Elsevier

Molecular and Cellular Endocrinology

Volume 419, 5 January 2016, Pages 252-258
Molecular and Cellular Endocrinology

Protein Kinase C Delta restrains growth in ACTH-secreting pituitary adenoma cells

https://doi.org/10.1016/j.mce.2015.10.025Get rights and content

Highlights

  • PRKCD influences the mechanisms regulating pituitary adenoma cell viability.

  • PRKCD is involved in POMC/ACTH, E-cadherin, β-catenin, GSK3β and EGFR expression regulation.

  • human corticotroph adenoma samples displaying lower PRKCD levels have higher EGFR protein levels.

  • PRKCD directly influences EGFR-mediated proliferative effects in corticotroph adenoma cells.

  • PRKCD might restrain corticotroph adenoma cells from acquiring an aggressive behavior.

Abstract

Protein Kinase C Delta (PRKCD) has been highlighted among disrupted pathways in corticotroph adenomas. PRKCD is expressed at low level in human corticotroph adenomas and controls cell cycle in vitro. Therefore, PRKCD may play an important role in the development/progression of corticotroph adenomas, warranting further studies to understand the role of PRKCD and related pathways in restraining pituitary cell growth. We evaluated PRKCD role in influencing cell behavior in terms of cell viability, hormone expression and protein expression profile, by silencing PRKCD in AtT-20/D16v-F2 cells. PRKCD silencing increases cell viability, enhances hormone expression and induces morphological changes associated with deregulation of adhesion molecules. PRKCD silencing is associated with an increase in Epithelial Growth Factor Receptor (EGFR) expression, a marker of tumor aggressive behavior, and sensitivity to anti-EGFR molecules. PRKCD might restrain corticotroph adenoma cells from acquiring an aggressive behavior, candidating PRKCD as a possible molecular target for the treatment of corticotroph adenomas.

Introduction

The mechanisms underlying the pathogenesis of adrenocorticotroph hormone (ACTH) secreting pituitary adenomas is a matter of great interest, since clinical effects of uncontrolled hypercortisolism are very grim and associated with high morbidity and mortality (van Haalen et al., 2015). Pituitary surgery is always referred to as the first-line therapy, but it is burdened with a high recurrence/persistence rate (Fleseriu, 2012). Therefore, effective pharmacological treatments are still needed, and are even more necessary for patients with high surgical risk and/or acute hypercortisolism. Currently available medical treatment for Cushing's disease (CD) allows to achieve disease control in a subset of patients (Eckstein et al., 2014), representing a great therapeutic advantage. However, some are not adequately controlled, therefore current research aims at discovering new effective drugs and identifying novel mechanisms that may represent promising pharmacological targets. Along this line, the research for new putative molecular targets has become very intensive, highlighting the importance of abnormal cell signaling (Dworakowska and Grossman, 2012). Recently, PRKCD has been highlighted among the pathways that are disrupted in ACTH-secreting pituitary adenomas (Gentilin et al., 2013a). PRKCD is a serine–threonine kinase that regulates proliferation, cell cycle, differentiation and apoptosis in several cellular models (Kikkawa et al., 2002). Alterations in its activity and/or expression levels are crucial events in the development of a malignant phenotype in many tumors (Koike et al., 2006, Haughian et al., 2006, D'Costa et al., 2006, Castilla et al., 2013). Recently, we demonstrated that PRKCD is expressed at low level in human ACTH-secreting pituitary adenomas and in a murine ACTH-secreting pituitary adenoma cell line, where it mediates the powerful effects of microRNA 26a on cell cycle, down-regulating cyclin E and cyclin A expression (Gentilin et al., 2013a). These results support the hypothesis that PRKCD may play an important role in the development and progression of ACTH pituitary adenomas, and therefore prompt us to investigate the role of PRKCD and related pathways in restraining corticotroph cell growth.

The aim of this study was to evaluate the role of PRKCD in influencing corticotroph cells behavior in terms of cell viability, proopiomelanocortin (POMC) expression and protein expression profile, by silencing PRKCD in a murine ACTH-secreting pituitary adenoma cell line.

Section snippets

Cell culture

The mouse ACTH secreting pituitary adenoma cell line, AtT-20/D16v-F2, was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). The cell line was maintained as previously reported (Tagliati et al., 2010, Gentilin et al., 2013b).

Transfection with PRKCD shRNA

Cells were transfected with PRKCD shRNA plasmid (OriGene, Rockville, MD, USA), by using Nucleofector II (Amaxa, Gaithersburg, MD, USA) according to the manufacturer's protocols, as previously reported (Gentilin et al., 2013a). Then, stably

PRKCD silencing increases AtT-20/D16v-F2 cell viability

AtT-20/D16v-F2 cells were transfected with a shPRKCD plasmid (sh cells). As shown in Fig. 1, sh cells showed lower PRKCD protein levels (∼60%) as compared to control cells.

For each cell line, cell viability was monitored after 24 and 48 h (Fig. 2A). Cell lines displayed unique growth profiles over time, since sh cells doubling time is significantly shorter (26.7 h) as compared to control cells (51.8 h; http://www.doubling-time.com/compute.php). Cell viability was significantly greater in sh

Discussion

Our study shows that PRKCD influences corticotroph cell viability, POMC and ACTH expression, as well as E-cadherin/EGF pathway, suggesting that PRKCD plays an important role in restraining corticotroph cell proliferation and function. Several studies investigated the importance of PRKC family in carcinogenesis and maintenance of malignant phenotype (Haughian et al., 2006, D'Costa et al., 2006, Castilla et al., 2013, Molè et al., 2012), that may be influenced by activation/expression of

Conflicts of interest

None.

Acknowledgments

This work was supported by grants from the Italian Ministry of Education, Research and University (FIRB RBAP11884M, RBAP1153LS, 2010TYCL9B_002), Fondazione Cassa di Risparmio di Ferrara, and Associazione Italiana per la Ricerca sul Cancro (AIRC) in collaboration with Laboratorio in rete del Tecnopolo “Tecnologie delle terapie avanzate” (LTTA) of the University of Ferrara. The funding sources had no involvement in study design, in the collection, analysis and interpretation of data, in the

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