Medullary Thyroid Carcinoma Model Research Articles

Medullary Thyroid Carcinoma Arises in the Absence of Prolactin Signaling

Prolactin, a pituitary hormone, exerts pleiotropic effects in various cells. These effects are mediated by a membrane receptor highly expressed in many tissues. To analyze prolactin effects on the thyroid gland, we first identified prolactin receptor (PRLR) mRNAs by in situ hybridization. To further evaluate the physiologic relevance of PRLR actions in the thyroid in vivo, we used PRLR knockout mice. Whereas the histologic structure of thyroid of PRLR-null mice was not disturbed, we show that T4 levels are lower in null animals (13.63 +/- 2.98 versus 10.78 +/- 2.25 pmol/L in null mice), confirming that prolactin participates in the control of thyroid metabolism. To further investigate thyroid effects in mice, we measured body temperature and thyroid-stimulating hormone in young and adult male and/or female PRLR-null mice and their normal siblings. Surprisingly, in null animals, we saw medullary thyroid carcinoma (MTC) arising from parafollicular C cells producing calcitonin. The incidence of these carcinomas attained 41% in PRLR-null mice, whereas this malignant tumor occurs sporadically or as a component of the familial cancer syndrome in humans. This finding suggests that PRLR-null mice could represent a valuable animal model for MTC, which could be compared with existing MTC models. These observations suggest a possible link between the appearance of this carcinoma and the absence of prolactin signaling.

Gene therapy of a rat follicular thyroid carcinoma model with adenoviral vectors transducing murine interleukin-12.

Interleukin-12 (IL12) is a heterodimeric cytokine that plays an important role in the development of cellular immunity. We previously reported the antitumor activity of mouse IL12 (mIL12) transduced by adenovirus in a medullary thyroid carcinoma model. In this study, a rat thyroid follicular cancer cell line (RTC-R2) was employed to develop tumors after sc injection in Wistar rats. In five of five animals, RTC-R2 cells infected with mIL12 transducing adenovirus (AdCMVmIL12) in vitro failed to be tumorigenic in vivo in syngenic rats, whereas four of five animals developed tumors after injection of luciferase transducing adenovirus (AdCMVLuc)-infected cells. After intratumoral treatment with AdCMVmIL12 at 1 x 10(9) plaque-forming units per rat, 90% (26/29) of animals bearing small (<100 mm(3)) tumors were apparently cured. Larger tumors treated by injection of AdCMVmIL12 became significantly smaller than AdCMVLuc-treated animals, and growth stabilized. Challenge studies showed that only 3 of 28 animals previously treated and cured with AdCMVmIL12 developed a tumor after sc reinjection of RTC-R2 cells, whereas all animals developed tumors in naïve animals. Thus, AdCMVmIL12-treated animals developed long-term antitumor immunity. We also studied animals with two tumors, injecting virus in one. Tumors regressed at both sites in five of six animals after treatment of one tumor with AdCMVmIL12, and in the sixth animal one site tumor regressed and another tumor continued to grow. In AdCMVLuc-treated animals, both tumors regressed in only one animal, and the reminder continued to grow. To detect toxicity to liver and other tissues after administration of AdCMVmIL2, the vector was administrated intratumorally or iv at the dose of 2 x 10(9) plaque-forming units per rat. No change in behavior was observed in any of the treated animals. Rats were killed at different time after virus administration. An overt increase of spleen size was observed 7 d after infection in all animals treated with AdCMVmIL12. All animals given virus IV had some lymphocyte infiltration in the sinusoids and triads of the liver, whereas AdCMVmIL12 injected intratumorally did not cause this effect. Spleens of some virus-treated animals showed decreased white pulp, with apparently increased hematopoiesis. No specific changes were found in lungs and kidneys. Iv administration of AdCMVmIL12 induced a moderate increase of glutamic-oxalacetic transaminase and glutamic-pyruvic transaminase, whereas AdCMVmIL12 injected intratumorally did not. This study confirms the efficient antitumor activity of an adenovirus expressing mIL12 after in vivo delivery in an animal model and indicates the possibility of application to patients because of the low toxicity.

Combretastatin A-4 and doxorubicin combination treatment is effective in a preclinical model of human medullary thyroid carcinoma.

Medullary thyroid carcinoma (MTC), both in patients and in preclinical models, is resistant to chemotherapy. In this study, we show that the anti-neovascular agent combretastatin A-4 phosphate prodrug (CA4P) in combination with doxorubicin was effective in curtailing tumor growth in a preclinical model of human MTC. This combination of combretastatin and doxorubicin extended the doubling time of established MTC tumors in nude mice to 29 days, compared to 12 days in untreated controls. This suggests that a combination of combretastatin and a cytotoxic chemotherapeutic agent may be an effective treatment for MTC.

Genetic immunotherapy of established tumours with adenoviral vectors transducing murine interleukin-12 (mIL12) subunits in a rat medullary thyroid carcinoma model.

Interleukin-12 (IL12) is a heterodimeric cytokine that plays an important role in the development of cellular immunity. Studies have demonstrated antitumour activity after systemic administration of recombinant IL12. As with other cytokines, with increasing dosage and longer exposure, systemic toxicity is observed. To reduce systemic toxicity and obtain local production of IL12, we developed a replication defective adenovirus transducing two subunits of the murine IL12 (AdCMVmIL12) gene. Two separate cassettes, expressing the p35 or p40 subunit of mIL12, under the control of human cytomeglavirus immediate early promoter, were inserted into the early1 (E1) region of adenovirus 5. Biological activity of virally expressed mIL12 was demonstrated in vitro through its ability to induce proliferation of mouse ConA blast cells. Rat medullary thyroid carcinoma (MTC) cells infected with AdCMVmIL12 lost their tumorigenicity in their syngenic WAG/Rij rat hosts. Efficient antitumour activity was found after direct injection of the AdCMVmIL12 vector into rMTC tumours. After intratumoural treatment with AdCMVmIL12, 86% of tumour bearing animals were apparently cured, and almost all remaining tumours were stabilized. Challenge studies showed that most animals cured after the first treatment remained tumour free after reinjection of wild type rMTC cells, indicating that long-term antitumour immunity developed. This study demonstrates the construction of an adenoviral vector expressing a functional heterodimeric mIL12 and its efficient antitumour activity after in vivo delivery in an animal model of medullary thyroid carcinoma.

Roles of trk family neurotrophin receptors in medullary thyroid carcinoma development and progression.

Although initiating mutations in the ret protooncogene have been found in familial and sporadic medullary thyroid carcinoma (MTC), the molecular events underlying subsequent tumor progression stages are unknown. We now report that changes in trk family neurotrophin receptor expression appear to be involved in both preneoplastic thyroid C cell hyperplasia and later tumor progression. Only a subset of normal C cells expresses trk family receptors, but, in C cell hyperplasia, the affected cells consistently express trkB, with variable expression of trkA and trkC. In later stages of gross MTC tumors, trkB expression was substantially reduced, while trkC expression was increased and often intense. In a cell culture model of MTC, exogenous trkB expression resulted in severely impaired tumorigenicity and was associated with 11-fold lower levels of the angiogenesis factor vascular endothelial growth factor. These results suggest that trk family receptor genes participate in MTC development and progression, and, in particular, that trkB may limit MTC tumor growth by inhibition of angiogenesis.

Medullary thyroid carcinomas in transgenic mice expressing a Polyoma carboxyl-terminal truncated middle-T and wild type small-T antigens.

Medullary thyroid carcinoma (MTC) is a rare human tumor affecting the calcitonin-secreting c-cells of the thyroid. Here we report that two independent strains of transgenic mice expressing a Polyomavirus (Py) truncated middle-T antigen (deltaMT), consisting of the amino-terminal 304 amino acids, and the full length Py small-T antigen, developed multifocal bilateral MTCs with 100% penetrance. Occasionally one strain also developed mammary and bone tumors. Furthermore, offspring from both transgenic lines displayed pronounced waviness of the whiskers and fur, previously associated with defective epidermal growth factor receptor signaling. Transgene transcription, driven by the homologous early promoter/enhancer, and the corresponding translation products were detected in tumors and in many other organs which did not develop pathologies. The subcellular distribution of deltaMT and its interactions with the adapter proteins of the SHC family have also been analysed. Our study describes a novel murine model of MTC and provides evidence that the N-terminal 304 amino acid fragment of Py middle-T antigen, possibly in co-operation with small-T antigen, acts as a potent oncogene in c-cells of the thyroid.

Immune response against medullary thyroid carcinoma (MTC) induced by parental and/or interleukin-2-secreting MTC cells in a rat model of human familial medullary thyroid carcinoma.

The existence of inherited aggressive forms of medullary thyroid carcinoma (MTC), and their resistance to all classical therapies, make it a prime candidate for adoptive immunotherapy. As a prelude to a vaccine for the protection of family members at risk of developing the disease, we investigated the immunological antitumour response provoked by the 6/23 rMTC cell line, compared to that of the same cell engineered to secrete interleukin-2 (rMTC-IL2), in an animal model of familial human MTC, the inbred strain of Wag/Rij rats. The rMTC cells developed a tumour that invaded the whole neck 15 days after orthotopic injection (into the thyroid), while the rMTC-IL2 cells were progressively rejected. Co-injection of rMTC-IL2 with the parental cells induced the rejection of the rMTC transplants. When injected, both tumoral cell types showed a similar positive immunoreaction with anti-MHC class I (major histocompatibility complex class I) antibodies. They both recruited natural killer cells and eosinophils at the site of injection. In addition, CD8+ T lymphocytes infiltrated the rMTC-IL2 cells, and eosinophil recruitment was amplified. Neutrophils, macrophages and CD4+ T lymphocytes were scarce. Our results suggest that the CD8+ T lymphocytes are implicated in the anti-tumour reaction elicited by the IL-2-transfected cells. As these effectors are known to induce a specific immunological response, including memory, such a protocol should be tested as a vaccine on the young population genetically at risk of developing a MTC.

Calcitonin secretion, C cell differentiation and proliferation during the spontaneous development of murine medullary thyroid carcinoma.

Medullary thyroid carcinoma (MTC), a C cell neoplasm, synthesizes large amounts of calcitonin (CT), its biological marker. However, in some cases with a poor prognosis, MTC is associated with low basal CT levels owing to a decrease in the thyroid CT content. Using a murine model of human MTC, we studied the relationships between CT biosynthesis, C cell proliferation, and the circulating CT level during MTC progression. Cell proliferation was revealed by autoradiography of radioactive thymidine incorporation in dividing nuclei, after CT or CT mRNA detection by immunocytochemistry (ICC) or in situ hybridization (ISH). All rat thyroids showed a severe hyperplasia of C cells containing significant amounts of CT and CT mRNA, and a very low mitotic index. Tumours were found in 68% of the thyroids. In the strongly immunoreactive small nodules (ICC+), many labelled nuclei were observed. Subsequently some nodular cells, still containing detectable CT mRNA (ISH+), were not detected by immunocytochemistry (ICC-) owing to a dramatic decrease in secretory granules. Their mitotic index increased, and a rise of the basal CT plasma level was noted. These ISH+, ICC- tumour MTC cells represent a modified aggressive tumour C cell population exhibiting an increased ability to proliferate and were detected by the rise in the basal circulating CT level.