MicroRNAs (miRNAs) act at the posttranscriptional level to control gene expression in virtually every biological process, including oncogenesis. Here, we report the identification of a set of miRNAs that are differentially regulated in childhood adrenocortical tumors (ACT), including miR-99a and miR-100. Functional analysis of these miRNAs in ACT cell lines showed that they coordinately regulate expression of the insulin-like growth factor-mammalian target of rapamycin (mTOR)-raptor signaling pathway through binding sites in their 3′-untranslated regions. In these cells, the active Ser 2448 -phosphorylated form of mTOR is present only in mitotic cells in association with the mitotic spindle and midbody in the G 2 -M phases of the cell cycle. Pharmacologic inhibition of mTOR signaling by everolimus greatly reduces tumor cell growth in vitro and in vivo. Our results reveal a novel mechanism of regulation of mTOR signaling by miRNAs, and they lay the groundwork for clinical evaluation of drugs inhibiting the mTOR pathway for treatment of adrenocortical cancer. Cancer Res; 70(11); 4666-75. ©2010 AACR.

TASK1 (KCNK3) and TASK3 (KCNK9) are two-pore domain potassium channels highly expressed in adrenal glands. TASK1/TASK3 heterodimers are believed to contribute to the background conductance whose inhibition by angiotensin II stimulates aldosterone secretion. We used task1-/- mice to analyze the role of this channel in adrenal gland function. Task1-/- exhibited severe hyperaldosteronism independent of salt intake, hypokalemia, and arterial 'low-renin' hypertension. The hyperaldosteronism was fully remediable by glucocorticoids. The aldosterone phenotype was caused by an adrenocortical zonation defect. Aldosterone synthase was absent in the outer cortex normally corresponding to the zona glomerulosa, but abundant in the reticulo-fasciculata zone. The impaired mineralocorticoid homeostasis and zonation were independent of the sex in young mice, but were restricted to females in adults. Patch-clamp experiments on adrenal cells suggest that task3 and other K+ channels compensate for the task1 absence. Adrenal zonation appears as a dynamic process that even can take place in adulthood. The striking changes in the adrenocortical architecture in task1-/- mice are the first demonstration of the causative role of a potassium channel in development/differentiation.

Signaling by the Wnt family of secreted glycolipoproteins plays key roles in embryonic development of organisms ranging from nematodes to mammals and is also implicated in several types of human cancers. Canonical Wnt signaling functions by regulating the translocation of β-catenin to the nucleus, where it controls key gene expression programs through interaction with Tcf/Lef and other families of transcription factors. Wnts can also act through non-canonical pathways that do not involve β-catenin activation, but implicate small GTPases/JNK kinase and intracellular calcium. Here we review recent studies that have revealed the expression of several components of Wnt/β-catenin signaling in the adrenal cortex and discovered a key role for this pathway in the regulation of proliferation/differentiation of progenitor cells and in tumorigenesis of that endocrine organ.

The first-in-class inhibitor of ALK, c-MET and ROS1, crizotinib (Xalkori), has shown remarkable clinical efficacy in treatment of ALK-positive non-small cell lung cancer. However, in neuroblastoma, activating mutations in the ALK kinase domain are typically refractory to crizotinib treatment, highlighting the need for more potent inhibitors. The next-generation ALK inhibitor PF-06463922 is predicted to exhibit increased affinity for ALK mutants prevalent in neuroblastoma. We examined PF-06463922 activity in ALK-driven neuroblastoma models in vitro and in vivo. In vitro kinase assays and cell-based experiments examining ALK mutations of increasing potency show that PF-06463922 is an effective inhibitor of ALK with greater activity towards ALK neuroblastoma mutants. In contrast to crizotinib, single agent administration of PF-06463922 caused dramatic tumor inhibition in both subcutaneous and orthotopic xenografts as well as a mouse model of high-risk neuroblastoma driven by Th-ALKF1174L/MYCN. Taken together, our results suggest PF-06463922 is a potent inhibitor of crizotinib-resistant ALK mutations, and highlights an important new treatment option for neuroblastoma patients.

Anaplastic lymphoma kinase (ALK) is an important molecular target in neuroblastoma. Although tyrosine kinase inhibitors abrogating ALK activity are currently in clinical use for the treatment of ALK-positive (ALK(+)) disease, monotherapy with ALK tyrosine kinase inhibitors may not be an adequate solution for ALK(+) neuroblastoma patients. Increased expression of the gene encoding the transcription factor MYCN is common in neuroblastomas and correlates with poor prognosis. We found that the kinase ERK5 [also known as big mitogen-activated protein kinase (MAPK) 1 (BMK1)] is activated by ALK through a pathway mediated by phosphoinositide 3-kinase (PI3K), AKT, MAPK kinase kinase 3 (MEKK3), and MAPK kinase 5 (MEK5). ALK-induced transcription of MYCN and stimulation of cell proliferation required ERK5. Pharmacological or RNA interference-mediated inhibition of ERK5 suppressed the proliferation of neuroblastoma cells in culture and enhanced the antitumor efficacy of the ALK inhibitor crizotinib in both cells and xenograft models. Together, our results indicate that ERK5 mediates ALK-induced transcription of MYCN and proliferation of neuroblastoma, suggesting that targeting both ERK5 and ALK may be beneficial in neuroblastoma patients.

Postmitotic neurons are produced from a pool of cycling progenitors in an orderly fashion that requires proper spatial and temporal coordination of proliferation, fate determination, differentiation and morphogenesis. This probably relies on complex interplay between mechanisms that control cell cycle, specification and differentiation. In this respect, we have studied the possible implication of GATA2, a transcription factor that is involved in several neuronal specification pathways, in the control of the proliferation of neural progenitors in the embryonic spinal cord. Using gain-and loss-of-function manipulations, we have shown that Gata2 can drive neural progenitors out of the cycle and, to some extent, into differentiation. This correlates with the control of cyclin D1 transcription and of the expression of the p27/Kip1 protein. Interestingly, this functional aspect is not only associated with silencing of the Notch pathway but also appears to be independent of proneural function. Consistently, GATA2 also controls the proliferation capacity of mouse embryonic neuroepithelial cells in culture. Indeed, Gata2 inactivation enhances the proliferation rate in these cells. By contrast, GATA2 overexpression is sufficient to force such cells and neuroblastoma cells to stop dividing but not to drive either type of cell into differentiation. Furthermore, a non-cell autonomous effect of Gata2 expression was observed in vivo as well as in vitro. Hence, our data have provided evidence for the ability of Gata2 to inhibit the proliferation of neural progenitors, and they further suggest that, in this regard, Gata2 can operate independently of neuronal differentiation.

High-risk neuroblastoma (NB) is responsible for a disproportionate number of childhood deaths due to cancer. One indicator of highrisk NB is amplification of the neural MYC (MYCN) oncogene, which is currently therapeutically intractable. Identification of anaplastic lymphoma kinase (ALK) as an NB oncogene raised the possibility of using ALK tyrosine kinase inhibitors (TKIs) in treatment of patients with activating ALK mutations. 8-10% of primary NB patients are ALK-positive, a figure that increases in the relapsed population. ALK is activated by the ALKAL2 ligand located on chromosome 2p, along with ALK and MYCN, in the "2p-gain" region associated with NB. Dysregulation of ALK ligand in NB has not been addressed, although one of the first oncogenes described was v-sis that shares > 90% homology with PDGF. Therefore, we tested whether ALKAL2 ligand could potentiate NB progression in the absence of ALK mutation. We show that ALKAL2 overexpression in mice drives ALK TKI-sensitive NB in the absence of ALK mutation, suggesting that additional NB patients, such as those exhibiting 2p-gain, may benefit from ALK TKI-based therapeutic intervention.

Task1 and Task3 potassium channels (Task: tandem of P domains in a weak inward rectifying K ϩ channel-related acid-sensitive K ϩ channel) are believed to control the membrane voltage of aldosterone-producing adrenal glomerulosa cells. This study aimed at understanding the role of Task3 for the control of aldosterone secretion. The adrenal phenotype of Task3 Ϫ/Ϫ mice was investigated using electrophysiology, adrenal slices, and blood pressure measurements. Primary adrenocortical cells of Task3 Ϫ/Ϫ mice were strongly depolarized compared with wild-type (Ϫ52 vs.Ϫ79 mV), and in fresh adrenal slices Ca 2ϩ signaling of Task3 Ϫ/Ϫ glomerulosa cells was abnormal.In living Task3 Ϫ/Ϫ mice, the regulation of aldosterone secretion showed specific deficits: Under low Na ϩ and high K ϩ diets, protocols known to increase aldosterone, and under standard diet, Task3 inactivation was compensated and aldosterone was normal. However, high Na ϩ and low K ϩ diets, two protocols known to lower aldosterone, failed to lower aldosterone in Task3 Ϫ/Ϫ mice. The physiological regulation of aldosterone was disturbed: aldosterone-renin ratio, an indicator of autonomous aldosterone secretion, was 3-fold elevated at standard and high Na ϩ diets. Isolated adrenal glands of H igh blood pressure is one of the major cardiovascular risk factors (1). The pathogenesis of arterial hypertension, however, is very complex and encompasses environmental, genetic, vascular, and endocrine factors. Among the latter, inappropriately high aldosterone secretion is a common cause of salt and water retention resulting in hypertension. Recently, data from human genetics (2-4) pointed to a critical role of K ϩ channel defects as a cause of hyperaldosteronism.In adrenal glomerulosa cells, depolarization is considered to be the first step of a chain of events leading to aldosterone secretion (5). Glomerulosa cells have a very high K ϩ conductance leading to a hyperpolarized membrane potential close to the K ϩ equilibrium potential.