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[Cancer Research 63, 4777-4780, August 15, 2003]
© 2003 American Association for Cancer Research

Advances in Brief

Modulation of Medullary Thyroid Carcinoma Penetrance Suggests the Presence of Modifier Genes in a RET Transgenic Mouse Model1

Aaron N. Cranston2 and Bruce A. J. Ponder

University of Cambridge and Cancer Research UK Department of Oncology, CIMR, Hills Road, Cambridge, CB2 2XY, United Kingdom


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
We described previously a thyroid phenotype for transgenic mice expressing an activated Ret oncogene driven from a human calcitonin promoter. Medullary thyroid carcinomas (MTC), a tumor of the thyroid parafollicular C cells, occur in this transgenic line with a pathology analogous to that seen in patients with multiple endocrine neoplasia type 2 (MEN2). When the transgene was introgressed onto four different genetic backgrounds, between 0 and 98% of transgenic progeny developed thyroid tumors by 10 months of age, indicating that tumor penetrance could be modulated by genetic background. Furthermore, tumors on the CBA/ca and C57BL/6J backgrounds were significantly larger than those arising in BALB/c transgenic mice. These results are relevant to human MEN2 disease, because this model system may be used to study genes modifying thyroid tumor penetrance in the dominantly inherited human cancer syndrome.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
The RET proto-oncogene encodes a receptor tyrosine kinase and, in conjunction with the GFR {alpha} coreceptors,3 binds the GDNF family of neurotrophic factors (reviewed in Refs. 1, 2, 3 ). Ligand-induced dimerization results in autophosphorylation of RET and the initiation of downstream signaling cascades (4) . Gain-of-function mutations in RET have been identified as the causative factor in the inherited cancer syndrome, MEN2, and familial medullary thyroid carcinoma (5, 6, 7, 8) . Previously, we created a number of transgenic lines of mice expressing the 3' long isoform of hRET carrying an activating mutation at codon 634 in which arginine was substituted for cysteine, analogous to the most common human MEN2A mutation, and driven from a human calcitonin promoter (9) . One of these lines, CT-2A-3, expressed a single copy of the transgene from a single insertion site. A proportion of transgenic mice from this line developed MTC. Characterization of the pathological lesions by immunohistochemistry confirmed similarities between this transgenic mouse model and the MEN2A human cancer syndrome. We observed that the number of mice developing MTC increased over time and that the proportion of mice with bilateral MTC was different depending on the strain background (9) , raising the possibility of modifier effects attributable to genetic background. However, a thorough analysis of strain background effects was not conducted. The existence of modifying genes in the human population may be inferred from genetic studies that show the MEN2 cancer syndrome to be characterized by variable phenotypic expression both within and between families (5) . The transgenic lines presented here may therefore provide a suitable model system in which to identify and elucidate the effects of modifier genes in the etiology of this dominantly inherited cancer syndrome.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
Mice.
To address the question of potential modifier genes, we introgressed our transgene onto four different genetic backgrounds by backcrossing our mixed background (C57BL/6J; FVB/N;CBA/ca) CT-2A-3 line (see Ref. 9 ) to CBA/ca, C57BL/6J (B6) and FVB/N and also by outcrossing it to BALB/c. Each line was maintained with the transgene in the heterozygous state and backcrossed to each of the four inbred strains for four further sequential generations (N). The N5 progeny were then aged for 43 weeks (~10 months). According to the equation, 1 - (1/2)N-1, where N is the Nth generation (10) , each of these lines is predicted to be 94% homozygous for the inbred strain alleles. We called these lines, CT-2A-3 CBA(N5), CT-2A-3 B6(N5), CT-2A-3 FVB(N5), and CT-2A-3 BALB(N5). Animals were handled according to the appropriate United Kingdom Home Office License, UKCCCR guidelines (11) , veterinary best practice, and local university regulations.

Histology and Immunohistochemistry.
At 43 weeks of age, mice were necropsied, and the thyroids were removed en bloc from age-matched transgenic and nontransgenic littermate mice. The esophagus and trachea were trimmed close to the thyroid glands. The thyroid glands were measured in three dimensions using Vernier scale calipers, and the entire thyroid block weighed to quantitate thyroid volume and mass, respectively. To demonstrate that increases in thyroid volume were indeed attributable to the development of MTC, immunohistochemistry using a rabbit polyclonal antibody to calcitonin (DAKO; 1:3000) was performed on transverse histological sections as described previously (9) .


   RESULTS AND DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
Of the original CT-2A line, ~65% of the transgenics developed MTC, indicating that the tumor phenotype was not fully penetrant (9) . These mice ranged in age from 6 months to 2 years and segregated alleles from three genetic backgrounds (CBA/B6/FVB). We expected that alleles from different genetic backgrounds would differ in the effects on tumor penetrance. We chose an experimental end point of 43 weeks (10 months) to assess tumor development across the various strain backgrounds.

When the transgene was introgressed to N5 on four different genetic backgrounds, the tumor phenotype, assessed macroscopically at 43 weeks of age (Fig. 1)Citation , varied in penetrance from 0% in the FVB/N background (0 of 16) to almost complete penetrance (47 of 48; 98%) in the CBA/ca background (Fig. 2A)Citation . We confirmed that these tumors were in fact medullary thyroid carcinomas by immunohistochemical staining for calcitonin (Fig. 3)Citation . By 43 weeks, the majority of transgenic mice on a C57BL/6J background (9 of 14; 64%) but only ~1 in 7 (4 of 29; 14%) of the BALB/c transgenics had developed MTC. These differences are highly significant (P < 0.0001). Generally, tumors were bilateral, although only one lobe had detectable tumor in 8 and 3% of CBA/ca and BALB/c transgenic mice, respectively. As expected, no tumors were observed at the same age in any of the nontransgenic mice on any of the strain backgrounds (CBA/ca NTg:19; C57BL/6J NTg: 12; BALB/c NTg: 26; FVB/N NTg: 11).



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  		Fig. 1. Gross thyroid morphology illustrating the obvious size difference of thyroid glands taken from representative nontransgenic (left) and transgenic thyroids (right) affected by tumor in mice from the CT-2A (N5) CBA/ca line. Scale bars in A–C are ~2 mm in length.

 


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  		Fig. 2. A, the percentage tumor penetrance of transgenic CT-2A mice on various semicongenic (N5) backgrounds that develop thyroids at 43 weeks of age. B, a scatter plot for measurements made on thyroid tumors in terms of volume (white circles) and weight (gray circles). C, a strong correlation between thyroid tumor volume and weight across all strain backgrounds.

 


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  		Fig. 3. Immunohistochemical detection of calcitonin positivity in transgenic CT-2A thyroids from the backcrossed lines, CBA/ca, C57BL/6J, and BALB/c; CT-2A (N5) mice on the FVB/N background did not develop tumors. Positive calcitonin staining (brown; DAB) demonstrates definitively that the tumors are medullary thyroid carcinomas (MTC). A–E refer to mice from the CT-2A (N5) CBA/ca line; F–K refer to mice from the CT-2A (N5) C57BL/6J line; L and M refer to mice from the CT-2A (N5) BALB/c line. A and B illustrate bilateral MTC in mouse 02/370 at x5 magnification; C shows a higher power magnification (x20) of a field of view from A; D and E show bilateral MTC in a second animal (02/371) at x5 magnification. F and G illustrate bilateral MTC in mouse 02/356 at x5 magnification; H shows a high power magnification (x40) of calcitonin-positive cells in the thyroid lobe shown in G; I shows a bilateral MTC at x5 magnification in a second animal, 02/420, for which the contralateral lobe is not shown, but in which a small isthmus of normal thyroid, not involved by MTC, can be seen; J shows a x20 magnification of this normal isthmus; K shows the presence of a polyp in the trachea of this mouse (02/420) at x20 magnification. L and M show the thyroid lobes of mouse 02/437 at x5 magnification; N is a high power (x40) magnification of a tight cluster of C-cells apparent in one of these lobes (M). O and P illustrate the presence of monolateral MTC in another mouse, 02/471; the affected lobe is shown at x5 magnification in O, whereas the unaffected contralateral lobe is shown in P (x5 magnification). Scale bars at x5 magnification are ~0.4 mm in length, whereas those at x20 and x40 magnifications are ~100 and 50 µM, respectively.

 
To quantify the phenotypic differences, we measured thyroid weights and volumes (Fig. 2B)Citation . To see if there was a correlation, one was plotted against the other (Fig. 2C)Citation . The correlation coefficient (r) was calculated to be 0.63, whereas the SD of residuals from the line was 17.98 (n = 35), indicating that there was not a significant departure from linearity (P = 0.27), and demonstrating, as expected, a strong correlation between thyroid weight and volume. One caveat is that there may be an inherent bias in the thyroid measurements. The trachea accounts for a higher proportion relative to the thyroid gland of the nontransgenic thyroid blocks so the size of the smaller thyroids is quite possibly overestimated relative to the cancerous thyroids. This effect should be small and would in any case result in an underestimation of size difference. Measurements made on nontransgenic thyroids and unaffected thyroids from transgenics, along with those that were cancerous, are summarized in Table 1Citation . Individual measurements (volume and mass) are shown as a scatter plot in Fig. 2BCitation . We compared the volumes of nontransgenic thyroids from each backcrossed line using an unpaired Student’s t test and found that nontransgenic thyroids from the CBA/ca line were larger than those of the BALB/c and FVB/N lines (P = 0.0003 and 0.0015, respectively); similarly, thyroids from nontransgenic CT-2A C57BL/6J mice were significantly larger than BALB/c and FVB/N nontransgenic thyroids (P = 0.0054 and 0.0171, respectively). There was no significant difference in size between CBA/ca and C57BL/6J or between BALB/c and FVB/N nontransgenic thyroids (P = 0.92 and 0.412, respectively). It is interesting to speculate that the larger thyroids of the CBA/ca and C57BL/6J lines contain more C-cells and may therefore offer more potential target cells for Ret-mediated transformation, but this would imply that these aspects are, in some way, coregulated. However, it is more likely that it reflects a modifier in those strains, which affects some aspect of the thyroid that facilitates the development or growth of MTC.


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  		Table 1 Thyroid gland volume (mm3) and weight (mg) in each of the CT-2A (N5) backcrossed lines

The top panel refers to nontransgenic thyroids for each of the CT-2A (N5) backcrossed lines. The middle panel refers to transgenic mice that did not present with thyroid tumors, and the bottom panel refers to transgenic mice that presented with thyroid tumours.

 
MTCs have also been reported in another transgenic mouse model of MEN2A (12) . This model differs from ours in several respects: (a) the mice were on a mixed B6/DBA background; (b) the transgene consisted of the RET short isoform driven off a rat CGRP/CT promoter; and (c) presumably differed in terms of transgene integration site, expression, and copy number. Three lines of mice displayed overt thyroid tumor phenotypes, but unfortunately, there is insufficient data relating to tumor penetrance and size to draw conclusions about strain-related effects from this study.

Taken together, the results presented here suggest that the thyroid C-cells in CBA/ca and, to a lesser extent, C57BL/6J mice are relatively highly susceptible to Ret transformation, whereas those of FVB/N mice are generally resistant to Ret-driven tumorigenesis. In support of this, we also observed a very low penetrance of MTC (7%) on an FVB/N background in an independent founder line (CT-2A-1) expressing the same transgene (9) . Furthermore, we recently produced mice transgenic for the MEN2B mutation (M918T), under the control of the same calcitonin promoter, on an inbred FVB/N genetic background. Of seven independent transgene-expressing founder lines, none developed a detectable thyroid phenotype by 18 months of age.4

A strain background effect has been proposed for a line of c-mos transgenic mice in which a full-length mos cDNA was driven from a Moloney virus LTR and which developed thyroid and adrenal tumors similar to those of the MEN2 cancer syndrome (13) . Interestingly, only one line generated on an FVB/N genetic background developed medullary thyroid neoplasia characteristic of MEN2 patients. Outcrossing the lines to the BALB/c strain resulted, overall, in an increase in the tumor phenotype in all three F1 lines, albeit with variable penetrance (2, 52, and 33%). This result is consistent with a genetic background effect, with the variable penetrance between lines explained by differences in transgene integration site and/or copy number and/or expression. In our study, because each of our N5 backcrossed lines were derived from the same transgenic founder line (CT-2A-3), transgene copy number and integration site effects with concomitant differences in transgene expression cannot account for the range of tumor penetrances observed across the different strains.

The results from our detailed strain analysis indicate that activated RET driven from a calcitonin promoter is capable of transforming thyroid C-cells with different efficiencies in different mouse genetic backgrounds such that CBA/ca > C57BL/6J > BALB/c > FVB/N. We therefore conclude that the differences are caused by differences in modifying alleles in these mouse strains. Such putative modifying alleles may act directly on the Ret signaling pathway or on other aspects of the transformation of C-cells and progression to MTC, including possibly through effects on the thyroid gland that are reflected in larger thyroids, which we observed in the more susceptible strains. We cannot, however, exclude the possibility that the modifying effects on the MTC phenotype result from modulation of the activity of the calcitonin promoter. The existence of modifying genes in the human population may be inferred from genetic studies that show the MEN2 cancer syndrome to be characterized by variable phenotypic expression both within and between families (5) . The transgenic lines presented here may therefore provide a suitable model system in which to identify and elucidate the effects of modifier genes in the etiology of this dominantly inherited cancer syndrome.


   ACKNOWLEDGMENTS
 
We thank Louise Howard for histology. B. A. J. P. is a Gibb Fellow of Cancer Research UK.


   FOOTNOTES
 
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.

1 Supported by Cancer Research UK. Back

2 To whom requests for reprints should be addressed, at University of Cambridge and Cancer Research UK Department of Oncology, CIMR, Hills Road, Cambridge, CB2 2XY, United Kingdom. Phone: 44 (0) 1223-336900; Fax: 44 (0) 1223-336902; E-mail: arron.cranston{at}ntlworld.com Back

3 The abbreviations used are: MEN2, multiple endocrine neoplasia type 2; MTC, medullary thyroid carcinoma; GFR, GDNF family receptors; GDNF, glial cell line-derived neurotrophic factor. Back

4 A. Cranston, unpublished results. Back

Received 6/ 3/03. Revised 6/ 3/03. Accepted 7/ 2/03.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 

  1. Airaksinen M. S., Titievsky A., Saarma M. GDNF family neurotrophic factor signaling: four masters, one servant?. Mol. Cell. Neurosci., 13: 313-325, 1999.[Medline]
  2. Saarma M., Sariola H. Other neurotrophic factors: glial cell line-derived neurotrophic factor (GDNF). Microsc. Res. Tech., 45: 292-302, 1999.[Medline]
  3. Baloh R. H., Enomoto H., Johnson E. M., Jr., Milbrandt J. The GDNF family ligands and receptors–implications for neural development. Curr. Opin. Neurobiol., 10: 103-110, 2000.[Medline]
  4. Santoro M., Melillo R. M., Carlomagno F., Visconti R., De Vita G., Salvatore G., Lupoli G., Fusco A., Vecchio G. Molecular biology of the MEN2 gene. J. Intern. Med., 243: 505-508, 1998.[Medline]
  5. Ponder, B. A. The phenotypes associated with RET mutations in the multiple endocrine neoplasia type 2 syndrome. Cancer Res., 59: 1736s-1741s.
  6. Santoro M., Carlomagno F., Melillo R. M., Billaud M., Vecchio G., Fusco A. Molecular mechanisms of RET activation in human neoplasia. J. Endocrinol. Investig., 22: 811-819, 1999.[Medline]
  7. Hansford J. R., Mulligan L. M. Multiple endocrine neoplasia type 2 and RET: from neoplasia to neurogenesis. J. Med. Genet., 37: 817-827, 2000.[Abstract/Free Full Text]
  8. Jhiang S. M. The RET proto-oncogene in human cancers. Oncogene, 19: 5590-5597, 2000.[Medline]
  9. Reynolds L., Jones K., Winton D. J., Cranston A., Houghton C., Howard L., Ponder B. A., Smith D. P. C-cell and thyroid epithelial tumours and altered follicular development in transgenic mice expressing the long isoform of MEN 2A RET. Oncogene, 20: 3986-3994, 2001.[Medline]
  10. Silver L. M. Mouse Genetics–Concepts and Applications362 Oxford University Press 1995.
  11. Anon. United Kingdom Co-ordinating Committee on Cancer Research (UKCCCR) Guidelines for the Welfare of Animals in Experimental Neoplasia, Ed. 2. Br. J. Cancer, 77: 1-10, 1998.
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