Protein kinase A activation enhances ß-catenin transcriptional activity through nuclear localization to PML bodies.

The Protein Kinase A (PKA) and Wnt signaling cascades are fundamental pathways involved in cellular development and maintenance. In the osteoblast lineage, these pathways have been demonstrated functionally to be essential for the production of mineralized bone. Evidence for PKA-Wnt crosstalk has been reported both during tumorigenesis and during organogenesis, and the nature of the interaction is thought to rely on tissue and cell context. In this manuscript, we analyzed bone tumors arising from mice with activated PKA caused by mutation of the PKA regulatory subunit Prkar1a. In primary cells from these tumors, we observed relocalization of ß-catenin to intranuclear punctuate structures, which were identified as PML bodies. Cellular redistribution of ß-catenin could be recapitulated by pharmacologic activation of PKA. Using 3T3-E1 pre-osteoblasts as a model system, we found that PKA phosphorylation sites on ß-catenin were required for nuclear re-localization. Further, ß-catenin's transport to the nucleus was accompanied by an increase in canonical Wnt-dependent transcription, which also required the PKA sites. PKA-Wnt crosstalk in the cells was bi-directional, including enhanced interactions between ß-catenin and the cAMP-responsive element binding protein (CREB) and transcriptional crosstalk between the Wnt and PKA signaling pathways. Increases in canonical Wnt/ß-catenin signaling were associated with a decrease in the activity of the non-canonical Wnt/Ror2 pathway, which has been shown to antagonize canonical Wnt signaling. Taken together, this study provides a new understanding of the complex regulation of the subcellular distribution of ß-catenin and its differential protein-protein interaction that can be modulated by PKA signaling.

Follicular thyroid cancers demonstrate dual activation of PKA and mTOR as modeled by thyroid-specific deletion of Prkar1a and Pten in mice.

CONTEXT: Thyroid cancer is the most common form of endocrine cancer, and it is a disease whose incidence is rapidly rising. Well-differentiated epithelial thyroid cancer can be divided into papillary thyroid cancer (PTC) and follicular thyroid cancer (FTC). Although FTC is less common, patients with this condition have more frequent metastasis and a poorer prognosis than those with PTC. OBJECTIVE: The objective of this study was to characterize the molecular mechanisms contributing to the development and metastasis of FTC. DESIGN: We developed and characterized mice carrying thyroid-specific double knockout of the Prkar1a and Pten tumor suppressor genes and compared signaling alterations observed in the mouse FTC to the corresponding human tumors. SETTING: The study was conducted at an academic research laboratory. Human samples were obtained from academic hospitals. PATIENTS: Deidentified, formalin-fixed, paraffin-embedded (FFPE) samples were analyzed from 10 control thyroids, 30 PTC cases, five follicular variant PTC cases, and 10 FTC cases. INTERVENTIONS: There were no interventions. MAIN OUTCOME MEASURES: Mouse and patient samples were analyzed for expression of activated cAMP response element binding protein, AKT, ERK, and mammalian target of rapamycin (mTOR). Murine FTCs were analyzed for differential gene expression to identify genes associated with metastatic progression. RESULTS: Double Prkar1a-Pten thyroid knockout mice develop FTC and recapitulate the histology and metastatic phenotype of the human disease. Analysis of signaling pathways in FTC showed that both human and mouse tumors exhibited strong activation of protein kinase A and mTOR. The development of metastatic disease was associated with the overexpression of genes required for cell movement. CONCLUSIONS: These data imply that the protein kinase A and mTOR signaling cascades are important for the development of follicular thyroid carcinogenesis and may suggest new targets for therapeutic intervention. Mouse models paralleling the development of the stages of human FTC should provide important new tools for understanding the mechanisms of FTC development and progression and for evaluating new therapeutics.

Knockdown of PRKAR1A, the gene responsible for Carney complex, interferes with differentiation in osteoblastic cells.

PRKAR1A is the gene encoding the type 1A regulatory subunit of protein kinase A, and it is the cause of the inherited human tumor syndrome Carney complex. Data from our laboratory has demonstrated that Prkar1a loss causes tumors in multiple cell lineages, including neural crest cells and osteoblasts. We have proposed that one mechanism by which tumorigenesis occurs is through the failure of terminal differentiation. In the present study, we directly test the effects of Prkar1a reduction on osteogenic differentiation in mouse and human cells in vitro. We found that Prkar1a levels noticeably increased during osteoblastic differentiation, indicating a positive correlation between the expression of Prkar1a and osteogenic potential. To validate this hypothesis, we generated stable Prkar1a knockdown in both mouse and human cells. These cells displayed significantly suppressed bone nodule formation and decreased expression of osteoblast markers such as osteocalcin and osteopontin. These observations imply that the antiosteogenic effect of Prkar1a ablation is not species or cell line specific. Furthermore, because Runt-related transcription factor-2 (Runx2) is a key mediator of osteoblast differentiation, we reasoned that the function of this transcription factor may be inhibited by Prkar1a knockdown. Chromatin immunoprecipitation and luciferase assays demonstrated that Prkar1a ablation repressed DNA binding and function of Runx2 at its target genes. Additionally, we determined that this effect is likely due to reductions in the Runx2-cooperating transcription factors forkhead box O1 and activating transcription factor 4. Taken together, this study provides direct evidence that ablation of Prkar1a interferes with signaling pathways necessary for osteoblast differentiation.

RAC1: an emerging therapeutic option for targeting cancer angiogenesis and metastasis.

Angiogenesis and metastasis are well recognized as processes fundamental to the development of malignancy. Both processes involve the coordination of multiple cellular and chemical activities through myriad signaling networks, providing a mass of potential targets for therapeutic intervention. This review will focus on one master regulator of cell motility, RAC1, and the existing data with regard to its role in cell motility, including particular roles for tumor angiogenesis and invasion/metastasis. We also emphasize the preclinical investigations carried out with RAC1 inhibitors to evaluate the therapeutic potential of this target. Herein, we explore potential future directions as well as the challenges of targeting RAC1 in the treatment of cancer. Recent insights at the molecular and cellular levels are paving the way for a more directed and detailed approach to target mechanisms of RAC1 regulating angiogenesis and metastasis. Understanding these mechanisms may provide insight into RAC1 signaling components as alternative therapeutic targets for tumor angiogenesis and metastasis.

Thyroid-specific ablation of the Carney complex gene, PRKAR1A, results in hyperthyroidism and follicular thyroid cancer.

Thyroid cancer is the most common endocrine malignancy in the population, and the incidence of this cancer is increasing at a rapid rate. Although genetic analysis of papillary thyroid cancer (PTC) has identified mutations in a large percentage of patients, the genetic basis of follicular thyroid cancer (FTC) is less certain. Thyroid cancer, including both PTC and FTC, has been observed in patients with the inherited tumor predisposition Carney complex, caused by mutations in PRKAR1A. In order to investigate the role of loss of PRKAR1A in thyroid cancer, we generated a tissue-specific knockout of Prkar1a in the thyroid. We report that the resulting mice are hyperthyroid and developed follicular thyroid neoplasms by 1 year of age, including FTC in over 40% of animals. These thyroid tumors showed a signature of pathway activation different from that observed in other models of thyroid cancer. In vitro cultures of the tumor cells indicated that Prkar1a-null thyrocytes exhibited growth factor independence and suggested possible new therapeutic targets. Overall, this work represents the first report of a genetic mutation known to cause human FTC that exhibits a similar phenotype when modeled in the mouse. In addition to our knowledge of the mechanisms of human follicular thyroid tumorigenesis, this model is highly reproducible and may provide a viable mechanism for the further clinical development of therapies aimed at FTC.

Mouse models of endocrine tumours.

Since the onset of the genomic era, there has been tremendous progress in identifying the genetic causes of endocrine tumours. Although this knowledge is valuable in its own right, understanding the molecular basis of tumourigenesis allows the development of new therapies targeted at the causative defects. Understanding the connection between genotype and phenotype is a complex process, which can only be partially understood from the analysis of primary tumours or from the studies of cells in vitro. To bridge this gap, genetically modified mice have been developed to allow molecular dissection of the relevant defects in an intact organism. In this article, we discuss the status of genetic modelling for hereditary and sporadic endocrine tumourigenesis with a goal towards providing a view of how this technology will be of future benefit to clinicians developing specifically targeted therapies for endocrine tumours.

Influence of genetic polymorphisms in GSTM1, GSTM3, GSTT1 and GSTP1 on allograft outcome in renal transplant recipients.

INTRODUCTION: Glutathione S-transferases (GSTs) are important in protection against xenobiotic compounds and toxicity caused by immunosuppressants in renal transplant recipients. In the present study we hypothesize that genetic variability in GSTM1, GSTM3, GSTP1 and GSTT1 genes may be associated with allograft outcome. METHODS: The study included 223 controls and 273 transplant recipients categorized into 184 stable graft function (SGF), 57 rejection episodes (RE) and 32 delayed graft function (DGF). The polymorphism was studied using multiplex PCR and PCR-RFLP. RESULTS: GSTM1 null genotype showed a 3.35-fold higher risk for rejection in SGF vs. RE category [95% confidence interval (CI) 1.27-8.84, p = 0.014]. Mutant (G) allele of GSTP1 was associated with a 5.52-fold risk for DGF (95% CI 1.37-22.17, p = 0.016). Kaplan-Meier analysis revealed significantly lower mean time to first RE in null genotype as compared with GSTM1 present patients (Log p = 0.002). The dose adjusted C(2) levels in null genotype was higher as compared with GSTM1 present patients at one (p = 0.007) and three months (p = 0.027) post transplantation. CONCLUSION: Patients with variant genotype of GSTM1 and GSTP1 were at higher risk for rejection and delayed functioning of the allograft, respectively, supporting the hypothesis for involvement of GST isoform variants in allograft outcome in renal transplant recipients.

Preferential induction of EphB4 over EphB2 and its implication in colorectal cancer progression.

The receptor tyrosine kinase EphB2 is expressed by colon progenitor cells; however, only 39% of colorectal tumors express EphB2 and expression levels decline with disease progression. Conversely, EphB4 is absent in normal colon but is expressed in all 102 colorectal cancer specimens analyzed, and its expression level correlates with higher tumor stage and grade. Both EphB4 and EphB2 are regulated by the Wnt pathway, the activation of which is critically required for the progression of colorectal cancer. Differential usage of transcriptional coactivator cyclic AMP-responsive element binding protein-binding protein (CBP) over p300 by the Wnt/beta-catenin pathway is known to suppress differentiation and increase proliferation. We show that the beta-catenin-CBP complex induces EphB4 and represses EphB2, in contrast to the beta-catenin-p300 complex. Gain of EphB4 provides survival advantage to tumor cells and resistance to innate tumor necrosis factor-related apoptosis-inducing ligand-mediated cell death. Knockdown of EphB4 inhibits tumor growth and metastases. Our work is the first to show that EphB4 is preferentially induced in colorectal cancer, in contrast to EphB2, whereby tumor cells acquire a survival advantage.

The role of Ephs, Ephrins, and growth factors in Kaposi sarcoma and implications of EphrinB2 blockade.

Kaposi sarcoma (KS) is associated with human herpesvirus (HHV)-8 and is dependent on the induction of vascular endothelial growth factors (VEGFs). VEGF regulates genes that provide arterial or venous identity to endothelial cells, such as the induction of EphrinB2, which phenotypically defines arterial endothelial cells and pericytes, and represses EphB4, which defines venous endothelial cells. We conducted a comprehensive analysis of the Eph receptor tyrosine kinases to determine which members are expressed and therefore contribute to KS pathogenesis. We demonstrated limited Eph/Ephrin expression; notably, the only ligand highly expressed is EphrinB2. We next studied the biologic effects of blocking EphrinB2 using the extracellular domain of EphB4 fused with human serum albumin (sEphB4-HSA). sEphB4-HSA inhibited migration and invasion of the KS cells in vitro in response to various growth factors. Finally, we determined the biologic effects of combining sEphB4-HSA and an antibody to VEGF. sEphB4-HSA was more active than the VEGF antibody, and combination of the 2 had at least additive activity. sEphB4-HSA reduced blood vessel density, pericyte recruitment, vessel perfusion, and increased hypoxia, with an associated increase in VEGF and DLL4 expression. The combination of sEphB4-HSA and VEGF antibody is a rational treatment combination for further investigation.

Do IL-4 intron 3 VNTR and IL-6 (-174) G/C variants reflect ethnic variation? A comparative study between the global and North Indian populations.

Variations in the production and activity of cytokines have been reported by several investigators which influence the susceptibility and/or resistance to various infectious agents and cancer. Differences in the cytokine production between individuals are often caused by single nucleotide polymorphism (SNP) in the promoter or coding regions of cytokine genes. Although the SNP cytokine gene variations are basically mutations, they are designated as polymorphisms, because these changes do not modify the alleles to rare or abnormal variants. The two important cytokine genes IL-4 and IL-6 of 343 unrelated healthy individuals from North India were compared with the published polymorphism of other populations. It was seen that our population differs from South Indian population as well as from other Caucasian populations except, Taiwanese population at IL-4 locus and Spanish and Polish population at the IL-6 gene locus. This study may be helpful for predicting clinical outcome of various infectious and immunoregulated disorders as well as explore for risk alleles for various cancers.

Predisposition of genetic polymorphism with the risk of urolithiasis.

Urolithiasis is a relevant clinical problem with a subsequent burden for health system. The aim of this review is to provide recent progress made using genetic polymorphisms to define pathophysiology, to identify persons at risk for kidney stone disease and to predict treatment response. Population case-control studies are useful both as an alternative and an adjunct as compared to family studies. These involve either whole genome scanning or candidate gene approaches. While whole genome scanning is likely to be widely used in future, at present, candidate gene studies are more feasible. When performing candidate gene case-control studies factors such as study design, methods for recruitment of case and controls, selection of candidate genes, functional significance of polymorphisms chosen for study and statistical analysis require close attention to ensure that only genuine associations are detected. Some of the significant genes that play role in stone formation include calcitonin receptor gene (CTR), vitamin D receptor (VDR), Urokinase, Interleukin, (IL-1ß, IL-Ra), E-Cadherin, Androgen & oestrogen receptor gene, vascular endothelial growth factor (VEGF) and Arginine p21. In our case-control study we studied CTR, VDR, Urokinase, IL-1ß(-511 and +3954), IL-Ra from north India and predict that VDR, IL-ß (-511) and IL-1Ra gene may be used as a possible genetic marker for earlier detection in patients who are at risk for calcium oxalate stone disease. Further, linkage disequilibrium and haplotype structure of a certain candidate gene is important for association analysis. When a certain polymorphic allele has been found to be associated with disease, it is further explained on basis of LD and haplotype structure by one or more other alleles. Once it is determined which haplotype carries the risk allele, by means of molecular biological functional analyses, the variants on that haplotype allele truly causing the effect can be determined.

Analysis of polymorphisms of tumor necrosis factor-alpha and polymorphic xenobiotic metabolizing enzymes in inflammatory bowel disease: study from northern India.

BACKGROUND: Tumor necrosis factor (TNF)-alpha is a proinflammatory cytokine associated with inflammatory diseases, while GSTM1 and T1 enzymes catalyze detoxification of products of oxidative stress and hence reduce inflammation. Thus, both may play important roles in the pathogenesis of inflammatory bowel disease (IBD). The present study aimed to evaluate the effect of polymorphism of the TNF-alpha promoter at the -308 site, GSTM1 and GSTT1 in patients with IBD and healthy controls from northern India. METHOD: Genotyping was performed in 114 patients with IBD (22 Crohn's disease [CD] and 92 ulcerative colitis [UC]) in TNF-alpha and 105 (20 CD and 85 UC) in GSTM1 and T1 and 164 healthy controls using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and multiplex PCR methods. RESULTS: Patients with IBD were comparable to healthy controls in relation to age and gender. Genotypic and allelic frequencies of TNF-alpha were comparable among patients with IBD and healthy controls. GSTM1 null genotype was more frequent in UC than in healthy controls (52/85 vs 49/164; P < 0.001) and GSTT1 null genotype was more frequent both in UC and CD as compared to healthy controls (77/85 and 18/20 vs 26/164, respectively; P < 0.001 for both). Frequency of combined null genotype in GSTM1 and T1 was more frequently associated with IBD than healthy controls (4/20 vs 8/164; P = 0.029, OR = 4.875 and 28/85 vs 8/164; P < 0.001, OR = 9.579, respectively). CONCLUSIONS: 'Null' genotypes of GSTM1 and T1 are associated with IBD and the combination of the two GST genotypes further increases the risk, possibly due to gene-gene interaction. TNF-alpha is unlikely to be an important determinant of susceptibility to IBD in the Indian population.

Association of vitamin-D receptor (Fok-I) gene polymorphism with bladder cancer in an Indian population.

OBJECTIVE: To explore the association of vitamin-D receptor (VDR) genotypes and haplotypes (variants at the Fok-I, and Taq-I sites) with the risk of bladder cancer, as vitamin D is antiproliferative and reported to induce apoptosis in human bladder tumour cells in vitro. PATIENTS, SUBJECTS AND METHODS: A case-control study using polymerase chain reaction-restriction fragment length polymorphism was conducted in 130 patients with bladder cancer and 346 normal healthy individuals in a north Indian population. Patients were also categorized according to grade and stage of tumour. RESULTS: There was a significant difference in genotype and allelic distribution of VDR (Fok-I) polymorphism in the patients (P = 0.033 and = 0.017, respectively). The FF genotype was associated with twice the risk for bladder cancer (odds ratio 2.042, 95% confidence interval, CI, 0.803-5.193). There was no significant difference in genotypic distribution or allelic frequencies of the VDR (Taq-I) polymorphism (P = 0.477 and 0.230) when compared with the controls. The stage and grade of the bladder tumours had no association with VDR (Fok-I and Taq-I) genotypes. There was a significant difference in the frequency distribution of the haplotypes FT and fT (P < 0.001); these haplotypes had a protective effect in the control group (odds ratio 0.167, 95% CI 0.096-0.291, and 0.079, 0.038-0.164). CONCLUSION: These data suggest that VDR (Fok-I) polymorphism is associated with the risk of bladder cancer. Further, the results for the haplotype FT and fT indicate that patients with this haplotype have a lower risk of developing bladder cancer than those with other haplotypes.

Genotype and haplotype determination of IL1B (g. -511C>T and g. +3954C>T) and (IL1RN) in pediatric nephrolithiasis.

BACKGROUND: Nephrolithiasis is a multifactorial and polygenic disorder characterized by presence of stones in urinary tract. Interleukin1 (IL1) plays role in process of bone loss/hypercalciuria and is involved in formation of kidney stones. We investigated the association between IL1B promoter region and exon-5 (g.-511C>T and g. +3954C>T) polymorphism and variable number of tandem repeats in IL1 receptor antagonist, IL1RN (IVS2) with risk of stone formation in childhood nephrolithiasis in north Indian population. METHODS: Control group of 60 healthy pediatric individuals (age range =4-16 y) and 50 pediatric nephrolithiasis patients (age range =2-14 y) were studied. Polymorphism was detected by PCR based restriction analysis. Haplotypes for IL1B and IL1RN were constructed using Arlequin v2.0 software. RESULTS: Distribution of IL1RN gene polymorphism demonstrated significant difference (p=0.023). Pediatric patients had significantly higher frequency of allele I in IL1RN (16% vs. 1.7%). The distribution of IL1B (g. -511C>T and g. +3954C>T) genotypes in patients and controls were similar (p=0.263 and 0.694 respectively). There was a significant difference in haplotype frequencies between pediatric patients and control group (p<0.05). Haplotype T-E1-I showed>7-folds risk for nephrolithiasis (p=0.033; OR=7.07, 95% CI=1.16-42.84). CONCLUSIONS: Significant association was observed for allele I(*) of IL1RN however, no association was observed for IL1B. Haplotype T-E1-I was significantly associated with higher risk of pediatric nephrolithiasis. These findings suggest that the IL1RN and haplotyping may be an influential marker for susceptibility to pediatric nephrolithiasis.

Association of interleukin-1beta gene and receptor antagonist polymorphisms with calcium oxalate urolithiasis.

BACKGROUND AND PURPOSE: Genetic polymorphisms of the interleukin-1beta (IL-1beta) promoter region (-511) and exon 5 +3954 and a variable number of tandem repeats in the IL receptor antagonist (IL-1RA) gene have been proposed as markers for calcium oxalate urolithiasis. Because the prevalence of these polymorphisms could be influenced by racial variation/ethnicity, we explored the association of IL-1 gene-cluster polymorphisms with stone formation in a north India population. PATIENTS AND METHODS: The case-control study involved 150 stone-free control subjects (mean age 46.5 +/- 10.5 years) and 130 patients (mean age 40.0 +/- 11.5 years) with calcium oxalate urolithiasis. Biallelic polymorphisms of two loci, IL-1beta (-511) and IL-1beta (+3954), as well as the penta-allelic variable number of tandem repeats of IL-1RA, were genotyped by polymerase chain reaction-based restriction analysis. Haplotypes were constructed for the IL-1 gene cluster using SNP Analyzer software. RESULTS: We observed a significant association between stone disease and IL-1beta -511 and IL-1RA polymorphisms (P < 0.001 and P = 0.039, respectively), whereas no association was observed for IL-1beta +3954 (P = 0.408). The frequency of the TT (-511) and I/II (410/240; IL-1RA) genotypes was higher in patients than in control subjects (50/130 v 16/150 and 55/130 v 38/150, respectively), whereas the frequencies of the haplotypes were similar (P = 0.485). Significant linkage disequilibrium showed that three genes were strongly linked (P < 0.0001). Patients with a combination of high IL-1beta (-511 and +3954) and low IL-1RA genotypes were at significantly higher risk for urolithiasis (P < 0.001; odds ratio = 5.448, 0.013, and 2.560, respectively). CONCLUSION: Our study demonstrated a strong association of IL-1RA and IL-1beta-511 and suggested that differences in the IL-1 gene cluster could be linked to the risk of urolithiasis. A combination of IL-1beta and IL-1RA associations exhibiting gene-gene interaction further substantiates the finding of risk.