. (A) KRAS/BRAF mutational status correlates with favourable overall survival in patients with ovarian carcinoma. (B) p-ERK1/2 expression does not correlate with shorter overall survival in patients with ovarian carcinoma.

Figure 4

Western blot analysis. Expression of phosphorylated ERK1/2 (p-ERK1/2) is undetectable in all CI-1040-treated samples. A similar amount of protein was loaded in CI-1040 and DMSO-treated samples as evidenced by a similar intensity of total ERK1/2. D, DMSO treatment, C, and CI-1040 treatment.

Figure 5

Effects of CI-1040 on cell proliferation. Cells were counted after 72 h of CI-1040 or DMSO (control) treatment. The mutational status of KRAS and BRAF for each sample is shown under the cell lines and primary cancer cell cultures. Ovarian cancers with mutations in either KRAS or BRAF are more sensitive to growth inhibition by CI-1040 than those with wild-type (WT) sequences.

Figure 6

Detection of apoptotic cells and proliferation cells. (A) The CI-1040-treated ES2 cells, but not KF28 cells, show morphological features typical of apoptosis. (B) Apoptotic cells are quantified by counting them under a fluorescent microscope. (C) Treatment with CI-1040 decreases DNA synthesis as measured by BrdUrd uptake in ES2 cells, but not KF28 cells. (D) Proliferation is estimated by counting BrdUrd-stained cells under a fluorescent microscope. The experiment was performed 72 h after CI-1040 or DMSO treatment.

Figure 7

Effects of CI-1040 in a mouse xenograft model. (A) CI-1040-treated cells produced small tumour nodules in the peritoneal cavity. However, the diluent control-treated cells grew much larger i.p. tumours in KRAS mutant MDAH2774 cells. (B) In contrast, there were no differences in tumour weights between CI-1040-treated cells and control-treated cells in wild-type (WT) KRAS/BRAF SKOV3 cells. Tumours were excised and weighed. The data are expressed as the total tumour weight from each mouse. (C and D) Immunohistochemical staining of phosphorylated extracellular-regulated kinase 1/2 (p-ERK1/2) in tumours with KRAS mutant MDAH2774 cells. (C) Intense immunoreactivity is present in both the nucleus and the cytoplasm in CI-1040-untreated tumour. (D) Immunoreactivity is absent in both the nucleus and the cytoplasm in CI-1040-untreated tumour cells.