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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2004-2198
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The Journal of Clinical Endocrinology & Metabolism Vol. 90, No. 7 4366-4370
Copyright © 2005 by The Endocrine Society

BRIEF REPORT

AKT Is Highly Phosphorylated in Pheochromocytomas But Not in Benign Adrenocortical Tumors

Martin Fassnacht1, Dirk Weismann1, Silke Ebert, Patrick Adam, Martina Zink, Felix Beuschlein, Stefanie Hahner and Bruno Allolio

Endocrine and Diabetes Unit (M.F., D.W., S.E., M.Z., S.H., B.A.), Department of Internal Medicine I, and Institute of Pathology (P.A.), University of Würzburg, 97080 Würzburg, Germany; and Endocrine and Diabetes Unit (F.B.), Department of Medicine II, University of Freiburg, 179106 Freiburg, Germany

Address all correspondence and requests for reprints to: Dr. Martin Fassnacht, Department of Medicine, Endocrine and Diabetes Unit, University of Würzburg, 97080 Würzburg, Germany. E-mail: Fassnacht_m{at}medizin.uni-wuerzburg.de.


   Abstract
Top
 Abstract
Introduction
 Patients and Methods
 Results
 Discussion
 References
 
Context: Activation of AKT plays a major role in a variety of human neoplasias. In mice, a heterozygous deletion of the Pten gene is associated with increased activation of AKT and with development of pheochromocytomas.

Objective: The objective of this study was the investigation of the role of AKT in the pathogenesis of pheochromocytomas and adrenocortical tumors.

Design, Setting, and Participants: Total AKT and phosphorylated AKT (pAKT) in 15 pheochromocytomas, nine aldosterone-producing adenomas, nine cortisol-producing adenomas, eight adrenocortical carcinomas (ACC), and 15 normal adrenals were investigated by Western blot analysis. Immunohistochemistry for total AKT and pAKT was performed in pheochromocytomas (n = 8), ACC (n = 4), and normal adrenal glands (n = 2). In addition, in pheochromocytomas PTEN protein expression and PTEN loss of heterozygosity were analyzed.

Main Outcome Measures: Determination of pAKT/total AKT ratio in adrenal tissues was the main outcome.

Results: In comparison to normal adrenals, total AKT expression was elevated in both pheochromocytomas (193 ± 22%) and ACC (176 ± 36%). The pAKT/AKT ratio was significantly increased in pheochromocytomas (338 ± 49% vs. 100 ± 11%) but not in ACC, aldosterone-producing adenomas, and cortisol-producing adenomas. No loss of heterozygosity of PTEN and no decrease in PTEN protein was detected in pheochromocytomas. Immunohistochemistry showed strong and homogeneous AKT and pAKT staining in pheochromocytomas and focal staining in ACC.

Conclusion: Our findings provide evidence for increased activation of AKT in pheochromocytomas but not in adrenocortical adenomas.


   Introduction
Top
 Abstract
 Introduction
Patients and Methods
 Results
 Discussion
 References
 
UNDERSTANDING OF THE pathogenesis of pheochromocytoma has been guided by mutations that occur as part of hereditary tumor syndromes: mutations in the VHL gene (von Hippel-Lindau syndrome), the RET protooncogene [multiple endocrine neoplasia type 2 (MEN2)], and mutations in subunits of the succinate dehydrogenase complex (1). Although mutations described in these hereditary tumor syndromes are also found in about 25% of seemingly sporadic pheochromocytomas (2), in the majority of sporadic cases, the molecular events leading to tumor development remain unknown.

The AKT/protein kinase-B pathway is a major pathway involved in the regulation of apoptosis and cell survival and has, therefore, become a focus of cancer research in recent years (3). AKT is activated in response to a variety of growth factors via the phosphatidylinositol-3-kinase (PI3K) pathway. Only phosphorylated AKT (pAKT) is able to regulate downstream targets. AKT activation is inhibited by phosphatase and tensin homolog (PTEN). PTEN dephosphorylates PI3K-generated 3'-phosphorylated phosphatidylinositides, thereby limiting the activity of the PI3K pathway. Dysregulation of the PI3K-AKT pathway has been found in a wide spectrum of human neoplasias (4, 5), and increased AKT activity as the result of PTEN inactivation by loss of heterozygosity (LOH) and somatic mutations has been demonstrated in a variety of tumor types including prostate, endometrium, and lung cancer (6, 7, 8).

Observations in Pten knockout mice indicate that the PI3K/AKT pathway might be involved in the molecular pathogenesis of adrenal tumors. Heterozygous Pten +/– mice show increased tumor incidence consistent with the role of Pten as a tumor suppressor gene. Intriguingly, about 25% of Pten +/– mice developed adrenal pheochromocytoma (9, 10). This is in keeping with the observation that in the pheochromocytoma cell line PC12 survival is crucially dependent on PI3K activity (11).

The pathogenesis of adrenocortical tumors is still poorly understood. Most frequently, mutations in the p53 gene have been described, but only few adrenocortical mitogens have been identified (12, 13, 14, 15, 16, 17, 18, 19). A role of IGF-II for the pathogenesis of adrenocortical carcinoma (ACC) is suggested by the frequent development of ACC in the Beckwith-Wiedemann syndrome (associated with overexpression of IGF-II) (20) and the overexpression of IGF-II in most sporadic ACC (21, 22). IGF-II exerts its biological activity through the IGF-I receptor. Because AKT is a major downstream target of IGF-I receptor signaling, we hypothesized that activation of AKT may be involved in the molecular pathogenesis of ACC.

The aim of this study was, therefore, to investigate a potential role of AKT activation in the pathogenesis of both pheochromocytomas and adrenocortical tumors.


   Patients and Methods
Top
 Abstract
 Introduction
 Patients and Methods
Results
 Discussion
 References
 
Patients and tumors

Tissue specimens from 42 patients with adrenal tumors were studied [25 females and 17 males; median age, 49 yr (range, 23–71 yr)] with a median tumor size of 4.9 cm (range, 1.1–14 cm) and compared with 15 nonneoplastic, normal adrenal glands (NAG). We investigated 15 adrenal pheochromocytomas (10 sporadic, three MEN2, one von Hippel-Lindau syndrome, and one malignant), nine aldosterone-producing adenomas (APA), nine cortisol-producing adenomas (CPA), and eight ACC. Nonneoplastic adrenal tissue was obtained from patients who underwent ipsilateral adrenalectomy as part of surgery for renal cell cancer. DNA and protein from adrenal tissue were extracted using TriZol reagent (Invitrogen, Karlsruhe, Germany). DNA from leukocytes was isolated from whole blood using the QIAGEN DNA blood isolation kit (QIAGEN, Hilden, Germany). All tissue specimens were collected after written informed consent and with approval of the ethical committee of the University of Würzburg.

Western blotting

Western blotting was performed as described previously (23). Antibodies against the following antigens were used: pAKT(Ser473), total AKT, PTEN (all from Cell Signaling Technology, Frankfurt, Germany), ß-actin (Sigma-Aldrich, Deisenhofen, Germany), and a horseradish peroxidase-labeled goat antirabbit IgG as second antibody. Autoradiographs were scanned, and the density of specific bands was measured using Image Gauge software (Fuji, Dusseldorf, Germany). Interblot differences were calculated by comparing the densities with a reference sample included in each blot. The results are given as percentage of levels in normal adrenals (100%) after normalization to ß-actin and total AKT, respectively.

Immunohistochemistry

Immunohistochemistry was performed in formalin-fixed and paraffin-embedded specimens (eight pheochromocytomas, four ACC, and two NAG). Before staining, the tissue was deparaffinized in xylene for 2 min and rehydrated in ethanol. Antigen retrieval was performed in target retrieval solution (S1700; Dako, Carpinteria, CA) in a pressure cooker for 8 min. Slides were then incubated in blocking reagent (Histostain-Plus Bulk Kit; Zymed, San Francisco, CA) for 10 min before application of the primary antibody against pAKT(Ser473) (Cell Signaling Technology) or total AKT (Abcam, Cambridge, UK). Tissue sections were incubated with antibody at room temperature for 60 min, followed by a biotinylated antirabbit second antibody for 30 min at room temperature and a peroxidase-linked streptavidin conjugate (Histostain-Plus Bulk Kit) for another 30 min. Positive cells were visualized by diaminobenzidine (Sigma) substrate. Finally, the nuclei were counterstained with hematoxylin.

Negative controls were carried out by treating the slides with a nonimmune serum instead of the primary antibody, yielding a nearly complete loss of staining with only some faint background.

Grading was performed as described elsewhere with vascular endothelium used as internal control (24, 25). Endothelium was set to 2+ on a scale from 0+ (no staining) to 2+ (strong staining). The tissue was scored independently by two investigators (D.W. and P.A.).

10q23 Allelotyping for LOH analysis of PTEN

Heterozygosity for PTEN was determined in matching germline and tumor DNA samples in 15 patients with pheochromocytoma. Two markers covering the flanking regions of the PTEN gene, D10S1765 and D10S541 (UniSTS:7674 and UniSTS:83094, respectively), and two intragenic-intronic polymorphic markers, IVS4 + 109 ins/del TCTT and IVS8 + 32 T/G, were used. Analysis was performed as described elsewhere previously (26, 27) using 4% low-melt agarose for separation by gel electrophoresis.

Statistical analysis

All results are expressed as means ± SEM. Significance of differences was evaluated by ANOVA using the StatView 4.51 software. A value of P < 0.05 was considered statistically significant with post hoc analysis carried out by Fisher’s projected least significant difference test.


   Results
Top
 Abstract
 Introduction
 Patients and Methods
 Results
Discussion
 References
 
In benign adrenocortical tumors, total AKT expression did not differ from that in NAG. However, mean AKT protein expression in ACC (176 ± 36%) and pheochromocytomas (193 ± 22%) was significantly elevated in comparison with normal adrenal glands (100 ± 7%) (Fig. 1Go).



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  		FIG. 1. AKT expression and phosphorylation in normal adrenals, adrenocortical tumors, and pheochromocytomas. For Western blot analysis, specific polyclonal antibodies detecting pAKT (Ser473; 1:1000), total AKT (all three isoforms; 1:1000), and ß-actin (1:5000) for internal control were used. A sample of one NAG served as reference (Ref) on each blot of adrenal tumors. Representative blots for pheochromocytomas (A) and adrenal carcinomas (B) are shown. A clear increase of pAKT in pheochromocytoma (A) is visible. Photodensitometric analysis [nine APA, nine CPA, eight ACC, and 15 pheochromocytomas (Pheo) compared with 15 normal adrenals] reveals elevation of total AKT/ß-actin ratio in ACC and Pheo (C). The pAKT/total AKT ratio (D) and the pAKT/ß-actin ratio (E) are significantly elevated only in pheochromocytoma. Values are given as percentage (mean ± SEM) of levels in normal adrenal (100%) after normalization to ß-actin and total AKT, respectively. *, P < 0.01; **, P < 0.001, in comparison with NAG.

 
In adrenocortical tumors (APA, CPA, and ACC), no increase in AKT activation (pAKT/total AKT) was detectable in Western blot analysis. In contrast, with one exception, all pheochromocytomas showed a phosphorylation level above the mean of NAG. The pAKT/ß-actin ratio was about 6-fold (620 ± 104%) higher than that of normal adrenal tissue, and despite the increase of total AKT, the pAKT/total AKT ratio was more than 3-fold (338 ± 49%) elevated (Fig. 1Go). A subanalysis of the three MEN2 pheochromocytomas revealed comparable results.

Immunohistochemical studies (Fig. 2Go and Table 1Go) confirmed the high level of AKT expression and AKT phosphorylation in pheochromocytomas. In addition, these studies revealed foci with strong staining for AKT and pAKT in ACC in parallel with unstained areas. In contrast, no pAKT staining was visible in the nonmalignant adrenal medulla. However, AKT and pAKT were detectable in the adrenal cortex with the strongest staining in the zona reticularis. Because the cortex is naturally the predominant component of total NAG extracts, the lack of pAKT staining in the normal medulla emphasizes the significance of the increased pAKT/total AKT ratio in the neoplastic medulla in the Western blot analysis.



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  		FIG. 2. Expression of pAKT and total AKT by immunohistochemistry in NAG (1), pheochromocytoma (2), and ACC (3). First row (a) shows the pAKT-stained sections, second row (b) total AKT, third row (c) the negative control (incubation with nonimmune serum), and the fourth row (d) hematoxylin-eosin staining. In normal adrenal tissue, the zona reticularis (1a, brace) is notably stained for total AKT (2+) and pAKT (2+), whereas the expression of both total AKT and pAKT in zona fasciculata and zona granulosa is weaker (1+) (1a and 1b). In the normal medulla, a faint staining for total AKT is visible but not for pAKT. In pheochromocytoma (2a and 2b), strong homogeneous staining for total AKT (2+) and pAKT (2+) but only focal expression of total AKT (2+) and pAKT (2+) is found in ACC (3a). The regions with no total AKT and pAKT staining in ACC correspond to stromal and necrotic tissue. A summary of the grading is given in Table 1Go. Magnification: normal adrenal, x40; pheochromocytomas and ACC, x100.

 

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  		TABLE 1. Summary of NAG, pheochromocytomas, and ACC for staining of total AKT and pAKT in immunohistochemistry

 
Because deletion of Pten leads to a high incidence of pheochromocytomas in mice, we performed allelotyping analysis in 15 patients with pheochromocytomas. All 15 patients were informative for a possible LOH of PTEN for at least one marker. However, none of the cases carried LOH of PTEN (data not shown).

Because down-regulation of PTEN can also occur through transcriptional silencing or protein instability (28, 29), we also investigated PTEN protein levels in pheochromocytomas and normal adrenal tissue. However, the relative amount of PTEN protein in pheochromocytomas was not decreased but increased in comparison with normal adrenals (219 ± 26.5% vs. 100 ± 8.7%; P < 0.001) (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
References
 
During recent years, the association between AKT and tumorigenesis has received increasing attention. AKT is critical for proliferation and antiapoptotic signaling pathways, and increased activation of AKT has been found to be involved in a variety of neoplasia.

The major finding of our study is the observation of increased expression and activation of AKT in pheochromocytomas but not in benign adrenocortical tumors. In human neoplasia, elevated AKT activity is frequently the consequence of dysregulation of PI3K signaling caused by inactivation of PTEN, which normally limits the activity of the PI3K pathway (4). However, using LOH analysis, we found no evidence for PTEN inactivation in pheochromocytomas. Because PTEN protein levels were not reduced, we can rule out PTEN suppression by transcriptional silencing or protein instability as has been found in other human tumors (6, 30, 31). Instead, increased PTEN protein levels are suggestive of a compensatory up-regulation. However, we cannot exclude point mutations of PTEN, although point mutations on both alleles in all our pheochromocytomas seem to be unlikely. Because AKT mutations seem to play no major role in human tumorigenesis (32), these findings support the concept of alterations in the upstream regulation of AKT activity.

It seems likely that the consistent activation of AKT is of importance for the molecular pathogenesis of both sporadic and MEN2-associated pheochromocytomas. In mice heterozygous for the deletion of the tumor suppressor gene Pten, the constitutively increased phosphorylation of AKT was associated with a high incidence of pheochromocytomas (14 of 59) (9).

Receptor tyrosine kinases such as IGF-I receptor, vascular endothelial growth factor receptor, and platelet-derived growth factor receptor play an important role in the upstream regulation of AKT kinase activity and may, thus, be involved in activation of AKT in pheochromocytoma. In the pheochromocytoma cell line PC12 it has been demonstrated that growth factor-mediated survival is abrogated by inhibition of PI3K activity. Thus, it is likely that increased phosphorylation of AKT in pheochromocytoma is related to enhanced receptor tyrosine kinase signaling. In support of this view is the report by Segouffin-Cariou and Billaud (33) demonstrating that the RET mutation in MEN2 leads to activation of the PI3K-AKT pathway. Moreover, transfection of the MEN2 RET oncogene into rat pheochromocytoma PC12 cells promotes serum-free survival via the PI3K-AKT pathway (34). In keeping with these data, pheochromocytomas derived from patients with MEN2 also exhibited an elevated pAKT/AKT ratio in our study.

At present, it remains unclear which of the downstream targets of pAKT plays a major role in the molecular pathogenesis of pheochromocytoma. Enhanced AKT leads to increased phosphorylation of a variety of AKT substrates such as nuclear factor-{kappa}B, Forkhead, Bcl-2/Bcl-X-associated death promoter (BAD) glycogen synthase kinase-3, caspase-9, S6 kinase, and mammalian target of rapamycin (4, 35, 36, 37, 38).

In other tumors, activation of AKT is frequently associated with a malignant phenotype (39, 40, 41, 42). However, with one exception, we found no evidence for malignancy in our series of pheochromocytomas. Increased activation of AKT in benign tumors is unusual and might support the hypothesis that AKT activation is an early event in the molecular pathogenesis of this tumor type.

In contrast, benign adrenocortical tumors demonstrated no enhanced activation of AKT compared with normal adrenals. In ACC, activation of the IGF cascade has been consistently observed with overexpression of IGF-II mRNA in 90% of cases (21). We, therefore, expected increased activation of AKT via enhanced IGF-I receptor signaling. However, although total AKT was significantly elevated, no increased ratio of pAKT/AKT was found using Western blot analysis. Increased AKT protein, however, is usually not related to tumorigenesis, because only transfection of constitutively activated but not wild-type AKT is tumorigenic (32). However, immunohistochemistry consistently revealed areas of strong pAKT staining in the ACC tissues studied, indicating local activation of this pathway. In addition, it has to be considered that an impairment of this pathway could be relevant only in a subset of ACC. In the human adrenocortical cancer cell line NCI-H295 highly phosphorylated AKT is detectable even in starved cells, indicating a constitutive activation of this pathway in these cells (Hahner, S., M. Fassnacht, and B. Allolio, unpublished data). To clarify the role of the PI3K/AKT pathway in ACC, additional studies taking into account the inhomogeneous structure of ACC are required.

In conclusion, in this study we provide evidence for activation of AKT in both sporadic and MEN2 pheochromocytomas but not in benign adrenocortical adenomas. This activation is not associated with LOH of the PTEN gene and, therefore, most likely is caused by signaling dysregulation upstream of PI3K. The downstream targets of AKT activation remain to be elucidated.


   Footnotes
 
This work was supported by the Deutsche Forschungsgemeinschaft (Grant Al 203/7-3,4 to M.F. and B.A.).

This work was part of an initiative of the German Adrenal Network GANIMED.

First Published Online April 26, 2005

1 M.F. and D.W. contributed equally to this work. Back

Abbreviations: ACC, Adrenocortical carcinomas; APA, aldosterone-producing adenomas; CPA, cortisol-producing adenomas; LOH, loss of heterozygosity; MEN2, multiple endocrine neoplasia type 2; NAG, normal adrenal glands; pAKT, phosphorylated AKT; PI3K, phosphatidylinositol-3-kinase; PTEN, phosphatase and tensin homolog.

Received November 9, 2004.

Accepted April 14, 2005.


   References
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 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
 References
 

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M. Bielinska, H. Parviainen, S. Kiiveri, M. Heikinheimo, and D. B. Wilson
Review Paper: Origin and Molecular Pathology of Adrenocortical Neoplasms
Veterinary Pathology, March 1, 2009; 46(2): 194 - 210.
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