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Lawrence S Kirschner

Publications and source records attributed to Lawrence S Kirschner.

13 recordsLinked to original sources
PubMed2025

In vivo and in vitro analysis of functional effects of the SDHD H50R variant.

Germline mutations in the four genes (SDHA, SDHB, SDHC and SDHD) encoding the succinate dehydrogenase (SDH) holoenzyme are known to predispose towards the development of tumor including pheochromocytomas/paragangliomas (PPGLs), gastrointestinal stromal tumors (GISTs), clear cell renal cancers (RCC) and possibly others. Mutations in these genes have also been described in patients with Cowden syndrome, which includes tumors of the breast, brain and thyroid gland. Although nonsense mutations are clearly pathogenic, the functional consequences of many missense mutations are unclear. It has previously been reported that the missense mutations SDHDG12S and SDHDH50R predispose to thyroid and breast cancers, although this characterization has been disputed. To address this question, we developed mouse models to test tumorigenicity of these variants. The reference mouse genome codes for a serine at residue 12 in Sdhd, so this variant was not pursued further. To assess the role of SDHDH50R (H50R), we generated a knock-in mouse allele for this variant and studied its effects in vivo as well as in vitro in mouse embryonic fibroblasts. Unlike null alleles for Sdhd, the H50R allele did not produce embryonic lethality when homozygous. There was no statistically significant difference in survival or tumor formation in homozygous or heterozygous animals compared to littermate controls. In vitro studies similarly failed to detect significant differences in proliferation, colony formation or metabolic function. Based on our analysis of this allele's function both in vivo and in vitro, we conclude that the SDHDH50R allele is most likely a non-pathogenic polymorphism.

Animals↗
PubMed2005

Disruption of protein kinase a regulation causes immortalization and dysregulation of D-type cyclins.

Phosphorylation is a key event in cell cycle control, and dysregulation of this process is observed in many tumors, including those associated with specific inherited neoplasia syndromes. We have shown previously that patients with the autosomal dominant tumor predisposition Carney complex carry inactivating mutations in the PRKAR1A gene, which encodes the type 1A regulatory subunit of protein kinase A (PKA), the cyclic AMP-dependent protein kinase. This defect was associated with dysregulation of PKA signaling, and genetic analysis has suggested that complete loss of the gene may be required for tumorigenesis. To determine the mechanism by which dysregulation of PKA causes tumor formation, we generated in vitro primary mouse cells lacking the Prkar1a protein. We report that this genetic disruption of PKA regulation causes constitutive PKA activation and immortalization of primary mouse embryonic fibroblasts (MEFs). At the molecular level, knockout of Prkar1a leads to up-regulation of D-type cyclins, and this increase occurs independently of other pathways known to increase cyclin D levels. Despite the immortalized phenotype, known mediators of cellular senescence (e.g., p53 and p19ARF) seem to remain intact in Prkar1a-/- MEFs. Mechanistically, cyclin D1 mRNA levels are not altered in the knockout cells, but protein half-life is markedly increased. Using this model, we provide the first direct genetic evidence that dysregulation of PKA promotes important steps in tumorigenesis, and that cyclin D1 is an essential target of PKA.

Animals↗
PubMed2005

Emerging treatment strategies for adrenocortical carcinoma: a new hope.

CONTEXT: Adrenocortical carcinoma (ACC) is a rare cancer but one that has devastating consequences for affected patients. Surgery is the mainstay of therapy, although the high frequency of metastatic disease implies that it is frequently noncurative. Traditional cytotoxic chemotherapy for ACC has generally produced disappointing responses, implying the need for the new therapies for this disease. EVIDENCE ACQUISITION: Review articles and primary literature were identified by extensive PubMed searching to obtain papers evaluating the current state of knowledge regarding ACC, as well as assessing the development of new therapeutic modalities for the treatment of cancer. When needed, additional articles were identified from the reference lists of the papers obtained from the primary screen. EVIDENCE SYNTHESIS: Multiple new modalities that may enhance the future treatment of ACC were identified. They include the following: combating drug resistance, targeting tumor vasculature, inhibiting signaling pathways with small molecules, and using gene and/or immunotherapy. This review provides a brief summary of the progress and prospects of each of these modalities and focuses on emerging data and treatments that may alter the course of this disease within the next few years. CONCLUSIONS: Despite the current grim outlook, the recent applications of emerging technology to the study of ACC and the development of newer, "targeted" therapies for cancer suggest the possibility of a new hope for patients with this disease, although these therapies will need to be evaluated by rigorous clinical trials to verify their effectiveness.

Adrenal Cortex Neoplasms↗
PubMed2005

A mouse model for the Carney complex tumor syndrome develops neoplasia in cyclic AMP-responsive tissues.

Carney complex is an autosomal dominant neoplasia syndrome characterized by spotty skin pigmentation, myxomatosis, endocrine tumors, and schwannomas. This condition may be caused by inactivating mutations in PRKAR1A, the gene encoding the type 1A regulatory subunit of protein kinase A. To better understand the mechanism by which PRKAR1A mutations cause disease, we have developed conventional and conditional null alleles for Prkar1a in the mouse. Prkar1a(+/-) mice developed nonpigmented schwannomas and fibro-osseous bone lesions beginning at approximately 6 months of age. Although genotype-specific cardiac and adrenal lesions were not seen, benign and malignant thyroid neoplasias were observed in older mice. This spectrum of tumors overlaps that seen in Carney complex patients, confirming the validity of this mouse model. Genetic analysis indicated that allelic loss occurred in a subset of tumor cells, suggesting that complete loss of Prkar1a plays a key role in tumorigenesis. Similarly, tissue-specific ablation of Prkar1a from a subset of facial neural crest cells caused the formation of schwannomas with divergent differentiation. These observations confirm the identity of PRKAR1A as a tumor suppressor gene with specific importance to cyclic AMP-responsive tissues and suggest that these mice may be valuable tools not only for understanding endocrine tumorigenesis but also for understanding inherited predispositions for schwannoma formation.

Alleles↗
PubMed2004

Down-regulation of regulatory subunit type 1A of protein kinase A leads to endocrine and other tumors.

Mutations of the human type Ialpha regulatory subunit (RIalpha) of cyclic AMP-dependent protein kinase (PKA; PRKAR1A) lead to altered kinase activity, primary pigmented nodular adrenocortical disease, and tumors of the thyroid and other tissues. To bypass the early embryonic lethality of Prkar1a(-/-) mice, we established transgenic mice carrying an antisense transgene for Prkar1a exon 2 (X2AS) under the control of a tetracycline-responsive promoter. Down-regulation of Prkar1a by up to 70% was achieved in transgenic mouse tissues and embryonic fibroblasts, with concomitant changes in kinase activity and increased cell proliferation, respectively. Mice developed thyroid follicular hyperplasia and adenomas, adrenocortical hyperplasia, and other features reminiscent of primary pigmented nodular adrenocortical disease, histiocytic and epithelial hyperplasias, lymphomas, and other mesenchymal tumors. These were associated with allelic losses of the mouse chromosome 11 Prkar1a locus, an increase in total type II PKA activity, and higher RIIbeta protein levels. This mouse provides a novel, useful tool for the investigation of cyclic AMP, RIalpha, and PKA functions and confirms the critical role of Prkar1a in tumorigenesis in endocrine and other tissues.

Adenoma↗
PubMed2004

Gene array analysis of macronodular adrenal hyperplasia confirms clinical heterogeneity and identifies several candidate genes as molecular mediators.

Corticotropin (ACTH)-independent macronodular adrenal hyperplasia (AIMAH) is a heterogeneous condition in which cortisol secretion may be mediated by gastrointestinal peptide (GIP), vasopressin, catecholamines and other hormones. We studied the expression profile of AIMAH by genomic cDNA microarray analysis. Total RNA was extracted from eight tissues (three GIP-dependent) and compared to total RNA obtained from adrenal glands from 62 normal subjects. Genes had to be altered in 75% of the patients, and be up- or downregulated at a cutoff ratio of at least 2.0; 82 and 31 genes were found to be consistently up- and downregulated, respectively. Among the former were regulators of transcription, chromatin remodeling, and cell cycle and adhesion. Downregulated sequences included genes involved in immune responses and insulin signaling. Hierarchical clustering correlated with the two main AIMAH diagnostic groups: GIP-dependent and non-GIP-dependent. The genes encoding the 7B2 protein (SGNE1) and WNT1-inducible signaling pathway protein 2 (WISP2) were specifically overexpressed in the GIP-dependent AIMAH. For these, and six more genes, the data were validated by semiquantitative amplification in samples from a total of 32 patients (the original eight, six more cases of AIMAH, and 18 other adrenocortical hyperplasias and tumors) and the H295R adrenocortical cancer cell line. In conclusion, our data confirmed AIMAH's clinical heterogeneity by identifying molecularly distinct diagnostic subgroups. Several candidate genes that may be responsible for AIMAH formation and/or progression were also identified, suggesting pathways that affect the cell cycle, adhesion and transcription as possible mediators of adrenocortical hyperplasia.

Adrenal Cortex Neoplasms↗
PubMed2004

A mouse model for Carney complex.

Mice with complete inactivation of the type Ialpha regulatory subunit (RIalpha) of cyclic (c) AMP-dependent protein kinase (PKA) (coded by the Prkar1a gene) die early in embryonic life. To bypass the early embryonic lethality of Prkar1a-/- mice, we established transgenic mice carrying an antisense transgene for Prkar1a exon 2 (X2AS) under the control of a tetracycline-responsive promoter. Mice developed thyroid follicular hyperplasia and adenomas, adrenocortical hyperplasia, and other features reminiscent of PPNAD, and histiocytic and epithelial hyperplasias, lymphomas, and other mesenchymal tumors. This mouse provides a useful tool for the investigation of cAMP, RIalpha, and PKA functions and confirms Prkar1a's critical role in tumorigenesis in endocrine and other tissues.

Animals↗
PubMed2003

Molecular and functional analysis of PRKAR1A and its locus (17q22-24) in sporadic adrenocortical tumors: 17q losses, somatic mutations, and protein kinase A expression and activity.

Germ-line protein kinase A (PKA) regulatory-subunit type-Ialpha (RIalpha; PRKAR1A)-inactivating mutations and loss-of-heterozygosity (LOH) of its 17q22-24 locus have been found in Cushing syndrome (CS) caused by primary pigmented nodular adrenocortical disease (PPNAD). We examined whether somatic 17q22-24, PRKAR1A, or PKA changes are present in 44 sporadic adrenocortical tumors (29 adenomas and 15 cancers); 26 of these tumors were responsible for CS. A probe containing the PRKAR1A gene-mapped by fluorescent in situ hybridization to 17q22-24-and corresponding microsatellite markers were used to study allelic losses; PRKAR1A was sequenced in all samples. 17q22-24 losses were seen in 23 and 53% of adenomas and cancers, respectively. In three tumors, somatic, PRKAR1A-inactivating mutations were identified: (a) a nonsense mutation in exon 6 (A751G); (b) a splicing mutation (9IVS-1G/A); and (c) a transition (1050T>C) followed by a 22-bp deletion, also in exon 9; all predicted premature RIalpha protein terminations. Quantitative message and protein studies showed RIalpha down-regulation in tumors with genetic changes; their cortisol secretion pattern was similar to that of PPNAD, and they had higher PKA activity by enzymatic studies. We conclude that somatic allelic losses of the 17q22-24 region, PRKAR1A-inactivating mutations or down-regulation, and corresponding PKA activity changes are present in at least some sporadic adrenocortical tumors, especially those with a PPNAD-like clinical presentation of CS.

Adrenal Cortex Neoplasms↗
PubMed2002

Molecular analysis of the cyclic AMP-dependent protein kinase A (PKA) regulatory subunit 1A (PRKAR1A) gene in patients with Carney complex and primary pigmented nodular adrenocortical disease (PPNAD) reveals novel mutations and clues for pathophysiology: augmented PKA signaling is associated with adrenal tumorigenesis in PPNAD.

We studied 11 new kindreds with primary pigmented nodular adrenocortical disease (PPNAD) or Carney complex (CNC) and found that 82% of the kindreds had PRKAR1A gene defects (including seven novel inactivating mutations), most of which led to nonsense mRNA and, thus, were not expressed in patients' cells. However, a previously undescribed base substitution in intron 6 (exon 6 IVS +1G-->T) led to exon 6 skipping and an expressed shorter PRKAR1A protein. The mutant protein was present in patients' leukocytes and tumors, and in vitro studies indicated that the mutant PRKAR1A activated cAMP-dependent protein kinase A (PKA) signaling at the nuclear level. This is the first demonstration of an inactivating PRKAR1A mutation being expressed at the protein level and leading to stimulation of the PKA pathway in CNC patients. Along with the lack of allelic loss at the PRKAR1A locus in most of the tumors from this kindred, these data suggest that alteration of PRKAR1A function (not only its complete loss) is sufficient for augmenting PKA activity leading to tumorigenesis in tissues affected by CNC.

Adenocarcinoma↗
PubMed2002

Regulatory subunit type I-alpha of protein kinase A (PRKAR1A): a tumor-suppressor gene for sporadic thyroid cancer.

The tumor-suppressor gene encoding the cyclic AMP-dependent protein kinase A type I-alpha regulatory subunit PRKAR1A has been mapped to chromosome 17 (17q22-24) and is mutated in Carney complex, a familial neoplasia syndrome that is associated with thyroid tumors. Other genes implicated in cyclic nucleotide-dependent signaling have been investigated in thyroid tumorigenesis. We studied protein kinase A (PKA) activity in noninherited follicular thyroid adenomas and follicular, papillary, and undifferentiated (anaplastic) thyroid carcinomas. We then examined these and additional thyroid tumors for losses of the 17q22-24 PRKAR1A region, mutations of the PRKAR1A gene, and expression of its peptide product. Total PKA activity was markedly increased in carcinomas over that in adenomas, whereas the ratio of free vs. total PKA activity was decreased in cancer. Consistent with these findings, the 17q22-24 region was frequently lost in cancer but not in benign adenomas. A novel inactivating mutation of the PRKAR1A gene (leading to premature termination of the predicted protein) was found in an aggressive thyroid cancer. The tumor with PRKAR1A gene mutation, as well as the tumors with 17q allelic losses, showed decreased PRKAR1A expression by immunostaining. We conclude that PRKAR1A, the most abundant regulatory subunit of protein kinase A and a principal cyclic AMP-signaling modulator, acts as a tumor-suppressor gene in sporadic thyroid cancer. Published 2002 Wiley-Liss, Inc.

Adenoma↗
PubMed2002

Sequence analysis of the PRKAR1A gene in sporadic somatotroph and other pituitary tumours.

OBJECTIVE: Carney complex (CNC) is an autosomal dominant multiple neoplasia syndrome featuring cardiac, endocrine, cutaneous and neural tumours, as well as a variety of pigmented lesions of the skin and mucosa. Pituitary GH-secreting tumours are found in approximately 10% of patients with CNC. One of the genes responsible for CNC, the PRKAR1A gene located on human chromosome 17q22-24, has recently been cloned. This represents a putative tumour suppressor gene, coding for the type 1alpha regulatory subunit of protein kinase A (PKA), which is found to be mutated in approximately half of the patients with CNC. However, it is currently unclear as to whether similar mutations occur in sporadic pituitary tumours. We have therefore investigated a series of GH-secreting and other pituitary tumours for sequence abnormalities in the PRKAR1A gene. The mRNA produced by the PRKAR1A undergoes decay if it codes for a truncated protein; we therefore also determined PRKAR1A mRNA levels in the tumours, and compared them with known mutant PRKAR1A-carrying lymphocyte samples. METHODS: We extracted RNA from a series of pituitary tumours, reverse transcribed it to cDNA, and directly sequenced the PRKAR1A coding sequence in 17 GH-secreting, three prolactin-secreting, three ACTH-secreting, one FSH-secreting and 10 nonfunctioning pituitary tumours. Lymphocyte and tumour tissue RNA from two patients with CNC was used as positive controls. Using duplex polymerase chain reaction (PCR) with the PRKAR1A and the "housekeeping" gene GAPDH, we determined the relative expression of the PRKAR1A gene in the unknown as well as in the positive control samples. RESULTS AND CONCLUSION: No mutations were found in any of the exons sequenced. Relative mRNA expression was not decreased in any of the sporadic pituitary tumour samples. The present data thus do not suggest a major role for the PRKAR1A tumour suppressor gene in sporadic GH-secreting or other pituitary tumours.

Adenoma↗
PubMed2002

Signaling pathways in adrenocortical cancer.

Adrenocortical carcinoma is a rare tumor that carries a very poor prognosis. Despite efforts to develop new therapeutic regimens to treat this disease, surgery remains the mainstay of treatment. Laboratory studies of adrenocortical cancers have revealed a wide variety of signaling pathways that can be altered in these neoplasms. Although ACTH signaling through adenylyl cyclase and protein kinase A is important for normal adrenal cellular physiology, there is evidence to suggest that this pathway may inhibit the growth of adrenocortical tumors, and that inactivation of the ACTH receptor may promote tumor formation. Although multiple signal transduction pathways are essential for normal adrenal growth and hormone secretion, efforts to identify events required for neoplastic transformation have met with limited success. Alterations that have frequently been observed in adrenocortical carcinoma include up-regulation of the IGF-II system, as well as mutations in TP53 and RAS. Current studies aim to elucidate the mechanisms of tumor growth by studying proproliferative signaling pathways, such as those involving Akt/PKB and the mitogen-activated protein kinases (MAPKs). Although studies of single pathways have been helpful in guiding investigations, new tools to study the integration and multiplicity of signaling pathways hold the hope of improved understanding of the signaling pathway alterations in adrenocortical cancer.

Adrenal Cortex Neoplasms↗