Abstract
Background: Akt is a regulator of cell proliferation, tumorigenesis and apoptosis. This study evaluated the incidence of Akt activation in a subset of neuroendocrine tumors (NETs) and its correlation to clinical pathological parameters. Materials and Methods: A subset of 46 samples from patients with enteropancreatic NETs (26 male and 20 female) were selected for evaluation. The tissue slides were stained with a mouse monoclonal antiphospho-Akt antibody using the avidin-biotin-complex method. Results: The tumors were from the small (n=18) and large bowel (n=9) and from the pancreas (n=16). Activation of Akt was detected in 61% (28/46) of cases. No statistical correlation was found between the p-Akt score and tumor grade (p=0.72), tumor size (p=0.72) and the presence of metastases (p=0.52). Conclusion: This study shows activation of Akt in a subset of enteropancreatic NET for the first time. This finding suggests a role of p-Akt in NET carcinogenesis.
Enteropancreatic neuroendocrine tumors (NETs) are usually subclassified as pancreatic, gastric or intestinal NETs. If well-differentiated, these tumors display indolent behavior and may secrete neuropeptides resulting in characteristic clinical symptoms such as flushing, diarrhea, hypoglycemia, or peptic ulcerations. The treatment of these tumors includes surgical resection cytotoxic therapy, use of somatostatin analogs and locoregional hepatic therapy (1-3). In cases with metastatic disease, standard cytotoxic therapy offers limited benefit and curative surgery is seldom an option (1).
The modest efficacy of current systemic treatment regimens has led to an interest in the development of novel therapeutic approaches for patients with advanced NETs. These include the use of agents targeting genes and gene products involved in neuroendocrine tumorigenesis.
Gastrointestinal (GI) NETs overexpress not only vascular endothelial growth factor (VEGF) (4), but also other growth factors, such as basic fibroblast growth factor (bFGF) (5), transforming growth factor (TGF) -α and -β (6-8), platelet-derived growth factor (PDGF) (9), insulin-like growth factor (IGF-I) (10), PDGF receptor (PDGFR) (9), IGF-I receptor (IGF-IR) (11), epidermal growth factor receptor (EGFR) (8, 12), VEGF receptor (VEGFR) (4, 13), and stem cell factor receptor (KIT) (14). In addition, several molecular signaling pathways have been shown to play important roles in regulating the growth of NETs (15-19).
For example, mTOR, a threonine kinase that mediates downstream signaling from a number of pathways, including the VEGF and IGF signaling pathways, is implicated in NET growth (20).
Disruption of these signaling pathways in different experimental models has resulted in inhibition of neuroendocrine cell growth (20-22). Recently, a number of clinical trials using receptor tyrosine kinase inhibitors and monoclonal antibodies targeting specific growth factors have been offered to patients with advanced NETs (23, 24).
Activation of the phosphatidylinositol 3-kinase PI3K/Akt pathway has been identified as a major regulator of cell proliferation, tumorigenesis and apoptosis. Only rare reports of Akt activation in NETs are found in the literature (12). This finding may have important therapeutic implication in light of the recent demonstration of the inhibitory action of octreotide on phosphoinositide-dependent kinase 1 PDK1 and Akt (25).
Using immunohistochemistry (IHC), the present study evaluated the incidence of Akt activation in a subset of NETs of the GI tract and pancreas. The IHC scores were correlated to clinical pathological parameters.
Clinical/pathological findings.
Patients and Methods
Selection of cases. Human tissues. A subset of 46 NETs of the GI tract and pancreas were selected for evaluation. All of the tumors evaluated were NETs identified from the H. Lee Moffitt Cancer Center Anatomic Pathology Division's CoPath database, representing surgical resection specimens obtained between 1999 and 2007. All of the specimens were preserved in 10% buffered formalin prior to embedding in paraffin. Each case was independently reviewed by two pathologists (D.C., M.G.), and a block representative of the tumor was selected for inclusion in the study.
IHC. Four-μm sections were cut with a Leica microtome (Leica Microsystems Inc, Bannockburn, IL, USA) and transferred to adhesive-coated slides. For each block, three slides (one test slide, one positive and one negative control) were stained for p-Akt, using a mouse monoclonal anti-p-Akt antibody. The slides were dewaxed by heating at 55°C for 30 minutes and by three washes, for 5 minutes each, with xylene. Tissues were rehydrated by a series of 5-minute washes in 100%, 95%, and 80% ethanol and distilled water. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 20 minutes. After blocking with universal blocking serum (OmniMap; Ventana Medical Systems, Inc., Tucson, AZ, USA) for 30 minutes, the samples were then incubated with a mouse monoclonal anti-p-Akt antibody (clone 587F11; 1:100 dilution; Cell Signaling Technology, Beverly, MA, USA) at 4°C overnight. This antibody identifies the phosphorylated form of Akt at serine 473. The sections were then incubated with biotin-labeled secondary antibody and streptavidin-horseradish peroxidase for 30 minutes each (Ventana Medical Systems, Inc.). The samples were developed with 3,3′-diaminobenzidine tetrahydrochloride substrate (Ventana Medical Systems, Inc.) and counterstained with hematoxylin (product #760-2021; Ventana Medical Systems, Inc.). The slides were dehydrated and coversliped. Following the manufacturer's recommendations, standard cell conditioning was used for antigen retrieval. Negative controls were included by omitting the p-Akt antibody during the primary antibody incubation.
The p-Akt-stained slides were independently examined by two independent pathologists (M.G., D.C.). If needed, a consensus score was reached for each case. The positive reaction of p-Akt was scored into the following four-grade scale according to the intensity of staining: 0, 1+, 2+ and 3+. The percentages of p-Akt-positive cells were also scored into the following four-grade scale: 0 (0%), 1+ (1-33), 2+ (34-66) and 3+ (more than 66%). In cases with a discrepancy between duplicated scores, the highest score between the two tissue cores was taken as the final score. The product of intensity and percentage scores was used as the final staining score. The final staining score was defined as follows: 0, negative; 1-3, weak; 4-6, moderate and 7-9, strong.
The procedures followed in this study were conducted in accordance with the ethical standards of the responsible committee on human experimentation and with the Declaration of Helsinki.
Statistical analysis. Descriptive statistics for the p-Akt final scores were generated. The Spearman's correlation coefficient was used to assess the correlation between the p-Akt final staining score and tumor grade. All statistical analyses were performed using SAS software (version 9.1).
Results
Patient demographics. The patient demographics are detailed in Table I. The patients, 26 males and 20 females, had an average age of 61 years (range, 34-81 years). All tumors were NETs, as confirmed by the histopathological features and IHC (cytokeratin CAM 5.2 and chromogranin and/or synaptophysin). Average tumor size was 4.5 cm (range, 0.2-11.5 cm). Eighteen tumors were located in the small bowel, nine in the large bowel, one in the stomach, one in the gallbladder, one in the appendix, and sixteen in the pancreas. Seventeen tumors were well-differentiated, fifteen were moderately differentiated and fourteen were poorly differentiated.
IHC results. All p-Akt positively stained cases had cytoplasmic staining. The cytoplasmic staining was diffusely granular with intensity variations within the same lesion of some cases. Cases with variable staining were graded based on the predominant staining intensity and the percentage of positive stain was determined based on the amount of the lesion demonstrating the predominant intensity.
Activation of Akt was detected in 61% (28/46) of the cases. In 39% (18/46) of the tumors, p-Akt was weak (IHC score, 1-3) while in 22% (10/46) of them it was moderate (IHC score, 4-6). Strong p-Akt (IHC score, 7-9) was not seen in any of the tumors studied and 39% (18/46) of NET were p-Akt negative. When tumor grade was considered, of the well-differentiated tumors, five, four and eight cases showed weak, moderate and negative p-Akt staining, respectively. Of the moderately differentiated tumors, two, eight and five cases showed moderate, weak and negative p-Akt staining, respectively. In the poorly differentiated tumors, staining for p-Akt was negative in five cases, weak in six cases and moderate in three cases.
A primary moderately differentiated GI NET staining positively for p-Akt (A), and a metastatic GI NET to the liver showing weak p-Akt staining (B). A primary (C) and a metastatic (D) GI NET negative for p-Akt are also shown (IHC; magnification, ×40).
Statistical analysis. No statistical correlation was found between the p-Akt score and tumor grade (p=0.72), tumor size (p=0.72), and the presence of metastases (p=0.52).
Discussion
Using IHC, the present study demonstrated the activation of p-Akt in a subset of enteropancreatic NETs. Despite the lack of significant correlation with tumor grade, size and the presence of metastases, this finding is relevant as it identifies a potential target for novel gene therapies with inhibitors capable of suppressing this critical signaling transduction pathway.
The involvement of this oncogene in the carcinogenetic process of a variety of cell types is well-known. Akt activation induces tumorigenesis by promoting growth signal autonomy, insensitivity to antiproliferative signals, inhibition of apoptosis, unlimited replicative potential, angiogenesis, invasion and metastasis (26-30).
Recent clinical and laboratory investigations have pointed to Akt activation as a pivotal step during neuroendocrine cell carcinogenesis. It has been shown that the growth of human carcinoid cells in vitro is suppressed by a PI3K-Akt inhibitor. The same compound was also found to inhibit the expression of neuroendocrine markers (human achaete-scute homolog 1- ASCL1 and chromogranin) in the treated cells (31).
There is only one previous clinical report demonstrating the activation of Akt in 76% of 98 NETs of the major subtypes (12). In that study, the authors observed expression of EGFR and of the downstream signaling molecule ERK1/2. They reported the correlation of the p-Akt and p-ERK1/2 histological score with the histological score for activated EGFR. Also, they reported no correlation between p-Akt and tumor grade (12). Similarly, in the present study there was no statistical correlation between the p-Akt score and tumor grade and size and the presence of metastases.
Taken together, these are important findings pointing to a novel molecular pathway of carcinogenesis in NETs that may be the target for new treatment modalities for patients with advanced NET.
It has been shown that aberrant activation of the PI3K/Akt/mTOR pathway increases the expression of cell-cycle regulatory proteins in pancreatic NETs (18, 19, 32) and that the mTOR inhibitors are capable of down-regulating the PI3K/Akt/mTOR pathway decreasing the growth of NETs (33). In one phase II study of patients with progressive NETs treated with the mTOR inhibitor temsirolimus, it was noted that higher baseline tumor levels of mTOR predicted better outcomes (34). In addition, the use of this compound was found to increase the levels of Akt activity. A similar increase in p-Akt has been described with the use of rapamycin, limiting the antitumor activity of this compound (35). Another study has demonstrated that the somatostatin analogue octreotide produces antiproliferative activity on neuroendocrine cells (pituitary tumor cells) by inducing the expression of Zac1, a tumor suppressor gene. The product of this gene induced cell-cycle arrest and apoptosis, by targeting the PI3K/Akt survival pathway (36). It was reported that octreotide treatment decreased the tyrosine phosphorylation levels of the PI3K regulatory subunit p85, induced dephosphorylation of PDK1 and Akt and activated glycogen synthase kinase 3beta (GSKbeta). This is an important finding suggesting that the combination therapy with ocreotide and mTOR inhibitor RAD001 (everolimus) blocks the proliferation and interact with the Akt-mTOR-p70S6K pathway in NET cells (37).
In line with such previous studies, the present finding of p-Akt activation in a subset of enteropancreatic NET suggests the role of p-Akt in NET carcinogenesis and provides a rationale for using p-Akt inhibitors as an alternative adjuvant treatment in a subset of NETs.
Acknowledgements
We thank the Histology Section of the Tissue Core and the Analytic Microscopy Core at the Moffitt Cancer Center and Research Institute for the support in performing and analyzing the IHC stains. We also thank Andrea Dattilo for helping during the preparation of the manuscript.
- Received September 30, 2010.
- Revision received November 3, 2010.
- Accepted November 5, 2010.
- Copyright© 2010 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
