Notch3 interactome analysis identified WWP2 as a negative regulator of Notch3 signaling in ovarian cancer.

The Notch3 signaling pathway is thought to play a critical role in cancer development, as evidenced by the Notch3 amplification and rearrangement observed in human cancers. However, the molecular mechanism by which Notch3 signaling contributes to tumorigenesis is largely unknown. In an effort to identify the molecular modulators of the Notch3 signaling pathway, we screened for Notch3-intracellular domain (N3-ICD) interacting proteins using a human proteome microarray. Pathway analysis of the Notch3 interactome demonstrated that ubiquitin C was the molecular hub of the top functional network, suggesting the involvement of ubiquitination in modulating Notch3 signaling. Thereby, we focused on functional characterization of an E3 ubiquitin-protein ligase, WWP2, a top candidate in the Notch3 interactome list. Co-immunoprecipitation experiments showed that WWP2 interacted with N3-ICD but not with intracellular domains from other Notch receptors. Wild-type WWP2 but not ligase-deficient mutant WWP2 increases mono-ubiquitination of the membrane-tethered Notch3 fragment, therefore attenuating Notch3 pathway activity in cancer cells and leading to cell cycle arrest. The mono-ubiquitination by WWP2 may target an endosomal/lysosomal degradation fate for Notch3 as suggested by the fact that the process could be suppressed by the endosomal/lysosomal inhibitor. Analysis of The Cancer Genome Atlas dataset showed that the majority of ovarian carcinomas harbored homozygous or heterozygous deletions in WWP2 locus, and there was an inverse correlation in the expression levels between WWP2 and Notch3 in ovarian carcinomas. Furthermore, ectopic expression of WWP2 decreased tumor development in a mouse xenograft model and suppressed the Notch3-induced phenotypes including increase in cancer stem cell-like cell population and platinum resistance. Taken together, our results provide evidence that WWP2 serves as a tumor suppressor by negatively regulating Notch3 signaling in ovarian cancer.

Genome-wide reprogramming of the chromatin landscape underlies endocrine therapy resistance in breast cancer.

The estrogen receptor (ER)α drives growth in two-thirds of all breast cancers. Several targeted therapies, collectively termed endocrine therapy, impinge on estrogen-induced ERα activation to block tumor growth. However, half of ERα-positive breast cancers are tolerant or acquire resistance to endocrine therapy. We demonstrate that genome-wide reprogramming of the chromatin landscape, defined by epigenomic maps for regulatory elements or transcriptional activation and chromatin openness, underlies resistance to endocrine therapy. This annotation reveals endocrine therapy-response specific regulatory networks where NOTCH pathway is overactivated in resistant breast cancer cells, whereas classical ERα signaling is epigenetically disengaged. Blocking NOTCH signaling abrogates growth of resistant breast cancer cells. Its activation state in primary breast tumors is a prognostic factor of resistance in endocrine treated patients. Overall, our work demonstrates that chromatin landscape reprogramming underlies changes in regulatory networks driving endocrine therapy resistance in breast cancer.

Discovery of MK-1084: An Orally Bioavailable and Low-Dose KRASG12C Inhibitor.

Oncogenic mutations in the RAS gene account for 30% of all human tumors; more than 60% of which present as KRAS mutations at the hotspot codon 12. After decades of intense pursuit, a covalent inhibition strategy has enabled selective targeting of this previously "undruggable" target. Herein, we disclose our journey toward the discovery of MK-1084, an orally bioavailable and low-dose KRASG12C covalent inhibitor currently in phase I clinical trials (NCT05067283). We leveraged structure-based drug design to identify a macrocyclic core structure, and hypothesis-driven optimization of biopharmaceutical properties to further improve metabolic stability and tolerability.

Sequential and gamma-secretase-dependent processing of the betacellulin precursor generates a palmitoylated intracellular-domain fragment that inhibits cell growth.

Betacellulin (BTC) belongs to the family of epidermal growth factor (EGF)-like growth factors that are expressed as transmembrane precursors and undergo proteolytic ectodomain shedding to release soluble mature ligands. BTC is a dual-specificity ligand for ErbB1 and ErbB4 receptors, and can activate unique signal-transduction pathways that are beneficial for the function, survival and regeneration of pancreatic beta-cells. We have previously shown that BTC precursor (proBTC) is cleaved by ADAM10 to generate soluble ligand and a stable, transmembrane remnant (BTC-CTF). In this study, we analyzed the fate of the BTC-CTF in greater detail. We demonstrated that proBTC is cleaved by ADAM10 to produce BTC-CTF, which then undergoes intramembrane processing by presenilin-1- and/or presenilin-2-dependent gamma-secretase to generate an intracellular-domain fragment (BTC-ICD). We found that the proBTC cytoplasmic domain is palmitoylated and that palmitoylation is not required for ADAM10-dependent cleavage but is necessary for the stability and gamma-secretase-dependent processing of BTC-CTF to generate BTC-ICD. Additionally, palmitoylation is required for nuclear-membrane localization of BTC-ICD, as demonstrated by the redistribution of non-palmitoylated BTC-ICD mutant to the nucleoplasm. Importantly, a novel receptor-independent role for BTC-ICD signaling is suggested by the ability of BTC-ICD to inhibit cell growth in vitro.

Discovery and characterization of novel peptide inhibitors of the NRF2/MAFG/DNA ternary complex for the treatment of cancer.

Pathway activating mutations of the transcription factor NRF2 and its negative regulator KEAP1 are strongly correlative with poor clinical outcome with pemetrexed/carbo(cis)platin/pembrolizumab (PCP) chemo-immunotherapy in lung cancer. Despite the strong genetic support and therapeutic potential for a NRF2 transcriptional inhibitor, currently there are no known direct inhibitors of the NRF2 protein or its complexes with MAF and/or DNA. Herein we describe the design of a novel and high-confidence homology model to guide a medicinal chemistry effort that resulted in the discovery of a series of peptides that demonstrate high affinity, selective binding to the Antioxidant Response Element (ARE) DNA and thereby displace NRF2-MAFG from its promoter, which is an inhibitory mechanism that to our knowledge has not been previously described. In addition to their activity in electrophoretic mobility shift (EMSA) and TR-FRET-based assays, we show significant dose-dependent ternary complex disruption of NRF2-MAFG binding to DNA by SPR, as well as cellular target engagement by thermal destabilization of HiBiT-tagged NRF2 in the NCI-H1944 NSCLC cell line upon digitonin permeabilization, and SAR studies leading to improved cellular stability. We report the characterization and unique profile of lead peptide 18, which we believe to be a useful in vitro tool to probe NRF2 biology in cancer cell lines and models, while also serving as an excellent starting point for additional in vivo optimization toward inhibition of NRF2-driven transcription to address a significant unmet medical need in non-small cell lung cancer (NSCLC).

Discovery of cell active macrocyclic peptides with on-target inhibition of KRAS signaling.

Macrocyclic peptides have the potential to address intracellular protein-protein interactions (PPIs) of high value therapeutic targets that have proven largely intractable to small molecules. Here, we report broadly applicable lessons for applying this modality to intracellular targets and specifically for advancing chemical matter to address KRAS, a protein that represents the most common oncogene in human lung, colorectal and pancreatic cancers yet is one of the most challenging targets in human disease. Specifically, we focused on KRpep-2d, an arginine-rich KRAS-binding peptide with a disulfide-mediated macrocyclic linkage and a protease-sensitive backbone. These latter redox and proteolytic labilities obviated cellular activity. Extensive structure-activity relationship studies involving macrocyclic linker replacement, stereochemical inversion, and backbone α-methylation, gave a peptide with on-target cellular activity. However, we uncovered an important generic insight - the arginine-dependent cell entry mechanism limited its therapeutic potential. In particular, we observed a strong correlation between net positive charge and histamine release in an ex vivo assay, thus making this series unsuitable for advancement due to the potentially fatal consequences of mast cell degranulation. This observation should signal to researchers that cationic-mediated cell entry - an approach that has yet to succeed in the clinic despite a long history of attempts - carries significant therapy-limiting safety liabilities. Nonetheless, the cell-active molecules identified here validate a unique inhibitory epitope on KRAS and thus provide valuable molecular templates for the development of therapeutics that are desperately needed to address KRAS-driven cancers - some of the most treatment-resistant human malignancies.

De-risking Drug Discovery of Intracellular Targeting Peptides: Screening Strategies to Eliminate False-Positive Hits.

Nonspecific promiscuous compounds can mislead researchers and waste significant resources. This phenomenon, though well-documented for small molecules, has not been widely explored for the peptide modality. Here we demonstrate that two purported peptide-based KRas inhibitors, SAH-SOS1 A and cyclorasin 9A5, exemplify false-positive molecules-in terms of both their binding affinities and cellular activities. Through multiple gold-standard biophysical techniques, we unambiguously show that both peptides lack specific binding to KRas and instead induce protein unfolding. Although these peptides inhibited cellular proliferation, the activities appeared to be off-target on the basis of a counterscreen with KRas-independent cell lines. We further demonstrate that their cellular activities are derived from membrane disruption. Accordingly, we propose that to de-risk false-positive molecules, orthogonal binding assays and cellular counterscreens are indispensable.

Brain development in mice lacking L1-L1 homophilic adhesion.

A new mouse line has been produced in which the sixth Ig domain of the L1 cell adhesion molecule has been deleted. Despite the rather large deletion, L1 expression is preserved at normal levels. In vitro experiments showed that L1-L1 homophilic binding was lost, along with L1-alpha5beta1 integrin binding. However, L1-neurocan and L1-neuropilin binding were preserved and sema3a responses were intact. Surprisingly, many of the axon guidance defects present in the L1 knockout mice, such as abnormal corticospinal tract and corpus callosum, were not observed. Nonetheless, when backcrossed on the C57BL/6 strain, a severe hydrocephalus was observed and after several generations, became an embryonic lethal. These results imply that L1 binding to L1, TAG-1, or F3, and L1-alpha5beta1 integrin binding are not essential for normal development of a variety of axon pathways, and suggest that L1-L1 homophilic binding is important in the production of X-linked hydrocephalus.

Malignant ascites-derived exosomes of ovarian carcinoma patients contain CD24 and EpCAM.

OBJECTIVE: CD24 is an established marker for poor prognosis in ovarian and other carcinomas. Acquisition of cytoplasmic CD24, as opposed to membranous expression, has been correlated with a higher invasiveness of tumor cells. Exosomes are small vesicles of endosomal origin that are often secreted by tumor cells. Given the emerging role of exosomes in tumor progression, we investigated whether cytoplasmic CD24 expression is correlated with the secretion of CD24 in exosomes. METHODS: We used CD24 transfected carcinoma cell lines, ovarian carcinoma cell lines and malignant ascites fluid of ovarian carcinoma patients. Exosomes were isolated via ultracentrifugation and sucrose density fractionation and subsequently examined by Western blot analysis and gelatine zymography. In tissue sections CD24 was detected by immunohistochemical staining. RESULTS: We show that CD24 is released by transfected as well as endogenously expressing cells in vesicles that represent exosomes. CD24 was also identified in exosomes isolated from ascites fluid of ovarian carcinoma patients. In 16 ovarian carcinomas analyzed no correlation between CD24 in tumor tissue sections and the appearance of CD24 in exosomes was detected. Furthermore, we provide evidence that the epithelial cell adhesion molecule (EpCAM), known to be overexpressed in ovarian carcinomas, is secreted in exosomes. The ascites exosomes contain gelatinolytic enzymes. CONCLUSIONS: Our study identifies CD24 and EpCAM as cargo proteins of exosomes of cultured cell lines and malignant ascites. The exosome-associated proteolytic activity in the tumor vicinity might augment tumor invasion into the stroma.

A role for exosomes in the constitutive and stimulus-induced ectodomain cleavage of L1 and CD44.

Ectodomain shedding is a proteolytic mechanism by which transmembrane molecules are converted into a soluble form. Cleavage is mediated by metalloproteases and proceeds in a constitutive or inducible fashion. Although believed to be a cell-surface event, there is increasing evidence that cleavage can take place in intracellular compartments. However, it is unknown how cleaved soluble molecules get access to the extracellular space. By analysing L1 (CD171) and CD44 in ovarian carcinoma cells, we show in the present paper that the cleavage induced by ionomycin, APMA (4-aminophenylmercuric acetate) or MCD (methyl-beta-cyclodextrin) is initiated in an endosomal compartment that is subsequently released in the form of exosomes. Calcium influx augmented the release of exosomes containing functionally active forms of ADAM10 (a disintegrin and metalloprotease 10) and ADAM17 [TACE (tumour necrosis factor a-converting enzyme)] as well as CD44 and L1 cytoplasmic cleavage fragments. Cleavage could also proceed in released exosomes, but only depletion of ADAM10 by small interfering RNA blocked cleavage under constitutive and induced conditions. In contrast, cleavage of L1 in response to PMA occurred at the cell surface and was mediated by ADAM17. We conclude that different ADAMs are involved in distinct cellular compartments and that ADAM10 is responsible for shedding in vesicles. Our findings open up the possibility that exosomes serve as a platform for ectodomain shedding and as a vehicle for the cellular export of soluble molecules.

Exosomes: from biogenesis and secretion to biological function.

Exosomes are small microvesicles that are released from late endosomal compartments of cultured cells. Recent work has shown that exosome-like vesicles are also found in many body fluids such as blood, urine, ascites and amnionic fluid. Although the biological function of exosomes is far from being fully understood, exosomes may have general importance in cell biology and immunology. The present review aims to address some of the facets of exosome research with particular emphasis on the immunologist's perspective: (i) exosomes as a novel platform for the ectodomain shedding of membrane proteins by ADAMs and (ii) recent findings on the role of exosomes in tumor biology and immune regulation.

L1 on ovarian carcinoma cells is a binding partner for Neuropilin-1 on mesothelial cells.

The progression of ovarian cancer is driven by a variety of cellular factors that are incompletely understood. Binding of tumor cells to normal cells and to soluble factors influence tumor growth, angiogenesis and the stimulation of vascular permeability leading to ascites production. L1 adhesion molecule is overexpressed in ovarian carcinoma and is associated with bad prognosis. One receptor for L1 is Neuropilin-1 (NRP-1) that is also known as a receptor for VEGF(165). In the nervous system a complex of NRP-1 and L1 transmits signals by the neurorepellant Sem3A that is critical for the control of neurite outgrowth. NRP-1 has also been detected in human carcinomas but its function remains unknown. Here, we have examined NRP-1 expression in ovarian carcinoma cell lines and tissue. We report that little NRP-1 protein was detected in primary ovarian carcinoma tissues or established cell lines although mRNA for soluble and transmembrane NRP-1 were detected by RT-PCR. Instead, we observed strong expression of NRP-1 in mesothelial cells, which form the lining of the peritoneum. NRP-1 could serve as an isolation marker for primary mesothelial cells present in ascites fluid. We demonstrate that ovarian cancer cells expressing L1 can bind to NRP-1 overexpressing cells and mesothelial cells. Likewise, soluble L1 isolated from ascites of patients or produced as a fusion protein could bind to NRP-1 overexpressing cells and a direct interaction was demonstrated at the protein level. These findings suggest that L1 can support the binding of ovarian carcinoma cells to mesothelial cells via NRP-1. The L1-NRP-1 binding pathway could contribute to the growth of ovarian carcinomas and to reciprocal signalling between mesothelial cells and tumors.

Cleavage of L1 in exosomes and apoptotic membrane vesicles released from ovarian carcinoma cells.

PURPOSE: The L1 adhesion molecule (CD171) is overexpressed in human ovarian and endometrial carcinomas and is associated with bad prognosis. Although expressed as a transmembrane molecule, L1 is released from carcinoma cells in a soluble form. Soluble L1 is present in serum and ascites of ovarian carcinoma patients. We investigated the mode of L1 cleavage and the function of soluble L1. EXPERIMENTAL DESIGN: We used ovarian carcinoma cell lines and ascites from ovarian carcinoma patients to analyze soluble L1 and L1 cleavage by Western blot analysis and ELISA. RESULTS: We find that in ovarian carcinoma cells the constitutive cleavage of L1 proceeds in secretory vesicles. We show that apoptotic stimuli like C2-ceramide, staurosporine, UV irradiation, and hypoxic conditions enhance L1-vesicle release resulting in elevated levels of soluble L1. Constitutive cleavage of L1 is mediated by a disintegrin and metalloproteinase 10, but under apoptotic conditions multiple metalloproteinases are involved. L1 cleavage occurs in two types of vesicles with distinct density features: constitutively released vesicles with similarity to exosomes and apoptotic vesicles. Both types of L1-containing vesicles are present in the ascites fluids of ovarian carcinoma patients. Soluble L1 from ascites is a potent inducer of cell migration and can trigger extracellular signal-regulated kinase phosphorylation. CONCLUSIONS: We suggest that tumor-derived vesicles may be an important source for soluble L1 that could regulate tumor cell function in an autocrine/paracrine fashion.

High Levels of Expression of P-glycoprotein/Multidrug Resistance Protein Result in Resistance to Vintafolide.

Targeting surface receptors overexpressed on cancer cells is one way to specifically treat cancer versus normal cells. Vintafolide (EC145), which consists of folate linked to a cytotoxic small molecule, desacetylvinblastine hydrazide (DAVLBH), takes advantage of the overexpression of folate receptor (FR) on cancer cells. Once bound to FR, vintafolide enters the cell by endocytosis, and the reducing environment of the endosome cleaves the linker, releasing DAVLBH to destabilize microtubules. Vintafolide has shown efficacy and improved tolerability compared with DAVLBH in FR-positive preclinical models. As the first FR-targeting drug to reach the clinic, vintafolide has achieved favorable responses in phase II clinical trials in FR-positive ovarian and lung cancer. However, some FR-positive patients in these clinical trials do not respond to vintafolide. We sought to identify potential biomarkers of resistance to aid in the future development of this and other FR-targeting drugs. Here, we confirm that high P-glycoprotein (P-gp) expression was the strongest predictor of resistance to DAVLBH in a panel of 359 cancer cell lines. Furthermore, targeted delivery of DAVLBH via the FR, as in vintafolide, fails to overcome P-gp-mediated efflux of DAVLBH in both in vitro and in vivo preclinical models. Therefore, we suggest that patients whose tumors express high levels of P-gp be excluded from future clinical trials for vintafolide as well as other FR-targeted therapeutics bearing a P-gp substrate. Mol Cancer Ther; 15(8); 1998-2008. ©2016 AACR.

L1 expression as a predictor of progression and survival in patients with uterine and ovarian carcinomas.

BACKGROUND: Ovarian and uterine carcinomas are the most common cause of cancer-related deaths in gynecological malignant diseases. We aimed to assess whether the L1 adhesion molecule, an important mediator for cell migration for neural and tumour cells, is expressed in these carcinomas. METHODS: We investigated L1 expression by immunohistochemistry, RT-PCR, and Western blot analysis of tumour samples. Soluble L1 in the serum was detected by ELISA and immunoprecipitation. FINDINGS: We detected the L1 adhesion molecule in ovarian and uterine tumours in a stage-dependent manner. In a retrospective study L1 was found in 46 of 58 ovarian carcinomas and 20 of 72 uterine adenocarcinomas. L1 expression was an excellent predictor of poor outlook (p<0.00001). Patients with L1 positive uterine tumours were at high risk for progression even in the endometrioid-type tumours, which usually have a favourable prognosis. In uterine tumours, expression of L1 in curettage samples enabled us to identify aggressive tumours before the operation. Soluble L1 was specifically detected in serum samples from patients with ovarian and uterine tumours. ADAM10, which was implicated in previous studies as L1 sheddase, was expressed in tumours in which soluble L1 was present in the serum. INTERPRETATION: L1 is overexpressed in ovarian and uterine carcinomas and is associated with short survival. L1 can serve as a new marker for prediction of clinical outcome and could be helpful to identify patients with uterine tumours who are at high risk for recurrent disease. L1 expression and cleavage could promote dissemination of tumours by facilitating cell migration.

ADAM10-mediated cleavage of L1 adhesion molecule at the cell surface and in released membrane vesicles.

Cells can release membrane components in a soluble form and as membrane vesicles. L1, an important molecule for cell migration of neural and tumor cells, is released by membrane-proximal cleavage, and soluble L1 promotes cell migration. Release of L1 is enhanced by shedding inducers such as phorbol ester and pervanadate, but it is also enhanced by depletion of cellular cholesterol with methyl-beta-cyclodextrin (MCD). How such different compounds can induce shedding is presently unknown. We show here that ADAM10 is involved in L1 cleavage, which occurs at the cell surface and in the Golgi apparatus. MCD and pervanadate treatment induced the release of microvesicles containing full-length L1 and the active form of ADAM10. L1 cleavage occurred in isolated vesicles. L1-containing microvesicles could trigger haptotactic cell migration. Only the neural L1 form carrying the RSLE signal for clathrin-dependent endocytosis was recruited and cleaved in vesicles. Phorbol ester treatment activated L1 cleavage predominantly at the cell surface. Our results provide evidence for two pathways of L1 cleavage, based on ADAM10 localization, that can be activated differentially: 1) direct cleavage at the cell surface, and 2) release and cleavage in secretory vesicles most likely derived from the Golgi apparatus. The findings establish a novel role for ADAM10 as a vesicle-based protease.

Discovery of biomarkers predictive of GSI response in triple-negative breast cancer and adenoid cystic carcinoma.

UNLABELLED: Next-generation sequencing was used to identify Notch mutations in a large collection of diverse solid tumors. NOTCH1 and NOTCH2 rearrangements leading to constitutive receptor activation were confined to triple-negative breast cancers (TNBC; 6 of 66 tumors). TNBC cell lines with NOTCH1 rearrangements associated with high levels of activated NOTCH1 (N1-ICD) were sensitive to the gamma-secretase inhibitor (GSI) MRK-003, both alone and in combination with paclitaxel, in vitro and in vivo, whereas cell lines with NOTCH2 rearrangements were resistant to GSI. Immunohistochemical staining of N1-ICD in TNBC xenografts correlated with responsiveness, and expression levels of the direct Notch target gene HES4 correlated with outcome in patients with TNBC. Activating NOTCH1 point mutations were also identified in other solid tumors, including adenoid cystic carcinoma (ACC). Notably, ACC primary tumor xenografts with activating NOTCH1 mutations and high N1-ICD levels were sensitive to GSI, whereas N1-ICD-low tumors without NOTCH1 mutations were resistant. SIGNIFICANCE: NOTCH1 mutations, immunohistochemical staining for activated NOTCH1, and HES4 expression are biomarkers that can be used to identify solid tumors that are likely to respond to GSI-based therapies.

Gauging NOTCH1 Activation in Cancer Using Immunohistochemistry.

Fixed, paraffin-embedded (FPE) tissues are a potentially rich resource for studying the role of NOTCH1 in cancer and other pathologies, but tests that reliably detect activated NOTCH1 (NICD1) in FPE samples have been lacking. Here, we bridge this gap by developing an immunohistochemical (IHC) stain that detects a neoepitope created by the proteolytic cleavage event that activates NOTCH1. Following validation using xenografted cancers and normal tissues with known patterns of NOTCH1 activation, we applied this test to tumors linked to dysregulated Notch signaling by mutational studies. As expected, frequent NICD1 staining was observed in T lymphoblastic leukemia/lymphoma, a tumor in which activating NOTCH1 mutations are common. However, when IHC was used to gauge NOTCH1 activation in other human cancers, several unexpected findings emerged. Among B cell tumors, NICD1 staining was much more frequent in chronic lymphocytic leukemia than would be predicted based on the frequency of NOTCH1 mutations, while mantle cell lymphoma and diffuse large B cell lymphoma showed no evidence of NOTCH1 activation. NICD1 was also detected in 38% of peripheral T cell lymphomas. Of interest, NICD1 staining in chronic lymphocytic leukemia cells and in angioimmunoblastic lymphoma was consistently more pronounced in lymph nodes than in surrounding soft tissues, implicating factors in the nodal microenvironment in NOTCH1 activation in these diseases. Among carcinomas, diffuse strong NICD1 staining was observed in 3.8% of cases of triple negative breast cancer (3 of 78 tumors), but was absent from 151 non-small cell lung carcinomas and 147 ovarian carcinomas. Frequent staining of normal endothelium was also observed; in line with this observation, strong NICD1 staining was also seen in 77% of angiosarcomas. These findings complement insights from genomic sequencing studies and suggest that IHC staining is a valuable experimental tool that may be useful in selection of patients for clinical trials.

Defining NOTCH3 target genes in ovarian cancer.

NOTCH3 gene amplification plays an important role in the progression of many ovarian and breast cancers, but the targets of NOTCH3 signaling are unclear. Here, we report the use of an integrated systems biology approach to identify direct target genes for NOTCH3. Transcriptome analysis showed that suppression of NOTCH signaling in ovarian and breast cancer cells led to downregulation of genes in pathways involved in cell-cycle regulation and nucleotide metabolism. Chromatin immunoprecipitation (ChIP)-on-chip analysis defined promoter target sequences, including a new CSL binding motif (N1) in addition to the canonical CSL binding motif, that were occupied by the NOTCH3/CSL transcription complex. Integration of transcriptome and ChIP-on-chip data showed that the ChIP target genes overlapped significantly with the NOTCH-regulated transcriptome in ovarian cancer cells. From the set of genes identified, we showed that the mitotic apparatus organizing protein DLGAP5 (HURP/DLG7) was a critical target. Both the N1 motif and the canonical CSL binding motif were essential to activate DLGAP5 transcription. DLGAP5 silencing in cancer cells suppressed tumorigenicity and inhibited cellular proliferation by arresting the cell cycle at the G(2)-M phase. In contrast, enforced expression of DLGAP5 partially counteracted the growth inhibitory effects of a pharmacologic or RNA interference-mediated NOTCH inhibition in cancer cells. Our findings define direct target genes of NOTCH3 and highlight the role of DLGAP5 in mediating the function of NOTCH3.

Identification of PBX1 target genes in cancer cells by global mapping of PBX1 binding sites.

PBX1 is a TALE homeodomain transcription factor involved in organogenesis and tumorigenesis. Although it has been shown that ovarian, breast, and melanoma cancer cells depend on PBX1 for cell growth and survival, the molecular mechanism of how PBX1 promotes tumorigenesis remains unclear. Here, we applied an integrated approach by overlapping PBX1 ChIP-chip targets with the PBX1-regulated transcriptome in ovarian cancer cells to identify genes whose transcription was directly regulated by PBX1. We further determined if PBX1 target genes identified in ovarian cancer cells were co-overexpressed with PBX1 in carcinoma tissues. By analyzing TCGA gene expression microarray datasets from ovarian serous carcinomas, we found co-upregulation of PBX1 and a significant number of its direct target genes. Among the PBX1 target genes, a homeodomain protein MEOX1 whose DNA binding motif was enriched in PBX1-immunoprecipicated DNA sequences was selected for functional analysis. We demonstrated that MEOX1 protein interacts with PBX1 protein and inhibition of MEOX1 yields a similar growth inhibitory phenotype as PBX1 suppression. Furthermore, ectopically expressed MEOX1 functionally rescued the PBX1-withdrawn effect, suggesting MEOX1 mediates the cellular growth signal of PBX1. These results demonstrate that MEOX1 is a critical target gene and cofactor of PBX1 in ovarian cancers.