SUMMARY While VEGF-targeted therapies are showing promise, new angiogenesis targets are needed to make additional gains. Here, we show that increased Zeste homologue 2 (EZH2) expression in either tumor cells or in tumor vasculature is predictive of poor clinical outcome. The increase in endothelial EZH2 is a direct result of VEGF stimulation by a paracrine circuit that promotes angiogenesis by methylating and silencing vasohibin1 (VASH1). EZH2 silencing in the tumor-associated endothelial cells inhibited angiogenesis mediated by reactivation of VASH1, and reduced ovarian cancer growth, which is further enhanced in combination with EZH2 silencing in tumor cells. Collectively, these data support the potential for targeting EZH2 as an important therapeutic approach. SIGNIFICANCE In this work, we identify EZH2 as a key regulator of tumor angiogenesis. The increase in endothelial EZH2 is a direct result of VEGF stimulation and indicates the presence of a paracrine circuit that promotes angiogenesis. EZH2 silencing in the tumor-associated endothelial cells using siRNA, packaged in the chitosan delivery system, resulted in significant growth inhibition in an orthotopic ovarian cancer model. EZH2 silencing in tumor endothelial cells resulted in decreased angiogenesis that was mediated by increased levels of the angiogenesis inhibitor, vasohibin1 (VASH1). Combined, these data provide a significant conceptual advance in our understanding of the regulation of angiogenesis in ovarian carcinoma and support the potential for targeting EZH2 as a therapeutic approach.

SUMMARY IκB kinase β (IKKβ) is involved in tumor development and progression through activation of the nuclear factor (NF)–κB pathway. However, the molecular mechanism that regulates IKKβ degradation remains largely unknown. Here, we show that a Cullin 3 (CUL3)–based ubiquitin ligase, Kelch-like ECH-associated protein 1 (KEAP1), is responsible for IKKβ ubiquitination. Depletion of KEAP1 led to the accumulation and stabilization of IKKβ and to up-regulation of NF-κB–derived tumor angiogenic factors. A systematic analysis of the CUL3, KEAP1, and RBX1 genomic loci revealed a high percentage of genome loss and missense mutations in human cancers that failed to facilitate IKKβ degradation. Our results suggest that the dysregulation of KEAP1-mediated IKKβ ubiquitination may contribute to tumorigenesis.

Poly (ADP-ribose) polymerase (PARP) inhibitors have emerged as promising therapeutics for many diseases, including cancer, in clinical trials1. One PARP inhibitor, olaparib (Lynparza™, AstraZeneca), was recently approved by the FDA to treat ovarian cancer with BRCA mutations. BRCA1 and BRCA2 play essential roles in repairing DNA double strand breaks, and a deficiency of BRCA proteins sensitizes cancer cells to PARP inhibition2,3. Here we show that receptor tyrosine kinase c-Met associates with and phosphorylates PARP1 at Tyr907. Phosphorylation of PARP1 Tyr907 increases PARP1 enzymatic activity and reduces binding to a PARP inhibitor, thereby rendering cancer cells resistant to PARP inhibition. Combining c-Met and PARP1 inhibitors synergized to suppress growth of breast cancer cells in vitro and xenograft tumor models. Similar synergistic effects were observed in a lung cancer xenograft tumor model. These results suggest that PARP1 pTyr907 abundance may predict tumor resistance to PARP inhibitors, and that treatment with a combination of c-Met and PARP inhibitors may benefit patients bearing tumors with high c-Met expression who do not respond to PARP inhibition alone.

EGF induces the translocation of EGF receptor (EGFR) from the cell surface to the nucleus where EGFR activates gene transcription through its binding to an AT-rich sequence (ATRS) of the target gene promoter. However, how EGFR, without a DNA-binding domain, can bind to the gene promoter is unclear. In the present study, we show that RNA helicase A (RHA) is an important mediator for EGFRinduced gene transactivation. EGF stimulates the interaction of EGFR with RHA in the nucleus of cancer cells. The EGFR/RHA complex then associates with the target gene promoter through binding of RHA to the ATRS of the target gene promoter to activate its transcription. Knockdown of RHA expression in cancer cells abrogates the binding of EGFR to the target gene promoter, thereby reducing EGF/EGFR-induced gene expression. In addition, interruption of EGFR-RHA interaction decreases the EGFR-induced promoter activity. Consistently, we observed a positive correlation of the nuclear expression of EGFR, RHA, and cyclin D1 in human breast cancer samples. These results indicate that RHA is a DNA-binding partner for EGFR-mediated transcriptional activation in the nucleus.cyclin D1 | nuclear translocation | inducible nitric oxide synthase | transcription C ell surface EGF receptor (EGFR) has been shown to be localized in the nucleus (1-4). Nuclear EGFR has been demonstrated to contribute to cancer cell resistance to cetuximab and radiation treatment (5, 6) and to be negatively correlated with overall survival of patients with multiple cancer types (7-11). Moreover, nuclear EGFR interacts with signal transducer and activator of transcription 3 (STAT3), signal transducer and activator of transcription 5A (STAT5A), E2F1, DNA-dependent protein kinase (DNA-PK), and proliferating cell nuclear antigen (PCNA) and plays important roles in cell transformation, proliferation, and DNA repair and replication (12-16). Nuclear EGFR regulates gene expression by binding to an AT-rich sequence (ATRS) of the gene's promoter (13,16,17). Additionally, a recent unbiased protein-DNA interactome study indicates that EGFR is a DNAbinding protein (18). However, EGFR does not contain a DNAbinding domain, and evidence supporting direct binding of EGFR to the specific DNA sequence is lacking. Thus, identifying the DNA-binding partner for EGFR is crucial for understanding how EGFR regulates gene transcription in the nucleus.RNA helicase A (RHA), the human homolog of Drosophila maleless (MLE) that increases the transcription of male X-linked genes (19), is a multifunctional protein and is conserved in Drosophila and mammals (20)(21)(22). RHA belongs to the aspartateglutamate-alanine-aspartate (DEAD) box family of proteins and has the ability to bind to RNA and DNA (23,24). RHA regulates gene transcription by interacting with transcription factors (22) or by binding directly to the target gene promoter (25). Moreover, Drosophila MLE activates rox2 transcription by binding to an ATrich region of the gene promoter (26). Interestingly, this AT-rich region contains the previo...

Purpose Results of multiple clinical trials suggest that EGFR tyrosine kinase inhibitors (TKIs) exhibit negative effects on platinum-based chemotherapy in lung cancer patients with wild type (wt) EGFR, but the underlying molecular mechanisms are still uncertain. Studies that identify the mechanism of how TKIs negatively affect patients with wt EGFR are important for future development of effective strategies to target lung cancer. Thus, we returned to in vitro study to investigate and determine a possible explanation for this phenomenon. Experimental Design We investigated the effects of TKIs and cisplatin on caspase-independent cell death (CID) and the role of CID in the efficacy of each drug and the combination. Furthermore, we studied the mechanism how EGFR signaling pathway is involved in CID. Finally, based on the identified mechanism, we tested the combinational effects of cisplatin plus SAHA or erastin on CID. Results We found that gefitinib inhibited cisplatin-induced CID but not caspase-dependent apoptotic cell death. In wt EGFR cells, gefitinib not only inhibited CID but also failed to induce apoptosis, therefore, compromising the efficacy of cisplatin. Inhibition of EGFR-ERK/AKT by gefitinib activates FOXO3a which in turn reduces reactive oxygen species (ROS) and ROS-mediated CID. To overcome this, we showed that SAHA and erastin, the inducers of ROS-mediated CID, strongly enhance the effect of cisplatin in wt EGFR cells. Conclusion TKI-mediated inhibition of CID plays an important role of the efficacy of chemotherapy. Moreover, FOXO3a is a key factor in the negative effects of TKI by eliminating cisplatin-induced ROS.