Kate E Lines

miR‐135b‐ and miR‐146b‐dependent silencing of calcium‐sensing receptor expression in colorectal tumors

Studies have shown that the calcium‐sensing receptor (CaSR) mediates the antitumorigenic effects of calcium against colorectal cancer (CRC). Expression of the CaSR in colorectal tumors is often reduced. We have reported previously that silencing of CaSR in CRC is caused in part by methylation of CaSR promoter 2 and loss of histone acetylation. We investigated the impact of aberrant microRNA expression on loss of CaSR expression. A microarray study in two Caco‐2 subclones (Caco2/AQ and Caco2/15) that have similar genetic background, but different CaSR expression levels (Caco2/AQ expressing more CaSR than Caco2/15), identified 22 differentially expressed microRNAs that potentially target the CaSR. We validated these results by performing gain‐ and loss‐of‐function studies with the top candidates: miR‐9, miR‐27a, miR‐135b, and miR‐146b. Modulation of miR‐135b or miR‐146b expression by mimicking or inhibiting their expression regulated CaSR protein levels in two different colon cancer cell lines: Caco2/AQ (moderate endogenous CaSR expression) and HT29 (low endogenous CaSR levels). Inhibition of miR‐135b and miR‐146b expression led to high CaSR levels and significantly reduced proliferation. In samples of colorectal tumors we observed overexpression of miR‐135b and miR‐146b, and this correlated inversely with CaSR expression (miR‐135b: r = −0.684, p < 0.001 and miR‐146b: r = −0.448, p < 0.001), supporting our in vitro findings. We demonstrate that miR‐135b and miR‐146b target the CaSR and reduce its expression in colorectal tumors, reducing the antiproliferative and prodifferentiating actions of calcium. This provides a new approach for finding means to prevent CaSR loss, developing better treatment strategies for CRC.

Pasireotide Therapy of Multiple Endocrine Neoplasia Type 1-Associated Neuroendocrine Tumors in Female Mice Deleted for an Men1 Allele Improves Survival and Reduces Tumor Progression.

Pasireotide, a somatostatin analog, is reported to have anti-proliferative effects in neuroendocrine tumors (NETs). We therefore assessed the efficacy of pasireotide for treating pancreatic and pituitary NETs that develop in a mouse model of multiple endocrine neoplasia type 1 (MEN1). Men1+/− mice were treated from age 12 mo with 40 mg/kg pasireotide long-acting release formulation, or PBS, intramuscularly monthly for 9 mo. The Men1+/− mice had magnetic resonance imaging at 12 and 21 mo, and from 20 mo oral 5-bromo-2-deoxyuridine for 1 mo, to assess tumor development and proliferation, respectively. NETs were collected at age 21 mo, and proliferation and apoptosis assessed by immunohistochemistry and TUNEL assays, respectively. Pasireotide-treated Men1+/− mice had increased survival (pasireotide, 80.9% vs PBS, 65.2%; P < .05), with fewer mice developing pancreatic NETs (pasireotide, 86.9% vs PBS, 96.9%; P < .05) and smaller increases in pituitary NET volumes (pre-treated vs post-treated, 0.803 ± 0.058 mm3 vs 2.872 ± 0.728 mm3 [pasireotide] compared with 0.844 ± 0.066 mm3 vs 8.847 ±1.948 mm3 [PBS]; P < .01). In addition, pasireotide-treated mice had fewer pancreatic NETs compared with PBS-treated mice (2.36 ± 0.25 vs 3.72 ± 0.32, respectively; P < .001), with decreased proliferation in pancreatic NETs (pasireotide, 0.35 ± 0.03% vs PBS, 0.78 ± 0.08%; P < .0001) and pituitary NETs (pasireotide, 0.73 ±0.07% vs PBS, 1.81 ± 0.15%; P < .0001), but increased apoptosis in pancreatic NETs (pasireotide, 0.42 ± 0.05% vs PBS, 0.19 ± 0.03%; P < .001) and pituitary NETs (pasireotide, 14.75 ± 1.58% vs PBS, 2.35 ± 0.44%; P < .001). Thus, pasireotide increased survival and inhibited pancreatic and pituitary NET growth, thereby indicating its potential as an anti-proliferative and pro-apoptotic therapy.

Epigenetic pathway inhibitors represent potential drugs for treating pancreatic and bronchial neuroendocrine tumors.

Cancer is associated with alterations in epigenetic mechanisms such as histone modifications and methylation of DNA, and inhibitors targeting epigenetic mechanisms represent a novel class of anti-cancer drugs. Neuroendocrine tumors (NETs) of the pancreas (PNETs) and bronchus (BNETs), which may have 5-year survivals of <50% and as low as 5%, respectively, represent targets for such drugs, as >40% of PNETs and ~35% of BNETs have mutations of the multiple endocrine neoplasia type 1 (MEN1) gene, which encodes menin that modifies histones by interacting with histone methyltransferases. We assessed 9 inhibitors of epigenetic pathways, for their effects on proliferation, by CellTiter Blue assay, and apoptosis, by CaspaseGlo assay, using 1 PNET and 2 BNET cell lines. Two inhibitors, referred to as (+)-JQ1 (JQ1) and PFI-1, targeting the bromo and extra terminal (BET) protein family which bind acetylated histone residues, were most effective in decreasing proliferation (by 40–85%, P<0.001) and increasing apoptosis (by 2–3.6 fold, P<0.001) in all 3 NET cell lines. The anti-proliferative effects of JQ1 and PFI-1 remained present for at least 48 hours after removal of the compound. JQ1, but not PFI-1, had cell cycle effects, assessed by propidium iodide staining and flow cytometry, resulting in increased and decreased proportions of NET cells in G1, and S and G2 phases, respectively. RNA Sequencing analysis revealed that these JQ1 effects were associated with increased histone 2B expression, and likely mediated through altered activity of bromodomain-containing (Brd) proteins. Assessment of JQ1 in vivo, using a pancreatic beta cell-specific conditional Men1 knockout mouse model that develops PNETs, revealed that JQ1 significantly reduced proliferation (by ~50%, P<0.0005), assessed by bromodeoxyuridine incorporation, and increased apoptosis (by ~3 fold, P<0.0005), assessed by terminal deoxynucleotidyl transferase dUTP nick end labelling, of PNETs. Thus, our studies demonstrate that BET protein inhibitors may provide new treatments for NETs.

Animal models of pituitary neoplasia.

Pituitary neoplasias can occur as part of a complex inherited disorder, or more commonly as sporadic (non-familial) disease. Studies of the molecular and genetic mechanisms causing such pituitary tumours have identified dysregulation of >35 genes, with many revealed by studies in mice, rats and zebrafish. Strategies used to generate these animal models have included gene knockout, gene knockin and transgenic over-expression, as well as chemical mutagenesis and drug induction. These animal models provide an important resource for investigation of tissue-specific tumourigenic mechanisms, and evaluations of novel therapies, illustrated by studies into multiple endocrine neoplasia type 1 (MEN1), a hereditary syndrome in which ∼30% of patients develop pituitary adenomas. This review describes animal models of pituitary neoplasia that have been generated, together with some recent advances in gene editing technologies, and an illustration of the use of the Men1 mouse as a pre clinical model for evaluating novel therapies.

A MEN1 pancreatic neuroendocrine tumour mouse model under temporal control.

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disorder characterised by occurrence of parathyroid tumours and neuroendocrine tumours (NETs) of the pancreatic islets and anterior pituitary. The MEN1 gene, encoding menin, is a tumour suppressor, but its precise role in initiating in vivo tumourigenesis remains to be elucidated. The availability of a temporally controlled conditional MEN1 mouse model would greatly facilitate the study of such early tumourigenic events, and overcome the limitations of other MEN1 knockout models, in which menin is lost from conception or tumour development occurs asynchronously. To generate a temporally controlled conditional mouse model, we crossbred mice with the MEN1 gene floxed by LoxP sites (Men1L/L), and mice expressing tamoxifen-inducible Cre recombinase under the control of the rat insulin promoter (RIP2-CreER), to establish a pancreatic β-cell-specific NET model under temporal control (Men1L/L/RIP2-CreER). Men1L/L/RIP2-CreER mice aged ~3 months were given tamoxifen in the diet for 5 days, and pancreata harvested 2–2.5, 2.9–3.5 and 4.5–5.5 months later. Control mice did not express Cre and did not receive tamoxifen. Immunostaining of pancreata from tamoxifen-treated Men1L/L/RIP2-CreER mice, compared to control mice, showed at all ages: loss of menin in all islets; increased islet area (>4.2-fold); increased proliferation of insulin immunostaining β-cells (>2.3-fold) and decreased proliferation of glucagon immunostaining α-cells (>1.7-fold). There were no gender and apoptotic or proliferation differences, and extra-pancreatic tumours were not detected. Thus, we have established a mouse model (Men1L/L/RIP2-CreER) to study early events in the development of pancreatic β-cell NETs.

Molecular genetic advances in pituitary tumor development

Pituitary adenomas are a heterogeneous group of tumors that may occur as part of a complex syndrome or as an isolated endocrinopathy and both forms can be familial or non-familial. Studies of syndromic and non-syndromic pituitary adenomas have yielded important insights about the molecular mechanisms underlying tumorigenesis. Thus, syndromic forms, including multiple endocrine neoplasia type 1 (MEN1), MEN4, Carney Complex and McCune Albright syndrome, have been shown to be due to mutations of the tumor-suppressor protein menin, a cyclin-dependent kinase inhibitor (p27Kip1), the protein kinase A regulatory subunit 1-α, and the G-protein α-stimulatory subunit (Gsα), respectively. Non-syndromic forms, which include familial isolated pituitary adenoma (FIPA) and sporadic tumors, have been shown to be due to abnormalities of: the aryl hydrocarbon receptor-interacting protein; Gsα; signal transducers; cell cycle regulators; transcriptional modulators and miRNAs. The roles of these molecular abnormalities and epigenetic mechanisms in pituitary tumorigenesis, and their therapeutic implications are reviewed.

Current and emerging therapies for PNETs in patients with or without MEN1

Pancreatic neuroendocrine tumours (PNETs) might occur as a non-familial isolated endocrinopathy or as part of a complex hereditary syndrome, such as multiple endocrine neoplasia type 1 (MEN1). MEN1 is an autosomal dominant disorder characterized by the combined occurrence of PNETs with tumours of the parathyroids and anterior pituitary. Treatments for primary PNETs include surgery. Treatments for non-resectable PNETs and metastases include biotherapy (for example, somatostatin analogues, inhibitors of receptors and monoclonal antibodies), chemotherapy and radiological therapy. All these treatments are effective for PNETs in patients without MEN1; however, there is a scarcity of clinical trials reporting the efficacy of the same treatments of PNETs in patients with MEN1. Treatment of PNETs in patients with MEN1 is challenging owing to the concomitant development of other tumours, which might have metastasized. In recent years, preclinical studies have identified potential new therapeutic targets for treating MEN1-associated neuroendocrine tumours (including PNETs), and these include epigenetic modification, the β-catenin–wingless (WNT) pathway, Hedgehog signalling, somatostatin receptors and MEN1 gene replacement therapy. This Review discusses these advances.

Mice deleted for cell division cycle 73 gene develop parathyroid and uterine tumours: model for the hyperparathyroidism-jaw tumour syndrome

The hyperparathyroidism-jaw tumour (HPT-JT) syndrome is an autosomal dominant disorder characterized by occurrence of parathyroid tumours, often atypical adenomas and carcinomas, ossifying jaw fibromas, renal tumours and uterine benign and malignant neoplasms. HPT-JT is caused by mutations of the cell division cycle 73 (CDC73) gene, located on chromosome 1q31.2 and encodes a 531 amino acid protein, parafibromin. To facilitate in vivo studies of Cdc73 in tumourigenesis we generated conventional (Cdc73+/−) and conditional parathyroid-specific (Cdc73+/L/PTH-Cre and Cdc73L/L/PTH-Cre) mouse models. Mice were aged to 18-21 months and studied for survival, tumour development and proliferation, and serum biochemistry, and compared to age-matched wild-type (Cdc73+/+ and Cdc73+/+/PTH-Cre) littermates. Survival of Cdc73+/− mice, when compared to Cdc73+/+ mice was reduced (Cdc73+/−=80%; Cdc73+/+=90% at 18 months of age, P<0.05). Cdc73+/−, Cdc73+/L/PTH-Cre and Cdc73L/L/PTH-Cre mice developed parathyroid tumours, which had nuclear pleomorphism, fibrous septation and increased galectin-3 expression, consistent with atypical parathyroid adenomas, from 9 months of age. Parathyroid tumours in Cdc73+/−, Cdc73+/L/PTH-Cre and Cdc73L/L/PTH-Cre mice had significantly increased proliferation, with rates >fourfold higher than that in parathyroid glands of wild-type littermates (P<0.0001). Cdc73+/−, Cdc73+/L/PTH-Cre and Cdc73L/L/PTH-Cre mice had higher mean serum calcium concentrations than wild-type littermates, and Cdc73+/− mice also had increased mean serum parathyroid hormone (PTH) concentrations. Parathyroid tumour development, and elevations in serum calcium and PTH, were similar in males and females. Cdc73+/− mice did not develop bone or renal tumours but female Cdc73+/− mice, at 18 months of age, had uterine neoplasms comprising squamous metaplasia, adenofibroma and adenomyoma. Uterine neoplasms, myometria and jaw bones of Cdc73+/− mice had increased proliferation rates that were 2-fold higher than in Cdc73+/+ mice (P<0.05). Thus, our studies, which have established mouse models for parathyroid tumours and uterine neoplasms that develop in the HPT-JT syndrome, provide in vivo models for future studies of these tumours.

Molecular Genetic Studies of Pancreatic Neuroendocrine Tumors: New Therapeutic Approaches

Pancreatic neuroendocrine tumors (PNETs) arise sporadically or as part of familial syndromes. Genetic studies of hereditary syndromes and whole exome sequencing analysis of sporadic NETs have revealed the roles of some genes involved in PNET tumorigenesis. The multiple endocrine neoplasia type 1 (MEN1) gene is most commonly mutated. Its encoded protein, menin, has roles in transcriptional regulation, genome stability, DNA repair, protein degradation, cell motility and adhesion, microRNA biogenesis, cell division, cell cycle control, and epigenetic regulation. Therapies targeting epigenetic regulation and MEN1 gene replacement have been reported to be effective in preclinical models.