pre-B-cell Leukemia Homeobox Research Articles

Integrated multi-omics analysis of PBX1 in mouse adult neural stem- and progenitor cells identifies a transcriptional module that functionally links PBX1 to TCF3/4.

Developmental transcription factors act in networks, but how these networks achieve cell- and tissue specificity is still poorly understood. Here, we explored pre-B cell leukemia homeobox 1 (PBX1) in adult neurogenesis combining genomic, transcriptomic, and proteomic approaches. ChIP-seq analysis uncovered PBX1 binding to numerous genomic sites. Integration of PBX1 ChIP-seq with ATAC-seq data predicted interaction partners, which were subsequently validated by mass spectrometry. Whole transcriptome spatial RNA analysis revealed shared expression dynamics of Pbx1 and interacting factors. Among these were class I bHLH proteins TCF3 and TCF4. RNA-seq following Pbx1, Tcf3 or Tcf4 knockdown identified proliferation- and differentiation associated genes as shared targets, while sphere formation assays following knockdown argued for functional cooperativity of PBX1 and TCF3 in progenitor cell proliferation. Notably, while physiological PBX1-TCF interaction has not yet been described, chromosomal translocation resulting in genomic TCF3::PBX1 fusion characterizes a subtype of acute lymphoblastic leukemia. Introducing Pbx1 into Nalm6 cells, a pre-B cell line expressing TCF3 but lacking PBX1, upregulated the leukemogenic genes BLK and NOTCH3, arguing that functional PBX1-TCF cooperativity likely extends to hematopoiesis. Our study hence uncovers a transcriptional module orchestrating the balance between progenitor cell proliferation and differentiation in adult neurogenesis with potential implications for leukemia etiology.

PBX1 and PBX3 transcription factors regulate SHH expression in the Frontonasal Ectodermal Zone through complementary mechanisms.

Sonic hedgehog (SHH) signaling from the frontonasal ectodermal zone (FEZ) is a key regulator of craniofacial morphogenesis. Along with SHH, pre-B-cell leukemia homeobox (PBX) transcription factors regulate midfacial development. PBXs act in the epithelium during fusion of facial primordia, but their specific interactions with SHH have not been fully investigated. We hypothesized that PBX1/3 regulate SHH expression in the FEZ by activating or repressing transcription. The hypothesis was tested by manipulating PBX1/3 expression in chick embryos and profiling epigenomic landscapes at early developmental stages. PBX1/3 expression was perturbed in the chick face beginning at stage 10 (HH10) using RCAS viruses, and the resulting SHH expression was assessed at HH22. Overexpressing PBX1 expanded SHH expression, while overexpressing PBX3 decreased SHH expression. Conversely, reducing PBX1 expression decreased SHH expression, but reducing PBX3 induced ectopic SHH expression. We performed ATAC-seq and mapped binding of PBX1 and PBX3 with ChIP-seq on the FEZ at HH22 to assess direct interactions of PBX1/3 with the SHH locus. These multi-omics approaches uncovered a 400 bp PBX1-enriched element within intron 1 of SHH (chr2:8,173,222-8,173,621). Enhancer activity of this element was demonstrated by electroporation of reporter constructs in ovo and luciferase reporter assays in vitro . When bound by PBX1, this element upregulates transcription, while it downregulates transcription when bound by PBX3. The present study identifies a cis- regulatory element, named SFE1, that interacts with PBX1/3 to modulate SHH expression in the FEZ and establishes that PBX1 and PBX3 play complementary roles in SHH regulation during embryonic development.

Regulation of the STAT3 pathway by lupus susceptibility gene Pbx1 in T cells

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease in which poorly characterized genetic factors lead to the production of proinflammatory or autoreactive T cells. Pre-B cell leukemia homeobox 1 (PBX1) is a transcription factor whose dominant negative isoform (PBX1-D) is overexpressed in the CD4+ T cells of SLE patients and lupus-prone mice. Pbx1-D overexpression favors the expansion of proinflammatory T cells and impairs regulatory T (Treg) cell development. Here we show that Pbx1 deficiency and Pbx1-D overexpression decreased STAT3 expression and activation in T cells. Accordingly, Pbx1 deficiency in T cells and Pbx1-D overexpression reduced STAT3-dependent TH17 cell polarization in vitro, but it had no effect in vivo at steady state. STAT3-dependent follicular helper T (TFH) cell polarization in vitro and splenic TFH cell frequency were not affected by either Pbx1 deficiency or Pbx1-D overexpression. Pbx1 deficiency also increased the expression of cell cycle arrest and pro-apoptotic genes, with an increased apoptosis in T cells. Our results suggest a complex interplay between PBX1 and STAT3, which may contribute to lupus pathogenesis through dysregulation of the cell cycle and apoptosis.

Molecular Mechanisms of Lupus Susceptibility Allele PBX1D.

Pre-B cell leukemia homeobox 1 (PBX1) controls chromatin accessibility to a large number of genes in various cell types. Its dominant negative splice isoform, PBX1D, which lacks the DNA and Hox-binding domains, is expressed more frequently in the CD4+ T cells from lupus-prone mice and patients with systemic lupus erythematosus than healthy control subjects. PBX1D overexpression in CD4+ T cells impaired regulatory T cell homeostasis and expanded inflammatory CD4+ T cells. In this study, we showed that PBX1 message expression is downregulated by activation in CD4+ T cells as well as in B cells. PBX1D protein was less stable than the normal isoform, PBX1B, and it is degraded through the ubiquitin-proteasome-dependent pathway. The DNA binding domain lacking in PBX1D has two putative ubiquitin binding sites, K292 and K293, that are predicted to be in direct contact with DNA. Mutation of K292-293 reduced PBX1B stability to a level similar to PBX1D and abrogated DNA binding. In addition, contrary to PBX1B, PBX1D is retained in the cytoplasm without the help of the cofactors MEIS or PREP1, indicating a different requirement for nuclear translocation. Overall, these findings suggest that multiple post-transcriptional mechanisms are responsible for PBX1D loss of function and induction of CD4+ T cell inflammatory phenotypes in systemic lupus erythematosus.

The PBX1/miR-141-miR-200a/EGR2/SOCS3 Axis; Integrative Analysis of Interaction Networks to Discover the Possible Mechanism of MiR-141 and MiR-200a-Mediated Th17 Cell Differentiation.

Overexpression of miR-141 and miR-200a is known to be associated with the differentiation of T helper 17 (Th17) cells, which are key players in the pathophysiology of autoimmune disorders. However, the function and governing mechanism of these two microRNAs (miRNAs) in Th17 cell skewing are poorly defined. The aim of the present study was to identify the common upstream transcription factors and downstream target genes of miR-141 and miR-200a to obtain a better insight into the possible dysregulated molecular regulatory networks driving miR-141/miR-200a-mediated Th17 cell development. A consensus-based prediction strategy was applied for in-silico identification of potential transcription factors and putative gene targets of miR-141 and miR-200a. Thereafter, we analyzed the expression patterns of candidate transcription factors and target genes during human Th17 cell differentiation by quantitative real-time PCR and examined the direct interaction between both miRNAs and their potential target sequences using dual-luciferase reporter assays. According to our miRNA-based and gene-based interaction network analyses, pre-B cell leukemia homeobox (PBX1) and early growth response 2 (EGR2) were respectively taken into account as the potential upstream transcription factor and downstream target gene of miR-141 and miR-200a. There was a significant overexpression of the PBX1 gene during the Th17 cell induction period. Furthermore, both miRNAs could directly target EGR2 and inhibit its expression. As a downstream gene of EGR2, the suppressor of cytokine signaling 3 (SOCS3) was also downregulated during the differentiation process. These results indicate that activation of the PBX1/miR-141-miR-200a/EGR2/SOCS3 axis may promote Th17 cell development and, therefore, trigger or exacerbate Th17-mediated autoimmunity.

Identifying the Potential Roles of PBX4 in Human Cancers Based on Integrative Analysis.

PBX4 belongs to the pre-B-cell leukemia homeobox (PBX) transcription factors family and acts as a transcriptional cofactor of HOX proteins participating in several pathophysiological processes. Recent studies have revealed that the dysregulation of PBX4 is closely related to multiple diseases, especially cancers. However, the research on PBX4’s potential roles in 33 cancers from the Cancer Genome Atlas (TCGA) is still insufficient. Therefore, we performed a comprehensive pan-cancer analysis to explore the roles of PBX4with multiple public databases. Our results showed that PBX4 was differentially expressed in 17 types of human cancer and significantly correlated to the pathological stage, tumor grade, and immune and molecular subtypes. We used the Kaplan–Meier plotter and PrognoScan databases to find the significant associations between PBX4 expression and prognostic values of multiple cancers. It was also found that PBX4 expression was statistically related to mutation status, DNA methylation, immune infiltration, drug sensitivity, and immune checkpoint blockade (ICB) therapy. Additionally, we found that PBX4 was involved in different functional states of multiple cancers from the single-cell resolution perspective. Enrichment analysis results showed that PBX4-related genes were enriched in the cell cycle process, MAPK cascade, ncRNA metabolic process, positive regulation of GTPase activity, and regulation of lipase activity and mainly participated in the pathways of cholesterol metabolism, base excision repair, herpes simplex virus 1 infection, transcriptional misregulation in cancer, and Epstein–Barr virus infection. Altogether, our integrative analysis could help in better understanding the potential roles of PBX4 in different human cancers.

Lupus Susceptibility Gene Pbx1 Regulates STAT3 in T cells via JAK2/STAT3 Signaling Pathway

Abstract Systemic Lupus Erythematosus (SLE) is an autoimmune disease in which poorly characterized genetic factors lead to the production of autoreactive or inflammatory T cells. Pre-B cell leukemia homeobox 1 (Pbx1) is a transcription factor whose dominant negative splice isoform (Pbx1-D) is overexpressed in CD4 T cells of lupus patients and lupus-prone mice as compared to the normal isoform (Pbx1-B). Pbx1-D overexpression impairs Treg cell development and function while favoring the production of follicular helper T cells. Based on previous studies showing that Pbx1 promotes cell proliferation via JAK2/STAT3 signaling pathway in a renal cell carcinoma, we hypothesized that Pbx1-D decreases T cell proliferation and viability by attenuating the JAK2/STAT3 signaling pathway. U3A STAT3 reporter cells transfected with Pbx1-B plasmid have a greater luciferase expression, and hence pSTAT3 transcriptional activity in comparison to cells transfected with Pbx1-D or control plasmids. We also observed that Jurkat T cells stably overexpressing Pbx1-B increases STAT3 gene expression as compared to Pbx1-D. Furthermore, Pbx1 silencing in Jurkat T cells leads to the attenuation of STAT3 message. These results suggest a mechanism by which Pbx1 contributes to impaired T cells in lupus pathogenesis is via the JAK2/STAT3 pathway. Current studies are conducted to dissect the mechanism by which Pbx1 regulates this pathway in primary T cells. Supported by a grant from the NIH (R01 AI045050) to LM.

USF1-ATRAP-PBX3 Axis Promote Breast Cancer Glycolysis and Malignant Phenotype by Activating AKT/mTOR Signaling

Angiotensin II type 1 receptor-associated protein (ATRAP) is widely expressed in different tissues and organs, although its mechanistic role in breast cancer remains unclear. Here, we show that ATRAP is highly expressed in breast cancer tissues. Its aberrant upregulation promotes breast cancer aggressiveness and is positively correlated with poor prognosis. Functional assays revealed that ATRAP participates in promoting cell growth, metastasis, and aerobic glycolysis, while microarray analysis showed that ATRAP can activate the AKT/mTOR signaling pathway in cancer progression. In addition, ATRAP was revealed to direct Ubiquitin-specific protease 14 (USP14)-mediated deubiquitination and stabilization of Pre-B cell leukemia homeobox 3 (PBX3). Importantly, ATRAP is a direct target of Upstream stimulatory factor 1 (USF1), and that ATRAP overexpression reverses the inhibitory effects of USF1 knockdown. Our study demonstrates the broad contribution of the USF1/ATRAP/PBX3 axis to breast cancer progression and provides a strong potential therapeutic target.

Regulation of the JAK2/STAT3 Signaling Pathway by the Lupus Susceptibility Gene Pbx1

Abstract Systemic Lupus Erythematosus (SLE) is an autoimmune disease in which poorly characterized genetic factors lead to the production of autoreactive or inflammatory T cells. Pre-B cell leukemia homeobox 1 (Pbx1) is a transcription factor whose dominant negative splice isoform (Pbx1-D) is overexpressed in CD4 T cells of lupus patients and lupus-prone mice as compared to the normal isoform (Pbx1-B). Pbx1-D overexpression impairs Treg cell development and function while favoring the production of follicular helper T cells. Based on previous studies showing that Pbx1 promotes cell proliferation via JAK2/STAT3 signaling pathway in a renal cell carcinoma, we hypothesized that Pbx1-D decreases T cell proliferation and viability by attenuating the JAK2/STAT3 signaling pathway. Pbx1-B or Pbx1-D expression plasmids increased pSTAT3 protein levels but there was no difference between the two isoforms in 293T cells. U3A STAT3 reporter cells transfected with Pbx1-B plasmid have greater luciferase expression, and hence pSTAT3 transcriptional activity in comparison to cells transfected with Pbx1-D or control plasmids. We also observed that Pbx1-B increases the expression of Jak2 and Stat3 message as compared to Pbx1-D. These results suggest a mechanism by which Pbx1 contributes to impaired T cells in lupus pathogenesis is via the JAK2/STAT3 pathway. Current studies are conducted to dissect the mechanism by which Pbx1 regulates this pathway in primary T cells. Supported by a grant from the NIH (RO1AI04505021) to LM

MiR-129-5p suppresses proliferation, migration, and induces apoptosis in pancreatic cancer cells by targeting PBX3.

Pancreatic cancer (PC) is the seventh most frequent cause of cancer-related deaths worldwide with a high mortality. MicroRNAs (miRNAs) act as important regulators for the development of PC and participate in the progression of PC. miR-129-5p was reported to regulate the progression of tumors, such as thyroid cancer and gastric cancer. However, the function of miR-129-5p in PC is still unclear. In this study, the down-regulation of miR-129-5p was detected in PC tissues and PC cells. miR-129-5p was overexpressed or knocked down in AsPC-1 and BxPC-3 cells. The results showed that miR-129-5p overexpression suppressed proliferation, migration and invasion, and induced apoptosis of PC cells, whereas miR-129-5p knockdown showed opposite effects. In addition, we found that pre-B-cell leukemia homeobox 3 (PBX3) overexpression promoted proliferation, migration and invasion, but reduced apoptosis of PC cells. PBX3 was identified as a target of miR-129-5p by informatics analysis and dual luciferase reporter assay. Finally, our results indicated that miR-129-5p suppressed cell proliferation and migration by targeting PBX3. This study demonstrated that miR-129-5p could function as a tumor suppressor in the progression and development of PC by targeting PBX3, providing a reliable prognostic factor and a new therapeutic strategy for PC.

Abstract 5215: Targeting of HOX-PBX binding in glioblastoma multiforme as a novel therapeutic treatment

Abstract The HOX genes encode a family of transcription factors that play an essential role in embryonic patterning. They are also aberrantly expressed in numerous cancers, including glioblastoma (GBM). Three Amino Acid Loop Extension Homeobox (TALE) proteins act as important co-factors for HOX proteins, modulating their binding affinities to genomic targets. TALE proteins include the Pre-B-cell leukemia homeobox (PBX) proteins 1-4, which bind anterior HOX proteins, facilitating their nuclear entry and limiting their degradation. HTL00-1 is a 2nd generation hexapeptide drug that inhibits HOX-PBX dimer formation, and has been shown to induce rapid apoptosis in cancer cells, but not normal cells, through the rapid upregulation of genes including cFos, DUSP1, and EGR1. We have found that both HOX and TALE genes are markedly dysregulated in primary GBM tumors as well as in murine (GL261), adult (LN18, U87-MG, U251-MG) and pediatric (KNS42, SF188) GBM cell lines, all of which are sensitive to HTL-001. These genes were even more highly expressed in experimentally induced GBM cancer stem cells (CSCs) compared with parental lines, with a corresponding increase in sensitivity to HTL-001. We also investigated the in vivo activity of HTL-001 in Black 6 mice carrying GL-261 subcutaneous and orthotropic tumors with twice weekly intraperitoneal delivery. HTL-001 was shown to cross the blood brain barrier using Alexa660 labelled peptide, and significant anti-tumor activity was observed in both subcutaneous and orthotropic models with increased survival (p<0.02 and p<0.0078, respectively). Resected tumors from HTL-001 treated mice showed marked evidence of apoptosis, tumor vasculature disruption and focal necrosis compared to untreated tumors. Taken together, our findings indicate that HOX-PBX inhibition is a potential therapeutic target for adult and pediatric GBM patients. Citation Format: Richard Morgan, Monika Primon, Steven Shnyder, Susan Short, Balveen Kaur, Bangxing Hong, Izhar Bagwan, William Rogers, Hardev S. Pandha. Targeting of HOX-PBX binding in glioblastoma multiforme as a novel therapeutic treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5215.

Effect of pre-B-cell leukemia homeobox 3 gene small interfering RNA on apoptosis of glioma cells in vitro

Objective To investigate the effect of pre-B-cell leukemia homeobox 3 (PBX3) gene small interfering RNA (siRNA) on the viability and apoptosis of glioma cells. Methods According to Lipofectamine™ 2000, the specific siRNA of PBX3 (PBX3-siRNA group) was transfected into human glioma U87 cells, and the negative control siRNA (negative group) was transfected, and the blank group was set up, cell was transfected for 48 h, the expression of p38, p-p38, p53, cysteinyl aspartate specific proteinase-3 (Caspase-3) and cleaved Caspase-3 protein were detected by Western blotting. The cell apoptosis rate was detected by flow cytometry. Methyl thiazol tetrazolium (MTT) was used to detect the cell viability after PBX3-siRNA was transfected cells for 24, 48 and 72 h. Results The expression of PBX3 in U87 cells transfected with PBX3-siRNA was significantly lower than that in blank group (Protein relative expression were 0.096±0.010 and 0.467±0.051, P=0.000). Compared with the blank group, the cell viability in PBX3-siRNA group (24, 48, 72 h were 0.336±0.034, 0.549±0.057, 0.671±0.073, P24 h=0.001, P48 h=0.004, P72 h=0.004) decreased significantly PBX3-siRNA was transfected for 24-72 h, the apoptosis rate increased significantly, the expression of p-p38 protein(0.031±0.006) decreased significantly, and the expression of p53(0.388±0.041) and Cleaved-Caspase-3 protein (0.205±0.018) increased significantly (Pp-p38=0.000, Pp53=0.000, PCleaved-Caspase-3=0.000). There was no significant difference in the expression of p38 and Caspase-3 between the three groups (Pp38=0.987, PCaspase-3=0.871). Conclusion PBX3 gene siRNA can inhibit glioma cell viability and induce apoptosis, which may be related to downregulation of p38MAPK signal. Key words: Glioma; Pre-B-cell leukemia homeobox 3 gene; Apoptosis; p38 mitogen-activated protein kinases signal

Metabolic Functions of the Murine Lupus Susceptibility Gene Pbx1

Abstract Systemic Lupus Erythematosus (SLE) is an autoimmune disease that affects many organ systems. Poorly characterized genetic factors contribute to SLE, in part through the production of autoreactive or inflammatory T cells. Pre-B cell leukemia homeobox 1 (Pbx1) is a transcription factor whose Pbx1-d dominant negative splice isoform is overexpressed in CD4T cells of lupus patients as well as in the NZM2410 lupus-prone mouse as compared to the normal Pbx1-b isoform. Based on gene expression studies comparing murine CD4 T cells overexpressing Pbx1-d to controls, we hypothesize that Pbx1-d enhances cellular metabolism in T cells through the HIF1α and mTORc1 pathways. CD4 T cells expressing Pbx1-d present a higher cellular metabolism and show a higher mTORc1 activation than normal control T cells. Using mesenchymal stem cells, we showed that transfection of Pbx1-d was sufficient to increase glycolysis, a pathway linked to T cell activation. We found that Ddit4, an mTORc1 inhibitor, shows a lower expression in the Pbx1-d-expressing CD4 T cells than in normal T cells. We also discovered that Pbx1-d preferentially binds to the promoter of Ddit4, as well as Egln1 and Egln3, two HIF1a inhibitors. These results suggest that a mechanism by which the Pbx1-d allele contributes to lupus pathogenesis is to enhance CD4 T cell metabolism. Future work will define how Pbx1 controls the immune system and how the function of this transcription factor is linked to cellular metabolism.

MiR-320 inhibits the growth of glioma cells through downregulating PBX3

BackgroundMiR-320 is downregulated in multiple cancers, including glioma and acts as tumor suppressor through inhibiting tumor cells proliferation and inducing apoptosis. PBX3 (Pre-B cell leukemia homeobox 3), a putative target gene of miR-320, has been reported to be upregulated in various tumors and promote tumor cell growth through regulating MAKP/ERK pathway. This study aimed to verify whether miR-320 influences glioma cells growth through regulating PBX3.MethodsTwenty-four human glioma and paired adjacent nontumorous tissues were collected for determination of miR-320 and PBX3 expression using RT-qPCR and western blot assays. Luciferase reporter assay was performed to verify the interaction between miR-320 and its targeting sequence in the 3′ UTR of PBX3 in glioma cells U87 and U251. Increased miR-320 level in U87 and U251 cells was achieved through miR-320 mimic transfection and the effect of which on glioma cells growth, proliferation, cell cycle, apoptosis and activation of Raf-1/MAPK pathway was determined using MTT, colony formation, flow cytometry and western blot assays. PBX3 knockdown was performed using shPBX3 and the influence on MAPK pathway activation was evaluated.ResultsMiR-320 downregulation and PBX3 upregulation was found in glioma tissues. Luciferase reporter assays identified miR-320 directly blinds to the 3′ UTR of PBX3 in glioma cells. MiR-320 mimic transfection suppressed glioma cells proliferation, and induced cell cycle arrest and apoptosis. Both miR-320 overexpression and PBX3 knockdown inhibited Raf-1/MAPK activation.ConclusionMiR-320 may suppress glioma cells growth and induced apoptosis through the PBX3/Raf-1/MAPK axis, and miR-320 oligonucleotides may be a potential cancer therapeutic for glioma.

MEIS homeodomain proteins facilitate PARP1/ARTD1-mediated eviction of histone H1.

Pre-B-cell leukemia homeobox (PBX) and myeloid ecotropic viral integration site (MEIS) proteins control cell fate decisions in many physiological and pathophysiological contexts, but how these proteins function mechanistically remains poorly defined. Focusing on the first hours of neuronal differentiation of adult subventricular zone-derived stem/progenitor cells, we describe a sequence of events by which PBX-MEIS facilitates chromatin accessibility of transcriptionally inactive genes: In undifferentiated cells, PBX1 is bound to the H1-compacted promoter/proximal enhancer of the neuron-specific gene doublecortin (Dcx) Once differentiation is induced, MEIS associates with chromatin-bound PBX1, recruits PARP1/ARTD1, and initiates PARP1-mediated eviction of H1 from the chromatin fiber. These results for the first time link MEIS proteins to PARP-regulated chromatin dynamics and provide a mechanistic basis to explain the profound cellular changes elicited by these proteins.

MicroRNA-98 Attenuates Cell Migration and Invasion in Glioma by Directly Targeting Pre-B Cell Leukemia Homeobox 3.

Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults. The extraordinary invasion of human GBM into adjacent normal brain tissues contributes to treatment failure. However, the mechanisms that control this process remain poorly understood. Increasing evidence has demonstrated that microRNAs are strongly implicated in the migration and invasion of GBM. In this study, we found that microRNA-98 (miR-98) was markedly downregulated in human glioma tissues and cell lines. Functional experiments indicated that restored expression of miR-98 attenuated glioma cell invasion and migration, whereas depletion of miR-98 promoted glioma cell invasion and migration. Subsequent investigation showed that pre-B-cell leukemia homeobox 3 (PBX3), an important transcription factor that controls tumor invasion, was a direct and functional target of miR-98 in GBM cells. Consistently, an orthotopic mouse model also demonstrated the suppressive effects of miR-98 overexpression on tumor invasion and PBX3 expression. Silencing of PBX3 using small interfering RNA inhibited the migratory and invasive capacities of glioma cells, whereas reintroduction of PBX3 into glioma cells reversed the anti-invasive function of miR-98. Furthermore, depletion of PBX3 phenocopied the effects of miR-98 overexpression in vivo. Finally, quantitative real-time polymerase chain reaction results showed that miR-98 was negatively correlated with PBX3 expression in 24 glioma tissues. Thus, we propose that PBX3 modulation by miR-98 has an important role in regulating GBM invasion and may serve as therapeutic target for GBM.

A PBX1 transcriptional network controls dopaminergic neuron development and is impaired in Parkinson's disease.

Pre-B-cell leukemia homeobox (PBX) transcription factors are known to regulate organogenesis, but their molecular targets and function in midbrain dopaminergic neurons (mDAn) as well as their role in neurodegenerative diseases are unknown. Here, we show that PBX1 controls a novel transcriptional network required for mDAn specification and survival, which is sufficient to generate mDAn from human stem cells. Mechanistically, PBX1 plays a dual role in transcription by directly repressing or activating genes, such as Onecut2 to inhibit lateral fates during embryogenesis, Pitx3 to promote mDAn development, and Nfe2l1 to protect from oxidative stress. Notably, PBX1 and NFE2L1 levels are severely reduced in dopaminergic neurons of the substantia nigra of Parkinson's disease (PD) patients and decreased NFE2L1 levels increases damage by oxidative stress in human midbrain cells. Thus, our results reveal novel roles for PBX1 and its transcriptional network in mDAn development and PD, opening the door for new therapeutic interventions.

PBX3 is a putative biomarker of aggressive prostate cancer.

There is a great need to identify new and better prognostic and predictive biomarkers to stratify prostate cancer patients for optimal treatment. The aims of this study were to characterize the expression profile of pre-B cell leukemia homeobox (PBX) transcription factors in prostate cancer with an emphasis on investigating whether PBX3 harbours any prognostic value. The expression profile of PBX3 and PBX1 in prostate tissue was determined by immunohistochemical and immunoblot analysis. Furthermore, the expression of PBX3 transcript variants was analyzed by RT-PCR, NanoString Technologies®, and by analyzing RNA sequence data. The potential of PBX3 to predict prognosis, either at mRNA or protein level, was studied in four independent cohorts. PBX3 was mainly expressed in the nucleus of normal prostate basal cells, while it showed cytosolic expression in prostatic intraepithelial neoplasia and cancer cells. We detected four PBX3 transcript variants in prostate tissue. Competing risk regression analysis revealed that high PBX3 expression was associated with slower progression to castration resistant prostate cancer (sub-hazard ratio (SHR) 0.18, 95% CI: 0.081-0.42, p values < 0.001). PBX3 expression had a high predictive accuracy (area under the curve (AUC) = 0.82) when combined with Gleason score and age. Patients undergoing radical prostatectomy, with high levels of PBX3 mRNA, had improved prostate cancer specific survival compared to patients expressing low levels (SHR 0.21, 95% CI: 0.46-0.93, p values < 0.001, and AUC = 0.75). Our findings strongly indicate that PBX3 has potential as a biomarker, both as part of a larger gene panel and as an immunohistochemical marker, for aggressive prostate cancer.

Transcription factor PREP1 induces EMT and metastasis by controlling the TGF-β-SMAD3 pathway in non-small cell lung adenocarcinoma.

Pre-B-cell leukemia homeobox (Pbx)-regulating protein-1 (Prep1) is a ubiquitous homeoprotein involved in early development, genomic stability, insulin sensitivity, and hematopoiesis. Previously we have shown that Prep1 is a haploinsufficient tumor suppressor that inhibits neoplastic transformation by competing with myeloid ecotropic integration site 1 for binding to the common heterodimeric partner Pbx1. Epithelial-mesenchymal transition (EMT) is controlled by complex networks of proinvasive transcription factors responsive to paracrine factors such as TGF-β. Here we show that, in addition to inhibiting primary tumor growth, PREP1 is a novel EMT inducer and prometastatic transcription factor. In human non-small cell lung cancer (NSCLC) cells, PREP1 overexpression is sufficient to trigger EMT, whereas PREP1 down-regulation inhibits the induction of EMT in response to TGF-β. PREP1 modulates the cellular sensitivity to TGF-β by inducing the small mothers against decapentaplegic homolog 3 (SMAD3) nuclear translocation through mechanisms dependent, at least in part, on PREP1-mediated transactivation of a regulatory element in the SMAD3 first intron. Along with the stabilization and accumulation of PBX1, PREP1 induces the expression of multiple activator protein 1 components including the proinvasive Fos-related antigen 1 (FRA-1) oncoprotein. Both FRA-1 and PBX1 are required for the mesenchymal changes triggered by PREP1 in lung tumor cells. Finally, we show that the PREP1-induced mesenchymal transformation correlates with significantly increased lung colonization by cells overexpressing PREP1. Accordingly, we have detected PREP1 accumulation in a large number of human brain metastases of various solid tumors, including NSCLC. These findings point to a novel role of the PREP1 homeoprotein in the control of the TGF-β pathway, EMT, and metastasis in NSCLC.

Potential role of hematopoietic pre-B-cell leukemia transcription factor-interacting protein in oral carcinogenesis.

Hematopoietic pre-B-cell leukemia transcription factor-interacting protein (HPIP) is a corepressor of pre-B-cell leukemia homeobox (PBX) 1 and is known to play a role in hematopoiesis. Recently, HPIP was demonstrated to promote breast cancer cell proliferation and hepatocellular carcinoma growth. Moreover, it has been revealed that homeobox and PBX proteins, the expression of which is regulated by HPIP, play key roles in cancer of various organs, including oral squamous cell carcinoma (OSCC). Nevertheless, there has not been any study regarding the role of HPIP in OSCC. This study investigated the expression of HPIP in normal oral mucosa, epithelial precursor lesion (OEPL), and OSCC, and the functional roles of HPIP in OSCC cells and normal keratinocytes. Immunohistochemical analysis of HPIP, Ki-67, and involucrin was performed in OSCC specimens, and the change in involucrin expression following RNA interference treatment against HPIP was examined by quantitative RT-PCR and Western blot analysis in SCC9 and NHEK cells undergoing extracellular calcium-induced differentiation. Matrigel transwell and cell proliferation assays for both cell lines transfected with HPIP siRNA were also conducted. HPIP expression increased in OEPL and OSCC specimens. In vitro analysis revealed that HPIP suppressed differentiation and proliferation of SCC9 cells and transwell migration of NHEK cells, while HPIP promoted invasion of SCC9 and proliferation of NHEK cells. However, HPIP has no significant effect on NHEK cell differentiation. HPIP may play a critical role in oral carcinogenesis and is thus a potential target for anticancer therapy, with particular emphasis on its involvement in differentiation and migration/metastasis.