HUMAN MUTATION Mutation in Brief #923 (2006) Online

MUTATION IN BRIEF

Somatic Mutations of GUCY2F, EPHA3, and NTRK3 in Human Cancers Laura D. Wood1, Eric S. Calhoun1, Natalie Silliman1, Janine Ptak1, Steve Szabo1, Steve M. Powell2, Gregory J. Riggins1,3, Tian-Li Wang1, Hai Yan4, Adi Gazdar5, Scott E. Kern1, Len Pennacchio6, Kenneth W. Kinzler1, Bert Vogelstein1, and Victor E. Velculescu1*

1The Ludwig Center for Cancer Genetics and Therapeutics and The Howard Hughes Medical Institute at the Johns Hopkins Kimmel Cancer Center, Baltimore, Maryland; 2Divison of Gastroenterology/Hepatology, University of Virginia Health System, Charlottesville, Virginia; 3Department of Neurosurgery, Johns Hopkins Medical Institutions, Baltimore, Maryland; 4Department of Pathology, Duke University Medical Center, Durham, North Carolina; 5Hamon Center for Therapeutic Oncology Research and Department of Pathology, University of Texas Southwestern Medical Center, Dallas, Texas; 6U.S. Department of Energy Joint Genome Institute, Walnut Creek, California

*Correspondence to: Victor E. Velculescu, M.D., Ph.D., The Ludwig Center for Cancer Genetics and Therapeutics at the Johns Hopkins Kimmel Cancer Center, 1650 Orleans Street, Room 5M05, Baltimore, Maryland 21231; E- mail: [email protected]

Communicated by Richard Wooster

Tyrosine kinases are major regulators of cascades involved in cellular proliferation and have important roles in tumorigenesis. We have recently analyzed the tyrosine kinase family for alterations in human colorectal cancers and identified somatic mutations in seven members of this gene family. In this study we have used high- throughput sequencing approaches to further evaluate this subset of for genetic alterations in other human tumors. We identified somatic mutations in GUCY2F, EPHA3, and NTRK3 in breast, lung, and pancreatic cancers. Our results implicate these tyrosine kinase genes in the pathogenesis of other tumor types and suggest that they may be useful targets for diagnostic and therapeutic intervention in selected patients. Published 2006 Wiley- Liss, Inc.

KEY WORDS: tyrosine kinases; human cancer; genetic mutation; GUCY2F; EPHA3; NTKR3

INTRODUCTION Tyrosine kinases are important regulators of signal transduction pathways, including those important for cell growth and apoptosis. Several studies have systematically evaluated the sequences of kinase genes in distinct types of human tumors (Bardelli, et al., 2003; Davies, et al., 2005; Parsons, et al., 2005; Stephens, et al., 2005). In our previous study of tyrosine kinase genes in colorectal tumors, we identified mutations in a small subset of genes in this family, including the EPHA3, FES, GUCY2F, KDR, MLK4, NTRK2, and NTRK3 genes (Bardelli, et al., 2003). In the current study, we further analyzed this subset of genes in other tumor types and identified somatic mutations in three different genes in breast, lung, and pancreatic cancers.

Received 12 April 2005; accepted revised manuscript 23 May 2006.

PUBLISHED 2006 WILEY-LISS, INC. DOI: 10.1002/humu.9452

2 Wood et al.

METHODS Primers for polymerase chain reaction (PCR) and sequencing reactions were synthesized by MWG (High Point, NC, USA; http://www.mwg-biotech.com) and were identical to those previously described (Bardelli, et al., 2003). All exons encoding the kinase domains of EPHA3 (MIM# 179611; GenBank Accession NM_005233.3), FES (MIM# 190030; GenBank Accession NM_002005.2), KDR (MIM# 191306; NM_002253.1), MLK4 (GenBank Accession NM_032435.1), NTRK2 (MIM# 600456; GenBank Accession NM_006180.3), and NTRK3 (MIM# 191316; GenBank Accession NM_001012338.1) were PCR amplified and directly sequenced in a panel of 94 tumors comprising 23 lung carcinomas, 11 breast carcinomas, 12 pancreatic carcinomas, 12 gastric carcinomas, 12 ovarian carcinomas, 12 glioblastomas, and 12 medulloblastomas (Supplementary Tables 1 and 2; see Appendix). For GUCY2F (MIM# 300041; GenBank Accession NM_001522.1), all exons (including those not in the kinase domain) were sequenced because the kinase domain of this gene is thought to be inactive (Lucas, et al., 2000) and previous analyses identified mutations throughout the gene sequence (Bardelli, et al., 2003). All tumor samples were obtained in accordance with the Health Insurance Portability and Accountability Act regulations. PCR amplification and sequencing reactions were performed using 384 capillary automated sequencing apparatuses (Spectrumedix, State College, PA, USA; http://www.spectrumedix.com) as previously described (Wang, et al., 2002). In all cases, sequence traces were assembled and analyzed to identify potential somatic mutations using the Mutation Surveyor software package (SoftGenetics, State College, PA, USA; http://www.softgenetics.com). Of the 71 exons sequenced, 94% were successfully analyzed. Any exon with a potential somatic mutation was independently amplified and sequenced in the corresponding tumor and normal DNA to confirm the observed alteration. Statistical evaluation of mutation frequencies was performed using TRAB (Wang, et al., 2002) (http://astor.som.jhmi.edu/~gp/software/trab/). To assess evolutionary conservation of mutated residues, human protein sequences were compared to those of other available species. For GUCY2F these included sequences from cow, mouse, and rat; for EPHA3 these included sequences from chicken, mouse, and rat; and for NTRK3 these included sequences from chicken, mouse, rat, pig, macaque, and chimpanzee. Sequences were aligned using the Clustal Method (DNASTAR, Inc., Madison,WI, USA; http://www.dnastar.com) and identical residues were considered conserved.

RESULTS Analysis of the seven tyrosine kinase genes in 94 human tumors identified a total of 6 nonsynonymous somatic mutations: two missense changes in EPHA3, both in lung tumors, and 4 missense changes in GUCY2F, affecting two pancreatic tumors, one lung tumor, and one breast tumor (Table 1, examples in Fig. 1). One synonymous somatic mutation in EPHA3 was also detected in a lung tumor. Because of the high frequency of GUCY2F mutations in the 12 pancreatic tumors examined and because of the previously identified importance of NTRK3 in pancreatic cancer (Miknyoczki, et al., 1999a; Miknyoczki, et al., 1999b), we further analyzed the sequence of these two genes in 48 additional pancreatic tumors (Supplementary Table 3). These analyses identified one nonsynonymous somatic mutation in NTRK3 and no additional changes in GUCY2F (Table 1, Fig. 1).

Table 1. Somatic mutations detected in GUCY2F, EPHA3, and NTRK3 Tumor type and Tumor type and NCBI Tumor Mutation Gene name Other names fraction affected fraction affected accession sample (nucleotide and amino acid) (this study) (other studies) pancreas (2/60) Px147 c.2015A>C (p.Lys672Thr) GUC2DL, GC-F, Px117 c.3187A>C (p.Lys1063Gln) colon (10/182), lung GUCY2F RetGC-2, ROS-GC2, NM_001522.1 lung (1/23) NCI-H2009 c.3155A>G (p.Lys1052Arg)‡ (3/89), breast (1/81) CYGF breast (1/11) HCC2157 c.29G>C (p.Arg10Pro)*‡ lung (3/23) HCC15 c.1979C>A (p.Thr660Lys) ETK, ETK1, TYRO4, colon (2/182), EPHA3 NM_005233.3 HCC515 c.2798C>T (p.Thr933Met)* HEK, HEK4 lung (4/89) HCC515 c.2832C>A (p.Ala944Ala)* colon (6/182), lung NTRK3 TRKC NM_002530.2 pancreas (1/60) Px21 c.1795C>T (p.His599Tyr)* (2/89), breast (1/81)

For each tumor type, the fraction of samples with somatic mutations is indicated. The nucleotide position of each mutation corresponds to the position of that change in the coding sequence of each gene, where position 1 is the A of the ATG. Mutations indicated by a star are homozygous, presumably due to loss of heterozygosity (LOH) of the wild-type allele in the tumor. All other mutations are heterozygous. All mutations affected evolutionarily conserved residues except the G to C change in GUCY2F at nucleotide position 29. Mutations described in previous studies include the following reports: colon cancer (Bardelli et al., 2003), breast cancer (Stephens et al., 2005), and lung cancer (Davies et al., 2005). The mutations indicated by a "‡" in GUCY2F in breast and lung tumors were identified in the same samples previously described in the Stephens et al. and Davies et al. studies, respectively. NTRK3, EPHA3, and GUCY2F Mutations in Cancer 3

GUCY2F EPHA3 NTRK3

Normal

C Tumor

c.2015A>C c.2798C>T c.1795C>T (p.Lys672Thr) (p.Thr933Met) (p.His599Tyr)

Figure 1. Examples of somatic mutations in GUCY2F, EPHA3, and NTRK3. Representative examples of mutations in the three genes. In each case the bottom sequence chromatogram was obtained from tumor DNA, and the top chromatogram was obtained from normal DNA from the same patient. Arrows indicate the location of missense somatic mutations, and the nucleotide and amino acid alterations are indicated below the traces.

DISCUSSION One of the questions that can be addressed by studies such as the one described above is whether certain genes play a role in specific tumor types or are more generally important in tumorigenesis. The results presented here suggest that some tyrosine kinases may play unique roles in specific tumors. For example, while MLK4 is mutated in >5% of colorectal tumors (Bardelli, et al., 2003), no mutations were detected in MLK4 in any of seven different tumor types examined in this study (p<0.05, chi-square test). Additionally, no mutations in MLK4 were identified in independent studies analyzing protein kinases in lung, breast, and testicular tumors (Davies, et al., 2005; Stephens, et al., 2005; Bignell, et al., 2006). Thus, while MLK4 may play a role in colorectal tumorigenesis, it is less likely to play a role in the other tumor types examined to date. This conclusion should be tempered by the fact that in this study only a limited number of tumors of each type were analyzed and only the kinase domain of each gene was examined, potentially missing rare mutations or mutations that occur outside the kinase domain. In contrast, GUCY2F, EPHA3, and NTRK3 are implicated in several other tumor types. We found that GUCY2F was somatically mutated in breast, lung, and pancreatic tumors, EPHA3 was altered in lung tumors, and NTRK3 was mutated in pancreatic tumors. These results are consistent with the recent reports of mutations of GUCY2F and NTRK3 in lung and breast tumors and mutations of EPHA3 in lung cancers (Davies, et al., 2005; Stephens, et al., 2005). While the number of mutations in these genes is relatively small, two levels of evidence suggest that the identified nonsynonymous mutations were functionally important rather than passenger (nonfunctional) alterations. First, the somatic mutation frequencies observed in these genes (4 nonsynonymous somatic mutations in ~1 Mb of DNA from non-lung tumors, and 4 somatic mutations in ~0.25 Mb of DNA from lung tumors) are significantly higher than the background somatic mutation frequencies previously observed in different tumors (~1 nonsynonymous somatic mutation per Mb DNA for most tumors examined to date, p<0.05; ~3 somatic mutations per Mb of DNA for lung tumors, p<0.05) (Wang, et al., 2002; Davies, et al., 2005; Stephens, et al., 2005; Bignell, et al., 2006). Second, the specific locations of observed mutations suggest that they are functionally important. Six of the seven observed changes occurred in residues that were evolutionarily identical in all species analyzed. Although further analyses will be needed to examine the function of these mutated genes in tumorigenesis, previous studies of GUCY2F, EPHA3, and NTRK3 suggest potentially interesting roles for these genes in neoplasia. GUCY2F is one of six receptor guanylate that catalyze the synthesis of cGMP, a second messenger involved in multiple signaling cascades (Lucas, et al., 2000). Although no mutations in other guanylate cyclases have been identified, previous studies suggested that these genes may play a role in regulating cellular growth. For example, activation of guanylate C (GC-C), which is expressed throughout the gastrointestinal tract, suppresses proliferation of colon cancer cells in vitro and inhibits polyp formation in a mouse model of familial adenomatous polyposis (FAP) (Shailubhai, et al., 2000; Pitari, et al., 2001; Pitari, et al., 2003). Conversely, 4 Wood et al. inactivation of the guanylin gene, which encodes a ligand for GC-C, results in decreased cGMP levels in the colonic mucosa and increased proliferation of the colonic epithelium (Steinbrecher, et al., 2002). Accordingly, decreased expression of guanylin has been observed in mouse and human intestinal adenomas (Steinbrecher, et al., 2000). These results suggest a role for cGMP signaling in inhibiting tumorigenesis and suggest that mutations of GUCY2F disrupt this signaling. The mutations in EPHA3 highlight another gene family that appears to have a significant role in tumorigenesis. EPHA3 is a member of the Eph family of receptors, the largest subfamily of protein tyrosine kinases, consisting of 14 members (Manning, et al., 2002). In addition to mutations in EPHA3 that we identified in colorectal and lung tumors, mutations in ephrin receptors have also been identified in breast cancer (EPHA1, EPHA6, EPHA10) (Stephens, et al., 2005) and in lung cancer (EPHA3, EPHA5, EPHA6, EPHB2, EPHB3, EPHB4) (Davies, et al., 2005). Eph receptors are thought to be involved in the development of the mammalian nervous and vascular systems and have been shown to be important in the processes of cell adhesion and angiogenesis (Dodelet and Pasquale, 2000). Interestingly, soluble forms of ephrin A receptors (EPHA2 and EPHA3) appear to inhibit tumor angiogenesis and tumor progression (Brantley, et al., 2002; Cheng, et al., 2003), suggesting that specific inhibition of ephrin receptors may be therapeutically useful. NTRK3, also known as TrkC, is one of three high-affinity neurotrophin receptors which regulate growth, differentiation, and apoptosis of neurons (Nakagawara, 2001). The NTRK1 gene was originally cloned as an oncogenic gene fusion with the tropomyosin gene in a colon carcinoma (Martin-Zanca, et al., 1986; Martin-Zanca, et al., 1989). Rearrangements of NTRK3 and ETV6 have also been identified in congenital fibrosarcomas and secretory breast carcinomas (Knezevich, et al., 1998; Tognon, et al., 2002). Through our study and those of others, point mutations in NTRK3 have been now identified in colorectal, lung, breast, and pancreatic tumors (Bardelli, et al., 2003; Davies, et al., 2005; Stephens, et al., 2005). At least some of these mutations appear to affect the activation loop of the kinase domain, potentially resulting in constitutive kinase activity that may be required for continued tumor growth. If these predictions are confirmed experimentally, pharmacologic inhibition of NTRK3 should be considered as an approach to treat tumors containing such mutations. Previous reports have shown that inhibition of neurotrophin receptors can reduce cancer cell growth in vitro (Weeraratna, et al., 2000), and decrease metastases and increase survival in mouse models in vivo (Miknyoczki, et al., 1999a; Weeraratna, et al., 2001), suggesting that this approach may be realistic.

ACKNOWLEDGMENTS This work was supported by The Pew Charitable Trusts, and NIH grants CA121113, CA062924, and CA057345. A portion of this research was conducted at the E.O. Lawrence Berkeley National Laboratory and performed under Department of Energy Contract DE-AC02-05CH11231, University of California.

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APPENDIX

Supplementary Table 1. Tyrosine kinase gene regions screened

Gene name NCBI accession Domain screened Exons screened EPHA3 NM_005233.3 kinase domain 10-17 FES NM_002005.2 kinase domain 13-19 GUCY2F NM_001522.1 whole gene 1-20 KDR NM_002253.1 kinase domain 17-25 MLK4 NM_032435.1 kinase domain 1-5 NTRK2 NM_006180.3 kinase domain 14-19 NTRK3 NM_002530.2 kinase domain 13-18

Supplementary Table 2. Tumor samples analyzed for seven tyrosine kinase genes Tumor tissue Tumor type Tumor name Sample type Lung Adenocarcinoma NCI-H1395 Cell line Lung Adenocarcinoma NCI-H1437 Cell line Lung Adenocarcinoma NCI-H2009 Cell line Lung Adenocarcinoma NCI-H2122 Cell line Lung Adenocarcinoma NCI-H2087 Cell line Lung Small cell carcinoma NCI-H2171 Cell line Lung Small cell carcinoma NCI-H2195 Cell line Lung Small cell carcinoma NCI-H1184 Cell line Lung Small cell carcinoma NCI-H209 Cell line Lung Small cell carcinoma NCI-H2107 Cell line Lung Small cell carcinoma NCI-H128 Cell line Lung Adenocarcinoma HCC193 Cell line Lung Squamous carcinoma HCC15 Cell line Lung Adenocarcinoma HCC78 Cell line Lung Adenocarcinoma HCC366 Cell line Lung Adenocarcinoma HCC515 Cell line Lung Large cell carcinoma HCC20 Cell line Lung Non-Small cell carcinoma HCC2429 Cell line Lung Small cell carcinoma NCI-H1450 Cell line Lung Small cell carcinoma HCC33 Cell line Lung Small cell carcinoma HCC970 Cell line Lung Small cell carcinoma NCI-H1607 Cell line Lung Small cell carcinoma NCI-H2028 Cell line 8 Wood et al.

Supplementary Table 2 (Continued). Tumor tissue Tumor type Tumor name Sample type Breast Ductal carcinoma Hs 578T Cell line Breast Ductal carcinoma HCC1008 Cell line Breast Ductal carcinoma HCC1954 Cell line Breast Ductal carcinoma HCC38 Cell line Breast Ductal carcinoma HCC1143 Cell line Breast Ductal carcinoma HCC1187 Cell line Breast Ductal carcinoma HCC1395 Cell line Breast Ductal carcinoma HCC1599 Cell line Breast Ductal carcinoma HCC1937 Cell line Breast Ductal carcinoma HCC2157 Cell line Breast Ductal carcinoma HCC2218 Cell line Pancreas Ductal adenocarcinoma Px55 Xenograft Pancreas Ductal adenocarcinoma Px56 Xenograft Pancreas Ductal adenocarcinoma Px94 Xenograft Pancreas Ductal adenocarcinoma Px117 Xenograft Pancreas Ductal adenocarcinoma Px120 Xenograft Pancreas Ductal adenocarcinoma Px139 Xenograft Pancreas Ductal adenocarcinoma Px147 Xenograft Pancreas Ductal adenocarcinoma Px149 Xenograft Pancreas Ductal adenocarcinoma Px153 Xenograft Pancreas Ductal adenocarcinoma Px171 Xenograft Pancreas Ductal adenocarcinoma Px184 Xenograft Pancreas Ductal adenocarcinoma Px188 Xenograft Gastric Intestinal adenocarcinoma GX14 Xenograft Gastric Diffuse adenocarcinoma GX38 Xenograft Gastric Diffuse adenocarcinoma GX40 Xenograft Gastric Diffuse adenocarcinoma GX49 Xenograft Gastric Intestinal adenocarcinoma GX52 Xenograft Gastric Intestinal adenocarcinoma GX57 Xenograft Gastric N/A GX58 Xenograft Gastric Diffuse adenocarcinoma GX73 Xenograft Gastric Intestinal adenocarcinoma GX80 Xenograft Gastric N/A GX96 Xenograft Gastric Intestinal adenocarcinoma GX101 Xenograft Gastric N/A GX112 Xenograft Ovarian Serous carcinoma 196 Primary tumor Ovarian Serous carcinoma 215 Primary tumor Ovarian Serous carcinoma 310 Primary tumor Ovarian Serous carcinoma 452 Primary tumor Ovarian Serous carcinoma 463 Primary tumor Ovarian Serous carcinoma 541 Primary tumor Ovarian Serous carcinoma 608 Primary tumor Ovarian Serous carcinoma 660 Primary tumor Ovarian Serous carcinoma 713 Primary tumor Ovarian Serous carcinoma 1120 Primary tumor Ovarian Serous carcinoma 12736 Primary tumor Ovarian Serous carcinoma 2549 Primary tumor Brain Glioblastoma H534C Cell line Brain Glioblastoma H542C Cell line Brain Glioblastoma H566C Cell line Brain Glioblastoma H693X Xenograft Brain Glioblastoma H717X Xenograft Brain Glioblastoma H561X Xenograft Brain Glioblastoma H395X Xenograft Brain Glioblastoma H317X Xenograft Brain Glioblastoma H457X Xenograft Brain Glioblastoma H839P Primary tumor Brain Glioblastoma H860P Primary tumor Brain Glioblastoma H522P Primary tumor Brain Medulloblastoma H385 Primary tumor Brain Medulloblastoma H556 Xenograft Brain Medulloblastoma H690 Xenograft Brain Medulloblastoma H692 Primary tumor Brain Medulloblastoma H876 Primary tumor Brain Medulloblastoma H931 Primary tumor Brain Medulloblastoma H1413 Primary tumor Brain Medulloblastoma H1414 Primary tumor Brain Medulloblastoma H1456 Primary tumor Brain Medulloblastoma H1483 Primary tumor Brain Medulloblastoma H1922 Primary tumor Brain Medulloblastoma H2138 Primary tumor NTRK3, EPHA3, and GUCY2F Mutations in Cancer 9

Supplementary Table 3. Additional tumor samples analyzed for GUCY2F and NTRK3 Tumor tissue Tumor type Tumor Name Source of DNA Pancreas Ductal adenocarcinoma PX 16 Xenograft Pancreas Ductal adenocarcinoma PX 17 Xenograft Pancreas Ductal adenocarcinoma PX 19 Xenograft Pancreas Ductal adenocarcinoma PX 21 Xenograft Pancreas Ductal adenocarcinoma PX 24 Xenograft Pancreas Ductal adenocarcinoma PX 27 Xenograft Pancreas Ductal adenocarcinoma PX 29 Xenograft Pancreas Ductal adenocarcinoma PX 61 Xenograft Pancreas Ductal adenocarcinoma PX 66 Xenograft Pancreas Ductal adenocarcinoma PX 67 Xenograft Pancreas Ductal adenocarcinoma PX 72 Xenograft Pancreas Ductal adenocarcinoma PX 74 Xenograft Pancreas Ductal adenocarcinoma PX 75 Xenograft Pancreas Ductal adenocarcinoma PX 76 Xenograft Pancreas Ductal adenocarcinoma PX 88 Xenograft Pancreas Ductal adenocarcinoma PX 90 Xenograft Pancreas Ductal adenocarcinoma PX 92 Xenograft Pancreas Ductal adenocarcinoma PX 101 Xenograft Pancreas Mucinous cystadenocarcinoma PX 104 Xenograft Pancreas Ductal adenocarcinoma PX 105 Xenograft Pancreas Ductal adenocarcinoma PX 122 Xenograft Pancreas Ductal adenocarcinoma PX 123 Xenograft Pancreas Ductal adenocarcinoma PX 132 Xenograft Pancreas Ductal adenocarcinoma PX 133 Xenograft Pancreas Ductal adenocarcinoma PX 141 Xenograft Pancreas Ductal adenocarcinoma PX 143 Xenograft Pancreas Ductal adenocarcinoma PX 150 Xenograft Pancreas Ductal adenocarcinoma PX 175 Xenograft Pancreas Ductal adenocarcinoma PX 178 Xenograft Pancreas Ductal adenocarcinoma PX 182 Xenograft Pancreas Ductal adenocarcinoma PX 186 Xenograft Pancreas Ductal adenocarcinoma PX 191 Xenograft Pancreas Ductal adenocarcinoma PX 192 Xenograft Pancreas Ductal adenocarcinoma PX 198 Xenograft Pancreas Ductal adenocarcinoma PX 226 Xenograft Pancreas Ductal adenocarcinoma PX 227 Xenograft Pancreas Ductal adenocarcinoma PX 240 Xenograft Pancreas Ductal adenocarcinoma PX 248 Xenograft Pancreas Biliary adenocarcinoma PX 258 Xenograft Pancreas Duodenal adenocarcinoma PX 264 Xenograft Pancreas Ductal adenocarcinoma PX 271 Xenograft Pancreas Ductal adenocarcinoma PX 281 Xenograft Pancreas Ductal adenocarcinoma PX 283 Xenograft Pancreas Ductal adenocarcinoma PX 289 Xenograft Pancreas Ductal adenocarcinoma PX 328 Xenograft Pancreas Ductal adenocarcinoma PX 338 Xenograft Pancreas Ductal adenocarcinoma PX 346 Xenograft Pancreas Ductal adenocarcinoma PX 348 Xenograft Ff