Supplementary Figure 1 from CREB3L2-PPARγ Fusion Mutation Identifies a Thyroid Signaling Pathway Regulated by Intramembrane Proteolysis

Supplementary Figure 1 from CREB3L2-PPARγ Fusion Mutation Identifies a Thyroid Signaling Pathway Regulated by Intramembrane Proteolysis

OBJECTIVE:Tumours in the adrenocortex are common human tumours. Malignancy is however, rare, the yearly incidence being 0.5-2 per million inhabitants, but associated with a very aggressive behaviour. Adrenocortical tumours are often associated with altered hormone production with a variety of clinical symptoms. The aggressiveness of carcinomas together with the high frequency of adenomas calls for a deeper understanding of the underlying biological mechanisms and an improvement of the diagnostic possibilities. METHODS:Microarray gene expression analysis was performed in tumours of adrenocortex with emphasis on malignancy as well as hormonal activity. The sample set consisted of 17 adenomas, 11 carcinomas and 4 histological normal adrenocortexes. RNA from these was hybridised according to a reference design on microarrays harbouring 29 760 human cDNA clones. Confirmation was performed with quantitative real time-PCR and western blot analysis. RESULTS:Unsupervised clustering to reveal relationships between samples based on the entire gene expression profile resulted in two subclusters; carcinomas and non-cancer specimens. A large number of genes were accordingly found to be differentially expressed comparing carcinomas to adenomas. Among these were IGF2, FGFR1 and FGFR4 in growth factor signalling the most predominant and also the USP4, UBE2C and UFD1L in the ubiquitin-proteasome pathway. Moreover, two subgroups of carcinomas were identified with different survival outcome, suggesting that survival prediction can be made on the basis of gene expression profiles. Regarding adenomas with aldosterone overproduction, OSBP and VEGFB were among the most up-regulated genes compared with the other samples. CONCLUSIONS:Adrenocortical carcinomas are associated with a distinct molecular signature apparent in their gene expression profiles. Differentially expressed genes were identified associated with malignancy, survival as well as hormonal activity providing a resource of candidate genes for an exploration of possible drug targets and diagnostic and prognostic markers.

Objective : Due to increased use of imaging techniques, adrenal incidentalomas are frequently detected. The majority are non-hyperfunctioning adrenocortical tumors. We have previously shown that expression of the gene CYP17 , coding for the enzyme in the cortisol pathway, correlates with cortisol release from adrenocortical tumors in vitro. The aim of this study was to compare clinical data with mRNA expression of CYP17 and CYP11B1 in adrenocortical tumors from patients with and without Cushing’s syndrome and to identify adrenal tumors that may cause subclinical Cushing’s syndrome. Design : A retrospective study of 34 patients undergoing adrenalectomy due to an adrenal tumor. Methods : Clinical data were collected. In the adrenal gland the mRNA expression of the genes CYP17 and CYP11B1 was studied with in situ hybridisation technique. Results : The median ratio of CYP17/CYP11B1 expression in tumors from patients with Cushing’s syndrome was significantly higher than the median ratio in the non-hyperfunctioning tumors. Tumors from 2 patients with subclinical Cushing’s syndrome had ratios within the upper range for non-hyperfunctioning tumors. Conclusions : The ratio between the expression of the genes CYP17 and CYP11B1 in tumors from patients with Cushing’s syndrome is significantly higher than in the non-hyperfunctioning tumors. This indicates that 17α-hydroxylase is a major determinant of cortisol overproduction. The patients with subclinical Cushing’s syndrome in this study are too few to draw any firm conclusions although the results suggest that subclinical Cushing’s syndrome may be identified post-operatively with this method.

Abstract The discovery of gene fusion mutations, particularly in leukemia, has consistently identified new cancer pathways and led to molecular diagnostic assays and molecular-targeted chemotherapies for cancer patients. Here, we report our discovery of a novel CREB3L2-PPARγ fusion mutation in thyroid carcinoma with t(3;7)(p25;q34), showing that a family of somatic PPARγ fusion mutations exist in thyroid cancer. The CREB3L2-PPARγ fusion encodes a CREB3L2-PPARγ fusion protein that is composed of the transactivation domain of CREB3L2 and all functional domains of PPARγ1. CREB3L2-PPARγ was detected in <3% of thyroid follicular carcinomas. Engineered overexpression of CREB3L2-PPARγ induced proliferation by 40% to 45% in primary human thyroid cells, consistent with a dominant oncogenic mechanism. Wild-type CREB3L2 was expressed in the thyroid as a bZIP transcription factor with a transmembrane domain that has flanking S1P and S2P proteolytic cleavage sites. Native CREB3L2 was cleaved to nuclear CREB3L2 by regulated intramembrane proteolysis in normal thyroid cells that expressed the S1P and S2P proteases. Nuclear CREB3L2 stimulated transcription 8-fold from the EVX1 cyclic AMP (cAMP) response element in the absence of cAMP, whereas CREB3L2-PPARγ inhibited transcription 6-fold from EVX1 in the same experiments. CREB3L2-PPARγ also inhibited 4-fold the expression of thyroglobulin, a native cAMP-responsive gene, in primary thyroid cells treated with thyroid-stimulating hormone. Our findings identify a novel CREB3L2-PPARγ gene fusion mutation in thyroid carcinoma and reveal a thyroid signaling pathway that is regulated by intramembrane proteolysis and disrupted in cancer. [Cancer Res 2008;68(17):7156–64]

BACKGROUND AND AIMS:Differentiation between the two major subgroups of primary aldosteronism, bilateral hyperplasia and aldosterone producing adenoma is essential since therapy in the former is medical and in the latter surgical. The aim of the present study was to evaluate the clinical utility of adrenocortical scintigraphy in the management of primary aldosteronism.MATERIAL AND METHODS:[131I] norcholesterol (NP-59) scintigraphy with dexamethasone suppression for subclassification and lateralization of primary aldosteronism was evaluated in 49 patients with long-term follow-up after diagnosis and treatment.RESULTS:Thirty-three patients with the diagnosis of aldosterone producing adenoma were operated with adrenalectomy. Preoperative scintigraphy showed lateralized isotope uptake in 27/33 patients while 6 showed no uptake. Twenty-two were cured and three significantly improved. Thus, in 25/33 (76%), scintigraphy showed the correct side as the patients benefited of surgery. Two patients did not improve. Fourteen patients with a probable diagnosis of bilateral hyperplasia had normal scintigraphies.CONCLUSIONS:In the present retrospective study we found limited sensitivity of NP-59 scintigraphy. However, when a lateralized scintigraphic uptake is achieved it has a high accuracy. Scintigraphy may be used as an adjunct in cases where adrenal venous sampling is inconclusive.

OBJECTIVE: Primary aldosteronism (PA) is characterized by hypertension, hypokalemia and suppressed renin-angiotensin system caused by autonomous aldosterone production. The aim of this study was to localize mRNA expression of the genes coding for steroidogenic enzymes in adrenals from a group of patients with PA and relate this to clinical work-up, histopathology and outcome of adrenalectomy. DESIGN: This was a retrospective study of 27 patients subjected to adrenalectomy for PA. METHODS: Clinical data were collected and follow-up of all patients was performed. Paraffin-embedded specimens were analyzed by the in situ hybridization technique, with oligonucleotide probes coding for the steroidogenic enzyme genes. RESULTS: The resected adrenals had the histopathologic diagnosis of adenoma (11), adenoma and/or hyperplasia (15) or hyperplasia (1). CYP11B2 expression (indicating aldosterone production) was found in a dominant adrenal nodule from 22 patients. Fourteen of these had additional CYP11B2 expression in the zona glomerulosa. All 22 patients were cured of PA by adrenalectomy. One of these patients, who had additional high expression of CYP11B2 in the zona glomerulosa, was initially cured, but the condition had recurred at follow-up. Two patients had a mass shown on computed tomography without CYP11B2 but with CYP11B1 and CYP17 expression (indicating cortisol production). Instead their adrenals contained small nodules with CYP11B2 expression. These patients were not cured. CONCLUSIONS: Clinical data, endocrinologic evaluation and histopathology in combination with mRNA in situ hybridization of steroidogenic enzyme genes provide improved opportunities for correct subclassification postoperatively of patients with primary aldosteronism. At present, the in situ hybridization method is of special value for analysis of cases not cured by adrenalectomy.

The clinical and histopathological distinction between benign and malignant pheochromocytomas and paragangliomas is difficult, and reliable diagnostic markers are lacking. Here we have evaluated the prognostic value of human telomerase reverse transcriptase (hTERT) gene expression detected by reverse transcription PCR (RT-PCR); telomerase activity (TA) measured by TRAP (telomeric repeat amplification protocol) assay; immunohistochemical staining for Ki-67/MIB-1; and the mRNA expression of matrix metalloproteinase (MMP)–2 and EMMPRIN (extracellular matrix metalloproteinase inducer) analyzed by in situ hybridization in 32 primary pheochromocytomas or abdominal paragangliomas. hTERT was expressed in 7/11 malignant tumors (defined as presence of metastasis and/or extensive local invasion) as compared with in 2/21 benign tumors. All of the benign tumors showed <1% proliferative activity, as measured by Ki-67/MIB-1 staining. In all three patients with malignant tumors who developed metastases and/or invasive local recurrence during follow-up, the tumors were positive for either hTERT expression or Ki-67/MIB-1 immunoreactivity. TA was not a significant discriminator between benign and malignant tumors, and the value of EMMPRIN and MMP-2 as predictive markers was limited. In conclusion, the findings imply that the combined use of Ki-67/MIB-1 and hTERT, in addition to histopathology, provides a highly specific tool to identify benign pheochromocytoma and abdominal paraganglioma cases that are not at risk of developing recurrent or metastatic disease.

The adrenal cortex synthesizes and releases steroid hormones, mainly mineralocorticoids and glucocorticoids. There is a functional zonation of the adrenal cortex and steroid synthesis is thoroughly regulated. Overproduction of aldosterone, primary aldosteronism, may be much more common than previously known and may be responsible for 10% of essential hypertension. Primary aldosteronism is characterized by autonomous production of aldosterone, suppressed renin activity, hypokalemia, and hypertension. The two most common forms are unilateral adenoma and bilateral hyperplasia. In spite of thorough clinical workup and careful histopathology it is often difficult to differentiate between adenoma and hyperplasia. The gene CYP11B2 encodes the steroid synthesizing enzymes for aldosterone production, while the genes CYP17 and CYP11B1 are needed for cortisol production. Most normal controls show expression of CYP11B2 in zona glomerulosa. Expression of CYP11B1 and CYP17 is seen in zona fasciculata and reticularis, whereas the expression of CYP21 is present in all three cortical layers. Adenomas from patients with primary aldosteronism show considerable variation in the expression of CYP11B2. Adenomas from patients with Cushing's syndrome have a strong expression of CYP11B1 and CYP17. In a patient material of 29 cases of primary aldosteronism, 4 patients had small nodules detected with expression of CYP11B2 gene. These nodules were not visualized on CT, whereas adrenal masses seen on CT in these patients showed CYP11B1 and CYP17 gene expression. This suggests that these small nodules are responsible for the aldosterone production and this is characteristic of nodular hyperplasia in patients with primary aldosteronism. In conclusion, this method to visualize mRNA gene expression of steroidogenic enzymes, and especially expression of CYP11B2, has increased the knowledge of adrenal pathophysiology. The results emphasize the value to include functional studies (venous sampling and/or scintigraphy) in the preoperative work up of patients with primary aldosteronism.

Adenomas of the adrenal cortex cause different disorders depending on the main steroid synthesized and released. The aim of this research is to increase our understanding of the pathophysiology of steroidogenesis in adrenocortical disorders by comparing the release of steroids from adrenocortical adenomas in vitro with the messenger RNA (mRNA) expression of steroid synthesizing enzymes. Fourteen patients with adrenal tumors were included in the present study; nine were diagnosed with primary aldosteronism and three with Cushing’s syndrome. Two patients had an adrenal tumor discovered on computed tomography (CT) during workup for an unrelated disease. Serum cortisol, plasma aldosterone, and urinary catecholamines were normal. Tissue was taken for in vitro steroid release, and aldosterone and cortisol in the medium after a 1-hour incubation were determined. Oligonucleotide probes with sequences complementary to mRNAs encoding for the steroid synthesizing enzymes 11β-hydroxylase (CYP11B1), 18-hydroxylase (CYP11B2), 17α-hydroxylase (CYP17), and 21-hydroxylase (CYP21) were synthesized (Genset, Paris, France) and in situ hybridization was performed. Moderate expression of CYP11B2 and low expression of CYP11B1 were seen in the zona glomerulosa. The zona fasciculata of the control adrenals expressed a high signal of CYP11B1, whereas the expression of CYP11B2 was very low. There was considerable variation in aldosterone release from the aldosteronomas, whereas the tumors from the Gushing patients showed no detectable release of aldosterone. In contrast, tumors from patients with primary aldosteronism, Cushing’s syndrome, and no hyper-function all had the ability to synthesize and release cortisol in vitro. The highest cortisol release was found in tumors from patients with Cushing’s syndrome, but also the nonhyperfunctioning tumors and some of the aldosteronomas released significant amounts of cortisol. The two patients with highest release of aldosterone in vitro showed the highest expression of CYP11B2 and the lowest expression of CYP11B1 and CYP17. The remaining aldosteronomas had low expression of CYP11B2, similar to the two other groups. Expression of CYP11B1 was high as expected in the Gushing adenomas, but also the two nonhyperfunctioning tumors and some of the aldosteronomas showed a moderate expression. Adenomas from Cushing’s syndrome, nonhyperfunctioning adenomas, and some of the aldosterone- producing adenomas had moderate to high expression of CYP17. This paper presents new means for functional characterization of adrenocortical tumors. Diagnosis of an aldosteronoma is often difficult, and with the advent of these methods it is possible to determine the functional capacity of a tumor, once it is removed. This is of special interest if the patient remains hypertensive postoperatively, and it is not clear whether the patient indeed had a functioning tumor.

Purpose: Extracellular matrix metalloproteinase inducer (EMMPRIN) has been shown to stimulate fibroblasts to production of matrix metalloproteinases (MMPs). MMPs comprise a family of proteolytic enzymes implicated in the degradation of extracellular matrix which has been proposed to be one of the essential steps in tumor invasion and metastases. In the present study we investigated the expression and location of mRNAs for EMMPRIN, matrix metalloproteinase-2 (MMP-2), and membrane-type 1 matrix metalloproteinase (MT1-MMP) in mesenchymal tumors with different tendencies to recur or metastasize. Subjects: Eight malignant fibrous histiocytomas (MFH), seven aggressive fibromatosis (AF), and six benign fibrous tumors (BF). Method: The mRNA-expression of EMMPRIN, MMP-2 and MT1-MMP were studied using mRNA in situ hybridization technique. Results: The mRNA-expression of EMMPRIN, MMP-2 and MT1-MMP respectively were found at varying frequency and level in all tumor types. The mRNAs corresponding to EMMPRIN and MMP-2 were seen in neoplastic cells as well as in endothelial cells both inside and outside the tumor pseudo-capsule, whereas MT1-MMP was seen only within the tumors. The estimated mRNA levels of EMMPRIN and MMP-2 covariated significantly. Overall, the highest expression was found in the MFH tumors and the lowest levels in the BF tumors. Discussion: These findings suggest that the MMP-inducer EMMPRIN and the extracellular matrix degrading system involving the metalloproteinases MMP-2 and MT1-MMP is frequently activated in mesenchymal tumors. The covariation between EMMPRIN and MMP-2 support previous findings that EMMPRIN may be an inducer of MMP-2. The high levels of MMP-2 mRNA in MFH indicate a relationship between the proteolytic activity of MMP-2 and the tumor aggressiveness.

Invasive breast cancer varies widely in biologic aggressiveness, from fairly indolent tumors to rapidly disseminating carcinomas. Matrix metalloproteinases have enzymatic activity and assist in tumor invasion by degrading basement membranes and extracellular matrix. The extracellular matrix metalloproteinase inducer EMMPRIN is thought to stimulate fibroblasts to produce the zymogen pro-gelatinase A. The membrane type 1-matrix metalloproteinase (MT1-MMP) is thought to assist in tumor invasion and metastasis by activating pro-gelatinase A, which shows enhanced expression in various tumors. Overexpression of gelatinase A has shown to correlate with a malignant phenotype in many tumor forms. The aim of the study was to investigate the mRNA expression pattern of MT1-MMP, gelatinase A, and EMMPRIN in breast tumors. Formalin-fixed paraffin-embedded breast tissue samples from 18 patients operated on with breast-conserving surgery for invasive breast carcinoma <20 mm between 1977 and 1985 were analyzed using the mRNA in situ hybridization technique. Most of the patients were node-negative (15/18) and underwent postoperative irradiation to the breast (16/18). The median age at diagnosis was 52 years (21–83 years). At the time of the study 11 patients were alive, 4 without recurrence; 7 patients had been operated for ipsilateral breast tumor recurrences, and 2 had distant metastases. The median follow-up was 112 months (102–193 months). Seven patients died of disseminated breast cancer; their median follow-up was 43 months (22–116 months). 35 S-labeled antisense and sense mRNA probes transcribed from linearized plasmids containing cDNA for the matrix metalloproteinases gelatinase A and MT1-MMP and the glycoprotein EMMPRIN were hybridized to 5 μm paraffin-embedded tissue sections. Several invasive carcinomas were surrounded by normal tissue and carcinoma in situ lesions. Gelatinase A, MT1-MMP, and EMMPRIN mRNA expression were detected in all of the carcinomas. The gelatinase A mRNA expression was mainly localized to stromal cells at moderate to high levels surrounding the invading carcinoma cells but was also seen in single cells at low levels in in situ lesions and in some normal glandular cells. MT1-MMP and EMMPRIN were expressed in all of the carcinomas and were mainly localized to tumor cells; but they were also seen to some extent in single cells at low levels in in situ lesions and in normal glandular cells. No differences in levels of expression for gelatinase A, MT1-MMP, or EMMPRIN were seen in patients who survived compared to patients who died from metastatic disease. The co-expression of gelatinase A, MT1-MMP, and EMMPRIN mRNA in invasive breast carcinoma supports the theory that these proteins interact and are important for the invasive phenotype in breast carcinoma. Hence EMMPRIN may be a central factor for stimulation of gelatinase A activation. Specific inhibitors for individual MMP members could in the future be target-specific events in breast tumor progression. Inhibition of EMMPRIN could be such a target.

In an attempt to understand the mechanism behind the invasion and metastasis in adrenocortical cancer we performed mRNA in situ hybridization on 30 tumors for three matrix metalloproteinases (MMPs): gelatinase A, membrane type 1 matrix metalloproteinase (MT1-MMP), and collagenase-3. All are known to participate in the invasion and metastasis of other tumor forms by degrading the extracellular matrix. Thirteen of sixteen cancers, but only one of fourteen benign lesions showed expression of gelatinase A, which was localized in stromal cells. MT1-MMP is thought to assist in tumor invasion and metastasis by activating the zymogen gelatinase A. Of 14 malignant tumors analyzed, 12 showed MT1-MMP mRNA expression, which in 7 cases was detected in both neoplastic and stromal cells. The benign tumors showed MT1-MMP expression in only 3 of 11 cases, and it was restricted to tumor cells. Fourteen tumors (11 cancers, 3 adenomas) were also analyzed for collagenase-3 mRNA, but no expression was detected. In conclusion, our data show that gelatinase A mRNA is expressed in most malignant adrenocortical tumors but not in the benign tumors. Gelatinase A mRNA expression is restricted to stromal cells, whereas its activator, MT1-MMP, is expressed in both stromal and neoplastic cells. Inhibition of gelatinase A and other proteinases may in the future become important as a form of cancer treatment.

Secretion of PTH is regulated by extracellular calcium via calcium receptors (CaR) on the parathyroid cell surface. Recent studies have shown a decreased expression of CaR messenger RNA (mRNA) and CaR protein in pathological parathyroids. We studied the expression of CaR mRNA in pairs of adenoma and adenoma-associated normal gland from the same patients (n = 17) and in biopsies of normal parathyroid glands of normocalcemic subjects (n = 4) using in situ hybridization with oligonucleotide probes on frozen sections. No down-regulation of CaR mRNA caused by hypercalcemia could be demonstrated in the normal adenoma-associated parathyroids when compared with the normal parathyroids of normocalcemic subjects. In contrast, CaR mRNA in the adenomas was significantly reduced to 64% (median; range 41-98) of the corresponding normal adenoma-associated glands. No correlation was seen between CaR mRNA in the adenoma and preoperative serum calcium, PTH, or weight of the adenoma. Loss of heterozygosity studies were performed on adenomas using markers for the locus of the CaR gene on chromosome 3q. No allelic loss was demonstrated, excluding allelic loss as the cause for decreased CaR mRNA expression in the adenomas. It is concluded that the lowered levels of CaR mRNA in parathyroid adenomas may contribute to the increased set point of PTH secretion. In large adenomas the increased cell mass seems to be more important for the increased secretion of PTH.

Thyroid tumors vary widely in biologic behavior; they range from benign adenomas to rapidly growing anaplastic carcinomas. Among thyroid neoplasms, the follicular tumor is especially suited as a model for studies of tumor cell invasion; the distinction between adenomas and carcinomas relies mainly on the presence of capsular and vascular invasion. Matrix metalloproteinases play an important role in tumor cell invasion, as they are able to degrade basement membrane and extracellular matrix components. Twenty-nine thyroid tumors of varying type and aggressiveness were selected for analysis of relative molecular weight 72,000-dalton type IV collagenase (gelatinase A) expression by mRNA in situ hybridization. Strong gelatinase A mRNA expression was seen in 10 of 14 follicular carcinomas, in none of six follicular adenomas, in all four anaplastic carcinomas, and in four of five papillary carcinomas. The expression was restricted to fibroblasts in the stroma adjacent or close to invading tumor cells. Twelve of the tumors were also investigated for expression of stromelysin 3 mRNA, no expression of which was detected in any tumor. The findings suggest that gelatinase A contributes to the invasive process and spread of aggressive thyroid tumors.

Awake rats were bled 2.5 per cent of their body weight during 15 min. This caused a 50-fold increase of plasma adrenaline and a 15-fold increase of plasma noradrenaline after 90 min. Treatment with naloxone or morphine did not significantly affect blood pressure or plasma catecholamine levels. The results suggest that the action of naloxone in hemorrhagic hypotension is not mediated via the sympatho-adrenal system.