Minireview: Role of genetic changes of faciogenital dysplasia protein 1 in human disease.

The FGD1 gene encodes for a guanine exchange factor (GEF) protein that specifically activates the Rho GTPase Cdc42. For cellular migration, Cdc42 is a key molecular switch that regulates cytoskeleton restructuring, gene transcription, cellular morphology, extension, and cell adhesion. In the past decade, germline mutations in the FGD1 gene have been associated with a rare X-linked disorder known as faciogenital dysplasia (FGDY). Malformations are consistent with a loss of cellular migration during embryonic development. Insertion and deletion mutations in FGD1 result in a frameshift causing inactivation of fgd1 protein. Since Cdc42 is a key molecular switch in cytoskeletal restructuring and cell adhesion, the loss of fgd1 is postulated to attenuate Cdc42-mediated cellular migration in embryonic development. In metastatic tumors, Cdc42 modulates migration and invasiveness. Fgd1 overexpression has been found in infiltrating and poorly differentiated breast and invasive prostate tumors. Amplification at Xp11.21, the FGD1 gene locus, has been reported in several cancers. Sequencing analyses in numerous types of cancer have found missense mutations in the FGD1 gene in metastatic tumors. FGDY and cancer studies suggest that the germline and somatic changes downregulate or upregulate the FGD1 gene playing a key role in the development of diseases.

The calcitriol analogue EB1089 impairs alveolarization and induces localized regions of increased fibroblast density in neonatal rat lung.

The active form of vitamin D3, 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3, or calcitriol), is a potent mitogen for fibroblasts cultured from rat lungs at postnatal day 4 (P4), during the peak of septation (P3 to P7). In light of the key role of fibroblasts in alveolar septation, the authors conducted studies to measure the extent to which 1,25-(OH)2D3 affects lung maturation in vivo, as well as its ability to influence the stimulatory activity of all-trans retinoic acid (RA). To identify a calcitriol analogue with maximal mitogenic activity and low systemic toxicity, two compounds with reduced calcemic activity (EB1089 and CB1093) and a superagonist (MC1288) were evaluated in neonatal rat lung fibroblast cultures. All 3 analogues were more potent mitogens than 1,25-(OH)(2)D3 itself (MC1288 approximately CB1093 > EB1089 > 1,25-(OH)2D3). In addition, each was more effective than 1,25-(OH)2D3(EB1089 > CB1093 > MC1288 > 1,25-(OH)2D3) in the activation of a vitamin D response element from the platelet-derived growth factor (PDGF)-A gene, whose expression is essential for normal alveolarization. Daily administration of EB1089 to rats 4 to 12 days of age caused an increase in mean alveolar chord length (P < .0001), and also elicited prominent regions of fibroblast hypercellularity, as defined in terms of a vimentin-positive, factor VIII-negative phenotype. EB1089 and RA each induced the expression of 2 important lung structural proteins, collagen and elastin. Regions of fibroblast hypercellularity induced by EB1089 were strongly positive for expression of the alveolarization-relevant growth factors, PDGF-AA and vascular endothelial growth factor (VEGF). These studies demonstrate that 1,25-(OH)2D3 disrupts the overall alveolarization process in the neonatal lung, although it stimulates expression of some proteins associated with lung morphogenesis.

Retinoic acid inducibility of the human PDGF-a gene is mediated by 5'-distal DNA motifs that overlap with basal enhancer and vitamin D response elements.

Retinoic acid (RA) upregulates expression of PDGF ligands and receptors in neonatal rat lung fibroblasts, a process likely to promote maturation of the lung alveolus and possibly microstructures of other organs. A mutational analysis of the gene encoding the PDGF-A ligand has identified a complex retinoic acid response element (RARE) located far upstream of the transcription start site, in a 5'-distal enhanceosome region previously shown to harbor basal and vitamin D-inducible enhancer activity. Maximal RA responsiveness (fourfold) was conferred by nucleotide sequence located between -7064 and -6787, with a variety of deletion and point mutations revealing the importance of at least three nuclear receptor half-sites within the enhancer region (-6851 to -6824), as well as nucleotides located further upstream. Recombinant human retinoic acid receptor/retinoid-X receptor heterodimers bound with high affinity and sequence specificity to multiple regions within the RARE, as demonstrated by electrophoretic mobility shift and DNase I footprinting assays. The addition of RARE activity to previously described functions of the 5'-distal enhanceosome underscores the importance of this region as a key integration point for regulatory control of PDGF-A expression.

A 5'-distal enhanceosome in the PDGF-A gene is activated in choriocarcinoma cells via ligand-independent binding of vitamin D receptor and constitutive jun kinase signaling.

Overexpression of platelet-derived growth factor A-chain (PDGF-A) is clearly linked to autocrine and paracrine stimulation of malignant growth in many human cancers. We have shown previously that PDGF-A overexpression in choriocarcinoma, hepatoma and lung carcinoma cell lines is driven by the activity of a 66 bp enhancer element (ACE66) located approximately 7 kb upstream of the PDGF-A transcription start site. In this study, the ACE66 element is shown to be activated in JEG-3 choriocarcinoma cells through synergistic interactions between consensus DNA motifs for binding of vitamin D receptor, AP1 and ELK1. Binding of the vitamin D/retinoid-X receptor (VDR/RXRalpha) heterodimer to the ACE66 element was reconstituted in vitro with recombinant VDR/RXRalpha and with JEG-3 nuclear extract, and was verified in living JEG-3 cells by chromatin immunoprecipitation analysis. Transcriptional activity of the ACE66 element, as well as occupancy of the element by VDR/RXRalpha, was shown to be independent of stimulation with the hormonal VDR ligand, 1,25-dihydroxyvitamin D3. The jun kinase pathway of mitogen-activated protein kinase (MAPK) signaling was shown to activate the ACE66 enhancer, most likely through activation of factors binding to the AP1 element. These results identify a novel mechanism of transcriptional enhancement involving ligand-independent activity of the VDR/RXR heterodimer and MAPK signaling pathways that appears to play an important role in the overexpression of PDGF in many different settings of human malignancy.

Association of FGD1 polymorphisms with early-onset breast cancer.

Recent cancer studies have suggested that the faciogenital dysplasia 1 (FGD1) gene may play a role in the development of tumor cells. Somatic alterations in the FGD1 gene and increased Fgd1 protein expression have been observed in many breast tumor cases. The present study sequenced the FGD1 gene in tumor DNA from 46 breast cancer patients using Ion Torrent sequencing. Three synonymous polymorphisms and one missense polymorphism were detected with next-generation sequencing; however, no somatic mutations were observed. The Thr697 variant was identified in 18 patients with an average age at diagnosis of 55 years, which was a lower average age than patients without the polymorphism. In addition, a higher frequency of Thr697 was observed in African-American patients. The Pro712 was observed in 15 breast cancer patients with an average age of 58 years, and was observed as a haplotype with the Thr697 variant in 28% of the breast cancer patients studied. The missense polymorphism (Ala226Thr) was identified in a 40-year-old female patient who had a recurrence of cancer. These polymorphisms (Ala226Thr, Thr697 and Pro712) may be associated with an earlier onset of breast cancer.

Application of genomic principles to pharmacotherapy of cancer.

OBJECTIVES: To teach first-year (P1) pharmacy students to apply the principles of pharmacogenomics underlying clinical pharmacotherapeutics to cancer patients. DESIGN: Using polymerase chain reaction (PCR) and high-resolution melting analysis of deoxyribonucleic acid (DNA) from colorectal cancer cell lines to determine the presence of somatic mutations for an oncogenic marker, students formulated the proper course of treatment for a patient with similar tumor genomics. ASSESSMENT: In a postintervention survey, students highly rated the effectiveness of the laboratory session for learning pharmacogenomics, and subsequent examination scores reflected retention of principles and understanding of clinical application. CONCLUSION: The pharmacogenomic laboratory exercise prepared students to understand how genetic markers give clinical insight into the appropriate application of drugs in oncology pharmacotherapy. Further, the session inspired their interest in learning more about pharmacogenomics and their professional roles in personalized medicine.

NM23-H1 and NM23-H2 repress transcriptional activities of nuclease-hypersensitive elements in the platelet-derived growth factor-A promoter.

The platelet-derived growth factor (PDGF)-A promoter is regulated by a number of GC-rich regulatory elements that possess non-B-form DNA structures. Screening of a HeLa cDNA expression library with the C-rich strand of a PDGF-A silencer sequence (5'-S1 nuclease-hypersensitive site (SHS)) yielded three cDNA clones encoding NM23-H1, a protein implicated as a suppressor of metastasis in melanoma and breast carcinoma. Recombinant human NM23-H1 cleaved within the 3'-portions of both 5'-SHS strands in either single-stranded or duplex forms. In contrast, NM23-H2, known as a transcriptional activator with a DNA cleavage function, cleaved within the 5'-portions of both strands, revealing that NM23-H1 and NM23-H2 cleave at distinct sites of the 5'-SHS and by different mechanisms. NM23-H1 and NM23-H2 also cleaved within the PDGF-A basal promoter region, again exhibiting preferences for cleavage within the 5'- and 3'-portions of the element, respectively. Transient transfection analyses in HepG2 cells revealed that both NM23-H1 and -H2 repressed transcriptional activity driven by the PDGF-A basal promoter (-82 to +8). Activity of the negative regulatory region (-1853 to -883), which contains the 5'-SHS, was also inhibited modestly by NM23-H1 and NM23-H2. These studies demonstrate for the first time that NM23-H1 interacts both structurally and functionally with DNA. They also indicate a role for NM23 proteins in repressing transcription of a growth factor oncogene, providing a possible molecular mechanism to explain their metastasis-suppressing effects.