Bon Q. Trinh

Homeodomain protein DLX4 counteracts key transcriptional control mechanisms of the TGF-Β cytostatic program and blocks the antiproliferative effect of TGF-Β

The antiproliferative activity of transforming growth factor-β (TGF-β) is essential for maintaining normal tissue homeostasis and is lost in many types of tumors. Gene responses that are central to the TGF-β cytostatic program include activation of the cyclin-dependent kinase inhibitors, p15Ink4B and p21WAF1/Cip1, and repression of c-myc. These gene responses are tightly regulated by a repertoire of transcription factors that include Smad proteins and Sp1. The DLX4 homeobox patterning gene encodes a transcription factor that is absent from most normal adult tissues, but is expressed in a wide variety of malignancies, including lung, breast, prostate and ovarian cancers. In this study, we demonstrate that DLX4 blocks the antiproliferative effect of TGF-β. DLX4 inhibited TGF-β-mediated induction of p15Ink4B and p21WAF1/Cip1 expression. DLX4 bound and prevented Smad4 from forming complexes with Smad2 and Smad3, but not with Sp1. However, DLX4 also bound and inhibited DNA-binding activity of Sp1. In addition, DLX4 induced expression of c-myc independently of TGF-β/Smad signaling. The ability of DLX4 to counteract key transcriptional control mechanisms of the TGF-β cytostatic program could explain, in part, the resistance of tumors to the antiproliferative effect of TGF-β.

Dual Functions of the Homeoprotein DLX4 in Modulating Responsiveness of Tumor Cells to Topoisomerase II-Targeting Drugs

Topoisomerase II (TOP2)-targeting poisons such as anthracyclines and etoposide are commonly used for cancer chemotherapy and kill tumor cells by causing accumulation of DNA double-strand breaks (DSB). Several lines of evidence indicate that overexpression of TOP2A, the gene encoding topoisomerase IIα, increases sensitivity of tumor cells to TOP2 poisons, but it is not clear why some TOP2A-overexpressing (TOP2A-High) tumors respond poorly to these drugs. In this study, we identified that TOP2A expression is induced by DLX4, a homeoprotein that is overexpressed in breast and ovarian cancers. Analysis of breast cancer datasets revealed that TOP2A-high cases that also highly expressed DLX4 responded more poorly to anthracycline-based chemotherapy than TOP2A-high cases that expressed DLX4 at low levels. Overexpression of TOP2A alone in tumor cells increased the level of DSBs induced by TOP2 poisons. In contrast, DLX4 reduced the level of TOP2 poison-induced DSBs irrespective of its induction of TOP2A. DLX4 did not stimulate homologous recombination-mediated repair of DSBs. However, DLX4 interacted with Ku proteins, stimulated DNA-dependent protein kinase activity, and increased erroneous end-joining repair of DSBs. Whereas DLX4 did not reduce levels of TOP2 poison-induced DSBs in Ku-deficient cells, DLX4 stimulated DSB repair and reduced the level of TOP2 poison-induced DSBs when Ku was reconstituted in these cells. Our findings indicate that DLX4 induces TOP2A expression but reduces sensitivity of tumor cells to TOP2 poisons by stimulating Ku-dependent repair of DSBs. These opposing activities of DLX4 could explain why some TOP2A-overexpressing tumors are not highly sensitive to TOP2 poisons.

The homeoprotein DLX4 controls inducible nitric oxide synthase-mediated angiogenesis in ovarian cancer.

BackgroundHomeobox genes encode transcription factors that control patterning of virtually all organ systems including the vasculature. Tumor angiogenesis is stimulated by several homeobox genes that are overexpressed in tumor cells, but the mechanisms of these genes are poorly understood. In this study, we investigated the mechanisms by which DLX4, a homeobox gene that is associated with increased tumor microvessel density, stimulates ovarian tumor angiogenesis.MethodsExpression of DLX4 and nitric oxide synthases was analyzed in publicly available transcriptional profiles of ovarian cancer clinical specimens. Levels of inducible nitric oxide synthase (iNOS) were evaluated by quantitative RT-PCR, flow cytometry and nitric oxide assays using ovarian cancer cell lines in which DLX4 was overexpressed or knocked down. Signal Transducer and Activator of Transcription 1 (STAT1) expression and activity were evaluated by luciferase reporter assays, immunofluorescence staining, Western blot and immunoprecipitation. Endothelial cell growth and tumor angiogenesis were evaluated in in vitro assays and xenograft models.ResultsWe identified that DLX4 induces expression of iNOS, an enzyme that stimulates angiogenesis by generating nitric oxide. Analysis of datasets of two independent patient cohorts revealed that high DLX4 expression in ovarian cancer is strongly associated with elevated expression of iNOS but not of other nitric oxide synthases. Studies using STAT1-expressing and STAT1-deficient cells revealed that DLX4 interacts with STAT1 and induces iNOS expression in part by stimulating STAT1 activity. Expression of DLX4 in ovarian cancer cells stimulated endothelial cell growth in vitro and increased microvessel density in xenograft models, and these stimulatory effects of DLX4 were abrogated when its induction of iNOS was inhibited.ConclusionThese findings indicate that DLX4 promotes ovarian tumor angiogenesis in part by stimulating iNOS expression.

The homeobox gene DLX4 regulates erythro-megakaryocytic differentiation by stimulating IL-1β and NF-κB signaling.

ABSTRACT Megakaryocyte and erythroid development are tightly controlled by a repertoire of cytokines, but it is not clear how cytokine-activated signaling pathways are controlled during development of these two lineages. Here, we identify that expression of DLX4, a transcription factor encoded by a homeobox gene, increases during megakaryopoiesis but decreases during erythropoiesis. Enforced expression of DLX4 in CD34+ stem and progenitor cells and in bipotent K562 cells induced lineage markers and morphologic features of megakaryocytes and repressed erythroid marker expression and hemoglobin levels. Converse results were obtained when DLX4 was knocked down. Gene Ontology and Gene Set Enrichment Analyses of genome-wide changes in gene expression revealed that DLX4 induces a megakaryocytic transcriptional program and inhibits an erythroid transcriptional program. DLX4 also induced gene signatures that are associated with nuclear factor κB (NF-κB) signaling. The ability of DLX4 to promote megakaryocyte development at the expense of erythroid generation was diminished by blocking NF-κB activity or by repressing IL1B, a transcriptional target of DLX4. Collectively, our findings indicate that DLX4 exerts opposing effects on the megakaryocytic and erythroid lineages in part by inducing IL-1β and NF-κB signaling. Summary: The homeobox gene DLX4 promotes megakaryocyte development at the expense of erythroid generation by inducing IL-1 and NF-κB signaling.

Abstract 731: Transcriptional control of inflammatory signaling in the megakaryocyte and erythroid lineages: implications for the tumor microenvironment

Thrombocytosis, characterized by high platelet counts, and anemia, caused by low numbers of red blood cells, are associated with poor outcomes in various solid tumors and also occur in several myeloid disorders. The megakaryocyte and erythroid lineages give rise to platelets and red blood cells, respectively, but the mechanisms that cause increased megakaryopoiesis and concurrently decreased erythropoiesis in malignancies are poorly understood. We and others have previously identified that DLX4, a developmental transcription factor encoded by a homeobox gene, is aberrantly expressed in several types of solid tumors and myeloid malignancies but the mechanisms of DLX4 are poorly understood. In this study, we identified that DLX4 expression increases during megakaryopoiesis and decreases during erythropoiesis. Overexpression and knockdown experiments using normal CD34+ stem/progenitor cells and bipotent K562 cells demonstrated that DLX4 induces lineage markers and morphologic features of megakaryocytes, and concomitantly represses erythroid marker expression and hemoglobin levels. Gene Ontology and Gene Set Enrichment Analyses of genome-wide changes in gene expression induced by DLX4 showed that DLX4 induces a megakaryocytic transcriptional program and inhibits an erythroid transcriptional program. Bioinformatic analysis also revealed that DLX4 induces gene signatures that are associated with NF-kappaB signaling. The ability of DLX4 to promote megakaryocyte development and to inhibit erythroid differentiation was diminished by blocking NF-kappaB activity or by repressing IL1B, a transcriptional target of DLX4. These findings indicate that DLX4 promotes megakaryocyte development at the expense of erythroid generation in part by inducing inflammatory signaling. Studies are ongoing to investigate the transcriptional control by homeobox genes of inflammatory signaling in thrombocytosis and anemia that are associated with solid tumors and myeloid disorders. Citation Format: Bon Q. Trinh, Nicolas Barengo, Sang Bae Kim, Ju-Seog Lee, Patrick A. Zweidler-McKay, Honami Naora. Transcriptional control of inflammatory signaling in the megakaryocyte and erythroid lineages: implications for the tumor microenvironment. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 731.

Abstract A25: Transcriptional control of inflammatory signaling in the megakaryocyte and erythroid lineages

Thrombocytosis, characterized by high platelet counts, and anemia, caused by low numbers of red blood cells, are associated with poor outcomes in solid tumors and occur in several types of myeloid disorders. The megakaryocyte and erythroid lineages give rise to platelets and red blood cells, respectively, but the mechanisms that cause increased megakaryopoiesis and concurrently decreased erythropoiesis are poorly understood. In this study, we identified that expression of DLX4, a transcription factor encoded by a homeobox gene, increases during megakaryopoiesis and decreases during erythropoiesis. Overexpression and knockdown experiments using normal CD34+ stem/progenitor cells and bipotent K562 cells demonstrated that DLX4 induces lineage markers and morphologic features of megakaryocytes, and concomitantly represses erythroid marker expression and hemoglobin levels. Gene Ontology and Gene Set Enrichment Analyses of genome-wide changes in gene expression induced by DLX4 showed that DLX4 induces a megakaryocytic transcriptional program and inhibits an erythroid transcriptional program. Bioinformatic analysis also revealed that DLX4 induces gene signatures that are associated with NF-kappaB signaling. The ability of DLX4 to promote megakaryocyte development and to inhibit erythroid differentiation was diminished by blocking NF-kappaB activity or by repressing IL1B, a transcriptional target of DLX4. These findings indicate that DLX4 promotes megakaryocyte development at the expense of erythroid generation in part by inducing inflammatory signaling. Studies are ongoing to investigate the transcriptional control by homeobox genes of inflammatory signaling in thrombocytosis and anemia that are associated with solid tumors and myeloproliferative diseases. Citation Format: Bon Q. Trinh, Nicolas Barengo, Sang Bae Kim, Ju-Seog Lee, Patrick A. Zweidler-McKay, Honami Naora. Transcriptional control of inflammatory signaling in the megakaryocyte and erythroid lineages. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr A25.

Abstract 4176: The homeobox gene DLX4 stimulates inducible nitric oxide synthase-mediated angiogenesis in ovarian cancer

Increasing evidence indicates that the free radical gas nitric oxide plays an important role in stimulating angiogenesis. However, the mechanisms that control nitric oxide-mediated angiogenesis in tumors are poorly understood. We previously identified that DLX4, a transcription factor encoded by a homeobox gene, stimulates ovarian tumor progression and correlates with poor outcomes of ovarian cancer patients. In this study, we identified that high DLX4 expression in ovarian cancer specimens is strongly associated with elevated expression of inducible nitric oxide synthase (iNOS), an enzyme that generates nitric oxide. DLX4 induced iNOS expression in ovarian cancer cells in vitro and in xenograft models. We identified that DLX4 interacts with STAT1 and induces iNOS expression in a STAT1-dependent manner. In in vitro studies, inhibition of iNOS abrogated the stimulatory effects of DLX4 on nitric oxide production, vascular endothelial growth factor expression and endothelial cell growth. Furthermore, inhibition of iNOS abrogated the stimulatory effects of DLX4 on tumor microvessel density and ascites formation in ovarian cancer xenograft models. Collectively, our findings indicate that DLX4 contributes to ovarian tumor progression in part by promoting nitric oxide-mediated angiogenesis. This is the first report that functionally links the aberrant expression of a developmental patterning gene in tumors with free radical gas generation. Note: This abstract was not presented at the meeting. Citation Format: Bon Q. Trinh, Song Yi Ko, Dhwani Haria, Nicolas Barengo, Honami Naora. The homeobox gene DLX4 stimulates inducible nitric oxide synthase-mediated angiogenesis in ovarian cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4176. doi:10.1158/1538-7445.AM2015-4176

Homeobox Genes and Their Functional Significance in Ovarian Tumorigenesis

It is widely recognized that many pathways that control normal embryonic patterning are deregulated in human cancers. Mutations or aberrant expression of components of the Wnt, Hedgehog and Notch signaling pathways have been demonstrated to play pivotal roles in tumorigenesis. Homeobox genes constitute an evolutionarily conserved gene super-family that represents another important class of patterning regulators. These genes encode transcription factors that are essential for controlling cell differentiation and specification of the body plan during embryonic development. Although many homeobox genes have been reported to be aberrantly expressed in ovarian cancer, the functional significance of these genes in ovarian tumorigenesis has only emerged in recent years. This chapter discusses recent research studies that demonstrate that homeobox genes have diverse functions in the biology of ovarian cancer. These functions include specifying patterns of histologic differentiation of ovarian cancers, controlling growth and survival of tumor cells, and promoting tumor angiogenesis, cell-cell interactions and tumor cell invasiveness. This chapter discusses how studies of homeobox genes provide insights into our understanding of the cell-of-origin of ovarian cancers, the striking morphologic heterogeneity of these tumors, and the unique clinical behavior of ovarian cancer.

Abstract B15: The homeobox patterning gene DLX4 confers resistance to transforming growth factor-β signaling in tumors

Rationale: Transforming growth factor-β (TGF-β) inhibits cell proliferation and also induces epithelial-to-mesenchymal transition (EMT). The antiproliferative effect of TGF-β is orchestrated by a cytostatic program of gene responses that induce G 1 arrest and are controlled by Smad-dependent mechanisms, whereas TGF-β induces EMT by both Smad and non-Smad mechanisms. In contrast to normal cells, many tumors are resistant to the antiproliferative effect of TGF-β. This resistance has been attributed to TGF-β receptor or Smad mutations in several types of tumors, particularly those of gastrointestinal origin. However, the rareness of these mutations in many other tumors (e.g., prostate, lung cancers) indicates that resistance to TGF-β also stems from other aberrations. The transcription factor encoded by the homeobox gene DLX4 is aberrantly expressed in a wide range of malignancies. Increasing evidence indicates that crosstalk between members of the TGF super-family and DLX genes is important for controlling normal embryonic patterning. The objective of this study is to determine whether and how DLX4 modulates the TGF-β signaling pathway in tumors. Experimental procedure: The effect of DLX4 on TGF-β-mediated growth inhibition and EMT was investigated by cell cycle and morphological analyses in different types of nontumorigenic and malignant epithelial cells. Molecular mechanisms of DLX4 in modulating TGF-β signaling were determined by protein and chromatin immunoprecipitation and analysis of transcriptional activity of TGF-β target genes. Results: Our studies demonstrate that DLX4 blocks the antiproliferative effect of TGF-β by disabling key transcriptional control mechanisms of the TGF-β cytostatic program. In particular, DLX4 blocked the ability of TGF-β to induce p15 Ink4B and p21 WAF1/Cip1 transcription by directly interacting with Smad4 and Sp1. Binding of DLX4 to Smad4 prevented Smad4 from forming transcriptional complexes with Smad2 and Smad3, whereas binding of DLX4 to Sp1 inhibited DNA-binding activity of Sp1. DLX4 also induced expression of c-myc, a repressor of p15 Ink4B and p21 WAF1/Cip1 transcription. On the other hand, DLX4 enabled epithelial cells to retain epithelial morphology and partially prevented TGF-β-mediated downregulation of E-cadherin and induction of N-cadherin. Conclusion: Our studies demonstrate that DLX4 blocks the antiproliferative effect of TGF-β and partially inhibits TGF-β-mediated EMT. The ability of DLX4 to counteract key transcriptional control mechanisms of the TGF-β cytostatic program could explain in part the resistance of tumors to the anti-proliferative effect of TGF-β in the absence of mutations in core components of the TGF-β signaling pathway. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr B15.