Endocrine Related Cancer current issue

Emerging roles of chemokines in prostate cancer

Vindrieux, D., Escobar, P., Lazennec, G. — Thu, 20 Aug 2009 07:48:39 PDT

Prostate cancer (PCa) represents the second leading cause of death among all cancer types in men in Europe and North America. Among the factors suspected to control PCa, incidence and progression, chemokines, and their receptors are now intensively studied. Chemokines are produced by tumor cells and also by the stromal microenvironment, both in the primary tumor site and in distant metastatic locations. The wide and differential distribution of chemokines and their receptors account for the pleiotropic actions of chemokines in PCa, including the modulation of growth, angiogenesis, invasion, metastasis, and hormone escape. This review will focus on the roles and the mechanisms of action and regulation of chemokines in the different steps of PCa development and will discuss the novel strategies that are currently envisioned to target chemokines in PCa.

Target-based therapies in breast cancer: current status and future perspectives

Thu, 20 Aug 2009 07:48:39 PDT

Identification of molecular alterations in key proteins involved in breast cancer cell proliferation and survival resulted in the development of a new treatment strategy with target-based agents. The anti-ErbB-2 monoclonal antibody (mAb) trastuzumab and the dual epidermal growth factor receptor/ErbB-2 tyrosine kinase inhibitor lapatinib are effective in patients with breast cancer that overexpresses ErbB-2. The anti-vascular endothelial growth factor-A mAb bevacizumab is approved in combination with taxanes for treatment of unselected patients with metastatic breast cancer. In addition, preclinical data suggest that signaling inhibitors can prevent or overcome resistance to endocrine therapy in estrogen receptor positive (ER+) breast cancer. However, the majority of signaling inhibitors explored in breast cancer patients has shown little activity, at least when used as monotherapy; and the results of clinical trials in ER+ breast cancer of combinations of signaling inhibitors and endocrine therapies are rather disappointing. Negative findings are likely due to mechanisms of intrinsic or acquired resistance to target-based agents. Breast carcinoma is a complex and heterogeneous disease and several different molecular alterations are involved in its pathogenesis and progression. The redundancy of oncogenic pathways activated in cancer cells, the heterogeneity of the mechanisms of resistance, and the plasticity of tumor cells that are capable to adapt to different growth conditions, significantly hamper the efficacy of each signaling inhibitor in breast cancer. Therefore, a comprehensive approach that takes into account the complexity of the disease is definitely required to improve the efficacy of target-based therapy in breast cancer.

Mechanisms of bone metastases of breast cancer

Suva, L. J, Griffin, R. J, Makhoul, I. — Thu, 20 Aug 2009 07:48:39 PDT

Cancer development is a multi-step process driven by genetic alterations that elicit the progressive transformation of normal human cells into highly malignant derivatives. The altered cell proliferation phenotype of cancer involves a poorly characterized sequence of molecular events, which often result in the development of distant metastasis. In the case of breast cancer, the skeleton is among the most common of metastatic sites. In spite of its clinical importance, the underlying cellular and molecular mechanisms driving bone metastasis remain elusive. Despite advances in our understanding of the phenotype of cancer cells, the increased focus on the contribution of the tumor microenvironment and the recent revival of interest in the role of tumor-propagating cells (so called cancer stem cells) that may originate or be related to normal stem cells produced in the bone marrow, many important questions remain unanswered. As such, a more complete understanding of the influences of both the microenvironment and the tumor phenotype, which impact the entire multi-step metastatic cascade, is required. In this review, the importance of tumor heterogeneity, tumor-propagating cells, the microenvironment of breast cancer metastasis to bone as well as many current endocrine therapies for the prevention and treatment of metastatic breast cancer is discussed.

New therapeutic advances in the management of progressive thyroid cancer

Woyach, J. A, Shah, M. H — Thu, 20 Aug 2009 07:48:39 PDT

The spectrum of thyroid cancers ranges from one of the most indolent to one of the most aggressive solid tumors identified. Conventional therapies for thyroid cancers are based on the histologic type of thyroid cancers such as papillary or follicular thyroid cancer (differentiated thyroid cancer (DTC)), medullary thyroid cancer (MTC), or anaplastic thyroid cancer (ATC). While surgery is one of the key treatments for all such types of thyroid cancers, additional therapies vary. Effective targeted therapy for DTC is a decades-old practice with systemic therapies of thyroid stimulating hormone suppression and radioactive iodine therapy. However, for the iodine-refractory DTC, MTC, and ATC there is no effective systemic standard of care treatment. Recent advances in understanding pathogenesis of DTC and development of molecular targeted therapy have dramatically transformed the field of clinical research in thyroid cancer. Over the last five years, incredible progress has been made and phases I–III clinical trials have been conducted in various types of thyroid cancers with some remarkable results that has made an impact on lives of patients with thyroid cancer. Such history-making events have boosted enthusiasm and interest among researchers, clinicians, patients, and sponsors and we anticipate ongoing efforts to develop more effective and safe therapies for thyroid cancer.

Radiation-induced hypopituitarism

Fernandez, A., Brada, M., Zabuliene, L., Karavitaki, N., Wass, J. A H — Thu, 20 Aug 2009 07:48:39 PDT

The hypothalamic–pituitary unit is a particularly radiosensitive region in the central nervous system. As a consequence, hypopituitarism commonly develops after radiation treatments for sellar and parasellar neoplasms, extrasellar brain tumours, head and neck tumours, and following whole body irradiation for systemic malignancies. Increasing tumour-related survival rates provide an expanding population at risk of developing hypopituitarism. In this population, long-term monitoring tailored to the individual risk profile is required to avoid the sequelae of untreated pituitary hormonal deficiencies and resultant decrease in the quality of life. This review analyses the pathogenesis, prevalence and consequences of radiation-induced hypopituitarism (RIH) in diverse subgroups at risk. Also discussed is the impact of modern radiotherapy techniques in the prevalence of RIH, the spectrum of endocrine disorders and radiation-induced brain conditions that also occur in patients with RIH.

Mouse models of altered protein kinase A signaling

Kirschner, L. S, Yin, Z., Jones, G. N, Mahoney, E. — Thu, 20 Aug 2009 07:48:40 PDT

Protein kinase A (PKA) is an evolutionarily conserved protein which has been studied in model organisms from yeast to man. Although the cAMP–PKA signaling system was the first mammalian second messenger system to be characterized, many aspects of this pathway are still not well understood. Owing to findings over the past decade implicating PKA signaling in endocrine (and other) tumorigenesis, there has been renewed interest in understanding the role of this pathway in physiology, particularly as it pertains to the endocrine system. Because of the availability of genetic tools, mouse modeling has become the pre-eminent system for studying the physiological role of specific genes and gene families as a means to understanding their relationship to human diseases. In this review, we will summarize the current data regarding mouse models that have targeted the PKA signaling system. These data have led to a better understanding of both the complexity and the subtlety of PKA signaling, and point the way for future studies, which may help to modulate this pathway for therapeutic effect.

Novel mechanism of IGF-binding protein-3 action on prostate cancer cells: inhibition of proliferation, adhesion, and motility

Thu, 20 Aug 2009 07:48:40 PDT

IGF-binding protein-3 (IGFBP-3) is a modulator of the IGF-signaling pathway and was described as an anti-cancer agent in prostate cancer. The molecular mechanisms underlying these effects remained, however, largely undefined. We analyzed the influence of recombinant IGFBP-3 on cell proliferation of PC3, Du145, and LNCaP prostate cancer cells. As expected, IGFBP-3 inhibited IGF-stimulated cell proliferation by blocking IGF-mediated proliferation signals, but we observed an IGF-independent inhibitory effect of IGFBP-3 on prostate cancer cell proliferation in long-term cultures. We further investigated the influence of IGFBP-3 on adhesion, motility, and invasion of prostate cancer cells using adhesion assays, live-cell imaging techniques, and matrigel invasion measurements. There was a clear inhibitory effect of IGFBP-3 on tumor cell adhesion to extracellular matrix components in the presence and absence of IGF, whereas cell–cell adhesion was not affected. The same inhibitory effect of IGFBP-3 was determined on cell motility when real-time cell movements were followed. In addition, IGFBP-3 was able to inhibit tumor cell invasion through matrigel. In summary, we show that IGFBP-3 inhibits proliferation, adhesion, migration, and invasion processes of prostate tumor cells. These newly described mechanisms of IGFBP-3 can be of importance for tumor progression and support a role of IGFBP-3 in therapeutic settings.

Regulation of thrombospondin-1 by natural and synthetic progestins in human breast cancer cells

Hyder, S. M, Liang, Y., Wu, J., Welbern, V. — Thu, 20 Aug 2009 07:48:40 PDT

Our recent studies show that progestins induce vascular endothelial growth factor (VEGF) in breast cancer cells that express mutant p53 protein. Here, we show that natural and synthetic progestins also induce thrombospondin-1 (TSP-1) mRNA and protein in T47-D and BT-474 breast cancer cells. Antiprogestin RU-486 inhibits the induction of VEGF and TSP-1 by progestins, suggesting that this effect of progestin is mediated by the progesterone receptor (PR). Actinomycin-D, but not puromycin, also blocks progestin-dependent induction of TSP-1. A putative progestin-response element was identified in the human TSP-1 promoter, which is consistent with the hypothesis that a progestin–PR complex might directly regulate transcription of the TSP-1 gene in human cells. Conditioned medium from progestin-treated breast cancer cells stimulates endothelial cell proliferation in the absence though not in the presence of antibody to TSP-1, indicating that TSP-1 secreted by breast cancer cells could be pro-angiogenic. Since tumor cell-derived TSP-1 has the potential to promote angiogenesis in the tumor microenvironment, it could be a potential target for breast cancer therapy.

Estrogen regulation of vascular endothelial growth factor in breast cancer in vitro and in vivo: the role of estrogen receptor {alpha} and c-Myc

Thu, 20 Aug 2009 07:48:40 PDT

The role of c-Myc in estrogen regulation of vascular endothelial growth factor (VEGF) and of the vasculature function has been investigated in breast cancer cells and tumors. The studies were performed on MCF7 wild-type cells and MCF7-35im clone, stably transfected with an inducible c-Myc gene. In vitro and ex vivo methods for investigating molecular events were integrated with in vivo magnetic resonance imaging of the vascular function. The results showed that the c-Myc upregulation by estrogen is necessary for the transient induction of VEGF transcription; however, overexpression of c-Myc alone is not sufficient for this induction. Furthermore, both c-Myc and the activated estrogen receptor (ER) were shown to co-bind the VEGF promoter in close proximity, indicating a novel mechanism for estrogen regulation of VEGF. Studies of long-term estrogen treatment and overexpression of c-Myc alone demonstrated regulation of stable VEGF expression levels in vitro and in vivo, maintaining steady vascular permeability in tumors. However, withdrawal of estrogen from the tumors resulted in increased VEGF and elevated vascular permeability, presumably due to hypoxic conditions that were found to dominate VEGF overexpression in cultured cells. This work revealed a cooperative role for ER and c-Myc in estrogen regulation of VEGF and the ability of c-Myc to partially mimic estrogen regulation of angiogenesis. It also illuminated the differences in estrogen regulation of VEGF during transient and long-term sustained treatments and under different microenvironmental conditions, providing a complementary picture of the in vitro and in vivo results.

The aryl hydrocarbon receptor as a target for estrogen receptor-negative breast cancer chemotherapy

Thu, 20 Aug 2009 07:48:40 PDT

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and the relatively non-toxic selective aryl hydrocarbon receptor (AhR) modulator 6-methyl-1,3,8-trichlorodibenzo-furan (MCDF) induced CYP1A1-dependent ethoxyresorufin O-deethylase activity and inhibited proliferation of seven estrogen receptor (ER) negative breast cancer cell lines. MCDF, TCDD and structurally related 2,3,7,8-tetrachlorodibenzofuran, 1,2,3,7,8-pentachlorodibenzo-p-dioxin, 2,3,4,7,8-pentachlorodibenzofuran, and 3,3',4,4',5-pentachlorobiphenyl induced CYP1A1 and inhibited proliferation of BT-474 and MDA-MB-468 cells. In BT474 and MDA-MB-468 cells transfected with a small inhibitory RNA for the AhR, the antiproliferative activity of the chlorinated aromatic compounds was reversed, whereas for MCDF, only partial reversal was observed, suggesting that this compound acts through both AhR-dependent and AhR-independent pathways in these two cell lines. MCDF also inhibited tumor growth in athymic nude mice in which MDA-MB-468 cells were injected directly into the mammary fat pad. These results suggest that the AhR is a potential drug target for treatment of ER-negative breast cancer.

Hydrogen peroxide induces DNA single- and double-strand breaks in thyroid cells and is therefore a potential mutagen for this organ

Thu, 20 Aug 2009 07:48:40 PDT

DNA double-strand breaks (DSBs) are considered as one of the primary causes of cancer but their induction by hydrogen peroxide (H₂O₂) is still controversial. In this work, we studied whether the high levels of H₂O₂ produced in the thyroid to oxidize iodide could induce DNA modifications. Scores of DNA damage, in terms of strand breaks, were obtained by comet assay (alkaline condition for single-strand breaks (SSBs) and neutral condition for DSBs). We demonstrated that in a rat thyroid cell line (PCCl3), non-lethal concentrations of H₂O₂ (0.1–0.5 mmol/l) as well as irradiation (1–10 Gy) provoked a large number of SSBs (~2–3 times control DNA damage values) but also high levels of DSBs (1.2–2.3 times control DNA damage values). We confirmed the generation of DSBs in this cell line and also in human thyroid in primary culture and in pig thyroid slices by measuring phosphorylation of histone H2AX. l-Buthionine-sulfoximine, an agent that depletes cells of glutathione, decreased the threshold to observe H₂O₂-induced DNA damage. Moreover, we showed that DNA breaks induced by H₂O₂ were more slowly repaired than those induced by irradiation. In conclusion, H₂O₂ causes SSBs and DSBs in thyroid cells. DSBs are produced in amounts comparable with those observed after irradiation but with a slower repair. These data support the hypothesis that the generation of H₂O₂ in thyroid could also play a role in mutagenesis particularly in the case of antioxidant defense deficiency.

The role of AUF1 in thyroid carcinoma progression

Thu, 20 Aug 2009 07:48:40 PDT

AUF1/heterogeneous nuclear ribonucleoprotein D is an adenylate–uridylate-rich elements (AREs) -binding protein, which regulates the mRNA stability of many genes related to growth regulation, such as proto-oncogenes, growth factors, cytokines, and cell cycle-regulatory genes. Several studies demonstrated AUF1 involvement in the processes of apoptosis, tumorigenesis, and development by its interactions with ARE-bearing mRNAs. We report here that AUF1 may be involved in thyroid carcinoma progression. Investigations on thyroid tissues revealed that cytoplasmic expression of AUF1 in malignant tissues was increased when compared with benign thyroid tissues. In thyroid carcinoma cell lines, AUF1 was mostly detectable in the nucleus; however, in dividing cells, its increased production was also observed in the cytoplasm. We found AUF1 in complexes with ARE-bearing mRNAs, previously described to be crucial for proliferation and cell cycle of thyroid carcinoma. Total or exon-selective knockdown of AUF1 led to growth inhibition accompanied by induction of cell cycle inhibitors and decreased levels of cell cycle promoters. Our data demonstrate the existence of a complex network between AUF1 and mRNAs encoding proteins related to cell proliferation. AUF1 may control the balance between stabilizing and destabilizing effects, both of which are exerted on cell cycle machinery in thyroid carcinoma. Although we cannot exclude participation of other factors, thyroid carcinoma may recruit cytoplasmic AUF1 to disturb the stability of mRNAs encoding cyclin-dependent kinase inhibitors, leading to uncontrolled growth and progression of tumor cells. Thus, AUF1 may be considered as a new, additional marker for thyroid carcinoma.

Impact of connexin32 deletion on E7 or RET/PTC3 oncogene-driven growth and neoplastic transformation of the thyroid gland

Prost, G., Bernier-Valentin, F., Croset, M., Rousset, B. — Thu, 20 Aug 2009 07:48:40 PDT

Connexins (Cx) form gap junctions and allow direct cell-to-cell communication. Cx through gap junctions or by themselves play regulatory roles on cell growth and differentiation. Using genetically modified mice, we previously found that Cx32 acts as a down-regulator of growth in normal thyroid gland. In this study, we examined the impact of Cx32 ablation on oncogene-driven thyroid growth and neoplastic transformation. Cx32 knockout (Cx32-KO) mice were crossed with transgenic mice expressing, selectively in the thyroid gland, either the E7 or RET/PTC3 (RP3) oncogene. As already described, Cx32-KO mice had no detectable thyroid alteration in physiological conditions and mice expressing E7 or RP3 exhibited time-dependent thyroid hypertrophy and variable changes in expression of differentiation. The thyroid of E7 mice evolved towards a large colloid goitre whereas RP3 mice developed a hyperplastic thyroid of variable size, and the largest glands (about 40% of total) represented a profound tissue remodeling with proliferative papillary formations. E7-induced thyroid hypertrophy was reduced by about 40% in Cx32-KO mice as compared with wild-type (WT) littermates. On the contrary, thyroid hypertrophy induced by thyrotropin stimulation (in response to goitrogen treatment) was enhanced by about 40% in Cx32-KO mice as compared with WT mice. Thyroid hypertrophy of RP3 mice and the proportion of glands showing extensive tissue remodeling were drastically reduced in mice devoid of Cx32. Our data show that Cx32, which negatively controls thyroid growth activated by thyrotropin via the cAMP pathway, would act as a positive effector of thyroid growth triggered by oncogenes acting through other signaling cascades.

Midgut neuroendocrine tumours with liver metastases: results of the UKINETS study

Thu, 20 Aug 2009 07:48:40 PDT

We intended to identify the prognostic factors and the results of interventions on patients with liver metastatic midgut carcinoids. Five institutions that are part of United Kingdom and Ireland neuroendocrine tumour (NET) group took part in this study. Patients were included if they had histology proven NET of midgut origin and liver metastases at the time of the study. Clinical and biochemical data were collected retrospectively from hospital charts, pathology reports, radiology reports and biochemistry records for each patient. Three hundred and sixty patients were included in the study. The median survival from date of diagnosis was 7.69 years (confidence interval (CI) 6.40–8.99) and 5.95 years (CI 5.02–6.88) from date of diagnosis of liver metastases. On univariate analysis, increasing age at diagnosis, increasing urinary hydroxyindole acetic acid levels, increasing plasma chromogranin A levels, high Ki67, high tumour volume and treatment with chemotherapy were identified as factors associated with a significantly poorer outcome. Resection of liver metastases, resection of small bowel primary, treatment with somatostatin analogue therapy and treatment with peptide receptor therapy were associated with improved prognosis. Multivariate analysis revealed that age at diagnosis (P=0.014), Ki67 level (P=0.039) and resection of primary (P=0.015) were independent predictors of survival. This is the largest study to our knowledge looking specifically at the prognosis and clinical course of patients with liver metastatic midgut NETs. For the first time, we have shown that Ki67 and resection of primary are independent predictors of survival for this group of patients.

Integrative molecular bioinformatics study of human adrenocortical tumors: microRNA, tissue-specific target prediction, and pathway analysis

Thu, 20 Aug 2009 07:48:40 PDT

MicroRNAs (miRs) are involved in the pathogenesis of several neoplasms; however, there are no data on their expression patterns and possible roles in adrenocortical tumors. Our objective was to study adrenocortical tumors by an integrative bioinformatics analysis involving miR and transcriptomics profiling, pathway analysis, and a novel, tissue-specific miR target prediction approach. Thirty-six tissue samples including normal adrenocortical tissues, benign adenomas, and adrenocortical carcinomas (ACC) were studied by simultaneous miR and mRNA profiling. A novel data-processing software was used to identify all predicted miR–mRNA interactions retrieved from PicTar, TargetScan, and miRBase. Tissue-specific target prediction was achieved by filtering out mRNAs with undetectable expression and searching for mRNA targets with inverse expression alterations as their regulatory miRs. Target sets and significant microarray data were subjected to Ingenuity Pathway Analysis. Six miRs with significantly different expression were found. miR-184 and miR-503 showed significantly higher, whereas miR-511 and miR-214 showed significantly lower expression in ACCs than in other groups. Expression of miR-210 was significantly lower in cortisol-secreting adenomas than in ACCs. By calculating the difference between dCT_miR-511 and dCT_miR-503 (delta cycle threshold), ACCs could be distinguished from benign adenomas with high sensitivity and specificity. Pathway analysis revealed the possible involvement of G2/M checkpoint damage in ACC pathogenesis. To our knowledge, this is the first report describing miR expression patterns and pathway analysis in sporadic adrenocortical tumors. miR biomarkers may be helpful for the diagnosis of adrenocortical malignancy. This tissue-specific target prediction approach may be used in other tumors too.

Expression of excision repair cross complementing group 1 and prognosis in adrenocortical carcinoma patients treated with platinum-based chemotherapy

Thu, 20 Aug 2009 07:48:40 PDT

Therapeutic progress in adrenocortical carcinoma (ACC) is severely hampered by its low incidence. Platinum-based chemotherapies are the most effective cytotoxic treatment regimens in ACC but response rates remain <50%. In other tumor entities, expression of excision repair cross complementing group 1 (ERCC1) predicts resistance to platinum compounds. Therefore, we correlated ERCC1 protein expression and clinical outcome. We have retrolectively established adrenal tissue microarrays and analyzed prospectively samples from 163 ACCs, 15 benign adrenal adenomas, and 8 normal adrenal glands by immunohistochemistry for ERCC1 protein expression. Detailed clinical data were available by the German ACC Registry. ERCC1 protein was highly expressed in all normal adrenal glands, 14 benign tumors (93%) and in 75 ACCs (47%). In ACC, no differences in baseline parameters were found between patients with and without ERCC1 expression. Detection of ERCC1 was not correlated with survival in patients who never received platinum-based chemotherapy. In platinum-treated patients (n=45), objective response to platinum compounds was observed in 3/21 patients (14.3%) with high ERCC1 expression and in 7/24 patients (29.2%) with low ERCC1 expression (P=0.23). ERCC1 expression was strongly correlated with overall survival after platinum treatment (median: eight months in patients with high ERCC1 versus 24 months in low ERCC1 expression, hazard ratio (HR) 2.95 (95% confidence interval (CI) 1.4–6.2), P=0.004). Multivariate analysis confirmed that high ERCC1 expression was a predictive factor for poor prognosis in platinum treated patients (HR 2.2, 95% CI 1.0–4.5, P=0.038). Our findings suggest that ERCC1 expression is the first factor for predicting survival in ACC patients treated with platinum-based chemotherapy.

Glucose transporter GLUT1 expression is an stage-independent predictor of clinical outcome in adrenocortical carcinoma

Thu, 20 Aug 2009 07:48:40 PDT

Owing to the rarity of adrenocortical carcinoma (ACC) no prognostic markers have been established beyond stage and resection status. Accelerated glycolysis is a characteristic feature of cancer cells and in a variety of tumour entities key factors in glucose metabolism like glucose transporter 1 and 3 (GLUT1 and -3), transketolase like-1 enzyme (TKTL1) and pyruvate kinase type M2 (M2-PK) are overexpressed and of prognostic value. Therefore, we investigated the role of these factors in ACC. Immunohistochemical analysis was performed on tissue microarrays of paraffin-embedded tissue samples from 167 ACCs, 15 adrenal adenomas and 4 normal adrenal glands. Expression was correlated with baseline parameters and clinical outcome. GLUT1 and -3 were expressed in 33 and 17% of ACC samples respectively, but in none of the benign tumours or normal adrenals glands. By contrast, TKTL1 and M2-PK were detectable in all benign tissues and the vast majority of ACCs. GLUT1 expression was strongly associated with prognosis in univariate and multivariate analysis (P<0.01), whereas GLUT3, TKTL1 and M2-PK did not correlate with clinical outcome. Patients with strong GLUT1 staining showed a considerably higher overall mortality (hazard ratio (HR) 6.34 (95% confidence interval 3.10–12.90) compared with patients with no GLUT1 staining. When analysing patients in their early stages and advanced disease separately, similar results were obtained. HR for survival was 5.31 (1.80–15.62) in patients with metatastic ACC and in patients after radical resection the HR for disease-free survival was 6.10 (2.16–16.94). In conclusion, GLUT1 is a highly promising stage-independent, prognostic marker in ACC.

Molecular characterization of novel germline deletions affecting SDHD and SDHC in pheochromocytoma and paraganglioma patients

Thu, 20 Aug 2009 07:48:40 PDT

A major cause of paraganglioma and pheochromocytoma is germline mutation of the tumor suppressor genes SDHB, SDHC, and SDHD, encoding subunits of succinate dehydrogenase (SDH). While many SDH missense/nonsense mutations have been identified, few large deletions have been described. We performed multiplex ligation-dependent probe amplification deletion analysis in 126 point mutation-negative patients, and here we describe four novel deletions of SDHD and SDHC. Long-range PCR was used for the fine mapping of deletions. One patient had a 10 kb AluSg–AluSx-mediated deletion including SDHD exons 1 and 2, the entire TIMM8B gene, and deletion of exons of C11orf57. A second patient had a deletion of SDHD exons 1 and 2 and exon 1 of the TIMM8B gene. A third patient showed a deletion of exon 2 of SDHD, together with a 235 bp MIRb–Tensin gene insertion. In a fourth patient, a deletion of exons 5 and 6 of the SDHC gene was found, only the second SDHC deletion currently known. The deletions of the TIMM8B and C11orf57 genes are the first to be described, but do not appear to result in an additional phenotype in these patients. Four of the eight breakpoints occurred in Alu sequences and all three SDHD deletions showed an intron 2 breakpoint. This study underlines the fact that clinically relevant deletions may encompass neighboring genes, with the potential to modify phenotype. Gene deletions of SDHD and SDHC represent a substantial proportion of all mutations, and must be considered in paraganglioma patients shown to be negative for mutations by sequencing.

Hypermethylation of the IGF2 differentially methylated region 2 is a specific event in insulinomas leading to loss-of-imprinting and overexpression

Thu, 20 Aug 2009 07:48:40 PDT

Prediction of the evolution of endocrine pancreatic tumors remains difficult based on histological criteria alone. We have previously demonstrated that epigenetic changes are an early event in a mouse model developing insulinomas. Particularly, overexpression of the imprinted IGF2 was caused by the hypermethylation of CpGs in the differentially methylated region 2 (DMR2). Here, we investigated whether IGF2 hypermethylation is also observed in human insulinomas and whether this alteration is common to other human endocrine tumors of the pancreas and the digestive tract. We analyzed the methylation status of 40 CpGs located in the DMR0 and DMR2 of the IGF2 as well as in the H19 DMR by pyrosequencing in a cohort of 62 patients with pancreatic or small intestine endocrine tumors. Altered methylation patterns were observed in all tumor types for the different regions of IGF2, but not for H19. However, hypermethylation of the IGF2 DMR2 was specific for insulinomas and did not occur in any of the other types of tumors which were characterized by a loss of methylation in this region. Gain of methylation in the IGF2 DMR2 in insulinomas correlated with loss-of-imprinting and promoter 4 mediated overexpression of IGF2 at the RNA and protein level. Furthermore, a decreasing degree of methylation in the different regions of IGF2 correlated well with increasing degree of malignancy according to the WHO classification of pancreatic endocrine tumors (PETs), suggesting that methylation of IGF2 might be a useful biomarker for classification and staging of PETs.

High-resolution genomic profiling reveals gain of chromosome 14 as a predictor of poor outcome in ileal carcinoids

Andersson, E., Sward, C., Stenman, G., Ahlman, H., Nilsson, O. — Thu, 20 Aug 2009 07:48:40 PDT

Ileal carcinoids are malignant neuroendocrine tumours of the small intestine. The aim of this study was to obtain a high-resolution genomic profile of ileal carcinoids in order to define genetic changes important for tumour initiation, progression and survival. Forty-three patients with ileal carcinoids were investigated by high-resolution array-based comparative genomic hybridization. The average number of copy number alterations (CNAs) per tumour was 7.1 (range 1–22), with losses being more common than gains (ratio 1.4). The most frequent CNA was loss of chromosome 18 (74%). Other frequent CNAs were gain of chromosome 4, 5, 14 and 20, and loss of 11q22.1–q22.2, 11q22.3–q23.1 and 11q23.3, and loss of 16q12.2–q22.1 and 16q23.2-qter. Two distinct patterns of CNAs were found; the majority of tumours was characterized by loss of chromosome 18 while a subgroup of tumours had intact chromosome 18, but gain of chromosome 14. Survival analysis, using a series of Poisson regressions including recurrent CNAs, demonstrated that gain of chromosome 14 was a strong predictor of poor survival. In conclusion, high-resolution profiling demonstrated two separate patterns of CNAs in ileal carcinoids. The majority of tumours showed loss of chromosome 18, which most likely represents a primary event in the development and pathogenesis of tumours. A different genetic pathway is operative in a subgroup of tumours; this is characterized by gain of chromosome 14 and is strongly associated with poor prognosis. Predictive fluorescence in situ hybridization analysis of chromosome 14 status in patients with ileal carcinoids is suggested.

Circulating angiopoietin-2 is elevated in patients with neuroendocrine tumours and correlates with disease burden and prognosis

Srirajaskanthan, R, Dancey, G, Hackshaw, A, Luong, T, Caplin, M E, Meyer, T — Thu, 20 Aug 2009 07:48:40 PDT

Angiogenesis is an essential process in the development and growth of tumours. There are a large number of angiogenic mediators including the angiopoietin (Ang) family and vascular endothelial growth factor, which play an important role in both physiological and pathological angiogenesis. This study examines serum levels of Ang-1 and Ang-2 in patients with neuroendocrine tumour (NET) compared healthy controls. ELISA for Ang-1 and Ang-2 was performed in 47 patients with histologically proven NETs and 44 healthy controls. Immunohistochemical staining for Ang-2 was performed in patients to demonstrate cellular location of Ang-2. Serum Ang-2 levels were significantly elevated in patients compared controls (median 4756 vs 2495 pg/ml, P<0.001), while there was no significant difference in Ang-1 levels. The ratio of Ang-2:Ang-1 was significantly elevated in patients compared controls (0.13 vs 0.066, P<0.001). Serum Ang-2 levels were significantly elevated in patients with distant metastases compared with those without metastasis (median 5080 vs 3360 pg/ml, P=0.01). There was also a significant increase between Ang-2 levels and volume of liver metastases (P=0.014). Time to disease progression was worse in patients with serum Ang-2 levels >4756 pg/ml (P=0.04). Serum Ang-2 but not Ang-1 is elevated in NET patients. Ang-2 may be a useful serum marker for monitoring and assessment of prognosis in patients with NETs.

Clinical and imaging follow-up after exhaustive liver resection of endocrine metastases: a 15-year monocentric experience

Thu, 20 Aug 2009 07:48:40 PDT

Liver metastases are common in gastroenteropancreatic neuroendocrine tumors and significantly impair survival. Hepatic resection is the only potential curative treatment. The records of 41 consecutive patients undergoing exhaustive resection of liver-only endocrine metastases and followed between 1992 and 2006 were reviewed. Patient's outcome and diagnostic accuracy of somatostatin receptor scintigraphy (SRS) and morphological imaging (MI) for detection of recurrences during post-operative follow-up were assessed. All identified primary had been resected. MI studies including abdominal computed tomography (CT) and/or liver magnetic resonance imaging and thoracic CT if indicated were performed every 6 months; SRS timing was decided by referring clinician. Tumor recurrences were confirmed by pathology or subsequent imaging studies. The results of 136 MI and SRS examinations performed within a 30-day interval from each other were retrospectively compared. Median post-operative follow-up was 51 months (7–165). Recurrences developed in 32 patients (78%), mainly in the liver (n=24) after a median of 19 months (2–79). Five-year overall and disease-free survival rates were 79 and 3% respectively. For recurrence detection, sensitivity, specificity, and accuracy were 89, 94, and 91% for SRS, 68, 91, and 74% for MI respectively. In 11 out of 32 patients (34%), abdominal or extra-abdominal metastases were detected 15.5 months earlier by SRS than MI. In conclusion, despite exhaustive liver surgery for endocrine metastases, hepatic or extra-hepatic recurrences are frequent and develop early. SRS is highly accurate for the detection of recurrences during post-operative follow-up and permitted early diagnosis in one third of patients; therapeutic implications of this early diagnosis remain to be determined.

Activation of a prometastatic gene expression program in hypoxic neuroblastoma cells

Poomthavorn, P., Wong, S. H X, Higgins, S., Werther, G. A, Russo, V. C — Thu, 20 Aug 2009 07:48:40 PDT

The hypoxia inducible factor-1 (HIF1) is a key regulator of oxygen homeostasis, modulating cell survival, and growth in cells exposed to hypoxia. In this study, neuroblastoma (NB) cells SH-SY5Y and SK-N-MC were employed to determine the mechanisms regulating adaptation to hypoxia. NB cells were cultured in a serum-free medium in the presence or absence of CoCl₂ (100 µM, hypoxia mimic) for up to 48 h. SH-SY5Y and SK-N-MC cell numbers were not affected by CoCl₂ treatment, while mitochondrial activity was reduced by ~50% in SH-SY5Y cells and by ~70% in SK-N-MC cells. Intracellular accumulation of HIF1 protein was detected as early as 30 min of post-hypoxia, followed by the increase of mRNA for vascular endothelial growth factor (VEGF) and nuclear accumulation of the ID1–2 transcription factors by 4 h. In hypoxic SH-SY5Y NB cells, real-time PCR analysis showed that the genes involved in maintenance of cell–cell and cell–matrix interactions (i.e. adenomatosis polyposis coli, E-cadherin, catenin, EphB2, fibronectin-1, HTATIP2, tissue inhibitor of metalloprotease-4) were down-regulated by up to 90%, while genes involved in enhancement of metastatic behavior (integrin a7b1, hepatocyte growth factor receptor, transforming growth factor-β1, VEGF, kisspeptin, interleukin-1β) were dramatically up-regulated above 200%. These changes were all consistent with the induction of epithelial–mesenchymal transition. We have thus demonstrated that NB cell adaptation to hypoxia, in addition to the modulation of HIF1 and VEGF expression and nuclear translocation of ID1 and ID2 transcription factors, involve in the activation of a gene expression program consistent with the pro-metastatic events. These processes are probably responsible for the NB cell transition from an adherent phenotype to a highly migratory, invasive and aggressive NB cell type.

Androgen, progesterone, and FSH receptor polymorphisms in ovarian cancer risk and outcome

Ludwig, A. H., Murawska, M., Panek, G., Timorek, A., Kupryjanczyk, J. — Thu, 20 Aug 2009 07:48:40 PDT

Genes encoding hormone receptors are among candidate genes modulating the risk of ovarian cancer. We aimed to assess a frequency of PGRG+331A, FSHRAla307Thr, and FSHRSer680Asn polymorphic variants, and the length of (CAG)n and (GGN)n repeat tracts in the androgen receptor gene (AR) with respect to ovarian cancer risk and outcome. We genotyped 215 ovarian cancer patients and 352 unaffected control subjects. Statistical analysis was performed with the logistic regression model with adjustment for age. Clinical importance of the polymorphic variants was evaluated in multivariate models on 69 patients treated with taxane–platinum chemotherapy, with respect to TP53 status. Longer AR (GGN)n and (CAG)n repeat tracts decreased the risk of ovarian cancer. For (GGN)n, each additional repeat decreased the risk by 17% (P=0.011) or 27% (P=0.002), while the presence of at least 23 repeats decreased the risk by 41% (P=0.032) or 68% (P=0.008), for the shorter or longer allele respectively. The risk of disease was also decreased by 11% with each additional (CAG)n repeat (P=0.006 for the longer allele). FSHRAla307Ala or FSHRSer680Ser polymorphisms increased ovarian cancer risk by 1.8 times (P=0.042). In all 69 patients, longer AR (CAG)n repeats decreased the risk of recurrence (P=0.031). In the group with TP53 accumulation, longer AR (CAG)n repeats decreased the risk of recurrence (P=0.003) and death (P=0.03), while the FSHRSer680Ser polymorphism increased the risk of recurrence (P=0.037). Progesterone receptor polymorphisms analyzed did not show any associations. Our results support both the androgen and gonadotropin hypotheses of ovarian cancer development.

Mammalian target of rapamycin inhibitors rapamycin and RAD001 (everolimus) induce anti-proliferative effects in GH-secreting pituitary tumor cells in vitro

Thu, 20 Aug 2009 07:48:40 PDT

The effect of mammalian target of rapamycin (mTOR) inhibitors on pituitary tumors is unknown. Akt overexpression was demonstrated in pituitary adenomas, which may render them sensitive to the anti-proliferative effects of these drugs. The objective of the study was to evaluate the anti-proliferative efficacy of the mTOR inhibitor, rapamycin, and its orally bioavailable analog RAD001 on the GH-secreting pituitary tumor GH3 and MtT/S cells and in human GH-secreting pituitary adenomas (GH-omas) in primary cell cultures. Treatment with rapamycin or RAD001 significantly decreased the number of viable cells and cell proliferation in a dose- and time-dependent manner. This was reflected by decreased phosphorylation levels of the downstream mTOR target p70S6K. Rapamycin treatment of GH3 cells induced G0/G1 cell cycle arrest. In other tumor cell types, this was attributed to a decrease in cyclin D1 levels. However, rapamycin did not affect cyclin D1 protein levels in GH3 cells. By contrast, it decreased cyclin D3 and p21/CIP, which stabilizes cyclin D/cyclin-dependent kinase 4 (cdk4) complexes. Rapamycin inhibited FCS-induced retinoblastoma phosphorylation and subsequent E2F-transcriptional activity. In response to decreased E2F activity, the expression of the E2F-regulated genes cyclin E and cdk2 was reduced. Our results showed that mTOR inhibitors potently inhibit pituitary cell proliferation, suggesting that mTOR inhibition may be a promising anti-proliferative therapy for pituitary adenomas. This therapeutic manipulation may have beneficial effects particularly for patients harboring invasive pituitary tumors resistant to current treatments.

Expression of aryl hydrocarbon receptor (AHR) and AHR-interacting protein in pituitary adenomas: pathological and clinical implications

Thu, 20 Aug 2009 07:48:40 PDT

Germline mutations of the aryl hydrocarbon receptor (AHR)-interacting protein (AIP) gene confer a predisposition to pituitary adenomas (PA), usually in the setting of familial isolated PA. To provide further insights into the possible role of AIP in pituitary tumour pathogenesis, the expression of AIP and AHR was determined by real-time RT-PCR and/or immunohistochemistry (IHC) in a large series of PA (n=103), including 17 with AIP mutations (AIP^mut). Variable levels of AIP and AHR transcripts were detected in all PA, with a low AHR expression (P<0.0001 versus AIP). Cytoplasmic AIP and AHR were detected by IHC in 84.0 and 38.6% of PA respectively, and significantly correlated with each other (P=0.006). Nuclear AHR was detected in a minority of PA (19.7%). The highest AIP expression was observed in somatotrophinomas and non-secreting (NS) PA, and multivariate analysis in somatotrophinomas showed a significantly lower AIP immunostaining in invasive versus non-invasive cases (P=0.019). AIP expression was commonly low in other secreting PA. AIP immunostaining was abolished in a minority of AIP^mut PA, with a frequent loss of cytoplasmic AHR and no evidence of nuclear AHR. In contrast, AIP overexpression in a subset of NS PA could be accompanied by nuclear AHR immunopositivity. We conclude that down-regulation of AIP and AHR may be involved in the aggressiveness of somatotrophinomas. Overall, IHC is a poorly sensitive tool for the screening of AIP mutations. Data obtained on AHR expression suggest that AHR signalling may be differentially affected according to PA phenotype.

Lanreotide promotes apoptosis and is not radioprotective in GH3 cells

Ning, S., Knox, S. J, Harsh, G. R, Culler, M. D, Katznelson, L. — Thu, 20 Aug 2009 07:48:40 PDT

Somatostatin analogs are a mainstay of medical therapy in patients with GH producing human pituitary tumors, and it has been suggested that somatostatin analogs may be radioprotective. We utilized GH secreting rat GH3 cells to investigate whether a somatostatin analog may limit the effects of radiation on proliferation and apoptosis in vitro and on tumor growth in vivo. Treatment with lanreotide alone at doses of either 100 or 1000 nM for 48 h reduced clonogenic survival by 5–10%. Radiation alone produced a dose-dependent survival curve with a SF2 of 48–55%, and lanreotide had no effect on this curve. The addition of lanreotide resulted in a 23% increase in the proportion of apoptotic sub-G1 cells following irradiation (P<0.01). In a mouse GH3 tumor xenograft model, lanreotide 10 mg/kg moderately inhibited the growth of GH3 tumors, with a 4x tumor growth delay (TGD) time that ranged from 4.5 to 8.3 days. Fractionated local tumor radiation alone significantly inhibited tumor growth and produced a TGD of 35.1±5.7 days for 250 cGy fractions. The combination of lanreotide, either antecedent to or concurrent, with radiation of 250, 200 or 150 cGy/fraction for 5 days inhibited tumor growth and produced the TGD times that were similar to radiation alone (P>0.05). Pretreatment with lanreotide had the most significant radiosensitizing effect. These studies demonstrate that the somatostatin analog lanreotide is not radioprotective in GH3 cells, and further studies are necessary to determine the impact of lanreotide on apoptosis.

Genome-wide scan identifies novel modifier loci of acromegalic phenotypes for isolated familial somatotropinoma

Thu, 20 Aug 2009 07:48:40 PDT

Isolated familial somatotropinoma (IFS) accounts for 18% of familial isolated pituitary adenoma (FIPA) cases. Recently, germline mutations of the aryl hydrocarbon receptor-interacting protein gene (AIP) have been found in families with pituitary adenoma predisposition, FIPA, and IFS. In this study, we investigate the AIP mutation status and perform a genome-wide scan to search for the modifier regions of acromegalic phenotypes in an IFS family of 31 aborigines from Borneo. Complete endocrine diagnosis and data could not be collected due to logistical and cultural reasons. AIP mutation screening was carried out by direct sequencing and the genome-wide scan was performed using 400 microsatellites. Non-parametric linkage analysis was performed to obtain the logarithm of odds (LOD) scores. A novel AIP frameshift mutation in exon 4 (c.500delC) (p.P167HfsX3) was identified in all members with acromegalic features, as well as in 15 members without acromegalic features, revealing incomplete penetrance of AIP. The data showed that patients with the same mutation may express acromegalic features of differing severity, suggesting the existence of modifier genes. The highest LOD score of 2.2 was obtained near D19S571 (19q13.41). We also found weak linkages on chromosomes 3q28, 8q12.1, and 21q22.13, with LOD scores of 1.1, 1.8, and 1.4 respectively. Our results show the first genome-wide scan that identifies novel modifier loci for acromegalic phenotypes in an IFS family. Identification of modifier loci may provide further insight into the disease mechanism and explain the clinical variability observed in its patients.