Following their successful implementation for the treatment of metastatic breast cancer, the ‘third-generation’ aromatase inhibitors (anastrozole, letrozole, and exemestane) have now become standard adjuvant endocrine treatment for postmenopausal estrogen receptor-positive breast cancers. These drugs are characterized by potent aromatase inhibition, causing >98% inhibition of estrogen synthesis in vivo. A recent meta-analysis found no difference in anti-tumor efficacy between these three compounds. As of today, aromatase inhibitor monotherapy and sequential treatment using tamoxifen followed by an aromatase inhibitor for a total of 5 years are considered equipotent treatment options. However, current trials are addressing the potential benefit of extending treatment duration beyond 5 years. Regarding side effects, aromatase inhibitors are not found associated with enhanced risk of cardiovascular disease, and enhanced bone loss is prevented by adding bisphosphonates in concert for those at danger of developing osteoporosis. However, arthralgia and carpal tunnel syndrome preclude drug administration among a few patients. While recent findings have questioned the use of aromatase inhibitors among overweight and, in particular, obese patients, this problem seems to focus on premenopausal patients treated with an aromatase inhibitor and an LH-RH analog in concert, questioning the efficacy of LH-RH analogs rather than aromatase inhibitors among overweight patients. Finally, recent findings revealing a benefit from adding the mTOR inhibitor everolimus to endocrine treatment indicate targeted therapy against defined growth factor pathways to be a way forward, by reversing acquired resistance to endocrine therapy.
Per Eystein Lønning and Hans Petter Eikesdal
Stefano Caramuta, Linkiat Lee, Deniz M Özata, Pinar Akçakaya, Hong Xie, Anders Höög, Jan Zedenius, Martin Bäckdahl, Catharina Larsson, and Weng-Onn Lui
Deregulation of microRNA (miRNA) expression in adrenocortical carcinomas (ACCs) has been documented to have diagnostic, prognostic, as well as functional implications. Here, we evaluated the mRNA expression of DROSHA, DGCR8, DICER (DICER1), TARBP2, and PRKRA, the core components in the miRNA biogenesis pathway, in a cohort of 73 adrenocortical tumors (including 43 adenomas and 30 carcinomas) and nine normal adrenal cortices using a RT-qPCR approach. Our results show a significant over-expression of TARBP2, DICER, and DROSHA in the carcinomas compared with adenomas or adrenal cortices (P<0.001 for all comparisons). Using western blot and immunohistochemistry analyses, we confirmed the higher expression of TARBP2, DICER, and DROSHA at the protein level in carcinoma cases. Furthermore, we demonstrate that mRNA expression of TARBP2, but not DICER or DROSHA, is a strong molecular predictor to discriminate between adenomas and carcinomas. Functionally, we showed that inhibition of TARBP2 expression in human NCI-H295R ACC cells resulted in a decreased cell proliferation and induction of apoptosis. TARBP2 over-expression was not related to gene mutations; however, copy number gain of the TARBP2 gene was observed in 57% of the carcinomas analyzed. In addition, we identified that miR-195 and miR-497 could directly regulate TARBP2 and DICER expression in ACC cells. This is the first study to demonstrate the deregulation of miRNA-processing factors in adrenocortical tumors and to show the clinical and biological impact of TARBP2 over-expression in this tumor type.
Nimrod B Kiss, Andreas Muth, Adam Andreasson, C Christofer Juhlin, Janos Geli, Martin Bäckdahl, Anders Höög, Bo Wängberg, Ola Nilsson, Håkan Ahlman, and Catharina Larsson
Recurrent alterations in promoter methylation of tumor suppressor genes (TSGs) and LINE1 (L1RE1) repeat elements were previously reported in pheochromocytoma and abdominal paraganglioma. This study was undertaken to explore CpG methylation abnormalities in an extended tumor panel and assess possible relationships between metastatic disease and mutation status. CpG methylation was quantified by bisulfite pyrosequencing for selected TSG promoters and LINE1 repeats. Methylation indices above normal reference were observed for DCR2 (TNFRSF10D), CDH1, P16 (CDKN2A), RARB, and RASSF1A. Z-scores for overall TSG, and individual TSG methylation levels, but not LINE1, were significantly correlated with metastatic disease, paraganglioma, disease predisposition, or outcome. Most strikingly, P16 hypermethylation was strongly associated with SDHB mutation as opposed to RET/MEN2, VHL/VHL, or NF1-related disease. Parallel analyses of constitutional, tumor, and metastasis DNA implicate an order of events where constitutional SDHB mutations are followed by TSG hypermethylation and 1p loss in primary tumors, later transferred to metastatic tissue. In the combined material, P16 hypermethylation was prevalent in SDHB-mutated samples and was associated with short disease-related survival. The findings verify the previously reported importance of P16 and other TSG hypermethylation in an independent tumor series. Furthermore, a constitutional SDHB mutation is proposed to predispose for an epigenetic tumor phenotype occurring before the emanation of clinically recognized malignancy.
Colette Meyer, Andrew H Sims, Kevin Morgan, Beth Harrison, Morwenna Muir, Jianing Bai, Dana Faratian, Robert P Millar, and Simon P Langdon
GNRH significantly inhibits proliferation of a proportion of cancer cell lines by activating GNRH receptor (GNRHR)-G protein signaling. Therefore, manipulation of GNRHR signaling may have an under-utilized role in treating certain breast and ovarian cancers. However, the precise signaling pathways necessary for the effect and the features of cellular responses remain poorly defined. We used transcriptomic and proteomic profiling approaches to characterize the effects of GNRHR activation in sensitive cells (HEK293-GNRHR, SCL60) in vitro and in vivo, compared to unresponsive HEK293. Analyses of gene expression demonstrated a dynamic response to the GNRH superagonist Triptorelin. Early and mid-phase changes (0.5–1.0 h) comprised mainly transcription factors. Later changes (8–24 h) included a GNRH target gene, CGA, and up- or downregulation of transcripts encoding signaling and cell division machinery. Pathway analysis identified altered MAPK and cell cycle pathways, consistent with occurrence of G2/M arrest and apoptosis. Nuclear factor kappa B (NF-κB) pathway gene transcripts were differentially expressed between control and Triptorelin-treated SCL60 cultures. Reverse-phase protein and phospho-proteomic array analyses profiled responses in cultured cells and SCL60 xenografts in vivo during Triptorelin anti-proliferation. Increased phosphorylated NF-κB (p65) occurred in SCL60 in vitro, and p-NF-κB and IκBε were higher in treated xenografts than controls after 4 days Triptorelin. NF-κB inhibition enhanced the anti-proliferative effect of Triptorelin in SCL60 cultures. This study reveals details of pathways interacting with intense GNRHR signaling, identifies potential anti-proliferative target genes, and implicates the NF-κB survival pathway as a node for enhancing GNRH agonist-induced anti-proliferation.
Christine M Friedenreich, Annie R Langley, Thomas P Speidel, David C W Lau, Kerry S Courneya, Ilona Csizmadi, Anthony M Magliocco, Yutaka Yasui, and Linda S Cook
Markers of insulin resistance such as the adiponectin:leptin ratio (A:L) and the homeostasis model assessment ratio (HOMA-IR) are associated with obesity and hyperinsulinemia, both established risk factors for endometrial cancer, and may therefore be informative regarding endometrial cancer risk. This study investigated the association between endometrial cancer risk and markers of insulin resistance, namely adiponectin, leptin, the A:L ratio, insulin, fasting glucose, and the HOMA-IR. We analyzed data from 541 incident endometrial cancer cases and 961 frequency age-matched controls in a population-based case–control study in Alberta, Canada from 2002 to 2006. Participants completed interview-administered questionnaires were assessed for anthropometric measures, and provided 8-h fasting blood samples either pre- or postoperatively. Blood was analyzed for concentrations of leptin, adiponectin, and insulin by immunoassay, and fasting plasma glucose levels were determined by fluorimetric quantitative determination. Compared with the lowest quartile, the highest quartile of insulin and HOMA-IR was associated with 64% (95% confidence intervals (CI): 1.12–2.40) and 72% (95% CI: 1.17–2.53) increased risks of endometrial cancer, respectively, and the highest quartile of adiponectin was associated with a 45% (95% CI: 0.37–0.80) decreased risk after multivariable adjustments. Null associations were observed between fasting glucose, leptin and A:L, and endometrial cancer risk. This population-based study provides evidence for a role of insulin resistance in endometrial cancer etiology and may provide one possible pathway whereby obesity increases the risk of this common cancer. Interventions aimed at decreasing both obesity and insulin resistance may decrease endometrial cancer risk.
Kristen K Rumer, Miriam D Post, Rhea S Larivee, Martina Zink, Jill Uyenishi, Anita Kramer, Deanna Teoh, Kevin Bogart, and Virginia D Winn
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.
Tsai-Der Chuang, Harekrushna Panda, Xiaoping Luo, and Nasser Chegini
MicroRNA-200c (miR-200c) through repression of specific target genes has been associated with cellular transition, tumorigenesis, and tissue fibrosis. We explored the expression and functional aspects of miR-200c in genesis of leiomyomas (LYO), benign uterine tumors with fibrotic characteristic. Using LYO and matched myometrium (MYO; n=76) from untreated and from patients exposed to hormonal therapies (GNRH agonist (GNRHa), Depo-Provera, and oral contraceptives), we found that miR-200c was expressed at significantly lower levels (P<0.05) in LYO as compared with MYO. These levels were lower in LYO from African Americans as compared with Caucasians, patients experiencing abnormal uterine bleeding and those exposed to GNRHa therapy. Gain-of-function of miR-200c in isolated leiomyoma smooth muscle cells (LSMCs), myometrial smooth muscle cells (MSMCs), and leiomyosarcoma cell line (SKLM-S1) repressed ZEB1/ZEB2 mRNAs and proteins, with concurrent increase in E-cadherin (CDH1) and reduction in vimentin expression, phenotypic alteration, and inhibition of MSMC and LSMC proliferations. We further validated TIMP2, FBLN5, and VEGFA as direct targets of miR-200c through interaction with their respective 3′ UTRs, and other genes as determined by microarray analysis. At tissue levels, LYO expressed lower levels of TIMP2 and FBLN5 mRNAs but increased protein expressions, which to some extent altered due to hormonal exposure. Given the regulatory functions of ZEBs, VEGFA, FBLN5, and TIMP2 on cellular activities that promote cellular transition, angiogenesis, and matrix remodeling, we concluded that altered expression of miR-200c may have a significant impact on the outcome of LYO growth, maintenance of their mesenchymal and fibrotic characteristics, and possibly their associated symptoms.
Luqman Sulaiman, Inga-Lena Nilsson, C Christofer Juhlin, Felix Haglund, Anders Höög, Catharina Larsson, and Jamileh Hashemi
In this study, we genetically characterized parathyroid adenomas with large glandular weights, for which independent observations suggest pronounced clinical manifestations. Large parathyroid adenomas (LPTAs) were defined as the 5% largest sporadic parathyroid adenomas identified among the 590 cases operated in our institution during 2005–2009. The LPTA group showed a higher relative number of male cases and significantly higher levels of total plasma and ionized serum calcium (P<0.001). Further analysis of 21 LPTAs revealed low MIB1 proliferation index (0.1–1.5%), MEN1 mutations in five cases, and one HRPT2 (CDC73) mutation. Total or partial loss of parafibromin expression was observed in ten tumors, two of which also showed loss of APC expression. Using array CGH, we demonstrated recurrent copy number alterations most frequently involving loss in 1p (29%), gain in 5 (38%), and loss in 11q (33%). Totally, 21 minimal overlapping regions were defined for losses in 1p, 7q, 9p, 11, and 15q and gains in 3q, 5, 7p, 8p, 16q, 17p, and 19q. In addition, 12 tumors showed gross alterations of entire or almost entire chromosomes most frequently gain of 5 and loss of chromosome 11. While gain of 5 was the most frequent alteration observed in LPTAs, it was only detected in a small proportion (4/58 cases, 7%) of parathyroid adenomas. A significant positive correlation was observed between parathyroid hormone level and total copy number gain (r=0.48, P=0.031). These results support that LPTAs represent a group of patients with pronounced parathyroid hyperfunction and associated with specific genomic features.
Su Jung Oh, Holger H H Erb, Alfred Hobisch, Frédéric R Santer, and Zoran Culig
Antihormonal and chemotherapy are standard treatments for nonorgan-confined prostate cancer. The effectivity of these therapies is limited and the development of alternative approaches is necessary. In the present study, we report on the use of the multikinase inhibitor sorafenib in a panel of prostate cancer cell lines and their derivatives which mimic endocrine and chemotherapy resistance. 3H-thymidine incorporation assays revealed that sorafenib causes a dose-dependent inhibition of proliferation of all cell lines associated with downregulation of cyclin-dependent kinase 2 and cyclin D1 expression. Apoptosis was induced at 2 μM of sorafenib in androgen-sensitive cells, whereas a higher dose of the drug was needed in castration-resistant cell lines. Sorafenib stimulated apoptosis in prostate cancer cell lines through downregulation of myeloid cell leukemia-1 (MCL-1) expression and Akt phosphorylation. Although concentrations of sorafenib required for the antitumor effect in therapy-resistant sublines were higher than those needed in parental cells, the drug showed efficacy in cells which became resistant to bicalutamide and docetaxel respectively. Most interestingly, we show that sorafenib has an inhibitory effect on androgen receptor (AR) and prostate-specific antigen expression. In cells in which AR expression was downregulated by short interfering RNA, the treatment with sorafenib increased apoptosis in an additive manner. In summary, the results of the present study indicate that there is a potential to use sorafenib in prostate cancers as an adjuvant therapy option to current androgen ablation treatments, but also in progressed prostate cancers that become unresponsive to standard therapies.
Evangelia-Ourania Fourkala, Alexey Zaikin, Matthew Burnell, Aleksandra Gentry-Maharaj, Jeremy Ford, Richard Gunu, Christina Soromani, Guido Hasenbrink, Ian Jacobs, Anne Dawnay, Martin Widschwendter, Hella Lichtenberg-Fraté, and Usha Menon
Postmenopausal women with elevated serum sex steroids have an increased risk of breast cancer. Most of this risk is believed to be exerted through binding of the sex steroids to their receptors. For the first time, we investigate the association of estrogen receptor (ER) and androgen receptor (AR) serum bioactivity (SB) in addition to hormone levels in samples from women with breast cancer collected before diagnosis. Two hundred postmenopausal women participating in the UK Collaborative Trial of Ovarian Cancer Screening who developed ER-positive breast cancer 0.6–5 years after sample donation were identified and matched to 400 controls. ER and AR bioassays were used to measure ERα, ERβ, and AR SB. Androgen and estrogen levels were measured with immunoassays. Subjects were classified according to quintiles of the respective marker among controls and the associations between SB and hormones with breast cancer risk were determined by logistic regression analysis. ERα and ERβ SB were significantly higher before diagnosis compared with controls, while estrogens showed no difference. Women had a twofold increased breast cancer risk if ERα SB (odds ratio (OR), 2.114; 95% confidence interval (CI), 1.050–4.425; P=0.040) was in the top quintile >2 years before diagnosis or estrone (OR, 2.205; 95% CI, 1.104–4.586; P=0.029) was in the top quintile <2 years before diagnosis. AR showed no significant association with breast cancer while androstenedione (OR, 3.187; 95% CI, 1.738–6.044; P=0.0003) and testosterone (OR, 2.145; 95% CI, 1.256–3.712; P=0.006) were significantly higher compared with controls and showed a strong association with an almost threefold increased breast cancer risk independent of time to diagnosis. This study provides further evidence on the association of androgens and estrogens with breast cancer. In addition, it reports that high ER but not AR SB is associated with increased breast risk >2 years before diagnosis.