Polymorphic genetic control of tumor invasion in a mouse model of pancreatic neuroendocrine carcinogenesis Matthew G. H. Chuna,b,c,d, Jian-Hua Maoc,1, Christopher W. Chiub,c, Allan Balmainc, and Douglas Hanahana,b,c,2,3 aDepartment of Biochemistry and Biophysics, bDiabetes Center, and cHelen Diller Family Comprehensive Cancer Center, University of California, San Francisco, CA 94143; and dProgram in Biological Sciences, University of California, San Francisco, CA 94158 Contributed by Douglas Hanahan, August 31, 2010 (sent for review August 2, 2010) Cancer is a disease subject to both genetic and environmental hallmark capability for invasive growth in the RT2 mouse model influences. In this study, we used the RIP1-Tag2 (RT2) mouse of cancer. model of islet cell carcinogenesis to identify a genetic locus that influences tumor progression to an invasive growth state. RT2 Results mice inbred into the C57BL/6 (B6) background develop both non- PNET Progression to Invasive Carcinoma Is Modulated by Genetic invasive pancreatic neuroendocrine tumors (PNET) and invasive Background. Following anecdotal observations that PNETs de- carcinomas with varying degrees of aggressiveness. In contrast, veloping in RT2 mice inbred into the C3H background were RT2 mice inbred into the C3HeB/Fe (C3H) background are compar- predominantly noninvasive, we carefully examined the distribu- atively resistant to the development of invasive tumors, as are RT2 tion of the distinctive invasive phenotypes in de novo PNETs C3HB6(F1) hybrid mice. Using linkage analysis, we identified a 13- arising in RT2 mice inbred into either the B6 or C3H genetic Mb locus on mouse chromosome 17 with significant linkage to the backgrounds, as well as in C3HB6(F1) hybrids (F1), to determine development of highly invasive PNETs. A gene residing in this whether the parameter of tumor invasiveness was indeed af- locus, the anaplastic lymphoma kinase (Alk), was expressed at fected by genetic background (Fig. 1 A–C). significantly lower levels in PNETs from invasion-resistant C3H The development of invasive carcinoma lesions (IC) was mice compared with invasion-susceptible B6 mice, and pharmaco- strongly suppressed in RT2 C3H mice. Whereas IC lesions con- logical inhibition of Alk led to reduced tumor invasiveness in RT2 stitute more than half of all tumors in RT2 B6 animals at 14 wk, B6 mice. Collectively, our results demonstrate that tumor invasion less than 15% of all tumors could be classified as invasive in RT2 is subject to polymorphic genetic control and identify Alk as a ge- C3H mice (Fig. 1D). This reduction occurred in both the focally fi netic modi er of invasive tumor growth. invasive IC1 and the widely invasive IC2 subclasses of invasive RT2 tumor lesions (Fig. 1E). The development of IC lesions was also fi anaplastic lymphoma kinase | cancer modi er genes | malignant suppressed in RT2 F1 mice, and the overall distribution of invasive progression | pancreas cancer | transgenic mouse 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 ancer is a complex disease governed by environmental and is resistant to the development of invasive RT2 PNETs, whereas Cgenetic factors, including genetic mutations and polymor- the F1 phenotype demonstrates that the resistant C3H back- phisms that modulate cancer susceptibility (1). Although many ground is dominant over the susceptible B6 background. investigations have focused on identifying factors that affect in- We also examined other parameters of PNET tumorigenesis itial tumor development (2, 3), data from both human and in the B6 and C3H backgrounds to determine whether additional mouse studies have demonstrated that genetic polymorphisms phenotypes were similarly affected by genetic background. The can modulate multiple aspects of tumorigenesis, such as tumor average tumor burden per animal was significantly higher in both – progression (4 6) and response to therapy (7). RT2 C3H and RT2 F1 mice as compared with RT2 B6 mice, In this study, we investigated the effects of genetic background whereas the average number of macroscopic tumors per animal on tumor progression to an invasive growth state, motivated by was higher in RT2 C3H mice as compared with RT2 B6 and RT2 a provocative observation that mice carrying the same oncogenic F1 mice (Fig. S1). However, there were no significant differences transgene but differing in genetic background developed tumors with regard to either the rate of tumor proliferation or tumor that were markedly distinctive in their invasiveness. This model, apoptosis (Fig. S1). There was no indication that the driving on- the RIP1-Tag2 (RT2) mouse model of islet cell carcinogenesis, cogene was responsible for these phenotypic differences because develops multiple pancreatic neuroendocrine tumors (PNET) in the levels of the Tag oncoprotein were similar in tumors isolated a relatively synchronous and predictable multistage progression – from RT2 mice in the different genetic backgrounds (Fig. S2), pattern by 12 14 wk of age owing to the expression of the SV40 consistent with a previous assessment (10). Additionally, the ex- 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 Author contributions: M.G.H.C. and D.H. designed research; M.G.H.C. and C.W.C. per- that arise in this genetic context display a spectrum of invasive formed research; M.G.H.C., J.-H.M., C.W.C., A.B., and D.H. analyzed data; and M.G.H.C. phenotypes and can be classified as noninvasive islet tumors (IT), and D.H. wrote the paper. focally invasive type-1 carcinomas (IC1), and broadly invasive The authors declare no conflict of interest. type-2 carcinomas (IC2) (9). Surprisingly, we observed that when Freely available online through the PNAS open access option. RT2 mice were inbred into a second strain, C3HeB/Fe (C3H), the 1Present address: Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, tumors that arose were predominantly noninvasive, despite being CA 94720. otherwise similar in their tumorigenesis phenotype. The impli- 2Present address: Swiss Institute for Experimental Cancer Research, Swiss Federal Institute cation that the invasive phenotype was influenced by genetic of Technology Lausanne, Lausanne CH-1015, Switzerland. background prompted our investigation, which was aimed at 3To whom correspondence should be addressed. E-mail: dh@epfl.ch. fi assessing the hypothesis that a polymorphic modi er locus (or loci) This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. mediated the susceptibility or resistance to the acquisition of the 1073/pnas.1012705107/-/DCSupplemental. 17268–17273 | PNAS | October 5, 2010 | vol. 107 | no. 40 www.pnas.org/cgi/doi/10.1073/pnas.1012705107 Downloaded by guest on September 25, 2021 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 quanti- tative 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% confi- dence 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 fre- quencies in the development of this class of tumors in RT2 B6 and RT2 C3H mice (Fig. S5 and Dataset S2). – Fig. 1. PNET invasion is dependent on genetic background. (A C)H&E Additionally, we observed significant linkage to the X chro- 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, mosome to the development of IT lesions and to the metric of and Ex indicates exocrine pancreas. Dashed lines demarcate tumor mar- tumor number (Fig. S5 and Dataset S2). In both situations, the gins. (Scale bars, 200 μm.) (D) Quantification of tumor invasiveness rep- linked region essentially spanned the entire chromosome, which resented as the percentage of IT tumors or total IC tumors (IC1 + IC2) in complicated our efforts to analyze this region in further detail. RT2 mice on the B6, C3H,andF1 genetic backgrounds at 14 wk of age. A We therefore proceeded to investigate the genes in the minimal minimum of 117 tumors per group was graded. *P < 0.001 by Fisher’sexact region of chromosome 17 that showed significant linkage to the test. (E)SameasD except IC lesions are separated into the IC1 and IC2 development of IC2 tumors. subclasses. *P < 0.001 by the χ2 test. GENETICS Anaplastic Lymphoma Kinase Resides in the Chromosome 17 Minimal B6 C3H pression of cadherin 1 (Cdh1, also known as E-cadherin), a known Region and Is Differentially Expressed in the and Genetic regulator of invasion in the RT2 model as well as other cancers Backgrounds. It has previously been suggested that genetic poly- fl (11), was not obviously different (Fig. S2). morphisms can in uence the levels of gene expression in the context of phenotypic modifiers of complex traits (16, 17). We Invasive Modifier Does Not Act in the Bone Marrow–Derived Tissue therefore asked whether any of the genes located within the Compartment. Because bone marrow–derived (BMD) inflam- minimal chromosome 17 region might be differentially expressed matory cells that supply matrix-degrading enzymes such as ca- thepsin 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 func- tionality of BMD cells.