Effects Of HOXB4 Research Articles

HOXB4 promotes the malignant progression of ovarian cancer via DHDDS

BackgroundHomeobox B4 (HOXB4) is correlated with poor prognosis of various cancer types. However, how HOXB4 promotes ovarian cancer (OV) progression remains unclear.MethodsThe Cancer Genome Atlas (TCGA) database indicated that a high level of HOXB4 in OV was correlated with poor prognosis. The biological functions of HOXB4 were confirmed by colony formation, migration, and invasion assays. The effect of HOXB4 on the expression of EMT cell markers was determined. The transcriptional target of HOXB4 was DHDDS, which was detected by a ChIP assay. A xenograft tumor model was generated in nude mice to detect the role of HOXB4 in tumor proliferation and metastasis.ResultsThe results showed that HOXB4 protein levels were higher in OV tissues than in normal tissues and correlated with poor prognosis of OV. HOXB4 reduction inhibited the proliferation and invasion ability of OV cells in vitro. Conversely, these effects were enhanced by the upregulation of HOXB4 in OV cells. The binding of HOXB4 to two DNA motifs regulated DHDDS expression and contributed to the malignant progression of OV. The role of HOXB4 in contributing to tumor development in vivo was verified in mice. Further results indicated that HOXB4 induced Snail and Zeb1 expression.ConclusionOverall, HOXB4 overexpression was remarkably correlated with poor prognosis of OV. Mechanistically, HOXB4 enhances the proliferation and invasion of tumor cells by activating DHDDS, thereby promoting the malignant progression of OV.

The HOXB4 Homeoprotein Differentially Promotes Ex Vivo Expansion of Early Human Lymphoid Progenitors

The HOXB4 homeoprotein is known to promote the expansion of mouse and human hematopoietic stem cells (HSCs) and progenitors of the myeloid lineages. However, the putative involvement of HOXB4 in lymphopoiesis and particularly in the expansion of early lymphoid progenitor cells has remained elusive. Based on the ability of the HOXB4 protein to passively enter hematopoietic cells, our group previously designed a long-term culture procedure of human HSCs that allows ex vivo expansion of these cells. Here, this method has been further used to investigate whether HOXB4 could cause similar expansion on cells originating from CD34(+) hematopoietic progenitor cells (HPCs) committed at various levels toward the lymphoid lineages. We provide evidence that HOXB4 protein delivery promotes the expansion of primitive HPCs that generate lymphoid progenitors. Moreover, HOXB4 acts on lymphomyeloid HPCs and committed T/natural killer HPCs but not on primary B-cell progenitors. Our results clarify the effect of HOXB4 in the early stages of human lymphopoiesis, emphasizing the contribution of this homeoprotein in the maintenance of the intrinsic lymphomyeloid differentiation potential of defined HPC subsets. Finally, this study supports the potential use of HOXB4 protein for HSC and HPC expansion in a therapeutic setting and furthers our understanding of the mechanisms of the molecular regulation of hematopoiesis.

Thrombopoietin and the Hematopoietic Stem Cell

The molecular mechanisms that underlie the favorable effects of thrombopoietin on stem cell survival, self-renewal, and expansion are unknown. On the basis of known effects of HoxB4, HoxA9, and vascular endothelial cell growth factor on stem cells, we explored whether TPO might affect these pathways. We found that TPO enhances the transcription of HoxB4, induces the nuclear localization of HoxA9, and upregulates VEGF expression by reducing the ubiquination of hypoxia inducible factor.

456. HOXB4 Overexpression Expands Short-Term Repopulating Cells in Nonhuman Primates and Dogs

Human HOXB4 overexpression is able to extensively expand hematopoietic repopulating stem cells in murine models. Here we examined the effect of HOXB4 in two commonly used large-animal models using competitive repopulation assay. Three macaques and five dogs were transplanted with peripheral blood CD34+ cells transduced with a HOXB4/GFP or a control YFP gammaretroviral vector after lethal irradiation. Two dogs and one monkey died from transplant related complications. Follow-ups for the other three dogs and two monkeys are eight months and nine months, respectively. For all animals pretransplant gene transfer efficiencies in CD34+ cells were similar between HOXB4/GFP and YFP transduced cells: 45% (range 36-55%) and 38% (range 36- 40%) in macaque cells, and 48% (range 23-69%) and 51% (range 31-62%) in dog cells. In all 3 monkeys the initial gene transfer levels in vivo one week after transplantation were higher with the control YFP vector (average, 32% vs 25%). A similar trend was observed for three of the five dogs (average, 21% vs 11% for YFP vs GFP). In all the animals, dogs and monkeys, we observed a dramatic increase in HOXB4/GFP marked cells from 5-30% to 10-62% during the early engraftment period resulting in a significant difference between HOXB4/GFP and YFP marked granulocytes. The peak ratio between HOXB4-overexpressing to YFP-expressing in the three surviving dogs was approximately four at 5-6 weeks post-transplant. In the monkeys the peak ratio was up to ten at 5 weeks post-transplant. Interestingly, in all the dogs the HOXB4/GFP transduced cells decreased to within 2 fold of the control vector by week 15. Taqman PCR analysis of samples at the latest time point in all animals confirmed that the decrease in gene-marked cells was not due to transgene silencing. Subset analysis showed that the ratios of HOXB4-overexpressing cells to YFP-expressing cells were obviously higher for granulocytes than for lymphocytes in macaques and dogs, indicating that HOXB4 overexpression may inhibit lymphoid differentiation or/and promote higher level expansion of myeloid progenitors than lymphoid progenitors. In conclusion, our preclinical studies show that HOXB4 overexpression in CD34+ cells can expand hematopoietic cells that boost short-term engraftment.

705. AMD3100-Mobilized CD34+ Cells Are Phenotypically Different and Better Targets for Retroviral Transduction Than G-CSF-Mobilized CD34+ Cells in Rhesus Macaques

Successful ex vivo and in vivo expansion of transgene modified hematopoietic stem cells (HSC) have great application potential for clinical gene therapy. Overexpression of the human HoxB4 gene induces ex vivo expansion and self-renewal of murine HSC. Since mouse and human differ substantially, we have started to study the effect of HoxB4 on HSC in a large animal model. Here we investigated if human HoxB4 enhances ex vivo expansion and in vivo selection of CD34+ cells from mobilized dog peripheral blood (PB), cytokine primed baboon and macaque bone marrow (BM), human cord blood (CB) and mobilized human PB. CD34+ cells were transduced with Phoenix-GALV or RD114-pseudotype MSCV-HoxB4-ires-GFP or the control vector, MSCV-ires-YFP. After 4 weeks culture, the percentage of HoxB4-overexpressing cells significantly increased for dog, baboon and macaque cells. HoxB4 also imparts a noteworthy effect on expansion of progenitors. HoxB4 expression resulted in an up to 2-fold expansion for human PB, 4-fold expansion for human CB, 5 to 7-fold expansion for baboon and macaque, and 28-fold expansion for dog cells in CFUs when compared with YFP-transduced cells. Furthermore, in vivo competitive repopulation experiments in NOD/SCID mouse model were performed to test the effects of HoxB4 on HSC. The engraftment level for GFP+ cells was 6-fold higher for PB cells, and 10-fold higher for CB cells compared with YFP+ cells, indicating a significant growth advantage of HoxB4-transduced cells. To examine whether HoxB4 expression promotes expansion of HSC in large animal model, myeloblated dogs were transplanted with HoxB4- and YFP- transduced cells. Preliminary data show that the ratio of GFP+ cells to YFP+ cells increased from 0.9 at day 0 to ratio of 9 at day 20 post-transplant, demonstrating a remarkable in vivo selection of HoxB4-transduced cells in clinically relevant model. To explore the mechanisms that underlie the apparently more pronounced effects on cells from dog, macaque and baboon compared to human cells, we monitored the HoxB4 expression in these cells at the RNA level by real-time RT-PCR and at the protein level by western blot. Comparable level of HoxB4 and YFP transcripts was detected in dog and mouse transduced cells, while copies of HoxB4 transcripts were only 10–30% those of YFP transcripts for human and baboon transduced cells. Likewise, HoxB4 protein expression in human and baboon cells decreased continuously with time, in parallel with the differentiation of these cells. However, protein expression in transduced mouse and dog cells was stable, which was associated with inhibition of cell differentiation. We also found that HoxB4 was tyrosine phosphorylated in transduced mouse and dog cells. Our results suggest that the higher level of HoxB4 protein expressed inhibit the differentiation and promote self-renewal of HSC, while lower level confers a less pronounced effects on self-renewal. Further insights into the regulation of transduced HoxB4 in human cells might pave the road to its successful application in clinical gene therapy.