| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Experimental Therapeutics, Molecular Targets, and Chemical Biology |
1 Institute of Medical Biology, 2 Bioinformatics Institute, and 3 Institute of Molecular and Cell Biology, Singapore, Singapore; 4 Department of Pathology, Technical University of Munich, Munich, Germany; 5 Max-Planck-Institute for Biochemistry, Martinsried, Germany; 6 Department of Oncology, University of Chieti Medical School, Chieti, Italy; and 7 Department of Urology, Klinikum Darmstadt, Darmstadt, Germany
Requests for reprints: Jens E. Ruhe, U3 Pharma AG, Bunsenstrasse 1, 82152 Martinsried, Germany. E-mail: ruhe{at}u3pharma.com.
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResults and DiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
|---|
Because of the established relevance of PTKs for cancer initiation and progression, extensive screens for genomic sequence changes of the cancer kinome have been undertaken and yielded a diversity of sporadic alterations (1–8). Besides sporadic mutations that accumulate in the developing tumor due to cancer genome instability, DNA sequence polymorphisms have been linked to cancer aggressiveness and susceptibility (9). The fibroblast growth factor receptor 4 (FGFR4) G388R single nucleotide polymorphism (SNP), for instance, was found to be associated with increased cell motility in vitro as well as lymph node metastasis, advanced tumor stage, and reduced survival of breast cancer patients (10). Moreover, this SNP was shown to mediate resistance to adjuvant therapy in primary breast cancer, and its clinical relevance was also shown for soft tissue sarcoma, and head and neck, colon, and prostate cancers (11). Recent genome-wide association studies to identify SNPs connected with cancer susceptibility reflect the increasing recognition of polymorphisms as cancer-relevant parameters (12–14).
Through cDNA-based tyrosine kinase transcriptome (TKT) sequence evaluation, we performed a comprehensive characterization and analysis of widely used cancer cell lines with regard to variations in the primary structure of all expressed tyrosine kinases. For 254 established tumor cell lines of 19 tissue origins, we determined the profiles of both somatic mutations and germ line polymorphisms detectable in the transcripts of 90 PTK genes (15). The localization within the respective protein sequence, sequence comparisons with related genes, or the tissue distribution indicated potential functional relevance for individual genetic alterations that may define the cell line specific signaling characteristics and/or properties related to oncogenesis.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
|---|
RNA isolation and cDNA synthesis. Total RNA was extracted from exponentially growing cells using guanidinium thiocyanate (16). Subsequently, mRNA was isolated via oligo(dT) cellulose and reverse transcribed using oligo(dT) primers (17).
Genes, PCR, and sequencing. With purified cDNAs prepared from 261 cell lines and 15 control tissues as templates, overlapping fragments covering the entire coding region of all 58 receptor tyrosine kinase (RTK) and 32 cytoplasmic tyrosine kinase genes (15) were PCR amplified in at least two independent experiments. For each fragment, PCR conditions were specifically optimized using pooled cDNAs in a first step, and cDNAs from six individual cell lines in a second step. Thereby, the efficiencies of three proofreading polymerases (Novagen, Roche, Invitrogen) in combination with or without additives (DMSO, betaine, TMA oxalate), various primer pairs and different annealing temperatures were compared, and best parameters applied to the full panel of samples.
PCR products for all genes except IRR, MUSK, FGR, and SRMS, for which we obtained no amplicons, were subjected to direct sequencing of both forward and reverse strands. For selected alterations, homologous fragments from cDNAs of 165 primary tumors were PCR amplified using the same primer pairs. To further verify potential germ line origin of given variants, additional primers were designed to PCR amplify the respective region from genomic DNA of 90 blood samples from healthy donors.
Primers for amplification and sequencing of cDNA (Supplementary Table S3) and genomic DNA (Supplementary Table S4) were designed using the Primer3 program8 and refer to National Center for Biotechnology Information (NCBI)9 reference sequence files with accession numbers provided in Supplementary Table S5.
Data analysis. Sequence differences to the NCBI9 reference sequence were identified via manual inspection of aligned electropherograms assisted by the Mutation Surveyor software package. In addition to nonsynonymous genetic alterations, we detected numerous silent sequence variations that are not presented and further analyzed here. It is noteworthy that 63 sequence differences to the NCBI9 database occurred in all cDNA samples we analyzed and represent either other isoforms or variants that might actually be wild-type rather than genetic alterations (Supplementary Table S6).
|
Results and Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
|---|
|
Based on these data, we determined the absolute number and distribution of TKT-linked somatic mutations and germ line polymorphisms within the entire tumor cell line panel. For polymorphisms, we observed a Gaussian-like distribution with an average of 12.3 sequence variations per cancer cell line. In contrast, somatic mutations were unevenly distributed (Fig. 1C). No somatic alterations were detected in the TKT of 119 cancer cell lines, consistent with kinome mutations entirely absent in subsets of recently screened breast cancer, lung carcinoid, and testicular germ-cell tumor samples (1–3). In contrast, high frequencies of 9 to 14 somatic mutations in the transcribed tyrosine kinomes of LNCaP, Jurkat, MeWo, MKN-1, HCT-15, and DLD-1 might reflect a mutator phenotype (1). They are in agreement with sequence data of 24 cancer genes in the NCI-60 cell line panel that also showed HCT-15 to be one of the most frequently mutated tumor cell lines (18). With intermediate mutation rates for the other tumor cell lines, our data indicate an accumulation of somatic mutations in PTK transcripts of various cancer cell lines which may contribute to the progression characteristics of certain cancers.
In the entire panel of cancer cell lines and controls, we identified 389 nonsynonymous genetic variations within 39.85 of 72.08 MB of PTK coding sequence that were amplifiable and represent the detectable TKT of all samples. As alternative to the allocation to cell line–specific variant profiles, we grouped these sequence differences by genes and PTK subfamilies. Each variation was thereby specified regarding the spectrum of affected cell lines as well as the zygosity status and the presumable somatic versus germ line origin. These data are shown for FGFR4 (Fig. 2 ) that will be discussed below. The full information for all transcript variants and PTK genes can be obtained from Supplementary Table S8. Sequence specifications of frame shift alterations and insertions are provided in Supplementary Table S9.
|
The expressed PTK variants may define cell line–specific signaling characteristics and cancer-related cell properties. In the following sections, we therefore address tissue distribution and localization of each polymorphism and somatic mutation within the respective protein sequence. Based on these data and the current literature, we discuss potential functional and/or clinical relevance for some of the identified genetic variants.
Characterization of 155 PTK gene sequence polymorphisms. According to our definition of somatic and germ line sequence variants, 155 of the 389 identified alterations were classified as sequence polymorphisms. They include 131 SNPs, 16 germ line deletions, and 8 insertions. Their overall frequencies and localization in distinct protein domains are summarized in Fig. 3A and B . Moreover, the occurrence frequency of each polymorphism in individual tumor types or control samples was determined. Occurrence frequency was thereby defined as the fraction of carriers of a given sequence variant and the number of cell lines with the same tissue origin that express the corresponding gene regardless of its genotype (paired numbers in Fig. 3C and Supplementary Table S10). It therefore reflects the expression aspect of respective genes and alterations, as addressed by our cDNA analysis.
|
To verify the in vivo relevance of the sequence variations detected in tumor cell lines, we analyzed cDNA from 165 primary breast, kidney, and prostate cancer specimens as well as blood DNA from 90 healthy individuals for the occurrence of a representative subset of our identified genetic alterations. This subset was defined as all nonconservative sequence changes that were found at least twice in our panel of cell lines and control samples with at least one cell line originating from breast, kidney, or prostate cancer. All but 2 of the 46 polymorphisms that fulfilled these criteria could be verified in patient sample cDNAs or blood DNA (Supplementary Table S11), hence confirming the in vivo relevance of sequence variations in tumor cell lines.
Cancer relevance of identified polymorphisms. Some of the identified polymorphisms have previously been associated with nonproliferative diseases. Respective functional modulations may, because of the pleiotropic effects of many PTKs, also be relevant for cancer. This is exemplified by the V722I transversion in the pseudokinase domain of Janus-activated kinase 3 that we identified as a rare heterozygous polymorphism in the head and neck cancer cell lines SCC-10A and SCC-10B (Fig. 3C). First reported in patients with autosomal recessive T-B+ SCID syndrome (19), its recent detection in an acute megakaryoblastic leukemia patient and the capacity to transform Ba/F3 cells (20) support a potential role in cancer. Another example is NTRK1 R780Q, which we found in the colon, ovarian, and head and neck cancer cell lines Caco2, SK-OV-8, and SCC-9 (Fig. 3C), respectively. This SNP affects the same arginine residue whose replacement with proline was shown to be associated with "congenital insensitivity to pain with anhidrosis" and abrogation of catalytic tyrosine kinase activity in vitro (21). Assuming a similar loss-of-function for the NTRK1 Q780 isotype, this variant may exert antiapoptotic and hence pro-oncogenic effects, as expression of NTRK1 wild-type was associated with induction of apoptosis and a favorable prognosis of neuroblastoma patients (22).
Cancer relevance was also established for MET T1010I, which represents a biomarker for MET inhibitor efficacy (23) and was originally reported as a somatic gain-of-function mutation in small cell lung cancer and non–small cell lung cancer (NSCLC; ref. 24) and malignant pleural mesotheliomas (23). Its detection in 4 of our 90 blood control DNAs (Supplementary Table S11), however, confirmed previous hints for potential germ line occurrence (25). Moreover, the identification of MET T1010I in the prostate carcinoma cell line TSU-PR1 and a primary prostate tumor as well as in the brain, breast, colon, hematopoietic, and skin cancer cell lines IHR-32, DAL, LS-123, U-266, and Colo-829, respectively (Fig. 3C and Supplementary Tables S10 and S11), suggests enhanced MET signaling in these tumor cell lines and expands the currently reported spectrum of affected tumor types.
Polymorphism frequencies in cancer cells versus normal tissues. Differential occurrence rates of sequence polymorphisms in particular cancer types and/or normal tissues may indicate tumor suppressive or promoting effects. To address the potential relevance of all polymorphism for certain tumor types, we compared their occurrence frequencies in tissue types and control samples (Fig. 3C and Supplementary Table S10). Only some examples are displayed in Fig. 4 . For epidermal growth factor receptor (EGFR) R521K, a relative overrepresentation of the EGFR K521 allele in cDNAs of normal control samples (55%), colon (52%), and head and neck (69%) tumor cell lines was detected (Fig. 4, left). This indicates a possible tumor suppressive activity of the EGFR K521 isotype that apparently is not relevant to colon cancer and head and neck cancer. An attenuated growth response to EGFR ligands and reduced induction of the proto-oncogenes FOS, JUN, and MYC in EGFR K521, but not EGFR R521–expressing cells (26), and an increased risk of local recurrence after chemoradiation treatment for rectal cancer patients with at least one EGFR R521 allele (27) support these conclusions. Similarly, a clearly differential occurrence of TYK2 F362 allele carriers was observed in brain (75%)–derived and hematopoietic/lymphoid system (67%)–derived tumor cell lines compared with control tissues (31%) or other tumor types (Fig. 4, middle). The novel polymorphism TNK M598delinsEVRSHX was found at low frequencies in control samples (5%) and cancer cells of several tissue origins, but occurred in 62% of blood-derived, 55% of skin-derived, and, even more prominent, 80% of brain-derived tumor cell lines (Fig. 4, right). In contrast to EGFR R521K, the underrepresentation of the TYK2 F362 allele and the TNK insertion in control samples indicates a tumor-promoting function with particular relevance for leukemia, melanoma, and glioma. It can be expected that, as for EGFR R521K (27) or FGFR4 G388R (10), the correlation with clinical parameters will assign therapeutic and/or predictive value to many of such unequally distributed alleles.
|
Overall, these examples point at the potential role of sequence polymorphisms as genetic parameters that may contribute to a patient-specific definition of disease predisposition, rate of progression, or responsiveness to therapeutic agents. In conjunction with simple detectability in blood samples, this renders polymorphisms to be highly valuable biomarkers for diagnostic patient characterization.
Identification of 234 somatic PTK gene mutations. Of all sequence differences, 234 were undetectable in any of the control samples or public databases and were thus defined as somatic mutations. However, because of the lack of cell line–specific normal tissue controls, we cannot exclude the possibility that some actually represent rare germ line polymorphisms. The somatic mutations are composed of 210 missense and 2 nonsense single nucleotide substitutions as well as 19 deletions and 3 insertions. Although the majority (186) occurred once, 53 were found two to five times, and 3 in 6 to 10 tumor cell lines (Fig. 5A ). Among the twice-occurring somatic mutations, 20 were detected in cell lines originating from the same tumor donor (Supplementary Table S1). They may be considered single mutations, thus adding up to a total of 206 nonrecurring mutational events. As for the polymorphisms, we present all somatic TKT alterations in the context of the respective protein domains and tumor types, and display the ratio of affected and expression-positive cell lines for each tissue origin (Fig. 5B and C; Supplementary Table S12).
|
|
Similar to EGFR L858R and gefitinib, the KIT N822K mutation that we confirmed in the acute myelogenous leukemia cell line KASUMI-1 (32) was reported to mediate sensitivity to Gleevec (33). The enhanced in vitro receptor activation shown for these EGFR and KIT mutations (30, 34) might be related to their location within the regulatory activation loop. In this respect, the sporadic variations FLT3 R849H, TEK A1006T, ABL G417E, ARG K450R, and TEC W531R, which we detected homozygously or heterozygously in BM-1604, SK-MEL-2, MM-Leh, Caki-2, and Jurkat (Fig. 5C), are particularly intriguing as they are located in the activation loop as well. By inference, these mutations may also have a higher probability to modulate the tyrosine kinase catalytic activity and/or related signaling pathways within the respective tumor cell lines.
The 18 somatic mutations we identified in intracellular juxtamembrane domains (Supplementary Table S12) might affect functionally important elements that mediate down-regulation of RTK activity. The in-frame deletion MET D981_E1027del as a result of exon 14 skipping, for instance, leads to the loss of c-Cbl E3-ligase binding, decreased ubiquitination, and prolonged ligand-dependent cell signaling in vitro and in vivo (35). Although we confirmed MET D981_E1027del in the NSCLC cell line NCI-H596, its homozygous detection in breast and stomach cancer cell lines MDA-MB-415 and Hs746T, respectively (Fig. 5C), provides evidence for its occurrence in tumor types other than the reported NSCLC (35, 36). Presuming enhanced sensitivity to anti-MET therapeutics that MET D981_E1027del was suggested to mediate (35), our findings extend the potential clinical relevance for this deletion.
The somatic mutations we identified within the extracellular domain of two FGFR family members, FGFR1 P252S and FGFR4 Y367C (Figs. 5C and 6B), possibly augment receptor activation by influencing ligand binding and receptor dimerization, respectively. The highly conserved FGFR1 P252 residue we found to be heterozygously exchanged with hydrophilic serine in the melanoma cell line MeWo has previously been shown to be replaced by threonine in lung cancer (2) and arginine in patients with Pfeiffer syndrome (37). The crystal structure of the homologous activating FGFR2 mutant, FGFR2 P252R, revealed the formation of three additional hydrogen bonds with complexed fibroblast growth factor 2 (FGF2). They were predicted to increase the receptor affinity for its specific ligand as well as to allow binding of a different set of ligands (38). Because the hydroxyl group of the P252-replacing serine residue in FGFR1 also has a high potential to form additional hydrogen bonds, the somatic FGFR1 P252S substitution may represent a gain-of-function mutation with analogous functional consequences as for FGFR2 P252R. This is particularly intriguing in the context of studies demonstrating that blockage of FGFR1 or basic fibroblast growth factor function was associated with suppressed proliferation and survival of melanoma cells (39).
The novel FGFR4 Y367C mutation was detected as a homozygous genotype in the breast cancer cell line MDA-MB-453, and the affected Y367 residue in the extracellular juxtamembrane domain is highly conserved throughout the FGF family. Remarkably, homologous substitutions in FGFR1 (Y372C), FGFR2 (Y375C), and FGFR3 (Y373C) were shown to cause various osteogenic deficiency syndromes (40) through the formation of intermolecular disulfide bonds that force receptor dimerization and activation. Ligand-independent, constitutive receptor activation has been confirmed in vitro for FGFR1 Y372C (41) and FGFR3 Y373C (42). Furthermore, the oncogenic potential of the FGFR3 Y373C variant has been shown and was suggested to contribute to tumor progression of multiple myeloma (43). Thus, it is most likely that Y367C as the homologous FGFR4 variant also results in basal receptor activation, which strongly indicates an important role of this mutant in cancer.
Nonsense substitutions abrogate tumor suppressor activity. Down-regulation of tumor-suppressive activity is expected for the two nonsense substitutions we detected in EPHB2 and CSK (Fig. 5C). Q722X-mediated truncation and kinase inactivation of EPHB2 in the two prostate cancer cell lines BM-1604 and DU-145 supports mutational inactivation to be involved in progression of prostate cancer as proposed by Huusko et al. (44). They showed suppressed growth and colony formation of DU-145 cells upon reconstitution with functional EPHB2. The heterozygous CSK Q26X nonsense substitution detected in the colon cancer cell lines DLD-1 and HCT-15 is consistent with reduced protein levels of this negative regulator of SRC family kinases that were reported for 
60% of human colon cancer cases with elevated SRC activity (45). These data indicate a significant role of CSK nonsense mutations in the development and/or progression of colon carcinoma and therefore strongly suggest the inclusion of SRC kinase inhibitors in the therapeutic regimen of this prevalent malignancy.
The examples discussed above represent only a partial extract of our overall data. Other genetic alterations affecting less investigated PTKs such as members of the AATYK, DDR, EPH, ROR, ROS, or FRK families, as well as tyrosine kinases that more recently captured scientific attention such as HER3 or ACK1, have been found (Supplementary Tables S2–S6). Their identification will support novel functional investigations toward the understanding of the therapeutic value of these kinases.
Low redundancy of PTK gene mutations in human tumors. In agreement with results from previous studies (1–7, 46), our analysis of 254 cancer cell lines and additional primary tumors indicates that mutational patterns might be quite unique for the majority of human tumors, and that the frequency of specific somatic mutations in PTKs is low. Data mining of public databases and the literature revealed that only nine of all sporadic alterations identified in our study have been described before (Supplementary Table S12). Among them are KIT N822K and VEGFR1 R781Q as the only two alterations that were picked up in the currently most comprehensive mutational kinome analysis of human cancer samples (7). The low redundancy of somatic mutations is furthermore reflected by the nonrecurrence that we found for 206 of the 234 mutational events within our panel of tumor cell lines.
Consistent with this picture, none of the seven somatic representatives of our exemplary subset of nonconservative andmore frequent alterations found in at least one breast, kidney, or prostate cancer cell line could be detected in any of the 165 primary breast, kidney, and prostate cancer specimens (Supplementary Table S11). In fact, two genetic alterations, YES K113Q and TYRO3 E489K, were found in blood controls and therefore must be considered rare germ line alterations.
Despite the low redundancy of individual mutations, 70 of the tyrosine kinase genes turned out to carry at least one somatic mutation. Although most of our mutations require further experimental evaluation to determine their cancer relevance and in some cases may turn out to represent "passenger" rather than "driver" mutations, this broad incidence of sporadic alterations underscores the central importance of the entire PTK family in oncogenesis. Moreover, it provides further compelling support for the development of multitargeted kinase inhibitors or combination of complementary therapeutics as cancer treatments that may be adapted to the pathologic and genetic parameters of an individual patient. The extensive characterization of established tumor cell lines with respect to transcriptional profiles of genetic variations in this currently most promising cancer target family will aid in the selection of suitable cell systems, data interpretation and target validation, and thereby support preclinical development of novel targeted cancer drugs.
|
Acknowledgments |
|---|
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
We thank Gunaretnam Rajagopal and Lim Yun Ping from A*STAR BII for strong support; the BioComputing Center at BII and Chew Ah Keng from the DNA sequencing facility at the Singapore Institute of Molecular and Cell Biology for expert assistance; and Philip Yeo and Hwai Loong Kong, without whom this project would not have been possible.
|
Footnotes |
|---|
Current address for J.E. Ruhe: U3 Pharma AG, Martinsried, Germany.
J.E. Ruhe, S. Streit, and S. Hart contributed equally to this work.
8 http://www.genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi 
12 http://snp500gov.nci.nih.gov
13 http://tykiva.bii.a-star.edu.sg
14 http://www.ncbi.nlm.nih.gov/Genbank
15 http://bioinf.uta.fi/KinMutBase
16 http://bioinf.uta.fi/base_root
Received 7/17/07. Revised 9/21/07. Accepted 10/ 9/07.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
|---|
transforming growth factor binding and transmembrane signaling. Proc Natl Acad Sci U S A 1994;91:10217–21.This article has been cited by other articles:
|
|
 |
|
  O. Simma, E. Zebedin, N. Neugebauer, C. Schellack, A. Pilz, S. Chang-Rodriguez, K. Lingnau, E. Weisz, E. M. Putz, W. F. Pickl, et al. Identification of an Indispensable Role for Tyrosine Kinase 2 in CTL-Mediated Tumor Surveillance Cancer Res., January 1, 2009; 69(1): 203 - 211. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Cancer Research | Clinical Cancer Research |
| Cancer Epidemiology Biomarkers & Prevention | Molecular Cancer Therapeutics |
| Molecular Cancer Research | Cancer Prevention Research |
| Cancer Prevention Journals Portal | Cancer Reviews Online |
| Annual Meeting Education Book | Meeting Abstracts Online |
