Signaling Pathways in Adrenocortical Cancer
LAWRENCE S. KIRSCHNER
Unit on Genetics and Endocrinology, DEB, NICHD, National Instutes of Health, Bethesda, Maryland 20892-1862, USA
ABSTRACT: Adrenocortical carcinoma is a rare tumor that carries a very poor prognosis. Despite efforts to develop new therapeutic regimens to treat this dis- ease, surgery remains the mainstay of treatment. Laboratory studies of adrenocortical cancers have revealed a wide variety of signaling pathways that can be altered in these neoplasms. Although ACTH signaling through adenylyl cyclase and protein kinase A is important for normal adrenal cellular physiol- ogy, there is evidence to suggest that this pathway may inhibit the growth of adrenocortical tumors, and that inactivation of the ACTH receptor may pro- mote tumor formation. Although multiple signal transduction pathways are es- sential for normal adrenal growth and hormone secretion, efforts to identify events required for neoplastic transformation have met with limited success. Alterations that have frequently been observed in adrenocortical carcinoma in- clude up-regulation of the IGF-II system, as well as mutations in TP53 and RAS. Current studies aim to elucidate the mechanisms of tumor growth by studying proproliferative signaling pathways, such as those involving Akt/PKB and the mitogen-activated protein kinases (MAPKs). Although studies of single pathways have been helpful in guiding investigations, new tools to study the in- tegration and multiplicity of signaling pathways hold the hope of improved un- derstanding of the signaling pathway alterations in adrenocortical cancer.
KEYWORDS: adrenal cancer; adrenocortical carcinoma; growth factors; signal transduction; G proteins; protein kinase A (PKA); mitogen-activated protein kinase (MAPK); ACTH receptor; p53; ras; insulin-like growth factor;EGF
INTRODUCTION
Cancers of the endocrine glands are rare entities, with only thyroid cancer found among the top 50 causes of cancer deaths. The reasons for this observation may lie in the highly differentiated nature of these tissues and the fact that endocrine cells reach the end of their proliferative potential early in life. Again, with the exception of thyroid carcinoma, endocrine cancers tend to be resistant to therapy other than surgical excision. For this reason, the prognosis for a patient diagnosed with an en- docrine cancer is generally poor.
Because of its low incidence and resultant low emphasis as a health care issue, experimental investigations of endocrine carcinomas have not been common. Most
Address for correspondence: Lawrence S. Kirschner, M.D., Ph.D., Division of Endocrinology, Ohio State University, 491D McCampbell Hall, 1581 Dodd Drive, Columbus, OH 43210. Voice: 614-292-2995.
studies to date have studied small numbers of tumors, investigating a single molecule or pathway to look for changes associated with malignancy.
Adrenocortical carcinoma (ACC) is a good example of this paradigm. There are good clinical studies describing its behavior in patients, but relatively few laboratory investigations aimed at examining its molecular etiology. In this review, I will at- tempt to summarize the data from these published investigations, focusing on this in- formation as a means to describe current understanding of the alterations in signal transduction that may lead to the development of adrenocortical malignancy.
Because studies of ACC are limited by the rarity of tumor specimens, many in- vestigators have turned to model systems in an attempt to address questions of adrenocortical cell proliferation and carcinogenesis. Although these systems can provide vital insights into adrenal cell function, each also has certain limitations, which must be kept in mind when the data are analyzed.
Although the majority of currently published studies have analyzed only single pathways of signal transduction, it is now becoming clear that most cellular respons- es involve multiple pathways. For this reason, studies examining the integration of signals and the interplay of downstream pathways are becoming more important than understanding the effects of any individual molecular alteration. As we usher in the new age of analysis, including the tools of genomics and proteomics, better tech- niques for addressing these questions are becoming available, with the expectation that the answers will also become clearer. Like all good science, though, better un- derstanding may pave the way to new, as yet unappreciated, questions.
CLINICAL ASPECTS OF ADRENAL CANCER
In terms of tumor epidemiology, adrenocortical cancer (ACC) is a rare cancer, ac- counting in large studies for 0.04 to 0.2% of all cancer deaths.1,2 The incidence of the disease appears highest in the fifth decade,1-3 although there is also another peak of incidence in children under 5 years of age.3,4 Most series agree that adrenal cancer is more common in women than in men, with a ratio of approximately 1.5:1.1,3,5
The clinical presentation of ACC can be variable, and patients can present with symptoms of a specific hormonal syndrome or can present with nonspecific symp- toms resulting from an abdominal mass. In larger case series, 45-75% of adult pa- tients present with a hormonal oversecretion syndrome,1,3,6 of which Cushing syndrome is the most common.1,4 Other hormonal hypersecretion syndromes asso- ciated with ACC include virilization (from androgen-producing tumors), feminiza- tion (estrogen-producing tumors), and hyperaldosteronism. Multiple hormones can be produced by a single tumor, leading to a mixed clinical picture (e.g., Cushing syn- drome plus virilization).2,6 In children, there is a greater tendency for tumors to present with a hormonal syndrome, and virilization is more common in this age group.4
Of the patients presenting without a hormonal syndrome, nonspecific abdominal or dorsal pain is the most common presentation, being the presenting symptom in up to 30% of cases.1,5 In a recent study of ACC from Poland, this diagnosis was found in 6.8% of patients found to have an adrenal mass,7 in line with previous estimates that 0-25% of incidentalomas will be ACCs.8,9 Of all adrenal cancers, incidentally detected lesions form approximately 30% of the group.10
Although ACC is a rare tumor, it tends to be quite aggressive and carries a very poor prognosis, which appears to depend little on the initial presentation. Approxi- mately 50% of adults found to have ACC will not survive beyond two years of the diagnosis, and the 5-year mortality rate hovers around 80%.1 Other than improved surgical management,11 the prognosis for an individual diagnosed with ACC has not changed significantly over the past 40 years.2,3 Adrenalectomy for benign lesions is now most commonly performed via a laparoscopic approach, and a low-level suspi- cion for cancer does not preclude this type of operation.12 However, in cases where adrenal cancer is expected, an open approach provides a better opportunity for mar- gin-free resection and resection of isolated metastases, and thus is the preferred sur- gical approach. 13-16
Medical therapy of ACC consists mainly of mitotane (o, p`-DDD, 1,1 dichlo- rodiphenyldichloroethane), which has therapeutic effects in approximately 30% of patients.3,6 Data to support that mitotane significantly improves survival are lacking, however, despite the fact that it appears to decrease hormone secretion in steroid- producing tumors. Efforts to use other chemotherapeutic agents, either alone or in combination, have generally not been more effective than mitotane alone,17,18 al- though a recent study suggested that combination chemotherapy plus mitotane in- duced at least partial response in up to 54% of patients. 19
ALTERATIONS IN SIGNAL TRANSDUCTION PATHWAYS
G Protein-Coupled Receptors
The ACTH-cAMP/PKA Pathway
Corticotropin (ACTH) signaling has long been one of the classic paradigms in en- docrinology, and this pathway has been well worked out at the molecular level.20 Treatment of adrenal cells with ACTH leads to the binding of this hormone to its re- ceptor, termed MC2R, which belongs to the seven-transmembrane G protein-cou- pled receptor (GPCR) family. Activation of the receptor causes dissociation of the heterotrimeric stimulatory G protein (Gs), leading to the release of the alpha-subunit (Gsg) and stimulation of adenylyl cyclase. This enzyme in turn causes the produc- tion of cyclic AMP (cAMP) from ATP. Cyclic AMP binds to the regulatory subunits of PKA, causing release of the catalytic subunits with subsequent transduction of the signal via phosphorylation of proteins both in the cytoplasm and in the nucleus.
There was ample reason to suspect that this pathway might be involved in adrenal tumorigenesis. First, adrenal cells under the constant stimulation of ACTH in pa- tients (such as in those with Cushing disease) have enlarged adrenals.21 Second, GPCR gain-of-function mutations have been well characterized in other endocrine tumors,22 most notably activation of the TSH receptor in toxic thyroid adenomas.23 Last, in the inherited syndrome Carney complex (CNC), the causative mutations were recently found to be inactivating mutations of the PRKARIA gene, leading pre- sumably to an increase in PKA activity.24 Patients with this disease get multiple, hy- perfunctioning adrenal nodules, suggesting that the pathway may play a role in proliferation and/or hyperfunction in the adrenal cortex.25,26 Of note, adrenocortical carcinomas have not been shown to be associated with the complex, despite the well- described (benign) adrenal pathology.
To investigate the role of ACTH signaling in ACC, adrenal tumors were screened for mutations in MC2R. This receptor was initially cloned from humans in 1992,27 and the number of investigations published is small. To date, there are only 38 adre- nal tumors that have been studied (25 adenomas and 13 carcinomas), and no activat- ing mutations of the ACTH receptor have been detected.28,29 Failure to detect such mutations is likely not a cause for these findings, given that inactivating mutations of the ACTH receptor have been described in patients with the syndrome of ACTH insensitivity.30
The role of Gsg is less clear. Although mutations have been sought in adrenal tu- mors in this gene, there is only a single report of an activating mutation in one aldos- teronoma from Japan.31 Efforts to detect mutations in other series of both adrenal adenomas and adrenal carcinomas have not yielded any positive findings.32,33
Mutations in the subunits of PKA itself have not been described other than the mutations seen in CNC, and, as mentioned above, it is not clear if these alterations play a role in adrenal cancer. Studies of the cAMP/PKA pathway in vitro suggest a reason why activation of the ACTH receptor pathway may not be involved in car- cinogenesis. It has long been known that ACTH is the most potent stimulus for steroid secretion in adrenal cells isolated either from rodent or bovine species.20 The same observation is made in the Y1 cell line, an adrenocortical cancer cell line ini- tially derived from a mouse adrenocortical tumor (www.atcc.org, cat. CCL-79). In each of these systems, treatment of cells with ACTH leads to a rapid and dose-de- pendent increase in steroidogenesis. However, analysis of the proliferative effects of ACTH has not been so clear. Studies of the proliferative effects of ACTH suggest that this hormone leads to decreased proliferation, or at least growth arrest.34,35 This growth arrest is thought to be the result of ACTH’s action as a factor that promotes differentiation, rather than proliferation. At the molecular level, treatment of cells with ACTH appears to have a biphasic effect on quiescent cells. Treatment for short periods of time (less than 2 hours) leads to cells exiting GO and entering the G1 phase of the cell cycle.35 However, prolonged treatment (18-24 hours) leads to an arrest of cells before S phase, so that the overall effect is an inhibition of cell cycle progres- sion.34 At the molecular level, ACTH or cAMP treatment leads to a rise in c-fos and c-jun mRNA, but appears to inhibit the accumulation of c-myc.34,36,37 This blockade of c-myc appears sufficient to prevent cells from entering the cell cycle.37
Furthermore, there is ample evidence in adrenal cells that ACTH, acting through the PKA pathway, is able to inhibit proliferative signals initiated through other sig- naling pathways.38 One proposed mechanism of this effect was demonstrated in Y1 cells, where ACTH treatment led to a dephosphorylation of the Akt/PKB kinase, a key modulator of cell cycle progression.39,40 In Y1 adrenal cells, ACTH has also been suggested to play a role in suppressing signal transduction via the Mek kinase, a key player in activation of the MAP kinases.41 These observations support the no- tion that ACTH is a differentiation-maintaining factor and is therefore anticarcino- genic. As this concept has become more appreciated, studies have been performed to address the potential role that loss of the ACTH response (via loss of MC2R) may play as a permissive factor for adrenal carcinogenesis. In a recent small study, loss- of-heterozygosity (LOH) at the MC2R locus was rarely detected in benign adrenal lesions, but was present in two of four adrenal cancers. This allelic loss of the ACTH receptor was correlated with reduced levels of the receptor in malignant tumors.42
Other G Protein-Coupled Systems
In addition to ACTH, many other hormones signal in the adrenal cortex via GPCRs. For example, the recently described family of hormones known as orexins also stimulates the adrenal gland through the OR1 receptor.43 These signals appear to be mediated by stimulation of adenylyl cyclase and the PKA system.
In addition to the hormones described above, there are several other hormones that bind to GPCRs in the adrenal glands, but whose actions are modulated through signaling systems other than AC-PKA system.
The best characterized of these hormones is angiotensin-II (A-II), which is though to act primarily in the adrenal glomerulosa to stimulate release of aldosterone from the normal adrenal.44,45 It has also been shown that A-II increases adrenal cell proliferation in tissue culture systems from human, rat, and bovine cells.44,46,47 At the receptor level, A-II has two molecules that mediate its signal, AT-1R and AT-2R. In the adrenals, it is the former of these that is present and modulates signaling through this pathway.44 In tissue culture studies, the AT-1R has been demonstrated to couple through multiple G proteins, including both the inhibitory G subunit Gi2 and the phospholipase C coupled subunit Gq.45 In bovine adrenal cells, the prolifer- ative effect of A-II was shown to be pertussis toxin (PTX) insensitive, implying that the majority of the signaling occurred through Gq.48 However, similar experiments carried out in a slightly different culture system suggest that there were both PTX- sensitive and -insensitive components to the observations, implicating Gq- and Gi- mediated signaling.49 Gq is known to transduce signals through protein kinase C (PKC), the diacylglycerol- and calcium-sensitive kinase that is also stimulated by phorbol esters (e.g., TPA). Effects mediated by this kinase can be blocked by using the specific PKC inhibitors Ro-8339 or calphostin. When adrenal cells were treated with these agents, most of the proliferative effect of A-II was blocked, although there appeared to be some persistence of signaling.45 The signaling mechanism for G;2 is less clear, although there is mounting evidence that this G protein complex signals through its By subunits through the ras pathway to activate the MAP kinase signaling pathway (ref. 50, and see below).
Other investigators have examined the role that the endothelins (ETs) play in ad- renal hormone secretion. Because ETs are involved in vascular tone and blood pres- sure control, there has been significant interest in studying the role of these hormones in the regulation of aldosterone secretion. It has clearly been demonstrated that ET-1 stimulates aldosterone secretion both in normal adrenals51,52 and in aldos- terone producing adenomas (APAs).53 The mechanism of these effects is quite com- plex, but appears to involve a multiplicity of pathways, including those of protein kinase C (PKC), cyclooxygenase, PI3-kinase, and phospholipase C (PLC).51,52
There has also been significant interest in the role of adrenomedullin, a peptide hormone initially identified from pheochromocytomas and thought to play a role in interaction between the cortex and the medulla.54 This protein, which signals in the adrenal cortex through the CGRP-1 receptor, also induced proliferation via the MAPK pathway.55 As with A-II, there appear to be multiple signaling mechanisms involved, as the proliferative effect is sensitive not only to modulators of the MAPK pathway, but also to molecules which modulate the PKC/PLC and PKA pathways.56
Despite the evidence for their involvement in adrenocortical cell proliferation provided from these studies, no mutations of the receptors for any of these hormones
have been described in adrenal tumors. Interestingly, mutations in the Go;2 subunit have been described in a small number of tumors, although these results were found only in a single study.32 In that investigation, 11 adrenocortical tumors were studied, and activating mutations of Ga;2 were detected in three tumors. In two adrenal ade- nomas, an activating mutation of Gaj2 was present in the heterozygous state. In the third tumor, an adrenal cancer, only the mutated allele was detected, indicating loss of the normal allele (either by LOH or gene conversion). In order to verify these find- ings, the mutated form of Go;2 was introduced into tissue culture cells. In NIH3T3 fibroblast cells, the mutant allele led to a marked reduction of cAMP in the cells, al- though no growth effect was noted.57 However, in a different fibroblast cell line, Rat- 1, the same construct not only decreased cAMP levels, but also led to enhanced pro- liferation, including the gain of the ability to grow in soft agar.58 The authors inter- preted these findings to suggest a tissue specificity of the transforming effect of this oncogene, dubbed the gip2 oncogene; unfortunately, no studies introducing the con- struct into adrenal cells have been carried out. Subsequent investigations of the Go;2 gene from other groups have not detected mutations in over 60 additional tumors. 33,59,60
The Role of Ectopic Receptors
Interestingly, there are a number of reports suggesting that ectopic GPCRs may play a role in adrenal tumorigenesis. The pioneering work of Lacroix and colleagues has clearly demonstrated that ectopic expression of a small number of Gs-coupled GPCRs can lead to the phenomenon of massive macronodular adrenocortical disease (MMAD, also known as ACTH-independent macronodular adrenal hyperplasia, AIMAH).61-67 In this condition, there is marked bilateral enlargement of the adrenal glands associated with hypersecretion of cortisol under the control of an aberrant hormone receptor. In all of the cases described to date, the receptors involved are thought to recapitulate the hormone stimulation by ACTH, but there is obvious dis- ruption of the negative feedback loop. As in states associated with ACTH excess (e.g., Cushing disease or ectopic ACTH production), there is massive hypertrophy of the adrenals, although there have been no cases of malignant transformation reported.
Similarly, ectopic expression of a specific receptor can lead to the development of a single adenoma, as has been shown in a small number of cases. In older studies, the coupling of ectopic receptors to the adenylate cyclase system has clearly been demonstrated in in vitro studies using extracts from adrenal adenomas.68,69 In a sin- gle study, similar observations were made about the ectopic coupling of the ß-adren- ergic receptor to PKA,70 although large studies of this phenomenon have not been performed to date. MMAD and the concept of signaling through ectopic GPCRs are discussed elsewhere in this volume.
Growth Factor Signaling Pathways
Insulin-like Growth Factors
The insulin-like growth factor (IGF) system is perhaps the best characterized in terms of being involved in adrenocortical tumorigenesis,71 although it has many
roles in normal cell growth and development.72-74 There are two distinct forms of IGFs, known as IGF-I and IGF-II. IGF-I, coded for by a gene at 12q22-q24.1, is thought to be the major signal elucidated by growth hormone (GH) in postnatal life. IGF-II, located at 11p15.5, is felt to play a much more important role during fetal life, and its importance as a growth mediator is minimal after birth. The two proteins signal through two receptors, which bind IGF-I and IGF-II with similar affinities. They also bind insulin, albeit at much lower affinity. The IGF1R has a structure anal- ogous to that of the insulin receptor, being composed of two heterodimeric chains that possess an intrinsic tyrosine kinase activity. The IGF2R is a single polypeptide chain that also functions as the receptor for mannose-6-phosphate, a key sugar moi- ety involved in intracellular trafficking of lysosomal enzymes. The mechanism by which the IGF2R signals is not as clear, although mutations of the gene have been shown to be involved in hepatocellular carcinoma. 75
Studies of the IGF system were initially suggested by correlation with the Beckwith-Wiedemann syndrome (BWS) (www.ncbi.nlm.nih.gov/omim OMIM #130650). This syndrome is characterized by generalized overgrowth, macroglossia, and exomphalos. Adrenal carcinoma is also a feature of the syndrome. Genetically, the disease had been mapped to the 11p15.5 region harboring not only the IGF-II gene, but the genes coding for insulin (INS), p57Kip2 and H19, a transcript of un- known function that is not translated into protein.
With this background, IGF-I and -II levels were investigated in a variety of adre- nal lesions. In early studies, no changes in IGF-I were detected, although marked el- evation of IGF-II was detected.76-80 This change was observed both by analysis of mRNA levels and at the protein level by immunohistochemistry.76 These findings were infrequently seen in benign lesions, but were prominent in over 60% of adreno- cortical carcinomas. Studies searching for potential receptors for IGF-II in ACC have looked at levels of both the IGF1R and IGF2R, as IGF-II can signal through both of these. There have been consistent findings of elevation in the IGF1R, without marked changes in IGF2R levels. Again, this has been observed at both the level of mRNA and protein, but also at the functional level by receptor binding studies.79 In more recent studies, transgenic mice expressing IGF-II postnatally were generated and were demonstrated to have adrenocortical hyperplasia, although frank malignan- cy was not observed.81 This observation suggests that IGF-II is important for the ab- normal proliferation of adrenal cells, but that additional steps are required for transformation to neoplasia.
At the genetic level, the IGF-II gene and its surrounding genomic region (includ- ing H19 and p57Kip2) are known to be subject to regulation by genomic imprinting. Imprinting is a specific form of methylation that occurs early in life (often in embry- onic stages) leading to stable patterns of activation or inactivation of genes.82 This locus, like most that are subject to imprinting, shows specific parent-of-origin effects of imprinting on gene expression. Specifically, the IGF-II gene is maternally im- printed, such that expression in the adult comes almost exclusively from the paternal allele. In contrast, the other two genes in the region, H19 and p57Kip2, are paternally imprinted, such that expression derives solely from the maternal allele.
In genetic studies to address the mechanism of IGF-II overexpression in adrenal lesions, it has been observed that there are genetic changes at the IGF-II locus. Most commonly, LOH is observed, leading to specific deletion of the maternal allele.83
This is often accompanied (although the exact mechanism of the genetic changes is not clear) by gain of a second copy of the paternal allele, a process called uniparental disomy (UPD), such that there are two copies of the father’s allele and none of the mother’s.77 Not only is the genetic sequence of the mother lost or changed to the fa- ther’s, but also the imprinting status reflects the chromosome of origin. In this man- ner, the replacement of the maternal allele with the paternal allele leads to the presence of a double dose of paternal allele, with the effect that both copies of the gene are now expressed. Whether the level of expression is similar to what would be predicted solely on the basis of two copies of the paternal allele is not clear, but this disruption of the normal genomic structure of the locus clearly plays a role.
Conversely, as mentioned above, the H19 gene and p57Kip2 are normally ex- pressed solely from the maternal allele. When this region of the genome is lost and/ or replaced by the paternal region, the expression of these two genes is also lost, or at least markedly reduced. The role of H19 is not clear, although it has been posited to function as a tumor suppressor, so its loss is of unknown functional consequence. However, p57Kip2 is known to function in the suppression of cell cycle progression. Indeed, it is loss of this function that is thought to explain why mutations of this gene lead to BWS. Although it has been studied in only a small number of tumors to date, down-regulation and/or loss of both H19 and p57Kip2 may contribute to the pheno- type of adrenal cancers that are found to have alterations in the 11p15.5 locus.78,84
Other Tyrosine Kinase Family Growth Factors
Growth factors that signal through tyrosine kinase mechanisms are widely varied, and the signals can be mediated either directly through a receptor with tyrosine ki- nase activity (such as the IGF1R) or via accessory signaling molecules, which me- diate the transduction of the signaling through accessory kinases (such as the IGF2R).
One of the most influential general growth factors is epithelial growth factor (EGF), which was one of the first peptide growth factors to be identified, and its cog- nate receptor, the EGF receptor (EGFR). This latter protein was initially described as a growth-promoting oncogene known as the v-erb-B oncogene, which causes avi- an erythroblastic leukemia.85 The EGF system has been studied in a small number of adrenal tumors, and expression of EGF receptor was found to be present in over 90% of adrenocortical carcinomas.86-88 However, the levels were not markedly dif- ferent from those observed in both cortisol- and aldosterone-producing adrenal ade- nomas, suggesting that the EGFR system may be a nonspecific growth factor that plays a permissive but nonspecific role in adrenocortical transformation.87,88 Inter- estingly, EGF itself does not appear to be expressed in adrenal tumors, suggesting either that EGF functions as an endocrine hormone (i.e., as opposed to a locally act- ing factor) or that the EGFR is stimulated by another compound. A good candidate ligand for this receptor is the growth factor TGF-a, which is expressed in adrenocor- tical lesions; again, there was not a marked difference between benign and malignant tumors, suggesting that the growth-promoting effect may not be specific for tumors. 88
Last, the role of cytokines has also been assessed in the adrenal, although studies specific for adrenocortical cancer are quite rare. It was described a number of years ago that a number of interleukins could affect steroid hormone production and pro-
liferation of adrenocortical cells. Specific studies have examined the role of IL-3 and IL-6 in this process. Cytokines signal through a heterodimeric receptor complex consisting of an a-chain that is specific for the cytokine (but that lacks kinase activ- ity) and a common cytokine transducing component known as gp130. IL-3 is able to mediate steroid production through its receptor, and the effect has been shown to be dependent on an intact lipoxygenase pathway, as treatment of adrenal cells with in- domethacin is able to block the action of this cytokine. Similarly, IL-6 has similar effects in adrenocortical cells, but the effect is mediated instead through the cyclo- oxygenase pathway.89,90 Neither of these cytokines has been shown to have a spe- cific role in adrenal tumorigenesis.
In contrast to these in vitro studies, two recent studies have suggested a similar action may be occurring in vivo. In one case, a benign adrenal adenoma was found to express receptors for IL-1, which were thought to mediate both the growth and cortisol hypersecretion found in the patient.91 Within the last few months, a similar report has appeared for an adrenal cancer92 in which a tumor was found to secrete a variety of cytokines of the CXC family, including Gro-a and -B, as well as NAC70 and others. When archived ACCs were examined for the same cytokines, expression levels were also found at appreciable levels in six of seven samples tested. When the index tumor was explanted into nude mice, it grew aggressively, recapitulating its behavior in the patient from whom it was isolated. In contrast, blockade of the cy- tokines using specific antisera led to a marked reduction in tumor growth, implying a direct connection between cytokine signaling and tumor aggressiveness. Although this last manuscript clearly proved that these pathways could play a significant role in adrenocortical cancer, more studies will need to be done to see how common these pathways are.
Intracellular Pathways of Signaling in Adrenal Cancer
Intracellular mediators of signaling pathways are multiple, and sorting out the role of any particular molecule is difficult. Before attempting to integrate the signal- ing pathways into a larger scheme, it is worthwhile to review those molecules in which mutations have been shown to be associated with adrenal cancer. These are few, and so I will discuss each briefly.
TP53 Mutations
The first intracellular molecule clearly shown to be associated with adrenal can- cer is the tumor suppressor gene TP53. TP53 gene mutations have been known for a long time to cause cancer, either through inactivation or, more commonly, through a dominant-negative effect. The direct connection between TP53 and adrenal cancer was made when it was discovered that patients with the Li-Fraumeni cancer syn- drome harbored mutations in this gene.93 Li-Fraumeni syndrome (LFS) is an inher- ited cancer syndrome consisting primarily of soft-tissue sarcomas, tumors of the brain and breast, as well as a wide variety of other malignant tumors, including adrenocortical cancer (www.ncbi.nlm.nih.gov/omim OMIM #151623). The TP53 gene, which mediates an important cell cycle checkpoint control, is inactivated in these tumors. It is largely a target for many proliferative systems, so it is not possible to place this gene in the setting of one particular signaling cascade. However, it is certainly worth mentioning that large percentages of patients with ACC have muta-
tions in TP53.94 Indeed, it has recently been demonstrated that in southern Brazil, where the incidence of pediatric ACC is markedly higher than anywhere else in the world, the majority of these tumors possess mutations in TP53. In fact, 35/36 unre- lated patients with ACC contained an identical point mutation in codon 337 (R337H), suggesting a potential mutation hot spot, as well as the possible involve- ment of a specific environmental factor in adrenal tumors of that region.95
RAS Mutations
The ras gene family is another important mediator of cancers, and mutations in this family of genes have been found in a wide variety of malignant tumors. The ras family is composed of three family members, termed v-Harvey-ras (Ha-ras, or HRAS), Kirsten-ras (Ki-ras, or KRAS), and NRAS. These genes are localized in hu- mans to 11p15.5, 12p12.1, and 1p13.2, respectively. The first two were identified from murine sarcoma virus, and the third was identified as an oncogene from human sarcomas. They are the founding members of a large family of small GTP binding proteins and their cohorts.
The RAS proteins themselves can be oncogenic, and they have been analyzed in large numbers of cancers in the body. In tumors containing RAS mutations, there is frequent alteration in codons 12, 13, or 61, leading to a decrease in the intrinsic GTP- ase activity of the protein and increased proliferative signals. Initial studies of adre- nal tumors did not identify RAS mutations, although only the mutations in the three common codons were evaluated.96 When a more general screening approach was used, mutations were detected in each of the RAS genes. To date, mutations of KRAS97 or HRAS98 have been detected in small numbers of ACCs, whereas muta- tions in NRAS have been seen in both adenomas and carcinomas.60 To study the ef- fects of the KRAS mutations, tumor-derived mutant forms of the protein were tested for intrinsic GTPase activity. Like other oncogenic mutations of RAS, the adrenal mutants were found to have a decrease in intrinsic GTPase activity.99 Interestingly, transfection of normal adrenocortical cells with KRAS mutants led to a marked in- crease in expression of steroidogenic enzymes, accompanied by a 20-30-fold in- crease in hormone secretion. 100
Transcription Factors
Clearly, mitogenic signals have their ultimate end point in the nucleus, at the level of altered gene transcription. Genes that are turned on (or off) in cancers represent a stable mechanism to perpetuate the signal to continue to proliferate. To generate these changes, there must also be changes in the presence or activity of the factors governing mRNA transcription. Small numbers of studies have examined alterations in transcription associated with adrenal cancers.
The GATA family of transcription factors is ubiquitous and has been shown to be involved in a variety of cellular processes, including both proliferation and differen- tiation. The two most important GATA family members for the endocrine glands are GATA-4 and GATA-6. It was reported in 1999101 that GATA-6 is present in fetal and postnatal adrenal glands of the mouse, whereas GATA-4 levels are low. In a mouse tumor model, adrenal tumorigenesis is accompanied by the loss of GATA-6 and marked up-regulation of GATA-4. In human tissues, GATA-4 was found to be absent from the normal adrenal, whereas it was easily detected in a series of benign and ma-
lignant adrenal tumors. The downstream effects of these alterations are unknown in the adrenal gland.
More recently, and perhaps more interestingly, the transcription factors associat- ed with cAMP signaling were evaluated in a small series of adrenal tumors.102 Spe- cifically, levels of the cAMP response element (CRE) binding protein (CREB), the CRE modulator (CREM), and the inducible cAMP early repressor (ICER) were eval- uated in adrenal adenomas and carcinomas. CREB is an interesting protein, as it has long been known to be subject to phosphorylation, which causes it to become active. This phosphorylation was initially attributed to PKA, but later studies demonstrated that CREB can be phosphorylated by many kinases, including MAPK. Less is known about the function of the other proteins in this pathway. In the study of Peri et al.102 CREM levels were found not to vary significantly between benign and malignant ad- renal tumors. However, whereas levels of CREB and ICER were steady in the ade- nomas, they were markedly reduced in about half of the carcinomas.
Antiapoptotic Signals
Study of bcl-2 in tumors suggests that alterations in this antiapoptotic factor do not play a significant role in tumors, nor did related family members mcl-1 or bax. The role of other family members (e.g., Bcl-x) was not studied.103 Recently, there was a report describing alterations in the human homologue of the C. Elegans Diminuto protein. This protein, which is thought to be involved in steroid biosynthe- sis in plants, has been suggested to play an antiapoptotic role in human neurons. This study demonstrated that it is up-regulated in adrenal tumors, suggesting that it may play a role in preventing apoptotic death of steroid secreting cells undergoing un- scheduled cell cycling.104 However, these data are very preliminary at present and will need to be verified in more systematic studies.
INTEGRATION OF SIGNALING PATHWAYS IN ADRENAL TUMORS
Although the large majority of studies aimed at examining changes in the signal- ing pathways in adrenal cancer have studied single pathways, it is now becoming clear that this approach provides only a limited view of the picture. In other experi- mental systems, recent work has turned towards elucidating the interaction of signal- ing pathways for growth and/or differentiation, and these interactions have been shown to be quite complex.105,106 Clearly, a better understanding of adrenal cancer will require a similar approach.
As of now, what can be said about alterations in signaling events responsible for adrenal cancer? The change with the strongest supporting evidence is for overex- pression of IGF-II and signal transduction through a tyrosine kinase-mediated IGF1R pathway. There may be a similar role for EGFR signaling, as well. Slightly downstream from these molecules is ras, where mutations have been found in sig- nificant numbers of ACC samples. The importance of cytokine overexpression for tumor proliferation has been demonstrated in only a single tumor, although the de- tailed characterization of that tumor’s behavior both in vitro and in vivo clearly showed that this pathway is sufficient to drive tumor growth.92
Activating mutations of MC2R are clearly not observed in ACC, although loss of this gene (by LOH or other means) may play a role in overcoming antiproliferative
signals. The role of activating mutations in the GPCR-associated G proteins Gsx and Gia2 is unclear, since there have been single reports of mutations, but they have not been confirmed.
How can these disparate lines of evidence be assembled into a coherent pathway that may explain the signaling pathways that are important in adrenal cancer? As in all cancers, carcinogenesis is a combination of two factors: hyperproliferation and dedifferentiation. Current thinking using Knudsen’s “two-hit” hypothesis states that hyperproliferation increases the chance of obtaining a secondary mutation, which then leads to cancer. This is especially true in terms of the necessary alterations that need to occur in tumor cells-namely, reactivation of telomerase and deregulation of cell cycle checkpoints.
In highly differentiated cells such as are found in the endocrine system, the most important ultimate regulator of proliferation is likely the mitogen-activated protein kinase (MAPK), also known as extracellular signal-regulated kinase (ERKs). There are two proteins in the family, ERK-1 and ERK-2, also known as p44 and p42, re- spectively. In the adrenal, as in most other tissues, activation of the MAPK system appears to be a prerequisite to enhanced proliferation.
Regulation of the MAPKs is extremely complex, and their ultimate activation can occur via many pathways. In adrenal cell culture systems (either cell lines -Y1 mouse adrenocortical cancer cells or H295R human adrenocortical cells) or in pri- mary cell cultures from human, mouse, or cow, each of the pathways described above has been implicated in the activation of the MAPK pathway.
The role of ACTH signaling in adrenal cancers appears quite complex, and our current understanding is far from clear. It is well established from patient data that stimulation of the adrenal by ACTH eventually leads to proliferation (manifested as hyperplastic adrenals), although neoplasia does not appear to occur. It has been dem- onstrated that ACTH can lead, via a PKA-independent pathway, to the activation of MAPKs after only a very short (<5-min) pulse of ACTH.41 This observation is con- gruent with the findings that transient ACTH treatment stimulates adrenal cells to leave GO and enter G1,34,39 although the role of the hormone in long-term prolifer- ation seems doubtful.
In fact, most data suggest that, on balance, ACTH is antiproliferative. Again, the data are not clear, but there appear to be two mechanisms for this action. First is the reduction in c-myc, most likely caused by increased protein degradation as well as blockade of synthesis.34 More recent studies have also demonstrated a second mode of action of ACTH. It appears to markedly stimulate dephosphorylation of the Akt/ PKB kinase.39,40 This kinase, which is also activated primarily by a PI3-K-depen- dent pathway, is a major component of mitogenic signaling. Dephosphorylation re- duces its activity and diminishes the signals for cell proliferation.
In the case of MMAD, another state with hyperplastic but nonmalignant adrenal growth, it has been demonstrated that the growth is due to the presence of ectopic GPCRs.61 It is also possible that, like the presence of PKA-coupled receptor expres- sion itself, these receptors may acquire the facility to couple with the growth- promoting effects of the MAPK pathway. Their retention of coupling to cAMP path- ways likely leads to a check on growth, and hence these tumors are not malignant.
For kinases that fall in the tyrosine kinase family (IGF2R, EGFR, and so on), their direct coupling to the MAPK provides adequate explanation for their role in the ab- normal proliferation seen in adrenal cancer. The same paradigm would be expected
to apply to cytokine signaling pathways, such as in the CXC cytokine-producing tu- mor described above.92 Experimentally, when cytokine signaling was blocked, so was proliferation.
Rather than gain of activity, dedifferentiation likely occurs by loss of molecules that keep proliferation in check. Chief among them may be p53, but loss of the MC2R (ACTH receptor) also may play a permissive role in the transformation of ad- renal cells from benign hyperplasia to malignancy.
SUMMARY
Adrenal cancer remains a rare and, unless caught in its early stages, almost uni- formly fatal disease. The large majority of studies of this disease have dealt with studying the presence or absence of a given mRNA or protein thought to be involved in the tumorigenic process. As the tools for molecular medicine improve, it will like- ly become possible to analyze large numbers of transcripts, which, in turn, will lead to the identification of new steps in the malignant pathways. In addition to this better understanding, these new approaches hold open the hope that new therapeutic strat- egies may be developed that will render this disease less burdensome.
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