Frank Wu

Activity of PXD101, a histone deacetylase inhibitor, in preclinical ovarian cancer studies

Histone deacetylase inhibitors represent a promising new class of anticancer agents. In the current investigation, we examined the activity of PXD101, a potent histone deacetylase inhibitor, used alone or in combination with clinically relevant chemotherapeutics (docetaxel, paclitaxel, and carboplatin), in preclinical in vitro and in vivo models of ovarian cancer. In vitro activity was examined in ovarian cancer and multidrug-resistant cell lines grown in monolayer culture, and in primary clinical ovarian cancer specimens grown in three-dimensional organoid culture. PXD101 was found to inhibit in vitro cancer cell growth at sub- to low micromolar IC50 potency, exhibited synergistic activity when used in combination with relevant chemotherapeutics, and effectively inhibited the growth of multidrug-resistant cells. In vivo, PXD101 displayed single-agent antitumor activity on human A2780 ovarian cancer s.c. xenografts which was enhanced via combination therapy with carboplatin. In support of these findings, PXD101 was shown to increase the acetylation of α-tubulin induced by docetaxel and the phosphorylation of H2AX induced by carboplatin. Taken together, these results support the clinical evaluation of PXD101 used alone or in combination therapy for the treatment of ovarian cancer. [Mol Cancer Ther 2006;5(8):2086–95]

Recombinant semaphorin 6A-1 ectodomain inhibits in vivo growth factor and tumor cell line-induced angiogenesis

The Semaphorins are a large family of transmembrane, GPI-anchored and secreted proteins that play an important role in neuronal and endothelial cell guidance. A human gene related to the class VI Semaphorin family, Semaphorin 6A-1 (Sema 6A-1) was identified by homology-based genomic mining. Recent implication of Sema 3 family members in tumor angiogenesis and our expression analysis of Sema 6A-1 suggested that class VI Semaphorin might effect tumor neovascularization. The mRNA expression of Sema 6A-1 was elevated in several renal tumor tissue samples relative to adjacent non-tumor tissue samples from the same patient. Sema 6A-1 transcript was also expressed in the majority of renal clear cell carcinoma (RCC) cell lines and to a lesser extent in endothelial cells. To test the role of Sema 6A-1 in tumor angiogenesis, we engineered, expressed and purified the Sema 6A-1 soluble extracellular domain (Sema-ECD). The purified Sema-ECD was screened in a variety of endothelial cell-based assay both in vitro and in vivo. In vitro, Sema-ECD blocked VEGF-mediated endothelial cell migration. These effects were explained in part by our observation in endothelial cells that Sema-ECD inhibited VEGF-mediated Src, FAK and ERK phosphorylation. In vivo, mouse Matrigel assays demonstrated that the intraperitoneal administration of recombinant Sema-ECD inhibited both bFGF/VEGF and tumor cell line-induced neovascularization. These findings reveal a novel therapeutic utility for Sema 6A-1 (Sema-ECD) as an inhibitor of growth factor as well as tumor-induced angiogenesis.

YY1 as a regulator of replication-dependent hamster histone H3.2 promoter and an interactive partner of AP-2.

In analyzing cis-regulatory elements important for cell cycle control of the replication-dependent hamster histone H3.2 gene, we discovered a binding site for the transcription factor YY1 embedded within GC-rich sequences between the two tandem CCAAT repeats proximal to the TATA element. Base mutations that specifically eliminated YY1 binding resulted in suppression of the S phase induction of the H3.2 promoter. In addition, we discovered that YY1 is an interactive partner of AP-2, which also binds the H3.2 promoter and regulates its cell cycle-dependent expression. The critical domains for YY1 and AP-2A interaction are mapped, revealing that the N-terminal portion of YY1 (amino acids 1–300) and the DNA-binding/dimerization region of AP-2A are required. Our results suggest that YY1, acting as a transcription factor binding to its site on the promoter, or through protein-protein interaction with AP-2, may be part of a regulatory network including key cell cycle regulators such as c-Myc and Rb in controlling growth- and differentiation-regulated gene expression.

Activator Protein-2 Overexpression Accounts for Increased Insulin Receptor Expression in Human Breast Cancer

Various studies have shown that the insulin receptor (IR) is increased in most human breast cancers, and both ligand-dependent malignant transformation and increased cell growth occur in cultured breast cells overexpressing the IR. However, although numerous in vivo and in vitro observations have indicated an important contributory role for the IR in breast cancer cell biology, the molecular mechanisms accounting for increased IR expression in breast tumors have not previously been elucidated. Herein, we did immunoblot analyses of nuclear protein from cultured breast cancer cells and normal and tumoral tissues from breast cancer patients combined with promoter studies by using a series of human wild-type and mutant IR promoter constructs. We provide evidence that IR overexpression in breast cancer is dependent on the assembly of a transcriptionally active multiprotein-DNA complex, which includes the high-mobility group A1 (HMGA1) protein, the developmentally regulated activator protein-2 (AP-2) transcription factor and the ubiquitously expressed transcription factor Sp1. In cultured breast cancer cells and human breast cancer specimens, the expression of AP-2 was significantly higher than that observed in cells and tissues derived from normal breast, and this overexpression paralleled the increase in IR expression. However, AP-2 DNA-binding activity was undetectable with the IR gene promoter, suggesting that transactivation of this gene by AP-2 might occur indirectly through physical and functional cooperation with HMGA1 and Sp1. Our findings support this hypothesis and suggest that in affected individuals, hyperactivation of the AP-2 gene through the overexpression of IR may play a key role in breast carcinogenesis.

CDP and AP-2 mediated repression mechanism of the replication-dependent hamster histone H3.2 promoter.

The replication‐dependent hamster histone H3.2 promoter contains two tandem CCAAT repeats located upstream of the TATA element. It has been shown that the NF‐Y/CBF complex binds to a single CCAAT motif with high affinity, whereas the CCAAT displacement protein (CDP) binds to at least two CCAAT motifs in close proximity. Here, we report that the two CCAAT motifs within the H3.2 promoter confer transcriptional repression of the promoter during the cell cycle. While we cannot detect direct association of CDP with Rb in vitro, we discover that CDP can bind AP‐2, a ubiquitous factor that interacts with Rb. The interaction domains between CDP and AP‐2 are mapped to the highly conserved cut repeats of CDP as well as the basic and dimerization region of AP‐2. Further, in transfection assays, CDP and AP‐2 act synergistically to suppress the H3.2 promoter. Together, these data support a repression mechanism mediated by CDP and AP‐2 that regulates H3.2 gene expression during the mammalian cell cycle. J. Cell. Biochem. 84: 699–707, 2002.