Open Access
AZION, INTERNAL . MED
Insulin-Like Growth Factor-Phosphatidylinositol 3 Kinase Signaling in Canine Cortisol-Secreting Adrenocortical Tumors
M.M.J. Kool, S. Galac, N. van der Helm, S. Corradini, H.S. Kooistra, and J.A. Mol
Background: Hypercortisolism is a common endocrine disorder in dogs, caused by a cortisol-secreting adrenocortical tumor (AT) in approximately 15% of cases. In adrenocortical carcinomas of humans, activation of the phosphatidylinositol 3 kinase (PI3K) signaling pathway by insulin-like growth factor (IGF) signaling represents a promising therapeutic target.
Objectives: To investigate the involvement of PI3K signaling in the pathogenesis of ATs in dogs and to identify pathway components that may hold promise as future therapeutic targets or as prognostic markers.
Animals: Analyses were performed on 36 canine cortisol-secreting ATs (11 adenomas and 25 carcinomas) and 15 normal adrenal glands of dogs.
Methods: mRNA expression analysis was performed for PI3K target genes, PI3K inhibitor phosphatase and tensin homo- log (PTEN), IGFs, IGF receptors, IGF binding proteins and epidermal growth factor receptors. Mutation analysis was per- formed on genes encoding PTEN and PI3K catalytic subunit (PIK3CA).
Results: Target gene expression indicated PI3K activation in carcinomas, but not in adenomas. No amino acid-changing mutations were detected in PTEN or PIK3CA and no significant alterations in IGF-II or IGFR1 expression were detected. In carcinomas, ERBB2 expression tended to be higher than in normal adrenal glands, and higher expression of inhibitor of differentiation 1 and 2 (ID1 and ID2) was detected in carcinomas with recurrence within 2.5 years after adrenalectomy.
Conclusions and Clinical Importance: Based on these results, ERBB2 might be a promising therapeutic target in ATs in dogs, whereas ID1 and 2 might be valuable as prognostic markers and therapeutic targets.
Key words: Adrenal; Dog; Hypercortisolism; Insulin-like growth factor; Phosphatidylinositol 3 kinase.
H ypercortisolism is 1 of the most common endo- crine disorders in dogs.1 Approximately, 15% of spontaneous cases of hypercortisolism in dogs are due to cortisol-secreting adrenocortical adenomas or carci- nomas.1 Therapeutic options for dogs with adrenocorti- cal tumors (ATs) are limited: complete adrenalectomy of the affected adrenal gland is the treatment of choice, provided no metastases are present at the time of pre- sentation.2,3 However, surgery is not possible or suc- cessful in all cases, and tumor recurrence and metastasis occur regularly.4 Options for medical management are limited to mitotane, a chemotherapeutic agent, or trilos- tane, which can only alleviate the clinical signs of hypercortisolism. The lack of reliable prognostic mark- ers further complicates treatment.
A pathway with the potential to provide both thera- peutic targets and prognostic markers is the phosphati- dylinositol-3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling pathway (Fig 1). The PI3K pathway is 1 of the most frequently activated
From the Department of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands (Kool, Galac, van der Helm, Kooistra, Mol); and the University of Bologna, Ozzano dell’Emilia, Italy (Corradini).
All research was performed at the Department of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, Yalelaan 108, 3584 CM, Utrecht, The Netherlands.
Part of this study was presented as a poster at the 2014 ACVIM conference.
Corresponding author: S. Galac, Department of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, Yalelaan 108, 3584 CM Utrecht, The Netherlands; e-mail: s.galac@uu.nl. Submitted August 29, 2014; Revised November 19, 2014; Accepted November 19, 2014.
Copyright @ 2015 by the American College of Veterinary Internal Medicine
DOI: 10.1111|jvim.12528
Abbreviations:
| ACC | adrenocortical carcinoma |
| AT | adrenocortical tumor |
| BCL2L1 | B-cell lymphoma 2 related protein |
| CCND1 | cyclin D1 |
| EGF | epidermal growth factor |
| EGFR | epidermal growth factor receptor |
| ERBB | erythroblastic leukemia viral oncogene homolog |
| GUSB | beta-glucuronidase |
| HPRT | hypoxantine phosphoribosiltransferase |
| ID | inhibitor of differentiation |
| IGFBP | insulin-like growth factor binding protein |
| IGF | insulin-like growth factor |
| IGFR | insulin-like growth factor receptor |
| INSR | insulin receptor |
| mTOR | mammalian target of rapamycin |
| p-AKT | phosphorylated AKT |
| PI3K | phosphatidylinositol 3 kinase |
| PIK3CA | phosphatidylinositol 3 kinase catalytic subunit |
| PTEN | phosphatase and tensin homolog |
| qPCR | quantitative RT-PCR |
| RPS19 | ribosomal protein S19 |
| RPS5 | ribosomal protein S5 |
| SGK1 | serum glucocorticoid regulated kinase 1 |
| SNAI1 | snail |
| SNAI2 | slug |
| SPRP | small proline rich protein |
| TRAIL | tumor necrosis factor superfamily member 10 |
| UTR | untranslated region |
| XIAP | X-linked inhibitor of apoptosis |
signal transduction pathways in cancers of humans, the activation of which also has been documented in ATs in humans.5-7 Pathway activation is initiated by recep- tor tyrosine kinases, such as the type 1 insulin-like growth factor (IGF) receptor (IGFR1) or dimers of the
EGFR ligands
IGFBP2
IGF-I
GF-II
IGFBP5
IGFBP3
IGFBP6
IGFBP4
EĞFR
ErbB2
ErbB3
ErbB4
ĮGFR1
ĮGFR2
PI3K
SGK1
P
P
AKT
PIP3
PIP2
XIAP
P
PTEN
Increased transcription
Decreased transcription
ID1
ID2
BCL2L1
CCND1
SNAI1
SNAI2
TRAIL
Cell survival
Cell proliferation
Cell growth
epidermal growth factor (EGF) receptor family (EGFR, ERBB2-4), and counteracted by the competitive PI3K inhibitor phosphatase and tensin homolog (PTEN). Upon activation of the PI3K pathway, phosphorylated AKT (p-AKT) and its downstream effectors stimulate cell proliferation, survival and growth by transcriptional and posttranslational mechanisms.5
The PI3K pathway contains multiple targets for ther- apeutic intervention, the choice of which depends on the mode of activation. In adrenocortical carcinomas (ACC) of humans, frequent overexpression of IGF-II and IGFRI indicates IGF-signaling as a likely mode for PI3K activation.8-10 Selective IGFR1 kinase inhibitors thus could be of benefit, and indeed have shown antitu- mor effects both in cell culture studies and preclinical and early phase clinical trials in humans with ACC.6,10 For EGFR-induced PI3K pathway activation, several specific inhibitors already have been approved for clini- cal use in humans.11 Activation of the PI3K pathway also may occur downstream of the receptors, for instance as a result of mutations in the genes encoding PTEN or the PI3K catalytic subunit (PIK3CA) or because of decreased expression of PTEN.12,13 In these cases, single or dual inhibitors of PIK3CA and mTOR could be employed.14 In a human ACC cell line, use of these compounds has resulted in decreased cell prolifer- ation and cortisol secretion.15,16
In ACC of humans, IGF-II is the most frequently and strongly overexpressed gene,17,18 whereas in adenomas
overexpression occurs only rarely.18,19 Additionally, high IGF-II expression in ACC of humans is associated with aggressive tumor behavior and increased risk of metasta- sis.20,21 Therefore, in humans IGF-II is a diagnostic and prognostic marker for ATs.
The aim of this study was to investigate involvement of the PI3K signaling pathway in the pathogenesis of cortisol-secreting ATs in dogs, to identify pathway com- ponents that may hold promise as future therapeutic targets or may serve as prognostic markers. Pathway activation was evaluated by means of target gene expression analysis, whereas mRNA expression analysis and mutation analysis were used to indicate mode of activation.
Materials and Methods Patient Material
Patient material used in this study consisted of 36 cortisol- secreting ATs from dogs and 15 whole tissue explants of normal canine adrenal glands. All normal adrenal glands from healthy dogs were available as archived tissue for comparison with AT tis- sue obtained from patients. The tumor group consisted of histo- logically confirmed ATs from patients with clinical signs of hypercortisolism, referred to the Department of Clinical Sciences of Companion Animals of the Faculty of Veterinary Medicine in Utrecht between 2001 and 2012. The diagnosis of ACTH-indepen- dent hypercortisolism because of a cortisol-secreting AT was based upon (1) increased urinary corticoid-to-creatinine ratios, that were
not suppressible with high doses of dexamethasone, (2) suppressed or undetectable basal plasma ACTH concentrations1 and (3) dem- onstration of an AT by ultrasonography, computed tomography or both.22 All dogs subsequently underwent unilateral adrenalec- tomy. The dogs’ ages at the time of surgery ranged from 2 to 12 years (mean, 9 years). Seven dogs were mongrels and the other dogs were of 22 different breeds. Eighteen of the dogs were male (8 castrated) and 18 female (12 spayed).
After resection, all ATs and normal adrenal glands were imme- diately put on ice for inspection, and material was saved for quan- titative RT-PCR (qPCR) analysis and histopathology. Fragments for RNA isolation were snap frozen in liquid nitrogen within 10 minutes after resection and stored at -80℃ until further use. The remaining part of the tissue was immersed in formalin for fix- ation and embedded in paraffin after 24-48 hours. Permission to use the AT tissue for this study was obtained from all patient owners and the study was approved by the Ethical Committee of Utrecht University.
Histopathology
Histopathological evaluation was performed on formalin-fixed and paraffin-embedded tissue slides of all samples and used to con- firm the diagnosis and classify the tumors. All histological evalua- tions were performed by a single pathologist. Classification was performed based on criteria described previously.23 Classification as a carcinoma was based on histological evidence of vascular invasion, peripheral fibrosis, capsular invasion, trabecular growth, hemorrhage, necrosis, and single cell necrosis. Typical histological characteristics of adenomas were hematopoiesis, fibrin thrombi, and cytoplasmic vacuolization. Based on these criteria, the tumor group consisted of 11 adenomas and 25 carcinomas.
Follow-up
Of the dogs in the tumor group, follow-up information was available for 15 dogs with histologically confirmed carcinomas: 7 of these dogs developed signs of hypercortisolism within 2.5 years after surgical removal of the tumor. Recurrence of hypercortiso- lism was confirmed by endocrine testing, and was caused by metastases in 6 of these dogs, and by regrowth of the AT in 1 dog. The remaining 8 dogs were in remission for at least 2.5 years after adrenalectomy.
Total RNA Extraction and Reverse Transcription
Total RNA for quantitative RT-PCR analysis was isolated from the adrenal tissue using the RNeasy mini kit,a according to manufacturer’s protocols. An additional DNAse step was per- formed to avoid genomic DNA contamination. RNA concentra- tions were measured on the NanoDrop ND-1000.b Synthesis of cDNA was performed using the iScript cDNA synthesis kit,“ according to the manufacturer’s protocols. For all samples, 1 cDNA reaction was performed without reverse transcriptase (RT-), to check for contamination with genomic DNA.
Quantitative RT-PCR
Primers for qPCR were designed to detect the mRNA expres- sion levels of PI3K pathway target genes (Table 1), PI3K pathway inhibitor PTEN and major components of the IGF and EGF axis (Table 2). Primer design was performed using Perl-primer v1.1.14 according to the parameters in the Bio-Rad iCycler manual, and primers were ordered from Eurogentec.d For all primer pairs, a temperature gradient was performed to determine the optimal
annealing temperature. Formation of the proper PCR products was confirmed by a sequencing reaction, using the ABI3130XL Genetic analyzere according to the manufacturer’s protocol.
For mRNA expression analysis of the EGF receptors, PI3K target genes and PTEN, a 10x diluted pool of cDNA samples was used to create a 4-fold standard dilution series. The remaining cDNA was diluted 5 times with milliQ water, to achieve a working stock. Reactions were performed on a CFX384 real-time PCR detection system.“ The following genes were measured as follows: EGF receptor (EGFR), erythroblastic leukemia viral oncogene homolog 2-4 (ERBB2-4), snail (SNAII), slug (SNAI2), B-cell lym- phoma 2 related protein (BCL2L1), cyclinD1 (CCND1), inhibitor of differentiation 1 and 2 (ID1 and ID2), tumor necrosis factor superfamily member 10 (TNFSF10 or TRAIL), serum glucocorti- coid regulated kinase 1 (SGK1), X-linked inhibitor of apoptosis (XIAP) and PTEN. To correct for differences in cDNA concentra- tion, ribosomal protein S5 (RPS5), RPS19, small proline rich pro- tein (SPRP) and hypoxantine phosphoribosiltransferase (HPRT) were used as reference genes.24,25
For mRNA expression analysis of IGF-I, IGF-II, IGFR1, IGFR2, IGF binding proteins (IGFBP 2-6) and the insulin recep- tor (INSR), an undiluted pool of cDNA samples was used to cre- ate a 4-fold standard dilution series. The remainder of the cDNA was diluted 10 times with milliQ water, to achieve a working stock. Reactions were performed on a MyIQ single color real-time PCR detection system.“ To correct for differences in cDNA con- centration, RPS5, RPS19, SPRP and beta-glucuronidase (GUSB) were used as reference genes.24,25
Detection was performed using SYBRgreen supermix and data were analyzed using CFX Manager 3.0° for the CFX384 real-time PCR data and using iQ5 software for the MyIQ single color real- time PCR data. The raw data were used to calculate the reaction efficiency. Reaction efficiencies between 90% and 110% were accepted. Analysis of the relative expression levels of the reference genes disclosed no significant differences among groups and refer- ence gene expression was shown to be stable using GeNorm soft- ware,21 justifying the use as reference genes. Calculation of normalized relative expression levels for each of the target genes was performed using the 2-AACT method.26
Mutation Analysis
Mutation analysis was performed on PTEN and PIK3CA. Primers for PCR amplification (Table 3) and sequence primers (Table 4) were designed using Perl-primer v1.1.14 according to BioRad iCycler parameters, and ordered from Eurogentec.d Sequence primers were located along the entire transcript, at a dis- tance of 300-500 bp apart, or closer together when needed for complete coverage. Amplification primers were located in the 3’ and 5’untranslated regions (UTRs) of the gene to ensure amplifica- tion of the complete coding region. For PTEN, the canine 3’ UTR is not annotated and was deduced from the human 3’ UTR and the canine genomic sequence. If a gene could not be amplified in 1 stretch, overlapping primer pairs were used. All amplification primers were tested on a pool of adrenal samples, to determine the optimal annealing temperature. Correct product formation was evaluated by means of gel electrophoresis and sequencing.
After PCR optimization, target genes in all samples were ampli- fied on a C1000 Touch thermal cyclere using Phusion Hot Start Flex DNA Polymerase. The PCR products were amplified for sequencing using the BigDye Terminator version 3.1 Cycle Sequencing Kit® and filtrated using Sephadex G-50 Superfine.h Sequencing reactions were performed on an ABI3130XL Genetic analyzer, according to the manufacturer’s instructions. The sequences obtained were aligned to the NCBI consensus mRNA sequence using DNAstar Lasergene core suite 9.1 SeqMan
| qPCR Primers (Position) | Sequence (5'-3') | Annealing Temperature (°C) | Product Length (bp) |
|---|---|---|---|
| cf_SNAI1 | |||
| Fw | CAA GAT GCA CAT CCG AAG C | 61.6 | 133 |
| Rv | CAG TGG GAG CAG GAA AAC | ||
| cf_SNAI2 | |||
| Fw | CTT CAC TCC GAC TCC AAA CG | 60 | 148 |
| Rv | TGG ATT TTG TGC TCT TGC AG | ||
| cf_BCL2L1 | |||
| Fw | GGG GTG GTG AGG TAC AAA AA | 61.6 | 112 |
| Rv | CTG GGT CTA GCG TCC AAA AG | ||
| cf_ID1 | |||
| Fw | CTC AAC GGC GAG ATC AG | 59.5 | 135 |
| Rv | GAG CAC GGG TTC TTC TC | ||
| cf_ID2 | |||
| Fw | GCT GAA TAA ATG GTG TTC GTG | 60.5 | 114 |
| Rv | GTT GTT CTC CTT GTG AAA TGG | ||
| cf_CCND1 | |||
| Fw | GCC TCG AAG ATG AAG GAG AC | 60 | 117 |
| Rv | CAG TTT GTT CAC CAG GAG CA | ||
| cf_TNFSF10 | |||
| Fw | GCT GAT CCT CAT CTT CAC TG | 62 | 90 |
| Rv | TCC TGC ATC TGC TTC AG | ||
| cf_SGK11 | |||
| Fw | TGG GCC TGA ACG ACT TTA TT | 62 | 124 |
| Rv | GAG GGG TTG GCA TTC ATA AG | ||
| cf_XIAP | |||
| Fw | ACT ATG TAT CÁC TTG AGG CTC TGG TTT C | 54 | 80 |
| Rv | AGT CTG GCT TGA TTC ATC TTG TGT TAT G |
Accession numbers used: SNAI1: XM_543048.1; SNAI2: XM_543048.1; CDH1: XM_536807.3; BCL2L1: NM_001003072.1; ID1: XM_847117.2; ID2: XR_134413.1; CCND1: NM_001005757.1; SGK11: XM_003432525.1; TNFSF10: NM_001130836; XIAP: XM_003435664.1.
PI3K, phosphatidylinositol 3 kinase; SNAI1, snail; SNAI2, slug; BCL2L1, B-cell lymphoma 2 related protein; ID1 and ID2, inhibitor of DNA binding 1 and 2; CCND1, CyclinD1; SGK11, serum/glucocorticoid regulated kinase 1; TNFSF10 or TRAIL, tumor necrosis factor superfamily member 10; XIAP, X-linked inhibitor of apoptosis; Fw, forward primer; Rv, reverse primer.
software. Mutations altering the amino acid sequence were con- firmed by repeat RNA extraction and sequencing in both sense and antisense directions.
Statistical Analyses
Statistical analyses were performed using SPSS20.ª Because of the non-normal distribution of most of the variables, the non- parametric Mann-Whitney U-test was used to compare mRNA expression levels among groups. The relative mRNA expression levels were compared between ATs (adenomas and carcinomas) and normal adrenals and between ATs with and without recurrent disease. For the first comparison, a Bonferroni correc- tion was applied and P < . 025 was considered significant, whereas for the latter comparison, a P < . 05 was considered sig- nificant.
Results
Relative mRNA Expression of PI3K Target Genes
To evaluate whether activation of the PI3K pathway was present in cortisol-secreting ATs of dogs, mRNA expression analysis was performed on a selection of 10 known target genes of the pathway. Based on the litera-
ture, PI3K activation would increase mRNA expression of ID1,27,28 ID2,29 SNAIL and SNAI2,30 CCNDI and BCL2L1.31 In contrast, lower mRNA expression of TRAIL31,32 would be expected (Fig 1). The activation of SGK1 and XIAP occurs by means of phosphoryla- tion; so for these genes, no effect on mRNA expression would be expected33,34 (Fig 1). In ACC of dogs, the rel- ative mRNA expression levels when compared to those in normal adrenal glands (Fig 2A) were significantly higher for ID1 (2.1-fold, P = . 021) and SNAI1 (1.8-fold, P = . 024), and significantly lower for SGK1 (0.5-fold, P = . 009). The relative mRNA expression of TRAIL (0.6-fold, P = . 028) tended to be lower. Thus, in carci- nomas 3 of the 7 transcriptionally regulated target genes showed a change in accordance with pathway activa- tion, whereas the mRNA expression of the other genes remained unchanged.
In adenomas, the mRNA expression of none of the genes showed a significant change consistent with PI3K pathway activation (Fig 2A). The only significant finding was a lower relative expression of CCND1 (0.42-fold, P = . 004) when compared to that of normal adrenal glands.
| qPCR Primers (Position) | Sequence (5'-3') | Annealing Temperature (°C) | Product Length (bp) |
|---|---|---|---|
| cf_EGFR | |||
| Fw | CTG GAG CAT TCG GCA | 53 | 107 |
| Rv | TGG CTT TGG GAG ACG | ||
| cf_ERBB2 | |||
| Fw | CGT GCT GGA CAA TGG AGA CC | 64 | 126 |
| Rv | CCG CTG AAT CAA GAC CCC TC | ||
| cf_ERBB3 | |||
| Fw | TAG TGG TGA AGG ACA ACG GCA G | 64 | 103 |
| Rv | GGT CTT GGT CAA TGT CTG GCA G | ||
| cf_ERBB4 | |||
| Fw | CAG TTC TTG TGT GCG TGC CTG | 70 | 121 |
| Rv | ATG ATC CTG TGC CGA TGC C | ||
| cf_PTEN | |||
| Fw | AGA TGT TAG TGA CAA TGA ACC T | 62 | 101 |
| Rv | GTG ATT TGT GTG TGC TGA TC | ||
| cf_IGF-I | |||
| Fw | TGT CCT CCT CGC ATC TCT T | 58 | 125 |
| Rv | GTC TCC GCA CAC GAA CTG | ||
| cf_IGF-II | |||
| Fw | CTT CTG GAG ACC TAC TGT GC | 61 | 128 |
| Rv | CTG CTT CCA GGT GTC GTA TTG | ||
| cf_IGFR1 | |||
| Fw | CAT GCC TTG GTC TCC CTG T | 60 | 129 |
| Rv | GGT GGT CCC AAT CCC AAA G | ||
| cf_IGFR2 | |||
| Fw | GAG TTC AGC CACG AGA C | 54 | 94 |
| Rv | GCA TTG TCA CCA TCA AGG | ||
| cf_IGFBP2 | |||
| Fw | GAT CTC CAC CAT GCA CCT TC | 60 | 127 |
| Rv | GCT GCC CGT TCA GAG ACA TCT TG | ||
| cf_IGFBP3 | |||
| Fw | CTG CAC ACG AAG ATG GAT GT | 61 | 127 |
| Rv | TAT TCC GTC TCC CGC TTG TA | ||
| cf_IGFBP4 | |||
| Fw | AGC CTG CAG CCC TCT GAC A | 59 | 120 |
| Rv | TGG TGC TGC GGT CTC GAA T | ||
| cf_IGFBP5 | |||
| Fw | TCG CAG AAA GAA GCT GAC C | 60 | 131 |
| Rv | GAA GCC TCC ATG TGT CTG C | ||
| cf_IGFBP6 | |||
| Fw | CAA TCC TGG TGG TGT CC | 54 | 136 |
| Rv | AGA AGC CCT TAT GGT CAC | ||
| cf_INSR | |||
| Fw | GTG ACA GAC TAT TTA GAT GTC CC | 60 | 166 |
| Rv | ACT CAG GGT TTG AAG AAG C |
Accession numbers used: EGFR: XM_533073.3; ERBB2: NM_001003217.1; ERBB3: XM_538226.4; ERBB4: XM_003640190.2; PTEN: NM_001003192.1; IGF-I: XM_848024.1; IGF-II: XM_858107.1; IGFR1: XM_853622.1; IGF-IIR: NM_001122602; IGFBP2: XM_545637.2; IGFBP3: XM_548740.2; IGFBP4: XM_845091.1; IGFBP5: XM_847792.1; IGFBP6: XM_844250; INSR: XM_542108.
EGFR, epidermal growth factor receptor; ERBB2-4, erythroblastic leukemia viral oncogene homolog 2-4; PTEN, phosphatase and ten- sin homolog; IGF-I and IGF-II, insulin-like growth factor 1 and 2; IGFR1 and IGFR2, IFG receptor type 1 and 2; IGFBP 2-6, IGF bind- ing protein 2-6; INSR, insulin receptor; Fw, forward primer; Rv, reverse primer.
When comparing dogs with recurrent disease within 2.5 years to dogs remaining in remission (Fig 2B), a sig- nificantly higher expression of both ID1 (2.0-fold, P = . 033) and ID2 (2.4-fold, P = . 019) was detected in dogs with recurrent disease. No significant changes were detected in the expression of other target genes.
Relative mRNA Expression of PTEN and the Components of IGF and EGF Signaling
To evaluate for a potential cause of PI3K activation, mRNA expression levels of the components of the IGF axis, the EGF receptors and PI3K-inhibitor PTEN were
| PCR Primers (Position) | Sequence (5'-3') | Annealing Temperature (°℃) | Product Length (bp) |
|---|---|---|---|
| cf_PTEN | |||
| Fw75 | TCC TCC TTC CTC TCC AG | 55 | 748 |
| Rv822 | TGA ACT TGT CTT CCC GTC | ||
| Fw718 | CAA TGT TCA GTG GCG GA | 55 | 743 |
| Rv1460 | CGA GAT TGG TCA GGA AGA G | ||
| cf_PIK3CA | |||
| F1 | TTT CTG CTT TGG GAC AGC | 55 | 1536 |
| R1537 | CTG GGA ACT TTA CCA CAC TG | ||
| Fw1438 | TGC TGA ACC CTA TTG GTG | 55 | 449 |
| Rv1887 | TAC AGT CCA GAA GCT CCA | ||
| Fw1516 | GCA GTG TGG TAA AGT TCC | 55 | 1843 |
| Rv3359 | CAG TCT TTG CCT GTT GAC | ||
Accession numbers used: PTEN: NM_001003192.1; PIK3CA: XM_545208.3. Fw, forward primer; Rv, reverse primer.
| Sequence Primers (Position) Sequence (5'-3') | |
|---|---|
| cf_PTEN | |
| Fw474 | CAC TGT AAA GCT GGA AAG GG |
| Fw1157 | TGT AGA GGA GCC ATC AAA CC |
| Rv249 | CAA AGG GTT CAT TCT CTG GG |
| Rv595 | TGT CTC TGG TCC TTA CTT CC |
| Rv1285 | CCT GTA TAC GCC TTC AAG TC |
| cf_PIK3CA | |
| Fw375 | GTA ATT GAG CCA GTA GGC |
| Fw707 | CAA CCA TGA CTG TGT TCC |
| Fw1153 | TCT ATC ATG GAG GAG AAC CC |
| Fw1483 | CTC CAT GCT TAG AGT TGG AG |
| Fw1767 | CTA CCC AAA CTG CTT CTG |
| Fw2183 | TAG GCA AGT TGA GGC TAT GG |
| Fw2582 | TGG TTG TCT GTC AAT CGG |
| Fw2806 | TGG GAA TTG GAG ATC GTC |
| Fw2963 | GAT TAG TAA AGG AGC CCA GG |
| Rv358 | AAA GCC GAA GGT CAC AAA GC |
| Rv730 | GTT CTG GAA CAC AGT CAT GG |
| Rv1236 | TAG CCA TTC ATT CCA CCT GG |
| Rv1750 | CAC AAT AGT GTC TGT GGC TC |
| Rv2205 | TTC CAT AGC CTC AAC TTG CC |
| Rv2642 | GTG TGA GAA TTT CGC ACC |
| Rv2993 | TTT GTG CAT TCC TGG GCT |
| Rv3271 | CAT GCT GCT TAA TGG TGT GG |
Accession numbers used: PTEN: NM_001003192.1; PIK3CA: XM_545208.3.
Fw, forward primer; Rv, reverse primer.
evaluated. Analysis of the components of the IGF axis in carcinomas (Fig 3A) disclosed a significantly higher expression of IGFBP2 (5.8-fold, P = . 001) and a signifi- cantly lower expression of IGFBP5 (0.5-fold, P = . 001). Likewise, in adenomas, IGFBP2 expression was
significantly higher (7.2-fold, P = . 013) and IGFBP5 expression was significantly lower (0.4-fold, P < . 001). No significant differences in the expression levels of IGF-II or the IGFR1 were detected.
Analysis of the EGF receptors in carcinomas identi- fied a tendency to higher ERBB2 expression (1.7-fold, P = . 027) and significantly lower expression of ERBB3 (0.4-fold, P = . 003). In adenomas, the only significant change was lower ERBB3 expression (0.2-fold, P = . 001).
When comparing dogs with recurrent disease within 2.5 years to dogs remaining in remission (Fig 3B), sig- nificantly higher expression of IGF-I (2.7-fold, P = . 042), IGFBP5 (6.8-fold, P = . 042) and INSR (2.3- fold, P = . 040) was detected.
Mutation Analysis of PTEN and PIK3CA
To determine whether inactivating mutations of PTEN or activating mutations of PIK3CA might be responsible for activation of the PI3K pathway, muta- tion analysis was performed. Mutation analysis of PTEN identified the presence of 1 silent mutation in codon 325 (CTC>CTT), which was present in 6 ATs and occurred in both homo- and hetero-zygous form. No amino acid-changing mutations were detected in of any of the ATs.
Mutation analysis of PIK3CA identified the presence of 3 different heterozygous silent mutations (codon 149 CCA>CCC, codon 438 TCT>TCA and codon 842 GTG>GTT) in 3 different ATs. No amino acid-chang- ing mutations were detected in any of the ATs.
Discussion
In this study, we aimed to investigate activation of the PI3K pathway in cortisol-secreting adrenocortical adenomas and carcinomas of dogs, to identify both potential therapeutic targets and prognostic markers for use in dogs with ATs. The presence of PI3K activation
A
7
Adenomas
6
Carcinomas
*
Relative expression
5
4
*
3
*
2
*
1
0
SNAI1
SNAI2
BCL2L1
CCND1
ID1
ID2
TRAIL
XIAP
SGK1
B
7
Non-recurrent
6
Recurrent
Relative expression
5
*
4
3
*
2
1
0
SNAI1
SNAI2
BCL2L1
CCND1
ID1
ID2
TRAIL
XIAP
SGK1
was assessed by means of target gene expression analy- sis. In adenomas, none of the target genes showed a sig- nificant change consistent with PI3K pathway activation, and there was even a significantly lower expression of CCND1. This may be explained by the fact that adrenocortical adenomas, based on histological characteristics and tumor behavior, are a highly differ- entiated tumor type, and repression of CCNDI expres- sion is thought to be a hallmark of cell differentiation.35 In line with this reasoning, lower CCND1 expression was detected in adrenocortical adenomas of humans when compared to carcinomas.36 In contrast, in ACC of dogs all target genes that showed significant alter- ation in mRNA expression were altered in accordance with PI3K activation.
The main modes of activation of the PI3K pathway are by intracellular alterations of signaling pathway components or through receptor tyrosine kinase signal- ing. With regard to intracellular pathway alterations, activating mutations of PIK3CA and inactivating muta- tions of PTEN are well-documented.14 In this study, no amino acid-changing mutations were detected in either PIK3CA or PTEN and no overall decreased expression of PTEN was detected in ATs.
With regard to receptor tyrosine kinase induced activa- tion, 1 of the most strongly documented changes that can activate the PI3K pathway in ACC of humans, is activa- tion of the IGF axis. Several studies have demonstrated that in ACC of humans, IGF-II is the most overexpressed gene.19,37 In the healthy individual, 1 of the alleles of the
A 25
20
*
Adenomas
*
15
Carcinomas
10
8
7
Relative expression
6
5
4
3
*
2
*
*
1
*
*
0
EGFR
ERBB2
ERBB3
ERBB4
IGFR1
IGFR2
INSR
IGF-I
IGF-II
IGFBP2
IGFBP3
IGFBP4
IGFBP5
IGFBP6
PTEN
B
60
50
Non-recurrent
40
30
Recurrent
20
10
8
7
Relative expression
6
5
*
3
*
*
2
1
0
EGFR
ERBB2
ERBB3
ERBB4
IGFR1
IGFR2
INSR
IGF-I
IGF-II
IGFBP2
IGFBP3
IGFBP4
IGFBP5
IGFBP6
PTEN
IGF-II locus is epigenetically silenced postnatally, but in ACC genetic and epigenetic alterations in the 11p15 locus cause both alleles to be active, resulting in IGF-II overex- pression.17 Likewise, high IGF-II protein expression is characteristic of ACC in humans.17,18 The mechanism by which high IGF-II expression leads to activation of the PI3K pathway is by binding IGFR1, which like IGF-II fre- quently shows increased expression in ACC of humans.8,9 Taken together, these data suggest that in ACC of humans changes in the IGF axis could be responsible for PI3K acti- vation. This notion is supported by studies in H295R human adrenocortical carcinoma cells, in which IGF-II - IGFR1 signaling results in an increase in p-AKT.6,7,38 Remarkably, this study did not identify a higher expression of either IGF-II or IGFR1 in ATs of dogs.
For IGFBP2, higher mRNA expression was detected in adenomas and carcinomas of dogs. The IGFBPs function as regulatory components of IGF signaling,39 and some studies have indicated that high IGFBP2 expression may contribute to ACC pathogenesis. In murine Y1 AT cells, long-term increased IGFBP2 expression enhanced the malignant phenotype40 and in IGF-II-overexpressing ACC of humans increased expression of IGFBP2 has been reported.20 However, the significance of high IGFBP2 expression in the absence of IGF-II overexpression is unknown. Theoretically, high IGFBP2 expression could lead to higher IGF-II availability and thus increase IGFR1/ PI3K signaling. However, the fact that IGFBP2 overex- pression also was detected in adenomas in the absence
of increased target gene expression does not support a functional role of IGFBP2 overexpression in PI3K acti- vation in dogs.
In contrast, IGFBP5 showed an overall lower expres- sion in ATs, but a 6.8-fold higher expression in those carcinomas showing recurrence within 2.5 years after adrenalectomy. In different types of cancer in humans, IGFBP5 overexpression has been noted as a prognostic marker. In particular, in breast cancer, high IGFBP5 expression is associated with a shorter recurrence-free, metastasis-free and overall survival and IGFBP5 overex- pression in breast cancer cell lines conferred resistance to IGFR1 inhibition.41 Our results therefore indicate the mRNA expression of IGFBP5 as a relevant prog- nostic factor in ACC in dogs.
An alternative mechanism for receptor tyrosine kinase- induced PI3K pathway activation is EGFR signaling. In this respect, the tendency toward higher ERBB2 mRNA expression in ACC of dogs is interesting. ERBB2 (also known as HER2) is a receptor tyrosine kinase that lacks a ligand-binding domain, and functions by heterodimer- ization with the other EGF receptors. Aberrant ERBB2 activation, for instance because of receptor overexpres- sion, results in activation of the PI3K pathway, growth stimulation and tumorigenesis in different tumor types, of which ERBB2-positive breast cancer is the most prom- inent example.11,42 Different drugs targeting ERBB2 have been approved for clinical use in cancer treatment, including antibodies that inhibit heterodimerization (trastuzumab and pertuzumab43,44) and tyrosine kinase inhibitors that affect both EGFR (ie, ERBB1) and ERBB2 activity (lapatinib and afatinib45,46). Recent stud- ies suggest that simultaneously targeting EGFR and ERBB2 further increases antitumor activity.11 Based on the higher expression of ERBB2, this receptor might be a promising new therapeutic target in dogs with ACC, either singly or in combination with EGFR inhibition.
Aside from therapeutic targets, we also aimed to investigate the pathway components for potential new prognostic markers. In this regard, aside from IGFBP5, SGK1, ID1 and ID2 also are worth mentioning. The rel- ative mRNA expression of SGK1 was significantly lower in carcinomas. At first glance, this seems surpris- ing, because SGK1 is activated by PI3K signaling only at the protein level, by phosphorylation.33 Recently, however, SGK1 microdeletions and low SGK1 mRNA expression have been reported in cortisol-secreting ATs of humans, but not in nonsecreting or aldosterone- secreting ATs.47-49 Low SGK1 protein expression was found to be an independent prognostic factor for shorter overall survival.48 Although this study did not identify significant differences in SGK1 mRNA expres- sion between dogs with and without recurrent disease, low SGK1 expression in cortisol-secreting carcinomas does suggest a functional role for SGK1. Additional studies involving SGK1 protein expression and overall survival analyses are needed to determine whether SGK1 expression might prove to be a prognostic mar- ker in these tumors.
In the group of dogs with recurrent disease, we did find an increase in both ID1 and ID2 expression. This
observation is in accordance with studies reporting an association between high ID1 and ID2 expression and poor prognosis in several different tumor types in humans.5º Inhibitor of differentiation proteins are thought to keep cells in a poorly differentiated, prolifer- ative state, and therefore a role for ID1 and 2 in the pathogenesis of malignant ATs in dogs appears likely. Based on our results, ID1 and 2 show promise as new prognostic markers for ACC in dogs. Future studies are needed to determine whether targeting IDs might also be feasible as a new therapeutic option.51,52
In conclusion, our results suggest the presence of PI3K activation in cortisol-secreting ACC in dogs, but not in adenomas. In contrast to 1 of the most prominent fea- tures of ACC in humans, no significant alterations in IGF-II or IGFR1 expression were detected. Therefore, our results suggest that inhibition of IGF signaling is not likely to prove successful in dogs with ATs. However, we did find higher expression of ERBB2, providing a preclin- ical rationale for studying the potential of ERBB2 inhibi- tion, as used in ERBB2-positive breast cancer, in dogs with ACC. Finally, the lower expression of SGK1 in car- cinomas and the higher expression of IGFBP5, ID1, and ID2 in ATs with early recurrence may represent an important step in the search for prognostic markers for cortisol-secreting ACC in dogs.
Footnotes
a Quiagen, Hilden, Germany
b NanoDrop Technologies, Wilmington, DE
” Bio-Rad, Hercules, CA
d Eurogentec, Maastricht, The Netherlands
e AB Applied biosystems, Carlsbad, CA f New England BioLabs Inc., Ipswich, MA
g Applied Biosystems, Foster City, CA
h Amersham, Buckinghamshire, UK IBM, Armonk, NY
Acknowledgments
This study was financially supported by a Morris Animal Foundation - Pfizer Animal Health veterinary fellowship for advanced study (grant ID: D09CA-913). The authors thank Dr. S. Klarenbeek for performing the histopathological evaluation of all ATs.
Conflict of Interest Declaration: The authors disclose no conflict of interest.
Off-label Antimicrobial Declaration: The authors declare no off-label use of antimicrobials.
References
1. Galac S, Reusch CE, Kooistra HS, Rijnberk A. Adrenals. In: Rijnberk A, Kooistra HS, eds. Clinical Endocrinology of Dogs and Cats. Second, revised and extended edition ed. Hannover: Schlütersche; 2010:93-154.
2. Schwartz P, Kovak JR, Koprowski A, et al. Evaluation of prognostic factors in the surgical treatment of adrenal gland
tumors in dogs: 41 Cases (1999-2005). J Am Vet Med Assoc 2008;232:77-84.
3. van Sluijs FJ, Sjollema BE, Voorhout G, et al. Results of adrenalectomy in 36 dogs with hyperadrenocorticism caused by adreno-cortical tumour. Vet Q 1995;17:113-116.
4. Barrera JS, Bernard F, Ehrhart EJ, et al. Evaluation of risk factors for outcome associated with adrenal gland tumors with or without invasion of the caudal vena aava and treated via adrenal- ectomy in dogs: 86 Cases (1993-2009). J Am Vet Med Assoc 2013;242:1715-1721.
5. Martini M, De Santis MC, Braccini L, et al. PI3K/AKT Sig- naling pathway and cancer: An updated review. Ann Med 2014;46:372-383.
6. Barlaskar FM, Spalding AC, Heaton JH, et al. Preclinical targeting of the type I insulin-like growth factor receptor in adrenocortical carcinoma. J Clin Endocrinol Metab 2009;94: 204-212.
7. Fassnacht M, Weismann D, Ebert S, et al. AKT is highly phosphorylated in pheochromocytomas but not in benign adreno- cortical tumors. J Clin Endocrinol Metab 2005;90:4366-4370.
8. Kamio T, Shigematsu K, Kawai K, Tsuchiyama H. Immu- noreactivity and receptor expression of insulinlike growth factor I and insulin in human adrenal tumors. An immunohistochemical study of 94 cases. Am J Pathol 1991;138:83-91.
9. Weber MM, Auernhammer CJ, Kiess W, Engelhardt D. Insulin-like growth factor receptors in normal and tumorous adult human adrenocortical glands. Eur J Endocrinol 1997;136:296-303.
10. Almeida MQ, Fragoso MC, Lotfi CF, et al. Expression of insulin-like growth factor-II and its receptor in pediatric and adult adrenocortical tumors. J Clin Endocrinol Metab 2008;93: 3524-3531.
11. Tebbutt N, Pedersen MW, Johns TG. Targeting the ERBB family in cancer: Couples therapy. Nat Rev Cancer 2013;13:663-673.
12. Martini M, Ciraolo E, Gulluni F, Hirsch E. Targeting PI3K in cancer: Any good news? Front Oncol 2013;3:108.
13. Hopkins BD, Hodakoski C, Barrows D, et al. PTEN func- tion: The long and the short of it. Trends Biochem Sci 2014;39:183-190.
14. Willems L, Tamburini J, Chapuis N, et al. PI3K and mTOR signaling pathways in cancer: New data on targeted thera- pies. Curr Oncol Rep 2012;14:129-138.
15. De Martino MC, van Koetsveld PM, Feelders RA, et al. The role of mTOR inhibitors in the inhibition of growth and corti- sol secretion in human adrenocortical carcinoma cells. Endocr Relat Cancer 2012;19:351-364.
16. Doghman M, Lalli E. Efficacy of the novel dual PI3-kinase/ mTOR inhibitor NVP-BEZ235 in a preclinical model of adreno- cortical carcinoma. Mol Cell Endocrinol 2012;364:101-104.
17. Gicquel C, Raffin-Sanson ML, Gaston V, et al. Structural and functional abnormalities at 11p15 are associated with the malignant phenotype in sporadic adrenocortical tumors: Study on a series of 82 tumors. J Clin Endocrinol Metab 1997;82:2559- 2565.
18. Schmitt A, Saremaslani P, Schmid S, et al. IGFII and MIB1 immunohistochemistry is helpful for the differentiation of benign from malignant adrenocortical tumours. Histopathology 2006;49:298-307.
19. Giordano TJ, Thomas DG, Kuick R, et al. Distinct tran- scriptional profiles of adrenocortical tumors uncovered by DNA microarray analysis. Am J Pathol 2003;162:521-531.
20. Boulle N, Logie A, Gicquel C, et al. Increased levels of insulin-like growth factor II (IGF-II) and IGF-binding protein-2 are associated with malignancy in sporadic adrenocortical tumors. J Clin Endocrinol Metab 1998;83:1713-1720.
21. Gicquel C, Boulle N, Logie A, et al. Involvement of the IGF system in the pathogenesis of adrenocortical tumors. Ann Endocrinol (Paris) 2001;62:189-192.
22. Galac S, Kool MM, Naan EC, et al. Expression of the ACTH Receptor, steroidogenic acute regulatory protein, and ste- roidogenic enzymes in canine cortisol-secreting adrenocortical tumors. Domest Anim Endocrinol 2010;39:259-267.
23. Labelle P, Kyles AE, Farver TB, De Cock HE. Indicators of malignancy of canine adrenocortical tumors: Histopathology and proliferation index. Vet Pathol 2004;41:490-497.
24. Brinkhof B, Spee B, Rothuizen J, Penning LC. Develop- ment and evaluation of canine reference genes for accurate quanti- fication of gene expression. Anal Biochem 2006;356:36-43.
25. Schlotter YM, Veenhof EZ, Brinkhof B, et al. A GeNorm algorithm-based selection of reference genes for quantitative real- time PCR in skin biopsies of healthy dogs and dogs with atopic dermatitis. Vet Immunol Immunopathol 2009;129:115-118.
26. Livak KJ, Schmittgen TD. Analysis of relative gene expres- sion data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 2001;25:402-408.
27. Perk J, Iavarone A, Benezra R. Id family of helix-loop-helix proteins in cancer. Nat Rev Cancer 2005;5:603-614.
28. Birkenkamp KU, Essafi A, van der Vos KE, et al. FOXO3a induces differentiation of Bcr-Abl-transformed cells through transcriptional down-regulation of Id1. J Biol Chem 2007;282:2211-2220.
29. Belletti B, Prisco M, Morrione A, et al. Regulation of Id2 gene expression by the insulin-like growth factor I receptor requires signaling by phosphatidylinositol 3-kinase. J Biol Chem 2001;276:13867-13874.
30. Lau MT, Leung PC. The PI3K/Akt/mTOR signaling path- way mediates insulin-like growth factor 1-induced E-cadherin down-regulation and cell proliferation in ovarian cancer cells. Can- cer Lett 2012;326:191-198.
31. Lam EW, Francis RE, Petkovic M. FOXO transcription factors: Key regulators of cell fate. Biochem Soc Trans 2006;34:722-726.
32. Allen JE, El-Deiry WS. Regulation of the human TRAIL gene. Cancer Biol Ther 2012;13:1143-1151.
33. Firestone GL, Giampaolo JR, O’Keeffe BA. Stimulus- dependent regulation of serum and glucocorticoid inducible protein kinase (SGK) transcription, subcellular localization and enzymatic activity. Cell Physiol Biochem 2003;13:1-12.
34. Dan HC, Sun M, Kaneko S, et al. Akt phosphorylation and stabilization of X-linked inhibitor of apoptosis protein (XIAP). J Biol Chem 2004;279:5405-5412.
35. Klein EA, Assoian RK. Transcriptional regulation of the cyclin D1 gene at a glance. J Cell Sci 2008;121:3853-3857.
36. Pereira SS, Morais T, Costa MM, et al. The emerging role of the molecular marker p27 in the differential diagnosis of adre- nocortical tumors. Endocr Connect 2013;2:137-145.
37. de Fraipont F, El Atifi M, Cherradi N, et al. Gene expres- sion profiling of human adrenocortical tumors using complemen- tary deoxyribonucleic acid microarrays identifies several candidate genes as markers of malignancy. J Clin Endocrinol Metab 2005;90:1819-1829.
38. Cantini G, Lombardi A, Piscitelli E, et al. Rosiglitazone inhibits adrenocortical cancer cell proliferation by interfering with the IGF-IR intracellular signaling. PPAR Res 2008;2008:904041.
39. Ribeiro TC, Latronico AC. Insulin-like growth factor sys- tem on adrenocortical tumorigenesis. Mol Cell Endocrinol 2011;351:96-100.
40. Hoeflich A, Fettscher O, Lahm H, et al. Overexpression of insulin-like growth factor-binding protein-2 results in increased tumorigenic potential in Y-1 adrenocortical tumor cells. Cancer Res 2000;60:834-838.
41. Becker MA, Hou X, Harrington SC, et al. IGFBP ratio confers resistance to IGF targeting and correlates with increased invasion and poor outcome in breast tumors. Clin Cancer Res 2012;18:1808-1817.
42. Hynes NE, MacDonald G. ErbB receptors and signaling pathways in cancer. Curr Opin Cell Biol 2009;21:177-184.
43. Hudis CA. Trastuzumab-Mechanism of action and use in clinical practice. N Engl J Med 2007;357:39-51.
44. Barthelemy P, Leblanc J, Goldbarg V, et al. Pertuzumab: Development beyond breast cancer. Anticancer Res 2014;34:1483- 1491.
45. Reid A, Vidal L, Shaw H, de Bono J. Dual inhibition of ErbB1 (EGFR/HER1) and ErbB2 (HER2/neu). Eur J Cancer 2007; 43:481-489.
46. Spicer JF, Rudman SM. EGFR inhibitors in non-small cell lung cancer (NSCLC): The emerging role of the dual irreversible EGFR/HER2 inhibitor BIBW 2992. Target Oncol 2010;5:245-255.
47. Ronchi CL, Leich E, Sbiera S, et al. Single nucleotide poly- morphism microarray analysis in cortisol-secreting adrenocortical adenomas identifies new candidate genes and pathways. Neoplasia 2012;14:206-218.
48. Ronchi CL, Sbiera S, Leich E, et al. Low SGK1 expression in human adrenocortical tumors is associated with ACTH-inde- pendent glucocorticoid secretion and poor prognosis. J Clin Endo- crinol Metab 2012;97:E2251-2260.
49. Ronchi CL, Sbiera S, Leich E, et al. Single nucleotide polymorphism array profiling of adrenocortical tumors-Evidence for an adenoma carcinoma sequence? PLoS One 2013;8:e73959.
50. Nair R, Teo WS, Mittal V, Swarbrick A. ID proteins regu- late diverse aspects of cancer progression and provide novel thera- peutic opportunities. Mol Ther 2014;000:000-000.
51. Sumida T, Murase R, Onishi-Ishikawa A, et al. Targeting Id1 reduces proliferation and invasion in aggressive human sali- vary gland cancer cells. BMC Cancer 2013;13:141.
52. Fong S, Debs RJ, Desprez PY. Id genes and proteins as promising targets in cancer therapy. Trends Mol Med 2004;10:387- 392.