Ulla Enberg

Gelatinase A, Membrane Type 1 Matrix Metalloproteinase, and Extracellular Matrix Metalloproteinase Inducer mRNA Expression: Correlation with Invasive Growth of Breast Cancer

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). 35S-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.

CREB3L2-PPARγ Fusion Mutation Identifies a Thyroid Signaling Pathway Regulated by Intramembrane Proteolysis

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-PPARgamma fusion mutation in thyroid carcinoma with t(3;7)(p25;q34), showing that a family of somatic PPARgamma fusion mutations exist in thyroid cancer. The CREB3L2-PPARgamma fusion encodes a CREB3L2-PPARgamma fusion protein that is composed of the transactivation domain of CREB3L2 and all functional domains of PPARgamma1. CREB3L2-PPARgamma was detected in <3% of thyroid follicular carcinomas. Engineered overexpression of CREB3L2-PPARgamma 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-PPARgamma inhibited transcription 6-fold from EVX1 in the same experiments. CREB3L2-PPARgamma 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-PPARgamma gene fusion mutation in thyroid carcinoma and reveal a thyroid signaling pathway that is regulated by intramembrane proteolysis and disrupted in cancer.

TRANSCRIPTIONAL PROFILING ENABLES MOLECULAR CLASSIFICATION OF ADRENOCORTICAL TUMOURS

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.

Stromal fibroblasts adjacent to invasive thyroid tumors: expression of gelatinase A but not stromelysin 3 mRNA.

Abstract. 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.

In vitro release of aldosterone and cortisol in human adrenal adenomas correlates to mRNA expression of steroidogenic enzymes for genes CYP11B2 and CYP17

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.RésuméLe tableau clínique des adénomes de la corticosurrénale varie selon le type dominant de stéroïde synthétisé et sécrété. Le but de cet article a été d’améliorer la compréhension de la physiopathogenèse des stéroïdes dans les maladies de la surrénale en comparant la sécrétion des stéroïdes à partir des adénomes de la corticosurrénale in vitro avec l’expression mRNA des enzymes de synthèse stéroídien. Quatorze patients ayant une tumeur de la surrénale ont été inclus dans cette étude. On a retenu le diagnostic d’hyperaldostéronisme primitif chez neuf alors que trois patients avaient un syndrome de Cushing. Deux patients avaient une tumeur de la surrénale découverte par la tomodensitométrie réalisée pour une pathologie sans rapport. Les taux sériques de cortisol, d’aldostérone et de catécholamines urinaires étaient normaux. On a déterminé la sécrétion de stéroïdes, d’aldostérone et de cortisol in vitro après une heure d’incubation du tissu prélevé. Des sondes d’oligonucléotide avec des séquences complémentaires à l’encodage mRNAs pour les enzymes de synthèse 11-beta-hydroxylase (CYP11B1), 18-hydroxylase (CYP11B2), 17 alpha-hydroxylase (CYP17) et 21-hydroxylase (CYP21) ont été synthétisés (Genset, Paris, France) et une hybridation in situ a été réalisée. On a trouvé une expression modérée de CYP11B2 et une expression basse de CYP11B1 dans la zone glomérulaire. La zone fasciculaire des surrénales de contróle a exprimé un signal intense de CYP11B1 alors que l’expression de CYP11B2 était basse. Il y avait une forte variation en ce qui concernait la sécrétion d’aldostérone à partir des aldostéronomes alors que les tumeurs des patients ayant un syndrome de Cushing ne sécrétaient pas d’aldostérone. En revanche, les tumeurs provenant des patients ayant un hyperaldostéronisme primitif, un syndrome de Cushing, sans hyperfonctionnement étaient toutes capables de synthétiser et de sécréter le cortisol in vitro. Le taux le plus élevé de cortisol a été retrouvé chez les patients ayant un syndrome de Cushing, mais également chez les patients porteurs de tumeurs non fonctionnelles et chez quelques patients ayant un aldostéronome. Les deux patients ayant le taux le plus élevé d’aldostérone in vitro avaient également la plus forte expression de CYP11B2, alors que celles de CYP11B1 et de CYP17 étaient la plus faible. Les autres patients porteurs d’aldostérome avaient une expression réduite de CYP11B2, similaire aux deux autres groupes. L’expression de CYP11B1 était élevé, comme attendue dans le syndrome de Cushing par adénome, mais également chez les deux tumeurs non hyperfonctionelles, alors que l’expression était modérée chez les patients ayant un aldostéronome. L’expression de CYP 17 était modérée à élevée chez les patients ayant un adénome avec syndrome de Cushing, un adénome non-hyperfonctionnel et dans le cas de quelques adénomes produisant de l’aldostérone. Dans cet article, on présente de nouvelles modalités pour caractériser la fonction des tumeurs de la corticosurrénale. Le diagnostic d’un aldostéronome est souvent difficile mais avec ces nouvelles modalités diagnostiques, il est possible désormais de connatre la capacité fonctionnelle de la tumeur, une fois enlevée. Ceci a un intérét particulier si le patient reste hypertendu en postopératoire lorsqu’ on ne savait pas en préopératoire si la tumeur était fonctionnelle.ResumenLos adenomas de la corteza suprarrenal ocasionan diferentes alteraciones clínicas según el principal esteroide que sintetizan y secretan. El propósito del présente trabajo fue incrementar el conocimiento sobre la fisiopatología de la estereidogénesis en las enfermedades adrenocorticoles comparando la liberation in vitro de esteroides por adenomas adrenocorticales con la expresión de mRNA de las enzimas sintetizadoras de esteroides. Catorce pacientes con tumores suprarrenales fueron incluidos en el estudio, nueve con el diagnóstico de aldosteronismo primario y très con síndrome de Cushing. En dos se encontró un tumor suprarrenal en tomografia computadorizada en el curso del estudio de una enfermedad no relacionada. El cortisol sérico, el nivel plasmático de aldosterona y las catecolaminas urinarias aparecieron dentro de límites normales. Se tomaron tejidos para la determination in vitro de liberación de esteriodes y de la presencia de aldosterona y cortisol en el medio luego de una hora de incubation. Se observé expresión moderada de CYP11B2 y baja expresion de CYP11B1 en la zona glomerulosaa. La zona fasciculata de las suprarrenales de control expresaron una elevada señal de CYP11B1, en tanto que la expresión de CYP11B2 apareció muy baja. Se observó una variación considerable en la liberación de aldosterona por los aldosteronomas, en tanto que los tumores de los pacientes con Cushing no demostraron liberación de aldosterona. En contraste, los tumores de pacientes con aldosteronismo primario, síndrome de Cushing y aquellos sin hiperfunción, todos exhibieron capacidad para sintetizar y liberar cortisol in vitro. La más alta liberación de cortisol fue hallada en tumores de pacientes con síndrome de Cushing, pero los tumores no hiperfuncionantes y algunos de los aldosteronomas mostraron liberación significante de cortisol. Los dos pacientes con la más alta liberación de aldosterona in vitro mostraron también la más elevada expresión de CYP11B2 y la más baja de CYP11B1 y CYP17. Los restantes aldosteronomas mostraron baja expresión de CYP11B2, similar a la de los otros dos grupos. La expresión de CYP11B1 apareció elevada, como era lo esperado, en los adenomas de Cushing, pero también en los tumores no hiperfuncionantes; algunos de los aldosteronomas exhibieron expresión moderada. Los adenomas del síndrome de Cushing, lo adenomas no hiperfuncionantes y algunos de los adenomas productores de aldosterona exhibieron expresión moderada a alta de CYP17. El présente artículo muestra nuevas maneras de hacer la caracterización funcional de los tumores suprarrenales. El diagnóstico de aldosteronoma frecuentemente es difícil y con el advenimiento de estos métodos es posible determinar la capacidad funcional del tumor que ha sido resecado. Esto es de interés especial cuando el patiente se mantiene hipertenso en el postoperatorio y no aparece claro si realmente tenía un tumor funcional.

Gelatinase A and Membrane-type 1 Matrix Metalloproteinase mRNA: Expressed in Adrenocortical Cancers but Not in Adenomas

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.

New Aspects on Primary Aldosteronism

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 Cushings 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.

Malignant Fibrous Histiocytoma, Aggressive Fibromatosis and Benign Fibrous Tumors Express mRNA for the Metalloproteinase Inducer EMMPRIN and the Metalloproteinases MMP-2 and MT1-MMP.

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.