Review paper: origin and molecular pathology of adrenocortical neoplasms.

Neoplastic adrenocortical lesions are common in humans and several species of domestic animals. Although there are unanswered questions about the origin and evolution of adrenocortical neoplasms, analysis of human tumor specimens and animal models indicates that adrenocortical tumorigenesis involves both genetic and epigenetic alterations. Chromosomal changes accumulate during tumor progression, and aberrant telomere function is one of the key mechanisms underlying chromosome instability during this process. Epigenetic changes serve to expand the size of the uncommitted adrenal progenitor population, modulate their phenotypic plasticity (i.e., responsiveness to extracellular signals), and increase the likelihood of subsequent genetic alterations. Analyses of heritable and spontaneous types of human adrenocortical tumors documented alterations in either cell surface receptors or their downstream effectors that impact neoplastic transformation. Many of the mutations associated with benign human adrenocortical tumors result in dysregulated cyclic adenosine monophosphate signaling, whereas key factors and/or signaling pathways associated with adrenocortical carcinomas include dysregulated expression of the IGF2 gene cluster, activation of the Wnt/beta-catenin pathway, and inactivation of the p53 tumor suppressor. A better understanding of the factors and signaling pathways involved in adrenal tumorigenesis is necessary to develop targeted pharmacologic and genetic therapies.

Gonadectomy-induced adrenocortical neoplasia in the domestic ferret (Mustela putorius furo) and laboratory mouse.

Sex steroid-producing adrenocortical adenomas and carcinomas occur frequently in neutered ferrets, but the molecular events underlying tumor development are not well understood. Prepubertal gonadectomy elicits similar tumors in certain inbred or genetically engineered strains of mice, and these mouse models shed light on tumorigenesis in ferrets. In mice and ferrets, the neoplastic adrenocortical cells, which functionally resemble gonadal steroidogenic cells, arise from progenitors in the subcapsular or juxtamedullary region. Tumorigenesis in mice is influenced by the inherent susceptibility of adrenal tissue to gonadectomy-induced hormonal changes. The chronic elevation in circulating luteinizing hormone that follows ovariectomy or orchiectomy is a prerequisite for neoplastic transformation. Gonadectomy alters the plasma or local concentrations of steroid hormones and other factors that affect adrenocortical tumor development, including inhibins, activins, and Müllerian inhibiting substance. GATA-4 immunoreactivity is a hallmark of neoplastic transformation, and this transcription factor might serve to integrate intracellular signals evoked by different hormones. Synergistic interactions among GATA-4, steroidogenic factor-1, and other transcription factors enhance expression of inhibin-alpha and genes critical for ectopic sex steroid production, such as cytochrome P450 17alpha-hydroxylase/17,20 lyase and aromatase. Cases of human adrenocortical neoplasia have been linked to precocious expression of hormone receptors and to mutations that alter the activity of G-proteins or downstream effectors. Whether such genetic changes contribute to tissue susceptibility to neoplasia in neutered ferrets and mice awaits further study.

Transcription factor GATA-4 is a marker of anaplasia in adrenocortical neoplasms of the domestic ferret (Mustela putorius furo).

Adrenocortical neoplasms are a common cause of morbidity in neutered ferrets. Recently we showed that gonadectomized DBA/2J mice develop adrenocortical tumors that express transcription factor GATA-4. Therefore, we screened archival specimens of adrenocortical neoplasms from neutered ferrets to determine whether GATA-4 could be used as a tumor marker in this species. Nuclear immunoreactivity for GATA-4 was evident in 19/22 (86%) of ferret adrenocortical carcinomas and was prominent in areas exhibiting myxoid differentiation. Normal adrenocortical cells lacked GATA-4 expression. Two other markers of adrenocortical tumors in gonadectomized mice, inhibin-alpha and luteinizing hormone receptor, were coexpressed with GATA-4 in some of the ferret tumors. No GATA-4 expression was observed in three cases of nodular hyperplasia, but patches of anaplastic cells expressing GATA-4 were evident in 7/14 (50%) of tumors classified as adenomas. We conclude that GATA-4 can function as a marker of anaplasia in ferret adrenocortical tumors.

Nude mice as a model for gonadotropin-induced adrenocortical neoplasia.

Certain inbred mice (e.g., DBA/2J, CE) develop sex steroid producing adrenocortical tumors following gonadectomy. This adrenal response is thought to result from an unopposed increase in circulating gonadotropins and/or a decrease in factor(s) of gonadal origin. To differentiate between these two possibilities, we utilized the NU/J strain of nude mice, which are immunologically compromised and therefore permissive to xenografts. One group of female nude mice was gonadectomized, while another group of females received xenografts of CHO cells stably transfected with human chorionic gonadotropin (hCG). After 1-2 months, subcapsular adrenocortical neoplasms containing sex steroid-producing cells were observed in both groups. We conclude that high levels of circulating gonadotropins are sufficient to induce adrenocortical tumorigenesis, even in the presence of intact gonads.

Transcription factors GATA-4 and GATA-6 in human adrenocortical tumors.

Transcription factors GATA-4 and GATA-6 are expressed during normal adrenocortical development in mice and humans, and in vitro studies have linked them to adrenal steroidogenesis. GATA-4 is highly expressed in the adrenocortical tumors of gonadectomized mice, whereas GATA-6 is down-regulated in the tumor area. Based on these findings we studied GATA-4 and GATA-6 expression in 39 human adrenocortical tumors using RT-PCR, Northern analysis and immunohistochemistry. 6/18 adenomas and 4/11 carcinomas were positive for GATA-4 mRNA. GATA-6 mRNA was expressed in 19/19 adenomas and 9/10 carcinomas, and GATA-6 immunoreactivity was remarkably lower in adrenocortical carcinomas than in adenomas (p < 0.05). Some of the steroidogenically active human adrenocortical cells (NCI-H295R) were weakly positive for GATA-4, whereas steroidogenically inactive cells (ACT-1) were totally GATA-4 negative. In contrast, both cell lines expressed GATA-6. GATA expression patterns similar to the animal models can thus be observed in human adrenocortical tumors, but the pathophysiological significance of these findings remains to be elucidated.

Transcription factors GATA-4 and GATA-6 during mouse and human adrenocortical development.

Our earlier work implicates transcription factors GATA-4 and GATA-6 in the murine adrenal. We have now studied their expression during mouse and human adrenal development in detail. GATA-4 and GATA-6 mRNAs are readily detectable from embryonic day 15 in mouse and gestational week 19 in human adrenal cortex. In postnatal adrenal, GATA-4 expression is down-regulated, whereas GATA-6 mRNA and protein continue to be abundantly present. In a human adrenocortical cell line NCI-H295R, GATA-6 mRNA is up-regulated by cAMP. This cell line does not express GATA-4. Our findings suggest that GATA-6 expression is hormonally controlled, and required throughout adrenal development from fetal to adult age. GATA-4, on the other hand, may serve a role in fetal adrenal gene regulation.

Adrenocortical tumorigenesis in transgenic mice: the role of luteinizing hormone receptor and transcription factors GATA-4 and GATA-61.

Transgenic (TG) mice, bearing the Simian Virus 40 T-antigen (Tag) under a 6-kb fragment of the murine inhibin alpha-subunit promoter (inhalpha), develop gonadal tumors of granulosa or Leydig cell origin with 100% penetrance by the age of 5-7 months. When these TG mice were gonadectomized prepubertally, between 21-25 days of life, adrenal gland tumors were observed in each mouse by the age of 5-7 months. No adrenal tumors were detected in any intact TG, gonadectomized or intact or control non-TG littermates. The adrenocortical tumors appeared to originate from the X-zone of the adrenal cortex. If functional gonadectomy was induced by GnRH antagonist treatment or by cross-breeding of the TG mice into hypogonadotropic hpg genetic background, neither gonadal nor adrenal tumorigenesis appeared. This prompted a hypothesis that adrenal tumor development in inhalpha/Tag TG mice is related to elevated gonadotropin secretion, which is the most obvious difference between the surgical and functional gonadectomy models. The adrenal tumors and a cell line (Calpha1) derived from them, was found to express luteinizing hormone receptor (LHR), but no FSHR, and hCG treatment stimulated their proliferation. No FSHR was found in the adrenal glands. On the basis of this it was suggested that expression of the potent oncogene T-antigen, allow LH in adrenocortical cells to function as a tumor promoter, and induction of high level functional LHR expression in adrenal tumors. Given the induction of expression and regulation of the GATA-4 and GATA-6 zinc finger family of transcription factors in the gonads by gonadotropins, it was in our interest to explore their expression in the adrenals. We utilized the inalpha/Tag TG mouse model and pathological human adrenal samples to explore the role of GATA-4 and GATA-6 in adrenocortical tumorigenesis. Abundant GATA-6 mRNA expression was found in normal control adrenal cortex during mouse development, whereas GATA-4 mRNA was undetectable. In striking contrast to this, GATA-6 was absent from murine adrenocortical tumors, while GATA-4 mRNA expression was dramatically upregulated in the murine adrenal tumors as well as in human adrenocortical carcinomas. Taken together, these results suggest different roles for GATA-4 and GATA-6 in the adrenal gland, and implicate GATA-4 in adrenal LHR expression and tumorigenesis. Immunohistochemical detection of GATA-4 may serve as a useful marker in differential diagnosis of human adrenal tumors. In addition, the inhalpha/Tag TG model will be helpful for exploring the molecular mechanisms underlying adrenocortical tumorigenesis, ectopic LHR expression in adrenals and the GATA-4/LHR interaction that is related to adrenal tumorigenesis in TG mice.

Reciprocal changes in the expression of transcription factors GATA-4 and GATA-6 accompany adrenocortical tumorigenesis in mice and humans.

While certain genetic changes are frequently found in adrenocortical carcinoma cells, the molecular basis of adrenocortical tumorigenesis remains poorly understood. Given that the transcription factors GATA-4 and GATA-6 have been implicated in gene expression and cellular differentiation in a variety of tissues, including endocrine organs such as testis, we have now examined their expression in the developing adrenal gland, as well as in adrenocortical cell lines and tumors from mice and humans. Northern blot analysis and in situ hybridization revealed abundant GATA-6 mRNA in the fetal and postnatal adrenal cortex of the mouse. In contrast, little or no GATA-4 expression was detected in adrenal tissue during normal development. In vivo stimulation with ACTH or suppression with dexamethasone did not affect the expression of GATA-4 or GATA-6 in the murine adrenal gland. To assess whether changes in the expression of GATA-4 or GATA-6 accompany adrenocortical tumorigenesis, we employed an established mouse model. When gonadectomized, inhibin alpha/SV40 T-antigen transgenic mice develop adrenocortical tumors in a gonadotropin-dependent fashion. In striking contrast to the normal adrenal glands, GATA-6 mRNA was absent from adrenocortical tumors or tumor-derived cell lines, while GATA-4 mRNA and protein were abundantly expressed in the tumors and tumor cell lines. Analogous results were obtained with human tissue samples; GATA-4 expression was detected in human adrenocortical carcinomas but not in normal tissue, adenomas, or pheochromocytomas. Taken together these results suggest different roles for GATA-4 and GATA-6 in the adrenal gland, and implicate GATA-4 in adrenal tumorigenesis. Immunohistochemical detection of GATA-4 may serve as a useful marker in the differential diagnosis of human adrenal tumors.