To study the role of human papillomavirus (HPV) infection in the development of genetic instability, we transduced normal human airway and anogenital epithelial cells with various combinations of HPV-16 E6, E7, and the reverse transcriptase component of telomerase (hTERT). Cell lines generated by co-expression of E7 with E6 and/or hTERT (i.e., E6/E7, E7/hTERT, and E6/E7/hTERT) exhibited extra copies of chromosome 20 and specific amplification of the 20q12-ter region, whereas those generated without E7 (i.e., hTERT alone or E6/hTERT) did not. Co-expression of hTERT and a dominant-negative version of cdk4 that has been shown to inactivate the retinoblastoma (pRb) pathway also resulted in 20q amplification. Interestingly, extra copies of chromosome 20 were observed in early passage keratinocytes that expressed E7 alone, and microarray expression analysis revealed that genes in the 20q region and on chromosome 5 were specifically upregulated in these cells. Our results indicate that chromosome 20q amplification is an early event that may be specifically caused by expression of E7 through inactivation of the pRb pathway in human epithelial cells.

TAZ and YAP are transcriptional coactivators negatively regulated by the Hippo pathway that have emerged as key oncoproteins in several cancers including sarcomas. We hypothesized that loss of expression of the Hippo kinases might be a mechanism of activating TAZ and YAP. By immunohistochemistry, TAZ/YAP activated clinical sarcoma samples demonstrated loss of MST1 (47%), MST2 (26%), LATS1 (19%), and LATS2 (27%). Western blot similarly demonstrated loss of MST1 (58%), MST2 (25%), and LATS2 (17%). Treatment with MG132 demonstrated an accumulation of MST2 in 25% of sarcoma cell lines, indicating that proteosomal degradation regulates MST2 expression. qRT-PCR in sarcoma cell lines demonstrated loss of expression of the Hippo kinases at the RNA level, most pronounced in MST1 (42%) and MST2 (25%). 5-azacytidine treatment in sarcoma cell lines modestly reversed expression of predominantly MST1 (8%) and MST2 (17%), indicating CpG island hypermethylation can silence expression of MST1 and MST2. Trichostatin A treatment reversed expression of MST1 (58%) and MST2 (67%), indicating histone deacetylation also plays a role in silencing expression of MST1 and MST2. Loss of expression of the Hippo kinases is frequent in sarcomas and is due to a variety of mechanisms including regulation at the post-translational level and epigenetic silencing.

Given the lack of any novel genes near either breakpoint, changes in potential regulatory elements may be the best model to explain the loss of PITX2 expression in these patients and hence the Rieger's syndrome phenotype.

BackgroundMedulloblastoma is the most common malignant brain tumor in children. Genetic profiling has identified four principle tumor subgroups; each subgroup is characterized by different initiating mutations, genetic and clinical profiles, and prognoses. The two most well-defined subgroups are caused by overactive signaling in the WNT and SHH mitogenic pathways; less is understood about Groups 3 and 4 medulloblastoma. Identification of tumor subgroup using molecular classification is set to become an important component of medulloblastoma diagnosis and staging, and will likely guide therapeutic options. However, thus far, few druggable targets have emerged. G-protein coupled receptors (GPCRs) possess characteristics that make them ideal targets for molecular imaging and therapeutics; drugs targeting GPCRs account for 30-40% of all current pharmaceuticals. While expression patterns of many proteins in human medulloblastoma subgroups have been discerned, the expression pattern of GPCRs in medulloblastoma has not been investigated. We hypothesized that analysis of GPCR expression would identify clear subsets of medulloblastoma and suggest distinct GPCRs that might serve as molecular targets for both imaging and therapy.ResultsOur study found that medulloblastoma tumors fall into distinct clusters based solely on GPCR expression patterns. Normal cerebellum clustered separately from the tumor samples. Further, two of the tumor clusters correspond with high fidelity to the WNT and SHH subgroups of medulloblastoma. Distinct over-expressed GPCRs emerge; for example, LGR5 and GPR64 are significantly and uniquely over-expressed in the WNT subgroup of tumors, while PTGER4 is over-expressed in the SHH subgroup. Uniquely under-expressed GPCRs were also observed. Our key findings were independently validated using a large international dataset.ConclusionsOur results identify GPCRs with potential to act as imaging and therapeutic targets. Elucidating tumorigenic pathways is a secondary benefit to identifying differential GPCR expression patterns in medulloblastoma tumors.

Purpose The gene that causes normal tension glaucoma (NTG) in a large pedigree was recently mapped to a region of chromosome 12q14 (GLC1P) that contains the genes TBK1, XPOT, RASSF3, and GNS. We sought to investigate the structure of the chromosome 12q14 duplication and explore the ocular expression of GLC1P locus genes. Methods The location of the chromosome 12q14 duplication in this pedigree was examined with fluorescent in situ hybridization (FISH) using probes for TBK1 and GNS. The expression pattern of XPOT, TBK1, RASSF3, and GNS was investigated with immunohistochemistry of human eyes. Results The karyotype of an NTG patient from pedigree GGO-414 was normal and FISH studies demonstrated that the duplicated DNA is organized as a tandem repeat on chromosome 12q14. Of the genes in or near the chromosome 12q14 duplication, TBK1 showed expression in the retina that is specific to the retinal ganglion cells and the retinal nerve fiber layer. Expression of RASSF3 and XPOT was relatively uniform throughout the retina, while GNS expression was expressed in a pattern consistent with Müller cells. Conclusions Previous studies demonstrated that chromosome 12q14 duplications are associated with NTG inherited as an autosomal dominant trait. FISH studies now demonstrate that the duplicated segments are tandemly organized on chromosome 12q14 in close proximity. The specific expression of TBK1 in human retinal ganglion cells compared to the widespread pattern of expression of neighboring genes provides additional evidence that TBK1 is the glaucoma gene in the chromosome 12q14 duplication within the GLC1P locus.