PTK7 proteolytic fragment proteins function during early Xenopus development.

Protein Tyrosine Kinase 7 (PTK7) is as a critical regulator of canonical and non-canonical Wnt-signaling during embryonic development and cancer cell formation. Disrupting PTK7 activity perturbs vertebrate nervous system development, and also promotes human cancer formation. Observations in different model systems suggest a complex cross-talk between PTK7 protein and Wnt signaling. During Xenopus laevis nervous system development, we previously showed that PTK7 protein positively regulates canonical Wnt signaling by maintaining optimal LRP6 protein levels, but PTK7 also acts in concert with LRP6 protein to repress non-canonical Wnt activity. PTK7 is a transmembrane protein, but studies in cancer cells showed that PTK7 undergoes "shedding" by metalloproteases to different proteolytic fragments. Some PTK7 proteolytic fragments are oncogenic, being localized to alternative cytoplasmic and nuclear cell compartments. In this study we examined the biological activity of two proteolytic carboxyl-terminal PTK7 proteolytic fragments, cPTK7 622-1070 and cPTK7 726-1070 during early Xenopus nervous system development. We found that these smaller PTK7 proteolytic fragments have similar activity to full-length PTK7 protein to promote canonical Wnt-signaling via regulation of LRP6 protein levels. In addition to cancer systems, this study shows in vivo proof that these smaller PTK7 proteolytic fragments can recapitulate full-length PTK7 protein activity in diverse systems, such as vertebrate nervous system development.

CDC174, a novel component of the exon junction complex whose mutation underlies a syndrome of hypotonia and psychomotor developmental delay.

Siblings of non-consanguineous Jewish-Ethiopian ancestry presented with congenital axial hypotonia, weakness of the abducens nerve, psychomotor developmental delay with brain ventriculomegaly, variable thinning of corpus callosum and cardiac septal defects. Homozygosity mapping identified a single disease-associated locus of 3.5 Mb on chromosome 3. Studies of a Bedouin consanguineous kindred affected with a similar recessive phenotype identified a single disease-associated 18 Mb homozygosity locus encompassing the entire 3.5 Mb locus. Whole exome sequencing demonstrated only two homozygous mutations within a shared identical haplotype of 0.6 Mb, common to both Bedouin and Ethiopian affected individuals, suggesting an ancient common founder. Only one of the mutations segregated as expected in both kindreds and was not found in Bedouin and Jewish-Ethiopian controls: c.1404A>G, p.[*468Trpext*6] in CCDC174. We showed that CCDC174 is ubiquitous, restricted to the cell nucleus and co-localized with EIF4A3. In fact, yeast-two-hybrid assay demonstrated interaction of CCDC174 with EIF4A3, a component of exon junction complex. Knockdown of the CCDC174 ortholog in Xenopus laevis embryos resulted in poor neural fold closure at the neurula stage with later embryonic lethality. Knockdown embryos exhibited a sharp reduction in expression of n-tubulin, a marker for differentiating primary neurons, and of hindbrain markers krox20 and hoxb3. The Xenopus phenotype could be rescued by the human normal, yet not the mutant CCDC174 transcripts. Moreover, overexpression of mutant but not normal CCDC174 in neuroblastoma cells caused rapid apoptosis. In line with the hypotonia phenotype, the CCDC174 mutation caused depletion of RYR1 and marked myopathic changes in skeletal muscle of affected individuals.

PTK7 modulates Wnt signaling activity via LRP6.

Protein tyrosine kinase 7 (PTK7) is a transmembrane protein expressed in the developing Xenopus neural plate. PTK7 regulates vertebrate planar cell polarity (PCP), controlling mesodermal and neural convergent-extension (CE) cell movements, neural crest migration and neural tube closure in vertebrate embryos. Besides CE phenotypes, we now show that PTK7 protein knockdown also inhibits Wnt/ß-catenin activity. Canonical Wnt signaling caudalizes the neural plate via direct transcriptional activation of the meis3 TALE-class homeobox gene, which subsequently induces neural CE. PTK7 controls meis3 gene expression to specify posterior tissue and downstream PCP activity. Furthermore, PTK7 morphants phenocopy embryos depleted for Wnt3a, LRP6 and Meis3 proteins. PTK7 protein depletion inhibits embryonic Wnt/ß-catenin signaling by strongly reducing LRP6 protein levels. LRP6 protein positively modulates Wnt/ß-catenin, but negatively modulates Wnt/PCP activities. The maintenance of high LRP6 protein levels by PTK7 triggers PCP inhibition. PTK7 and LRP6 proteins physically interact, suggesting that PTK7 stabilization of LRP6 protein reciprocally regulates both canonical and noncanonical Wnt activities in the embryo. We suggest a novel role for PTK7 protein as a modulator of LRP6 that negatively regulates Wnt/PCP activity.

Rare human papillomavirus 16 E6 variants reveal significant oncogenic potential.

The aim of this study was to determine whether low prevalence human papillomavirus (HPV) 16 E6 variants differ from high prevalence types in their functional abilities. We evaluated functions relevant to carcinogenesis for the rarely-detected European variants R8Q, R10G and R48W as compared to the commonly detected L83V. Human immortalized keratinocytes (NIKS) stably transduced with the E6 variants were used in most functional assays. Low and high prevalence E6 variants displayed similar abilities in abrogation of growth arrest and inhibition of p53 elevation induced by actinomycin D. Differences were detected in the abilities to dysregulate stratification and differentiation of NIKS in organotypic raft cultures, modulate detachment induced apoptosis (anoikis) and hyperactivate Wnt signaling. No distinctive phenotype could be assigned to include all rare variants. Like L83V, raft cultures derived from variants R10G and R48W similarly induced hyperplasia and aberrantly expressed keratin 5 in the suprabasal compartment with significantly lower expression of keratin 10. Unlike L83V, both variants, and particularly R48W, induced increased levels of anoikis upon suspension in semisolid medium. R8Q induced a unique phenotype characterized by thin organotypic raft cultures, low expression of keratin 10, and high expression of keratins 5 and 14 throughout all raft layers. Interestingly, in a reporter based assay R8Q exhibited a higher ability to augment TCF/ß-catenin transcription. The data suggests that differences in E6 variant prevalence in cervical carcinoma may not be related to the carcinogenic potential of the E6 protein.

Modeling Bainbridge-Ropers Syndrome in Xenopus laevis Embryos.

The Additional sex combs-like (ASXL1-3) genes are linked to human neurodevelopmental disorders. The de novo truncating variants in ASXL1-3 proteins serve as the genetic basis for severe neurodevelopmental diseases such as Bohring-Opitz, Shashi-Pena, and Bainbridge-Ropers syndromes, respectively. The phenotypes of these syndromes are similar but not identical, and include dramatic craniofacial defects, microcephaly, developmental delay, and severe intellectual disability, with a loss of speech and language. Bainbridge-Ropers syndrome resulting from ASXL3 gene mutations also includes features of autism spectrum disorder. Human genomic studies also identified missense ASXL3 variants associated with autism spectrum disorder, but lacking more severe Bainbridge-Ropers syndromic features. While these findings strongly implicate ASXL3 in mammalian brain development, its functions are not clearly understood. ASXL3 protein is a component of the polycomb deubiquitinase complex that removes mono-ubiquitin from Histone H2A. Dynamic chromatin modifications play important roles in the specification of cell fates during early neural patterning and development. In this study, we utilize the frog, Xenopus laevis as a simpler and more accessible vertebrate neurodevelopmental model system to understand the embryological cause of Bainbridge-Ropers syndrome. We have found that ASXL3 protein knockdown during early embryo development highly perturbs neural cell fate specification, potentially resembling the Bainbridge-Ropers syndrome phenotype in humans. Thus, the frog embryo is a powerful tool for understanding the etiology of Bainbridge-Ropers syndrome in humans.

HPV16 E6 augments Wnt signaling in an E6AP-dependent manner.

In this study we investigated the effect of HPV16 E6 on the Wnt/beta-catenin oncogenic signaling pathway. Luciferase reporter assays indicated that ectopically expressed E6 significantly augmented the Wnt/beta-catenin/TCF-dependent signaling response in a dose-dependent manner. This activity was independent of the ability of E6 to target p53 for degradation or bind to the PDZ-containing E6 targets. Epistasis experiments suggested that the stimulatory effect is independent of GSK3beta or APC. Coexpression, half-life determination, cell fractionation and immunofluorescence analyses indicated that E6 did not alter the expression levels, stability or cellular distribution of beta-catenin. Further experiments using E6 mutants defective for E6AP binding and E6AP knockdown cells indicated the absolute requirement of the ubiquitin ligase E6AP for enhancement of the Wnt signal by E6. Thus, this study suggests a role for the E6/E6AP complex in augmentation of the Wnt signaling pathway which may contribute to HPV induced carcinogenesis.

Human papillomavirus 16 E6 variants differ in their dysregulation of human keratinocyte differentiation and apoptosis.

L83V-related variants of human papillomavirus (HPV) 16 E6, exemplified by the Asian-American variant Q14H/H78Y/L83V, were shown to be more prevalent than E6 prototype in progressing lesions and cervical cancer. We evaluated functions relevant to carcinogenesis for the E6 variants L83V, R10/L83V and Q14H/H78Y/L83V as well as the prototype in a model of human normal immortalized keratinocytes (NIKS). All E6 expressing NIKS equally abrogated growth arrest and DNA damage responses. Organotypic cultures derived from these keratinocytes demonstrated hyperplasia and aberrantly expressed keratin 5 in the suprabasal compartment. In contrast, differentiation and induction of apoptosis varied. The E6 variant rafts expressed keratin 10 in nearly all suprabasal cells while the prototype raft showed keratin 10 staining in a subset of suprabasal cells only. In addition, E6 variant NIKS expressing R10G/L83V and Q14H/H78Y/L83V were more prone to undergo cell-detachment-induced apoptosis (anoikis) than NIKS expressing E6 prototype. The combined differentiation and apoptosis pattern of high-risk E6 variants, especially of Q14H/H78Y/L83V, may reflect a phenotype beneficial to carcinogenesis and viral life cycle.

Activities of human papillomavirus 16 E6 natural variants in human keratinocytes.

Genetic variations in the E6 oncogene have been associated with different risk for cancer progression. In the present study, the functional significance of human papillomavirus (HPV) polymorphism in the E6 oncogene was investigated. Ten HPV16 E6 variants containing amino acid substitutions in the N-terminal region of E6 were evaluated for different biological and biochemical activities in human keratinocytes, the target cells for HPV infection. Western blot analyses of primary foreskin human keratinocytes or immortalized human keratinocytes, stably transduced with the E6 variants, revealed reduced p53 and Bax levels in all E6 expressing cultures. The reduction induced by most E6 proteins was at similar levels and comparable to the reduction induced by the E6 prototype. The ability of the proteins to induce serum/calcium-differentiation resistant colonies in primary keratinocytes was more variable. Overall activities of the variants ranged between 0.24- and 2.18-fold of the E6 prototype activity. The I27R/L83V variant showed the lowest activity whereas the R8Q variant showed the highest activity. The L83V polymorphism previously associated with risk for cancer progression in some populations, showed significant activity, comparable to that of the E6 prototype, in reducing p53 and Bax levels. Furthermore, this variant showed enhancement in the ability to induce colonies resistant to serum/calcium-triggered differentiation, however, the difference from the prototype was not statistically significant. This, and augmentation of other described functions might result in differences in L83V pathogenicity.

HPV16 E6 natural variants exhibit different activities in functional assays relevant to the carcinogenic potential of E6.

Genetic studies have revealed natural amino acid variations within the human papillomavirus (HPV) type 16 E6 oncoprotein. To address the functional significance of E6 polymorphisms, 10 HPV16 E6 variants isolated from cervical lesions of Swedish women were evaluated for their activities in different in vitro and in vivo assays relevant to the carcinogenic potential of E6. Small differences between E6 prototype and variants, and among variants, were observed in transient expression assays that assessed p53 degradation, Bax degradation, and inhibition of p53 transactivation. More variable levels of activities were exhibited by the E6 proteins in assays that evaluated binding to the E6-binding protein (E6BP) or the human discs large protein (hDlg). The E6 prototype expressed moderate to high activity in the above assays. The L83V polymorphism, previously associated with risk for cancer progression in some populations, expressed similar levels of activity as that of the E6 prototype in most functional assays. On the other hand, L83V displayed more efficient degradation of Bax and binding to E6BP, but lower binding to hDlg. Results of this study indicate that naturally occurring amino acid variations in HPV16 E6 can alter activities of the protein important for its carcinogenic potential.