Polymorphic genetic control of tumor invasion in a mouse model of pancreatic neuroendocrine carcinogenesis

Abstract

Cancer is a disease subject to both genetic and environmental influences. In this study, we used the RIP1-Tag2 (RT2) mouse model of islet cell carcinogenesis to identify a genetic locus that influences tumor progression to an invasive growth state. RT2 mice inbred into the C57BL/6 (B6) background develop both noninvasive pancreatic neuroendocrine tumors (PNET) and invasive carcinomas with varying degrees of aggressiveness. In contrast, RT2 mice inbred into the C3HeB/Fe (C3H) background are comparatively resistant to the development of invasive tumors, as are RT2 C3HB6(F1) hybrid mice. Using linkage analysis, we identified a 13-Mb locus on mouse chromosome 17 with significant linkage to the development of highly invasive PNETs. A gene residing in this locus, the anaplastic lymphoma kinase (Alk), was expressed at significantly lower levels in PNETs from invasion-resistant C3H mice compared with invasion-susceptible B6 mice, and pharmacological inhibition of Alk led to reduced tumor invasiveness in RT2 B6 mice. Collectively, our results demonstrate that tumor invasion is subject to polymorphic genetic control and identify Alk as a genetic modifier of invasive tumor growth.

Cancer is a complex disease governed by environmental and genetic factors, including genetic mutations and polymorphisms that modulate cancer susceptibility (1). Although many investigations have focused on identifying factors that affect initial tumor development (2, 3), data from both human and mouse studies have demonstrated that genetic polymorphisms can modulate multiple aspects of tumorigenesis, such as tumor progression (4–6) and response to therapy (7).

In this study, we investigated the effects of genetic background on tumor progression to an invasive growth state, motivated by a provocative observation that mice carrying the same oncogenic transgene but differing in genetic background developed tumors that were markedly distinctive in their invasiveness. This model, the RIP1-Tag2 (RT2) mouse model of islet cell carcinogenesis, develops multiple pancreatic neuroendocrine tumors (PNET) in a relatively synchronous and predictable multistage progression pattern by 12–14 wk of age owing to the expression of the SV40 T antigen oncoprotein (Tag) in the pancreatic β cells (8). The tumorigenesis pathway has predominantly been studied in RT2 mice inbred into the C57BL/6 (B6) background, and the PNETs that arise in this genetic context display a spectrum of invasive phenotypes and can be classified as noninvasive islet tumors (IT), focally invasive type-1 carcinomas (IC1), and broadly invasive type-2 carcinomas (IC2) (9). Surprisingly, we observed that when RT2 mice were inbred into a second strain, C3HeB/Fe (C3H), the tumors that arose were predominantly noninvasive, despite being otherwise similar in their tumorigenesis phenotype. The implication that the invasive phenotype was influenced by genetic background prompted our investigation, which was aimed at assessing the hypothesis that a polymorphic modifier locus (or loci) mediated the susceptibility or resistance to the acquisition of the hallmark capability for invasive growth in the RT2 mouse model of cancer.

Results

PNET Progression to Invasive Carcinoma Is Modulated by Genetic Background.

Following anecdotal observations that PNETs developing in RT2 mice inbred into the C3H background were predominantly noninvasive, we carefully examined the distribution of the distinctive invasive phenotypes in de novo PNETs arising in RT2 mice inbred into either the B6 or C3H genetic backgrounds, as well as in C3HB6(F1) hybrids (F1), to determine whether the parameter of tumor invasiveness was indeed affected by genetic background (Fig. 1 A–C).

Fig. 1.

PNET invasion is dependent on genetic background. (A–C) H&E staining of a noninvasive IT PNET, a focally invasive IC1 PNET, and a broadly invasive IC2 PNET from an RT2 B6 mouse. T indicates tumor region, and Ex indicates exocrine pancreas. Dashed lines demarcate tumor margins. (Scale bars, 200 μm.) (D) Quantification of tumor invasiveness represented as the percentage of IT tumors or total IC tumors (IC1 + IC2) in RT2 mice on the B6, C3H, and F1 genetic backgrounds at 14 wk of age. A minimum of 117 tumors per group was graded. *P < 0.001 by Fisher's exact test. (E) Same as D except IC lesions are separated into the IC1 and IC2 subclasses. *P < 0.001 by the χ² test.

The development of invasive carcinoma lesions (IC) was strongly suppressed in RT2 C3H mice. Whereas IC lesions constitute more than half of all tumors in RT2 B6 animals at 14 wk, less than 15% of all tumors could be classified as invasive in RT2 C3H mice (Fig. 1D). This reduction occurred in both the focally invasive IC1 and the widely invasive IC2 subclasses of invasive RT2 tumor lesions (Fig. 1E). The development of IC lesions was also suppressed in RT2 F1 mice, and the overall distribution of invasive lesions in RT2 F1 mice was similar to that in RT2 C3H mice (Fig. 1 D and E). These data indicate that the C3H genetic background is resistant to the development of invasive RT2 PNETs, whereas the F1 phenotype demonstrates that the resistant C3H background is dominant over the susceptible B6 background.

We also examined other parameters of PNET tumorigenesis in the B6 and C3H backgrounds to determine whether additional phenotypes were similarly affected by genetic background. The average tumor burden per animal was significantly higher in both RT2 C3H and RT2 F1 mice as compared with RT2 B6 mice, whereas the average number of macroscopic tumors per animal was higher in RT2 C3H mice as compared with RT2 B6 and RT2 F1 mice (Fig. S1). However, there were no significant differences with regard to either the rate of tumor proliferation or tumor apoptosis (Fig. S1). There was no indication that the driving oncogene was responsible for these phenotypic differences because the levels of the Tag oncoprotein were similar in tumors isolated from RT2 mice in the different genetic backgrounds (Fig. S2), consistent with a previous assessment (10). Additionally, the expression of cadherin 1 (Cdh1, also known as E-cadherin), a known regulator of invasion in the RT2 model as well as other cancers (11), was not obviously different (Fig. S2).

Invasive Modifier Does Not Act in the Bone Marrow–Derived Tissue Compartment.

Because bone marrow–derived (BMD) inflammatory cells that supply matrix-degrading enzymes such as cathepsin proteases and heparanase are functionally implicated in the invasive phenotype in this model (12–14), we examined the possibility that the reduced invasiveness in RT2 C3H and RT2 F1 mice was due to deficiencies in the invasion-promoting functionality of BMD cells. We transferred bone marrow from B6 or F1 donor mice into RT2 F1 animals with the rationale that B6 but not F1 bone marrow would “rescue” the invasive phenotype in recipient RT2 F1 mice if the invasive modifier operated in this tissue compartment. RT2 F1 mice were chosen as recipients because they develop invasive PNETs at a reduced frequency (Fig. 1 D and E) and should also be capable of receiving bone marrow from either B6 or F1 donors without host/donor incompatibility complications. In brief, we did not observe any differences in the invasive phenotype or in any other parameter of RT2 tumorigenesis in RT2 F1 mice whose immune systems had been rendered B6 (Fig. S3). These results suggest that the polymorphic difference is operative in the cancer cells themselves or possibly in other cellular compartments of the stroma.

In light of the evident genetic differences in the frequency of developing invasive carcinomas in RT2 mice, we next sought to map the putative polymorphic locus/loci associated with susceptibility vs. resistance to the invasive phenotype using standard genetic linkage analysis.

Linkage Analysis Identifies a Region on Chromosome 17 That Is Associated with the Development of Invasive Carcinomas in RT2 Mice.

To identify the genetic locus/loci that modify the invasive phenotype in RT2 mice, we performed a genome-wide linkage study. One hundred forty-three RT2 N2 backcrossed mice, resulting from crossing RT2 F1 male mice with B6 female mice (Fig. S4), were scored for the incidence of IT, IC1, and IC2 tumor lesions in addition to the other parameters of RT2 tumorigenesis (Dataset S1). Constitutional tail DNA was genotyped across 561 SNPs that cover the mouse genome and discriminate between the B6 and C3H backgrounds (Dataset S1). Statistical analysis was subsequently performed using R/qtl to determine whether there was evidence of linkage to the development of invasive lesions or to any of the other RT2 tumor phenotypes. Log of odds (LOD) scores of ≥1.9 and ≥3.0 were considered suggestive and significant linkage, respectively (15).

Using the development of IT, IC1, or IC2 PNETs as quantitative traits, we observed significant linkage to four SNPs on chromosome 17 for the development of IC2 lesions, with a peak LOD score of 3.52 (Fig. 2A and Dataset S2). The 95% confidence interval was located from 63.7 to 76.4 Mb, a 13-Mb region that contains more than 50 annotated genes and one miRNA, mir-1195 (Fig. 2B). Interestingly, we did not identify any locus that was linked to the IC1 phenotype, despite the different frequencies in the development of this class of tumors in RT2 B6 and RT2 C3H mice (Fig. S5 and Dataset S2).

Fig. 2.

The IC2 tumor phenotype is linked to a region on chromosome 17. (A) LOD scores for the IC2 phenotype across the mouse genome. The IC2 phenotype shows significant linkage (LOD ≥3.0) to a region on chromosome 17. Dashed line demarcates LOD-3.0 significance cutoff. (B) Physical map of the 95% confidence interval on chromosome 17. Map was constructed using data from the University of California, Santa Cruz Genome Browser (http://genome.ucsc.edu/) and the National Center for Biotechnology Information MapViewer (http://www.ncbi.nlm.nih.gov/projects/mapview/) for the mouse genome. Red and green arrows indicate genes that are expressed at significantly higher and lower levels, respectively, in tumors isolated from RT2 C3H mice as compared with RT2 B6 mice.

Additionally, we observed significant linkage to the X chromosome to the development of IT lesions and to the metric of tumor number (Fig. S5 and Dataset S2). In both situations, the linked region essentially spanned the entire chromosome, which complicated our efforts to analyze this region in further detail. We therefore proceeded to investigate the genes in the minimal region of chromosome 17 that showed significant linkage to the development of IC2 tumors.

Anaplastic Lymphoma Kinase Resides in the Chromosome 17 Minimal Region and Is Differentially Expressed in the B6 and C3H Genetic Backgrounds.

It has previously been suggested that genetic polymorphisms can influence the levels of gene expression in the context of phenotypic modifiers of complex traits (16, 17). We therefore asked whether any of the genes located within the minimal chromosome 17 region might be differentially expressed between the parental strains and therefore contribute to the observed differences in the invasion phenotypes.

RNA from RT2 B6 and RT2 C3H tumors were profiled by quantitative PCR for the genes located within the minimal region on chromosome 17. This analysis revealed that a small subset of the resident genes—Alk, Dlgap1, Emilin2, Lbh, Ltbp1, Rab31, and Spdya—showed significant differential expression between the B6 and C3H genetic backgrounds at the mRNA level (Figs. 2B and 3A and Dataset S3).

Fig. 3.

Anaplastic lymphoma kinase (Alk) is differentially expressed in the B6 and C3H genetic backgrounds. (A) Real-time quantitative PCR values for Alk, Dlgap1, Emilin2, Lbh, Ltbp1, Rab31, and Spdya obtained using a TaqMan array to profile tumors isolated from RT2 mice on the B6 and C3H backgrounds. Seven independent tumors per genotype were analyzed in triplicate. Data shown are mean plus SE. *P < 0.05 and **P < 0.01 by the Mann–Whitney test. (B) Real-time quantitative PCR values for Alk in a pool of islets isolated from normal WT B6 or C3H mice or in tumors (T) isolated from RT2 mice on the B6, C3H, or F1 backgrounds. *P < 0.05 by the Mann–Whitney test. (C) Western analysis on tumor pool lysates (T) from RT2 B6 and RT2 C3H mice for Alk and β-actin. (D) Real-time quantitative PCR values for Alk during the stages of RT2 tumorigenesis [normal WT (N), hyperplastic (H), angiogenic (A), tumor (T)] on the B6 genetic background. All real-time quantitative PCR values are shown as the percentage expression of the ribosomal protein L19 (L19).

We were particularly intrigued by the Alk gene, which encodes the anaplastic lymphoma kinase. Alk mRNA levels were ~60% lower in RT2 C3H tumors vs. RT2 B6 tumors and ~40% lower in RT2 F1 tumors vs. RT2 B6 tumors, which was also reflected at the protein level (Fig. 3 A–C). Alk expression was also reduced in WT islets from C3H mice as compared with B6 mice, consistent with Alk being expressed at higher levels in the B6 background vs. the C3H background regardless of the neoplastic state of this tissue (Fig. 3B). Alk levels were higher in tumors compared with WT islets in both genetic backgrounds, and Alk expression showed a progressive increase during the course of RT2 tumorigenesis (Fig. 3 B and D). Notably, there are no polymorphisms in the exonic regions of the Alk gene that differentiate the B6 allele from the C3H allele (http://www.informatics.jax.org/strains_SNPs.shtml), and therefore the Alk protein is not intrinsically different in structure or function in these different genetic backgrounds. Interestingly, Alk belongs to the insulin-receptor superfamily of receptor tyrosine kinases (18), members of which are known to influence PNET tumorigenesis in RT2 mice, including tumor invasion (9, 19). Given this association and our observation that Alk expression levels were significantly different between the B6 and C3H backgrounds, we sought to explore the potential role that Alk might play in the development of invasive RT2 tumors.

Pharmacological Inhibitor of Alk Inhibits Invasion and Other Parameters of PNET Tumorigenesis.

We used a small molecule inhibitor of Alk kinase activity, NVP-TAE684 (TAE684) (20), in an experimental therapeutic trial in RT2 mice, aiming to assess the effects of reduced Alk activity on RT2 tumorigenesis, particularly with regard to the parameter of tumor invasion.

RT2 B6 mice were treated for 4 wk with TAE684 or vehicle using a previously defined dose regimen (20) beginning at 10 wk of age when incipient tumors are first observed in RT2 mice (21). RT2 B6 mice were used because they develop IC lesions at significantly higher levels than RT2 C3H mice, and they also express Alk in the pancreatic islets and PNETs at significantly higher levels than RT2 C3H mice (Figs. 1 D and E and 3 A–C). This is also the stage of RT2 tumorigenesis when there is an appreciable increase in Alk expression levels (Fig. 3D). TAE684 was well tolerated, and we did not observe any fluctuations in body mass in either TAE684- or vehicle-treated mice during the course of the trial (Fig. S6).

At the defined endpoint of the trial, TAE684-treated mice proved to have developed ~25% fewer macroscopic tumors than control mice (Fig. 4B); there was a concomitant trend toward reduced tumor burden in TAE684-treated mice, which, however, was not statistically significant (Fig. 4A).

Fig. 4.

Alk inhibitor NVP-TAE684 (TAE684) reduces tumor invasiveness in RT2 mice in an experimental therapeutic trial. (A) Tumor burden for RT2 B6 mice treated with TAE684 or vehicle from 10 to 14 wk of age. Data shown are mean plus SE. Data are not statistically different. (B) Tumor number for RT2 B6 mice treated with TAE684 or vehicle from 10 to 14 wk of age. Data shown are mean plus SE. *P < 0.05 by the Mann–Whitney test. (C) Quantification of tumor invasiveness represented as the percentage of IT tumors or total IC tumors (IC1 + IC2) in RT2 B6 mice treated with TAE684 or vehicle from 10 to 14 wk of age. A minimum of 83 tumors per group was graded. *P < 0.05 by Fisher's exact test. (D) Same as C except IC tumors are separated into the IC1 and IC2 subclasses. *P < 0.01 by the χ² test. (E) Western analysis on tumor pool lysates from TAE684- or vehicle-treated RT2 B6 mice for phospho-Alk, total Alk, phospho-Akt, total Akt, and β-actin.

Notably, TAE684-treated mice developed significantly fewer invasive lesions than control mice. There was a clear reduction in the frequency of total IC tumors (49.7% vs. 33.3% of total tumors in control vs. treated mice), which was accompanied by a concomitant increase in the frequency of IT tumors (50.3% vs. 66.7% of total tumors in control vs. treated mice), in TAE684-treated mice (Fig. 4C). This shift was due to a reduction in the frequencies of both the IC1 and IC2 subclasses of invasive RT2 PNETs (Fig. 4D).

TAE684 functions by interfering with Alk kinase activity (20), and tumors from treated RT2 mice showed reduced levels of phosphorylated Alk (Fig. 4E). We also observed a modest but appreciable reduction in the levels of phosphorylated Akt, one downstream Alk target, compared with controls (Fig. 4E), confirming that TAE684 inhibited Alk activity in the tumors of RT2 mice.

Discussion

A considerable body of research has identified polymorphic modifier loci scattered across the mouse genome that affect multiple aspects of cancer susceptibility and development (1, 22, 23). Our data demonstrate that tumor progression, specifically to an invasive growth state, is also subject to polymorphic genetic control. We identify a polymorphic locus on mouse chromosome 17 [syntenic to human chromosomes 2 (107.5–110 Mb), 5 (2.5–10 Mb), and 18 (29–34 Mb)], which influences the susceptibility of PNETs to progress from solid adenomatous tumors to invasive carcinomas.

Using a prototypical mouse model of multistage tumorigenesis, we observed that the propensity to develop an invasive phenotype is affected by genetic background. RT2 mice inbred into the B6 background develop PNETs of varying degrees of invasiveness, whereas RT2 mice inbred into the C3H background are largely resistant to the development of invasive tumors. Furthermore, RT2 F1 hybrid mice are also resistant, indicating that the C3H genetic background is dominant-suppressive over the invasion-prone B6 background. Linkage analysis of RT2 N2 backcross mice, produced from backcrossing RT2 F1 mice once to the susceptible B6 background, identified a locus on chromosome 17 that correlated with susceptibility (when the locus is homozygous B6) vs. resistance (when a C3H allele is present). Previous studies have documented that tumors isolated from RT2 mice undergo chromosomal gains and losses at different frequencies dependent on genetic background (10, 24). Notably, chromosome 17 is not affected by copy number abnormalities in either the B6 or C3H backgrounds, suggesting that this locus is of a class of genetic modifiers that is not altered during tumorigenesis.

The invasion modifier locus on chromosome 17 (63.7–76.4 Mb) contains more than 50 annotated genes. Additionally, one miRNA, mir-1195, resides in this locus, although there is no coding change between the B6 and C3H sequences for this miRNA (http://www.informatics.jax.org/strains_SNPs.shtml). Of the 50 genes in the modifier locus, 7 were found to be differentially expressed in the PNETs isolated from RT2 mice inbred into the B6 and C3H backgrounds. As a first step toward auditing candidate invasion modifier genes in this locus, we focused on the Alk receptor tyrosine kinase, motivated in part by a series of studies demonstrating that Alk is activated by mutation or chromosomal translocation in human hematopoietic and solid cancers, evidently converting it into an initiating oncogene (18, 25–27). On the basis of these and previous studies implicating Alk as an oncogene, several small-molecule inhibitors specific to Alk have been developed as potential therapeutics for these diseases (20, 28). Our use of one such kinase inhibitor to probe the possible roles of Alk in PNET tumorigenesis demonstrated that Alk promoted both tumor growth and progression; most notably, pharmacological inhibition of Alk activity reduced tumor invasiveness in RT2 B6 mice. These results are consistent with our observation that Alk is expressed at lower levels in the tumors of RT2 C3H mice, which are rarely invasive, as compared with the tumors of RT2 B6 mice, which consistently develop invasive PNETs. In comparing the B6 and C3H sequences, we did not identify any polymorphism in either the protein-coding or untranslated portions of the Alk mRNA that might suggest a basis for Alk’s invasion modifier effects and/or differential expression. However, there are four polymorphisms located within 10 kb of the 5′-flanking region and two within 10 kb of the 3′-flanking region, in addition to ~300 polymorphisms residing in the large intron 2 of the Alk gene, that distinguish the B6 and C3H alleles (http://www.informatics.jax.org/strains_SNPs.shtml), and one or more of these polymorphisms may account for the observed differences in allelic expression. Our results associating Alk with invasion are also congruent with a previous study demonstrating that single-chain variable fragment antibodies targeting Alk can reduce tumor cell invasion in an in vitro setting (29). Additionally, pharmacological inhibition of Alk hindered tumor formation in RT2 mice, in accordance with earlier studies examining the oncogenic properties of Alk (20, 25, 26, 30). Importantly and in contrast to the aforementioned studies in which Alk was the driving oncogene, our results demonstrate that Alk can also act as a tumor progression factor, being up-regulated during multistep tumorigenesis to collaborate with an initiating oncogene (in this case the SV40 Tag oncogene that abrogates the tumor-suppressing activities of pRb and p53). Thus, Alk inhibition may prove to be a useful therapy even in situations in which Alk is not the initiating oncogene, either as a result of mutation or other means.

Although our data implicate Alk levels as a determinant of RT2 tumor invasion, we envision that other polymorphic invasion modifier genes may reside in the chromosome 17 locus. The Alk inhibitor reduced tumor invasiveness, but not to the degree seen in the C3H background, which could reflect incomplete Alk inhibition or additional genetic components to the modifier effect. Indeed, several other genes residing in this locus also showed significant differential expression in RT2 tumors from the B6 and C3H genetic backgrounds (Fig. 3), and one of these genes, Ltbp1, contains a nonsynonymous coding change between the B6 and C3H backgrounds (http://www.informatics.jax.org/strains_SNPs.shtml). Ltbp1 encodes the latent TGF-β binding protein 1, a component of the TGF-β pathway (31), which is known to influence many aspects of cancer progression, including tumor invasion and metastasis (32). Additionally, it has recently been suggested that Emilin2, which encodes the elastin microfibril interfacer 2, is subject to DNA methylation leading to reduced gene expression in human breast cancers, and Emilin2 hypermethylation is associated with poorer clinical outcome, in particular relapse and poor survival (33). Last, elevated expression of Spdya (also known as Spy1), which encodes the speedy homolog A, accelerates tumorigenesis in a mouse model of breast cancer (34) and has also been associated with more aggressive human breast cancers (35). As such, other genes in this locus merit future investigation.

Although bone marrow–derived inflammatory cells have been shown to contribute to the invasiveness of RT2 PNETs (12, 13), it does not seem that their activity is modulated by the invasion modifier gene(s). Thus, invasive PNETs were still rare in RT2 F1 mice that received bone marrow from an invasion-permissive B6 donor (Fig. S3). Although we cannot rule out the possibility that this modifier locus operates in other stromal cell types or in another tissue compartment, it seems most likely that the invasive modifier acts in the cancer cells.

In addition to proinvasive inflammatory cells, other factors are known to influence progression to an invasive growth state in this prototypical model of multistage tumorigenesis. Loss of cell–cell adhesion complexes, including the adherens junctions mediated by Cdh1 (11) and desmosomes (36), are associated with the development of more-invasive tumors. Signaling through the type-1 insulin-like growth factor receptor (Igf1r) can also drive progression to an invasive state (9). The present study now establishes a unique dimension to this multifactorial invasive growth phenotype, involving a polymorphic genetic modifier that can alternatively override or allow these other functional effectors of invasive growth. It remains to be determined whether the chromosome 17 invasion modifier locus identified in this study modulates any of these functionalities or acts in a completely independent fashion.

Finally, it is pertinent to consider the translational implications of this newly identified invasion modifier. First, we suspect that this polymorphic modifier will prove operative in other cancer types but most likely not in all. Notably, the development of squamous carcinoma is under distinctive polymorphic control in mice. In this case, the B6 background is largely resistant to the development of invasive squamous carcinomas in three different oncogenic contexts—an activated Hras oncogene (37), the HPV16 oncogenes (38), and chemical carcinogens (39). Thus, the B6 background is permissive for invasive cancers in the pancreas but resistant for Hras-induced cancers in the skin. A major determinant of skin tumor resistance is a polymorphism in the Patched gene (Ptch1), located on mouse chromosome 13, that introduces a nonconservative coding sequence change at the C terminus of the protein (37). This polymorphism was not detected in the present linkage analysis of invasive pancreatic tumors. Therefore, both tumor types are governed by polymorphic modifiers of invasive cancer, albeit distinctive ones. Additionally, yet other phenotypic modifiers of metastasis are implicated in mouse models of breast cancer (40) and in human breast cancer (41). Given the neuroendocrine nature of the tumor type subject to the invasion modifier reported herein, we wonder whether similar tumor types such as small-cell lung cancer or brain cancers might also be affected by this genetic modifier. Interestingly, Alk has been implicated in glioblastoma (29), and as such, this tumor type could be subject to this polymorphic modifier.

Assessing the existence of polymorphic invasion modifiers in human cancers will be challenging. The availability of increasingly cost-effective DNA sequencing of individual genomes (both normal and cancerous) may afford inroads to identifying polymorphisms correlating with progression to invasive carcinomas, particularly in organs in which both noninvasive adenomas and invasive carcinomas are prevalent, such as the colon. Elucidation of such polymorphic modifiers could well contribute to the future of personalized medicine, whereby susceptibility vs. resistance alleles of invasion modifiers might be factored into the treatment for patients diagnosed with early-stage cancers.

Materials and Methods

Genetically Engineered Mice.

The generation and characterization of the RT2 mouse line has been previously described (8). The RT2 line has been backcrossed into the B6 (Charles River Laboratories) and the C3H (Jackson Laboratory) genetic backgrounds more than 20 times and is effectively inbred into these backgrounds. F1 hybrid mice and RT2 N2 mice were generated as described (Fig. S4). Beginning at 10 wk of age, all RT2 mice received 50% sugar food (Harlan Teklad) to relieve the effects of hypoglycemia caused by the insulin-secreting tumors. All mice used in this study were housed and maintained in accordance with the University of California, San Francisco institutional guidelines governing the care of laboratory mice.

Genomic DNA Preparation, SNP Genotyping, and Linkage Analysis.

Genomic DNA was isolated from mouse tails by proteinase K (Qiagen) digestion followed by phenol-chloroform extraction using standard methods (42). Four micrograms of genomic DNA per animal was SNP genotyped using the Illumina platform according to the manufacturer's specifications, and 143 RT2 N2 mice for which tumor phenotype data were available were genotyped by this method. A panel of primers that discriminate between the B6 and C3H genetic backgrounds across all 19 somatic chromosomes and the X chromosome was used (see Dataset S1 for a complete list of SNPs used in this study). Statistical analysis was performed using R/qtl (http://www.rqtl.org/) for the IT, IC1, IC2, tumor number, and tumor burden metrics. Because these metrics were not normally distributed, nonparametric tests were chosen in R/qtl. A LOD score of ≥3.0 was considered significant (15).

TAE684 Inhibitor Trial.

The characterization of the Alk inhibitor NVP-TAE684 (TAE684) has been previously described (20). TAE684 was resuspended in 10% 1-methyl-2-pyrrolidinone/90% PEG 300 (vol/vol) (Sigma), and male RT2 B6 mice were administered a 10-mg/kg dose once daily or vehicle solution alone by oral gavage from 10 to 14 wk of age. Body mass was monitored twice weekly to adjust dosing levels and to assess any toxicity caused by the treatment.