Analysis of the K-ras/B-raf/Erk signal cascade, p53 and CMAP as markers for tumor progression in colorectal cancer patients

Abstract

Abstract. Colorectal cancer patients may succumb to their disease because of local recurrence or formation of metastasis. To develop a prognostic tool for these fatal types of progression, 23 patients with colorectal carcinoma were included in this study for the detection at the time of surgery of the incidence of K-ras, B-raf and p53 mutations, the phosphorylation status of Erk and the expression of cystatin-like metastasis-associated protein (CMAP) in tumor, mucosa and liver samples. Polymerase chain reaction-restriction fragment length polymorphism and PCR-SSCP were used to detect the respective mutations. The results of these assays were complemented by sequencing the K-ras, B-raf and p53 mutations. A multiplex RT-PCR assay was used to detect the CMAP mRNA levels and the phosphorylation status of Erk in tumor samples was assessed by Western blot using a phospho-specific Erk antibody. The carcinomas were classified as stages T4 (70%), T3 (17%), T2 (9%) and T1 (4%) and thus represent a group of advanced colorectal carcinomas. The carcinomas (8 out of 23, 39.1%) were mutated in K-ras codons 12 or 13 and two patients had a B-raf (V599) mutation in their tumor. Of 22 tumors, 11 (50%) were positive for pErk, indicating the activation of the RAS/RAF/ERK signaling pathway. Of the 23 tumors, 13 (65.5%) showed an increased CMAP RNA level. Notably, 10 of these 13 patients have already died and two developed liver metastasis. Mutations in p53 were found in only 6 patients (26%), with 6 being detected in carcinoma, 1 in mucosa and 1 in liver tissue. These alterations were classified as non-sense (n=1), mis-sense (n=2) and frame-shift mutations (n=1) as well as intron polymorphisms (n=5). There was a significant correlation between Erk activation and K-ras codon 12 mutation (p=0.016), but not between K-ras codon 13 or B-raf mutations and Erk activation. Furthermore, there was a significant correlation of each positive marker with tumor stage (p=0.001).

Alterations in p53, BRCA1, ATM, PIK3CA, and HER2 genes and their effect in modifying clinicopathological characteristics and overall survival of Bulgarian patients with breast cancer

“…Another speciWcity of p53 mutational spectrum in Bulgarian patients with BC is the high frequency of the 14,766 T > C intronic polymorphisms accounting to 0.0483. The same polymorphic variant was previously found in three Bulgarian patients with Balkan Endemic Nephropathy (Krasteva and Georgieva 2006) and in one patient with colorectal carcinoma (Georgieva et al 2008).…”

Intrinsic resistance to the MEK1/2 inhibitor AZD6244 (ARRY‐142886) is associated with weak ERK1/2 signalling and/or strong PI3K signalling in colorectal cancer cell lines

“…In instances of coincident activation of the RAF and PI3K pathways, combinations of AZD6244 and PI3K/mTOR inhibitors may prove efficacious. 52 It is also important to stress that ERK1/2 activation did not simply correlate with the presence of a KRAS mutation; in some cells KRAS coupled well to ERK1/2 and in others poorly and this is also seen in human tumour samples 53,54 and mouse models. 55,56 This differential coupling to downstream effectors represents a challenge and suggests that biochemical analysis should be combined with mutational profiling in interpreting patient responses and informing patient recruitment to trials.…”

Phospho-ERK and AKT status, but not KRAS mutation status, are associated with outcomes in rectal cancer treated with chemoradiotherapy

“…There is strong evidence to suggest that KRAS and BRAF mutations are mutually exclusive [ 8 , 11 - 13 ]. Constitutive activation of BRAF leads to extracellular signal-regulated kinase (ERK) phosphorylation (p-ERK) and activation [ 14 ]. ERK is required for cell proliferation and allows for the evasion of apoptosis [ 14 ].…”