Selective Ablation of Tumor Suppressors in Parafollicular C Cells Elicits Medullary Thyroid Carcinoma

Abstract

Among the four different types of thyroid cancer, treatment of medullary thyroid carcinoma poses a major challenge because of its propensity of early metastasis. To further investigate the molecular mechanisms of medullary thyroid carcinoma and discover candidates for targeted therapies, we developed a new mouse model of medullary thyroid carcinoma based on our CGRP^CreER mouse line. This system enables gene manipulation in parafollicular C cells in the thyroid, the purported cells of origin of medullary thyroid carcinoma. Selective inactivation of tumor suppressors, such as p53, Rb, and Pten, in mature parafollicular C cells via an inducible Cre recombinase from CGRP^CreER led to development of murine medullary thyroid carcinoma. Loss of Pten accelerated p53/Rb-induced medullary thyroid carcinoma, indicating interactions between pathways controlled by tumor suppressors. Moreover, labeling differentiated parafollicular C cells by CGRP^CreER allows us to follow their fate during malignant transformation to medullary thyroid tumor. Our findings support a model in which mutational events in differentiated parafollicular C cells result in medullary thyroid carcinoma. Through expression analysis including RNA-Seq, we uncovered major signaling pathways and networks that are perturbed following the removal of tumor suppressors. Taken together, these studies not only increase our molecular understanding of medullary thyroid carcinoma but also offer new candidates for designing targeted therapies or other treatment modalities.

Keywords: CGRP; calcitonin; cancer biology; gene knock-out; genomics; medullary thyroid carcinoma; mouse genetics; parafollicular C cells; tumor suppressor gene.

FIGURE 1.

CGRP^CreER and Ascl1^CreER mouse lines enable gene manipulation selectively in parafollicular C cells. A, schematic diagram showing selective control of gene expression in parafollicular C cells using the CGRP^CreER mouse line. TM injection activates Cre and allows manipulation of gene expression. B–D, immunostaining of thyroid sections from wild-type adult mice. Round parafollicular C cells in the thyroid were identified by CT expression. E–S, immunostaining of thyroid sections from CGRP^CreER/+; ROSA26^mTmG/+ adult mice. Extensive labeling of parafollicular C cells in the thyroid by eGFP was observed as indicated by colocalization of eGFP (green) and SYP (red), CT (red), or ASCL1 (red) signals after three TM injections. No labeled parafollicular C cells were detected in CGRP^CreER/+; ROSA26^mTmG/+ adult mice without TM injection. NKX2.1 was detected in both columnar follicular and round parafollicular C cells. Follicular cells marked by NKX2.1 were not labeled by eGFP in CGRP^CreER/+; ROSA26^mTmG/+ thyroid. T–V, immunostaining of thyroid sections from Ascl1^CreER/+; ROSA26^mTmG/+ adult mice. Similarly, extensive labeling of parafollicular C cells in the thyroid by eGFP was observed upon TM injection. No apparent difference was discerned between CGRP^CreER/+ and Ascl1^CreER/+ mouse lines in this assay. W–Y, immunostaining of sections from the thyroid, adrenal medulla, and brain of Ascl1^CreER/+; ROSA26^mTmG/+ adult mice. Several neurons in the brain, parafollicular C cells, and scattered cells in the adrenal medulla also displayed Ascl1^CreER activity. Scale bars, 50 μm for B–M; 50 μm for N–P; 10 μm for Q–S; 25 μm for T–V; and 25 μm for W–Y.

FIGURE 2.

Ablation of tumor suppressors in parafollicular C cells results in murine medullary thyroid carcinoma. A–C, gross morphology of thyroid tissues dissected from representative WT, CGRP^CreER/+; p53^f/f; Rb^f/f (abbreviated as p53^−/−; Rb^−/−), CGRP^CreER/+; p53^f/f; Rb^f/f; Pten^f/f (abbreviated as p53^−/−; Rb^−/−; Pten^−/−), and CGRP^CreER/+; p53^f/f; Pten^f/f (abbreviated as p53^−/−; Pten^−/−) mice at different time points post-TM injection as indicated. Thyroid tumors increased in size with time, and p53/Rb/Pten TKO had a higher proliferation rate than p53/Rb DKO at the same stage. Loss of p53/Pten in parafollicular C cells did not seem to exert major effects on thyroid tumor development. D, PCR analysis of genomic DNA derived from tails of p53^f/f; Rb^f/f; Pten^f/f (control) animals and MTCs dissected from p53^−/−; Rb^−/−; Pten^−/− (TKO) mice. Floxed alleles of p53 (p53^f), Rb (Rb^f), and Pten (Pten^f) were converted to null alleles in thyroid tumors as indicated by the changes in PCR product size of genomic DNA. Gapdh serves as loading control. E, transverse section of medullary thyroid tumor in p53^−/−; Rb^−/−; Pten^−/− mice. F, survival curves of p53^−/−; Rb^−/−, p53^−/−; Rb^−/−; Pten^−/− and p53^−/−; Pten^−/− mice at different time points post-TM injection as indicated. E, esophagus; Tr, trachea; T, thyroid tumor.

FIGURE 3.

Histological and marker analysis of murine medullary thyroid carcinoma. A–J, hematoxylin-eosin (H&E) staining of thyroid sections derived from wild-type, CGRP^CreER/+; p53^f/f; Rb^f/f (abbreviated as p53^−/−; Rb^−/−), CGRP^CreER/+; p53^f/f; Rb^f/f; Pten^f/f (abbreviated as p53^−/−; Rb^−/−; Pten^−/−), and CGRP^CreER/+; p53^f/f; Pten^f/f mice (abbreviated as p53^−/−; Pten^−/−) at time points post-TM as indicated. Parafollicular C cell hyperplasia was evident in p53/Rb DKO thyroids at 2 months post-TM, and remnants of thyroid follicles could still be found at the periphery of the thyroid. By 5 months post-TM in p53/Rb DKO thyroids and by 2 months post-TM in p53/Rb/Pten TKO thyroids, the entire thyroid gland had been replaced by tumor cells, and almost no normal thyroid tissue could be found. By contrast, the histology of p53/Pten DKO thyroid is similar to that in wild type. K–Y, immunostaining of thyroid sections derived from wild-type, p53/Rb DKO, p53/Rb/Pten TKO, and p53/Pten DKO mice at time points post-TM as indicated. Tumor cells in p53/Rb DKO and p53/Rb/Pten TKO thyroids expressed markers of parafollicular C cells such as SYP and ASCL1 (MASH1) and were highly proliferative as judged by Ki67 staining. Z–K′, immunofluorescence of thyroid sections derived from p53^−/−; Rb^−/−; Pten^−/−; ROSA26^mTmG/+ mice at the time point post-TM as indicated. Tumor cells expressed markers of parafollicular C cells such as SYP and CT. Tumor cells also expressed NKX2.1, indicating the origin of tumors from thyroid tissues. Scale bars, 12.5 μm for A–E; 50 μm for F–Y; and 50 μm for Z–K′.

FIGURE 4.

Tracing of labeled parafollicular C cells during medullary thyroid tumor development. A–L, immunostaining of thyroid sections from CGRP^CreER/+; p53^f/f; Rb^f/f; ROSA26^mTmG/+ (abbreviated as p53^−/−; Rb^−/−; ROSA26^mTmG/+) and CGRP^CreER/+; p53^f/f; Rb^f/f; Pten^f/f; ROSA26^mTmG/+ (abbreviated as p53^−/−; Rb^−/−; Pten^−/−; ROSA26^mTmG/+) mice. Tamoxifen injection selectively ablated p53/Rb (A–F) or p53/Rb/Pten (G–I) in parafollicular C cells, which were simultaneously labeled by eGFP (from ROSA26^mTmG). Proliferating parafollicular C cells could be found even at 1 week after TM injection in the thyroid glands of both CGRP^CreER/+; p53^f/f; Rb^f/f; ROSA26^mTmG/+ (K) and CGRP^CreER/+; p53^f/f; Rb^f/f; Pten^f/f; ROSA26^mTmG/+ (L) mice. At 4 months post-TM in p53/Rb DKO thyroids, most, if not all, tumor cells were eGFP⁺, suggesting that these tumor cells were derived from eGFP-labeled parafollicular C cells. M, quantification of proliferating parafollicular C cells in p53/Rb DKO and p53/Rb/Pten TKO thyroids. Three mice were counted for each genotype at a given time point. A total 152 DKO and 178 TKO SYP⁺ cells were counted at 1 week post-TM, and a total of 1386 DKO and 1427 TKO SYP⁺ cells were counted at 2 months post-TM. The ratio of Ki67⁺; SYP⁺ and SYP⁺ cells is represented as means ± S.E. p value = 0.0017 (comparison between DKO and TKO at 1 week post-TM) and p value = 0.016 (comparison between DKO and TKO at 2 months post-TM).

FIGURE 5.

Active PI3K/AKT signaling in murine and human medullary thyroid carcinoma. A–C, immunostaining of human thyroid sections. Compared to normal human thyroid tissues (A), human medullary thyroid carcinoma had robust expression of neuroendocrine markers such as SYP (B). Among the 10 samples of human medullary thyroid carcinoma examined, eight of them had strong staining of phospho-AKT (Ser⁴⁷³) (pAKT) (C), whereas two had weaker pAKT staining. This is consistent with active PI3K/AKT signaling in human medullary thyroid carcinoma. D–F, similarly, thyroid tumors derived from p53/Rb DKO mice at 5 months post-TM also showed weak pAKT staining (D). By contrast, strong pAKT signals were detected in p53/Rb/Pten TKO thyroids even at 2 months post-TM (E and F). Scale bars, 50 μm for A–F.

FIGURE 6.

RNA-Seq analysis of medullary thyroid carcinoma induced by removal of tumor suppressors. A, top canonical pathways that were enriched in differentially expressed genes. Among the top pathways are cAMP-mediated signaling (p value = 8.32E-07, z score = 2.9), actin cytoskeleton signaling (p value = 3.31E-06, z score = −3.3), LXR/RXR activation (p value = 4.68E-06, z score = 0.949), and G protein-coupled receptor signaling (p value = 4.57E-06). Importantly, thyroid cancer signaling and thyroid hormone receptor/RXR activation are also enriched (p value = 6.61E-05, and p value = 7.94E-05, respectively) (not shown). B, qPCR analysis of RNA extracted from thyroid tumors derived from CGRP^CreER/+; p53^f/f; Rb^f/f (DKO) or CGRP^CreER/+; p53^f/f; Rb^f/f; Pten^f/f (TKO) adult mice injected with tamoxifen. Wild-type thyroid tissue was used as a control. Target genes for each major signaling pathway were assessed for changes in their expression in tumor versus non-tumor tissues. The numbers represent fold of changes in tumor tissues. Tumors from multiple mice were analyzed. The expression levels of Axin2, Hes1, and Notch1 genes in thyroid tumors were graphed as fold of changes relative to wild-type thyroid tissues.

FIGURE 7.

Heat map analysis, gene network analysis, and regulator effects analysis of medullary thyroid carcinoma. A, heat map analysis of the highly up-regulated (red) and down-regulated (blue) genes in thyroid tumors (TKO) compared with controls (CTL). Only genes at the top of the list were shown. B, gene network analysis of genes identify in RNA-Seq of thyroid tumors. GPCR signaling is the top network identified by IPA (score = 30). C, regulator effects analysis by IPA identified a top upstream regulator in Ccl11. Inhibition of Ccl11 could lower expression of Bmp6, Cxcl12, Dpp4, Fas, Fgf10, Icam1, Itga1, Itgam, Itgb1, Lep, and Vegfc and consequently disrupt multiple processes including adhesion of tumor cells, vascularization, and activation of leukocytes.