BRAF and KRAS mutations in stomach cancer - Nature

Oncogene (2003) 22, 6942–6945

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BRAF and KRAS mutations in stomach cancer Sug Hyung Lee1,2, Jong Woo Lee1,2, Young Hwa Soung1, Hong Sug Kim1, Won Sang Park1, Su Young Kim1, Jong Heun Lee1, Jik Young Park1, Yong Gu Cho1, Chang Jae Kim1, Suk Woo Nam1, Sang Ho Kim1, Jung Young Lee1 and Nam Jin Yoo*,1 1

Department of Pathology, College of Medicine, The Catholic University of Korea, 505 Banpo-dong, Socho-gu, Seoul 137-701, Korea

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 amino acid (V599 M) was different from the most common BRAF mutation (V599E). The remaining two mutations involved a conserved amino acid (D593G). One 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 in stomach cancer, and suggest that alterations of RAS pathway both by RAS and BRAF mutations contribute to the pathogenesis of stomach cancer. Oncogene (2003) 22, 6942–6945. doi:10.1038/sj.onc.1206749 Keywords: mutation; BRAF; RAS; stomach cancer; stomach

The RAS gene family consists of three closely related genes (HRAS, KRAS and NRAS), which have similar structures and encode p21 RAS (Kolch, 2000; Peyssonnaux and Eychene, 2001). These p21 RAS proteins, known to play an important role in the regulation of normal signal transduction, bind guanosine triphosphate (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 *Correspondence: NJ Yoo; E-mail: goldfi[email protected] 2 Contributed equally to this work Received 27 February 2003; revised 23 April 2003; accepted 23 April 2003

gene family consists of three members, each encoding serine/threonine kinases that are regulated by binding to RAS. RAS–RAF–MEK–ERK–MAP kinase pathway plays a critical role in cell proliferation, and is frequently activated in cancer cells. For example, approximately 10–20% of all human tumors contain mutated versions of RAS proteins, which activate the downstream pathway (Bos, 1989). Recently, Davies et al. (2002) identified somatic mutations of BRAF, one of the RAF members, in 66% of malignant melanomas and at a lower frequency in a wide range of human cancers. So far, all BRAF mutations have been reported within two kinase domains, and the most common mutation is a 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 elevated kinase activity and transforming activity in NIH3T3 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). 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 a low incidence of Ras gene mutation in gastric carcinoma (roughly 0–10%) (Jiang et al., 1989; Lee et al., 1995; Arber et al., 2000). As RAS-RAF–MEK–ERK–MAP kinase pathway is important for the development of human cancer, searching the mutation of genes in this pathway in addition to RAS is necessary. Although screening of BRAF mutation in human tumors has widely been performed, to date the data on BRAF 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 BRAF gene mutations in stomach cancers and in particular their relationship with KRAS mutations. Paraffin-embedded tissues of human stomach carcinomas were obtained from 319 patients. These samples consisted of 60 early gastric cancer (EGC) and 259 advanced gastric cancer (AGC). Histologically, the samples consisted of 170 diffuse-type and 149 intestinal-type gastric cancers. Through the microdissection technique, we selectively procured tumor cells and corresponding normal cells from histological sections

BRAF mutations in stomach cancer SH Lee et al

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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 Gloop 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 (P40.05). 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 (P40.05, 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

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 somatically. 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). 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 phosphorylation, 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

Table 1 Summary of BAD mutations identified in the stomach cancer BRAF mutationsa Nucleotide A1778G A1778G G1795A G1795A G1795A G1795A G1795A Nil Nil Nil Nil Nil Nil Nil Nil

KRAS mutationsa aab

Nucleotide

aab

D593G D593G V599M V599M V599M V599M V599M Nil Nil Nil Nil Nil Nil Nil Nil

Nil Nil Nil Nil Nil Nil G37A G37A G34T A15C G175A G175A G175A G175A G175A

Nil Nil Nil Nil Nil Nil G13D G13D G12V K5N A59T A59T A59T A59T A59T

Histologic type

TNM stage

EGC/AGC c

Diffuse Intestinal Diffuse Diffuse Intestinal Intestinal Diffuse Intestinal Intestinal Diffuse Intestinal Diffuse Intestinal Diffuse Diffuse

III IV I III II I III IV I II I III IV III I

AGC AGC AGC AGC AGC AGC AGC AGC AGC AGC AGC AGC AGC AGC EGC

a Numbering of cDNA of BRAF and KRAS was done with respect to the ATG start codon. baa, amino acid. cEGC, early gastric cancer; AGC, advanced gastric cancer

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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 941C and incubated for 30 cycles. Other procedures of PCR and SSCP 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 Biosystem, Foster City, CA, USA) according to the manufacturer’s recommendation. 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 (c). 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 (GenBank)

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mutation at the amino acid 599, raising the possibility that the V599 M mutation might be a stomach cancerspecific 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 and 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 the 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. Acknowledgements This work was supported by Grant (No. R02-2002-00000050-0) from the KOSEF. References Arber N, Shapira I, Ratan J, Stern B, Hibshoosh H, Moshkowitz M, Gammon M, Fabian I and Halpern Z. (2000). Gastroenterology, 118, 1045–1050. Bae JM, Won YJ, Jung KW, Suh KA, Ahn DH and Park JG. (1999). J. Korean Cancer Assoc., 30, 1175–1183. Bos JL. (1989). Cancer Res., 49, 4682–4689.


6945 Brose MS, Volpe P, Feldman M, Kumar M, Rishi I, Gerrero R, Einhorn E, Herlyn M, Minna J, Nicholson A, Roth JA, Albelda SM, Davies H, Cox C, Brignell G, Stephens P, Futreal PA, Wooster R, Stratton MR and Weber BL. (2002). Cancer Res., 62, 6997–7000. Davies H, Bignell GR, Cox C, Stephens P, Edkins S, Clegg S, Teague J, Woffendin H, Garnett MJ, Bottomley W, Davis N, Dicks E, Ewing R, Floyd Y, Gray K, Hall S, Hawes R, Hughes J, Kosmidou V, Menzies A, Mould C, Parker A, Stevens C, Watt S, Hooper S, Wilson R, Jayatilake H, Gusterson BA, Cooper C, Shipley J, Hargrave D, Pritchard-Jones K, Maitland N, Chenevix-Trench G, Riggins GJ, Bigner DD, Palmieri G, Cossu A, Flanagan A, Nicholson A, Ho JW, Leung SY, Yuen ST, Weber BL, Seigler HF, Darrow TL, Paterson H, Marais R, Marshall CJ, Wooster R, Stratton MR and Futreal PA. (2002). Nature, 417, 949–954. Jiang W, Kahn SM, Guillem JG, Lu SH and Weinstein IB. (1989). Oncogene, 4, 923–928. Kolch W. (2000). Biochem. J., 351 (Part 2), 289–305. Lee KH, Lee JS, Suh C, Kim SW, Kim SB, Lee JH, Lee MS, Park MY, Sun HS and Kim SH. (1995). Cancer, 75, 2794–2801. Naoki K, Chen TH, Richards WG, Sugarbaker DJ and Meyerson M. (2002). Cancer Res., 62, 7001–7003.

Neubauer A, Dodge RK, George SL, Davey FR, Silver RT, Schiffer CA, Mayer RJ, Ball ED, Wurster-Hill D and Bloomfield CD. (1994). Blood, 83, 1603–1611. Peyssonnaux C and Eychene A. (2001). Biol. Cell, 93, 53–62. Pollock PM, Harper UL, Hansen KS, Yudt LM, Stark M, Robbins CM, Moses TY, Hostetter G, Wagner U, Kakareka J, Salem G, Pohida T, Heenan P, Duray P, Kallioniemi O, Hayward NK, Trent JM and Meltzer PS. (2003). Nat. Genet., 33, 19–20. Rajagopalan H, Bardelli A, Lengauer C, Kinzler KW, Vogelstein B and Velculescu VE. (2002). Nature, 418, 934. Satyamoorthy K, Li G, Gerrero MR, Brose MS, Volpe P, Weber BL, Van Belle P, Elder DE and Herlyn M. (2003). Cancer Res., 63, 756–759. Shin MS, Park WS, Kim SY, Kanf SJ, Song KY, Park JY, Dong SM, PiJH, Oh RR, Lee JY, Yoo NJ and Lee SH. (1999). Am. J. Pathol., 154, 1785–1791. Smith ML, Snaddon J, Neat M, Cambal-Parrales M, Arch R, Lister TA and Fitzgibbon J. (2003). Leukemia, 17, 274–275. Yuen ST, Davies H, Chan TL, Ho JW, Bignell GR, Cox C, Stephens P, Edkins S, Tsui WW, Chan AS, Futreal PA, Stratton MR, Wooster R and Leung SY. (2002). Cancer Res., 62, 6451–6455.

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