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Strong nuclear staining of mutant TP53-R337H protein in normal tissue of carriers who developed ACC.

Mouse Cohorts and Associated Pathology

The TP53-R337H founder mutation exists at a high frequency throughout southern Brazil and represents one of the most common germline TP53 mutations reported to date. It was identified in pediatric adrenocortical tumors in families with a low incidence of cancer. The R337H mutation has since been found in association with early-onset breast cancers and Li–Fraumeni syndrome (LFS). To study this variability in tumor susceptibility, we generated a knockin mutant p53 mouse model (R334H). Endogenous murine p53-R334H protein was naturally expressed at high levels in multiple tissues and was functionally compromised in a tissue- and stress-specific manner. Mutant p53-R334H mice developed tumors with long latency and incomplete penetrance, consistent with many human carriers being at a low but elevated risk for cancer. These findings suggest the involvement of additional cooperating genetic alterations when TP53-R337H occurs in the context of LFS, which has important implications for genetic counseling and long-term clinical follow-up.Significance:A p53-R334H knockin mouse serves as an important model for studying the most common inherited germline TP53 mutation (R337H) that is associated with variable tumor susceptibility.

Supplemental Materials and Methods and Supplementary Figure Legends

Supplementary Tables 1-2, Figures 1-5 from Regulation of Insulin-like Growth Factor–Mammalian Target of Rapamycin Signaling by MicroRNA in Childhood Adrenocortical Tumors

Pediatric adrenocortical tumors (ACT) are rare and often fatal malignancies; little is known regarding their etiology and biology. To provide additional insight into the nature of ACT, we determined the gene expression profiles of 24 pediatric tumors (five adenomas, 18 carcinomas, and one undetermined) and seven normal adrenal glands. Distinct patterns of gene expression, validated by quantitative real-time PCR and Western blot analysis, were identified that distinguish normal adrenal cortex from tumor. Differences in gene expression were also identified between adrenocortical adenomas and carcinomas. In addition, pediatric adrenocortical carcinomas were found to share similar patterns of gene expression when compared with those published for adult ACT. This study represents the first microarray analysis of childhood ACT. Our findings lay the groundwork for establishing gene expression profiles that may aid in the diagnosis and prognosis of pediatric ACT, and in the identification of signaling pathways that contribute to this disease. [Cancer Res 2007;67(2):600–8]

Supplementary Figure 1 from Gene Expression Profiling of Childhood Adrenocortical Tumors

Supplementary Table 2 from Gene Expression Profiling of Childhood Adrenocortical Tumors

Supplementary Table 1 from Regulation of Insulin-like Growth Factor–Mammalian Target of Rapamycin Signaling by MicroRNA in Childhood Adrenocortical Tumors

Supplementary Table 5 from Gene Expression Profiling of Childhood Adrenocortical Tumors

Supplementary Table 4 from Gene Expression Profiling of Childhood Adrenocortical Tumors

Supplementary Table 3 from Gene Expression Profiling of Childhood Adrenocortical Tumors

Supplementary Figure 1 Legend from Gene Expression Profiling of Childhood Adrenocortical Tumors

Supplementary Table 1 from Gene Expression Profiling of Childhood Adrenocortical Tumors

MicroRNAs (miRNAs) act at the posttranscriptional level to control gene expression in virtually every biological process, including oncogenesis. Here, we report the identification of a set of miRNAs that are differentially regulated in childhood adrenocortical tumors (ACT), including miR-99a and miR-100. Functional analysis of these miRNAs in ACT cell lines showed that they coordinately regulate expression of the insulin-like growth factor–mammalian target of rapamycin (mTOR)–raptor signaling pathway through binding sites in their 3′-untranslated regions. In these cells, the active Ser2448-phosphorylated form of mTOR is present only in mitotic cells in association with the mitotic spindle and midbody in the G2-M phases of the cell cycle. Pharmacologic inhibition of mTOR signaling by everolimus greatly reduces tumor cell growth in vitro and in vivo. Our results reveal a novel mechanism of regulation of mTOR signaling by miRNAs, and they lay the groundwork for clinical evaluation of drugs inhibiting the mTOR pathway for treatment of adrenocortical cancer. Cancer Res; 70(11); 4666–75. ©2010 AACR.

The TP53-R337H founder mutation exists at a high frequency throughout southern Brazil and represents one of the most common germline TP53 mutations reported to date. It was identified in pediatric adrenocortical tumors in families with a low incidence of cancer. The R337H mutation has since been found in association with early-onset breast cancers and Li–Fraumeni syndrome (LFS). To study this variability in tumor susceptibility, we generated a knockin mutant p53 mouse model (R334H). Endogenous murine p53-R334H protein was naturally expressed at high levels in multiple tissues and was functionally compromised in a tissue- and stress-specific manner. Mutant p53-R334H mice developed tumors with long latency and incomplete penetrance, consistent with many human carriers being at a low but elevated risk for cancer. These findings suggest the involvement of additional cooperating genetic alterations when TP53-R337H occurs in the context of LFS, which has important implications for genetic counseling and long-term clinical follow-up. Significance: A p53-R334H knockin mouse serves as an important model for studying the most common inherited germline TP53 mutation (R337H) that is associated with variable tumor susceptibility.

Objective Adaptive changes in DHEA and sulfated-DHEA (DHEAS) production from adrenal zona reticularis (ZR) have been observed in normal and pathological conditions. Here we used three different cohorts to assess timing differences in DHEAS blood level changes and characterize the relationship between early blood DHEAS reduction and cell number changes in women ZR. Materials and methods DHEAS plasma samples (n = 463) were analyzed in 166 healthy prepubertal girls before pubarche ( Results We confirmed that overweight girls exhibited higher and earlier DHEAS levels and no difference was found compared with the average European and South American girls with a similar body mass index (BMI). Adrenopause onset threshold (AOT) defined as DHEAS blood levels 35% of the females >40 years old and associated with significantly reduced ZR cell number (based on PTEN and hematoxylin signals). ZR cell loss may in part account for lower DHEA/DHEAS expression, but most cells remain alive with lower DHEA/DHEAS biosynthesis. Conclusion The timely relation between significant reduction of blood DHEAS levels and decreased ZR cell number at the beginning of the 40s suggests that adrenopause is an additional burden for a significant number of middle-aged women, and may become an emergent problem associated with further sex steroids reduction during the menopausal transition.

Two major concerns associated with cancer development in Paraná state, South Brazil, are environmental pollution and the germline

A large number of previous reports described an effect of the pituitary hormone prolactin (PRL) on steroid hormone production by the adrenal cortex. However, those studies remained anecdotal and were never converted into a conceptual and mechanistic framework, let alone being translated into clinical care. In the light of our recently published landmark study where we described PRL signalling as a pivotal regulator of the sexually dimorphic adrenal phenotype in mouse and of adrenal androgen production in humans, we present here the overarching hypothesis that PRL signalling increases the activity of Steroidogenic Factor-1 (SF-1/NR5A1), a transcription factor that has an essential role in adrenal gland development and function, to regulate adrenal cortex growth and hormonal production in physiological and pathological conditions. PRL can then be considered as a bona fide adrenocorticotropic hormone synergizing with ACTH in the endocrine control of adrenal cortex function.

Growing attention is being paid to the association of adrenocortical carcinoma (ACC), a rare endocrine malignancy, to cancer predisposition syndromes caused by germline mutations in genes involved in the control of genome stability. Tumour cells with a defective DNA mismatch repair pathway have a high mutation burden, which results in the production of tumour-associated specific neoantigens and in an increase of the sensitivity to therapies that loosen the constraints of tumour attack by the immune system. The study by Landwehr et al. published in a recent issue of the European Journal of Endocrinology describes a patient with an aggressive ACC bearing a germline MUTYH mutation with loss of heterozygosity in the tumour and accumulation of 8-hydroxyguanine in its genomic DNA. The authors managed to establish a novel differentiated cell line from that tumour which bears the stigma of the defective DNA repair mechanism in its genome. The availability of this new cell model inside the expanding toolbox of the ACC cell lines will allow for novel experimental possibilities, in particular for the study of the tumour microenvironment and the response to immunotherapy.

The TP53-R337H founder mutation exists at a high frequency throughout southern Brazil and represents one of the most common germline TP53 mutations reported to date. It was identified in pediatric adrenocortical tumors in families with a low incidence of cancer. The R337H mutation has since been found in association with early-onset breast cancers and Li-Fraumeni syndrome (LFS). To study this variability in tumor susceptibility, we generated a knockin mutant p53 mouse model (R334H). Endogenous murine p53-R334H protein was naturally expressed at high levels in multiple tissues and was functionally compromised in a tissue- and stress-specific manner. Mutant p53-R334H mice developed tumors with long latency and incomplete penetrance, consistent with many human carriers being at a low but elevated risk for cancer. These findings suggest the involvement of additional cooperating genetic alterations when TP53-R337H occurs in the context of LFS, which has important implications for genetic counseling and long-term clinical follow-up. SIGNIFICANCE: A p53-R334H knockin mouse serves as an important model for studying the most common inherited germline TP53 mutation (R337H) that is associated with variable tumor susceptibility.

The SF-1 transcription factor target gene FATE1 encodes a cancer-testis antigen that has an important role in regulating apoptosis and response to chemotherapy in adrenocortical carcinoma (ACC) cells. Autoantibodies directed against FATE1 were previously detected in patients with hepatocellular carcinoma. In this study, we investigated the prevalence of circulating anti-FATE1 antibodies in pediatric and adult patients with adrenocortical tumors using three different methods (immunofluorescence, ELISA and Western blot). Our results show that a pervasive anti-FATE1 immune response is present in those patients. Furthermore, FATE1 expression is a robust prognostic indicator in adult patients with ACC and is associated with increased steroidogenic and decreased immune response gene expression. These data can open perspectives for novel strategies in ACC immunotherapy.

The XAF1-E134* variant increases the cancer risk for carriers of the TP53-R337H allele., Cancer risk is highly variable in carriers of the common TP53-R337H founder allele, possibly due to the influence of modifier genes. Whole-genome sequencing identified a variant in the tumor suppressor XAF1 (E134*/Glu134Ter/rs146752602) in a subset of R337H carriers. Haplotype-defining variants were verified in 203 patients with cancer, 582 relatives, and 42,438 newborns. The compound mutant haplotype was enriched in patients with cancer, conferring risk for sarcoma (P = 0.003) and subsequent malignancies (P = 0.006). Functional analyses demonstrated that wild-type XAF1 enhances transactivation of wild-type and hypomorphic TP53 variants, whereas XAF1-E134* is markedly attenuated in this activity. We propose that cosegregation of XAF1-E134* and TP53-R337H mutations leads to a more aggressive cancer phenotype than TP53-R337H alone, with implications for genetic counseling and clinical management of hypomorphic TP53 mutant carriers.

One of the crucial challenges in the clinical management of cancer is the resistance to chemotherapeutics. We recently demonstrated that the Hedgehog receptor Patched, which is overexpressed in many recurrent and metastatic cancers, is a multidrug transporter for chemotherapeutic agents such as doxorubicin. The present work provides evidences that Patched is expressed in adrenocortical carcinoma (ACC) patients, and is a major player of the doxorubicin efflux and the doxorubicin resistance in the human ACC cell line H295R. We discovered that methiothepin inhibits the doxorubicin efflux activity of Patched. This drug-like molecule enhances the cytotoxic, pro-apoptotic, antiproliferative and anticlonogenic effects of doxorubicin on ACC cells which endogenously overexpress Patched, and thereby mitigates the resistance of these cancer cells to doxorubicin. Moreover, we report that in mice the combination of methiothepin with doxorubicin prevents the development of xenografted ACC tumors more efficiently than doxorubicin alone by enhancing the accumulation of doxorubicin specifically in tumors without obvious undesirable side effects. Our results suggest that the use of an inhibitor of Patched drug efflux such as methiothepin in combination with doxorubicin could be a promising therapeutic option for adrenocortical carcinoma, and most likely also for other Patched-expressing cancers.

Endocrine tumors have the peculiarity to become clinically evident not only due to symptoms related to space occupation by the growing lesion, similarly to most other tumors, but also, and most often, because of their specific hormonal secretion, which significantly contributes to their pathological burden. Malignant endocrine tumors, in addition, have the ability to produce distant metastases. Here, we critically review the current knowledge about mechanisms and biomarkers characterizing the metastatic process in adrenocortical carcinoma (ACC), a rare endocrine malignancy with a high risk of relapse and metastatization even when the primary tumor is diagnosed and surgically removed at an early stage. We highlight perspectives of future research in the domain and possible new therapeutic avenues based on targeting factors having an important role in the metastatic process of ACC.

International audience; Adrenocortical carcinoma is a rare neoplasm with a poor prognosis. Very important advances have been made in the identification of the genetic determinants of adrenocortical carcinoma pathogenesis but our understanding is still limited about the mechanisms that determine cancer spread and metastasis. One major problem hindering preclinical experimentation for new therapies for adrenocortical carcinoma is represented by the lack of suitable animal models for metastatic disease. With the aim to overcome these limitations, in this study we tested several protocols in order to establish a mouse xenograft model of metastatic adrenocortical carcinoma. The most efficient method, based upon intrasplenic injection followed by splenectomy, produced metastases with high efficiency, whose development could be followed over time by bioluminescence measurements. We expect that the availability of this model will greatly improve the possibilities for preclinical testing of new treatments for advanced-stage disease.

Adrenocortical carcinoma (ACC) is a rare disease among children. Our goal was to identify prognostic biomarkers in 48 primary ACCs from children (2.83 ± 2.3 y; mean age ± SD) by evaluating the tumor stage and outcome for an age of diagnosis before or after 3 years, and association with ACC cluster of differentiation 8 positive (CD8+) cytotoxic T lymphocytes (CD8+-CTL) and Ki-67 immunohistochemical expression (IHC). Programmed death 1(PD-1)/Programmed death-ligand 1 (PD-L1) immunohistochemistry (IHC) in ACC was analyzed in a second, partially overlapping cohort (N = 19) with a similar mean age. All patients and control children were carriers of the germline TP53 R337H mutation. Survival without recurrence for less than 3 years and death unrelated to disease were excluded. Higher counts of CD8+-CTL were associated with patients diagnosed with ACC at a younger age and stage I, whereas a higher percentage of the Ki-67 labeling index (LI) and Weiss scores did not differentiate disease free survival (DFS) in children younger than 3 years old. No PD-1 staining was observed, whereas weakly PD-L1-positive immune cells were found in 4/19 (21%) of the ACC samples studied. A high CD8+-CTL count in ACC of surviving children is compelling evidence of an immune response against the disease. A better understanding of the options for enhancement of targets for CD8+ T cell recognition may provide insights for future pre-clinical studies.

ER-mitochondria contact sites represent hubs for signaling that control mitochondrial biology related to several aspects of cellular survival, metabolism, cell death sensitivity and metastasis, which all contribute to tumorigenesis. Altered ER-mitochondria contacts can deregulate Ca2+ homeostasis, phospholipid metabolism, mitochondrial morphology and dynamics. MAM represent both a hot spot in cancer onset and progression and an Achilles' heel of cancer cells that can be exploited for therapeutic perspectives. Over the past years, an increasing number of cancer-related proteins, including oncogenes and tumor suppressors, have been localized in MAM and exert their pro- or antiapoptotic functions through the regulation of Ca2+ transfer and signaling between the two organelles. In this review, we highlight the central role of ER-mitochondria contact sites in tumorigenesis and focus on chemotherapeutic drugs or potential targets that act on MAM properties for new therapeutic approaches in cancer.

Fragile X-associated tremor ataxia syndrome (FXTAS) is a rare disorder associated to the presence of the fragile X premutation, a 55–200 CGG repeat expansion in the 5′ UTR of the FMR1 gene. Two main neurological phenotypes have been described in carriers of the CGG premutation: (1) neurodevelopmental disorders characterized by anxiety, attention deficit hyperactivity disorder (ADHD), social deficits, or autism spectrum disorder (ASD); and (2) after 50 years old, the FXTAS phenotype. This neurodegenerative disorder is characterized by ataxia and a form of parkinsonism. The molecular pathology of this disorder is characterized by the presence of elevated levels of Fragile X Mental Retardation 1 (FMR1) mRNA, presence of a repeat-associated non-AUG (RAN) translated peptide, and FMR1 mRNA-containing nuclear inclusions. Whereas in the past FXTAS was mainly considered as a late-onset disorder, some phenotypes of patients and altered learning and memory behavior of a mouse model of FXTAS suggested that this disorder involves neurodevelopment. To better understand the physiopathological role of the increased levels of Fmr1 mRNA during neuronal differentiation, we used a small interfering RNA (siRNA) approach to reduce the abundance of this mRNA in cultured cortical neurons from the FXTAS mouse model. Morphological alterations of neurons were rescued by this approach. This cellular phenotype is associated to differentially expressed proteins that we identified by mass spectrometry analysis. Interestingly, phenotype rescue is also associated to the rescue of the abundance of 29 proteins that are involved in various pathways, which represent putative targets for early therapeutic approaches. Keywords: FXTAS, premutation, Fmr1 mRNA, dendritic arborization, Tia1, ROAA, Hnrnpll, Aldh4a1/P5CDH, dendrtic spine

Adrenocortical carcinoma (ACC) is a rare disease among children. Our goal was to identify prognostic biomarkers in 48 primary ACCs from children (2.83 &plusmn, 2.3 y, mean age &plusmn, SD) by evaluating the tumor stage and outcome for an age of diagnosis before or after 3 years, and association with ACC cluster of differentiation 8 positive (CD8+) cytotoxic T lymphocytes (CD8+-CTL) and Ki-67 immunohistochemical expression (IHC). Programmed death 1(PD-1)/Programmed death-ligand 1 (PD-L1) immunohistochemistry (IHC) in ACC was analyzed in a second, partially overlapping cohort (N = 19) with a similar mean age. All patients and control children were carriers of the germline TP53 R337H mutation. Survival without recurrence for less than 3 years and death unrelated to disease were excluded. Higher counts of CD8+-CTL were associated with patients diagnosed with ACC at a younger age and stage I, whereas a higher percentage of the Ki-67 labeling index (LI) and Weiss scores did not differentiate disease free survival (DFS) in children younger than 3 years old. No PD-1 staining was observed, whereas weakly PD-L1-positive immune cells were found in 4/19 (21%) of the ACC samples studied. A high CD8+-CTL count in ACC of surviving children is compelling evidence of an immune response against the disease. A better understanding of the options for enhancement of targets for CD8+ T cell recognition may provide insights for future pre-clinical studies.

Groundbreaking progress has been recently made in elucidating the signaling pathways that are altered in adrenocortical carcinoma (ACC), an endocrine malignancy that still has an unfavorable prognosis, and in understanding its genomic structure. These advances need now to be translated to create cellular and animal models more relevant to human disease in order to develop new and more effective diagnostic procedures and targeted therapies against this deadly malignancy.

Phosphodiesterases (PDEs) form a superfamily of enzymes that catalyze the hydrolysis of cyclic nucleotides adenosine 3&prime, 5&prime, cyclic monophosphate (cAMP) and guanosine 3&prime, cyclic monophosphate (cGMP) to their inactive 5&prime, monophosphates. cAMP plays a critical role as a second messenger in endocrine tissues, and activation of cAMP signaling has been reported in endocrine tumors. Germline variants in PDEs have been associated with benign cortisol-secreting adrenocortical adenomas and testicular germ cell cancer but not adrenocortical carcinoma. We performed whole genome sequencing (WGS) and whole exome sequencing (WES) of paired blood and tumor samples from 37 pediatric adrenocortical tumors (ACTs). Germline inactivating variants in PDEs were observed in 9 of 37 (24%) patients. Tumor DNA analysis revealed loss of heterozygosity, with maintenance of the mutated allele in all cases. Our results suggest that germline variants in PDEs and other regulators of the cAMP-signaling pathway may contribute to pediatric adrenocortical tumorigenesis, perhaps by cooperating with germline hypomorphic mutant TP53 alleles and uniparental disomy of chromosome 11p15 (Beckwith&ndash, Wiedemann syndrome).

Context Novel tumor markers are urgently needed to better stratify adrenocortical cancer (ACC) patients and improve therapies for this aggressive neoplasm. Objective To assess the diagnostic and prognostic value of the actin-bundling protein fascin-1 (FSCN1) in adrenocortical tumors. Design, Setting and Participants A local series of 37 malignant/37 benign adrenocortical tumors at Careggi University Hospital and two independent validation ACC cohorts (Cochin, TCGA) from the European Network for the Study of Adrenal Tumors were studied. Main Outcome Measures FSCN1 expression was quantified by immunohistochemistry, Western blot and quantitative RT-PCR in ACC specimens; overall and disease-free survival associated with FSCN1 expression were assessed by Kaplan-Meier analysis and compared with that of Ki67 labeling index and tumor stage. Results Despite the low diagnostic power, in the Florence ACC series, FSCN1 immunohistochemical detection appeared as an independent prognostic factor, also refining results obtained with staging and Ki67 labeling index. The robust prognostic power of FSCN1 levels was further confirmed in two independent ACC cohorts. A positive correlation was found between FSCN1 and steroidogenic factor-1 (SF-1), with a substantially higher expression of both factors in ACCs at advanced stages and with at least one of the three Weiss score parameters associated with invasiveness. Moreover, we demonstrated FSCN1 role in promoting cell invasion in a human ACC cell line only in the case of increased SF-1 dosage. Conclusions These findings show that FSCN1 is a novel independent prognostic marker in ACC and may serve as a potential therapeutic target to block tumor spread.

International audience; FMRP is an RNA-binding protein involved in synaptic translation. Its absence causes a form of intellectual disability, the Fragile X syndrome (FXS). Small neuroanatomical abnormalities, present both in human and mouse FMRP-deficient brains, suggest a subtle critical role of this protein in neurogenesis. Stable depletion of FMRP has been obtained in a mouse embryonic stem cell line Fmr1 (shFmr1 ES) that does not display morphological alterations, but an abnormal expression of a subset of genes mainly involved in neuronal differentiation and maturation. Inducing the differentiation of shFmr1 ES cells into the neuronal lineage results in an accelerated generation of neural progenitors and neurons during the first steps of neurogenesis. This transient phenotype is due to an elevated level of the Amyloid Precursor Protein (APP), whose mRNA is a target of FMRP. APP is processed by the BACE-1 enzyme, producing the β-amyloid (Aβ) peptide accelerating neurogenesis by activating the expression of Ascll. Inhibition of the BACE-1 enzyme rescues the phenotype of shFmr1 ES cells. Here we discuss the importance of the shFmr1 ES line not only to understand the physiopathology of FXS but also as a tool to screen biomolecules for new FXS therapies.

Context Adrenocortical carcinoma (ACC) is a rare endocrine malignancy with overall poor prognosis. The Ki67 labeling index (LI) has a major prognostic role in localized ACC after complete resection, but its estimates may suffer from considerable intra- and interobserver variability. VAV2 overexpression induced by increased Steroidogenic Factor-1 dosage is an essential factor driving ACC tumor cell invasion. Objective To assess the prognostic role of VAV2 expression in ACC by investigation of a large cohort of patients. Design, Setting, and Participants A total of 171 ACC cases (157 primary tumors, six local recurrences, eight metastases) from seven European Network for the Study of Adrenal Tumors centers were studied. Outcome Measurements H-scores were generated to quantify VAV2 expression. VAV2 expression was divided into two categories: low (H-score, Results VAV2 expression and Ki67 LI were significantly correlated with each other and with PFS and OS. Heterogeneity of VAV2 expression inside the same tumor was very low. Combined assessment of VAV2 expression and Ki67 LI improved patient stratification to low-risk and high-risk groups. Conclusion Combined assessment of Ki67 LI and VAV2 expression improves prognostic prediction in ACC.