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

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-1ß and NF-κB signaling.

The homeoprotein DLX4 stimulates NF-κB activation and CD44-mediated tumor-mesothelial cell interactions in ovarian cancer.

Ovarian cancers often highly express inflammatory cytokines and form implants throughout the peritoneal cavity. However, the mechanisms that drive inflammatory signaling and peritoneal metastasis of ovarian cancer are poorly understood. We previously identified that high expression of DLX4, a transcription factor encoded by a homeobox gene, is associated with reduced survival of ovarian cancer patients. In this study, we identified that DLX4 stimulates attachment of ovarian tumor cells to peritoneal mesothelial cells in vitro and increases the numbers of peritoneal implants in xenograft models. DLX4 induced expression of the cell surface molecule CD44 in ovarian tumor cells, and inhibition of CD44 abrogated the ability of DLX4 to stimulate tumor-mesothelial cell interactions. The induction of CD44 by DLX4 was attributed to increased activity of NF-κB that was stimulated by the inflammatory cytokine IL-1ß, a transcriptional target of DLX4. The stimulatory effects of DLX4 on CD44 levels and tumor-mesothelial cell interactions were abrogated when IL-1ß or NF-κB was inhibited in tumor cells. Furthermore, DLX4 expression levels strongly correlated with NF-κB activation and disease stage in clinical specimens of ovarian cancer. Collectively, these findings indicate that DLX4 induces CD44 by stimulating IL-1ß-mediated NF-κB activity, thereby promoting peritoneal metastasis of ovarian cancer.

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. CONCLUSION: These findings indicate that DLX4 promotes ovarian tumor angiogenesis in part by stimulating iNOS expression.

P-cadherin promotes ovarian cancer dissemination through tumor cell aggregation and tumor-peritoneum interactions.

UNLABELLED: More than 60% of patients who are diagnosed with epithelial ovarian cancer (EOC) present with extensive peritoneal carcinomatosis. EOC cells typically disseminate by shedding into the peritoneal fluid in which they survive as multicellular aggregates and then implant onto peritoneal surfaces. However, the mechanism that facilitates aggregation and implantation of EOC cells is poorly understood. The cell adhesion molecule P-cadherin has been reported to be induced during early progression of EOC and to promote tumor cell migration. In this study, P-cadherin not only promoted migration of EOC cells, but also facilitated the assembly of floating EOC cells into multicellular aggregates and inhibited anoikis in vitro. Furthermore, inhibiting P-cadherin by short hairpin RNAs (shRNA) or a neutralizing antibody prevented EOC cells from attaching to peritoneal mesothelial cells in vitro. In mouse intraperitoneal xenograft models of EOC, inhibition of P-cadherin decreased the aggregation and survival of floating tumor cells in ascites and reduced the number of tumor implants on peritoneal surfaces. These findings indicate that P-cadherin promotes intraperitoneal dissemination of EOC by facilitating tumor cell aggregation and tumor-peritoneum interactions in addition to promoting tumor cell migration. IMPLICATIONS: Inhibiting P-cadherin blocks multiple key steps of EOC progression and has therapeutic potential.

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.

HOXA9 promotes ovarian cancer growth by stimulating cancer-associated fibroblasts.

Epithelial ovarian cancers (EOCs) often exhibit morphologic features of embryonic Müllerian duct-derived tissue lineages and colonize peritoneal surfaces that overlie connective and adipose tissues. However, the mechanisms that enable EOC cells to readily adapt to the peritoneal environment are poorly understood. In this study, we show that expression of HOXA9, a Müllerian-patterning gene, is strongly associated with poor outcomes in patients with EOC and in mouse xenograft models of EOC. Whereas HOXA9 promoted EOC growth in vivo, HOXA9 did not stimulate autonomous tumor cell growth in vitro. On the other hand, expression of HOXA9 in EOC cells induced normal peritoneal fibroblasts to express markers of cancer-associated fibroblasts (CAFs) and to stimulate growth of EOC and endothelial cells. Similarly, expression of HOXA9 in EOC cells induced normal adipose- and bone marrow-derived mesenchymal stem cells (MSCs) to acquire features of CAFs. These effects of HOXA9 were due in substantial part to its transcriptional activation of the gene encoding TGF-ß2 that acted in a paracrine manner on peritoneal fibroblasts and MSCs to induce CXCL12, IL-6, and VEGF-A expression. These results indicate that HOXA9 expression in EOC cells promotes a microenvironment that is permissive for tumor growth.

Inhibition of ovarian cancer growth by a tumor-targeting peptide that binds eukaryotic translation initiation factor 4E.

PURPOSE: A critical step of protein synthesis involves the liberation of the mRNA cap-binding translation initiation factor eIF4E from 4EBP inhibitory binding proteins, and its engagement to the scaffolding protein eIF4G. eIF4E is a candidate target for cancer therapy because it is overexpressed or activated in many types of tumors and has tumorigenic properties. Our aim was to design and evaluate 4EBP-based peptides for their antitumor activity in ovarian cancer. EXPERIMENTAL DESIGN: The ability of peptides to bind and inhibit eIF4E was determined by immunoprecipitation and by assaying cap-dependent reporter synthesis. To target ovarian tumors, the lead candidate 4EBP peptide was fused to an analog of gonadotropin-releasing hormone (GnRH). Cellular uptake of peptide, and effects on cell viability and cell death were determined. The antitumor activity of fusion peptide was evaluated in female nude mice bearing i.p. ovarian tumor xenografts. RESULTS: 4EBP-based peptides bound eIF4E, prevented eIF4E from binding eIF4G, and inhibited cap-dependent translation. GnRH agonist-4EBP fusion peptide was taken up by, and inhibited the growth of, GnRH receptor-expressing tumor cells, but not receptor-negative cells. Intraperitoneal tumor burden was significantly smaller in mice treated with fusion peptide than in mice treated with saline (P < 0.001). Ascites was also reduced in peptide-treated mice. Significant cytotoxic effects to host tissues were not observed. On the other hand, treatment with GnRH agonist alone did not inhibit tumor growth or ascites. CONCLUSION: Because ovarian cancer is rarely cured by conventional chemotherapies, GnRH-4EBP fusion peptide may be of therapeutic potential for treatment of this disease.