[miR-181b-5p promotes cell proliferation and induces apoptosis in human acute myeloid leukemia by targeting PAX9].

Objective To investigate the effects of miR-181b-5p on cells proliferation and apoptosis in acute myeloid leukemia (AML) by targeting paired box 9 (PAX9). Methods The relationship between expression level of PAX9 and prognosis in AML patients was analyzed by gene expression profiling interactive analysis (GEPIA) database and The Cancer Genome Atlas (TCGA) database. Kasumi-1 and AML5 cells were transfected with empty vector (Vector group) or PAX9 (PAX9 group). The proliferation activity was detected by CCK-8 assay, and cells cycle and apoptosis were detected by flow cytometry. Expressions of cyclin-dependent kinase 2 (CDK2), cyclin B1 (CCNB1), B-cell lymphoma 2 (Bcl2) and Bcl2-associated X protein (BAX) were detected by Western blot analysis. The targeted microRNA (miRNA) by PAX9 was predicted by bioinformatics analysis, and the targeted effect was verified by luciferase reporter assay. The level of PAX9 mRNA was detected by real-time quantitative PCR, and expression of PAX9 protein was detected by Western blot analysis. Kasumi-1 and AML5 cells were transfected with miR-NC (miR-NC group) or miR-181b-5p (miR-181b-5p group). The cells were further transfected with PAX9 (miR-181b-5p combined with PAX9 group) in miR-181b-5p group. The proliferation, cycle and apoptosis of cells were detected by the above methods.Results GEPIA and TCGA databases showed that the expression of PAX9 was down-regulated in AML patients, which was correlated with poor prognosis. In Kasumi-1 and AML5 cells, compared with Vector group, proliferation activity of cells, percentage of cells in S phase, and expressions of CDK2, CCNB1 and Bcl2 proteins were decreased, while percentage of cells in G0/G1 phase, apoptosis rate and the expression of BAX protein were increased in PAX9 group. It was confirmed by double luciferase reporter assay that PAX9 was the target gene of miR-181b-5p. Compared with miR-NC group, proliferation activity of cells, percentage of cells in S phase, and expressions of CDK2, CCNB1 and Bcl2 proteins were increased, while percentage of cells in G0/G1 phase, apoptosis rate and the expression of BAX protein were decreased in miR-181b-5p group. Compared with miR-181b-5p group, proliferation activity of cells, percentage of cells in S phase, and expressions of CDK2, CCNB1 and Bcl2 proteins were decreased, while percentage of cells in G0/G1 phase, apoptosis rate and the expression of BAX protein were increased in miR-181b-5p combined with PAX9 group. Conclusion The miR-181b-5p can promote the proliferation of AML cells and delay apoptosis by inhibiting PAX9.

PAX9 in Cancer Development.

Paired box 9 (PAX9) is a transcription factor of the PAX family functioning as both a transcriptional activator and repressor. Its functional roles in the embryonic development of various tissues and organs have been well studied. However, its roles and molecular mechanisms in cancer development are largely unknown. Here, we review the current understanding of PAX9 expression, upstream regulation of PAX9, and PAX9 downstream events in cancer development. Promoter hypermethylation, promoter SNP, microRNA, and inhibition of upstream pathways (e.g., NOTCH) result in PAX9 silencing or downregulation, whereas gene amplification and an epigenetic axis upregulate PAX9 expression. PAX9 may contribute to carcinogenesis through dysregulation of its transcriptional targets and related molecular pathways. In summary, extensive studies on PAX9 in its cellular and tissue contexts are warranted in various cancers, in particular, HNSCC, ESCC, lung cancer, and cervical SCC.

PAX9 reactivation by inhibiting DNA methyltransferase triggers antitumor effect in oral squamous cell carcinoma.

Aberrant DNA hypermethylation is associated with oral carcinogenesis. Procaine, a local anesthetic, is a DNA methyltransferase (DNMT) inhibitor that activates anticancer mechanisms. However, its effect on silenced tumor suppressor gene (TSG) activation and its biological role in oral squamous cell carcinoma (OSCC) remain unknown. Here, we report procaine inhibited DNA methylation by suppressing DNMT activity and increased the expression of PAX9, a differentiation gene in OSCC cells. Interestingly, the reactivation of PAX9 by procaine found to inhibit cell growth and trigger apoptosis in OSCC in vitro and in vivo. Likely, the enhanced PAX9 expression after exposure to procaine controls stemness and differentiation through the autophagy-dependent pathway in OSCC cells. PAX9 inhibition abrogated procaine-induced apoptosis, autophagy, and inhibition of stemness. In OSCC cells, procaine improved anticancer drug sensitivity through PAX9, and its deficiency significantly blunted the anticancer drug sensitivity mediated by procaine. Additionally, NRF2 activation by procaine facilitated the antitumor response of PAX9, and pharmacological inhibition of NRF2 by ML385 reduced death and prevented the decrease in the orosphere-forming potential of OSCC cells. Furthermore, procaine promoted antitumor activity in FaDu xenografts in athymic nude mice, and immunohistochemistry data showed that PAX9 expression was significantly enhanced in the procaine group compared to the vehicle control. In conclusion, PAX9 reactivation in response to DNMT inhibition could trigger a potent antitumor mechanism to provide a new therapeutic strategy for OSCC.

PAX9 functions as a tumor suppressor gene for cervical cancer via modulating cell proliferation and apoptosis.

To investigate the effect of PAX9 on the progression of cervical cancer (CC). PAX9 expression was quantified in CC tissues and adjacent normal tissues, as well as human CC cell lines and human cervical epithelial cells (HCerEpiC). PAX9-overexpression lentiviral vectors were transfected into CC cell lines, followed by the measurement of proliferation and apoptosis and the quantification of apoptosis-related proteins. In vivo, mice were subcutaneously injected with CaSki cells transfected with PAX9-overexpression lentiviral vectors and control vectors. Then, the volume and weight of tumors were measured followed by hematoxylin and eosin (HE) staining, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, and immunohistochemistry. PAX9 expression in the CC tissues was lower than that in the adjacent normal tissues, which was correlated with the FIGO stage, tumor size, infiltration depth, parametrium invasion, lympho-vascular space invasion tumor-positive lymph nodes, and prognosis. Furthermore, PAX9 in CC cell lines was also lower than in HCerEpiC. PAX9 inhibits the CC cell proliferation and promotes the apoptosis, with the up-regulations of caspase-3, poly(ADP-ribose) polymerase (PARP), and Bax and the down-regulation of Bcl-2. In vivo experiments demonstrated that in the PAX9 group, the tumor weight and volume were lower than those in the vector group accompanying the decreased Ki-67, cleaved-caspase-3, and Bax expressions and the increased TUNEL and Bcl-2 expression. PAX9 was lowly expressed in the CC tissues and associated with the clinicopathological characteristics and prognosis. PAX9 could inhibit proliferation of CC cell lines and promote the apoptosis, thus suppressing the tumor growth in vivo, indicating its potential therapeutic role for CC treatment.

HES1 is a novel downstream modifier of the SHH-GLI3 Axis in the development of preaxial polydactyly.

Sonic Hedgehog/GLI3 signaling is critical in regulating digit number, such that Gli3-deficiency results in polydactyly and Shh-deficiency leads to digit number reductions. SHH/GLI3 signaling regulates cell cycle factors controlling mesenchymal cell proliferation, while simultaneously regulating Grem1 to coordinate BMP-induced chondrogenesis. SHH/GLI3 signaling also coordinates the expression of additional genes, however their importance in digit formation remain unknown. Utilizing genetic and molecular approaches, we identified HES1 as a downstream modifier of the SHH/GLI signaling axis capable of inducing preaxial polydactyly (PPD), required for Gli3-deficient PPD, and capable of overcoming digit number constraints of Shh-deficiency. Our data indicate that HES1, a direct SHH/GLI signaling target, induces mesenchymal cell proliferation via suppression of Cdkn1b, while inhibiting chondrogenic genes and the anterior autopod boundary regulator, Pax9. These findings establish HES1 as a critical downstream effector of SHH/GLI3 signaling in the development of PPD.

PAX7, PAX9 and RYK Expression in Cleft Affected Tissue.

Background and Objectives: Cleft lip with or without cleft palate is one of the most common types of congenital malformations. Transcription factors paired box 7 and 9 (PAX7, PAX9) and receptor-like tyrosine kinase (RYK) have been previously associated with the formation of orofacial clefts but their exact possible involvement and interactions in the tissue of specific cleft types remains uncertain. There is a limited number of morphological studies analyzing these specific factors in cleft affected tissue due to ethical aspects and the limited amount of available tissue material. This study analyses the presence of PAX7, PAX9, and RYK immunopositive structures within different cleft affected tissue to assess their possible involvement in cleft morphopathogenesis. Materials and Methods: Cleft affected tissue was collected from non-syndromic orofacial cleft patients during cleft correcting surgery (36 patients with unilateral cleft lip, 13 patients with bilateral cleft lip, 26 patients with isolated cleft palate). Control group oral cavity tissue was obtained from 7 patients without cleft lip and palate. To evaluate the number of immunopositive structures in the cleft affected tissue and the control group, a semiquantitative counting method was used. Non-parametric statistical methods (Kruskal-Wallis H test, Mann-Whitney U test, and Spearman's rank correlation) were used. Results: Statistically significant differences for the number of PAX7, PAX9, and RYK-positive cells were notified between the controls and the patient groups. Multiple statistically significant correlations between the factors were found in each cleft affected tissue group. Conclusions: PAX7, PAX9, and RYK have a variable involvement and interaction in postnatal morphopathogenesis of orofacial clefts. PAX7 is more associated with the formation of unilateral cleft lip, while PAX9 relates more towards the isolated cleft palate. The stable presence of RYK in all cleft types indicates its possible participation in different facial cleft formations.

Transcriptional epigenetic regulation of Fkbp1/Pax9 genes is associated with impaired sensitivity to platinum treatment in ovarian cancer.

BACKGROUND: In an effort to contribute to overcoming the platinum resistance exhibited by most solid tumors, we performed an array of epigenetic approaches, integrating next-generation methodologies and public clinical data to identify new potential epi-biomarkers in ovarian cancer, which is considered the most devastating of gynecological malignancies. METHODS: We cross-analyzed data from methylome assessments and restoration of gene expression through microarray expression in a panel of four paired cisplatin-sensitive/cisplatin-resistant ovarian cancer cell lines, along with publicly available clinical data from selected individuals representing the state of chemoresistance. We validated the methylation state and expression levels of candidate genes in each cellular phenotype through Sanger sequencing and reverse transcription polymerase chain reaction, respectively. We tested the biological role of selected targets using an ectopic expression plasmid assay in the sensitive/resistant tumor cell lines, assessing the cell viability in the transfected groups. Epigenetic features were also assessed in 189 primary samples obtained from ovarian tumors and controls. RESULTS: We identified PAX9 and FKBP1B as potential candidate genes, which exhibited epigenetic patterns of expression regulation in the experimental approach. Re-establishment of FKBP1B expression in the resistant OVCAR3 phenotype in which this gene is hypermethylated and inhibited allowed it to achieve a degree of platinum sensitivity similar to the sensitive phenotype. The evaluation of these genes at a translational level revealed that PAX9 hypermethylation leads to a poorer prognosis in terms of overall survival. We also set a precedent for establishing a common epigenetic signature in which the validation of a single candidate, MEST, proved the accuracy of our computational pipelines. CONCLUSIONS: Epigenetic regulation of PAX9 and FKBP1B genes shows that methylation in non-promoter areas has the potential to control gene expression and thus biological consequences, such as the loss of platinum sensitivity. At the translational level, PAX9 behaves as a predictor of chemotherapy response to platinum in patients with ovarian cancer. This study revealed the importance of the transcript-specific study of each gene under potential epigenetic regulation, which would favor the identification of new markers capable of predicting each patient's progression and therapeutic response.

PAX9 Determines Epigenetic State Transition and Cell Fate in Cancer.

Abnormalities in genetic and epigenetic modifications can lead to drastic changes in gene expression profiles that are associated with various cancer types. Small cell lung cancer (SCLC) is an aggressive and deadly form of lung cancer with limited effective therapies currently available. By utilizing a genome-wide CRISPR-Cas9 dropout screen in SCLC cells, we identified paired box protein 9 (PAX9) as an essential factor that is overexpressed in human malignant SCLC tumor samples and is transcriptionally driven by the BAP1/ASXL3/BRD4 epigenetic axis. Genome-wide studies revealed that PAX9 occupies distal enhancer elements and represses gene expression by restricting enhancer activity. In multiple SCLC cell lines, genetic depletion of PAX9 led to significant induction of a primed-active enhancer transition, resulting in increased expression of a large number of neural differentiation and tumor-suppressive genes. Mechanistically, PAX9 interacted and cofunctioned with the nucleosome remodeling and deacetylase (NuRD) complex at enhancers to repress nearby gene expression, which was reversed by pharmacologic HDAC inhibition. Overall, this study provides mechanistic insight into the oncogenic function of the PAX9/NuRD complex epigenetic axis in human SCLC and suggests that reactivation of primed enhancers may have potential therapeutic efficacy in treating SCLC expressing high levels of PAX9. SIGNIFICANCE: A genome-wide screen in small cell lung cancer reveals PAX9/NuRD-mediated epigenetic enhancer silencing and tumor progression, supporting the development of novel personalized therapeutic approaches targeting the PAX9-regulated network.

The clinical significance and correlative signaling pathways of paired box gene 9 in development and carcinogenesis.

Paired box 9 (PAX9) gene belongs to the PAX family, which encodes a family of metazoan transcription factors documented by a conserved DNA binding paired domain 128-amino-acids, critically essential for physiology and development. It is primarily expressed in embryonic tissues, such as the pharyngeal pouch endoderm, somites, neural crest-derived mesenchyme, and distal limb buds. PAX9 plays a vital role in craniofacial development by maintaining the odontogenic potential, mutations, and polymorphisms associated with the risk of tooth agenesis, hypodontia, and crown size in dentition. The loss-of-function of PAX9 in the murine model resulted in a short life span due to the arrest of cleft palate formation and skeletal abnormalities. According to recent studies, the PAX9 gene has a significant role in maintaining squamous cell differentiation, odontoblast differentiation of pluripotent stem cells, deregulation of which is associated with tumor initiation, and malignant transformation. Moreover, PAX9 contributes to promoter hypermethylation and alcohol- induced oro-esophageal squamous cell carcinoma mediated by downregulation of differentiation and apoptosis. Likewise, PAX9 activation is also reported to be associated with drug sensitivity. In summary, this current review aims to understand PAX9 function in the regulation of development, differentiation, and carcinogenesis, along with the underlying signaling pathways for possible cancer therapeutics.

Pax9 is essential for granulopoiesis but dispensable for erythropoiesis in zebrafish.

Paired Box (Pax) gene family, a group of transcription regulators have been implicated in diverse physiological processes. However, their role during hematopoiesis which generate a plethora of blood cells remains largely unknown. Using a previously reported single cell transcriptomics data, we analyzed the expression of individual Pax family members in hematopoietic cells in zebrafish. We have identified that Pax9, which is an essential regulator for odontogenesis and palatogenesis, is selectively localized within a single cluster of the hematopoietic lineage. To further analyze the function of Pax9 in hematopoiesis, we generated two independent pax9 knock-out mutants using the CRISPR-Cas9 technique. We found that Pax9 appears to be an essential regulator for granulopoiesis but dispensable for erythropoiesis during development, as lack of pax9 selectively decreased the number of neutrophils with a concomitant decrease in the expression level of neutrophil markers. In addition, embryos, where pax9 was functionally disrupted by injecting morpholinos, failed to increase the number of neutrophils in response to pathogenic bacteria, suggesting that Pax9 is not only essential for developmental granulopoiesis but also emergency granulopoiesis. Due to the inability to initiate emergency granulopoiesis, innate immune responses were severely compromised in pax9 morpholino-mediated embryos, increasing their susceptibility and mortality. Taken together, our data indicate that Pax9 is essential for granulopoiesis and promotes innate immunity in zebrafish larvae.

Tooth agenesis: What do we know and is there a connection to cancer?

Like all developmental processes, odontogenesis is highly complex and dynamically regulated, with hundreds of genes co-expressed in reciprocal networks. Tooth agenesis (missing one or more/all teeth) is a common human craniofacial anomaly and may be caused by genetic variations and/or environmental factors. Variants in PAX9, MSX1, AXIN2, EDA, EDAR, and WNT10A genes are associated with tooth agenesis. Currently, variants in ATF1, DUSP10, CASC8, IRF6, KDF1, GREM2, LTBP3, and components and regulators of WNT signaling WNT10B, LRP6, DKK, and KREMEN1 are at the forefront of interest. Due to the interconnectedness of the signaling pathways of carcinogenesis and odontogenesis, tooth agenesis could be a suitable marker for early detection of cancer predisposition. Variants in genes associated with tooth agenesis could serve as prognostic or therapeutic targets in cancer. This review aims to summarize existing knowledge of development and clinical genetics of teeth. Concurrently, the review proposes possible approaches for future research in this area, with particular attention to roles in monitoring, early diagnosis and therapy of tumors associated with defective tooth development.

Family-based association study of genetic analysis of paired box gene 9 polymorphisms in the peg-shaped teeth in the Jordanian Arab population.

OBJECTIVE: The aim of this study is to genotype thirteen Single Nucleotide Polymorphisms (SNPs) within the paired box gene 9 (PAX9) in 36 Jordanian Arab families with peg-shaped teeth, and also to investigate the association between the PAX9 gene and peg-shaped teeth disorder. METHODS: Genomic DNA samples were extracted from families according to distinguished processes. Then, DNA was amplified by polymerase chain reaction technique (PCR) using specified primers for the exons of the PAX9 gene. In addition, single nucleotide polymorphisms analysis was conducted using the DNA sequencing genotyping method to identify specific single nucleotide polymorphisms in the PAX9 gene associated with peg-shaped teeth. RESULTS: Thirteen single nucleotide polymorphisms in the PAX9 gene (Chromosome 14q13.3) were used; seven of them (rs104894467, rs104894469, rs28933373, rs28933970, rs28933971, rs28933972, and rs7143727) were non-polymorphic, and the other six were polymorphic (rs2073244, rs2073246, rs2295222, rs4904155, rs4904210, and rs12881240). Both rs12881240 and rs2295222 SNPs showed significant association with peg-shaped teeth disorder (P < 0.05). Moreover, the haplotype genetic analysis revealed that there is a genetic association with peg-shaped teeth disorder susceptibility (P < 0.05) in the Jordanian families of Arab descent. CONCLUSION: Our findings exhibited significant variations compared to the data recorded from other countries.

Systematic analysis of differentially methylated expressed genes and site-specific methylation as potential prognostic markers in head and neck cancer.

Head and neck cancer (HNC) remains one of the most malignant tumors with a significantly high mortality. DNA methylation exerts a vital role in the prognosis of HNC. In this study, we try to screen abnormal differential methylation genes (DMGs) and pathways in Head-Neck Squamous Cell Carcinoma via integral bioinformatics analysis. Data of gene expression microarrays and gene methylation microarrays were obtained from the Cancer Genome Atlas database. Aberrant DMGs were identified by the R Limma package. We conducted the Cox regression analysis to select the prognostic aberrant DMGs and site-specific methylation. Five aberrant DMGs were recognized that significantly correlated with overall survival. The prognostic model was constructed based on five DMGs (PAX9, STK33, GPR150, INSM1, and EPHX3). The five DMG models acted as prognostic biomarkers for HNC. The area under the curve based on the five DMGs predicting 5-year survival is 0.665. Moreover, the correlation between the DMGs/site-specific methylation and gene expression was also explored. The findings demonstrated that the five DMGs can be used as independent prognostic biomarkers for predicting the prognosis of patients with HNC. Our study might lay the groundwork for further mechanism exploration in HNC and may help identify diagnostic biomarkers for early stage HNC.

Familial oligodontia and regional odontodysplasia associated with a PAX9 initiation codon mutation.

OBJECTIVE: Tooth agenesis is one of the most common craniofacial developmental anomalies. In hypodontia, one to five teeth are missing, whereas oligodontia refers to the absence of at least six teeth, excluding the third molars. Mutations in several genes including MSX1, PAX9, AXIN2, and WNT10A have been shown to cause non-syndromic tooth agenesis. Regional odontodysplasia (RO), also known as "ghost teeth," is a rare developmental anomaly of tooth formation affecting both dentitions. Some possible causes of RO have been suggested, yet the etiology remains unknown. Because the phenotypes of both oligodontia and RO co-occur in one Finnish family, the aim here was to investigate the genetic etiology of the two conditions. MATERIALS AND METHODS: A mutation screening of the genes MSX1, PAX9, AXIN2, and WNT10A was performed for the family members of a RO patient and family history of oligodontia. RESULTS: An initiation codon mutation of the PAX9 gene was found in the proband and segregating with oligodontia in the family. CONCLUSIONS: The etiology of regional odontodysplasia (RO) may be genetic and the same genes can be involved both in RO and tooth agenesis. CLINICAL RELEVANCE: Our results give new insights into the etiology of regional odontodysplasia, yet further results are needed.

Genetic analysis: Wnt and other pathways in nonsyndromic tooth agenesis.

Tooth agenesis (TA) is one of the most common developmental anomalies that affects the number of teeth. An extensive analysis of publicly accessible databases revealed 15 causative genes responsible for nonsyndromic TA, along with their signaling pathways in Wnt/ß-catenin, TGF-ß/BMP, and Eda/Edar/NF-κB. However, genotype-phenotype correlation analysis showed that most of the causal genes are also responsible for syndromic TA or other conditions. In a total of 198 different mutations of the 15 genes responsible for nonsyndromic TA, 182 mutations (91.9%) are derived from seven genes (AXIN2, EDA, LRP6, MSX1, PAX9, WNT10A, and WNT10B) compared with the remaining 16 mutations (8.1%) identified in the remaining eight genes (BMP4, DKK1, EDAR, EDARADD, GREM2, KREMEN1, LTBP3, and SMOC2). Furthermore, specificity analysis in terms of the ratio of nonsyndromic TA mutations versus syndromic mutations in each of the aforementioned seven genes showed a 98.2% specificity rate in PAX9, 58.9% in WNT10A, 56.6% in MSX1, 41.2% in WNT10B, 31.4% in LRP6, 23.8% in AXIN2%, and 8.4% in EDA. These findings underscore an important role of the Wnt and Wnt-associated pathways in the genetic etiology of this heterozygous disease and shed new lights on the discovery of novel molecular mechanisms associated with tooth agenesis.

Novel mutations identified in patients with tooth agenesis by whole-exome sequencing.

OBJECTIVES: To identify potentially pathogenic mutations for tooth agenesis by whole-exome sequencing. SUBJECTS AND METHODS: Ten Chinese families including five families with ectodermal dysplasia (syndromic tooth agenesis) and five families with selective tooth agenesis were included. Whole-exome sequencing was performed using genomic DNA. Potentially pathogenic mutations were identified after data filtering and screening. The pathogenicity of novel variants was investigated by segregation analysis, in silico analysis, and functional studies. RESULTS: One novel mutation (c.441_442insACTCT) and three reported mutations (c.252delT, c.463C>T, and c.1013C>T) in EDA were identified in families with ectodermal dysplasia. The novel EDA mutation was co-segregated with phenotype. A functional study revealed that NF-κB activation was compromised by the identified mutations. The secretion of active EDA was also compromised detection by western blotting. Novel Wnt10A mutations (c.521T>C and c.653T>G) and EVC2 mutation (c.1472C>T) were identified in families with selective tooth agenesis. The Wnt10A c.521T>C mutation and the EVC2 c.1472C>T mutation were considered as pathogenic for affecting highly conserved amino acids, co-segregated with phenotype and predicted to be disease-causing by SIFT and PolyPhen2. Moreover, several reported mutations in PAX9, Wnt10A, and FGFR3 were also detected. CONCLUSIONS: Our study expanded our knowledge on tooth agenesis spectrum by identifying novel variants.

Bridging the molecular divide: alcohol-induced downregulation of PAX9 and tumour development.

The pathogenesis of oro-oesophaeal squamous cell carcinoma is causally linked to the consumption of alcohol. Beyond the carcinogenic effects of ethanol and its metabolites via DNA damage, the precise mechanisms by which alcohol drives tumourigenesis remain to be fully elucidated. A novel contributor now revealed is aberrant differentiation and proliferation mediated by suppression of PAX9, a key regulator of normal squamous maturation in oro-oesophageal tissues. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

PAX9 regulates squamous cell differentiation and carcinogenesis in the oro-oesophageal epithelium.

PAX9 is a transcription factor of the PAX family characterized by a DNA-binding paired domain. Previous studies have suggested a potential role of PAX9 in squamous cell differentiation and carcinogenesis of the oro-oesophageal epithelium. However, its functional roles in differentiation and carcinogenesis remain unclear. In this study, Pax9 deficiency in mouse oesophagus promoted cell proliferation, delayed cell differentiation, and altered the global gene expression profile. Ethanol exposure downregulated PAX9 expression in human oesophageal epithelial cells in vitro and mouse forestomach and tongue in vivo. We further showed that PAX9 was downregulated in human oro-oesophageal squamous cell carcinoma (OESCC), and its downregulation was associated with alcohol drinking and promoter hypermethylation. Moreover, ad libitum feeding with a liquid diet containing ethanol for 40 weeks or Pax9 deficiency promoted N-nitrosomethylbenzylamine-induced squamous cell carcinogenesis in mouse tongue, oesophagus, and forestomach. In conclusion, PAX9 regulates squamous cell differentiation in the oro-oesophageal epithelium. Alcohol drinking and promoter hypermethylation are associated with PAX9 silencing in human OESCC. PAX9 downregulation may contribute to alcohol-associated oro-oesophageal squamous cell carcinogenesis. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Multiethnic GWAS Reveals Polygenic Architecture of Earlobe Attachment.

The genetic basis of earlobe attachment has been a matter of debate since the early 20th century, such that geneticists argue both for and against polygenic inheritance. Recent genetic studies have identified a few loci associated with the trait, but large-scale analyses are still lacking. Here, we performed a genome-wide association study of lobe attachment in a multiethnic sample of 74,660 individuals from four cohorts (three with the trait scored by an expert rater and one with the trait self-reported). Meta-analysis of the three expert-rater-scored cohorts revealed six associated loci harboring numerous candidate genes, including EDAR, SP5, MRPS22, ADGRG6 (GPR126), KIAA1217, and PAX9. The large self-reported 23andMe cohort recapitulated each of these six loci. Moreover, meta-analysis across all four cohorts revealed a total of 49 significant (p < 5 × 10-8) loci. Annotation and enrichment analyses of these 49 loci showed strong evidence of genes involved in ear development and syndromes with auricular phenotypes. RNA sequencing data from both human fetal ear and mouse second branchial arch tissue confirmed that genes located among associated loci showed evidence of expression. These results provide strong evidence for the polygenic nature of earlobe attachment and offer insights into the biological basis of normal and abnormal ear development.

Genome-wide DNA methylation profiling integrated with gene expression profiling identifies PAX9 as a novel prognostic marker in chronic lymphocytic leukemia.

BACKGROUND: In chronic lymphocytic leukemia (CLL), epigenomic and genomic studies have expanded the existing knowledge about the disease biology and led to the identification of potential biomarkers relevant for implementation of personalized medicine. In this study, an attempt has been made to examine and integrate the global DNA methylation changes with gene expression profile and their impact on clinical outcome in early stage CLL patients. RESULTS: The integration of DNA methylation profile (n = 14) with the gene expression profile (n = 21) revealed 142 genes as hypermethylated-downregulated and; 62 genes as hypomethylated-upregulated in early stage CLL patients compared to CD19+ B-cells from healthy individuals. The mRNA expression levels of 17 genes identified to be differentially methylated and/or differentially expressed was further examined in early stage CLL patients (n = 93) by quantitative real time PCR (RQ-PCR). Significant differences were observed in the mRNA expression of MEIS1, PMEPA1, SOX7, SPRY1, CDK6, TBX2, and SPRY2 genes in CLL cells as compared to B-cells from healthy individuals. The analysis in the IGHV mutation based categories (Unmutated = 39, Mutated = 54) revealed significantly higher mRNA expression of CRY1 and PAX9 genes in the IGHV unmutated subgroup (p < 0.001). The relative risk of treatment initiation was significantly higher among patients with high expression of CRY1 (RR = 1.91, p = 0.005) or PAX9 (RR = 1.87, p = 0.001). High expression of CRY1 (HR: 3.53, p < 0.001) or PAX9 (HR: 3.14, p < 0.001) gene was significantly associated with shorter time to first treatment. The high expression of PAX9 gene (HR: 3.29, 95% CI 1.172-9.272, p = 0.016) was also predictive of shorter overall survival in CLL. CONCLUSIONS: The DNA methylation changes associated with mRNA expression of CRY1 and PAX9 genes allow risk stratification of early stage CLL patients. This comprehensive analysis supports the concept that the epigenetic changes along with the altered expression of genes have the potential to predict clinical outcome in early stage CLL patients.