Current and Future Medical Therapy, and the Molecular Features of Adrenocortical Cancer
Alicja Hubalewska-Dydejczyk*, Agata Jabrocka-Hybel, Dorota Pach, Aleksandra Gilis- Januszewska, and Grzegorz Sokołowski
Department of Endocrinology, Medical College, Jagiellonian University, 31-501 Krakow, Poland
Received: February 22, 2011; Accepted: March 28, 2011; Revised: August 22, 2011
Abstract: Adrenocortical carcinoma (ACC) is a rare neoplasm with very poor prognosis despite the recent development of aggressive antitumor therapies. The cause of adrenal cancer remains elusive, but some molecular mechanisms could be responsible for its development. Target-specific therapies have been developed for a number of human malignancies and have resulted in therapeutic benefits in some cancer patients. However, these therapies are only effective in cases in which the corresponding targets are expressed in tumor tissues. Molecular analysis has had a significant impact on the under- standing of the pathogenetic mechanism of ACC development and the evaluation of prognostic and predictive markers, among which alterations of the IGF system, the Wnt pathway, p53 and molecules involved in cancer cell invasion proper- ties and angiogenesis seem to be very promising. These molecular markers may not just play a role in the biology of these tumors and have prognostic implications, but can also be used as potential targets for treatment. The aim of this review is to summarize the genetic and molecular events implied in the pathogenesis of ACC and to highlight challenges to the de- velopment of anticancer agents in recent patents.
Keywords: Adrenocortical cancer, growth factors, insulin growth factor 1, molecular factors, targeted therapy, Wnt pathway.
INTRODUCTION
Adrenocortical tumors (ACT) are common tumors with a prevalence of at least 3% in the population over the age of 50 [1]. In contrast, adrenocortical cancer (ACC) is rare, with an estimated incidence of between 1 and 2 per million per year in adults in Europe and North America, and of very poor prognosis [2, 3]. Age distribution shows two peaks, the first occurring in early childhood, and the second between the ages of 40 and 50. There is a slightly higher ratio in females. Although most ACCs are of sporadic origin, they may also be part of a congenital and/or familial disease. The cause of adrenal cancer remains elusive, but some molecular mecha- nisms could be responsible for its development. Adrenal cancer occasionally develops in families with susceptibility to other types of neoplasms. Environmental factors have also been suggested to be significant in the development of ACC [4-6].
The clinical features of sporadic ACC are provoked by hypersecretion and/or tumor mass. Approximately, 50% of ACCs in adults and 90% in pediatric patients are function- ing. Cortisol hypersecretion is the most common endocrine presentation with rapidly progressing Cushing’s syndrome. Less frequently, tumors produce androgens and/or steroid precursors. Other biologically active steroids may also be oversecreted (mineralocorticoids - deoxycorticosterone DOC). Non-secretory ACCs are discovered by local symp- toms (abdominal discomfort, nausea, vomiting, abdominal
fullness, back pain) and distant metastases provoked by the tumor. The symptoms of non-functioning ACC usually occur in the very late stages and this is the reason why the tumor may be diagnosed as an advanced and metastatic disorder. Rarely, ACC is diagnosed during the diagnostic work-up for an adrenal incidentaloma. Other specific features may be associated with rare genetic diseases such as Li-Fraumeni and Wiedemann-Beckwith syndromes, where ACC is part of a more complex syndrome [7-10].
Careful endocrine assessment is mandatory prior to sur- gery in ACC (hormonal oversecretion, tumor markers). It is very important to exclude pheochromocytoma prior to sur- gery because imaging often cannot reliably differentiate be- tween ACC and pheochromocytoma. No marker has been shown to be specific and sensitive enough for the diagnosis of ACC [8, 11].
The size of the adrenal mass, as measured by CT (com- puted tomography) or MRI (magnetic resonance imaging) remains one of the best indicators of malignancy. According to the National Institutes of Health consensus conference, tumors larger than 6 cm are highly indicative of malignancy and should be removed. ACCs are inhomogeneous with ir- regular margins, hemorrhage, necrosis and irregular en- hancement of solid components. Sometimes calcifications are visible. Adrenal lesions with an attenuation value of more than 10HU (Hounsfield Units) in unenhanced CT or an enhancement washout of less than 50% and a delayed at- tenuation of more than 35HU (on 10- to 15-min delayed en- hanced CT) are indicative of malignancy [12-15].
Radiocholesterol scintigraphy, using 131 I-6-beta- iodomethylnorcholesterol (NP-59), has been used to deter-
*Address correspondence to this author at the Department of Clinical Endo- crinology, Jagiellonian University, Medical College, ul. Kopernika 17, 31- 501, Kraków, Poland; Tel: +48 (12) 424 75 00, +48 (12) 424 75 20; Fax: +48 (12) 424 73 99; E-mail: alahub@cm-uj.krakow.pl
mine whether an adrenal cortical tumor is benign. Decreased or no uptake of NP-59 is more likely in a cancer, and the majority of ACCs have no NP-59 uptake [16]. In both ani- mal and clinical studies 123I-metomidate revealed to be a suitable SPECT-tracer for detection of adrenocortical tissue and differential diagnosis of adrenal masses [17].
Recognizing the malignant nature of an adrenal cortical tumor often remains a challenge even for experienced pa- thologists. Pathologists have designed paradigms using a combination of various histological parameters. The most widely used is the Weiss score, composed of 9 different items. A score above 3 is most likely associated with a ma- lignant tumor [18-20].
ACC remains a disease with very poor prognosis. The highest probability of cure occurs when a localized tumor (stage 1 or 2 MacFarlane) can be subjected to complete re- moval (“curative” surgery). Almost half of patients are diag- nosed at later stages (invasive or metastatic disease) with a survival rate close to 0% at five years for metastatic ACC. The overall survival for patients with ACC is 38% at 5 years. When stratified for stage, it is 66% for stage 1 tumors, 58% for stage 2 tumors, 24% for stage 3 disease, and 0% for stage 4 disease. Clinically, it is accepted that curative resection, recent diagnosis and local stage give a more favorable prog- nosis. Even after “curative surgery” of localized ACC, recur- rence occurs in more than 50% of patients, even as many as 60-80%. The high relapse rate following radical operation offers a rationale for the use of adjuvant systemic therapies. Postoperative treatment with mitotane was associated with a better outcome in a recently published case-control study [8, 21-24].
The strongest molecular predictors of disease-free sur- vival are three molecular markers: 17p13 loss of heterozy- gosity, 11p15.5 loss of heterozygosity and IGF2 (Insulin Growth Factor) gene overexpression in ACC cases [10, 25]. New insights into ACC pathophysiology have made it possi- ble to test agents targeting epidermal growth factor receptor (EGFR), insulin growth factor receptor (IGFR), vascular endothelial growth factor (VEGF) and the vascular endothe- lial growth factor receptor (VEGFR) and in the future proba- bly Wnt/B-catenin pathway and others more specific path- ways. Several studies with these drugs are currently under- way [26, 27]. Advancement in the understanding of the pathophysiology of ACCs is essential for the development of more sensitive means of diagnosis and treatment, resulting in better clinical outcome.
The aim of this review is to summarize the currently ex- isting therapy, genetic and molecular mechanisms implied in the pathogenesis of ACC and to highlight challenges to the development of anticancer agents in recent patents.
1. CURRENTLY EXISTING TREATMENT
1.1. Surgery and Local Therapy
Surgery of the adrenal tumor is the basic treatment of stage 1-3 ACC. It can also be discussed in stage 4 patients. Only complete tumor removal may lead to long-term remis- sion. Open adrenalectomy is currently recommended, as laparoscopic removal of malignant adrenocortical tumors
could be associated with a high risk of peritoneal dissemina- tion and metastasis [28, 29]. Three cases of diffuse peritoneal dissemination and death in patients who underwent laparo- scopic adrenalectomy for adrenal cancer have been reported [30]. Additionally, laparoscopic adrenalectomy for clinically unsuspected adrenocortical cancer has been associated with a high recurrence rate [31]. Brix et al. conducted a retrospec- tive analysis of 152 patients with stage I-III ACC with a tu- mor ≤ 10cm registered with the German ACC Registry. For localized ACC with a diameter of ≤ 10cm, laparoscopic adrenalectomy by an experienced surgeon is not inferior to open adrenalectomy in respect of oncological outcome [32]. Lesion sizes of 12cm to 14cm have been cited as the upper limit for laparoscopic adrenalectomy in most studies [33]. At present, laparoscopic adrenalectomy is contraindicated for invasive malignant tumors [34]. En-block extensive resec- tions like nephrectomy, hepatectomy, pancreatectomy, and splenectomy are not well-suited to the laparoscopic tech- nique [35-37].
1.2. Radiation Therapy and Loco-Regional Therapy
Radiation therapy is usually considered somewhat inef- fective in controlling tumor growth. For optimal results, an experienced radiotherapist using modern treatment concepts with CT-planning, high-voltage radiation and multiple fields is required. These methods could help to prevent local recur- rence after surgical removal. For bone metastases, radiother- apy can be used as a palliative treatment to reduce pain and limit the risk of development of local complications [38].
Due to the low efficacy in general treatment of ACC and its metastases, there is frequently an indication for loco- regional treatment. Treatment can target a tumor directly through direct percutaneous access for radiofrequency abla- tion (RFA), or can target an organ through intra-arterial drug delivery and chemoembolization of liver metastases.
1.3. Medical Therapy
Mitotane (1,1 dichloro-2 (o-chlorophenyl)-2-(p-chloro- phenyl) etane) is an adrenolytic drug derived from the insec- ticide DDT, and is the only adrenal-specific drug approved for the treatment of advanced ACC. After non-curative sur- gery, and/or in case of inoperable recurrences or distant me- tastases, various chemotherapeutic regimens have been used over the years. O,p’DDD (Mitotane, Lysodren®) is consid- ered by most authors a reference treatment in spite of nu- merous side effects [39-41]. O,p’DDD is a highly lipophilic compound which concentrates in the adrenal glands. There, it provokes mitochondrial degeneration, and the subsequent destruction of the adrenal cortex. Tumor response to o,p’DDD has since been reported in several series [42, 43].
Mitotane is considered to be the drug of choice for pa- tients with inoperable, recurrent and metastatic disease. Mi- totane has been used for treating patients with ACC since the 1960s and is the only drug approved for ACC by the U.S. Food and Drug Administration and the European Medicines Evaluation Agency. An increase in survival [42, 44] and even long-term remission and cure of adrenocortical cancer [45, 46], have been attributed to mitotane. Unfortunately, because of the rarity of the tumour, no randomised or con- trolled studies have been performed to assess mitotane’s ef-
fect on patients’ survival in adrenocortical carcinoma. The toxicity of mitotane (gastrointestinal and central nervous side effects) has been a major obstacle for its use. Van Slooten et al. introduced monitoring of serum mitotane levels in order to reduce side effects [47]. From different studies, a thera- peutic threshold of mitotane (>14mg/l) is suggested [48-52]. Mitotane treatment resulting in lower serum levels was tan- tamount to not giving mitotane at all [53].
Despite radical surgery with curative intent, the majority will develop metastases within 6-24 months of resection. Because there is a high recurrence rate after “curative” sur- gery, and because O,p’DDD has an antitumoral effect, many authors have empirically used it as an adjuvant therapy, hop- ing to increase disease-free survival after complete surgery. With regard to adjuvant mitotane, small retrospective studies have provided insufficient and controversial results [54-60].
As a result, no recommendation regarding adjuvant treatment was made at the 2003 consensus conference on ACC held in Ann Arbor. Currently, an ADIUVO study (Effi- cacy of Adjuvant Mitotane Treatment in Prolonging Recur- rence-free Survival in Patients With Adrenocortical Carci- noma at Low-intermediate Risk of Recurrence) is being con- ducted, and its results will be published in 2014.
Substitutive treatment with hydrocortisone of secondary adrenal insufficiency after mitotane therapy is necessary. Mitotane increases the metabolic clearance of glucocorti- coids and the concentration of cortisol-binding globulin, therefore high-dose glucocorticoid replacement (50-80mg hydrocortisone daily) is needed [61].
In case of failure, or after recurrence, second line cyto- toxic chemotherapies should be proposed. Many other che- motherapeutic regimens have been tested over the years. Because ACC is a rare disease, only small groups of patients have been studied. Current success with cytotoxic chemo- therapy in ACC is unsatisfactory, with <50% of tumors re- sponding to treatment. In the literature, combined treatment with Cis-platine, Etoposide, Doxorubicin given with Mito- tane, and Streptozocin given with Mitotane have been de- scribed [62, 63]. During the 3rd World Congress on ACC and the 13th European Congress of Endocrinology, preliminary results for a FIRM-ACT trial were announced [64]. The study was randomized, prospective, controlled, open-labeled and multicenter. In the study, therapy using Etoposide, Doxorubicin, Cisplatin and Mitotane (EDP-M) was com- pared with Streptozotocin with Mitotane (Sz-M). Although a trend towards better overall survival in patients with ad- vanced ACC treated with EDP-M as first line therapy was observed, overall survival was not significantly different between groups. EDP-M significantly prolonged time to tu- mor progression compared with Sz-M [64]. ACCs tend to express the multidrug resistance gene MDR-1, which results in the production of P-glycoprotein, which is involved in the removal of the drug from cancer cells. This is why single- agent chemotherapy is not favored for ACCs [62].
In the overt clinical manifestation of hypercorticism, mi- totane treatment alone is frequently insufficient to inhibit glucocorticoid hypersecretion in all patients due to its slow action onset and its dose-limiting toxicity. In such cases,
adrenostatic drugs like ketoconazole, metyrapone, aminoglu- tethimide, and etomidate should be introduced to block ster- oidogenic enzymes [65].
Due to the many side effects of known adrenocorticolyti- cal agents, there is a recognized need for improved drugs for the treatment of adrenocortical hypersecretion and adreno- cortical tumor growth. MeSO2-DDE has the potential to be more effective in specifically targeting cells that produce glucocorticoid, which results in reduced dosage levels and subsequently lower unspecific toxicity compared with o,p’DDD (patent 2006). MeSO2-DDE reveals a higher thera- peutic potency than o,p’DDD, resulting in lower therapeutic doses than required for o,p’DDD. MeSO2-DDE has also a more targeted and selective effect in normal and cancerous adrenocortical tissue than is the case for o,p’DDD [66, 67].
Nevertheless, hopes for progress in a clinical setting are not supported by conventional chemotherapy but rather by the rapid development of medical strategies, either based on ACC biology (targeted therapy), or on the interactions be- tween adrenal cancer cells and their microenvironment (in- hibitors of tumor angiogenesis) Table 1.
2. PATHOGENESIS OF ACC AND FUTURE PROS- PECTS
Cancer is the end result of the clonal expansion of a population of cells which have acquired a number of non- lethal genetic alterations favoring uncontrolled cell prolifera- tion or inhibition of cell death. Many molecular techniques (comparative genomic hybridization - CGH or microsatellite analysis) can be used in genome-wide screening for chromo- somal alterations. A positive correlation between tumor size and the number of CGH changes has been observed in ade- nocortical tumors, suggesting that chromosomal alterations accumulate during tumor progression. Chromosomal altera- tions are observed in 28% of benign adrenal tumors and in 62% of cases of ACC [68].
Alternatively, small numbers of ACCs arise from a germ- line mutation predisposing to a familial cancer syndrome. Some of those in which ACC can occur are listed: Li- Fraumeni Syndrome, Beckwith-Weidemann Syndrome, Car- ney’s Complex or Multiple Endocrine Neoplasia Type 1.
Molecular analysis has had a significant impact on the understanding of the pathogenetic mechanism of ACC de- velopment and the evaluation of prognostic and predictive markers. These molecular markers may not only play a role in the biology of these tumors and have prognostic implica- tions, but can also be used as potential targets for treatment of this deadly cancer [69-71].
Recently, the tests that use the expression of biomarkers to predict adrenocortical carcinoma outcome were patented. The invention encompasses a method of predicting the disease outcome of an adrenocortical carcinoma patient on the basis of detecting the expression of a specific biomarker with known nucleic acid and protein sequences [72].
The most important genes involved in the pathogenesis of ACCs are introduced in Table 2.
| Official Title | Phase | Drug | Start of the Study |
|---|---|---|---|
| Phase II trial with Taxotere and Cisplatin in non-operable adrenocortical carcinoma | II | Cisplatin, taxotere | April 2006 |
| Sunitinib in refractory adrenocortical-carcinoma patients progressing after cytotoxic chemotherapy | II | Sunitinib | July 2007 |
| GALACCTIC: A study of OSI-906 in patients with locally advanced or me- tastatic adrenocortical carcinoma | III | OSI-906 | August 2009 |
| Gossypol acetic acid in treating patients with recurrent, metastatic, or primary adrenocortical cancer that cannot be removed by surgery | II | R-(-)-gossypol acetic acid | March 2009 |
| Mitotane with or without IMC-A12 in treating patients with recurrent, metas- tatic, or primary adrenocortical cancer that cannot be removed by surgery | II | Cixutumumab, mitotane | December 2008 |
| Efficacy of adjuvant mitotane treatment (ADIUVO) | III | Mitotane (adjuvant therapy) | April 2008 |
| Cisplatin-based chemotherapy and/or surgery in treating young patients with adrenocortical tumor | III | Filgrastim, pegfilgrastim cisplatin, doxorubicin hydrochloride, etoposide mitotane (up to 21 years) | September 2006 |
| Phase II study of antineoplastons A10 and AS2-1 in patients with carcinoma of the adrenal gland | II | antineoplaston A10 antineoplaston AS2-1 (stage IV of ACC) | August 1996 |
| Cixutumumab in treating patients with relapsed or refractory solid tumors | II | Cixutumumab (up to 30 years) | January 2009 |
| Seneca valley virus-001 in treating young patients with relapsed or refractory neuroblastoma, rhabdomyosarcoma, or rare tumors with neuroendocrine features | I | Seneca Valley virus-001 (NTX-010) (up to 21 years) | September 2009 |
| Sunitinib and hydroxychloroquine in treating patients with advanced solid tumors that have not responded to chemotherapy | I | Hydroxychloroquine, sunitinib malate | October 2010 |
| Multi-institutional phase II study of IMC-A12, a recombinant human IgG1/2 monoclonal antibody directed at the type I insulin-like growth factor receptor (IGF-1R), in adrenocortical carcinoma: A randomized trial comparing the activity of IMC-A12 with mitotane versus mitotane alone | II | IMC-A12, mitotane | December 2008 |
| Sorafenib plus paclitaxel metronomic chemotherapy in adreno-cortical- carcinoma patients progressing after 1st or 2nd line cytotoxic chemotherapy | II | Sorafenib, Paclitaxel | April 2008 |
| Phase II study of axitinib (AG-013736) with evaluation of the VEGF-pathway in metastatic, recurrent or primary unresectable adrenocortical cancer | II | Axitinib | September 2010 |
2.1. The Insulin-like Growth Factor 2, CDKN1C, and H19 Genes
The 11p15 region is organized into two different clusters: a telomeric domain including the insulin-like growth factor 2 (IGF-2) and H19 genes and a centromeric domain including the CDKNIC, KCNQ10TI and KCNQI genes. Beckwith- Weidemann Syndrome (characterized by macrosomia, mac- roglossia, organomegaly and development of embryonal tumors) is related to genetic and epigenetic changes in the imprinted 11p15 region resulting in an increased level of IGF-2 [73, 74].
The IGF-2 gene located at 11p15 encodes an important fetal growth factor. The H19 mRNA is not translated and this gene may modulate IGF-2 expression. Methylation of the H19 promoter has been shown to be involved in the abnor- mal expression of both H19 and IGF-2 in ACC. The CDKN1C gene encodes a G1 cyclin-dependent kinase in- hibitor from the CIP/KIP family involved in the G1/S phase of the cell cycle. Expression of H19 and CDKN1C genes are abolished in most ACCs. The abrogation of CDKN1C gene expression associated with an overexpression of G1 cyclins and G1 cyclin dependent kinases (CDK) leads to a break- down of cell cycle regulation [73, 74].
| Gene (chromosomal locus) | Evidence of involvement in sporadic ACCs | References |
|---|---|---|
| IGF2 (11p15) | Overexpression of IGF2 mRNA in ACCs, 11p15 LOH occurs in 67% of ACCs and 13% of ACAs | [84-89] |
| CTNNB1 gene (B-catenin) (3p21) | Abnormal accumulation of ß-catenin in ACCs. Somatic mutations of ß-catenin with similar frequencies in both groups: ACAs and ACCs, 27 and 31 %, respectively CTNNB1 mutations were mainly observed in larger and nonsecreting ACAs | [105] [108] |
| PRKAR1A (17q23-q24) | LOH of 17q22-24 occurs in 53% of ACCs, no mutations occur in ACCs | [111] |
| Ras genes | Mutations of KRAS (12p12) or HRAS (11p15) have been detected in a small number of ACCs, mutations in NRAS (1p13) have been seen in both ACCs and ACTs | [128] |
| TP53 (17p13) | Mutations of TP53 found in 25% of ACCs; 17p13 LOH occurs in up to 85% of ACCs | [134, 100] |
| GNAS (20q13) | No mutations in ACC patients, only ACAs and AIMAH | [135] |
| NR3C1 (5q31) (Glucocorticoid recep- tor) | Identification of the nuclear glucocorticoid receptor by immunohistochemical studies in 94% cases with ACC and only in 2% of cases with adenoma | [137] |
| MEN1 (11q13) | LOH of 11q13 occurs in 100% of ACCs, MEN1 mutation occurs in 7% of ACCs | [129] |
The insulin-like growth factor (IGF) system regulates many key aspects of cellular and whole-organism physiology and plays a crucial role in the regulation of growth and en- ergy metabolism. The IGF axis involves the coordinated function of three ligands (IGF-1, IGF-2, and insulin), four cell-surface receptors [IGF receptor type 1 (IGF-1R), IGF receptor type 2 (IGF-2R), insulin receptor (IR) and hybrid receptors of IGF and insulin], at least five adaptor proteins [insulin receptor substrate (IRS) 1-4 and Shc], six high- affinity binding proteins (IGFBPs 1-6), and binding protein proteases [75-77].
The IGF-1R is a transmembrane tyrosine kinase receptor (TKR) widely distributed in normal tissues that is responsi- ble for mediating IGF biological effects. The IGF-2R is a monomeric transmembrane protein, acting as a negative regulator of IGF activity [78].
The receptors’ substrates initiate different downstream signaling cascades that eventually lead to cell proliferation and survival. Two major signal transduction pathways are involved: the guanosine triphosphate (GTP)ase Ras/Raf/ extracellular signal-regulated kinase (ERK)/mitogen- activated protein kinase (MAPK) pathway, and the lipid kinase phosphatidylinositol 3-kinase (PI3K)/AKT pathway. Both pathways converge to activate the mammalian target of rapamycin (mTOR) [79] Fig. (1).
Several lines of evidence support the role of IGFs in can- cer development and progression. Excess energy balance leads to increased circulating insulin and decreased levels of IGFBP-1, and hyperinsulinemia and low IGFBP-1 levels have been associated with an increased risk of cancer inci- dence, relapse and death [80].
On the other hand, many preclinical studies have demon- strated that both insulin and IGFs are mitogenic to a variety of cell types, including different cancer cell lines [81-83].
Chromosome region 11p15 LOH (loss of heterozygosity) is associated with a higher risk of tumor recurrence and
could be used as a biological marker in ACC [71]. LOH of the 11p15 locus has been demonstrated more frequently in ACCs than in ACAs (adrenocortical adenoma) - 67% versus 13%, respectively [84].
IGF-2 mRNA is efficiently translated and malignant tu- mors contain large amounts of IGF-2 protein. The insulin- like growth factor system is involved in the development of the adrenal cortex and its role has been documented in ACTs. Many studies have demonstrated that IGF-2 is strongly overexpressed in malignant adrenocortical tumors, with such overexpression observed in approximately 90% of ACCs [85, 86]. Almeida et al. observed overexpression of IGF-2 transcripts in both pediatric adrenocortical carcinomas and adenomas. Otherwise, IGF-2 was mainly overexpressed in adult adrenocortical carcinomas. IGF-1R expression was significantly higher in pediatric adrenocortical carcinomas than adenomas whereas its expression was similar in adult adrenocortical carcinomas and adenomas. IGF-1R expres- sion was a predictor of metastases in pediatric adrenocortical tumors [87]. Several studies have shown that the IGF-2 gene is much more overexpressed in ACC in comparison with benign adrenocortical adenomas or normal adrenal glands [88, 89]. IGF-1 and IGF-2 receptors are present in adrenal tissues and strong overexpression of intact IGF-1 receptors has been shown in ACC [90]. IGF-2 is directly involved in the proliferation of the human ACC NCI H295R cell line [91]. The mitogenic effect of IGF-2 is dependent on the IGF- 1 receptor. Furthermore, IGFBP-2 levels have been shown to correlate with tumor stage in ACC [92].
The central role that the IGF system plays in initiating and promoting tumor progression makes it an attractive tar- get for cancer therapy [93]. Various strategies have been used to target components of this system in established ani- mal and human tumor cell lines (in vitro studies) and in ani- mal models of cancer (in vivo studies), and some of these strategies may be advancing to clinical stage [94]. Among them are down-regulation of IGF1R by antisense oligonu-
IGF-1 mAb:
CP721871
Anti-IGF-1R: XL228
AMG479 IMC-A12
IGF-1
IGF-2
OSI906
R1507
NDGA
BIIB022
IGF1 R
IGF2 R
SHC
GRB2
IRS-1
SOS
Anti-PI3K: PI103 BGT226 BEZ235 XL765
PIP2
PIP3
Anti-Akt: Perifosine GSK690693
GDP-RAS Inactive
GAP
GTP-RAS Active
Anti-RAS: Tipifarnib Lonafarnib
XL147
PI3K
Akt
BMS214662
Anti-MEK: AZD6244
Anti-RAF: Sorafenib RAF265 XL281 PLX4032
Anti-mTOR: Everolimus Deforolimus
RAF
RDEA119 XL518
MEK
mTOR
ERK1/2
NUCLEUS
S6K
ELK1
TRANSCRIPTION
cleotides, antisense RNA, small interfering RNA, triple he- lix-forming oligodeoxynucleotides, single chain antibody, fully humanized anti-IGF1R monoclonal antibodies (e.g. CP- 751,871, AVE1642/EM164, IMC-A12, SCH-717454, BIIB022, AMG 479, MK-0646/h7C10), and specific IGF1R tyrosine kinase inhibitors (e.g. BMS-536942, BMS-554417, NVP-AEW541, NVP-ADW742, AG1024, potent quinolinyl- derived imidazo (1,5-a)pyrazine PQIP, picropodophyllin PPP, nordihydroguaiaretic acid Insm-18/NDGA) [94]. Anti- cancer therapy using anti-IGF-1R antibodies or antigen- binding fragment was patented in 2010 [95]. OSI-906 is a small-molecule IGF-1R inhibitor and its clinical efficacy in ACC has been documented. Partial response, stable disease and, in some patients, no results were observed after using OSI-906 [96]. The available data suggest that targeting the IGF system in vivo may inhibit cancer progression and/or cause cancer regression directly by inducing apoptosis and cell growth arrest [95, 97]. In addition to these direct effects,
it has been shown that inhibition of IGF action could in- crease the efficacy of other therapeutic modalities. We are also considering combined therapy using anti-IGF system treatment with mitotane as an antiadrenal drug. Further, we consider the necessity of selecting indications characterized by pathological and molecular alterations, rational selection of medications for combined therapy based on signaling pathway interactions, and also patient selection based on analysis of predictive biomarkers and ineffectiveness of con- ventional methods of therapy [73, 74, 97-100].
2.2. Wnt Signaling Pathway
The Wnt signaling pathway is normally activated during embryonic development and plays an important role in adre- nal cortex development and is involved in the control of ad- renal steroidogenesis. ß-Catenin is the key component of this signaling pathway. ß-Catenin is a protein that in humans is encoded by the CTNNB1 gene. In both benign and malignant
ACT, ß-catenin accumulation may be observed. These al- terations seem to be very frequent in ACC, consistent with an abnormal activation of the Wnt-signaling pathway. GSK3-ß (glycogen synthase kinase 3-ß) is implied in the regulation of -catenin. Phosphorylated ß-catenin becomes ubiquitylated and is targeted for degradation by the protea- some. Following Wnt binding to a receptor complex com- posed of members of the Frizzled (Fz) family of seven transmembrane serpentine receptors and low density lipopro- tein receptor-related protein (LRP), the axin/APC/GSK-3 complex is inhibited. Hypophosphorylated ß-catenin accu- mulates in the cytoplasm and is translocated to the nucleus. After translocation to the nucleus, ß-catenin stimulates target genes expression, specific for tissue/organ [101, 102]. There are some specific “Wnts” for adrenal tissue: WNT4, WNT10b, WNT3a [103]. WNT4 increases activity of the steroidogenic enzymes. WNT3a and WNT10b molecules stimulate adrenocortical cells and production of aldosterone in vitro [104]. The upregulation of steroidogenic enzymes in response to Wnt-molecules is mediated by ß-catenin. This process activates steroidogenic factor-1 and expression of the StAR (Steroidogenic Acute Regulatory Protein) [103]. We
suspected that more aggressive form of adrenal carcinoma with coexisting cortisol overproduction could be Wnt/B- catenin dependent Fig. (2).
The Wnt pathway was analyzed by Gaujoux et al. in 26 ACAs and 13 ACCs. Abnormal accumulation of ß-catenin was found in 10 (38%) ACAs and 11 (77%) ACCs. In the same group the ß-catenin gene was screened for presence of mutations and such mutations were discovered with similar frequencies in both groups: ACAs and ACCs, 27 and 31 %, respectively [105].
Berthon et al. showed that constitutive activation of ß- catenin in the adrenal cortex of transgenic mice resulted in progressive steroidogenic and undifferentiated cell hyperpla- sia as well as dysplasia of the cortex and medulla. Over a 17 month period, transgenic adrenals developed malignant char- acteristics such as uncontrolled neovascularisation and loco- regional metastatic invasion [106].
The presence of a ß-catenin nuclear staining was signifi- cantly associated with a higher ENSAT (European Network for the Study of Adrenal Tumors) tumor stage (stages III and IV), higher Weiss score, more frequent necrosis, mitoses,
a)
b)
Wnt3a
Wnt10b
RTK
Adipocyte-derived Wnts
Wnt4
Frizzled
LRP
Frizzled
Wnt
LRP
cortisol 1
B-catenin
p120
VVV
a-catenin
Dsh
Axin
Dsh
Axin
APC GSK3
APC GSK3
Brc
B-catenin
B-catenin
For
B-catenin
B-catenin
ß-catenin
B-catenin
NUCLEUS
NUCLEUS
B-catenin
Target gene StAR
SF-1
SF-1
and CTNNB1|APC mutations. ß-catenin nuclear staining and the presence of CTNNBIIAPC mutations were both associ- ated with decreased overall and disease-free survival, and were independent predictive factors of survival in multivari- ate analysis [107]. Additionally, in 2011 Bonnet et al. showed abnormal cytoplasmic and/or nuclear ß-catenin im- munohistochemical staining in about half of ACAs. This suggests the activation of the Wnt/B-catenin pathway, which could be explained by activating mutations of CTNNB1 in 70% of the cases. CTNNB1 mutations were mainly observed in larger and nonsecreting ACAs, suggesting that the Wnt/B- catenin pathway activation could be associated with the de- velopment of less differentiated ACAs [108].
The complexities of the Wnt family and the Wnt signal- ing pathways have provided plentiful fruit for genetic, ge- nomic, and biochemical dissection. The misregulation of the Wnt signaling pathway is a contributing factor in a number of human diseases, as well as ACC. Manipulating within Wnt signaling pathway components could be promising in treating some Wnt-dependent diseases.
Pyrvinium is a Wnt pathway inhibitor and was patented as an anticancer therapeutic agent in 2009. Pyrvinium has historically been used in the treatment of enterobiasis caused by Enterobius vermicularis (pinworm). This drug stabilizes axin and destabilizes ß-catenin. In vivo it blocks Wnt signal- ing and cancer cell proliferation, induces apoptosis, and also blocks Wnt-mediated gene transcription [109].
The small-molecule inhibitor of the T cell factor (Tcf)/ B -catenin complex PKF115-584 dose-dependently inhibited ß -catenin-dependent transcription and proliferation of H295R adrenocortical tumor cells, which harbor mutations in CTNNB1 as well as the TP53 tumor suppressor gene. The drug had no effect on Hela cells, a cell line in which the ß - catenin pathway is not activated. PKF115-584 decreased the percentage of H295R cells in S-phase and increased the per- centage of apoptotic cells. Inhibitors of the Tcf/ ß-catenin complex may prove useful in the treatment of adrenocortical tumors in which multiple genetic alterations have accumu- lated [110].
PRKAR1A (regulatory subunit R1A of the protein kinase A) is the main mediator of cAMP signaling. The PRKARIA gene encodes the type 1A regulatory subunit of cAMP- dependent protein kinase A. One study found LOH of 17q22-24, the locus for PRKARIA, in 23% of ACAs and 53% of ACCs. Inactivating mutations in the PRKAR1A gene were found in 10% of ACAs and no mutations were found in ACCs [111]. The Wnt/B-catenin signaling pathway is acti- vated in ACTs presenting PRKAR1A mutations. This sug- gests a cross-talk between the cAMP and the Wnt/ ß-catenin signaling pathways. Nevertheless, further studies are neces- sary to determine the mechanisms of this cross-talk. The occurrence of CTNNB1 somatic mutations as a second event in ACT is associated with larger and/or more aggressive tu- mors [112].
2.3. Growth Factors
Various growth factors other than IGFs have been shown to regulate adrenal growth and function in normal adult and fetal adrenals, including fibroblast growth factor (FGF-2),
transforming growth factor-a (TGF-a), growth factor-ß1 (TGF-ß1), and vascular endothelial growth factor (VEGF) [113-115].
Angiogenesis has been shown to be an important process for new vessels formation, tumor growth, progression, and spread. Serum VEGF is elevated in patients with adrenal tumors undergoing surgery. VEGF expression in the tumor cells was positively correlated with scores of Weiss’ criteria. One month after surgery, there was a significant reduction in serum VEGF levels in patients with both cortical cancers and benign adrenocortical adenomas. However, whether serum VEGF provides a marker of long-term disease control or recurrence is not known [114].
VEGF is the predominant signal for both endothelial pro- liferation and migration into sites of neovascularization, and blocking this signal has been a major goal of research in this field [116]. Patients with ACC have significantly greater serum VEGF levels than patients with benign adrenal tumors [115]. Therefore, it would be rational to assess the effective- ness of angiogenesis inhibitors as single agents or in combi- nation with mitotane and/or IGF inhibition in future studies. Bevacizumab and other humanized anti-VEGF antibodies are further described as patents [117]. In 2010 adjuvant ther- apy also with using VEGF-specific antagonists was accepted [118]. The VEGF-specific antagonist and other therapeutic agents can be administered simultaneously or sequentially in an amount and for a time sufficient to reduce or eliminate the occurrence or recurrence of a tumor, a dormant tumor, or micrometastases. The results from a trial with Bevacizumab indicate that addition of this drug (AVASTIN®) to chemo- therapy significantly increased disease free survival (DFS) as compared to chemotherapy alone during the first year, which corresponds to the active treatment phase. The data show that this significant benefit was not associated with any in- creased toxicities or adverse effects. Also, combination of two anticancer therapeutic agents - antiVEGF antibody Bevacizumab and EGFR-tyrosine kinase inhibitor Vande- tanib - in therapy of solid cancers was patented in 2010 by Ryan [119]. In 2011, the methods of using anti-VEGF antibody for treating diseases and pathological conditions were patented. In particular, the invention provides an effective approach for treating cancers, partially based on the results that adding anti-VEGF antibody to a standard chemotherapy results in statistically and clinically significant improvements among cancer patients [120].
TGF-ß1 inhibits the proliferation of epithelial cells and regulates adult and fetal adrenal growth and functions. Two different studies demonstrated a reduced TGF-ß1 mRNA expression in ACC, but no difference in the expression of TGF-ß1 receptor was observed in ACC [115].
More than 80% of ACCs have a moderate to high inten- sity of EGFR. The expression of EGF receptors could be associated with tumor growth and/or metastatic potential in adrenocortical carcinoma. Interestingly, EGF itself does not appear to be expressed in adrenal tumors, suggesting either that EGF is functioning as an endocrine hormone or the EGFR is stimulated by another compound. Human TGFa is a native ligand co-overexpressed with its receptor EGFR in adrenocortical lesions [113, 115]. Cetuximab, gefitinib, and erlotinib, agents targeting EGFR, are clinically available and
have been reported to be administered in patients with differ- ent types of cancers, such as non-small cell lung cancer [121- 123]. The relative abundance of EGFR in ACC suggests that these agents may provide clinical benefits. Target drugs against EGFR have also been currently investigated in ad- vanced ACC (erlotinib, and a novel EGFR inhibitor - BMS- 690514) [124]. Current trials are testing the activity of sunit- inib, a multitargeted thyrosine kinase inhibitor that inhibits the receptors for VEGF and PDGF, KIT (Stem cell factor receptor), FLT-3 (FMS-like tyrosine kinase-3 receptor) and RET (Rearranged during transfection) [125]; and sorafenib, a multitargeted serin/thyrosine kinase inhibitor that inhibits RAF-1, the key enzyme in the RAS/RAF/MEK/ERK signal- ing pathway leading to cell proliferation, as well as VEGFR- 2 and PDGFR-ß involved in angiogenesis [126, 127].
In our opinion, therapy with the use of agents inhibiting growth factors and tumor angiogenesis in combination with mitotane could bring positive results in patients with ACC.
2.4. Ras Oncogene
Ras proteins are membrane-associated proteins involved in downstream signaling. The three Ras proteins (H, N and K) are one of the most commonly mutated oncogenes in hu- man cancers. Mutations of KRas or HRas have been detected in a small number of ACCs, whereas mutations in NRas have been seen in both adenomas and carcinomas. Further studies on Ras oncogene mutations in the diagnosis and therapy of ACC should be undertaken [128].
2.5. TP53 Gene and 17p13 Locus
The TP53 gene, a tumor suppressor gene, is located at 17p13.1 and encodes a 393 amino acid protein. It is involved in the control of cell proliferation. Acquired mutations of the TP53 gene are common tumor-specific alterations in hu- mans, and have been identified in most of the major types of cancer. These tumors have an early onset, affecting mostly children and young adults (described as Li-Fraumeni Syn- drome) [129]. Germline mutations in TP53 have also been observed in 50-80% of children with apparently sporadic ACC in North America and Europe [130, 131]. The inci- dence of pediatric ACC is about 10 times higher in Southern Brazil than in the rest of the world, and a specific germline mutation has been identified in exon 10 of the TP53 gene in almost all cases [132, 133]. In sporadic ACC in adults, so- matic mutations of TP53 are found in only 25% of cases and are located in four “hot spot regions” within exons 5 and 8 and are responsible for more aggressive tumors [134].
LOH at 17p13 has been demonstrated in ACC but not in ACA. LOH at 17p13 occurs in 85% malignant tumors and in <30% of benign adenomas. 17p13 LOH could be used as a molecular marker of malignancy of ACT [100].
2.6. The Guanine Nucleotide-Binding Protein, Alpha- Stimulating Activity Polypeptide Gene
Activating somatic mutations of the guanine nucleotide- binding protein, alpha-stimulating activity polypeptide (GNAS) gene, located on 20q13.2, are responsible for the McCune Albright syndrome. GNAS encodes the alpha
subunit of the stimulatory G protein (Gsa). In abnormal Gsa, there is lower GTPase activity, resulting in constitutive adenylate cyclase activation and consequent cAMP signal- ing. GNAS has been reported to rarely be mutated in sporadic ACAs. There have been no reports of GNAS mutations in sporadic ACCs [135].
2.7. ACTH Cyclic AMP/Protein kinase a Pathway
The ACTH (adrenocorticotropic hormone) receptor (ACTH-R) belongs to the superfamily of G-protein coupled seven transmembrane domain receptors. Few mutations have been found in this pathway in adrenocortical tumors [136]. ACTH-R expression seems down-regulated in ACC and de- creased expression of ACTH-R in ACC could take part in dedifferentiation of these aggressive tumors.
2.8. Glucocortcoid Receptor
Tacon et al. identified an increased expression of the nuclear glucocorticoid receptor (GR) by immunohistochemi- cal studies in 94% cases with ACC, whereas it was absent in 98% of cases with adenoma. The identification of GR over- expression in malignant ACTs offers mechanistic insights into carcinogenesis and identifies a potential therapeutic tar- get [137].
2.9. Steroidogenic Factor-1 (SF-1/Ad4BP; NR5A1)
The orphan nuclear factor, steroidogenic factor-1 (SF-1, also called Ad4BP, encoded by the NR5A1 gene) is an essen- tial regulator of tissue-specific gene expression in steroi- dogenic cells and of adrenogonadal development. SF-1 binds as a monomer to nuclear receptor half sites on DNA. The SF-1 expression pattern during development is restricted to tissues involved in steroidogenesis (adrenal cortex, testis, ovary) and reproductive function (pituitary gonadotropes, hypothalamic ventro-medial nucleus), plus the spleen [138, 139]. SF-1 overexpression was demonstrated in a small group of pediatric ACT [140]. Almeida et al. showed that the frequency of SF-1 overexpression and gene amplification was similar in ACAs and ACCs [141]. They also observed a greater frequency of SF-1 overexpression and gene amplifi- cation in pediatric compared to adult ACTs, suggesting an important role for SF-1 in pediatric adrenocortical tumori- genesis. Strong SF-1 expression significantly correlated with poor clinical outcome (tumor stage and recurrence ratio) [142].
Similar results were observed by other authors [140, 143]. SF-1 overexpression in human adrenocortical tumour cells has a significant impact on the expression of genes in- volved in steroid metabolism, cell cycle, apoptosis and cell adhesion. These properties suggest that this factor may have an important role during adrenal development and oncogene- sis.
Doghman et al. described how isoquinolinone com- pounds inhibit the constitutive transcriptional activity of SF- 1 (SF-1 inverse agonists). These compounds have the attrib- utes to inhibit the increase in proliferation triggered by an augmented SF-1 dosage in adrenocortical tumor cells and to reduce their steroid production [144].
ACC - Molecular Features
This latter property could be helpful in the therapy of adrenocortical tumors to alleviate symptoms of virilization and Cushing often associated with tumor burden [145].
SF-1 is a highly valuable immunohistochemical marker for determining the adrenocortical origin of an adrenal mass with high sensitivity and specificity. In addition, SF-1 ex- pression is of stage-independent prognostic value in patients with ACC [142].
2.10. Multiple Endocrine Neoplasms 1 Gene
The MEN1 (multiple endocrine neoplasms) gene func- tions as a tumor suppressor and a mutation in one of the nine coding exons predisposes to multiple endocrine neoplasia [146].
In 55% of individuals diagnosed with MEN1, ACTs (mainly ACAs) have also been reported; and ACCs have further been reported with MEN1, but with rare incidence. Only single cases with germline mutations in the MEN1 gene have recently been described. A new mutation at codon 443 in the coding region of exon 9 in the MENI gene (p.Ser443Tyr; c.1327C > A) was discovered in patients with MEN1 syndrome with ACC [147].
Griniatsos et al. described the p.E45V mutation in exon 2 of the MENI gene in a patient with bilateral adrenocortical carcinoma and MEN1; none of the/his family members re- vealed genetic changes [148].
Somatic mutations of the MEN1 gene in sporadic ACC are very rare. Loss of heterozygosity (LOH) at 11q13 has been described in greater than 90% of informative ACCs in three different series compared with an incidence of LOH at 11q13 in less than 30% of informative adenomas [129]. The frequent evidence of LOH at 11q13 in adrenal cancer in the absence of MENI gene defects hints at a role for a different tumor suppressor gene located in this chromosome band. Genes in this region may be important in the pathogenesis of adrenocortical carcinoma, and Fernandez-Ranvier et al. compared the expression profile of this region between be- nign and malignant adrenocortical tissue. They have identi- fied 25 genes located on chromosome 11q13 that are down- regulated in adrenocortical carcinoma and may be candidate tumor suppressor genes. Six of these genes (SERPING1, MRPL48, TM7SF2, DDB1, NDUSF8, PRDX5) were good diagnostic markers for distinguishing adrenocortical carci- noma from adenoma [149, 150].
3. MicroRNA EXPRESSION
MicroRNAs (miRs) are small non-protein-coding RNAs involved in the post-transcriptional negative regulation of gene expression. miRs may induce translational repression or target mRNA degradation [151]. Recent investigations on simultaneous miR-induced expression changes in the tran- scriptome and proteome have revealed mRNA degradation as the principal way miR acts in mammalian organisms [152, 153]. miR molecules are expressed in a tissue-specific fash- ion and may target various mRNAs in different organs. miRs have been implicated in the regulation of numerous cellular processes such as cell development, proliferation, differen- tiation, apoptosis [154, 155], and tumor development [154]. The identification of potential miR targets is of pivotal im-
portance for deciphering the molecular mechanisms of miR actions.
Soon et al. discovered that miR-335 and miR-195 were significantly downregulated in adrenocortical carcinomas compared with adrenocortical adenomas. Downregulation of miR-195 and upregulation of miR-483-5p in adrenocortical carcinomas were significantly associated with poorer dis- ease-specific survival. These findings indicate that deregula- tion of microRNAs is a recurring event in human adrenocor- tical carcinomas and that aberrant expression of miR-195 and miR-483-5p identifies a subset of poorer prognosis adrenocortical carcinomas [156]. By using directed quantita- tive RT-PCR analysis on a subset of these differentially ex- pressed miRs, Patterson et al. determined that miRs -100, - 125b, and -195 were significantly down-regulated, whereas miR-483-5p was significantly up-regulated in malignant as compared with benign tumors [157]. These observations were similar to the previous work by Soon et al. miR-483-5p expression can accurately categorize tumors as benign or malignant [157]. Tombol et al. discovered that the expres- sion of miR-210 and miR-375 was significantly lower in cortisol-secreting adenomas than in ACCs. miR-184 and miR-503 showed significantly higher, whereas miR-511 and miR-214 showed significantly lower expression in ACCs than in other groups. By calculating the difference between dCT(miR-511) and dCT(miR-503) (delta cycle threshold), ACCs could be distinguished from benign adenomas with high sensitivity and specificity [158]. The relative overex- pression of miR-210 and miR-184 and underexpression of miR-214 in ACC samples may thus be associated with re- duced apoptosis that is a common feature of malignancy [158]. Schmitz et al. in the one of the latest article suggest that miRNA profiling of miR-675 and miR-335 helps in dis- criminating ACCs from ACAs [159].
These pathways include novel, previously undescribed pathomechanisms of adrenocortical tumors, and associated gene products may serve as diagnostic markers of malig- nancy and therapeutic targets in the future [160]. miR bio- markers may be helpful for the diagnosis of adrenocortical malignancy.
CURRENT & FUTURE DEVELOPMENTS
ACC is a very rare disease in which systemic treatment has unsatisfactory results. An improved understanding of the molecular pathways that underline the development and pro- gression of this aggressive disease has provided a rationale for testing new target drugs in its management.
Some have hypothesized that the development and growth of ACCs is a multistep progression with accumula- tion of genetic alterations. Studies of the molecular abnor- malities in ACC have not yet revealed an ACC oncogenic pathway; however, there is evidence that the number of ge- netic alterations correlates with malignancy of ACC [161, 162].
Analyzing gene expression (transcriptome) in tissues is now reliable using industrial pangenomic microarrays. These transcriptome data observing the ACT phenotype could im- prove the outcome determination compared with standard clinical and pathological tools. This information could allow
for differentiation not only between adenoma and carcinoma but also between two sets of carcinoma which have very different prognoses.
There is abundant and specific expression of steroroi- dogenesis enzymes in the adrenal tissue so they represent a promising target for diagnostics and therapy in ACC. Io- dometomidate is a highly potent inhibitor of cortisol and aldosteron synthesis with affinity to CYP11B1- and CYP11B2-enzymes. Due to the high specificity of metomi- date uptake, it demonstrates potential for radiotherapy of ACC.
Due to the rarity of ACC, collaborative work performed in national and international networks will be important. Tumor biology and molecular alterations may be the key to determining the response to treatment and, in future, the mo- lecular approach will help with the selection of patients who could benefit from specific therapies. Thus, in the near future the combined strengths of endocrinologists, oncologists, ra- diologists and surgeons may provide new hope to patients suffering from ACC.
ACKNOWLEDGEMENTS
We would like to acknowledge the COST BM0607 ac- tion, especially the WG1 group, which has contributed to the study by making substantial contributions to the concept, and also the Polish Government for financial support.
CONFLICT OF INTEREST
The authors declare that they have no competing inter- ests.
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