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


METHODS
Expression 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.


RESULTS
We 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.


CONCLUSIONS
These findings indicate that DLX4 promotes ovarian tumor angiogenesis in part by stimulating iNOS expression.
Department of Molecular and Cellular Oncology, University of Texas MD Anderson Cancer Center, Houston, TX, 77030, USA. bqtrinh@mdanderson.org.
Bon
B
Trinh
Department of Molecular and Cellular Oncology, University of Texas MD Anderson Cancer Center, Houston, TX, 77030, USA. sko@mdanderson.org.
Song Yi
SY
Ko
Department of Molecular and Cellular Oncology, University of Texas MD Anderson Cancer Center, Houston, TX, 77030, USA. DDHaria@mdanderson.org.
Dhwani
D
Haria
Department of Molecular and Cellular Oncology, University of Texas MD Anderson Cancer Center, Houston, TX, 77030, USA. ngbareng@mdanderson.org.
Nicolas
N
Barengo
Department of Molecular and Cellular Oncology, University of Texas MD Anderson Cancer Center, Houston, TX, 77030, USA. hnaora@mdanderson.org.
Honami
H
Naora
D000818:Animals; D001201:Ascites / Q000473:pathology; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D042783:Endothelial Cells / Q000378:metabolism; D004790:Enzyme Induction; D005260:Female; D018398:Homeodomain Proteins / Q000378:metabolism*; D006801:Humans; D008819:Mice, Nude; D009389:Neovascularization, Pathologic / Q000201:enzymology* / Q000473:pathology; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism*; D010051:Ovarian Neoplasms / Q000098:blood supply* / Q000201:enzymology* / Q000473:pathology; D050794:STAT1 Transcription Factor / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism*; D042461:Vascular Endothelial Growth Factor A / Q000096:biosynthesis; D023041:Xenograft Model Antitumor Assays
C105304:DLX4 protein, human; D018398:Homeodomain Proteins; D050794:STAT1 Transcription Factor; C494079:STAT1 protein, human; D014157:Transcription Factors; D042461:Vascular Endothelial Growth Factor A; C496319:NOS2 protein, human; D052247:Nitric Oxide Synthase Type II