Thyroxine promotes lung cancer growth in an orthotopic mouse model.

Thyroid hormones are important for physiology and homeostasis. In addition to nuclear thyroid hormone receptors, the plasma membrane protein integrin αvß3 has been recognized as a receptor for both thyroxine (T4) and triiodothyronine (T3). Here, we studied whether thyroid hormone promotes growth of murine lung cancer via αvß3 in vivo. Murine Lewis lung carcinoma cells (3LL), stably transfected with luciferase, were injected into mouse lungs. Tumor growth in untreated mice was compared to hypothyroid mice and hypothyroid mice treated with T3 or T4 with or without the αvß3 inhibitor 3,5,3',5'-tetraiodothyroacetic acid (Tetrac). Tumor progression was determined by serial in vivo imaging of bioluminescence emitted from the tumor. Tumor weight was recorded at the end of the experiment. Neoangiogenesis was determined by immunohistochemistry for CD31. Tumor growth was reduced in hypothyroidism and increased by T4 treatment. Strikingly, only T4 but not T3 treatment promoted tumor growth. This T4 effect was abrogated by the αvß3 inhibitor Tetrac. Tumor weight and neoangiogenesis were also significantly increased only in T4-treated mice. The T4 effect on tumor weight and neoangiogenesis was abolished by Tetrac. In vitro, T4 did not stimulate 3LL cell proliferation or signaling pathway activation. We conclude that T4 promotes lung cancer growth in this orthotopic mouse model. The tumor-promoting effect is mediated via the plasma membrane integrin αvß3 and increased neoangiogenesis rather than direct stimulation of 3LL cells. These data suggest that such effects of levothyroxine may need to be considered in cancer patients on T4 substitution.

A 6-Base Pair in Frame Germline Deletion in Exon 7 Of RET Leads to Increased RET Phosphorylation, ERK Activation, and MEN2A.

CONTEXT: Multiple endocrine neoplasia type 2 (MEN2) is usually caused by missense mutations in the proto-oncogene, RET. OBJECTIVE: This study aimed to determine the mutation underlying MEN2A in a female patient diagnosed with bilateral pheochromocytoma at age 31 years and with medullary thyroid carcinoma (MTC) 6 years later. METHODS: Leukocyte DNA was used for exome and Sanger sequencing. Wild-type (WT) RET and mutants were expressed in HEK293 cells. Activation of MAPK/ERK and PI3K/AKT was analyzed by Western blotting and luciferase assay. The effect of RET mutants on cell proliferation was tested in a colony forming assay. RESULTS: Exome sequencing revealed a 6-nucleotide/2-amino acid in-frame deletion in exon 7 of RET (c.1512_1517delGGAGGG, p.505_506del). In vitro expression showed that phosphorylation of the crucial tyrosine 905 was much stronger in the p.505_506del RET mutant compared with WT RET, indicating ligand-independent autophosphorylation. Furthermore, the p.505_506del RET mutant induced a strong activation of the MAPK/ERK pathway and the PI3K/AKT pathway. Consequently, the p.505_506del RET mutant cells increased HEK293 colony formation 4-fold compared with WT RET. CONCLUSION: The finding of bilateral pheochromocytoma and MTC in our patient was highly suspicious of a RET mutation. Exome sequencing revealed a 6-base-pair deletion in exon 7 of RET, an exon not yet associated with MEN2. Increased ligand-independent phosphorylation of the p.505_506del RET mutant, increased activation of downstream pathways, and stimulation of cell proliferation demonstrated the pathogenic nature of the mutation. We therefore recommend screening the whole sequence of RET in MTC and pheochromocytoma patients with red flags for a genetic cause.

Diabetes insipidus as a rare cause of acute cognitive impairment in multiple sclerosis.

Multiple sclerosis (MS) is a complex neurodegenerative disease presenting with a diversity of clinical symptoms including palsy and cognitive impairment. We present a 59-year-old woman with a history of secondary progressive MS since 1987, who was referred to our department because of recent onset of confusion and polydipsia. Initial lab tests showed mildly elevated serum sodium levels and low urine osmolality. Under water deprivation, diuresis and low urine osmolality persisted and serum sodium levels rose above 150 mmol/l. Oral desmopressin resulted in normalisation of serum sodium as well as urine osmolarity, confirming a diagnosis of central diabetes insipidus. As drug-induced diabetes could be excluded, pituitary magnetic resonance imaging (MRI) was performed. A demyelinating lesion was detected in the hypothalamus. The patient was started on oral desmopressin treatment (0.2 mg/day). Fluid intake and serum sodium levels have since remained normal. In summary, we report the rare case of a patient presenting with diabetes insipidus due to progressive MS. Diabetes insipidus should be considered in MS patients who develop new onset of polydipsia.

Treatment of advanced medullary thyroid carcinoma with a combination of cyclophosphamide, vincristine, and dacarbazine: a single-center experience.

OBJECTIVE: Experience with chemotherapy in patients with medullary thyroid carcinomas (MTC) is limited. This retrospective study evaluated the outcome of a combination of cyclophosphamide, vincristine, and dacarbazine ('CVD-regimen'), which has previously been suggested for treatment of malignant pheochromocytomas. METHODS: 9 patients (5 males; age 55.0 ± 4.0 years) with MTC were enrolled. Prior to chemotherapy, progressive disease was established in all patients by use of WHO criteria. On day 1 of each cycle, patients started with cyclophosphamide 750 mg/m(2), vincristine 1.4 mg/m(2), and dacarbazine 600 mg/m(2); on day 2, patients received dacarbazine alone (600 mg/m(2)). Treatment cycles were repeated at 21-day intervals and 6 cycles were planned for each patient. The standard imaging procedure was computed tomography, and the primary end point was the objective tumor response rate. After chemotherapy, patients were followed up until progression. RESULTS: 9 patients underwent a total of 57 cycles (mean 6.3 ± 0.3 cycles). Treatment responses were: 0% complete response, 11% partial response, 56% stable disease, and 33% progressive disease. The median progression free survival was 13.6 months (range 5.8-24.2 months). The median change (baseline vs. end of treatment) of calcitonin was -19% (range -70% to +174%). Reversible myelosuppression and moderate gastrointestinal symptoms were the most common adverse events. CONCLUSION: Although objective tumor response rates were low, the CVD regimen allowed disease stabilization for a substantial period of time and had acceptable toxicity. After initial surgery, chemotherapy may therefore be considered as a medical treatment option.

Stanniocalcin 1 induction by thyroid hormone depends on thyroid hormone receptor ß and phosphatidylinositol 3-kinase activation.

CONTEXT: Thyroid hormone (TH) mediated changes in gene expression were thought to be primarily initiated by the nuclear TH receptor (TR) binding to a thyroid hormone response element in the promoter of target genes. A recently described extranuclear mechanism of TH action consists of the association of TH-liganded TRß with phosphatidylinositol 3-kinase (PI3K) in the cytosol and subsequent activation of the PI3K pathway. OBJECTIVE: The aim of this study was to examine the effect of TH, TRß and PI3K on stanniocalcin 1 (STC1) expression in human cells. DESIGN: We treated human skin fibroblasts with triiodothyronine (T3) in the absence or presence of the PI3K inhibitor LY294002, a dominant negative PI3K subunit, Δp85α, and the protein synthesis inhibitor cycloheximide (CHX). The role of the TRß was studied in cells from patients with resistance to thyroid hormone (RTH). STC-1 mRNA expression was measured by real-time PCR. RESULTS: We found an induction of STC1 by T3 in normal cells, but less in cells from subjects with RTH (2.7 ± 0.2 vs. 1.6 ± 0.04, P < 0.01). The effect of T3 was completely abrogated by blocking PI3K with LY294002 (3.9 ± 0.5 vs. 0.85 ± 0.5; P < 0.05) and greatly reduced after transfection of a dominant negative PI3K subunit, demonstrating dependency on the PI3K pathway. CONCLUSION: These results establish STC1 as a TH target gene in humans. Furthermore, we show that STC1 induction by TH depends on both TRß and PI3K activation.

A novel splice variant involving the 5' untranslated region of thyroid hormone receptor beta1 (TRbeta1).

The thyroid hormone receptor beta (TRbeta) gene generates two different proteins by use of a different promoter (beta1 and beta2). We now report a novel short TRbeta1 RNA splice variant in humans lacking 35 nucleotides at the 3' end of the non-coding exon 1 due to an alternative 5' splice donor site. This short variant was first identified in sequences of cDNA obtained from cultured human fibroblasts. Both variants were found in human fibroblasts, brain, pituitary, adrenal gland, placenta, muscle, thyroid and lymphocytes. These TRbeta1 variants possess splice donor sites with a sequence score slightly favoring the TRbeta1 long variant. Variant-specific real-time polymerase chain reaction (PCR) showed that their relative proportions were equal except in pituitary and muscle, in which the long form was 3- and 5-fold in excess. T3 treatment of fibroblasts grown in thyroid hormone depleted medium did not affect the absolute or relative expression of the two variants. Furthermore, the expression level in fibroblasts from patients with resistance to thyroid hormone with or without TRbeta gene mutations was not different to that in fibroblasts from normal controls.