Low Protein Expression of both ATRX and ZNRF3 as Novel Negative Prognostic Markers of Adult Adrenocortical Carcinoma.

Adrenocortical carcinoma (ACC) is a rare malignancy that is associated with a dismal prognosis. Pan-genomic studies have demonstrated the involvement of ATRX and ZNRF3 genes in adrenocortical tumorigenesis. Our aims were to evaluate the protein expression of ATRX and ZNRF3 in a cohort of 82 adults with ACC and to establish their prognostic value. Two pathologists analyzed immuno-stained slides of a tissue microarray. The low protein expression of ATRX and ZNRF3 was associated with a decrease in overall survival (OS) (p = 0.045, p = 0.012, respectively). The Cox regression for ATRX protein expression of >1.5 showed a hazard ratio (HR) for OS of 0.521 (95% CI 0.273-0.997; p = 0.049) when compared with ≤1.5; for ZNRF3 expression >2, the HR for OS was 0.441 (95% CI, 0.229-0.852; p = 0.015) when compared with ≤2. High ATRX and ZNRF3 protein expressions were associated with optimistic recurrence-free survival (RFS) (p = 0.027 and p = 0.005, respectively). The Cox regression of RFS showed an HR of 0.332 (95%CI, 0.111-0.932) for ATRX expression >2.7 (p = 0.037), and an HR of 0.333 (95%CI, 0.140-0.790) for ZNRF3 expression >2 (p = 0.013). In conclusion, low protein expression of ATRX and ZNRF3 are negative prognostic markers of ACC; however, different cohorts should be evaluated to validate these findings.

Mitotane liposomes for potential treatment of adrenal cortical carcinoma: ex vivo intestinal permeation and in vivo bioavailability.

The adrenal cortical carcinoma (ACC) treatment, for which mitotane (o,p'-DDD) is the drug of choice, still remains a challenge both because of the well-known solubility problems of the drug, and its serious side effects. Mitotane is currently administered as oral tablets. The loading of mitotane into nanocarriers has been suggested as a way to circumvent the low solubility of the drug and its limited oral bioavailability. In this work, we have developed liposomes containing mitotane to enhance its intestinal absorption and oral bioavailability. Liposomes were produced by spray-drying of a mixture of phospholipids and the developed formulation was optimized by studying the degree of crystallinity, spray-drying conditions, phospholipid/mitotane ratio, and influence of mannitol in the hydrating ethanolic solution. An optimal liposomal formulation was produced with a phospholipid:mitotane combination (3.34:1), exhibiting a mean hydrodynamic diameter around 1 µm and spherical shape. The produced mitotane liposomes were re-suspended by hydrating the spray-dried powders in a stirred tank, and tested their intestinal permeability (ex vivo) and relative bioavailability (in vivo), against a free drug solution (with or without Trigliceril®CM). Our results support the conclusion that the loading of mitotane in liposomes enhanced its intestinal absorption and relative bioavailability.

Angiotensin II stimulation promotes mitochondrial fusion as a novel mechanism involved in protein kinase compartmentalization and cholesterol transport in human adrenocortical cells.

In steroid-producing cells, cholesterol transport from the outer to the inner mitochondrial membrane is the first and rate-limiting step for the synthesis of all steroid hormones. Cholesterol can be transported into mitochondria by specific mitochondrial protein carriers like the steroidogenic acute regulatory protein (StAR). StAR is phosphorylated by mitochondrial ERK in a cAMP-dependent transduction pathway to achieve maximal steroid production. Mitochondria are highly dynamic organelles that undergo replication, mitophagy and morphology changes, all processes allowed by mitochondrial fusion and fission, known as mitochondrial dynamics. Mitofusin (Mfn) 1 and 2 are GTPases involved in the regulation of fusion, while dynamin-related protein 1 (Drp1) is the major regulator of mitochondrial fission. Despite the role of mitochondrial dynamics in neurological and endocrine disorders, little is known about fusion/fission in steroidogenic tissues. In this context, the present work aimed to study the role of angiotensin II (Ang II) in protein subcellular compartmentalization, mitochondrial dynamics and the involvement of this process in the regulation of aldosterone synthesis. We demonstrate here that Ang II stimulation promoted the recruitment and activation of PKCε, ERK and its upstream kinase MEK to the mitochondria, all of them essential for steroid synthesis. Moreover, Ang II prompted a shift from punctate to tubular/elongated (fusion) mitochondrial shape, in line with the observation of hormone-dependent upregulation of Mfn2 levels. Concomitantly, mitochondrial Drp1 was diminished, driving mitochondria toward fusion. Moreover, Mfn2 expression is required for StAR, ERK and MEK mitochondrial localization and ultimately for aldosterone synthesis. Collectively, this study provides fresh insights into the importance of hormonal regulation in mitochondrial dynamics as a novel mechanism involved in aldosterone production.

FGF2 Antiproliferative Stimulation Induces Proteomic Dynamic Changes and High Expression of FOSB and JUNB in K-Ras-Driven Mouse Tumor Cells.

Fibroblast growth factor 2 (FGF2) is a well-known cell proliferation promoter; however, it can also induce cell cycle arrest. To gain insight into the molecular mechanisms of this antiproliferative effect, for the first time, the early systemic proteomic differences induced by this growth factor in a K-Ras-driven mouse tumor cell line using a quantitative proteomics approach are investigated. More than 2900 proteins are quantified, indicating that terms associated with metabolism, RNA processing, replication, and transcription are enriched among proteins differentially expressed upon FGF2 stimulation. Proteomic trend dynamics indicate that, for proteins mainly associated with DNA replication and carbohydrate metabolism, an FGF2 stimulus delays their abundance changes, whereas FGF2 stimulation accelerates other metabolic programs. Transcription regulatory network analysis indicates master regulators of FGF2 stimulation, including two critical transcription factors, FOSB and JUNB. Their expression dynamics, both in the Y1 cell line (a murine model of adenocarcinoma cells) and in two other human cell lines (SK-N-MC and UM-UC-3) also susceptible to FGF2 antiproliferative effects, are investigated. Both protein expression levels depend on fibroblast growth factor receptor (FGFR) and src signaling. JUNB and FOSB knockdown do not rescue cells from the growth arrest induced by FGF2; however, FOSB knockdown rescue cells from DNA replication delay, indicating that FOSB expression underlies one of the FGF2 antiproliferative effects, namely, S-phase progression delay.

Is there a role for epithelial-mesenchymal transition in adrenocortical tumors?

PURPOSE: Epithelial-mesenchymal transition (EMT) is a biological dynamic process by which epithelial cells lose their epithelial phenotype and acquire mesenchymal invasive and migratory characteristics. This has been postulated as an essential step during cancer progression and metastasis. Although this is well described in other tumors, the role of EMT in adrenocortical tumors (ACT) has yet to be addressed. METHODS: The aim of this study was to evaluate the expression of EMT markers e-cadherin, vimentin, and fibronectin, along with EMT-transcription factors (EMT-TFs), TWIST1, SIP1, and SNAIL in 24 adrenocortical carcinoma (ACC), 19 adrenocortical adenomas (ACA), 27 childhood-onset adrenocortical tumors (CAT), and 12 normal adrenal glands. The association of EMT and EMT-TFs with clinical outcomes and pathology features were also evaluated. RESULTS: Cytoplasmic vimentin expression was increased among CAT samples when compared to ACC, ACA, and normal adrenal samples (p < 0.001). There was no difference in e-cadherin and fibronectin expression observed between groups. Nuclear and cytoplasmic expression of TWIST1 and SIP1 was stronger in CAT and ACC vs. ACA and normal tissue samples (all, p < 0.05). ACT, regardless of classification, exhibited increased SNAIL expression when compared to normal tissue (p < 0.05). A significant correlation was observed between vimentin and TWIST1 (r s = 0.44, p < 0.001); SIP1 (r s = 0.51, p < 0.001); and SNAIL (r s = 0.23, p < 0.05). TWIST1 and SIP1 expressions demonstrated a significant correlation (r s = 0.56, p < 0.001). High SIP1 expression was associated with a lower survival rate among ACC cases (p < 0.05). CONCLUSIONS: Vimentin, TWIST1, and SIP1 expressions are increased in aggressive ACT. Therefore, EMT may play a relevant role in adrenal tumorigenesis.

A Novel TP53 Mutation Associated with TWIST1 and SIP1 Expression in an Aggressive Adrenocortical Carcinoma.

Adrenocortical carcinomas (ACC) are very rare tumors related to TP53 mutations mostly in childhood onset cases. Epithelial-mesenchymal transition (EMT) transcription factors TWIST1 and Smad interacting protein 1 (SIP1) are related to poorer outcomes in other malignancies, but their role in ACC is unknown. We describe a case of an advanced metastatic ACC (Weiss-score of 9) in a patient at age 76. After primary tumor resection, mitotane therapy was started as palliation to low-volume liver metastasis. After a 2-year period of stable disease, the patient died due to brain metastasis. Somatic gene sequencing revealed a novel TP53 mutation in DNA extracted from paraffin-embedded tissue, a deletion of 8bp in exon 8 (c.811_818del8; GAGGTGCG/-) in homo or hemizygosis causing a subsequent frameshift and premature stop codon at position 302. Immunohistochemistry of P53 and p-Ser-15 P53 showed absent tumoral staining. In addition, immunohistochemical analysis showed an increased expression of the mesenchymal markers vimentin and fibronectin. At last, EMT transcription factors TWIST1 and SIP1 were also overexpressed in tumoral cells. This case report describes an aggressive ACC with not only a novel somatic mutation, but also a novel International Agency for Research on Cancer database 8 base-pair deletion in TP53 exon 8. In addition, the expression of EMT inducers TWIST1 and SIP1 have been reported for the first time in an ACC case. Further investigation is needed to clarify the biologic significance of this new TP53 mutation and its role in the EMT process.

IGF2 and IGF1R in pediatric adrenocortical tumors: roles in metastasis and steroidogenesis.

Deregulation of the IGF system observed in human tumors indicates a role in malignant cell transformation and in tumor cell proliferation. Although overexpression of the IGF2 and IGF1R genes was described in adrenocortical tumors (ACTs), few studies reported their profiles in pediatric ACTs. In this study, the IGF2 and IGF1R expression was evaluated by RT-qPCR according to the patient's clinical/pathological features in 60 pediatric ACT samples, and IGF1R protein was investigated in 45 samples by immunohistochemistry (IHC). Whole transcriptome and functional assays were conducted after IGF1R inhibition with OSI-906 in NCI-H295A cell line. Significant IGF2 overexpression was found in tumor samples when compared with non-neoplastic samples (P<0.001), significantly higher levels of IGF1R in patients with relapse/metastasis (P=0.031) and moderate/strong IGF1R immunostaining in 62.2% of ACTs, but no other relationship with patient survival and clinical/pathological features was observed. OSI-906 treatment downregulated genes associated with MAPK activity, induced limited reduction of cell viability and increased the apoptosis rate. After 24h, the treatment also decreased the expression of genes related to the steroid biosynthetic process, the protein levels of the steroidogenic acute regulatory protein (STAR), and androgen secretion in cell medium, supporting the role of IGF1R in steroidogenesis of adrenocortical carcinoma cells. Our data showed that the IGF1R overexpression could be indicative of aggressive ACTs in children. However, in vitro treatments with high concentrations of OSI-906 (>1µM) showed limited reduction of cell viability, suggesting that OSI-906 alone could not be a suitable therapy to abolish carcinoma cell growth.

Low DICER1 expression is associated with poor clinical outcome in adrenocortical carcinoma.

Low DICER1 expression was associated with poor outcome in several cancers. Recently, hot-spot DICER1 mutations were found in ovarian tumors, and TARBP2 truncating mutations in tumor cell lines with microsatellite instability. In this study, we assessed DICER1 e TRBP protein expression in 154 adult adrenocortical tumors (75 adenomas and 79 carcinomas). Expression of DICER1 and TARBP2 gene was assessed in a subgroup of 61 tumors. Additionally, we investigated mutations in metal biding sites located at the RNase IIIb domain of DICER1 and in the exon 5 of TARBP2 in 61 tumors. A strong DICER1 expression was demonstrated in 32% of adenomas and in 51% of carcinomas (p = 0.028). Similarly, DICER1 gene overexpression was more frequent in carcinomas (60%) than in adenomas (23%, p = 0.006). But, among adrenocortical carcinomas, a weak DICER1 expression was significantly more frequent in metastatic than in non-metastatic adrenocortical carcinomas (66% vs. 31%; p = 0.002). Additionally, a weak DICER1 expression was significantly correlated with a reduced overall (p = 0.004) and disease-free (p = 0.005) survival. In the multivariate analysis, a weak DICER1 expression (p = 0.048) remained as independent predictor of recurrence. Regarding TARBP2 gene, its protein and gene expression did not correlate with histopathological and clinical parameters. No variant was identified in hot spot areas of DICER1 and TARBP2. In conclusion, a weak DICER1 protein expression was associated with reduced disease-free and overall survival and was a predictor of recurrence in adrenocortical carcinomas.

DAX1 Overexpression in Pediatric Adrenocortical Tumors: A Synergic Role with SF1 in Tumorigenesis.

DAX1 transcription factor is a key determinant of adrenogonadal development, acting as a repressor of SF1 targets in steroidogenesis. It was recently demonstrated that DAX1 regulates pluripotency and differentiation in murine embryonic stem cells. In this study, we investigated DAX1 expression in adrenocortical tumors (ACTs) and correlated it with SF1 expression and clinical parameters. DAX1 and SF1 protein expression were assessed in 104 ACTs from 34 children (25 clinically benign and 9 malignant) and 70 adults (40 adenomas and 30 carcinomas). DAX1 gene expression was studied in 49 ACTs by quantitative real-time PCR. A strong DAX1 protein expression was demonstrated in 74% (25 out of 34) and 24% (17 out of 70) of pediatric and adult ACTs, respectively (χ(2)=10.1, p=0.002). In the pediatric group, ACTs with a strong DAX1 expression were diagnosed at earlier ages than ACTs with weak expression [median 1.2 (range, 0.5-4.5) vs. 2.2 (0.9-9.4), p=0.038]. DAX1 expression was not associated with functional status in ACTs. Interestingly, a positive correlation was observed between DAX1 and SF1 protein expression in both pediatric and adult ACTs (r=0.55 for each group separately; p<0.0001). In addition, DAX1 gene expression was significantly correlated with SF1 gene expression (p<0.0001, r=0.54). In conclusion, DAX1 strong protein expression was more frequent in pediatric than in adult ACTs. Additionally, DAX1 and SF1 expression positively correlated in ACTs, suggesting that these transcription factors might cooperate in adrenocortical tumorigenesis.

Expression of LIN28 and its regulatory microRNAs in adult adrenocortical cancer.

OBJECTIVE: LIN28 control cells reprogramming and pluripotency mainly through miRNA regulation and has been overexpressed in many advanced cancers. In this study, we evaluated the prognostic role of LIN28 and its regulatory miRNAs in a large cohort of adrenocortical tumours (ACTs). PATIENTS AND METHODS: LIN28 protein expression was assessed in 266 adults ACTs (78 adenomas and 188 carcinomas) from Brazil and Germany. LIN28A and LIN28B gene expression was analysed in 59 ACTs (31 adenomas and 28 carcinomas) and copy number variation in 39 ACTs. In addition, we determined the expression of let-7 family, mir-9, mir-30 and mir-125 in 28 carcinomas. RESULTS: LIN28A gene was overexpressed in aggressive ACCs when compared with adenomas and nonaggressive ACCs, but no LIN28A copy number variation was found in ACTs. Unexpectedly, weak LIN28 protein expression was significantly associated with reduced disease-free survival in ACC patients (P = 0·01), but for overall survival only a trend was detectable (P = 0·117). In the multivariate analysis, only Ki67 index ≥10% (HR 4·6, P = 0·000) and weak LIN28 protein expression (HR 2·0, P = 0·03) were independent predictors of recurrence in ACC patients. Interestingly, mir-9 expression, a negative LIN28A/B regulator, was significantly higher in aggressive than in nonaggressive ACCs [2076 (from 36 to 9307) vs 133·4 (from 2·4 to 5193); P = 0·011] and was highly associated with reduced overall (P = 0·01) and disease-free survival (P = 0·01). However, mir-9 prognostic role should be further evaluated in a larger cohort. CONCLUSION: Weak LIN28 protein expression was associated with recurrence in ACCs. Additionally, overexpression of mir-9, a negative LIN28A regulator, was associated with poor outcome.

Sonic hedgehog signaling is active in human adrenal cortex development and deregulated in adrenocortical tumors.

BACKGROUND: The sonic hedgehog (SHH) pathway plays a key role in rodent adrenal cortex development and is involved in tumorigenesis in several human tissues, but data in human adrenal glands are limited. OBJECTIVES: The objectives of the study were to analyze the involvement of the SHH pathway in human adrenal development and tumorigenesis and the effects of SHH inhibition on an adrenocortical tumor (ACT) cell line. PATIENTS AND METHODS: Expression of SHH pathway components was evaluated by immunohistochemistry in 51 normal adrenals (33 fetal) and 34 ACTs (23 pediatric) and by quantitative PCR in 81 ACTs (61 pediatric) and 19 controls (10 pediatric). The effects of SHH pathway inhibition on gene expression and cell viability in the NCI-H295A adrenocortical tumor cell line after cyclopamine treatment were analyzed. RESULTS: SHH pathway proteins were present in fetal and postnatal normal adrenals and showed distinct patterns of spatiotemporal expression throughout development. Adult adrenocortical carcinomas presented with higher expression of PTCH1, SMO, GLI3, and SUFU compared with normal adult adrenal cortices. Conversely, pediatric ACTs showed lower mRNA expression of SHH, PTCH1, SMO, GLI1, and GLI3 compared with normal pediatric adrenal cortices. In vitro treatment with cyclopamine resulted in decreased GLI3, SFRP1, and CTNNB1 mRNA expression and ß-catenin staining as well as decreased cell viability. CONCLUSIONS: The SHH pathway is active in human fetal and postnatal adrenals, up-regulated in adult adrenocortical carcinomas, and down-regulated in pediatric ACTs. SHH pathway antagonism impaired cell viability. The SHH pathway is deregulated in ACTs and might provide a new target therapy to be explored.

Treatment of aldosterone-secreting adrenocortical tumors in cats by unilateral adrenalectomy: 10 cases (2002-2012).

BACKGROUND: Primary hyperaldosteronism (PHA) in cats occurs as a consequence of excessive hormone production by an adrenocortical tumor. Median survival time, association between tumor type and prognosis, and the likelihood that cats require continued medical therapy after surgery have not been systematically evaluated. OBJECTIVES: To determine the median survival time of cats with PHA treated by unilateral adrenalectomy. To examine if tumor type, anesthesia time, or tumor location (left or right side) affect survival and if affected cats require continued postoperative treatment for persistent hypertension or hypokalemia. ANIMALS: Ten client-owned cats. METHODS: Retrospective study. Cats were diagnosed with PHA based on clinical signs, increased plasma aldosterone concentration, and advanced imaging. Cats underwent unilateral adrenalectomy. Survival time (days alive after surgery) was determined for each cat. Factors affecting median survival time were investigated, including histopathology, anesthesia time, and location (side) of the tumor. RESULTS: Eight of 10 cats survived to discharge from the hospital post adrenalectomy. Overall median survival was 1,297 days (range 2-1,582 days). The only significant factor affecting median survival time was anesthesia time >4 hours. Tumor type and location (side) did not significantly affect median survival time. No cats required continued medical treatment for PHA. CONCLUSIONS AND CLINICAL IMPORTANCE: Although PHA in cats is still considered an uncommon condition, it should be considered in middle to older aged cats with hypokalemic polymyopathy and systemic hypertension. Surgical correction by unilateral adrenalectomy is a viable approach to definitive treatment of PHA with no need for continued medical management.

Expression of STAT3 and IGF2 in adrenocortical carcinoma and its relationship with angiogenesis.

OBJECTIVE: The aim of this study was to determine the correlation between human adrenocortical carcinoma and the proteins involved in tumor angiogenesis, and to evaluate the angiogenic status of adrenocortical carcinoma. METHODS: The expression of signal transducer and activator of transcription 3 and insulin-like growth factor 2 as well as microvessel density was measured in a series of tissue samples from 44 human sporadic adrenocortical tumors by immunohistochemistry. These specimens were classified as adenomas (n = 20) and carcinomas (n = 24) according to the histological criteria defined by Weiss. RESULTS: A total of 19 of 24 (79.17 %) malignant cases showed positive staining for signal transducer and activator of transcription 3 and 4 of 20 (20.00 %) benign cases showed positive, the difference of signal transducer and activator of transcription 3 expression between adrenocortical adenomas and adrenocortical carcinomas was statistically significant (P < 0.001). Similarly, insulin-like growth factor 2 staining was seen in 70.83 % (17/24) of the malignant cases versus 25.00 % (5/20) of the benign, the difference of insulin-like growth factor 2 expression among two groups was statistically significant (P = 0.002). Malignant cases showed higher microvessel density compared to benign tumors (84.70 ± 12.44 vs 21.05 ± 8.07, P < 0.001). Signal transducer and activator of transcription 3 and insulin-like growth factor 2 expression were positively correlated with microvessel density in all specimens (r_s = 0.832, P < 0.001; r_s = 0.703, P = 0.001). CONCLUSIONS: This study has confirmed that adrenocortical carcinoma overexpress signal transducer and activator of transcription 3 and insulin-like growth factor 2; these results suggest that angiogenesis of human adrenocortical carcinoma may be mediated by these proteins and they could represent selective targets for the molecularly targeted treatments of adrenocortical carcinoma.

Adrenocortical carcinoma: prognostic indices based on clinical and immunohistochemical markers.

Adrenocortical carcinoma is a rare condition with an unpredictable prognosis as a rule. The authors retrospectively analyzed the clinical outcome of 46 patients (31 F, 15 M) during 16 years building up a numerical index for the prognosis, based on clinical and immunohistochemical data. Four indices were analyzed: J1= (Y + 2L + 4H)/T; J2 = (J1) square root of W/200; J3 = (O + Y + 2L + 4H)/T; J4 = (J3) square root W/200. Y = 1 when chronological age (CA) >33 mo, Y = 0 when CA < or =33 mo; L = 1 for right sided tumor and L = 0 for left sided tumor; H = 1 in presence of hypertension and H = 0 for normal blood pressure; T = length of disease in months; W = weight of tumor (g); O = 1 in the absence of p53 protein and O = 0 in the presence of p53. The chance of bad prognosis was observed when age is >33 mo, tumor is on the right side, systemic hypertension is present, tumor weight >250 g, in the absence of p53, J1, J2, J3 >0.4 (p <0.001) and J4 >0.5 (p <0.01). Clinical data and the mathematical model enabled us to establish probabilities of good prognosis in 78-96% and bad prognosis in 63-83%.