The Vault Complex Is Significantly Involved in Therapeutic Responsiveness of Endocrine Tumors and Linked to Autophagy under Chemotherapeutic Conditions.

Cancers display dynamic interactions with their complex microenvironments that influence tumor growth, invasiveness, and immune evasion, thereby also influencing potential resistance to therapeutic treatments. The tumor microenvironment (TME) includes cells of the immune system, the extracellular matrix, blood vessels, and other cell types, such as fibroblasts or adipocytes. Various cell types forming this TME secrete exosomes, and molecules thereby released into the TME have been shown to be important mediators of cellular communication and interplay. Specific stressors in the TME, such as hypoxia, starvation, inflammation, and damage, can furthermore induce autophagy, a fundamental cellular process that degrades and recycles molecules and subcellular components, and recently it has been demonstrated that the small non-coding vault RNA1-1 plays a role as a regulator of autophagy and the coordinated lysosomal expression and regulation (CLEAR) network. Here, we demonstrate for the first time that intra-tumoral damage following effective therapeutic treatment is linked to specific intracellular synthesis and subsequent exosomal release of vault RNAs in endocrine tumors in vitro and in vivo. While we observed a subsequent upregulation of autophagic markers under classical chemotherapeutic conditions, a downregulation of autophagy could be detected under conditions strongly involving inflammatory cascades.

Innovative multidimensional models in a high-throughput-format for different cell types of endocrine origin.

The adrenal gland provides an important function by integrating neuronal, immune, vascular, metabolic and endocrine signals under a common organ capsule. It is the central organ of the stress response system and has been implicated in numerous stress-related disorders. While for other diseases, regeneration of healthy organ tissue has been aimed at such approaches are lacking for endocrine diseases - with the exception of type-I-diabetes. Moreover, adrenal tumor formation is very common, however, appropriate high-throughput applications reflecting the high heterogeneity and furthermore relevant 3D-structures in vitro are still widely lacking. Recently, we have initiated the development of standardized multidimensional models of a variety of endocrine cell/tissue sources in a new multiwell-format. Firstly, we confirmed common applicability for pancreatic pseudo-islets. Next, we translated applicability for spheroid establishment to adrenocortical cell lines as well as patient material to establish spheroids from malignant, but also benign adrenal tumors. We aimed furthermore at the development of bovine derived healthy adrenal organoids and were able to establish steroidogenic active organoids containing both, cells of cortical and medullary origin. Overall, we hope to open new avenues for basic research, endocrine cancer and adrenal tissue-replacement-therapies as we demonstrate potential for innovative mechanistic insights and personalized medicine in endocrine (tumor)-biology.

Novel Insights into the Molecular Regulation of Ribonucleotide Reductase in Adrenocortical Carcinoma Treatment.

Current systemic treatment options for patients with adrenocortical carcinomas (ACCs) are far from being satisfactory. DNA damage/repair mechanisms, which involve, e.g., ataxia-telangiectasia-mutated (ATM) and ataxia-telangiectasia/Rad3-related (ATR) protein signaling or ribonucleotide reductase subunits M1/M2 (RRM1/RRM2)-encoded ribonucleotide reductase (RNR) activation, commonly contribute to drug resistance. Moreover, the regulation of RRM2b, the p53-induced alternative to RRM2, is of unclear importance for ACC. Upon extensive drug screening, including a large panel of chemotherapies and molecular targeted inhibitors, we provide strong evidence for the anti-tumoral efficacy of combined gemcitabine (G) and cisplatin (C) treatment against the adrenocortical cell lines NCI-H295R and MUC-1. However, accompanying induction of RRM1, RRM2, and RRM2b expression also indicated developing G resistance, a frequent side effect in clinical patient care. Interestingly, this effect was partially reversed upon addition of C. We confirmed our findings for RRM2 protein, RNR-dependent dATP levels, and modulations of related ATM/ATR signaling. Finally, we screened for complementing inhibitors of the DNA damage/repair system targeting RNR, Wee1, CHK1/2, ATR, and ATM. Notably, the combination of G, C, and the dual RRM1/RRM2 inhibitor COH29 resulted in previously unreached total cell killing. In summary, we provide evidence that RNR-modulating therapies might represent a new therapeutic option for ACC.

Targeting heterogeneity of adrenocortical carcinoma: Evaluation and extension of preclinical tumor models to improve clinical translation.

In recent years it has been recognized that clinical translation of novel therapeutic strategies for patients with adrenocortical carcinoma (ACC) often fails. These disappointing results indicate that the currently utilized tumor models only poorly reflect relevant pathophysiology and, thereby, do not predict clinical applicability of novel pharmacological approaches. However, also the development of new preclinical ACC models has remained a challenge with only one human cell line (NCI-H295R) and one recently established human pediatric xenograft model (SJ-ACC3) being available for this highly heterogeneous malignancy. Our current study furthermore reveals a poor reproducibility of therapeutic action between different clones of the most commonly used tumor model NCI-H295R. In an attempt to broaden the current preclinical armamentarium, we aimed at the development of patient-individual tumor models. During these studies, one xenograft (MUC-1) displayed marked engraftment and sustained tumor growth. MUC-1 tumor analysis revealed highly vascularized, proliferating and SF-1 positive xenografts. In a next step, we characterized all currently available human tumor models for ACC for Ki67, SF-1 and EGF-receptor status in comparison with MUC-1-xenografts. In addition, we established a primary culture, which is now viable over 31 passages with sustained nuclear SF-1 and cytoplasmic 3ßHSD immuno-positivity. Subsequent investigation of therapeutic responsiveness upon treatment with the current systemic gold standard EDP-M (etoposide, doxorubicin, cisplatin and mitotane) demonstrated maintenance of the clinically observed drug resistance for MUC-1 exclusively. In summary, we provide evidence for a novel patient-derived tumor model with the potential to improve clinical prediction of novel therapeutic strategies for patients with ACC.

The side population phenomenon enriches for designated adrenocortical progenitor cells in mice.

Somatic adrenal stem cells are believed to reside in the periphery of the adrenal cortex throughout life for organ maintenance. Herein, we used the side population (SP) phenomenon to enrich for these progenitors, which made up to 0.01-0.64% of the total cell count. Microarray analysis revealed an expression profile of SP cells, which clearly differed from that of non-SP cells. However, a promising adrenal specific stem cell marker could not be identified. In vitro, SP cells could be maintained in long-term culture, whereas non-SP cells did not proliferate. After 4 weeks of culturing, immunohistochemistry revealed the expression of steroidogenic enzymes such as 3ß-HSD, StAR, and P450SCC, suggesting spontaneous differentiation. Interestingly, the quantity of SP cells was significantly diminished in Pbx1 haploinsufficient mice, suggesting a stem cell deficit. By contrast, the subcapsular zone of ACTH-deficient Tpit(-/-) mice was significantly wider compared with wild-type adrenals (Tpit(-/-) 259±10.7 vs Tpit(+/-) 100±12.3%; P<0.01). Accordingly, the number of SP cells in these mice was significantly higher (Tpit(-/-) 0.45±0.16 vs Tpit(+/-) 0.13±0.04%; P<0.004). ACTH treatment of these animals reverted the subcapsular zone width and the SP fraction back to normal (130±10.2%; P=0.33 and 0.09%), providing indirect evidence for a stem cell 'arrest' in Tpit(-/-) mice and the role of ACTH in adrenocortical stem cell modulation and differentiation.

Identification of adrenal genes regulated in a potassium-dependent manner.

Potassium and angiotensin II are the main stimulators of aldosterone secretion from the adrenal cortex. As potassium-induced in vivo gene regulation in the adrenal cortex has not been studied in detail, we applied a stepwise screening approach: first, we investigated the effects of chronic potassium substitution in mice. Microarray analysis of adrenal glands revealed a set of genes (set A) that were counter-regulated in a high potassium (HP) and low potassium substitution group, while others (set B) were highly upregulated in the HP intake group. In a second step, time dependency of expression changes of these pre-defined genes was studied following short-term potassium stimulation experiments in vivo. Thirdly, dose dependency of potassium-induced gene regulation was investigated in vitro. Finally, to provide indirect evidence for the potential relevance of the detected changes for autonomous aldosterone secretion, expression analysis of aldosterone-producing adenomas was compared with normal adrenal glands. While most investigated genes were similarly regulated following long- and short-term potassium stimulation in vivo, observed changes were reproducible in NCI h295R adrenocortical cells mostly for the set of genes identified in the HP group (set B). Similarly, in Conn's adenomas, mostly genes from set B displayed changes in expression pattern in comparison to normal adrenal glands, while genes from set A were mostly unchanged. Thus, while in vivo models can help in identifying genes potentially involved in potassium-dependent aldosterone secretion, these findings also underline the necessity to interpret potassium-induced gene regulation on the basis of the experimental setting.

Side population does not define stem cell-like cancer cells in the adrenocortical carcinoma cell line NCI h295R.

Recent evidence suggests the existence of a stem cell-like subpopulation of cells in hematological and solid tumor entities, which determine the malignant phenotype of a given tumor through their proliferative potential and chemotherapy resistance. A recently used technique for the isolation of this cell population is through exclusion of the vital dye Hoechst 33342, which defines the so-called side population (SP). Herein we demonstrate the presence of SP cells in a variety of adrenal specimens, including primary cultures of human adrenocortical tumors and normal adrenal glands as well as established human and murine adrenocortical cancer cell lines by fluorescence-activated cell sorter analysis and confocal microscopy. On a functional level, SP cells from the human adrenocortical tumor cell line NCI h295R revealed an expression pattern consistent with a less differentiated phenotype, including lower expression of steroidogenic enzymes such as steroid acute regulatory protein (StAR) and side-chain cleavage enzyme (P450scc) in comparison with non-SP cells. However, proliferation between SP and non-SP cells did not differ (105.6 +/- 18.1 vs. 100.0 +/- 3.5%). Furthermore, re-sorting and tracing experiments revealed the capacity for both cell types to give rise to the original SP- and non-SP-containing cell population. Similarly to the baseline growth kinetics, no survival benefit was evident in SP cells after treatment with cytotoxic agents commonly used in adrenocortical carcinomas. Taken together, these findings provide evidence that Hoechst dye exclusion, in contrast to what has been reported for other tumor entities, is not a major tumor stem cell defining marker in adrenocortical NCI h295R tumor cells.

Expression and spatio-temporal distribution of differentiation and proliferation markers during mouse adrenal development.

Development of the adrenal cortex is dependent upon the specific regulation of cellular proliferation and differentiation. Although both intra-adrenal transcription factors and extra-adrenal peptide hormones have been demonstrated as indispensable for this regulatory process, the resulting distribution of proliferating and steroidogenic cell populations in the developing adrenal cortex has not been defined. Thus, we assessed expression and colocalization of a differentiation marker (3-beta-hydroxysteroid dehydrogenase, 3beta-HSD) and a proliferation marker (5-bromo-2'-deoxyuridine (BrdU) incorporation) at the various time points (embryonic day (E) 9.5 until 2 weeks post partum) during mouse adrenal development. In addition, adrenocorticotropin-hormone (ACTH) receptor (melanocortin-2-receptor (MC2-R)) expression was examined by in situ hybridization (ISH) and co-localized with 3beta-HSD. As demonstrated by immunohistochemistry (IHC) the number of BrdU positive cells within the adrenal cortex decreased during development, whereas the number of 3beta-HSD positive cells increased. While BrdU incorporation was evident in a scattered pattern throughout the adrenal gland up to day E13.5, at later time points BrdU positive cells assembled in a discrete subcapsular compartment possibly representing the stem cell layer of the adult adrenal cortex. Interestingly, only a small percentage of proliferating cells expressed 3beta-HSD, while the majority of 3beta-HSD positive cells co-stained for MC2-R expression by means of ISH. As demonstrated by semiquantitative RT-PCR, MC2-R mRNA levels increased from E11.5 until birth, while the highest adrenal secretory protease (AsP) expression was detected at E13.5 with a decrease thereafter. Taken together, these findings are in accordance with the concept of distinct cell populations present during adrenocortical development with a highly proliferative phenotype or differentiated steroidogenic properties.

Gonadectomy in mice of the inbred strain CE/J induces proliferation of sub-capsular adrenal cells expressing gonadal marker genes.

Mouse models of adrenal tumorigenesis have the potential to give insights in the dysregulation of adrenal growth and differentiation. The inbred mouse strain CE/J has been reported to develop adrenal tumors upon gonadectomy (GDX) similar to mice with targeted deletions of the inhibin alpha subunit (Inh-/-). We performed a detailed morphological and molecular characterization of adrenal glands from CE/J mice 8-50 weeks of age to define the cellular origin of these tumors as well as the spatial and temporal expression of marker genes associated with tumor growth. In contrast to the induction of x-zone growth upon GDX in Inh-/- mice, GDX in CE/J mice induced the appearance of sub-capsular nests of densely packed cells that progress into adrenal tumors. Staining for proliferative cell nuclear antigen confirms a substantial increased in cellular proliferation within this sub-capsular compartment and lack of apoptosis upon GDX. Induction of adrenal tumor growth was accompanied by transcriptional changes that otherwise define gonadal endocrine cells. These regulated genes included transcription factors such as GATA-4, WT-1, FOG-1, and steroidogenic factor-1 (SF-1), peptide hormones such as Mullerian-inhibiting substance (MIS), hormone receptors including luteinizing hormone and MIS receptor, and steroidogenic enzymes such as P450c17 and P450 aromatase. The functional significance of steroid enzyme expression was demonstrated by a gradual increase of adrenal androgens after GDX. Taken together these data suggest that adrenal tumors in gonad-ectomized CE/J mice are direct derivatives from cells of the proposed sub-capsular stem cell zone. The distinct expression pattern of this cell population is consistent with a defect in the differentiation of these cells into a cell population with functional properties that otherwise define a gonadal endocrine phenotype.

Peroxisome proliferator-activated receptor-gamma agonists suppress adrenocortical tumor cell proliferation and induce differentiation.

CONTEXT: Thiazolidinediones (TZDs) have been implemented into clinical practice for the treatment of type 2 diabetes mellitus as specific peroxisome proliferator-activated receptor (PPAR)-gamma ligands. Moreover, recent evidence has suggested that TZDs might have favorable effects in the treatment of a variety of tumors as differentiation-inducing agents. Adrenocortical carcinoma (ACC) is a rare tumor entity with poor prognosis due to its highly malignant phenotype and lack of effective treatment options. OBJECTIVE: The purpose of this study was to investigate effects of TZDs on adrenocortical cancer cells. RESULTS: PPARgamma mRNA expression was detectable in all adrenocortical tumors including ACCs at similar levels. Furthermore, incubation of the adrenocortical tumor cell line NCI h295 with the PPARgamma agonist rosiglitazone led to a decrease in cell viability, a decrease of cellular proliferation, and an increase in apoptosis as well as steroidogenesis. On the molecular level, NCI h295 cells expressed higher levels of ACTH receptor (melanocortin receptor-2) mRNA upon treatment, whereas cyclin E mRNA was reduced, thus reflecting a shift toward an expression pattern found in less aggressive adrenocortical tumors in vivo. Accordingly, luciferase experiments confirmed an increased promoter activity for the melanocortin receptor-2 after stimulation with rosiglitazone. Coincubation with the specific PPARgamma antagonist GW9662 demonstrated the inhibition of TZD-induced increase in steroidogenesis, whereas growth suppression upon TZD treatment was not affected by GW9662. CONCLUSIONS: Thus, both PPARgamma-dependent and PPARgamma-independent effects of TZD treatment are likely to contribute to the observed phenotypical effects on NCI h295 cells. Taken together, these data indicate that TZDs might have the potential to become an additional treatment option as differentiation-inducing agents in patients with ACC.