BRAF and KRAS mutations in stomach cancer

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Ras proteins control signaling pathways that are key regulators of several aspects of normal cell growth and malignant transformation. BRAF, which encodes a RAF family member in the downstream pathway of RAS, is somatically mutated in a number of human cancers. The activating mutation of BRAF is known to play a role in tumor development. As there have been no data on the BRAF mutation in stomach cancer, we analysed the genomic DNAs from 319 stomach carcinomas for the detection of somatic mutations of BRAF. Overall, we detected BRAF mutations in seven stomach carcinomas (2.2%). Five of the seven BRAF mutations involved Val 599, the previously identified hotspot, but the substituted

common BRAF mutation (V599E). The remaining two mutations involved a conserved amino acid (D593G). One

amino acid （V599 M） was different from the most

in stomach cancer, and suggest that alterations of RAS

tumor had both BRAF and KRAS mutations. This is the first report on BRAF mutation in stomach cancer, and the data indicate that BRAF is occasionally mutated

pathway both by RAS and BRAF mutations contribute to the pathogenesis of stomach cancer.

 

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The RAS gene family consists of three closely related genes (HRAS, KRAS and NRAS), which have similar structures and encode p21 RAS (Kolch, 2000; Peysson-naux and Eychene, 2001). These p21 RAS proteins, known to play an important role in the regulation of normal signal transduction, bind guanosine tripho-sphate (GTP) and guanosine diphosphate (GDT) with high affinity. When cells are stimulated by growth factor or other receptor-ligand interactions, RAS becomes activated by exchanging GDP for GTP. The activated RAS, in turn, excites the mitogen-activated protein (MAP) kinase pathway (RAS-RAF-MEK-ERK-MAP kinase pathway) by recruiting the cytosolic protein RAF (Kolch, 2000; Peyssonnaux and Eychene, 2001). RAF
gene family consists of three members, each encoding serine/threonine kinases that are regulated by binding t

RAS-RAF-MEK-ERK-MAP kinase pathway plays a critical role in cell proliferation, and is frequentls activated in cancer cells. For example, approximately

10-20% of all human tumors contain mutated version: of RAS proteins, which activate the downstream path way (Bos, 1989). Recently, Davies et al. (2002) identifiec somatic mutations of BRAF, one of the RAF members in 66% of malignant melanomas and at a lowes frequency in a wide range of human cancers. So far all BRAF mutations have been reported within twc kinase domains, and the most common mutation is : single substitution, V599E (Brose et al., 2002; Davies et al., 2002; Naoki et al., 2002; Rajagopalan et al., 2002 Yuen et al., 2002; Pollock et al., 2003; Satyamoorthy et al., 2003). Mutated BRAF proteins have elevatec kinase activity and transforming activity in NIH3T cells (Davies et al., 2002). Furthermore, RAS function is not required for the growth of cancer cell lines with the V599E mutation (Davies et al., 2002).

 

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Stomach cancer occurs with a high incidence in Asia and is one of the leading causes of cancer death in the world (Bae et al., 1999). Several studies have reported : low incidence of Ras gene mutation in gastric carcinom: (roughly 0-10%) (Jiang et al., 1989; Lee et al., 1995 Arber et al., 2000). As RAS-RAF-MEK-ERK-MAF kinase pathway is important for the development o human cancer, searching the mutation of genes in this pathway in addition to RAS is necessary. Although screening of BRAF mutation in human tumors ha: widely been performed, to date the data on BRAI mutation in primary stomach cancer tissues is lacking In the present study, to explore the possibility that the alterations of BRAF gene might play a role in stomach carcinogenesis, we investigated the occurrence of BRAI gene mutations in stomach cancers and in particula their relationship with KRAS mutations.

Paraffin-embedded tissues of human stomach carcinomas were obtained from 319 patients. These sample: consisted of 60 early gastric cancer (EGC) and 259 advanced gastric cancer (AGC). Histologically, the samples consisted of 17/0 diffuse-type and 149 intest-inal-type gastric cancers. Through the microdissectior technique, we selectively procured tumor cells anc corresponding normal cells from histological section:

 

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of the 319 stomach carcinomas as shown previously (Shin et al., 1999). Genomic DNA was isolated and analysed for mutations of BRAF and KRAS genes by polymerase chain reaction (PCR)-based single-strand conformation polymorphism (SSCP) analysis. Since all of the BRAF mutations have been so far detected in exons 11 and 15 that encode the kinase domains in G-loop and the activation segment of BRAF, respectively (Brose et al., 2002; Davies et al., 2002; Naoki et al.,

2002; Pollock et al., 2003; Rajagopalan et al., 2002;

Yuen et al., 2002; Satyamoorthy et al., 2003), we used two primer sets that amplified the two exons. We also analysed KRAS mutations at codons 12 and 13 in exon 2, and codons 59 and 61 in exon 4 that comprise most of the activating mutations of KRAS (Jiang et al., 1989;

Lee et al., 1995; Arber et al., 2000; Davies et al., 2002;

Yuen et al., 2002).

SSCP analysis of BRAF and KRAS identified seven and nine aberrant bands, respectively. Enrichment and DNA sequence analysis of these aberrantly migrating bands led to the identification of seven BRAF mutations (2.2%) and nine KRAS mutations (2.8%). All of the BRAF and eight of the nine KRAS mutations were observed in AGC (2.7 and 3.1%, respectively). In spite of the high occurrences of BRAF and KRAS mutations in AGC, this relationship was not statistically significant (P >0.05). Histologically, diffuse-type gastric cancers had four BRAF and five KRAS mutations, while intestinal-type gastric cancer had three BRAF and four KRAS mutations. In terms of cancer staging, no significant difference was observed in the frequency of KRAS and BRAF mutation in terms of TNM stage (P > 0.05, Table 1).

All of the BRAF mutations were identified in exon 15 (Table 1, Figure 1). Five of the BRAF mutations involved codon 599 (V599 M) and the remaining two involved codon 593 (D593G). The KRAS mutations consisted of four mutations in exon 2, and five mutations in exon 4. The mutations in exon 2 consisted of two G13D, one G12 V and one K5N, and the mutations

BRAF mutations in stomach cancer

SH Lee et al

in exon 4 consisted of five identical A59 T. Of note, one BRAF mutation (V599 M) was identified in a stomach cancer that also had a KRAS mutation (G13D). None of the corresponding normal samples showed evidence of mutations by SSCP (Figure 1), indicating that the mutations detected in the specimens had risen somati-cally. We repeated the experiments two times, including tissue microdissection, PCR, SSCP and sequencing analysis, to ensure the specificity of the results, and found that the data were consistent (data not shown).

 

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Whereas the malignant melanoma is the most common tumor with BRAF mutations (roughly 60%), this tumor is known to possess a much lesser frequency of RAS mutations. By contrast, leukemia has frequent mutations of RAS genes (Neubauer et al., 1994), but a low frequency of BRAF mutations has been reported (Davies et al., 2002; Smith et al., 2003). The differential occurrence of BRAF and RAS mutation in some human cancers led us to analyse BRAF mutation in stomach cancer in which RAS mutation is known to be an uncommon event. We found that in some stomach cancers BRAF gene is somatically mutated, and that the frequency of BRAF mutation was similar to that of KRAS (Table 1). These data, together with the earlier reports on BRAF mutations in human cancers, suggest that RAS RAF kinase pathway may be regulated in stomach cancer by somatic mutations of multiple components in this pathway.

Although V599, where more than 50% of the BRAF mutations have occurred (Davies et al., 2002), is not the site for phosphorylation, replacement of nonpolar amino acid valine at this amino acid by an acidic amino acid glutamic acid may mimic regulatory phosphoryla-tion,

resulting in constitutive activation of BRAF

(Davies et al., 2002). Several non-V599E BRAF mutation at the amino acid 599, including V599R, V599 K and V599D, have been detected (Davies et al., 2002;

Naoki et al., 2002; Rajagopalan et al., 2002; Pollock et al., 2003). In the current study, we observed a novel BRAF mutation V599 M, another non-V599E BRAF

 

[Cottonmouth]

Have you washed your vagina today?

 

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Have you washed your vagina today?

 

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Figure 1 Mutations of BRAF and KRAS genes in stomach cancers. Genomic DNA each from normal cells or tumor cells was amplified with two primer pairs covering exons 11 and 15 of BRAF gene. KRAS gene was amplified with two primer pairs covering exons 2 and 4. Radioisotope was incorporated into the PCR products for detection by autoradiogram.

The PCR reaction

mixture was denatured for 1 min at 94°C and incubated for 30

procedures of

analysis were

performed as described previously (Shin et al., 1999). After SSCP, DNAs showing mobility shifts were cut out from the dried gel, and reamplified for 30 cycles using the same primer sets. Sequencing of the PCR products was carried out using a capillary automatic sequencer (ABI Prism Genetic Analyzer, Applied Foster City, CA, USA) according to the manufacturer's recom-mendation. SSCP (a-d) and DNA sequencing analyses (e-h) of DNA from tumors (lane T) and normal tissues (lane N). Exon 15 (a and b) of BRAF, and exon 2 (c) and exon 4 (d) of KRAS were amplified. SSCPs of DNA from the tumors show wild-type bands and additional aberrant bands (arrows) as compared to SSCP from corresponding normal cells.

(e) Sequencing analysis from the

aberrant band in (a). There is a G to A transition at nucleotide 1795 of BRAF (arrow) in tumor tissue as compared to normal tissue. (f) Sequencing analysis from the aberrant band in (b). There is an A to G transition at nucleotide 1778 of BRAF (arrow) in tumor tissue as compared to normal tissue. (g) Sequencing analysis from the aberrant band in (e). There is a G to A transition at nucleotide 34 (arrow) of KRAS in tumor tissue as compared to normal tissue. (h) Sequencing analysis from the aberrant band in (d). There is a G to A transition at nucleotide 175 (arrow) of KRAS in tumor tissue as compared to normal tissue. Numbering of cDNA of BRAF and KRAS was done with respect to the ATG start codon

Oncogene

mutation at the amino acid 599, raising the possibility that the V599 M mutation might be a stomach cancer-specific BRAF mutation. In two stomach cancers, we also found D593G BRAF mutation, which has also been detected in colon tumors previously. In BRAF protein, several amino acids at the activation segment are conserved among the species, and these amino acids (D593, F594, G595, L596, T598, V599 and K600) were reported to be mutated in tumors (Brose et al., 2002;

Davies et al., 2002; Naoki et al., 2002; Rajagopalan et al., 2002; Yuen et al., 2002; Pollock et al., 2003;

Satyamoorthy et al., 2003). Currently, it is not known how the V599 M and D593G mutants alter the function of BRAF, and how they contribute to the development of stomach cancer. Clearly, further studies are required to characterize the functional consequences of these mutants in gastric carcinogenesis.

Previous documents on BRAF mutations revealed that the coincidence of BRAF and RAS mutations in the same tumor is not a rare event (Brose et al., 2002; Davies et al., 2002; Naoki et al., 2002; Rajagopalan et al., 2002;

Yuen et al., 2002; Pollock et al., 2003; Satyamoorthy et al., 2003). In agreement with these reports, one of the seven stomach cancers with BRAF mutation had a RAS mutation. Cells both with activating mutations of KRAS BRAF had a substantially higher BRAF kinase activity and ERK1/2 phosphorylation activity than those with BRAF mutation alone (Davies et al., 2002).

It is possible that the tumors with both BRAF and RAS mutations might have an accelerated course in the development or progression of the tumors. BRAF mutations have been detected in early stages of colon cancer and melanoma development (Yuen et al., 2002;

Pollock et al., 2003). By contrast, we detected BRAF mutations in AGC, but not in EGC. Although it was not statistically significant, this result suggested the role of mutant BRAF proteins in the relatively late stage of stomach cancer development. However, since number of stomach cancers with BRAF mutations is small, the common clinical features of the tumors with BRAF mutation remain unknown at this stage.

In summary, we have found seven BRAF mutations in 319 primary stomach cancers. Despite the low frequency of BRAF mutation in stomach cancer compared with that of malignant melanoma, our data suggest that alteration of RAS RAF kinase pathway by BRAF mutation together with RAS mutation may play an important role in gastric carcinogenesis.

 

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The median incidence of KRAS mutations was similar in studies from the East and the West (East: 6%, ranging from 0-20%; West 7.5%, ranging from 0-29%). KRAS amplifications were reported at an incidence of 1-9%. The median BRAF mutation incidence in GC was 0%, ranging from 0% to 12%.

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KRAS, BRAF and gastric cancer - Hewitt​

 

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These cancers include:

• Melanoma (about half of all melanomas have the BRAF gene mutation)
• Hairy cell leukemia.
• Non-Hodgkin lymphoma.
• Thyroid cancer.
• Ovarian cancer.
• Lung adenocarcinoma.
• Colorectal cancer.
• Certain brain cancers, including glioblastoma, pilocytic astrocytoma, and pediatric low-grade glioma.

https://www.hopkinsmedicine.org › ...

BRAF Mutation and Cancer | Johns Hopkins Medicine​