Article type: Review Title: Cell cycle regulation in adrenocortical carcinoma
Authors: Sofia S. Pereira1,2,3; Mariana P. Monteiro3; Isabelle Bourdeau4; André Lacroix4; Duarte Pignatelli1,2,5
Authors affiliation
1 Instituto de Investigação e Inovação em Saúde (I3S), Universidade do Porto, Portugal
2 Institute of Molecular Pathology and Immunology of the University of Porto (IPATIMUP), Porto, Portugal.
3 Clinical and Experimental Endocrinology, Department of Anatomy, Multidisciplinary Unit for Biomedical Research (UMIB), Instituto de Ciências Biomédicas Abel Salazar, University of Porto (ICBAS/UP), Porto, Portugal.
4 Endocrinology Division, Department of Medicine, Centre de Recherche du Centre hospitalier de l’Université de Montréal (CHUM), Montréal, Canada.
5 Department of Endocrinology, Hospital S.João, Porto, Portugal.
Corresponding Author:
Duarte Pignatelli, MD, PhD I3S - Cancer Signaling and Metabolism group R. Alfredo Allen, 4200-135 Porto, Portugal. Phone: +351 912880313
Short Title: Cell cycle in ACCs
ABSTRACT
Adrenocortical carcinomas are rather rare endocrine tumors that often have a poor prognosis. The reduced survival rate associated with these tumors is due to their aggressive biological behavior, combined with the scarcity of effective treatment options that are currently available. The recent identification of the genomic alterations present in ACC have provided further molecular mechanisms to develop consistent strategies for the diagnosis, prevention of progression and treatment of advanced ACCs. Taken together, molecular and genomic advances could be leading the way to develop personalized medicine in ACCs similarly to similar developments in lung or breast cancers.
In this review, we focused our attention to systematically compile and summarize the alterations in the cell cycle regulation that were described so far in ACC as they are known to play a crucial role in cell differentiation and growth. We have divided the analysis according to the major transition phases of the cell cycle, G1 to S and G2 to M. We have analyzed the most extensively studied checkpoints: the p53/Rb1 pathway, CDC2/cyclin B and Topoisomerases. We reached the conclusion that the most important alterations having a potential application in clinical practice are the ones related to p53/Rb1 and Topoisomerase 2. We also present a brief description of on-going clinical trials based on molecular alterations in ACC . The drugs have targeted the IGF receptor 1, Topoisomerase 2, PLK1, CDK inhibitors, p53 reactivation and CDC25.
Keywords: Adrenocortical tumors, adrenocortical carcinomas, cell cycle
INTRODUCTION
Adrenocortical carcinomas (ACCs) are rather rare tumors, and this constituted a major limitation to conduct wide and comprehensive molecular characterization studies. In spite of this, a relatively large number of studies identified a wide array of molecular alterations, but usually in a relatively limited number of ACC samples.
Molecular studies that aimed to characterize the cell cycle deregulations found in adrenocortical tumors are the subject of this systematic review. This comprehensive review aims to clarify some apparently contradictory results and rather complex molecular interactions. The data presented in this review intends to be a useful working resource for researchers interested in ACC to identify new diagnostic, prognostic and therapeutic tools to improve outcomes for ACC patients.
Adrenocortical carcinomas
ACCs have an annual incidence of 1 to 2 cases per million persons worldwide 1-5. ACCs can be functional, producing hormonal and metabolic syndromes that can occasionally accelerate their identification, or are discovered incidentally during imaging for unrelated clinical reasons 4,6. In some patients with very large tumors, the symptoms of abdominal discomfort or pain due to the mass effect will lead to adrenal imaging and diagnosis. The detection of non-functioning adrenocortical tumors (both benign and malignant) increased significantly over the last years, due the widespread use of computed tomography (CT), magnetic resonance imaging (MRI) and abdominal ultrasonography (US) 6,7.
The potentially dark prognosis of ACC is very different from adrenocortical adenomas which stresses the importance of differential diagnosis between the two entities8, 9. However, the evaluation of the potential malignant evolution of adrenocortical tumors is not always easy to assess. So far, the size and morphological characteristics of the lesion on the imaging studies is the initial strongest predictor of the adrenocortical malignant potential; according to the European Society of Endocrinology Clinical Practice Guideline 1º, unilateral adrenocortical tumors (ACTs) larger than 6 cms should be treated surgically. Those between 4-6 cms should
undergo additional imaging investigation to better define malignant potential. In addition to size, a threshold value of 10 Hounsfield units (HU) of unenhanced CT scan was shown to have a high specificity and sensibility for the differential diagnosis of adrenocortical malignant lesions 10, 11. After tumor removal, the pathological diagnosis includes evaluation of tumor macroscopic characteristics, again including size, but also the presence of an intact capsule, areas of necrosis and hemorrhage, adjacent organ invasion and lymph node metastasis. At the microscopic level, the modified Weiss scoring system helps in the assessment of malignancy potential whenever metastasis have not been identified 12, 13. A few molecular and immunohistochemistry markers are also helpful to define ACC diagnosis 11, 13, 14. The major alterations found in the ACCs are the overexpression of IGF-2 and the constitutive activation of the Wnt/B-catenin pathway. Although the former one was identified as a useful marker for ACC by several authors, it was not yet validated to be used in clinical practice. Ki-67 proliferation index has also been found to be an important marker for the prediction of recurrence 15, and it is used to guide the management of ACC patients.
The disease stage and margin-free resection are among the major prognostic factors in ACC 16, 17. The stage classification proposed by the European Network for the Study of Adrenal Tumors (ENSAT) in 2009, is the most accurate to define ACC prognosis 5, 17. This staging system, defines stage I and stage II as localized tumors with a size of ≤5 or >5 cm, respectively. Stage III ACCs present infiltration in surrounding tissue or regional lymph whereas stage IV is characterized by the presence of distant metastasis. Using ENSAT classification, the 5-year disease-specific survival rate is approximately 82% for stage I, 61% for stage II, 50% for stage III, and 13% for stage IV 17. The high proportion of ACC are diagnosed in an advanced stage leading to a poor prognosis4. The secretion of abnormal levels of steroids by ACCs also confers substantial morbidity 18.
The behavior of ACCs differs between patients. Almost 50% of stage I and II adrenal carcinomas will not be cured by surgery even when complete margin-free resection is achieved, while others with similar pathological characteristics are cured by surgery and patients remain
disease-free. Recent studies indicate that prognosis is related to significant variability of tumors at the molecular level 19.
Pan-genomic characterization of ACCs
Assié et al and Zheng et al recently performed comprehensive genomic characterizations of ACCs with the major goal of identifying stratification profiles of ACC patients with distinct clinical outcomes 20,21. The studies include data on gene expression, exome sequencing, miRNA expression, copy number alterations, single nucleotide polymorphisms, loss of heterozygosity (LOH), mutations, DNA methylation and even protein expression 20, 21. Much of this information is not yet translated into complete understanding of molecular pathways, but they have confirmed some previous findings and identified potentially very important novel contributing pathogenic alterations.
Using exome sequencing and SNP array analysis, the studies identified recurrent alterations in several genes implicated in the Wnt pathway, cell-cycle regulation, chromatin remodeling, and chromosome maintenance, as ACC drivers.
Indeed, the major findings of the integrated pan-genomic studies were the identification of distinct ACC molecular subgroups strongly associated with patient’s outcome. The subgroup of aggressive ACCs presented higher expression of cell-cycle related genes and accumulation of more mutations in the ACC drivers.
Based on the methylation on CpG islands in gene promoter regions, the aggressive ACC group was further divided into two other subgroups: a group of patients with intermediate outcome and a group with the poorer outcome which was characterized by having hypermethylation in those regions 20, 21
Cell Cycle
Since alterations in the cell cycle regulators were pointed-out as very important ACC drivers by the already mentioned pan-genomic studies but also by several smaller studies, we decided to
gather the current knowledge on cell cycle dysregulation in adrenocortical carcinomas in this review.
The cell cycle is a complex process that results in cell division. It includes four distinct phases going from the cell growth, DNA replication, partition and distribution of the duplicated chromosomes and cell division (Figure 1) 22. This process is regulated by complex intracellular and extracellular signaling cascades that coordinate the different phases of the cell cycle to ensure a successful cell division22, 23. Deregulation of the cell cycle is one of the most frequent events in tumor development. Therefore, elucidation of the underlying mechanisms of its alterations in tumorigenesis could lead to novel therapeutic targets24, 25.
Cell cycle regulation is generally grouped in three waves that correspond to the transition points of the cell cycle: G1 to S; G2 to M and M to G1 (Figure 1) 26. The majority of the cell cycle regulation alterations in ACCs were found to be present in the G1 to S and G2 to M transitions27.
Alterations in the regulation of G1-to-S phase found in adrenocortical carcinomas
Pan-genomic studies identified the p53-Rb1 pathway as the second most altered pathway found in the ACCs 20, 21, while the first one the Wnt B-catenin pathway is not related to the cell cycle. The p53-Rb1 pathway was already known as the major checkpoint of the G1 phase of the cell cycle. Briefly, in response to DNA damage, ataxia telangiectasia mutated (ATM) or ataxia telangiectasia and Rad3-related protein (ATR) are activated and phosphorylate p53 through Chk1/2. Phosphorylated p53 dissociates from the murine double minute 2 (Mdm2) and is thereby stabilized. Active p53 upregulates the endogenous p21 mRNA and protein levels 28. Overexpression of p21 blocks the phosphorylation of the retinoblastoma protein (pRb) by cyclin E/CDK2, preventing the E2F mediated gene expression induction required for cells to entry S phase 29.
Inactivating mutations or homozygous deletion of TP53 (16%), CDKN2A (11%) and RB1 (7%) and high levels of amplification of CDK4 (2%) and MDM2 (2%) were found to occur in 40 cases of the 122 ACCs analyzed by Assié et al 21. These alterations were mainly found in the
ACCs with poorer prognosis. Zheng et al 20 also reported deactivating alterations (mutations and deletions) in TP53 (21%), CDKN2A (16%) and RB1 (7%) and activating alteration in CDK4 (7%), MDM2 (7%) and CCNE1 (6%), the gene that encodes the Cyclin E. Altogether, the p53/Rb1 pathway was altered in 44.9 % of the analyzed ACCs by Zheng et al 20. Since the majority of the TP53 mutations observed affect the DNA-binding domain or the oligomerization domain, these alterations prevent the role of p53 as a tumor suppressor, leading to an important dysfunctionality of the G1 checkpoint. This point, together with a deregulation of the major molecules implicated in the G1 phase: Cyclin E, CDK4 and Rb1, will lead to an uncontrolled ACC cell proliferation.
Alterations in p53 pathway in ACCs
The study of the p53 and their regulators in ACC started many years ago, after the discovery that the Li-Fraumeni syndrome confers susceptibility to ACCs and is characterized by the presence of TP53 germline mutations in approximately 71% of families with that syndrome 30, 31 ENREF_58. Although germline TP53 mutations were later found to be rare in adult patients with ACC, somatic TP53 mutations were documented to be more frequent32-36. In a large cohort of adult Caucasian patients with ACC, a 3.9% prevalence of TP53 germline mutation was found 35; in contrast, 13% of adult patients (< 40 yo), carried a TP53 germline mutation35. Germline mutations in TP53 were observed in 50%-80% of children with sporadic ACC37-39. Many studies analyzed the presence of somatic TP53 mutation in ACCs and verified that its prevalence varies from 20 to 30% in sporadic ACCs32, 34, 40. The majority of patients with ACC and mutated TP53 had a poor outcome40.
Mutations of TP53 usually result in loss of p53 protein function. Several studies evaluated p53 expression in ACTs and it was demonstrated to be absent in the majority of ACAs 41, 42. Among ACCs, p53 expression has been found to be highly variable ranging between 5 to 52%, denoting the inadequacy of this marker to identify malignancy in ACT 41-45. Even in childhood ACCs, p53 expression demonstrated no prognostic significance46.
p53 and MDM2 (Mouse double minute 2) form an auto regulatory negative feedback loop allowing the maintenance of low cellular p53 levels in the absence of stress 47. p53 stimulates the expression of MDM2 and MDM2 inhibits p53 activity by promoting its degradation, blocking its transcriptional activity, and promoting its nuclear export 47,48. As already described, DNA damage promotes phosphorylation of p53 and MDM2, mediated by ATR and ATM, and avoid MDM2-p53 interaction and thus stabilizing p53 (Figure 2) 47. Protein p19ARF also stabilizes p53 by inhibiting the nuclear export of MDM2 by tethering MDM2 in the nucleolus 49
.
The amplification of the MDM2 gene was observed in a minority of the human ACCs, as reported by several studies, including the pan-genomic studies 50-53 20, 21. Using the immunohistochemistry technique, MDM2 protein expression showed no differences between ACAs and ACCs 42, 45. The underexpression of ATR gene was also observed in adrenocortical carcinomas and was associated with poor survival27, 53. Mutations in ATM gene were also described in ACCs by some authors50, 51
CDK, cyclins and CDKi status in ACCs
Cell cycle progression from G1 to S phase is driven by the cyclin-dependent kinase (CDK) family of serine/threonine kinases (CDK-2, -4 and -6), their regulatory partners, the cyclins (D and E) and their inhibitors, CDKi (p15, p16, p21, p27 and p57) 54-56.
In the pan-genomic studies 20,21 and in other smaller studies one of the most frequent DNA copy number changes in ACCs are gains in chromosome (chr)12, combined with the amplification of CDK4 (located at chr12q14.1) and CDK2 (located at chr12q13) 53, 57-60. Reinforcing its importance, gains in chr12q13.2 have been associated with ACCs poor survival rates53. Overexpression of CDK2 and CDK4 has also been reported in ACC 61. At the protein levels, CDK4 and CDK2 overexpression was detected in the human ACC cell line (H295R) and in the majority of the ACCs62. Schmitt et al verified that all the ACCs studied presented CDK4 positive staining, but the same occurred in the majority of adrenocortical adenomas (ACAs); however, ACAs had a very weak positivity when compared to ACCs 63.
Cyclin E is the regulatory subunit of the cyclin E-CDK2 complex, and together they control the progression through G1 phase23, 64, 65. Cyclin E dysregulation is often observed in several tumor cells; it is thought to be involved in the tumorigenesis process and to be an important prognosis marker for some tumors 64, 66, 67 . There are two subtypes of cyclin E, the cyclin E1 and the cyclin E2, encoded by the genes: CCNE1 at chr19q12, and CCNE2 at chr8q22.1, respectively 65 . CCNE1 and CCNE2 overexpression and amplification has been reported in ACCs by several authors, including in the recent pan-genomic studies 20, 61, 68-71. Overexpression of the cyclin protein was observed in ACCs and found to be significantly associated with the histologic grade and with shorter disease-free survival62, 67, 71
Cyclin-dependent kinase inhibitors (CDKi) are negative regulators of cell cycle progression and potentially act as tumor suppressors, through the inhibition of the cyclin/CDK complexes. The CDKi, p57 is encoded by the CDKN1C gene located at chr11p15.5, where IGF2 is also located5, 72. Genetic rearrangements at the chr11p15.5 are common in ACCs, leading to the well-known phenomenon of IGF2 overexpression 5, 73-75. The majority of ACCs and virilizing ACAs present low p57 and H19 expression and high IGF2 expression 73. In contrast to normal adrenal glands and most ACA where the p57 mRNA is highly expressed, suggesting that p57 has a physiological role in normal adrenal cortex growth, the combination of low p57 and H19 expression and high IGF-2 expression seems to be involved in ACT malignancy 73. Low expression of CDKN1C gene and p57 protein has been found in adult and childhood ACC. This was not due to mutations since no CDKN1C mutations were detected, suggesting that other mechanisms, such as abnormalities of imprinting or methylation, could be responsible for its low expression 8, 34, 62, 69, 76. Downregulation of p57 in ACCs was found to be associated with CDK2 increased activity 62.
CDKN2A can encode, by alternative splicing, for p16, a CDK4/6 inhibitor, and p14, the p53 stabilizer, whereas, CDKN2B encodes another CDK4/6 inhibitor, the p15 50, 77. Alterations in both genes, namely the deletion of CDKN2A and CDKN2B were observed in 14.3% and 10.7% of the analyzed ACCs, respectively. Allelic losses on chr9p21, where CDKN2A and CDKN2B are harbored were described 20, 78
p21 and p27 are the CDKi involved in the regulation of cyclin E-CDK2 and cyclin D-CDK4/6 complexes in the G1-S transition (Figure 1) 79, 80 p21 is encoded by CDKNIA gene and its expression is regulated by p53, while p27 is encoded by CDKN1B gene, a gene that is rarely mutated in the context of cancer 79-81. The p21 protein expression was observed in both benign and malignant ACT, although a significantly higher proportion ACCs presented positive expression 42. Babinska et al, also found an increased expression of p21 in ACCs when compared with ACAs and a significant correlation between its expression and the occurrence of metastasis 82. Still, other studies did not find significant differences between benign and malignant ACT and there is evidence that its expression is inconsistent regardless of other molecular abnormalities, similarly to the p53 expression 34, 45. Nakazumi et al observed p27 expression to be decreased in the ACCs when compared with ACAs 83. However, other authors have found opposite results, with increased p27 expression in ACCs when compared to ACAs 42, 45
.
RB-E2F complex status in ACCs
In G1 phase, in order to allow the transition to S phase, CDK-cyclin complexes are responsible for the inactivation of the pRb through phosphorylation 84. Activated pRb binds to the transactivation domain of E2F to form a pRB- E2F complex that changes the chromatin structure at the E2F-responsive promoter by recruiting histone deacetylase (HDAC) to the pRB- E2F complex (Figure 3) 84, 85. Then, this complex binds to the promoter of some genes such as DNA polymerase subunits, cyclin A and cyclin E, which are required for S phase entry, leading to the transcription repression of those genes, and cell cycle arrest in phase G1 84, 86.
Upregulation of genes with binding sites for E2F seems to be a common event in many tumor types and has been also observed in ACCs 71, 87. Gains in the chr20, namely chr20q and chr20q11, where E2F1 is harbored have been reported as a common event in ACCs 57, 58, 60 However, other studies, including the pan-genomic studies, have not reported gains in chr20 as a common occurrence in ACCs 20, 21, 88, 89
Inactivating mutations, deletions and allelic losses of RB gene were observed in several tumors and seem to be associated with an increase in cancer susceptibility 84, 90. Ragazzon et al found that the loss of pRB was exclusively found in the subgroup of aggressive tumors, suggesting that the RB1 has an important role in the last events of the ACC and could be used as a prognostic marker 91. Among the ACCs with pRB loss, the majority presented an allelic loss at the RB1 locus 91 . de Fraipont et al confirmed that mRNA expression of RB1 was reduced in the malignant ACTs 92. Gupta et al also reported differences in the pRB1 staining between benign and malignant ACTs whereas, among the ACTs studied by Vargas et al, both benign and malignant ACTs, were positive for pRB1, with no differences between these two groups 93, 94. c-Myc is a pivotal regulator of the cell cycle being able to activate and repress pathways affecting G1 to S phase progression in mammalian cells. c-Myc overexpression leads to the loss of CDK inhibition resulting in the inactivation of pRb through phosphorylation, release of E2F and cell cycle progression to S phase. Furthermore, c-Myc induces the activity of E2F, directly and subsequently activates transcriptional of DNA synthetic enzymes 54, 95.
Amplification of protooncogene c-myc is frequently observed in a wide variety of human neoplasms through a variety of mechanisms 95, 96. In contrast to the observation in the majority of other tumors, several studies revealed that c-myc is underexpressed in ACCs compared to ACAs and to the normal adrenal cortex 69-71, 97, 98. The location of c-myc protein has also been correlated with ACT malignancy, since ACCs express c-myc both in the cell cytoplasm and nuclei while ACAs only express it in the cell nuclei 99. Since overexpression of c-myc induces cell proliferation and ACCs are rapidly proliferating tumors, c-myc was expected to be overexpressed in ACCs, however the opposite was found to occur in this type of cancer 95, 97.
G2-to-M phase transition in adrenocortical carcinomas
In ACCs, alterations in the G2-to-M transition are less frequently reported than changes in the G1-to-S. In addition, these changes were mostly reported in isolated studies and rarely in an integrated approach.
The most important role of the G2 phase is to ensure that the chromosomes have been accurately replicated without mistakes or damages, in order to allow the cycle to progress to mitosis 100
G2 to M transition is mainly regulated by the cyclin-dependent kinase, Cdc2, also known as CDK1 101, 102. Cdc2 is able to form a complex with either Cyclin B or Cyclin A (Figure 1) 101, 103
.
Cdc2 is activated by a combination of some required steps: - the phosphorylation of Thr16 by the Cdk-activating kinase (CAK) in order to open the catalytic region of Cdc2; - the nuclear translocation of Cdc2/cyclin B1 complex by the polo-like kinase 1 (Plk1) phosphorylation of Ser147 on Cdc2 and - the dephosphorylation of Thr14 and Tyr15 by the division 25 (Cdc25) phosphatase family, Cdc25A, Cdc25B, and Cdc25C 104-107
Activation of Cdc25C is achieved by phosphorylation of Ser198 by Plk1, leading to nuclear export of the Cdc25C 107. After that, Cdc25C is hyperphosphorylated by the complex Cdc2- cyclinB1, leading to a positive feedback loop, increasing the Cdc2-cyclinB1 activity (Figure 4A) 104.
Cdc25 is a dual-specificity phosphatase that not only activates the complexes cyclin B-Cdc2, but also the cyclin A-CDK2 and cyclin E-CDK2 at key cell cycle transitions: the isoform Cdc25A regulates the G1/S transition whereas the isoforms Cdc25B and Cdc25C act at G2/M, controlling the entry into mitosis 108, 109. Inactivation of Cdc25C is reached through phosphorylation of Ser216, creating a binding site for the 14-3-3 protein. Then the Cdc25C goes to the cytoplasm preventing Cdc25C and Cdc2 interaction (Figure 4B) 104, 110. This phosphorylation is mediated by Chk1, Chk2, C-TAK1 and Plk3 and also through the complexes Cyclin B-cdc2 or Cyclin A-cdc2 formation 104.
Deregulation of Cdc25 isoforms expression and activity, leading to unrestrained proliferation, has been found in some tumors 108, 109. In ACCs, CDC25 isoforms gene overexpression have been described 27, 69, 70, in parallel with the gain of chromosome region ch5q31.2, where CDC25C is harbored 111, 112. The overexpression of CDC2 was also reported in ACCs, by several studies, with a higher expression in the more aggressive cases 68-71, 113. Overexpression
of Plk1, the kinase responsible for Cdc2/cyclin B1 complex nuclear translocation and Cdc25c phosphorylation, has been associated with tumor development and considered a possible prognostic marker for some tumors 14-119. Furthermore, Plk1 inhibitors are in preliminary tests for tumor treatment 120-122. Overexpression of PLK1 has been found in ACCs when compared with the non-functioning ACAs, cortisol-producing ACAs and with the normal adrenal glands 69. Plk1 inhibitors, such as the small molecule BI-2536, have been tested in adrenocortical carcinoma cell lines, SW-13 and H295R, and found to significantly reduce the tumor cell growth, suggesting that Plk1 inhibitors deserve to be further investigated as a potential therapeutic approach in ACCs 123, 124
Inactivation of the Cdc2-cyclin B complexes, responsible for mitosis initiation is a pre-requisite to exit mitosis (Figure 1) 101, 102. Cyclin B presents various isoforms: Cyclin B1 that is encoded by CCNB1, cyclin B2 that is encoded by CCNB2 and cyclin B3, that is the less well characterized cyclin B, encoded by CCNB3 125, 126 . Overexpression of CCNB1 or the correspondent chromosome gains were described in ACCs comparing to ACAs and normal adrenal glands 27, 69, 70, 113, 127. Cyclin B1 protein expression was also found to be increased in ACCs but in spite of a high specificity (100%), it had a low sensitivity (43%) for predicting malignancy. In consequence, Cyclin B1 is only useful for confirming malignancy but is not helpful for its exclusion it when it is negative 113.
CCNB2 overexpression was also found in ACCs, particularly in the more aggressive forms. However the correspondent chromosomal alterations were not observed 27, 69, 70, 127
The Myelin Transcription Factor 1 (Myt1) and Wee1 are proteins able to inhibit the Cdc2, through inhibitory phosphorylation at Thr14 and Tyr15 (Figure 4) 100, 109, 128, 129. Once activated, cyclin B-Cdc2 complexes phosphorylate Weel and Mytl to promote their inactivation, allowing an even larger amplification of Cdc2 activation 16. Weel is predominantly a nuclear protein that has been found to associate with centrosomes, whereas Myt1 is present in the cytoplasm bound to membrane structures 128, 130 . WEE1 gene overexpression has been found in ACCs when compared with ACAs and normal adrenal glands 68 , but there was no MYT1 gene overexpression in ACCs 69.
Topoisomerases (TOP) are enzymes that are involved in the biological processes that require strand unwinding, such as replication, transcription, and maintenance of genome stability, as they are able to introduce transient breaks in DNA 131, 132. The TOP1 introduces single strand breaks in the DNA and TOP2 introduces double strand breaks. Type II topoisomerases are required to segregate replicated chromosomes 131, 133. TOP2A, is a TOP2 isoform, present only in the S, G2, and M phases of the cell cycle of proliferating tissues 131, 133. Several authors confirmed TOP2A overexpression in ACCs 61, 68-71, 127 which was associated with higher ACC aggressiveness70. TOP2A protein overexpression was associated with significantly poorer overall and disease-free survival 93, 134-136. Iino et al have also suggested that TOP2A could be an even better proliferation marker than Ki-67, since some cells expressing TOP2A failed to express Ki-67 134. Chromosomal gains at chr17, where TOP2A is harbored, were also found in ACAs, suggesting that it may be an early event in the tumorigenesis of ACT 60.
Clinical implications for the diagnosis and treatment of adrenocortical carcinomas
Considerable advances in the understanding of adrenocortical neoplasia molecular pathology have occurred over the recent years. In spite of this, ACC continues to have a poor prognosis, as they are frequently diagnosed in advanced disease stages and harbor molecular alterations responsible for an aggressive biological behavior. Thus, much efforts are required to identify the main drivers in ACC pathogenesis in order to design the appropriate treatments that are still missing. Nonetheless, recent molecular studies allowed identification of two subsets of ACC tumors with distinctive cell proliferation rates and clinical prognosis 20, 21, 70. Tumors with worst prognosis depict a high histological grade and present a greater frequency of gene mutations, DNA methylation and a specific miRNA expression profile involving cell cycle and proliferation; more differentiated tumors tend to harbor metabolism instead of cell cycle related molecular alterations and have improved clinical outcomes, as evidenced by higher survival rates and lower ENSAT stage 20. There is, however, some overlap between the two tumor categories highlighting the need to pursue the distinctive mechanisms.
The morphological characteristics and the initial molecular studies in ACC supported the hypothesis that proliferation was the most important feature leading to tumor development and dissemination. Genomic analysis has allowed to identify particular gene subsets that are overexpressed while others are under-expressed, whereas more innovative investigations focusing on epigenetic analysis and microRNAs have highlighted additional determinants of tumor biological behavior. Concerning the cell cycle alterations, overexpression of the cell- cycle related genes can distinguish the ACC with good and poor prognosis, the last group being the one with an increased expression; in contrast, the adenomas rarely presented those types of alterations and thus those features are possible predictors of malignancy and prognosis 137, 138.
One of the most frequent alterations found in the ACC is LOH in chr11p15, where CDKN1C and IGF2 are localized. IGF2 expression has been demonstrated to be a useful marker for predicting malignancy after ACT surgical removal 139, 140. The influence of IGF2 overexpression in the ACC tumorigenesis is well established and reasonably understood. In contrast, CDKN1C under expression is frequently found in ACCs, but its role is not yet fully understood 73, 139. Besides its relevance as a diagnostic tool, these alterations have also raised the expectation of being possible treatment targets. Indeed, the use of IGF1-R inhibitors for ACC was tested in several clinical trials but so far failed to improve significantly patients’ progression-free survival and overall survival 141-143. Subsequently, as IGF1-R is a well-known mTOR pathway regulator, the combined use of simultaneous IGF1R and mTOR inhibitors to reduce ACC growth was tested. However, the only observable effect was disease stabilization for at least 6 months in 42% of the patients 144.
TOP2A, whose protein expression is associated with poor overall and disease-free survival 93, 134-136 is particularly interesting. In ACC, TOP2A seems to be a better proliferation marker even when compared to Ki-67, as some TOP2A expressing cells fail to express Ki-67 134. Roca et al evaluated the TOP2A gene and protein expression in a large series of 98 ACC patients and showed that a high TOP2A expression in ACC tumor is significantly associated with higher response to the combined chemotherapy EDP plus Mitotane (EDP-M) and with a longer time to tumor progression. These results suggest that TOP2A could also have a potential clinical
relevance as a predictor for the EDP-M treatment response 145. TOP2A inhibition has already been tested in in vitro and in clinical trials with some promising results 135. In vitro anti-cancer activity of 14 different agents targeting TOP2A were evaluated. Among these agents the one that demonstrated to have the highest anticancer activity was aclarubicin and this compound will probably be tested in future clinical trials for the treatment of locally advanced and metastatic ACC 135. Moreover, clinical trials including etoposide and doxorubicin, topoisomerase 2 enzyme inhibitors, in combination with cisplatin (EDP) used in advanced ACCs resulted in a better progression-free survival when compared to the mitotane and streptozotocin combination (NCT00094497) 5, 146, 147
PLK1 regulates multiple steps of cell division and DNA stability/repair. PLK1 expression levels are positively correlated with a poor survival in ACC patients, suggesting PLK1 as a good prognostic marker that could also be a good candidate for targeted therapy 124, 148 TKM- 080301 is a lipid nanoparticle formulation of a siRNA targeting PLK1. 8 ACC patients (NCT01262235) received two cycles of the treatment and 4 patients showed a good response in stabilizing disease including a 13% reduction of tumor size in one patient, thus suggesting that the role of PLK1 inhibition for ACC treatment should be further investigated 149.
The fact that the completed clinical trials produced insufficient outcomes, highlights that there is still much to be learned about the molecular pathology of these tumors. Thus, in addition to previous and ongoing clinical trials, other pharmacological targets based on the recent molecular findings related to the cell cycle alterations found in ACC are likely to start being tested in the near future. These include CDK inhibitors, drugs targeting p53 reactivation and CDC25 inhibitors.
The CDK/cyclin complexes have a critical role in the regulation of cell cycle transition that if disrupted can ultimately lead to uncontrolled proliferation. Thus, these are certainly molecular attractive targets for cancer treatment 25. The overexpression of CDK4/6/2 and 1, as well as the overexpression of cyclins involved in the G1-S transition (CCNE1/2) and in the G2-M (CCNB1/2) were recurrently observed in ACC CDK inhibitors are being tested for several advanced solid tumors, but there are no registered clinical trials aiming to assess
their efficacy in ACCs. Besides, despite the results of the initial clinical trials using CDK inhibitors being disappointing, the recent use of highly selective CDK inhibitors, specifically targeting CDK4 and CDK6, combined with patient stratification, showed a more substantial and promising clinical activity 25. Indeed, a small molecule that is a selective CDK4/6 inhibitor (SHR6390) is in clinical trial for advanced solid tumors, currently in a recruiting phase and thus could represent an opportunity to include patients with ACCs (NCT02684266).
TP53 was recognized as one of ACC driver genes by the pan-genomic studies 20,21. Inactivating mutations were found to be present in 21.3% of the ACCs 20, 21. So, the recovery of p53 “tumor suppressor gene” function, using MDM2 antagonist (such as RO5503781) and the reactivation of mutant TP53, using PRIMA-1MET, already tested for other malignancies 150, also have the potential in to be used in ACCs with TP53 inactivated.
Altered expression of CDC25 gene isoforms, repeatedly described in ACCs, could represent a potential treatment target for these tumors 27, 69, 70. However, despite the fact that several natural and synthetic molecules with distinct structural features targeting CDC25 with good pre-clinical results have been identified 151, 152, no registered clinical trials using CDC25 inhibitors for ACC are ongoing.
Finally, in addition to the specific molecules that were identified as potential targets for ACC treatment, there are a few other molecules that despite not having yet been considered for their therapeutic potential, will eventually prove very valuable as new diagnostic and prognostic molecular tools in the near future (Table 1, 2 and 3).
CONCLUSION
Our aim was to deliver an integrative review of what has been reported regarding cell cycle disruptions in ACC, so that this comprehensive and integrated knowledge could then be used to optimize disease diagnosis and treatment.
The rarity of the adrenocortical carcinoma represents an important limitation to accomplish a complete molecular characterization of cell cycle alterations. Despite this limitation, collaborative studies focusing in a wide array of molecular pathways have been performed. The results of these studies are summarized in this review, highlighting the complexity of the molecular alterations that may contribute for an aggressive biological behavior in ACC.
The molecular mechanisms underlying the traditional pathological features, have already proved to be useful molecular markers for differential diagnosis and tumor stratification and prognosis. In addition, targeting the mechanisms leading to uncontrolled cell proliferation may have a much required potential for ACC treatment. From a clinical perspective, these have already led to the implementation of targeted treatments aiming to be used alone or in combination with the classical adrenolytic drug mitotane. However, we must state that some genomic alterations were analyzed in a non-functional perspective and so up until now their functional impact, is most of the times, unknown.
In conclusion, the main goal for the future should be translating what was learned, and summarized in this review, together with the comprehensive information resulting from the pan- genomic studies into better tools to identify and evaluate ACC, as the final objective to develop specific medical therapy personalized for each patient to improve their disease free intervals and survival.
ACKNOWLEDGMENTS
The authors have no Conflict of Interest disclosure.
Portuguese Foundation for Science and Technology (FCT) funds UMIB (UID/Multi/00215/2013), I3S (POCI-01-0145-FEDER-007274) and Sofia S. Pereira through a PhD fellowship (SFRH/BD/89308/2012)
REFERENCES
1. Chagpar R, Siperstein AE & Berber E. Adrenocortical cancer update. Surg Clin North Am 2014 94 669-687.
2. Roman S. Adrenocortical carcinoma. Curr Opin Oncol 2006 18 36-42.
3. Libe R, Borget I, Ronchi CL, Zaggia B, Kroiss M, Kerkhofs T, Bertherat J, Volante M, Quinkler M, Chabre O, Bala M, Tabarin A, Beuschlein F, Vezzosi D, Deutschbein T, Borson-Chazot F, Hermsen I, Stell A, Fottner C, Leboulleux S, Hahner S, Mannelli M, Berruti A, Haak H, Terzolo M, Fassnacht M, Baudin E & network E. Prognostic factors in stage III-IV adrenocortical carcinomas (ACC): an European Network for the Study of Adrenal Tumor (ENSAT) study. Ann Oncol 2015 26 2119-2125.
4. Else T, Kim AC, Sabolch A, Raymond VM, Kandathil A, Caoili EM, Jolly S, Miller BS, Giordano TJ & Hammer GD. Adrenocortical carcinoma. Endocr Rev 2014 35 282-326.
5. Fassnacht M, Libe R, Kroiss M & Allolio B. Adrenocortical carcinoma: a clinician’s update. Nat Rev Endocrinol 2011 7 323-335.
6. Audenet F, Mejean A, Chartier-Kastler E & Roupret M. Adrenal tumours are more predominant in females regardless of their histological subtype: a review. World J Urol 2013 31 1037-1043.
7. Low G, Dhliwayo H & Lomas DJ. Adrenal neoplasms. Clin Radiol 2012 67 988-1000.
8. Soon PS, McDonald KL, Robinson BG & Sidhu SB. Molecular markers and the pathogenesis of adrenocortical cancer. Oncologist 2008 13 548-561.
9. Lafemina J & Brennan MF. Adrenocortical carcinoma: past, present, and future. J Surg Oncol 2012 106 586-594.
10. Fassnacht M, Arlt W, Bancos I, Dralle H, Newell-Price J, Sahdev A, Tabarin A, Terzolo M, Tsagarakis S & Dekkers OM. Management of adrenal incidentalomas: European Society of Endocrinology Clinical Practice Guideline in collaboration with the European Network for the Study of Adrenal Tumors. Eur J Endocrinol 2016 175 G1-G34.
11. Allolio B & Fassnacht M. Clinical review: Adrenocortical carcinoma: clinical update. J Clin Endocrinol Metab 2006 91 2027-2037.
12. Lau SK & Weiss LM. The Weiss system for evaluating adrenocortical neoplasms: 25 years later. Hum Pathol 2009 40 757-768.
13. Tissier F. Classification of adrenal cortical tumors: what limits for the pathological approach? Best Pract Res Clin Endocrinol Metab 2010 24 877-885.
14. Wachenfeld C, Beuschlein F, Zwermann O, Mora P, Fassnacht M, Allolio B & Reincke M. Discerning malignancy in adrenocortical tumors: are molecular markers useful? Eur J Endocrinol 2001 145 335-341.
15. Beuschlein F, Weigel J, Saeger W, Kroiss M, Wild V, Daffara F, Libe R, Ardito A, Al Ghuzlan A, Quinkler M, Osswald A, Ronchi CL, de Krijger R, Feelders RA, Waldmann J, Willenberg HS, Deutschbein T, Stell A, Reincke M, Papotti M, Baudin E, Tissier F, Haak HR, Loli P, Terzolo M, Allolio B, Muller HH & Fassnacht M. Major prognostic role of Ki67 in localized adrenocortical carcinoma after complete resection. J Clin Endocrinol Metab 2015 100 841-849.
16. Berruti A, Baudin E, Gelderblom H, Haak HR, Porpiglia F, Fassnacht M & Pentheroudakis G. Adrenal cancer: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Ann Oncol 2012 23 Suppl 7 vii131-138.
17. Fassnacht M, Johanssen S, Quinkler M, Bucsky P, Willenberg HS, Beuschlein F, Terzolo M, Mueller H-H, Hahner S, Allolio B, for the German Adrenocortical Carcinoma Registry G & the European Network for the Study of Adrenal T. Limited prognostic value of the 2004 International Union Against Cancer staging classification for adrenocortical carcinoma. Cancer 2009 115 243-250.
18. Abiven G, Coste J, Groussin L, Anract P, Tissier F, Legmann P, Dousset B, Bertagna X & Bertherat J. Clinical and biological features in the prognosis of adrenocortical cancer: poor outcome of cortisol-secreting tumors in a series of 202 consecutive patients. J Clin Endocrinol Metab 2006 91 2650-2655.
19. Faillot S & Assie G. ENDOCRINE TUMOURS: The genomics of adrenocortical tumors. Eur J Endocrinol 2016 174 R249-265.
20. Zheng S, Cherniack AD, Dewal N, Moffitt RA, Danilova L, Murray BA, Lerario AM, Else T, Knijnenburg TA, Ciriello G, Kim S, Assie G, Morozova O, Akbani R, Shih J, Hoadley KA, Choueiri TK, Waldmann J, Mete O, Robertson AG, Wu HT, Raphael BJ, Shao L, Meyerson M, Demeure MJ, Beuschlein F, Gill AJ, Sidhu SB, Almeida MQ, Fragoso MC, Cope LM, Kebebew E, Habra MA, Whitsett TG, Bussey KJ, Rainey WE, Asa SL, Bertherat J, Fassnacht M, Wheeler DA, Cancer Genome Atlas Research N, Hammer GD, Giordano TJ & Verhaak RG. Comprehensive Pan-Genomic Characterization of Adrenocortical Carcinoma. Cancer Cell 2016 29 723-736.
21. Assie G, Letouze E, Fassnacht M, Jouinot A, Luscap W, Barreau O, Omeiri H, Rodriguez S, Perlemoine K, Rene-Corail F, Elarouci N, Sbiera S, Kroiss M, Allolio B, Waldmann J, Quinkler M, Mannelli M, Mantero F, Papathomas T, De Krijger R, Tabarin A, Kerlan V, Baudin E, Tissier F, Dousset B, Groussin L, Amar L, Clauser E, Bertagna X, Ragazzon B, Beuschlein F, Libe R, de Reynies A & Bertherat J. Integrated genomic characterization of adrenocortical carcinoma. Nat Genet 2014 46 607-612.
22. Cooper GM. The Eukaryotic Cell Cycle. In The Cell: A Molecular Approach., edn Second edition: Sunderland (MA): Sinauer Associates, 2000.
23. Lim S & Kaldis P. Cdks, cyclins and CKIs: roles beyond cell cycle regulation. Development 2013 140 3079-3093.
24. Park MT & Lee SJ. Cell cycle and cancer. J Biochem Mol Biol 2003 36 60-65.
25. Asghar U, Witkiewicz AK, Turner NC & Knudsen ES. The history and future of targeting cyclin-dependent kinases in cancer therapy. Nat Rev Drug Discov 2015 14 130-146.
26. Bertoli C, Skotheim JM & de Bruin RA. Control of cell cycle transcription during G1 and S phases. Nat Rev Mol Cell Biol 2013 14 518-528.
27. Szabo PM, Tamasi V, Molnar V, Andrasfalvy M, Tombol Z, Farkas R, Kovesdi K, Patocs A, Toth M, Szalai C, Falus A, Racz K & Igaz P. Meta-analysis of adrenocortical tumour genomics data: novel pathogenic pathways revealed. Oncogene 2010 29 3163-3172.
28. el-Deiry WS, Tokino T, Velculescu VE, Levy DB, Parsons R, Trent JM, Lin D, Mercer WE, Kinzler KW & Vogelstein B. WAF1, a potential mediator of p53 tumor suppression. Cell 1993 75 817-825.
29. Adams PD, Sellers WR, Sharma SK, Wu AD, Nalin CM & Kaelin WG, Jr. Identification of a cyclin-cdk2 recognition motif present in substrates and p21- like cyclin-dependent kinase inhibitors. Mol Cell Biol 1996 16 6623-6633.
30. Hisada M, Garber JE, Fung CY, Fraumeni JF, Jr. & Li FP. Multiple primary cancers in families with Li-Fraumeni syndrome. J Natl Cancer Inst 1998 90 606-611.
31. McBride KA, Ballinger ML, Killick E, Kirk J, Tattersall MH, Eeles RA, Thomas DM & Mitchell G. Li-Fraumeni syndrome: cancer risk assessment and clinical management. Nat Rev Clin Oncol 2014 11 260-271.
32. Waldmann J, Patsalis N, Fendrich V, Langer P, Saeger W, Chaloupka B, Ramaswamy A, Fassnacht M, Bartsch DK & Slater EP. Clinical impact of TP53 alterations in adrenocortical carcinomas. Langenbecks Arch Surg 2012 397 209- 216.
33. Libe R, Groussin L, Tissier F, Elie C, Rene-Corail F, Fratticci A, Jullian E, Beck-Peccoz P, Bertagna X, Gicquel C & Bertherat J. Somatic TP53 mutations are relatively rare among adrenocortical cancers with the frequent 17p13 loss of heterozygosity. Clin Cancer Res 2007 13 844-850.
34. Barzon L, Chilosi M, Fallo F, Martignoni G, Montagna L, Palu G & Boscaro M. Molecular analysis of CDKN1C and TP53 in sporadic adrenal tumors. Eur J Endocrinol 2001 145 207-212.
35. Herrmann LJ, Heinze B, Fassnacht M, Willenberg HS, Quinkler M, Reisch N, Zink M, Allolio B & Hahner S. TP53 germline mutations in adult patients with adrenocortical carcinoma. J Clin Endocrinol Metab 2012 97 E476-485.
36. Ohgaki H, Kleihues P & Heitz PU. p53 mutations in sporadic adrenocortical tumors. Int J Cancer 1993 54 408-410.
37. Wasserman JD, Novokmet A, Eichler-Jonsson C, Ribeiro RC, Rodriguez- Galindo C, Zambetti GP & Malkin D. Prevalence and functional consequence of TP53 mutations in pediatric adrenocortical carcinoma: a children’s oncology group study. J Clin Oncol 2015 33 602-609.
38. Wagner J, Portwine C, Rabin K, Leclerc JM, Narod SA & Malkin D. High frequency of germline p53 mutations in childhood adrenocortical cancer. J Natl Cancer Inst 1994 86 1707-1710.
39. Varley JM, McGown G, Thorncroft M, James LA, Margison GP, Forster G, Evans DG, Harris M, Kelsey AM & Birch JM. Are there low-penetrance TP53 Alleles? evidence from childhood adrenocortical tumors. Am J Hum Genet 1999 65 995-1006.
40. Ragazzon B, Libe R, Gaujoux S, Assie G, Fratticci A, Launay P, Clauser E, Bertagna X, Tissier F, de Reynies A & Bertherat J. Transcriptome analysis reveals that p53 and {beta}-catenin alterations occur in a group of aggressive adrenocortical cancers. Cancer Res 2010 70 8276-8281.
41. Arola J, Salmenkivi K, Liu J, Kahri AI & Heikkila P. p53 and Ki67 in adrenocortical tumors. Endocr Res 2000 26 861-865.
42. Stojadinovic A, Brennan MF, Hoos A, Omeroglu A, Leung DH, Dudas ME, Nissan A, Cordon-Cardo C & Ghossein RA. Adrenocortical adenoma and carcinoma: histopathological and molecular comparative analysis. Mod Pathol 2003 16 742-751.
43. McNicol AM, Struthers AL, Nolan CE, Hermans J & Haak HR. Proliferation in Adrenocortical Tumors: Correlation with Clinical Outcome and p53 Status. Endocr Pathol 1997 8 29-36.
44. Reincke M, Karl M, Travis WH, Mastorakos G, Allolio B, Linehan HM & Chrousos GP. p53 mutations in human adrenocortical neoplasms: immunohistochemical and molecular studies. J Clin Endocrinol Metab 1994 78 790-794.
45. Pereira SS, Morais T, Costa MM, Monteiro MP & Pignatelli D. The emerging role of the molecular marker p27 in the differential diagnosis of adrenocortical tumors. Endocr Connect 2013 2 137-145.
46. Sbragia L, Oliveira-Filho AG, Vassallo J, Pinto GA, Guerra-Junior G & Bustorff-Silva J. Adrenocortical tumors in Brazilian children: immunohistochemical markers and prognostic factors. Arch Pathol Lab Med 2005 129 1127-1131.
47. Moll UM & Petrenko O. The MDM2-p53 interaction. Mol Cancer Res 2003 1 1001-1008.
48. Shi D & Gu W. Dual Roles of MDM2 in the Regulation of p53: Ubiquitination Dependent and Ubiquitination Independent Mechanisms of MDM2 Repression of p53 Activity. Genes Cancer 2012 3 240-248.
49. Tao W & Levine AJ. P19(ARF) stabilizes p53 by blocking nucleo-cytoplasmic shuttling of Mdm2. Proc Natl Acad Sci U S A 1999 96 6937-6941.
50. De Martino MC, Al Ghuzlan A, Aubert S, Assie G, Scoazec JY, Leboulleux S, Do Cao C, Libe R, Nozieres C, Lombes M, Pattou F, Borson-Chazot F, Hescot S, Mazoyer C, Young J, Borget I, Colao A, Pivonello R, Soria JC, Bertherat J, Schlumberger M, Lacroix L & Baudin E. Molecular screening for a personalized treatment approach in advanced adrenocortical cancer. J Clin Endocrinol Metab 2013 98 4080-4088.
51. Ross JS, Wang K, Rand JV, Gay L, Presta MJ, Sheehan CE, Ali SM, Elvin JA, Labrecque E, Hiemstra C, Buell J, Otto GA, Yelensky R, Lipson D, Morosini D, Chmielecki J, Miller VA & Stephens PJ. Next-generation sequencing of adrenocortical carcinoma reveals new routes to targeted therapies. J Clin Pathol 2014 67 968-973.
52. Letouze E, Rosati R, Komechen H, Doghman M, Marisa L, Fluck C, de Krijger RR, van Noesel MM, Mas JC, Pianovski MA, Zambetti GP, Figueiredo BC & Lalli E. SNP array profiling of childhood adrenocortical tumors reveals distinct pathways of tumorigenesis and highlights candidate driver genes. J Clin Endocrinol Metab 2012 97 E1284-1293.
53. Stephan EA, Chung TH, Grant CS, Kim S, Von Hoff DD, Trent JM & Demeure MJ. Adrenocortical carcinoma survival rates correlated to genomic copy number variants. Mol Cancer Ther 2008 7 425-431.
54. Zajac-Kaye M. Myc oncogene: a key component in cell cycle regulation and its implication for lung cancer. Lung Cancer 2001 34 Suppl 2 S43-46.
55. Bartek J, Bartkova J & Lukas J. The retinoblastoma protein pathway and the restriction point. Curr Opin Cell Biol 1996 8 805-814.
56. Sherr CJ. The Pezcoller lecture: cancer cell cycles revisited. Cancer Res 2000 60 3689-3695.
57. Barreau O, de Reynies A, Wilmot-Roussel H, Guillaud-Bataille M, Auzan C, Rene-Corail F, Tissier F, Dousset B, Bertagna X, Bertherat J, Clauser E & Assie G. Clinical and pathophysiological implications of chromosomal alterations in adrenocortical tumors: an integrated genomic approach. J Clin Endocrinol Metab 2012 97 E301-311.
58. Zhao J, Roth J, Bode-Lesniewska B, Pfaltz M, Heitz PU & Komminoth P. Combined comparative genomic hybridization and genomic microarray for detection of gene amplifications in pulmonary artery intimal sarcomas and adrenocortical tumors. Genes Chromosomes Cancer 2002 34 48-57.
59. Dohna M, Reincke M, Mincheva A, Allolio B, Solinas-Toldo S & Lichter P. Adrenocortical carcinoma is characterized by a high frequency of chromosomal
gains and high-level amplifications. Genes Chromosomes Cancer 2000 28 145- 152.
60. Zhao J, Speel EJ, Muletta-Feurer S, Rutimann K, Saremaslani P, Roth J, Heitz PU & Komminoth P. Analysis of genomic alterations in sporadic adrenocortical lesions. Gain of chromosome 17 is an early event in adrenocortical tumorigenesis. Am J Pathol 1999 155 1039-1045.
61. Ragazzon B, Assie G & Bertherat J. Transcriptome analysis of adrenocortical cancers: from molecular classification to the identification of new treatments. Endocr Relat Cancer 2011 18 R15-27.
62. Bourcigaux N, Gaston V, Logie A, Bertagna X, Le Bouc Y & Gicquel C. High expression of cyclin E and G1 CDK and loss of function of p57KIP2 are involved in proliferation of malignant sporadic adrenocortical tumors. J Clin Endocrinol Metab 2000 85 322-330.
63. Schmitt A, Saremaslani P, Schmid S, Rousson V, Montani M, Schmid DM, Heitz PU, Komminoth P & Perren A. IGFII and MIB1 immunohistochemistry is helpful for the differentiation of benign from malignant adrenocortical tumours. Histopathology 2006 49 298-307.
64. Hwang HC & Clurman BE. Cyclin E in normal and neoplastic cell cycles. Oncogene 2005 24 2776-2786.
65. Caldon CE & Musgrove EA. Distinct and redundant functions of cyclin E1 and cyclin E2 in development and cancer. Cell Div 2010 5 2.
66. Berrebi D, Leclerc J, Schleiermacher G, Zaccaria I, Boccon-Gibod L, Fabre M, Jaubert F, El Ghoneimi A, Jeanpierre C & Peuchmaur M. High cyclin E staining index in blastemal, stromal or epithelial cells is correlated with tumor aggressiveness in patients with nephroblastoma. PLoS One 2008 3 e2216.
67. Tissier F, Louvel A, Grabar S, Hagnere AM, Bertherat J, Vacher-Lavenu MC, Dousset B, Chapuis Y, Bertagna X & Gicquel C. Cyclin E correlates with malignancy and adverse prognosis in adrenocortical tumors. Eur J Endocrinol 2004 150 809-817.
68. Giordano TJ, Thomas DG, Kuick R, Lizyness M, Misek DE, Smith AL, Sanders D, Aljundi RT, Gauger PG, Thompson NW, Taylor JM & Hanash SM. Distinct transcriptional profiles of adrenocortical tumors uncovered by DNA microarray analysis. Am J Pathol 2003 162 521-531.
69. Tombol Z, Szabo PM, Molnar V, Wiener Z, Tolgyesi G, Horanyi J, Riesz P, Reismann P, Patocs A, Liko I, Gaillard RC, Falus A, Racz K & Igaz P. Integrative molecular bioinformatics study of human adrenocortical tumors: microRNA, tissue-specific target prediction, and pathway analysis. Endocr Relat Cancer 2009 16 895-906.
70. de Reynies A, Assie G, Rickman DS, Tissier F, Groussin L, Rene-Corail F, Dousset B, Bertagna X, Clauser E & Bertherat J. Gene expression profiling reveals a new classification of adrenocortical tumors and identifies molecular predictors of malignancy and survival. J Clin Oncol 2009 27 1108-1115.
71. Giordano TJ, Kuick R, Else T, Gauger PG, Vinco M, Bauersfeld J, Sanders D, Thomas DG, Doherty G & Hammer G. Molecular classification and prognostication of adrenocortical tumors by transcriptome profiling. Clin Cancer Res 2009 15 668-676.
72. Borriello A, Caldarelli I, Bencivenga D, Criscuolo M, Cucciolla V, Tramontano A, Oliva A, Perrotta S & Della Ragione F. p57(Kip2) and cancer: time for a critical appraisal. Mol Cancer Res 2011 9 1269-1284.
73. Liu J, Kahri AI, Heikkila P & Voutilainen R. Ribonucleic acid expression of the clustered imprinted genes, p57KIP2, insulin-like growth factor II, and H19, in adrenal tumors and cultured adrenal cells. J Clin Endocrinol Metab 1997 82 1766-1771.
74. Gicquel C, Raffin-Sanson ML, Gaston V, Bertagna X, Plouin PF, Schlumberger M, Louvel A, Luton JP & Le Bouc Y. 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.
75. Sidhu S, Gicquel C, Bambach CP, Campbell P, Magarey C, Robinson BG & Delbridge LW. Clinical and molecular aspects of adrenocortical tumourigenesis. ANZ J Surg 2003 73 727-738.
76. West AN, Neale GA, Pounds S, Figueredo BC, Rodriguez Galindo C, Pianovski MA, Oliveira Filho AG, Malkin D, Lalli E, Ribeiro R & Zambetti GP. Gene expression profiling of childhood adrenocortical tumors. Cancer Res 2007 67 600-608.
77. Dominguez-Brauer C, Brauer PM, Chen YJ, Pimkina J & Raychaudhuri P. Tumor suppression by ARF: gatekeeper and caretaker. Cell Cycle 2010 9 86-89.
78. Pilon C, Pistorello M, Moscon A, Altavilla G, Pagotto U, Boscaro M & Fallo F. Inactivation of the p16 tumor suppressor gene in adrenocortical tumors. J Clin Endocrinol Metab 1999 84 2776-2779.
79. Lloyd RV, Erickson LA, Jin L, Kulig E, Qian X, Cheville JC & Scheithauer BW. p27kip1: a multifunctional cyclin-dependent kinase inhibitor with prognostic significance in human cancers. Am J Pathol 1999 154 313-323.
80. Warfel NA & El-Deiry WS. p21WAF1 and tumourigenesis: 20 years after. Curr Opin Oncol 2013 25 52-58.
81. Nickeleit I, Zender S, Kossatz U & Malek NP. p27kip1: a target for tumor therapies? Cell Div 2007 2 13.
82. Babinska A, Sworczak K, Wisniewski P, Nalecz A & Jaskiewicz K. The role of immunohistochemistry in histopathological diagnostics of clinically “silent” incidentally detected adrenal masses. Exp Clin Endocrinol Diabetes 2008 116 246-251.
83. Nakazumi H, Sasano H, Iino K, Ohashi Y & Orikasa S. Expression of cell cycle inhibitor p27 and Ki-67 in human adrenocortical neoplasms. Mod Pathol 1998 11 1165-1170.
84. Giacinti C & Giordano A. RB and cell cycle progression. Oncogene 2006 25 5220-5227.
85. Takaki T, Fukasawa K, Suzuki-Takahashi I & Hirai H. Cdk-mediated phosphorylation of pRB regulates HDAC binding in vitro. Biochem Biophys Res Commun 2004 316 252-255.
86. Sun A, Bagella L, Tutton S, Romano G & Giordano A. From G0 to S phase: a view of the roles played by the retinoblastoma (Rb) family members in the Rb- E2F pathway. J Cell Biochem 2007 102 1400-1404.
87. Rhodes DR, Kalyana-Sundaram S, Mahavisno V, Barrette TR, Ghosh D & Chinnaiyan AM. Mining for regulatory programs in the cancer transcriptome. Nat Genet 2005 37 579-583.
88. Sidhu S, Marsh DJ, Theodosopoulos G, Philips J, Bambach CP, Campbell P, Magarey CJ, Russell CF, Schulte KM, Roher HD, Delbridge L & Robinson BG. Comparative genomic hybridization analysis of adrenocortical tumors. J Clin Endocrinol Metab 2002 87 3467-3474.
89. Kjellman M, Kallioniemi OP, Karhu R, Hoog A, Farnebo LO, Auer G, Larsson C & Backdahl M. Genetic aberrations in adrenocortical tumors detected using comparative genomic hybridization correlate with tumor size and malignancy. Cancer Res 1996 56 4219-4223.
90. Di Fiore R, D’Anneo A, Tesoriere G & Vento R. RB1 in cancer: different mechanisms of RB1 inactivation and alterations of pRb pathway in tumorigenesis. J Cell Physiol 2013 228 1676-1687.
91. Ragazzon B, Libe R, Assie G, Tissier F, Barreau O, Houdayer C, Perlemoine K, Audebourg A, Clauser E, Rene-Corail F, Bertagna X, Dousset B, Bertherat J & Groussin L. Mass-array screening of frequent mutations in cancers reveals RB1 alterations in aggressive adrenocortical carcinomas. Eur J Endocrinol 2014 170 385-391.
92. de Fraipont F, El Atifi M, Cherradi N, Le Moigne G, Defaye G, Houlgatte R, Bertherat J, Bertagna X, Plouin PF, Baudin E, Berger F, Gicquel C, Chabre O & Feige JJ. Gene expression profiling of human adrenocortical tumors using complementary deoxyribonucleic Acid microarrays identifies several candidate genes as markers of malignancy. J Clin Endocrinol Metab 2005 90 1819-1829.
93. Gupta D, Shidham V, Holden J & Layfield L. Value of topoisomerase II alpha, MIB-1, p53, E-cadherin, retinoblastoma gene protein product, and HER-2/neu immunohistochemical expression for the prediction of biologic behavior in adrenocortical neoplasms. Appl Immunohistochem Mol Morphol 2001 9 215- 221.
94. Vargas MP, Vargas HI, Kleiner DE & Merino MJ. Adrenocortical neoplasms: role of prognostic markers MIB-1, P53, and RB. Am J Surg Pathol 1997 21 556- 562.
95. Dang CV. MYC, metabolism, cell growth, and tumorigenesis. Cold Spring Harb Perspect Med 2013 3.
96. Nesbit CE, Tersak JM & Prochownik EV. MYC oncogenes and human neoplastic disease. Oncogene 1999 18 3004-3016.
97. Szabo PM, Racz K & Igaz P. Underexpression of C-myc in adrenocortical cancer: a major pathogenic event? Horm Metab Res 2011 43 297-299.
98. Liu J, Voutilainen R, Kahri AI & Heikkila P. Expression patterns of the c-myc gene in adrenocortical tumors and pheochromocytomas. J Endocrinol 1997 152 175-181.
99. Suzuki T, Sasano H, Nisikawa T, Rhame J, Wilkinson DS & Nagura H. Discerning malignancy in human adrenocortical neoplasms: utility of DNA flow cytometry and immunohistochemistry. Mod Pathol 1992 5 224-231.
100. DiPaola RS. To arrest or not to G(2)-M Cell-cycle arrest : commentary re: A. K. Tyagi et al., Silibinin strongly synergizes human prostate carcinoma DU145 cells to doxorubicin-induced growth inhibition, G(2)-M arrest, and apoptosis. Clin. cancer res., 8: 3512-3519, 2002. Clin Cancer Res 2002 8 3311-3314.
101. Kaldis P & Aleem E. Cell cycle sibling rivalry: Cdc2 vs. Cdk2. Cell Cycle 2005 4 1491-1494.
102. Nakayama Y & Yamaguchi N. Role of cyclin B1 levels in DNA damage and DNA damage-induced senescence. Int Rev Cell Mol Biol 2013 305 303-337.
103. Desai D, Wessling HC, Fisher RP & Morgan DO. Effects of phosphorylation by CAK on cyclin binding by CDC2 and CDK2. Mol Cell Biol 1995 15 345-350.
104. Schmit TL & Ahmad N. Regulation of mitosis via mitotic kinases: new opportunities for cancer management. Mol Cancer Ther 2007 6 1920-1931.
105. Porter LA & Donoghue DJ. Cyclin B1 and CDK1: nuclear localization and upstream regulators. Prog Cell Cycle Res 2003 5 335-347.
106. Lindqvist A, Rodriguez-Bravo V & Medema RH. The decision to enter mitosis: feedback and redundancy in the mitotic entry network. J Cell Biol 2009 185 193-202.
107. Toyoshima-Morimoto F, Taniguchi E & Nishida E. Plk1 promotes nuclear translocation of human Cdc25C during prophase. EMBO Rep 2002 3 341-348.
108. Frazer C & Young PG. Phosphorylation Mediated Regulation of Cdc25 Activity, Localization and Stability. 2012.
109. Karlsson-Rosenthal C & Millar JB. Cdc25: mechanisms of checkpoint inhibition and recovery. Trends Cell Biol 2006 16 285-292.
110. Abraham RT. Cell cycle checkpoint signaling through the ATM and ATR kinases. Genes Dev 2001 15 2177-2196.
111. Goh G, Scholl UI, Healy JM, Choi M, Prasad ML, Nelson-Williams C, Kunstman JW, Korah R, Suttorp AC, Dietrich D, Haase M, Willenberg HS, Stalberg P, Hellman P, Akerstrom G, Bjorklund P, Carling T & Lifton RP. Recurrent activating mutation in PRKACA in cortisol-producing adrenal tumors. Nat Genet 2014 46 613-617.
112. Lai F, Godley LA, Joslin J, Fernald AA, Liu J, Espinosa R, 3rd, Zhao N, Pamintuan L, Till BG, Larson RA, Qian Z & Le Beau MM. Transcript map and comparative analysis of the 1.5-Mb commonly deleted segment of human 5q31 in malignant myeloid diseases with a del(5q). Genomics 2001 71 235-245.
113. Soon PS, Gill AJ, Benn DE, Clarkson A, Robinson BG, McDonald KL & Sidhu SB. Microarray gene expression and immunohistochemistry analyses of adrenocortical tumors identify IGF2 and Ki-67 as useful in differentiating carcinomas from adenomas. Endocr Relat Cancer 2009 16 573-583.
114. Tokumitsu Y, Mori M, Tanaka S, Akazawa K, Nakano S & Niho Y. Prognostic significance of polo-like kinase expression in esophageal carcinoma. Int J Oncol 1999 15 687-692.
115. Takahashi T, Sano B, Nagata T, Kato H, Sugiyama Y, Kunieda K, Kimura M, Okano Y & Saji S. Polo-like kinase 1 (PLK1) is overexpressed in primary colorectal cancers. Cancer Sci 2003 94 148-152.
116. Knecht R, Elez R, Oechler M, Solbach C, von Ilberg C & Strebhardt K. Prognostic significance of polo-like kinase (PLK) expression in squamous cell carcinomas of the head and neck. Cancer Res 1999 59 2794-2797.
117. Wolf G, Elez R, Doermer A, Holtrich U, Ackermann H, Stutte HJ, Altmannsberger HM, Rubsamen-Waigmann H & Strebhardt K. Prognostic significance of polo-like kinase (PLK) expression in non-small cell lung cancer. Oncogene 1997 14 543-549.
118. Weichert W, Denkert C, Schmidt M, Gekeler V, Wolf G, Kobel M, Dietel M & Hauptmann S. Polo-like kinase isoform expression is a prognostic factor in ovarian carcinoma. Br J Cancer 2004 90 815-821.
119. Ito Y, Miyoshi E, Sasaki N, Kakudo K, Yoshida H, Tomoda C, Uruno T, Takamura Y, Miya A, Kobayashi K, Matsuzuka F, Matsuura N, Kuma K & Miyauchi A. Polo-like kinase 1 overexpression is an early event in the progression of papillary carcinoma. Br J Cancer 2004 90 414-418.
120. Mross K, Frost A, Steinbild S, Hedbom S, Rentschler J, Kaiser R, Rouyrre N, Trommeshauser D, Hoesl CE & Munzert G. Phase I dose escalation and pharmacokinetic study of BI 2536, a novel Polo-like kinase 1 inhibitor, in patients with advanced solid tumors. J Clin Oncol 2008 26 5511-5517.
121. McInnes C, Mazumdar A, Mezna M, Meades C, Midgley C, Scaerou F, Carpenter L, Mackenzie M, Taylor P, Walkinshaw M, Fischer PM & Glover D. Inhibitors of Polo-like kinase reveal roles in spindle-pole maintenance. Nat Chem Biol 2006 2 608-617.
122. Nogawa M, Yuasa T, Kimura S, Tanaka M, Kuroda J, Sato K, Yokota A, Segawa H, Toda Y, Kageyama S, Yoshiki T, Okada Y & Maekawa T. Intravesical administration of small interfering RNA targeting PLK-1 successfully prevents the growth of bladder cancer. J Clin Invest 2005 115 978- 985.
123. Linnehan CK, Coan KE, Kim J-H, Wandoloski M, Dastrup E, Rogers E, DelGiorno K, Gonzales P, Barrett MT & Von Hoff D. Inhibition of Polo-like kinase 1 as a strategy in the treatment of adrenocortical carcinoma. Cancer Research 2012 72 978-978.
124. Bussey KJ, Bapat A, Linnehan C, Wandoloski M, Dastrup E, Rogers E, Gonzales P & Demeure MJ. Targeting polo-like kinase 1, a regulator of p53, in the treatment of adrenocortical carcinoma. Clin Transl Med 2016 5 1.
125. Nieduszynski CA, Murray J & Carrington M. Whole-genome analysis of animal A- and B-type cyclins. Genome Biol 2002 3 RESEARCH0070.
126. Lozano JC, Perret E, Schatt P, Arnould C, Peaucellier G & Picard A. Molecular cloning, gene localization, and structure of human cyclin B3. Biochem Biophys Res Commun 2002 291 406-413.
127. Fernandez-Ranvier GG, Weng J, Yeh RF, Khanafshar E, Suh I, Barker C, Duh QY, Clark OH & Kebebew E. Identification of biomarkers of adrenocortical carcinoma using genomewide gene expression profiling. Arch Surg 2008 143 841-846; discussion 846.
128. Liu F, Stanton JJ, Wu Z & Piwnica-Worms H. The human Myt1 kinase preferentially phosphorylates Cdc2 on threonine 14 and localizes to the endoplasmic reticulum and Golgi complex. Mol Cell Biol 1997 17 571-583.
129. Liu F, Rothblum-Oviatt C, Ryan CE & Piwnica-Worms H. Overproduction of human Myt1 kinase induces a G2 cell cycle delay by interfering with the intracellular trafficking of Cdc2-cyclin B1 complexes. Mol Cell Biol 1999 19 5113-5123.
130. Baldin V & Ducommun B. Subcellular localisation of human wee1 kinase is regulated during the cell cycle. J Cell Sci 1995 108 ( Pt 6) 2425-2432.
131. Nitiss JL. DNA topoisomerase II and its growing repertoire of biological functions. Nat Rev Cancer 2009 9 327-337.
132. Wang JC. DNA topoisomerases. Annu Rev Biochem 1996 65 635-692.
133. Wendorff TJ, Schmidt BH, Heslop P, Austin CA & Berger JM. The structure of DNA-bound human topoisomerase II alpha: conformational mechanisms for coordinating inter-subunit interactions with DNA cleavage. J Mol Biol 2012 424 109-124.
134. Iino K, Sasano H, Yabuki N, Oki Y, Kikuchi A, Yoshimi T & Nagura H. DNA topoisomerase II alpha and Ki-67 in human adrenocortical neoplasms: a possible marker of differentiation between adenomas and carcinomas. Mod Pathol 1997 10 901-907.
135. Jain M, Zhang L, He M, Zhang YQ, Shen M & Kebebew E. TOP2A is overexpressed and is a therapeutic target for adrenocortical carcinoma. Endocr Relat Cancer 2013 20 361-370.
136. Ip JC, Pang TC, Glover AR, Soon P, Zhao JT, Clarke S, Robinson BG, Gill AJ & Sidhu SB. Immunohistochemical validation of overexpressed genes identified
by global expression microarrays in adrenocortical carcinoma reveals potential predictive and prognostic biomarkers. Oncologist 2015 20 247-256.
137. Angelousi A, Dimitriadis GK, Zografos G, Nolting S, Kaltsas G & Grossman A. Molecular targeted therapies in adrenal, pituitary and parathyroid malignancies. Endocr Relat Cancer 2017 24 R239-R259.
138. Assie G, Jouinot A & Bertherat J. The ‘omics’ of adrenocortical tumours for personalized medicine. Nat Rev Endocrinol 2014 10 215-228.
139. Libe R, Fratticci A & Bertherat J. Adrenocortical cancer: pathophysiology and clinical management. Endocr Relat Cancer 2007 14 13-28.
140. Gicquel C, Bertagna X, Gaston V, Coste J, Louvel A, Baudin E, Bertherat J, Chapuis Y, Duclos JM, Schlumberger M, Plouin PF, Luton JP & Le Bouc Y. Molecular markers and long-term recurrences in a large cohort of patients with sporadic adrenocortical tumors. Cancer Res 2001 61 6762-6767.
141. Cohen BD, Baker DA, Soderstrom C, Tkalcevic G, Rossi AM, Miller PE, Tengowski MW, Wang F, Gualberto A, Beebe JS & Moyer JD. Combination therapy enhances the inhibition of tumor growth with the fully human anti-type 1 insulin-like growth factor receptor monoclonal antibody CP-751,871. Clin Cancer Res 2005 11 2063-2073.
142. Haluska P, Worden F, Olmos D, Yin D, Schteingart D, Batzel GN, Paccagnella ML, de Bono JS, Gualberto A & Hammer GD. Safety, tolerability, and pharmacokinetics of the anti-IGF-1R monoclonal antibody figitumumab in patients with refractory adrenocortical carcinoma. Cancer Chemother Pharmacol 2010 65 765-773.
143. Fassnacht M, Berruti A, Baudin E, Demeure MJ, Gilbert J, Haak H, Kroiss M, Quinn DI, Hesseltine E, Ronchi CL, Terzolo M, Choueiri TK, Poondru S, Fleege T, Rorig R, Chen J, Stephens AW, Worden F & Hammer GD. Linsitinib (OSI-906) versus placebo for patients with locally advanced or metastatic adrenocortical carcinoma: a double-blind, randomised, phase 3 study. Lancet Oncol 2015 16 426-435.
144. Naing A, Kurzrock R, Burger A, Gupta S, Lei X, Busaidy N, Hong D, Chen HX, Doyle LA, Heilbrun LK, Rohren E, Ng C, Chandhasin C & LoRusso P. Phase I trial of cixutumumab combined with temsirolimus in patients with advanced cancer. Clin Cancer Res 2011 17 6052-6060.
145. Roca E, Berruti A, Sbiera S, Rapa I, Oneda E, Sperone P, Ronchi CL, Ferrari L, Grisanti S, Germano A, Zaggia B, Scagliotti GV, Fassnacht M, Volante M, Terzolo M & Papotti M. Topoisomerase 2alpha and thymidylate synthase expression in adrenocortical cancer. Endocr Relat Cancer 2017 24 299-307.
146. Libe R. Adrenocortical carcinoma (ACC): diagnosis, prognosis, and treatment. Front Cell Dev Biol 2015 3 45.
147. Fassnacht M, Terzolo M, Allolio B, Baudin E, Haak H, Berruti A, Welin S, Schade-Brittinger C, Lacroix A, Jarzab B, Sorbye H, Torpy DJ, Stepan V, Schteingart DE, Arlt W, Kroiss M, Leboulleux S, Sperone P, Sundin A, Hermsen I, Hahner S, Willenberg HS, Tabarin A, Quinkler M, de la Fouchardiere C, Schlumberger M, Mantero F, Weismann D, Beuschlein F, Gelderblom H, Wilmink H, Sender M, Edgerly M, Kenn W, Fojo T, Muller HH, Skogseid B & Group F-AS. Combination chemotherapy in advanced adrenocortical carcinoma. N Engl J Med 2012 366 2189-2197.
148. Liu X. Targeting Polo-Like Kinases: A Promising Therapeutic Approach for Cancer Treatment. Transl Oncol 2015 8 185-195.
149. Demeure MJ, Armaghany T, Ejadi S, Ramanathan RK, Elfiky A, Strosberg JR, Smith DC, Whitsett T, Liang WS, Sekar S, Carpten JD, Fredlund P, Niforos D, Dye A, Gahir S, Semple SC & Kowalski MM. A phase I/II study of TKM- 080301, a PLK1-targeted RNAi in patients with adrenocortical cancer (ACC). Journal of Clinical Oncology 2016 34 2547-2547.
150. Khoo KH, Verma CS & Lane DP. Drugging the p53 pathway: understanding the route to clinical efficacy. Nat Rev Drug Discov 2014 13 217-236.
151. Brenner AK, Reikvam H, Lavecchia A & Bruserud O. Therapeutic targeting the cell division cycle 25 (CDC25) phosphatases in human acute myeloid leukemia- -the possibility to target several kinases through inhibition of the various CDC25 isoforms. Molecules 2014 19 18414-18447.
152. Brezak MC, Kasprzyk PG, Galcera MO, Lavergne O & Prevost GP. CDC25 inhibitors as anticancer agents are moving forward. Anticancer Agents Med Chem 2008 8 857-862.
LEGEND OF THE FIGURES
Figure 1- Schematic representation of the mammalian cell cycle. Cell cycle is mainly regulated by cyclin-dependent kinase (CDK): CDK2, CDK4, CDK6 and Cdc2; cyclins (cyclin A, B, D and E) and CDK inhibitors (p151mk4B, p161mk4A, p18Ink4c, p19Ink4D, p21Cipl, p27kip1, p57Kip2. In red are represented the overexpressed cell cycle regulators and in green the underexpressed regulators in ACCs.
Figure 2- Schematic representation of p53 regulation. p53 transcriptional activity is inhibited by MDM2 that promote p53 ubiquititation. DNA damage leads to p53 phosphorylation via ATM/ATR, preventing is association with MDM2. Besides that, p19ARF also inhibit MDM2 preventing p53 ubiquitination. Stabilized p53 goes to the nucleus where is able to transcriptionally upregulate genes involved in Apoptosis and Cell cycle Arrest. Gene and proteins alteration already described in the ACCs are indicated.
Figure 3- Schematic representation of Rb regulation. Hyperphosphorylation of Rb by the CDK/Cyclin complexes prevents the binding of RB to the transcription factor E2F, allowing the transcription of genes required for S phase entry. Gene and proteins alteration already described in the ACCs are indicated.
Figure 4- Schematic representation of CDC2/cyclin B regulation. In unstressed cells, Plk1 phosphorylates Cdc25C, Wee1 and Myt1, leading to Cdc2/cyclin B activation and entry in the mitosis (A). In the presence of DNA damage, Chk1/2 are phosphorylated via ATM/ATR and are able to induce Cdc25C nuclear exclusion and to activate Wee1, leading to the inactivation of Cdc2/cyclin B and cell cycle arrest (B). Gene and proteins alteration already described in the ACCs are indicated.
| ACC | ACA | Ref. | |
|---|---|---|---|
| CDK4 | 1 64.7% cases were strongly positive | Į 13.6% cases were strongly positive | 63 a |
| Cyclin E | 1 2 fold | Į 2 fold | 71 a |
| î labeling index median of 15% | Į labeling index median of 0% | 67 a | |
| CCNE1 | 1 10.4 fold | Į 10.4 fold | 68 b |
| 1 3.56 fold | Į 3.56 fold | 70 b | |
| Cyclin D1 | 1 1.27% of stained area | Į 0.1% of stained area | 45 a |
| = 5.4% of positive cases | = 0% of positive cases | 42 a | |
| p53 | 1 59% of positive cases | Į 0% of positive cases | 41 a |
| 1 52.4% of positive cases | Į 11.8% of positive cases | 153 a | |
| = 5.4% of positive cases | = 0% of positive cases | 42 a | |
| = 7.39% of stained area | = 2.99% of stained area | 45 a | |
| CDKN1C | Į 26.3 % of positive cases | î 100.0% of positive cases | 62 c |
| p21 | 1 69.4 % of positive cases | Į 36.4 % of positive cases | 42 a |
| = 1.59% of stained area | = 1.25% of stained area | 45 a | |
| p27 | Į labeling index of 48.9% | î labeling index of 59.4% | 83 a |
| 1 94.4 % of positive cases | Į 68.8 % of positive cases | 42 a | |
| 1 9.37 % of stained | Į 3.89 % of stained area | 45 a |
| area | |||
|---|---|---|---|
| CDKN3 | 1 6.84 fold | Į 6.84 fold | 68 b |
| 1 5.28-fold | Į 5.28 fold | 70 b | |
| Î higher than 5-fold | Į lower that 5-fold | 71 b | |
| 1 2.69 fold | Į 2.69 fold | 113 b | |
| pRB1 | = 100% of positive cases | = 100% of positive cases | 94 a |
| Į 13.3% of cases with abundant staining | 1 73.3% of cases with abundant staining | 93 a |
î- higher; Į - lower, = - similar; ª Immunohistochemistry analysis; b Genome microarray analysis; € Northern Blot analysis
| ACC | ACA | Ref. | |
|---|---|---|---|
| CDC2 | 1 7.70 fold | Į 7.70 fold | 68 b |
| î higher than 5-fold | Į lower that 5-fold | 71 b | |
| ↑ | ↓ | 69 b | |
| 1 3.76 fold | Į 3.76 fold | 113 b | |
| 1 5.85 fold | Į 5.85 fold ☒ | 70 b | |
| Cyclin B1 | 1 43 % of positive cases | Į 0 % of positive cases | 113 a |
| CCNB1 | î higher than 8-fold | Į lower than 8-fold | 127 b |
| 1 7.05 fold | Į 7.05 fold | 70 b | |
| ↑ | ☒ ↓ | 69 b | |
| Î 2.88 fold | Į 2.88 fold ☒ | 113 b | |
| CCNB2 | 1 8.88 fold | Į 8.88 fold ☒ | 68 b |
| Î higher than 8-fold | Į lower than 8-fold | 127 b | |
| 1 5.61 fold | Į 5.61 fold | 70 b | |
| Î higher than 14-fold | Į lower than 14-fold | 69 b | |
| CDC25C | 1 2.22 fold | Į 2.22 fold | 70 b |
| ↑ | ☒ ↓ | 69 b | |
| TOP2A | î labeling index mean of 6.13 | Į labeling index mean of 0.72 | 134 a |
| Î labeling index mean of 37.5 | Į labeling index mean of 1.4 | 93 a | |
| ↑ | ☒ ↓ | 135 a | |
| ↑ | ☒ ↓ | 136 a | |
| TOP2A | 1 6.86 fold | Į 6.86 fold ☒ | 68 b |
| î higher than 5-fold | Į lower that 5-fold | 71 b | |
| 1 3.54 fold | Į 3.54 fold | 70 b | |
| î higher than 9-fold | Į lower that 9-fold | 69 b | |
| ↑ | ↓ | 135 c |
1- higher; Į - lower, = - similar; ª Immunohistochemistry analysis; b Genome microarray analysis; ” Real- time quantitative RT-PCR
1
Table 3 - Relevant cell cycle regulator to define clinical prognosis. Results from the Pan-
2 genomic studies Zheng et al and Assié et al are highlighted 20, 21
| Possible Prognostic Markers | Study |
|---|---|
| pRB1 loss | 91 |
| RB1 mutations | 20, 21 |
| chr17p13 LOH | 20, 21, 34, 41 |
| CCNE1 amplification | 28 |
| Cyclin E overexpression | 62 |
| CDK4 amplification | 20, 21 |
| CDKN2A deletions | 20, 21 |
| CCNB2 overexpression | 27, 69, 70, 127 |
| TOP2A overexpression | 93, 134-136 |
3 4
Cdc2
G2 checkpoint Check for: Cell size and DNA replication
p21Cip1 p27kip1 p57Kip2
Cyclin B
Spindle Assembly checkpoint Check for: Chromossome attachment to spindle
M Mitosis (nuclear division) and Cytokinesis (cytoplasm division)
CDK6/4
p15Ink4B p16Ink4A
Cyclin D
p18Ink4C p19Ink4D
p21Cip1 p27kip1 p57Kip2
Cdc2
CDK2
p21Cip1
Cyclin A
G2
Cell growth; organelles replication and preparation for mitosis
Cell growth; organelles replication and preparation for DNA synthesis
G1
Cyclin E
p27kip1 p57Kip2
G1 checkpoint Check for: Cell size, DNA damage, nutrients and growth factors
DNA synthesis and replication
GO
Cell cycle arrest
S
p21Cip1 p27kip1 p57Kip2
CDK2
Cyclin A
241x165mm (150 x 150 DPI)
DNA Damage
P
P
ATR
X
Activated oncogenes
ATM
p16
P
Chk1/2
Unstressed cells
P
p53
p53
MDM2
X
p53
p53 stabilization
p53 ubiquitination
P
Cytoplasm
p53
Nucleus
transcriptionally upregulates target genes
PUMA
p21Waf1
GADD45
BAX
NOXA
Reprimo
14-3-3 g
Apoptosis
Cell Cycle Arrest
☒ Gene underexpression
☒ Gene overexpression
☐ Protein underexpression
☐ Protein overexpression
☐ Mutated genes
☒ Chromosomal region loss
☒ Chromosomal region gain
209×242mm (150 x 150 DPI)
HDAC
X
RB
Transcription blocked
E2F1
DP-1
E2F site
Target genes : Cyclin E/A, Cdc2, E2F1/2/3, c-myc, etc
Cyclin E
CDK2
Hyperphosphorylated RB
X
Cyclin D
CDK4/6
X
RB
E2F1
DP-1
Transcription activated
E2F site
Target genes : Cyclin E/A, Cdc2, E2F1/2/3, c-myc, etc
☒ Gene overexpression
☐ Protein underexpression
☐ Protein overexpression
☐ Mutated genes
☒ Chromosomal region loss
☒ Chromosomal region gain
193×199mm (150 x 150 DPI)
A
Unstressed cells
P
CDC25C
X
CDC25C
X
Low CDK activity
High CDK activity
P
Plk1
P
Cdc2
Cdc2
M-phase
Cyclin B X
Cyclin B
P
Wee1
Wee1
Myt1
P
Myt1
B
Stressed cells
P
CDC25C
X
nuclear exclusion
CDC25C
X
14-3-3 ,3
ATR
Y
Low CDK activity
High CDK activity
P
DNA Damage
P
Chk1/2
P
Cdc2
Cdc2
P
ATM
Cyclin BX
Cyclin B
P
Wee1
☒ Gene underexpression
☒ Gene overexpression
☐ Protein underexpression
☐ Protein overexpression
☐ Mutated genes
☒ Chromosomal region loss
☒ Chromosomal region gain
252×236mm (150 x 150 DPI)