Characterization of neuroendocrine tumors in heterozygous mutant MENX rats: a novel model of invasive medullary thyroid carcinoma

  1. Natalia S Pellegata1⇑
  1. 1Institute for Diabetes and Cancer, Helmholtz Zentrum München, Neuherberg, Germany
  2. 2Institute of Experimental Genetics, Helmholtz Zentrum München, Neuherberg, Germany
  3. 3Research Unit Analytical Pathology, Helmholtz Zentrum München, Neuherberg, Germany
  4. 4German Center for Diabetes Research (DZD), Neuherberg, Germany
  5. 5Technische Universität München, Chair of Experimental Genetics, Freising, Germany
  6. 6Hereditary Endocrine Cancer Group, Spanish National Cancer Research Centre (CNIO) and ISCIII Center for Biomedical Research on Rare Diseases (CIBERER), Madrid, Spain
  7. 7Division of Neuroscience and Experimental Psychology, Faculty of Medicine, University of Manchester, Manchester, UK
  1. Correspondence should be addressed to N S Pellegata: natalia.pellegata{at}helmholtz-muenchen.de
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    Figure 1

    Phenotypic features of p27+/mut and p27mut/mut MENX rats. (A) The overall survival of p27+/+, p27+/mut and p27mut/mut rats is shown. Survival curves are Kaplan–Meier plots censored for deaths due to noncancerous causes. Hematoxylin and eosin (H&E) staining of adrenal (B, C and D), pituitary (F, G and H) and thyroid (J, K and L) glands of p27+/mut rats showing lesions at different stages. Original magnification: B, F, J: 40×; C, G, K: 20×; D, H, L: 400×. (E, I, M) Immunohistochemistry of advanced lesions with antibodies against L1CAM (E), SF1 (I) and calcitonin (M). Original magnification: 400×.

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    Figure 2

    Characterization of rat thyroid lesions. Expression of calcitonin in thyroid glands of p27+/+ (A) and p27+/mut (B and C) rats. Original magnification: 20×. Expression of calcitonin in thyroid glands of 2-month-old p27+/+ (D), p27+/mut (E) and p27mut/mut (F) rats. Original magnification: 200×. (G) Expression of calcitonin in a liver metastasis of a MTC in a p27+/mut rat. Original magnification: 400×. (H, I and J) Invasion of MTCs of p27+/mut rats in vasculature (H), muscles (I) and nerves (J). CD31 (H), H&E (I) and Masson’s trichrome stainings (J) were performed. Original magnification: I, 100×; H, J: 400×.

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    Figure 3

    Circulating levels of calcitonin. The levels of blood calcitonin were measured by ELISA in p27+/+, p27+/mut and p27mut/mut rats at the indicated ages. The number of animals was n = 6 per group. The genotype of the rat groups is reported. Shown is the mean (in pg/mL) ± s.e.m. #Not significant; **P = 0.003.

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    Figure 4

    Expression of p27 and DNA analysis in pituitary adenomas of p27+/mut rats. (A) Example of a rat adenoma retaining p27 expression. DNA was extracted from the tumor and from the adjacent non-tumorous area, and sequenced using primers for the rat Cdkn1b gene. Chromatograms corresponding to the indicated tissue areas are shown below, and indicate that both alleles (wild-type and mutant) are present in both areas. (B) The only rat adenoma (out of 8) with loss of p27 expression. Chromatograms corresponding to the indicated tissue areas show that the mutant allele is present in both areas while the signal for the wild-type allele is extremely reduced in the tumor indicating loss-of-heterozygosity (LOH).

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    Figure 5

    Gene expression signature of adrenal and pituitary tumors in p27+/mut and p27mut/mut MENX rats. (A and B) Most enriched Gene Ontology (GO) categories in rat adrenal and pituitary tumors. (A) Level 3 Biological Process GO annotations identified by comparing the p27+/mut with the p27+/+ dataset (left) and the p27mut/mut with the p27+/+ dataset (right) for the adrenal glands. (B) Level 3 Biological Process GO annotations identified by comparing the p27+/mut with the p27+/+ dataset (left) and the p27mut/mut with the p27+/+ dataset (right) for the pituitary gland. In colors are illustrated the GO terms in common between the ‘p27+/mut vs p27+/+’ and the ‘p27mut/mut vs p27+/+’ datasets in adrenal or pituitary tissues. In different shades of gray are illustrated the GO terms not shared in the above indicated comparisons.

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    Figure 6

    Expression of selected differentially expressed genes in human MTCs. qRT-PCR for TUBB2B, NREP, TUBB6, CA10, GREM2, HSPB1, PLA2G16, CLDN3 and SMAD9 was performed on human MTC samples with different RET mutation status (Table 2). The relative mRNA expression level of the target genes was normalized for input RNA using TBP as housekeeping gene and was calculated with the 2−ΔΔCt formula. Data were analyzed independently with six replicates each and are expressed as the mean ± s.e.m. Only the comparisons leading to a statistical significance are indicated in the graphs. WT, wild-type; *P < 0.05; **P < 0.01.

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  1. doi: 10.1530/ERC-17-0456 Endocr Relat Cancer 25 145-162
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