Association of mitotane with chylomicrons and serum lipoproteins: practical implications for treatment of adrenocortical carcinoma.

OBJECTIVE: Oral mitotane (o,p'-DDD) is a cornerstone of medical treatment for adrenocortical carcinoma (ACC). AIM: Serum mitotane concentrations >14  mg/l are targeted for improved efficacy but not achieved in about half of patients. Here we aimed at a better understanding of intestinal absorption and lipoprotein association of mitotane and metabolites o,p'-dichlorodiphenylacetic acid (o,p'-DDA) and o,p'-dichlorodiphenyldichloroethane (o,p'-DDE). DESIGN: Lipoproteins were isolated by ultracentrifugation from the chyle of a 29-year-old patient and serum from additional 14 ACC patients treated with mitotane. HPLC was applied for quantification of mitotane and metabolites. We assessed NCI-H295 cell viability, cortisol production, and expression of endoplasmic reticulum (ER) stress marker genes to study the functional consequences of mitotane binding to lipoproteins. RESULTS: Chyle of the index patient contained 197  mg/ml mitotane, 53  mg/ml o,p'-DDA, and 51  mg/l o,p'-DDE. Of the total mitotane in serum, lipoprotein fractions contained 21.7±21.4% (VLDL), 1.9±0.8% (IDL), 8.9±5.5% (LDL1), 18.9±9.6% (LDL2), 10.1±4.0% (LDL3), and 26.3±13.0% (HDL2). Only 12.3±5.5% were in the lipoprotein-depleted fraction. DISCUSSION: Mitotane content of lipoproteins directly correlated with their triglyceride and cholesterol content. O,p'-DDE was similarly distributed, but 87.9±4.2% of o,p'-DDA found in the HDL2 and lipoprotein-depleted fractions. Binding of mitotane to human lipoproteins blunted its anti-proliferative and anti-hormonal effects on NCI-H295 cells and reduced ER stress marker gene expression. CONCLUSION: Mitotane absorption involves chylomicron binding. High concentrations of o,p'-DDA and o,p'-DDE in chyle suggest intestinal mitotane metabolism. In serum, the majority of mitotane is bound to lipoproteins. In vitro, lipoprotein binding inhibits activity of mitotane suggesting that lipoprotein-free mitotane is the therapeutically active fraction.

Less common genotype variants of TP53 polymorphisms are associated with poor outcome in adult patients with adrenocortical carcinoma.

CONTEXT: The Li-Fraumeni tumor syndrome is strongly associated with adrenocortical carcinoma (ACC) and is caused by germline mutations in TP53 in 70% of cases. Also, TP53 polymorphisms have been shown to influence both cancer risk and clinical outcome in several tumor entities. We, therefore, investigated TP53 polymorphisms in a cohort of adult patients with ACC. OBJECTIVE: Evaluation of the role of TP53 polymorphisms in adult patients with ACC. SUBJECTS AND METHODS: Peripheral blood for DNA extraction was collected from 72 ACC patients. Polymorphism analysis was carried out by amplification and sequencing of exons and adjacent intron sections of TP53. Results were correlated with clinical data and the distribution of the polymorphisms was compared with published Caucasian control groups. RESULTS: Compared with control groups, genotype frequencies of analyzed TP53 polymorphisms among ACC patients were significantly different in three out of four polymorphisms: IVS2+38G>C (G/G, P=0.0248), IVS3ins16 (NoIns/NoIns, P<0.0001; NoIns/Ins, P<0.0001), and IVS6+62A>G (G/G, P<0.0001; G/A, P<0.0001). Overall, the survival of ACC patients, which harbored at least one of the less frequent genotype variants of four analyzed polymorphisms (n=23), was significantly inferior (median survival: 81.0 months in patients with the common homozygous genotypes vs 20.0 months in patients with the less frequent genotypes, HR 2.56, 95% CI 1.66-7.07; P=0.001). These results were confirmed by multivariable regression analysis (HR 2.84, 95% CI 1.52-7.17; P=0.037). CONCLUSION: Some TP53 polymorphisms seem to influence overall survival in ACC patients. This effect was observed for a combination of polymorphic changes rather than for single polymorphisms.

Siglec-6 is expressed in gestational trophoblastic disease and affects proliferation, apoptosis and invasion.

Sialic acid immunoglobulin-like lectin (Siglec)-6 is a transmembrane receptor that binds leptin. Leptin is an obesity-associated peptide hormone overexpressed in gestational trophoblastic disease (GTD). GTD encompasses several placental abnormalities that range from benign to malignant. Among GTD, molar placentas are characterized by excess proliferation, whereas gestational trophoblastic neoplasias (GTN) have characteristically aggressive invasion. We hypothesized that in GTD, Siglec-6 expression would increase with disease severity and that Siglec-6 and leptin would promote proliferation, inhibit apoptosis and/or promote invasion. Siglec-6 expression patterns were evaluated with particular attention to the diagnostic utility of Siglec-6 in GTD (controls: normal placentas (n=32), hydropic abortus placentas (n=7), non-GTD reproductive tract cancers (n=2); GTD: partial moles (PM; n=11), complete moles (n=24), GTN (n=6)). In normal placentas, Siglec-6 expression dramatically decreased after 8 weeks gestation. Complete molar placentas had significantly higher Siglec-6 expression than controls, but expression was not significantly different from PM. In GTN, Siglec-6 expression was low. These data suggest that Siglec-6 may have diagnostic utility for distinguishing complete moles from normal and hydropic abortus placentas. Functional studies in choriocarcinoma-derived BeWO cells demonstrated a complex interplay between Siglec-6 expression and leptin exposure. In cells lacking Siglec-6, leptin treatment promoted invasion, likely through interaction with LepR leptin receptor, without affecting proliferation or apoptosis. Siglec-6 expression promoted proliferation in a leptin-dependent manner, but protected cells from apoptosis and promoted invasion in a leptin-independent manner. We propose that Siglec-6 and leptin play a role in the aberrant properties characteristic of GTD, namely excess proliferation and invasion.

TP53 germline mutations in adult patients with adrenocortical carcinoma.

CONTEXT: Li-Fraumeni syndrome (LFS) is a cancer predisposition syndrome associated with germline mutations in TP53. According to the Chompret criteria for LFS, any patient with adrenocortical cancer (ACC), irrespective of age and family history, is at high risk for a TP53 germline mutation. However, whereas such mutations have been detected with high frequency in childhood ACC, a large cohort of adult patients with ACC has never been investigated for TP53 germline mutations. OBJECTIVE: The aim of the study was to evaluate the prevalence of TP53 germline mutations in adult patients with ACC. SUBJECTS AND METHODS: In 103 adult Caucasian patients with ACC, TP53 germline mutation analysis was performed. In patients with a TP53 germline mutation, tumor tissue was analyzed for loss of heterozygosity of TP53 and p53 immunohistochemistry. Family history and clinical course were also evaluated. RESULTS: In four patients, a total of five TP53 germline mutations were found. Two mutations occurred in exon 10 (R337H and I332M, respectively), outside the hot spot region. Here, three mutations are described for the first time in ACC, and one, which occurred combined with a second mutation (R202C) on the same allele, has never been reported before in the context of LFS. This combined mutation was associated with a remarkable family history of ACC also affecting the mother and uncle of the index patient. In the 23 patients with ACC below the age of 40 yr, 13% (95% confidence interval, 3.7-32.9%) carried a TP53 germline mutation, whereas such mutations were rare in older patients with ACC. CONCLUSION: Our findings indicate a need to revise the Chompret criteria. However, in younger adults (<40 yr old) with ACC, screening for TP53 germline mutations may be justified.

Sunitinib Inhibits Cell Proliferation and Alters Steroidogenesis by Down-Regulation of HSD3B2 in Adrenocortical Carcinoma Cells.

The multi-tyrosine kinase inhibitor sunitinib is used in the treatment of several solid tumors. Animal experiments pointed to an adrenotoxic effect of sunitinib. Therefore, we evaluated the expression of key targets of sunitinib in human adrenocortical carcinoma (ACC) tumor samples and investigated its in vitro effects in ACC cell lines. We carried out immunohistochemistry for vascular endothelial growth factor (VEGF) and its receptor (VEGF-R2) in 157 ACC samples and nine normal adrenal glands. VEGF and VEGF-R2 protein were expressed in 72 and 99% of ACC samples, respectively. Using NCI-H295 and SW13 ACC cell lines, we investigated the effects of sunitinib on cell proliferation. Sunitinib reduced dose-dependently cell viability of both NCI-H295 and SW13 cells (SW13: 0.1 µM 96 ± 7%, 1 µM 90 ± 9%*, 5 µM 62 ± 6%*, controls 100 ± 9%; *p < 0.05). To determine sunitinib effects on steroidogenesis, we measured steroid hormones in cell culture supernatant by gas chromatography-mass spectrometry. We observed a pronounced decrease of cortisol secretion (1 µM 90.1 ± 1.5%*, 5 µM 57.2 ± 0.3%*, controls 100 ± 2.4%) and a concomitant increase in the DHEA/4-androstenedione and 17-hydroxypregnenolone/17-hydroxyprogesterone ratios, indicating specific inhibition of 3ß-hydroxysteroid dehydrogenase (HSD3B2). In yeast microsomes transformed with HSD3B2, no direct inhibition of HSD3B2 by sunitinib was detected. Sunitinib induced down-regulation of HSD3B2 mRNA and protein in ACC cell lines (mRNA: 1 µM 44 ± 16%*; 5 µM 22 ± 2%*; 10 µM 19 ± 4%*; protein: 1 µM 82 ± 8%; 5 µM 63 ± 8%*; 10 µM 55 ± 9%*). CYP11B1 was down-regulated at mRNA but not at protein level and CYP11A1 remained unchanged. In conclusion, target molecules of sunitinib are expressed in the vast majority of ACC samples. Sunitinib exhibits anti-proliferative effects in vitro, and appears to specifically block adrenal steroidogenesis by down-regulation of HSD3B2, rendering it a promising option for treatment of ACC.

[123 I]Iodometomidate for molecular imaging of adrenocortical cytochrome P450 family 11B enzymes.

BACKGROUND: Due to advances in conventional imaging, adrenal tumors are detected with increasing frequency. However, conventional imaging provides only limited information on the origin of these lesions, which represent a wide range of different pathological entities. New specific imaging methods would therefore be of great clinical value. We, therefore, studied the potential of iodometomidate (IMTO) as tracer for molecular imaging of cytochrome P450 family 11B (Cyp11B) enzymes. METHODS: Inhibition of Cyp11B1 and Cyp11B2 by IMTO, etomidate, metomidate, and fluoroetomidate was investigated in NCI-h295 cells and in Y1 cells stably expressing hsCyp11B1 or hsCyp11B2. Pharmacokinetics and biodistribution after iv injection of [(123/125)I]IMTO were analyzed in mice in biodistribution experiments and by small-animal single-photon emission computed tomography (SPECT). Furthermore, four patients with known adrenal tumors (two metastatic adrenal adenocarcinomas, one bilateral adrenocortical adenoma, and one melanoma metastasis) were investigated with [(123)I]iodometomidate-SPECT. RESULTS: In cell culture experiments, all compounds potently inhibited both Cyp11B1 and Cyp11B2. Adrenals showed high and specific uptake of [(123/125)I]IMTO and were excellently visualized in mice. In patients, adrenocortical tissue showed high and specific tracer uptake in both primary tumor and metastases with short investigation time and low radiation exposure, whereas the non-adrenocortical tumor did not exhibit any tracer uptake. CONCLUSION: We have successfully completed the development of an in vivo detection system of adrenal Cyp11B enzymes by [(123)I]IMTO scintigraphy in both experimental animals and humans. Our findings suggest that [(123)I]IMTO is a highly specific radiotracer for imaging of adrenocortical tissue. Due to the general availability of SPECT technology, we anticipate that [(123)I]IMTO scintigraphy may become a widely used tool to characterize adrenal lesions.

AKT is highly phosphorylated in pheochromocytomas but not in benign adrenocortical tumors.

CONTEXT: Activation of AKT plays a major role in a variety of human neoplasias. In mice, a heterozygous deletion of the Pten gene is associated with increased activation of AKT and with development of pheochromocytomas. OBJECTIVE: The objective of this study was the investigation of the role of AKT in the pathogenesis of pheochromocytomas and adrenocortical tumors. DESIGN, SETTING, AND PARTICIPANTS: Total AKT and phosphorylated AKT (pAKT) in 15 pheochromocytomas, nine aldosterone-producing adenomas, nine cortisol-producing adenomas, eight adrenocortical carcinomas (ACC), and 15 normal adrenals were investigated by Western blot analysis. Immunohistochemistry for total AKT and pAKT was performed in pheochromocytomas (n = 8), ACC (n = 4), and normal adrenal glands (n = 2). In addition, in pheochromocytomas PTEN protein expression and PTEN loss of heterozygosity were analyzed. MAIN OUTCOME MEASURES: Determination of pAKT/total AKT ratio in adrenal tissues was the main outcome. RESULTS: In comparison to normal adrenals, total AKT expression was elevated in both pheochromocytomas (193 +/- 22%) and ACC (176 +/- 36%). The pAKT/AKT ratio was significantly increased in pheochromocytomas (338 +/- 49% vs. 100 +/- 11%) but not in ACC, aldosterone-producing adenomas, and cortisol-producing adenomas. No loss of heterozygosity of PTEN and no decrease in PTEN protein was detected in pheochromocytomas. Immunohistochemistry showed strong and homogeneous AKT and pAKT staining in pheochromocytomas and focal staining in ACC. CONCLUSION: Our findings provide evidence for increased activation of AKT in pheochromocytomas but not in adrenocortical adenomas.

N-terminal proopiomelanocortin acts as a mitogen in adrenocortical tumor cells and decreases adrenal steroidogenesis.

There is evidence that proopiomelanocortin (POMC)-derived peptides other than ACTH are involved in pituitary-dependent adrenal growth. We have synthesized the human N-terminal POMC fragment 1-28-POMC with the disulfide bridges in the correct position between cysteine residues 2-24 and 8-20 and studied the activity of these peptides in adrenocortical tumor cells in vitro. 1-28-POMC stimulated cell proliferation in human NCI-h295 and mouse Y-1 adrenal cancer cell lines and also in primary cultures of bovine adrenocortical cells in a concentration-dependent manner. 1-28-POMC led to rapid activation of the MAPKs extracellular signal-regulated kinases-1 and -2, but not c-Jun N-terminal kinase and p38, pathways. Steroid hormone production (cortisol, 17-hydroxyprogesterone, and dehydroepiandrosterone sulfate) in NCI-h295 cells was decreased by 1-28-POMC in a concentration-dependent fashion. However, protein levels of important regulators of steroidogenesis [steroidogenic factor-1, DAX-1 (dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X-chromosome 1), steroidogenic acute regulatory protein, and cytochrome P450 side-chain cleavage enzyme] remained unaffected by 1-28-POMC treatment. Our results provide evidence that synthetic 1-28-POMC induces adrenal tumor cell proliferation, inhibits adrenal steroidogenesis, and mediates its action by signaling via the extracellular signal-regulated kinase pathway. The distinct roles of 1-28-POMC and ACTH in the regulation of adrenal growth and steroidogenesis suggest that the adrenal cortex is under the dual opposing control of fragments from the same mother peptide POMC.