Maria Nesterova

INVOLVEMENT OF MICROTUBULES IN THE REGULATION OF BCL2 PHOSPHORYLATION AND APOPTOSIS THROUGH CYCLIC AMP-DEPENDENT PROTEIN KINASE

ABSTRACT The Bcl2 family of proteins plays a significant role in regulation of apoptosis. In this study, the microtubule-damaging drugs paclitaxel, vincristine, and vinblastine induced Bcl2 hyperphosphorylation and apoptosis in MCF-7 and MDA-MB-231 cells and reduced Bcl2-Bax dimerization. Paclitaxel or vincristine induced increased expression of Bax, while overexpression of Bcl2 in these cell lines counteracted the effects of low doses of these drugs. In addition, paclitaxel- and vincristine-induced activation of cyclic AMP (cAMP)-dependent protein kinase (protein kinase A [PKA]) induced Bcl2 hyperphosphorylation and apoptosis, which were blocked by the PKA inhibitor Rp diastereomers of cAMP (Rp-cAMP). This finding suggests that activation of PKA due to microtubule damage is an important event in Bcl2 hyperphosphorylation and induction of apoptosis. These microtubule-damaging drugs caused growth arrest in G2-M phase of the cell cycle and had no effect on p53 induction, suggesting that hyperphosphorylation mediated inactivation of Bcl2 and apoptosis without the involvement of p53. By comparison, the DNA-damaging drugs methotrexate and doxorubicin had no effect on Bcl2 hyperphosphorylation but induced p53 expression. Interestingly, paclitaxel or vincristine induced activation of caspase 3 and cleavage of poly(ADP-ribose) polymerase downstream of Bcl2 hyperphosphorylation. These data suggest that there may be a signaling cascade induced by agents that disrupt or damage the cytoskeleton that is distinct from (i.e., p53 independent), but perhaps related to (i.e., involves kinase activation and leads to apoptosis), the cellular response to DNA damage.

A single-injection protein kinase A-directed antisense treatment to inhibit tumour growth.

Expression of the RIα subunit of cAMP-dependent protein kinase type I is enhanced in human cancer cell lines, in primary tumours, in cells after transformation and in cells upon stimulation of growth. We have investigated the effect of sequence-specific inhibition of RIα gene expression on in vivo tumour growth. We report that single injection RIα antisense treatment results in a reduction in RIα expression and inhibition of tumour growth. Tumour cells behaved like untransformed cells by making less protein kinase type I. The RIα antisense, which produces a biochemical imprint for growth control, requires infrequent dosing to halt neoplastic growth in vivo.

The role of germline AIP, MEN1, PRKAR1A, CDKN1B and CDKN2C mutations in causing pituitary adenomas in a large cohort of children, adolescents, and patients with genetic syndromes.

Stratakis CA, Tichomirowa MA, Boikos S, Azevedo MF, Lodish M, Martari M, Verma S, Daly AF, Raygada M, Keil MF, Papademetriou J, Drori‐Herishanu L, Horvath A, Tsang KM, Nesterova M, Franklin S, Vanbellinghen J‐F, Bours V, Salvatori R, Beckers A. The role of germline AIP, MEN1, PRKAR1A, CDKN1B and CDKN2C mutations in causing pituitary adenomas in a large cohort of children, adolescents, and patients with genetic syndromes.

Dual Blockade of Cyclic AMP Response Element- (CRE) and AP-1-directed Transcription by CRE-transcription Factor Decoy Oligonucleotide GENE-SPECIFIC INHIBITION OF TUMOR GROWTH

Alteration of gene transcription by inhibition of specific transcriptional regulatory proteins has important therapeutic potential. Synthetic double-stranded phosphorothioate oligonucleotides with high affinity for a target transcription factor can be introduced into cells as decoy cis-elements to bind the factors and alter gene expression. The CRE (cyclic AMP response element)-transcription factor complex is a pleiotropic activator that participates in the induction of a wide variety of cellular and viral genes. Because the CRE cis-element, TGACGTCA, is palindromic, a synthetic single-stranded oligonucleotide composed of the CRE sequence self-hybridizes to form a duplex/hairpin. Herein we report that the CRE-palindromic oligonucleotide can penetrate into cells, compete with CRE enhancers for binding transcription factors, and specifically interfere with CRE- and AP-1-directed transcriptionin vivo. These oligonucleotides restrained tumor cell proliferation, without affecting the growth of noncancerous cells. This decoy oligonucleotide approach offers great promise as a tool for defining cellular regulatory processes and treating cancer and other diseases.

Clinical and Genetic Heterogeneity, Overlap with Other Tumor Syndromes, and Atypical Glucocorticoid Hormone Secretion in Adrenocorticotropin-Independent Macronodular Adrenal Hyperplasia Compared with Other Adrenocortical Tumors

OBJECTIVE ACTH-independent macronodular adrenal hyperplasia (AIMAH) is often associated with subclinical cortisol secretion or atypical Cushings syndrome (CS). We characterized a large series of patients of AIMAH and compared them with patients with other adrenocortical tumors. DESIGN AND PATIENTS We recruited 82 subjects with: 1) AIMAH (n = 16); 2) adrenocortical cortisol-producing adenoma with CS (n = 15); 3) aldosterone-producing adenoma (n = 19); and 4) single adenomas with clinically nonsignificant cortisol secretion (n = 32). METHODS Urinary free cortisol (UFC) and 17-hydroxycorticosteroid (17OHS) were collected at baseline and during dexamethasone testing; aberrant receptor responses was also sought by clinical testing and confirmed molecularly. Peripheral and/or tumor DNA was sequenced for candidate genes. RESULTS AIMAH patients had the highest 17OHS excretion, even when UFCs were within or close to the normal range. Aberrant receptor expression was highly prevalent. Histology showed at least two subtypes of AIMAH. For three patients with AIMAH, there was family history of CS; germline mutations were identified in three other patients in the genes for menin (one), fumarate hydratase (one), and adenomatosis polyposis coli (APC) (one); a PDE11A gene variant was found in another. One patient had a GNAS mutation in adrenal nodules only. There were no mutations in any of the tested genes in the patients of the other groups. CONCLUSIONS AIMAH is a clinically and genetically heterogeneous disorder that can be associated with various genetic defects and aberrant hormone receptors. It is frequently associated with atypical CS and increased 17OHS; UFCs and other measures of adrenocortical activity can be misleadingly normal.

Succinate Dehydrogenase (SDH) D Subunit (SDHD) Inactivation in a Growth-Hormone-Producing Pituitary Tumor: A New Association for SDH?

BACKGROUND Mutations in the subunits B, C, and D of succinate dehydrogenase (SDH) mitochondrial complex II have been associated with the development of paragangliomas (PGL), gastrointestinal stromal tumors, papillary thyroid and renal carcinoma (SDHB), and testicular seminoma (SDHD). AIM Our aim was to examine the possible causative link between SDHD inactivation and somatotropinoma. PATIENTS AND METHODS A 37-yr-old male presented with acromegaly and hypertension. Other family members were found with PGL. Elevated plasma and urinary levels of catecholamines led to the identification of multiple PGL in the proband in the neck, thorax, and abdomen. Adrenalectomy was performed for bilateral pheochromocytomas (PHEO). A GH-secreting macroadenoma was also found and partially removed via transsphenoidal surgery (TTS). Genetic analysis revealed a novel SDHD mutation (c.298_301delACTC), leading to a frame shift and a premature stop codon at position 133 of the protein. Loss of heterozygosity for the SDHD genetic locus was shown in the GH-secreting adenoma. Down-regulation of SDHD protein in the GH-secreting adenoma by immunoblotting and immunohistochemistry was found. A literature search identified other cases of multiple PGL and/or PHEO in association with pituitary tumors. CONCLUSION We describe the first kindred with a germline SDHD pathogenic mutation, inherited PGL, and acromegaly due to a GH-producing pituitary adenoma. SDHD loss of heterozygosity, down-regulation of protein in the GH-secreting adenoma, and decreased SDH enzymatic activity supports SDHDs involvement in the pituitary tumor formation in this patient. Older cases of multiple PGL and PHEO and pituitary tumors in the literature support a possible association between SDH defects and pituitary tumorigenesis.

Large Deletions of the PRKAR1A Gene in Carney Complex

Purpose: Since the identification of PRKAR1A mutations in Carney complex, substitutions and small insertions/deletions have been found in ∼70% of the patients. To date, no germ-line PRKAR1A deletion and/or insertion exceeded a few base pairs (up to 15). Although a few families map to chromosome 2, it is possible that current sequencing techniques do not detect larger gene changes in PRKAR1A–mutation-negative individuals with Carney complex. Experimental Design: To screen for gross alterations of the PRKAR1A gene, we applied Southern hybridization analysis on 36 unrelated Carney complex patients who did not have small intragenic mutations or large aberrations in PRKAR1A, including the probands from two kindreds mapping to chromosome 2. Results: We found large PRKAR1A deletions in the germ-line of two patients with Carney complex, both sporadic cases; no changes were identified in the remaining patients, including the two chromosome-2-mapping families. In the first patient, the deletion is expected to lead to decreased PRKAR1A mRNA levels but no other effects on the protein; the molecular phenotype is predicted to be PRKAR1A haploinsufficiency, consistent with the majority of PRKAR1A mutations causing Carney complex. In the second patient, the deletion led to in-frame elimination of exon 3 and the expression of a shorter protein, lacking the primary site for interaction with the catalytic protein kinase A subunit. In vitro transfection studies of the mutant PRKAR1A showed impaired ability to bind cyclic AMP and activation of the protein kinase A enzyme. The patient bearing this mutation had a more-severe-than-average Carney complex phenotype that included the relatively rare psammomatous melanotic schwannoma. Conclusions: Large PRKAR1A deletions may be responsible for Carney complex in patients that do not have PRKAR1A gene defects identifiable by sequencing. Preliminary data indicate that these patients may have a different phenotype especially if their defect results in an expressed, abnormal version of the PRKAR1A protein.

A cAMP-specific phosphodiesterase ( PDE8B ) that is mutated in adrenal hyperplasia is expressed widely in human and mouse tissues: a novel PDE8B isoform in human adrenal cortex

Bilateral adrenocortical hyperplasia (BAH) is the second most common cause of corticotropin-independent Cushing syndrome (CS). Genetic forms of BAH have been associated with complex syndromes such as Carney Complex and McCune–Albright syndrome or may present as isolated micronodular adrenocortical disease (iMAD) usually in children and young adults with CS. A genome-wide association study identified inactivating phosphodiesterase (PDE) 11A (PDE11A)-sequencing defects as low-penetrance predisposing factors for iMAD and related abnormalities; we also described a mutation (c.914A>C/H305P) in cyclic AMP (cAMP)-specific PDE8B, in a patient with iMAD. In this study we further characterize this mutation; we also found a novel PDE8B isoform that is highly expressed in the adrenal gland. This mutation is shown to significantly affect the ability of the protein to degrade cAMP in vitro. Tumor tissues from patients with iMAD and no mutations in the coding PDE8B sequence or any other related genes (PRKAR1A, PDE11A) showed downregulated PDE8B expression (compared to normal adrenal cortex). Pde8b is detectable in the adrenal gland of newborn mice and is widely expressed in other mouse tissues. We conclude that PDE8B is another PDE gene linked to iMAD; it is a candidate causative gene for other adrenocortical lesions linked to the cAMP signaling pathway and possibly for tumors in other tissues.

Frequent Phosphodiesterase 11A Gene (PDE11A) Defects in Patients with Carney Complex (CNC) Caused by PRKAR1A Mutations: PDE11A May Contribute to Adrenal and Testicular Tumors in CNC as a Modifier of the Phenotype

BACKGROUND Carney complex (CNC) is an autosomal dominant multiple neoplasia, caused mostly by inactivating mutations of the regulatory subunit 1A of the protein kinase A (PRKAR1A). Primary pigmented nodular adrenocortical disease (PPNAD) is the most frequent endocrine manifestation of CNC with a great inter-individual variability. Germline, protein-truncating mutations of phosphodiesterase type 11A (PDE11A) have been described to predispose to a variety of endocrine tumors, including adrenal and testicular tumors. OBJECTIVES Our objective was to investigate the role of PDE11A as a possible gene modifier of the phenotype in a series of 150 patients with CNC. RESULTS A higher frequency of PDE11A variants in patients with CNC compared with healthy controls was found (25.3 vs. 6.8%, P < 0.0001). Among CNC patients, those with PPNAD were significantly more frequently carriers of PDE11A variants compared with patients without PPNAD (30.8 vs. 13%, P = 0.025). Furthermore, men with PPNAD were significantly more frequently carriers of PDE11A sequence variants (40.7%) than women with PPNAD (27.3%) (P < 0.001). A higher frequency of PDE11A sequence variants was also found in patients with large-cell calcifying Sertoli cell tumors (LCCSCT) compared with those without LCCSCT (50 vs. 10%, P = 0.0056). PDE11A variants were significantly associated with the copresence of PPNAD and LCCSCT in men: 81 vs. 20%, P < 0.004). The simultaneous inactivation of PRKAR1A and PDE11A by small inhibitory RNA led to an increase in cAMP-regulatory element-mediated transcriptional activity under basal conditions and after stimulation by forskolin. CONCLUSIONS We demonstrate, in a large cohort of CNC patients, a high frequency of PDE11A variants, suggesting that PDE11A is a genetic modifying factor for the development of testicular and adrenal tumors in patients with germline PRKAR1A mutation.

Functional Phosphodiesterase 11A Mutations May Modify the Risk of Familial and Bilateral Testicular Germ Cell Tumors

Inactivating germline mutations in phosphodiesterase 11A (PDE11A) have been implicated in adrenal tumor susceptibility. PDE11A is highly expressed in endocrine steroidogenic tissues, especially the testis, and mice with inactivated Pde11a exhibit male infertility, a known testicular germ cell tumor (TGCT) risk factor. We sequenced the PDE11A gene-coding region in 95 patients with TGCT from 64 unrelated kindreds. We identified 8 nonsynonymous substitutions in 20 patients from 15 families: four (R52T, F258Y, G291R, and V820M) were newly recognized, three (R804H, R867G, and M878V) were functional variants previously implicated in adrenal tumor predisposition, and one (Y727C) was a known polymorphism. We compared the frequency of these variants in our patients to unrelated controls that had been screened and found negative for any endocrine diseases: only the two previously reported variants, R804H and R867G, known to be frequent in general population, were detected in these controls. The frequency of all PDE11A-gene variants (combined) was significantly higher among patients with TGCT (P = 0.0002), present in 19% of the families of our cohort. Most variants were detected in the general population, but functional studies showed that all these mutations reduced PDE activity, and that PDE11A protein expression was decreased (or absent) in TGCT samples from carriers. This is the first demonstration of the involvement of a PDE gene in TGCT, although the cyclic AMP signaling pathway has been investigated extensively in reproductive organ function and their diseases. In conclusion, we report that PDE11A-inactivating sequence variants may modify the risk of familial and bilateral TGCT.